Optical Biosensing with Nanostructured Surfaces

Universitat de Barcelona Departamet d'Electrònica

Optical Biosensing with Nanostructured Surfaces Presented by: Nasser Darwish Thesis advisor: Mauricio Moreno

Universitat de Barcelona Departamet d'Electrònica

Optical Biosensing with Nanostructured Surfaces Programa de doctorat:

Enginyeria i Tecnologia Electròniques

Bienni 2003/2005 Autor:

Nasser Darwish Miranda

Director de tesi:

Mauricio Moreno Sereno

Tutor de tesi:

Alejandro Pérez Rodríguez

El Dr. Mauricio Moreno Sereno, professor titular de la Facultat de Física de la Universitat de Barcelona, certica: Que la memòria Optical biosensing with Nanostructured Surfaces , que presenta Nasser Darwish Miranda per optar al grau de doctor, s'ha realitzat sota la seva direcció.

El Dr. Alejandro Pérez Rodríguez, catedràtic de la Facultat de Física de la Universitat de Barcelona, certica: Que la memòria Optical biosensing with Nanostructured Surfaces , que presenta Nasser Darwish Miranda per optar al grau de doctor, s'ha realitzat sota la seva tutoria.

Dr. Mauricio Moreno Sereno Director de tesi.

Alejandro Pérez Rodríguez Tutor de tesi.

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Dedicated to Pedro Gómez, my professor of optics

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Acknowledgements I must thank especially the following people for making this personal and professional project possible: My wife and my family, for their condence in the hard moments, Mauricio Moreno, for his support, leadership and, sometimes, patience, our project partners and host Professors for their collaboration and professionality, the Electronics Department, the mechanical workshop, the Physics Faculty, the University of Barcelona and all the people I've probably forgotten.

Supporting projects

Desarrollo de un microsistema biosensor óptico para medida de toxicidad en agua, MEC-CICyT: DPI2003-08060-C03-03. Microsistema multi-biosensor óptico, basado en redes de difracción, para detección de bacterias, MEC-CICyT: TEC2006-13109-C03-03/MIC. Microsistemas biológicos híbridos basados en la integración de dispositivos ópticos e impedimétricos para la detección de patógenos en aguas, MIEN - Ministerio de Industria y Energía: FIT-330100-2005-158 MC2ACCESS program, European contract nr026029, under the FP6 Research Infrastructures Action program.

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I was proceeding down the road. The trees on the right were passing me in orderly fashion at 60 MPH. Suddenly, one of them stepped out in my path. Boom! John Von Neumann

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Preface The word biosensing encompasses a series of measuring techniques devoted for the obteinance of relevant information about processes that take place in living organisms. Along the last decades binding events between biomolecules or between biomolecules and surfaces became intensively studied, thanks to progresses in micro and nanofabrication technologies that provided new insight into observation scales increasingly small. The attention that currently molecular binding events receive is a result of many of the current scientic challenges. From the medical point of view, cell transfection or implant rejection [Kasemo (1998)] start with the adsorption of proteins from the bloodstream. The immunological response also relies on molecular recognition between antigens and their specic antibodies, and this phenomenon is also one of the pillars of drug development [Fang (2006b)] and localized delivery [Jessel (2006)]. Many other areas enjoy the benets from this knowledge. Molecular recognition may also be used as a tool for mapping cell populations or even tissues. Examples of application of these procedures are regenerative medicine and neural computing. In the rst case, it is the ability to control the growth and diversication of stem cells which promises a way for repairing or replacing any damaged tissue or organ. Regarding the second case, neural connectivity studies search ways for repairing the nervous system and for implementing natural structures on biomimetic articial computers [Schmidt (2003)]. Environmental monitoring [Székács (2009); Srivastava (1996)], food safety [Homola (2006); Adányi (2006)] and articial photosynthesis [Bard (1995)] are just a few examples of other non medical applications. Most biorecognition events take place at interfaces, as a cellular membrane or the surface of an implant. Processes at surfaces or between species immersed in liquids are intrinsically dierent. On one hand, the rate of encounter between owing species and a static surface depends on parameters such as ow rate and diusion constants. Free analite to analite hybridization, on the other hand, takes place at the bulk stream and uid dynamics plays a less important role. Methods for monitoring free analite to analite binding existed since the beginnings of the analytical chemistry, but they 7

don't provide ways for distinguishing responses from the surface and from the bulk. These reasons justied the focus of this thesis on approaches for the surface to target problem. Another classication distinguishes between label and label-free systems. Unlike these last ones, which need some kind of sample preparation as the use of uorescent or radioactive tags [Van Weemen (1971); Engvall (1971); Yalow (1960)], label-free systems allow in many cases the direct analysis of raw samples, providing results in minutes instead of days. In all cases, a diculty of the application is the limited and, in most cases undesired recyclability of the sensors. As a consequence, disposable devices are clearly preferred, and the cost per assay becomes an important restriction. Optical detection techniques are intrinsically highly sensitive because of the wave nature of light. The electromagnetic radiation has easily observable properties like wavelength (color) or polarization. These properties have an immediate translation into the nanoscale, and hence represent an excellent link between the observer and the observed processes. Traditionally, optics meant alignment diculties and high precision mechanics. This made the optical alternative much more expensive than other existing techniques, in most cases less sensitive. Nevertheless, alignment gets signicantly simplied in waveguide-based systems, once a way for inserting light into them is provided. The reason is that waveguides provide a way for placing the radiant energy by themselves. Grating Coupling [Hutley (1982)] was a solution proposed for the light insertion problem in the communications environment along the 70s [Rosenblatt (1997)], but it was not adapted to biosensors until one decade after [Tiefenthaler (1989)]. Two great dierences when comparing grating coupling with other techniques are exchangeability and coupling allowance over large areas, which represent a chance for high throughput applications. Although many biosensing techniques have been explored along recent years, there is still room for research. The aim of this thesis is to provide answers to the following questions: a) not always the obtained resolutions or dynamic ranges satisfy the requirements of the applications, b) sampling rates may be too slow depending on the reaction kinetics, c) the cost per assay is too high compared with other techniques, d) the samples must be prepared in advance [Bilitewski (2000)] and e) the validity of the results relies on models instead of evidences [Vörös (2004)]. Along this thesis, dierent sensors based on dielectric and conductive waveguides were developed, together with their corresponding instrumentation. As a summary, this research can be dened as the application of optical (waveguide and diractive) techniques to the analysis of molecular binding events at surfaces. The underlying models are 8

described along chapter 1 in this dissertation. Applicability and reproducibility are key concepts for every sensing device. In this context an applicable technique provides the desered information with enough sensitivity and resolution. This process guarantees protection against the undesired false negative readouts. The optimization of the design parameters (chapter 2) makes this possible. Selectivity is also important, for the prevention of false positives readouts. This depends on surface chemistry, which is beyond the scope of this document. Nevertheless, it is possible to desing sensors that partially overcome this problem. The obtainment of reproducible readouts depends on both a smart operating conguration, for which eventual drifts along the measuring process can be understood and subtracted, and on the fabrication techniques, which should provide reliability and ease of calibration. For the rst problem, several design strategies were proposed and described along chapter 2. Taking into account that the diractive behavior requires sub-micrometric surface patterning, the fabrication must be carried out using nanofabrication techniques. Chapter 3 describes the fabrication of the devices using standard materials and techniques, which allows an accurate control of the results. The use of polymer based techniques is also explored for lowering the costs. The restriction to standard or low-cost nanofabrication technologies did not limit the sensing techniques available. In this research three techniques were studied: a) Optical Grating Coupler Biosensing (OGCB) [Vörös (2002)], b) Waveguide Fluorescence Excitation [Taitt (2005)] and c) Surface Plasmon Resonance (SPR) [Jönsson (1991)]. The developed instrumentation sets these devices up for the analysis of multiple or heterogeneous samples, with adjustable sensing ranges and improved sample rates. In chapter 4 these instruments are described, including the hardware fabricated for sample delivery, the optical arrangement needed by the sensors to operate and the acquisition and analysis tools that were developed. Surface analysis systems provide not only information about the presence of certain substances, but also about their reaction kinetics, which, for instance, allows the study of chemical anities. Kinetics depends also on temperature, sample rates and pH, and hence the described instruments should control these parameters accurately. In chapter 4 the application results obtained from realistic experiments are given, in which the developed instruments analyze buers or biological samples. Conclusions from the readouts are taken, so the ability of these instruments to provide relevant biochemical information is evaluated. 9

The overall performance of the dierent sensors and is nally discussed, together with their applicability for the desired applications, along the applications chapter and the conclusions (chapter 5). Then, the global results from the decisions taken at the development stage are discussed. This document nishes with a brief discussion about the given scientical contributions and future trends derived from them.

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Contents 1. An introduction to evanescent eld biosensing

1.1. Waveguides and optical biosensing . . . . . . . . . . . . . 1.1.1. Total internal reection (TIR) . . . . . . . . . . . . 1.1.2. Constructive self-interference . . . . . . . . . . . . 1.1.3. Eective refractive index . . . . . . . . . . . . . . . 1.1.4. Multilayer structures and numerical solutions . . . 1.1.5. Evanescent eld and system sensitivities . . . . . . 1.1.6. Coupling light into a waveguide . . . . . . . . . . . 1.2. Grating couplers . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. Single and dual grating couplers . . . . . . . . . . 1.2.2. Optical Grating Coupler Biosensors (OGCB) . . . 1.3. Surface Plasmon Resonance . . . . . . . . . . . . . . . . . 1.3.1. Excitation and detection of Surface Plasmons . . . 1.3.2. Surface Plasmon Resonance (SPR) biosensors . . . 1.4. Waveguide Fluorescence Excitation . . . . . . . . . . . . . 1.5. Molecular adlayers . . . . . . . . . . . . . . . . . . . . . . 1.5.1. The refractive index of an adlayer . . . . . . . . . . 1.6. Molecular recognition . . . . . . . . . . . . . . . . . . . . . 1.6.1. Functionalization and specicity . . . . . . . . . . 1.6.2. Information from the adsorption kinetics . . . . . . 1.6.3. Hydrodynamics and random sequential adsorption 1.7. Aims of this thesis . . . . . . . . . . . . . . . . . . . . . .

2. Design of a congurable sensing platform 2.1. Architectural levels . . . . . . . . . . 2.2. Materials and working conditions . . 2.3. Waveguide optimization . . . . . . . 2.3.1. Waveguide thickness . . . . . 2.3.2. Grating - SPR sensors . . . . 2.3.3. Thickness and fault-tolerance 2.3.4. Optical similarity . . . . . . . 2.3.5. Polymers or dielectrics . . . .

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Contents

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2.4. Design of Grating couplers . . . . . . . . . . . . . . . . . . . 75 2.4.1. Grating period: angular interrogation and dielectric OGCBs . . . . . . . . . . . . . . . . . . . . . . . . . 76 2.4.2. Grating period: spectral interrogation and SPR . . 79 2.4.3. Etching depth and duty cycle . . . . . . . . . . . . . 81 2.4.4. Size and distribution of the gratings . . . . . . . . . 84 2.5. Response to molecular adsorption . . . . . . . . . . . . . . 90 2.5.1. Interpretation of the sensor outcome . . . . . . . . . 90 2.5.2. Modeling sensor response . . . . . . . . . . . . . . . 91 2.5.3. Sensing beyond . . . . . . . . . . . . . . . . . . . . . 96 2.5.4. Field distribution and chemical passivation . . . . . 98 2.6. Multisensing and on-chip reference . . . . . . . . . . . . . . 99 2.7. Geometrical design constraints . . . . . . . . . . . . . . . . 100

3. Cost-ecient sensor fabrication and test

3.1. The selection of a technology . . . . . . . . . . . . . . . 3.2. Hard substrate Grating Couplers . . . . . . . . . . . . . 3.2.1. Summary of the technological process . . . . . . 3.2.2. Characterization . . . . . . . . . . . . . . . . . . 3.2.3. Validity of the Equivalent Layer Approximation . 3.3. Embossed biosensing devices . . . . . . . . . . . . . . . . 3.4. Polymer waveguides and grating couplers . . . . . . . . 3.4.1. Characterization . . . . . . . . . . . . . . . . . . 3.5. Embossed Grating SPR sensors . . . . . . . . . . . . . . 3.5.1. Spectral response of the Surface Plasmon Waves

4. Developed instruments and application results

4.1. General instrumentation blocks . . . . . . . . . . . . . . 4.1.1. Sample delivery system . . . . . . . . . . . . . . 4.1.2. Thermal module . . . . . . . . . . . . . . . . . . 4.1.3. Fluidic cell and gasket . . . . . . . . . . . . . . . 4.1.4. Illumination block . . . . . . . . . . . . . . . . . 4.2. Rotary grating couplers . . . . . . . . . . . . . . . . . . 4.2.1. Resonance tracking algorithm . . . . . . . . . . . 4.2.2. Preliminary results . . . . . . . . . . . . . . . . . 4.3. Fixed angle grating coupler . . . . . . . . . . . . . . . . 4.3.1. Multiple self-referenced detection . . . . . . . . . 4.3.2. Channel denition by software . . . . . . . . . . 4.3.3. Improving resolution . . . . . . . . . . . . . . . . 4.3.4. Chemical passivation and local functionalization 4.3.5. Application results . . . . . . . . . . . . . . . . . 12

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4.4. Waveguide uorescence excitation . . . 4.4.1. Modications of the OGCB and 4.5. Low-cost embossed SPR-G systems . . 4.5.1. SPR-G tests . . . . . . . . . . .

5. Conclusions

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5.1. Scientic contributions . . . . . . . . . . 5.1.1. Theory . . . . . . . . . . . . . . . 5.1.2. Design and fabrication . . . . . . 5.1.3. Instrumentation and application 5.2. Sensor comparison . . . . . . . . . . . . 5.3. Future trends. . . . . . . . . . . . . . . .

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A. Overview of the current relevant biointeraction analysis tools

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B. Biosensado óptico mediante supercies nanoestructuradas

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1. An introduction to evanescent eld biosensing The subject of this dissertation can be dened as the use of transduction systems to gather information about biological interactions. Among the different techniques proposed for this problem, in this work the approach based on integrated optics was chosen. The reason is that optics is:

Precise: The light frequency does not experiment changes during the propagation of light across dierent materials, and its wavelength and polarization are preserved in free space. Light remains free of disturbance because it interacts with matter but no with radiation. Finally, the optical paths are perfectly straight in the absence of strong temperature gradients, and ne focusing and collection systems exist and are cost eective. The problem of placing these components precisely is one of the challenges of this approach. Easy: Wavelength is uniquely associated with color and, as it happens to polarization, it can be distinguished with high accuracy using standard components. Optical detectors like photodiodes, PSDs or cameras are available everywhere and provide precise detection at low cost. When the visible range in being used, the optical paths can be observed at simple sight. This advantage has no electronic equivalent. Non-invasive: In the presence of material interfaces light may be reected, transmitted or diracted without altering the material structures. The visible spectrum has no eect on biological tissues, and the amount of radiative heat transfer may also be negligible depending on the requirements. As tissues are generally transparent to certain wavelengths, it is even possible to map organic structures without surgery, or to probe samples without modifying them.

Now that a framework has been proposed, the next paragraphs will dene it with more accuracy. The following questions will help to do it and will be kept in mind from now on:

Why to measure: On one hand, current medicine is the result of an evolution towards the personalized model. This term relies on the as15

CHAPTER 1.

AN INTRODUCTION TO EVANESCENT FIELD BIOSENSING

sistance of objective analyses and the personalization of the therapies. On the other hand, the evidence-based medicine bases diagnostics and therapies on accepted scientic literature. Early diagnosis and prevention of anaphylactic response to drugs are just two examples. Both personalized and evidence-based approaches have in common the use of quantitative tools, which may be either chemical, as clinical analysis or physical, like tomography imaging. These tools are a core part of current medicine, but do not cover many situations yet. As an example, while diabetes is early diagnosed, many genetic diseases are not detected before the apparition of the rst symptoms. Furthermore, a good diagnosis is useless if is obtained too late. The contrast between the eectiveness of the same therapy when is applied to dierent patients gives an idea about how does medicine, pharmacology, or

food safety need analytical tools to satisfy their future challenges. It is accepted that sensors are one of these tools [Saliterman (2006)].

What to measure: This question needs at least three answers. The rst one is obviously what process is being studied, the next is what are the relevant biological parameters that describe it and, as detection will be indirect, the last one is which physical transduction parameter may be used. Along the disease process, the concentration of specic molecules in biological uids and tissues vary in a quantity that is desired to measure. The reaction kinetics is even more relevant as it provides information about how much and in what conditions these substances are involved [Ramsden (1993)]. The process that better provides kinetic information is the surface molecular adsorption, and this is the rst answer. A diagnostic tool of this kind is expected to

detect relevant substances along adsorption events without interference, and in the lowest signicant concentrations that provide a conclusion. Lack of interference means high specicity,

and this problem was left to the end of the chapter. About transduction, the microscopic conguration and whatever sensor outcome need to be conceptually linked. It is clear that molecular adlayers have a thickness, and it is known that their physical parameters depend on their densities. Under these assumptions, and from the optical point of view, two parameters frequently used to study the formation of molecular layers are thickness and refractive index (see, for instance, [Vörös (2004)]).

How to measure: Dierent optical biosensors use dierent ways for putting the light into interaction with the external media and for re16

CHAPTER 1.

AN INTRODUCTION TO EVANESCENT FIELD

BIOSENSING

covering the eect of this interaction. These transduction principles will be studied, initially under the assumption that the other problems are solved. In concrete, Optical Grating Coupler Biosensing (OGCB), Surface Plasmon Resonance (SPR) and Surface Fluorescence Excitation will be described. These techniques are classied as Evanescent eld biosensing [Ramsden (1997)] and use optical waveguides as transduction elements. The need of accuracy is faced by optimization techniques and numerical methods which are also described along this part. Finally, all these methods require a nal answer: the information must be interpreted. Molecular recognition events depend not only on concentrations, but also on temperature, ow rate, diusion constants, molecular anities, and so on. The success of biosensing techniques relies on their ability to provide all these parameters, and this is only possible once the attachment process is well understood. The entire theory of optical biosensors relies on a series of assumptions, which are implicitly present in all the literature. For clarity, these assumptions will be summarized here, but their justication was left to the end of this chapter:

Under certain conditions the molecules adsorbed on a surface can be modeled as a homogeneous layer (adlayer ). The optical and geometrical parameters of such adlayers depend on the concentration of the adsorbed species. At low concentrations the thickness of the adlayer remains constant and the refractive index grows with mass concentration. For higher densities, saturation phenomena and variations of the thickness may appear. By the use of complementary molecular systems, a surface can be prepared for the adsorption of specic species. In such conditions, any observed change in the adlayer is due to specic adsorption.

The a fruitful example of this can be found in [Vörös (2004)]. Under the above assumptions, the following sections show how an adlayer on the surface of an optical waveguide induces changes that can be used as a transduction principle in a specic molecular detection system. Planar multilayer waveguides are studied at rst, as supporting structures. Then, several transductors based on these waveguides will be described, and nally, at the end of the chapter some space was left for the justication of the general assumptions. 17

CHAPTER 1. 1.1.


WAVEGUIDES AND OPTICAL BIOSENSING

BIOSENSING

Figure 1.0.1.: Evanescent eld optical biosensing: All systems of this kind are based on the same principle; a light beam propagates across a thin layer, with a fraction of its energy spread into the surroundings. This fraction acts as an optical probe for the external optical properties.

1.1. Waveguides and optical biosensing Optical waveguides are material structures with the ability to conne electromagnetic radiation inside them. Along the following explanation it will be assumed that the elds are already present and then the conditions that may produce their connement will be studied. These conditions are the total internal reection and the constructive self interference. From now on, homogeneous plane-wave illumination will be assumed. This is the most accurate solution of the Maxwell equations for distant or collimated light sources. Along the following formulation elds will be hence expressed as sums of plane waves Ψ+ exp(−ikr) + Ψ− exp(ikr).

1.1.1. Total internal reection (TIR) The Snell's refraction law [Born (1997)] predicts the conservation of the parallel component of a wave vector that crosses an interface between two dierent media (g. 1.1.1-up/left):

n2 sinθ2 = n1 sinθ1 18

(1.1.1)

CHAPTER 1. BIOSENSING

AN INTRODUCTION TO EVANESCENT FIELD 1.1.


As a consequence, the deection of light during this process results determined. Each θi represent the angle between the directions of propagation at the i-th medium and normal to the interface, and ni represent the refractive indices of the corresponding media. It can be observed that, for incidence angles above a critical value the Snell's law admit no solution. n2 [θ1 < asin( )] =⇒ [@θ2 |(n2 sinθ2 = n1 sinθ1 )] n1 Physically, no light crosses the interface, and the incidence above this angle leads to total internal reection, in which the rst and second media coincide, and eq. 1.1.1 turns into the well known law of reection, which states that the reection and incidence angles are equal:

θref = θinc As the arc sinus function is only dened for arguments within the [-1, 1] interval, total internal reection require a higher refractive index for the rst medium.

∃{asin(

n2 )} =⇒ [n1 > n2 ] n1

Finally, as it can be regarded as a sidewall condition, total internal reection may be used to create some kind of cavity in which its fulllment at several interfaces give rise to radiation connement. Apart from geometrical constraints, from the above discussion it may be concluded that:

Lemma 1. The connement medium must have a higher refractive index than its surroundings.

Depending on the geometry of the connement volume a preferred guidance direction may be selected. As an example, optical bers, which can be roughly described as high refractive wires, restrict light propagation to their axis of simetry. Along this thesis sensors were based on planar waveguides, i.e., consisting on stacked layers. The simplest structure (see g. 1.1.1, up/right) consists on a single layer (lm) surrounded by two media (called substrate and cover). Within this scheme, the numbers nf ≡ n1 and nc/s ≡ n2 will be identied respectively as the refractive indices of the connement volume and its surroundings. Although planar structures just restrict light propagation to a surface and hence solutions are not laterally conned, if limited beams (e.g. from Lasers) are used for illumination the coupled radiation will also exhibit a limited width (g. 1.1.1, down). 19

CHAPTER 1. 1.1.



BIOSENSING

1.1.2. Constructive self-interference Even if a light beam has been trapped inside a connement cavity and regardless its shape, guidance cannot still be assumed. In most cases light vanishes rapidly because of random interference. Only in some cases the geometry of the connement volume denes a resonant cavity, for which interference becomes constructive and leads to propagation modes. In practice this can be translated into equal phases at equivalent points. The simple but also relevant example of planar structures will be studied here. It was said that constructive (self-) interference require equivalent phases at equivalent points, which in planar structures are the upper and lower interfaces (g. 1.1.1). The phase shift after propagation between equivalent points, including the fraction due to reections at the interfaces must be a multiple of 2π . Assuming total internal reection, energy conservation requires preserved amplitudes, but no preserved phases, so these shifts have a key role in the guidance conditions. Their value is predicted by the Fresnel relations [Reitz (1992)], which are obtained from the interface conditions for the electromagnetic elds. It is important to note that, as a consequence, the orientation of the eld vectors has an explicit eect on the phase shifts upon reection. The Fresnel relations, limited to this case: (ref lected

= Aparalel exp(i∆ψparalel )

(ref lected Anormal

(incident Anormal exp(i∆ψnormal )

Aparalel

(incident

=

(1.1.2)

where

√

n2in sin2 θ−n2out ) √n2in nout2 cosθ2 nin sin θ−nout 2atan( ) nin cosθ

∆ψparalel = 2atan( ∆ψnormal =

(1.1.3)

In the above expressions parallel and normal refer to the plane of incidence, and A represents an arbitrary amplitude of either the electric or magnetic eld. From now on the discussion will be restricted to the electric eld, as the magnetic eld can be obtained from it. The situation in which the electric eld is perpendicular to the plane of incidence and hence parallel to the surface, is called transverse electric (TE). The opposite case, in which the magnetic eld is paralel to the interface, 20




Figure 1.1.1.: Refraction is governed by the law of Snell (up, left). If refractive indices verify n1 > n2 there exists a critical incidence angle θc above which transmission is replaced by Total Internal Reection. Multiple reections above θc lead to connement (up, right). A further restriction over the above geometry may be imposed by a laterally limited light source as a Laser beam (down). 21

CHAPTER 1. 1.1.



BIOSENSING

is known as transverse magnetic (TM) (g. 1.1.2). The word transverse here means that there's no projection of the magnitude onto the direction of propagation. For a three-layer structure as shown in g. 1.1.1 only two reections and the propagation in between must be considered. If ∆ψf c and ∆ψf s are the phase shifts introduced respectively at the lm-substrate and lm-cover interfaces (see eq. 1.1.3), the constructive self-interference condition can be expressed as:

−2π nf (2dcosθm ) + 2∆ψf c + 2∆ψf s = 2mπ λ

(1.1.4)

Where λ is the wavelength of the light, d is the thickness of the connement layer and nf is its refractive index. Inside this layer planar waves propagate at an angle θm with respect the normal of its faces, and the number m is known as the mode index. Dierent values of m lead to dierent solutions, or propagation modes. As phase shifts are dierent depending on whether the orientation of the elds is transverse electric or transverse magnetic, eq.1.1.4 splits into two dierent eigenvalue equations, which generate two families of propagation modes {T E0 ,T E1 ...T Em ...} and {T M0 ,T M1 ...T Mm ...} which are independent from each other. TE and TM modes are also called spolarized and p-polarized waves, respectively.

1.1.3. Eective refractive index From a geometrical point of view, the bounded light impinges both interfaces at angles ±θm . These angles are discretized by the resonance condition, and determine the propagation characteristics inside any waveguide. From an electromagnetic prospective, the energy is actually propagated along the direction of symmetry with a wave vector resulting from the projection of the physical one, (see again g. 1.1.1). From this projection, the eective propagation constant β and the eective refractive index nef f can also be dened:

βm = kf sinθm (1.1.5)

(m

nef f = nf sinθm 22




Figure 1.1.2.: Propagation modes: in the Transverse Electric or TE propagation mode (top) the electric eld remains perpendicular to the direction of propagation for both the non-coupled beam and the guided mode, while the magnetic intensity has a nonnull projection onto the guided wave vector. In the Transverse Magnetic or TM propagation mode the roles of the electric and magnetic eld vectors are exchanged. 23

CHAPTER 1. 1.1.



BIOSENSING

Here nf is the refractive index of the connement medium and θm is the reection angle. Considering the intrinsic relation

2π nef f λ0 it can be seen that a waveguide may be described equivalently using β or nef f . β=

Depending on the optogeometrical parameters several propagation modes may exist, each one with its corresponding eective refractive index. These eective indices decrease with mode number. About the dependencies of the eective refractive index, at simple sight the indices of cover and substrate appear in the expressions for the phase shifts due to reections at the interfaces (eq. 1.1.3). The substrate exists as a part of the structure but, in general, the role of the cover is shared by the external medium and by an eventual molecular adlayer, as the lm layer is directly exposed to it. Then, an uncovered waveguide senses the external refractive index. Along the following sections it will be shown that this is true within certain distance within the cover medium, and not only the interface.

1.1.4. Multilayer structures and numerical solutions The systems under study will be more realistically described as stacks of an arbitrary number of layers instead of only one. For instance, in this way the existence of adlayers may be taken into account. The study of such structures requires the analysis of the reections, transmissions and interferences that take place at every interface between their layers. It is hard to describe and solve the system using equations like eq. 1.1.4 for two reasons: rst, the analytical equations can be written only if the number of layers is known in advance and second, these equations can not be analytically solved in most cases. For the present study the Transfer Matrix method [Ghatak (1987); Shenoy (1988)] was chosen, as described by M.R. Ramadas in [Ramadas (1989)]. A general implementation of this method may assist the study of whatever stack system, including layers with grooves [Darwish (2007)] or absorptive materials. The method starts with the assumption of a set of complex propagation constants {βm,r +iβm,z }, solution of the waveguide equation. This equation may be expressed as: F (βm ) = 0 (1.1.6) 24




where m is a modal index and F (βr + iβz ) is a function on the propagation constant, still unknown. The function F −2 has peaks at the zeros of F, which will be assumed to be Lorentzian and, using the fact that even for highly absorptive waveguides the imaginary part of β is much smaller than the real one (see g. 1.1.3) it can be written:

k

1 A k2 = F (βγ ) [(β − βr )2 + βz2 ]

(1.1.7)

which expresses a proportionality between the function around its resonance and the inverse of the euclidean distance to the solution. Then, F (β) is expanded as a rst order Taylor series around βm and, taking eq. 1.1.6 into account,

F (β) = F (βm ) + (β − βm )(

dF )β = (β − βm )C dβ m

C is just a constant complex number. For systems with small losses imaginary part of β may be ignored. Then, by multiplying the result by its complex conjugate and taking the inverse, an analytical expression for eq. 1.1.7 may be proposed (eq. 1.1.8).

F (βr ) = (βr − βm,r + iβm,z )C kF (βr

)k2

= F (βr )F (βr

)∗

(1.1.8)

From the above expression, the parameter A may be interpreted as C −2 . Depending on the problem, this equation may be numerically solved in the real or the complex domains (g. 1.1.3). If only refractive indices are explored, the coordinate of each lorentzian peak provides the propagation constant of its corresponding modal index m, and its half width at half maximum (HWHM) is an estimation of the associated attenuation constant βm,z . If complex refractive indices are being considered, the eective propagation and attenuation constants are obtained from the real and imaginary coordinates, respectively. The necessary matrix description of the multilayer structure evaluates only real numbers as β is only evaluated along the real axis. The solutions for either the electric or magnetic elds have the same shape, although for simplicity, form now on, the discussion will be limited to the electric eld:

Em,i = Am,i cos(km,i (y −q di )) + Bm,i ξi sin(km,i (y − di )) km,i =

2 k02 n2i − βm

25

(1.1.9)

CHAPTER 1. 1.1.



BIOSENSING

5

10

TE

4

neff 0= 1.4750

10

nTE 1= 1.4823 eff

eigenfunction (adim.)

3

10

nTE2= 1.4877 eff

2

10

nTE 3= 1.4979 eff

1

10

0

10

−1

10

−2

10

−3

10 1.47

1.475

1.48

1.485

1.49

1.495

tested refractive indices (adim.)

Figure 1.1.3.: Eigenfunction examples: Up: A 5µm thickness layer of PMMA (n=1.492) surrounded by glass (n= 1.47) and water (n= 1.33). Down: A 140nm gold layer (n=0.197+j*3.466) surrounded by a glass substrate (n=1.47) and a cover material with a refractive index of 1.47. The eective indices correspond to the coordinates of the resonances, and the imaginary part of them correspond to the propagation losses.

26




di and ni are the thickness and refractive index of each layer (i is a layer index) and km,i is the corresponding vertical propagation constant, obtained from the eective propagation constant βm . The modal index m distinguishes between the dierent modal solutions which may exist. The number ξi takes values of 1/k or n2i /k for the TE or TM modes, respectively, because of the polarization dependent phase shifts at the interfaces (see section1.1.2). The physical solutions must be continuous at every layer boundary. The same restriction for the amplitudes Ai and Bi leads to the following expressions: Ai A1 = Si−1 Si−2 . . . S1 Bi B1

Si =

cos∆i ξi sin∆i (−1/ξi )sin∆i cos∆i

(1.1.10)

∆i = ki (di+1 − di ) It can be demonstrated [Ramadas (1989)] that both the amplitudes and the matrix elements remain real even when ki becomes imaginary, e.g. at the cover and substrate of the structure. It can be seen that if A1 and B1 are known, all the amplitudes can also be obtained from them. For dielectric systems, an additional requirement must be provided: The elds must vanish away the waveguide, otherwise there would be no chance of energy conservation. Applying this condition to the substrate and the cover, it is found that: B1 = (−i/ξs )A1 (1.1.11) Ac (β) + iξc Bc (β) = 0 The eld solution depends only on the arbitrary amplitude A1 and eq. 1.1.11 provides an analytical expression for 1.1.7, i.e., for the constant C in eq. 1.1.8. Finally, the method consists in checking a set of values of β between the ones corresponding to the highest index layer and the surroundings. Once xed A1 and, after obtaining the {Ai } and {Bi } sets, the function kF −2 k is evaluated. Plotting kF −2 k against β reveals the resonances, each one corresponding to a dierent propagation mode. When a peak is detected, the process is iteratively rened around it to get an accuracy limit below a measurable value. 27

CHAPTER 1. 1.1.



BIOSENSING

This method was extensively utilized along the design and analysis steps of the present research. Starting from this model, numerical derivatives and search algorithms helped to optimize the designs and fabrication parameters of the sensors. The calculations described from now on were programmed in the Matlab® environment.

1.1.5. Evanescent eld and system sensitivities As the phase shifts involved in the guidance condition (eq.1.1.4) arise from the Fresnel relations (eq. 1.1.2), the eld presence at both sides of any given interface is inherent in the formulation. This justies the basis of the evanescent eld sensing :

Lemma 2. In optical waveguides, the dependence of the eective index

on the cover medium occurs due to the interaction between this medium and the propagating evanescent eld. Equivalently, the evanescent eld

associated with a propagating mode in an optical waveguide may be used for the measurement of the optical parameters of the surrounding media. According with the last statement, it may be expected a stronger dependence on the volumes where the evanescent eld energy density is higher. This suggests the need of a further analysis of the eld distributions. As shown before, the described boundary conditions discretized the (in general complex) wave numbers βm of the plane-wave eld solutions. For a certain propagation mode, its modal index m may be suppressed, and the of eq. 1.1.9 may be written as: − Ei (y) = a+ i exp(iki y) + ai exp(−iki y)

(1.1.12)

In the lm layer ki is a real number, and the terms on the right side are trigonometric. In the surroundings, kc and ks are purely imaginary and the same terms become exponential. As the substrate and cover media extend − innitely across the OY axis, the amplitudes a+ c and as of the unbounded terms must be zero and, as a consequence, elds must vanish exponentially away the waveguide. Finally, the resulting expressions represent the evanescent elds : Ec = a− c exp(−kc y) (1.1.13) + Es = as exp(ks y) 28




The energy densities are proportional to the squared amplitudes of the elds, and from eq. 1.1.13 it can be seen that, beyond a distance of dp,s = (2ks )−1 into the substrate and a distance dp,c = (2kc )−1 into the cover, they decay by a factor e. For this reason, these distances are known as eld penetration depths and, as a convention, it is considered that evanescent eld sensors are (almost) blind away them. As the substrate medium remains unchanged, this term usually refers only the cover medium (dp ≡ dp,c ). Substituting the expression 1.1.9 for the propagation constant in the cover, the penetration depth can be analytically expressed as: q

dp = λ0 /[4π

n2ef f − n2c ]

(1.1.14)

Two conclusions may be extracted from the above expression: a) The working wavelength is an scaling factor for the system, and longer wavelengths produce longer penetration depths and b) larger dierences between nc and nef f (and hence higher refractive index contrasts between lm and the surrounding media) cause smaller penetration depths. According to it, a limit situation would consist on identical indices, and hence waveguide absence and no guidance at all. In gure 1.1.4 two examples of eld distributions in a planar waveguides can be observed. In the sensing conguration the convention is to use the name substrate for the thick material on which the waveguide relies and the name cover for the analite that ows in contact with the upper side of it. The wave-guiding layer (sometimes just called lm layer ) is the highly refractive slice in which the light beam gets coupled. In whatever realistic situation (see again g. 1.1.4) eld penetration depths are very small (usually between 100nm and 200nm). These numbers limit the application of evanescent eld techniques to monitor surface events. This is actually an advantage, which permits the study of surface events without other disturbances. Finally, it will be assumed that the surface event that will be studied is the adsorption of molecules from the cover medium.

1.1.6. Coupling light into a waveguide It may seem possible to couple light into a waveguide directly by just impinging its surface with beam at the appropriate angle, but this is not true. The guidance condition requires total internal reection inside the connement volume, but in this case the incidence angle should be above π/2 with respect the direction of propagation (g. 1.1.1). Written as a lemma: 29

CHAPTER 1. 1.1.



BIOSENSING

Figure 1.1.4.: Field solutions for a 65nm Si3 N4 waveguide (left) and a 900nm thick waveguide of PMMA (right): Although most energy remains conned into the waveguide, a fraction known as evanescent eld, extends away it. In these examples, the extinction distances were, respectively, 60nm and 80nm.

Lemma 3. It is impossible to couple a light beam into a waveguide by impinging it at any angle.

Three solutions have been proposed for this problem. The rst two will be briey described and the third, as a key element of this thesis is the subject of the next section.

Front face coupling: If the impingement surface is not parallel to

the guidance direction (specially if it is perpendicular) the refraction problem is skipped. This is the most frequent choice for optical bers. The core of a typical optical ber is at least several microns thick, so the entire beam can be collimated within the coupling area, and no signicant energy losses appear. The planar waveguides subject of this research have cores thicknesses between several tens and several hundreds of nanometers, and hence focusing a beam directly on their side would be both tricky and inecient. A lens-based collimation optics, typically a microscope objective may reach a 78% eciency [Shaklan (1988)], but its alignment diculties generally restricts its 30

CHAPTER 1.


BIOSENSING

1.2.

GRATING COUPLERS

application to the laboratory. Mechanical alignment is not only costly, but also a challenging source of problems for portable or disposable devices. Just as an example, it would be desired to overcame the alignment process each time a disposable device is inserted. An additional diculty arises from the roughness of the surface. Unlike bers, planar waveguides are usually made by means of planar technologies over hard substrates. Polishing the sides in this case is mandatory if signicant eciencies are desired, but this process rises the costs signicantly.

Prism Coupling: Guided modes may be excited in a thin lm by means of an evanescent eld generated in its vicinity. In the simplest case, a prism coupler generates this evanescent eld by total internal reection. As the faces of a prism are not parallel, it is always possible to get total internal reection on only of them (g. 1.1.5). Although prisms are an ecient way for coupling energy into waveguides [Rosenblatt (1997)] an reason against their use is the need of the prism itself; an additional optical component. In addition, the contact between prism and waveguide is also problematic: the tiny air gap that always exists between both elements makes evanescent eld coupling inecient unless both components are strongly pressed against each other, with a subsequent risk of breakage.

1.2. Grating couplers Diraction provides an alternative for deecting light, similar to prism coupling in the sense that overcomes the Snell's limitation. Still leaving a free interaction surface, diractive couplers let to choose between top or substrate coupling. In addition, its fabrication is fully compatible with planar technology and there exist some low cost alternatives. A grating is a diractive structure able to split an incoming light beam into several directions, according to the HuygensFresnel principle [(Huygens, 1690)]. Each point on the surface of a grating acts as a secondary light source shining towards every direction. Then, after the subsequent interference, only a few resonant directions arise, at which these re-emitted beams interfere constructively. Known as diracted beams, their number depend on the ratio between grating pitch and wavelength. 31

CHAPTER 1. 1.2.


GRATING COUPLERS

BIOSENSING

Figure 1.1.5.: Prism coupler with dielectric waveguides: A light beam is totally internally reected at the interface between prism and the air gap that always exist. Then, the evanescent eld stimulates a guided mode away this gap, into the dielectric lm. If a grating exists on the surface of a waveguide, a diracted beam may eventually fulll the resonance condition, which consists on the match between the parallel component of its wavenumber that of a coupled mode of the waveguide. Mathematically [Petit (1980) ]:

nef f = next sinθinc + m

λ Λ

(1.2.1)

where nef f and next are respectively the eective refractive indices of the waveguide and external medium, λ is the wavelength, Λ is the grating pitch m is the diractive order (labeling each diracted beam) and θinc is the angle between the incident beam and the normal to the surface. Figure 1.2.1 shows that the rst diraction order does not necessarily correspond to the rst coupled beam. When grating pitch and wavelength have comparable values the rst diracted beam lies in the coupling zone and the modes can be labeled equally. For a given waveguide, there exist two dierent coupling schemes:

For a certain wavelength some angles of incidence (just one, for single mode waveguides) allow resonance. This principle of operation is known as angular interrogation, and its application to the OGCB biosensing was rst described in [Tiefenthaler (1985)]. 32

CHAPTER 1.


BIOSENSING

1.2.

GRATING COUPLERS

For a xed incidence angle, if several wavelengths are available, one or some of them may fulll the resonance condition. These wavelengths may be scanned, for instance using a wavelength tunable light source [Wiki (2000); Jenq Nan (2006)] or provided together at the same time using a white source. In both cases the method is referred as spectral interrogation.

Figure 1.2.1.: Grating coupling: The coupling condition is the match between the wave number of a guided mode β and the wave || || number of a diraced beam βext + nK .βext is the paralel projection of the incoming wave number and K = (2π/Λ) is the wave number of the grating. For interpreting the grating equation it should be noted that, because of the Snell's law (eq. 1.1.1) the term next sinθinc is a medium invariant. This means that coupling angles can always be measured in air, using next ≡ 1 and forgetting all the other possible refractions. Another important thing is that the eective index depends on the system structure and on the wavelength, so translating coupling angles from one wavelength to another is not straightforward. Finally, if the eective indices were predicted, it 33

CHAPTER 1. 1.2.


GRATING COUPLERS

BIOSENSING

would be interesting to set a desired range of coupling angles, by selecting an appropriate grating pitch.

1.2.1. Single and dual grating couplers It has been shown that, if the rest of parameters are xed, the coupling resonances depend only on the waveguide eective refractive index. The use of grating couplers for biosensing consists on the study of this eective index through the coupling resonances, as described above. The most important alternatives of single and dual grating couplers will be described here.

Application to angular interrogation Basically there exist two alternatives for the angular interrogation of the coupling resonances; the standard (single grating) scheme [Lukosz (1995)] and the dual pad conguration [Clerc & Lukosz (1997)]. In the rst, also known as the rotary scheme (g. 1.2.2-up), the incidence angle varies continuously as the sensor is rotated, so the coupling resonances are cyclically found. Then, the angle at which these resonances appear can be related with the other variables (i.e., rst with the refractive index and then with surface mass concentration). In the dual-pad conguration (g. 1.2.2-down) a rst grating keeps an incoming beam constantly coupled, without the need to rotate the sensor. As the in-coupling angle must be kept constant, the in-coupling grating cannot be exposed to the studied medium. Then, a second grating (out-coupling pad) couples the radiation out, at an angle that depends on the local eective index of the exposed area. Finally, some detector with spatial resolution identies the impingement points.

Application to spectral interrogation Grating couplers also allow biosensing with spectral interrogation, which means the study of the spectral coupling resonances at a xed incidence angle (see the above paragraph). As only a single wavelength can be coupled at any xed angle, the dual-pad scheme is not applicable in this case. The reason is that, even if the coupling conditions were matched at the incoupling grating, the out coupling grating would couple a single wavelength out, which is actually angular interrogation. In contrast, the single grating conguration is commonly used in wavelength interrogation schemes [Wiki (2000)]. 34

CHAPTER 1.


BIOSENSING

1.2.

GRATING COUPLERS

Figure 1.2.2.: Dierent grating coupler congurations used for biosensing: The single grating coupler scheme (up) in which the incoupling angle is scanned, and the dual pad scheme (down), which measures the out-coupling angle. 35

CHAPTER 1. 1.3.


SURFACE PLASMON RESONANCE

BIOSENSING

1.2.2. Optical Grating Coupler Biosensors (OGCB) Commercially, Optical Grating Coupler Biosensors (OGCB) have been overshadowed by Surface Plasmon Resonance (SPR) sensors (sec. 1.3). It is convenient to mention here that SPR sensors monitor conductive surfaces while OGCBs analyze dielectric interfaces, so theoretically both techniques are not incompatible. Furthermore, grating couplers are able to scan TE and TM resonances simultaneously, which represent two independent data sources that can be obtained from the same experiment. In this way, OGCBs provide thicknesses and refractive indices without the need of assumptions, i.e. based on evidences instead of models [Vörös (2004)]. It will be seen that surface plasmons can only be excited by transverse magnetic illumination, so grating couplers are conceptually advantageous. From the point of view of the author, the SPR domination is most likely a matter of weight: at the time to spend over 50k¿ (year 2009) in a surface monitoring tool, SPR companies provide more than a decade of experience, full support and a huge community of users. In addition, the unique capabilities of grating gouplers may be scientically relevant, but frequently are not needed. For instance, it may be desired to detect a well known substance, and not to characterize it. Examples of OGCB systems are the OWLS instrument, from Microvacuum Ltd. [Adányi (2007)] and the EPIC® system, from Corning ® [Fang (2006a)]. The rst one is a single-channel rotary system that reported sensitivities of 5ng/mm2 of attached mass, while the second uses grating coupling and spectral interrogation in order to monitor up to 384 channels with declared sensitivities up to 0.5ng/cm2 . Bacteria detection series of studies were developed with a noncommercial version of this instrument [Horváth (2002a)]. The lack of commercial systems still leaves some room for research. Ideas like the chemical passivation [Homola (2001)] or the multi-sensing arrays [Dostalek (2005)], already present in other systems, were proposed along this thesis for dielectric grating couplers.

1.3. Surface Plasmon Resonance Surface Plasmon Polaritons (SPPs) admit two equivalent descriptions. From the optical point of view, SPPs can be considered as strongly conned propa36




gation modes (Fano modes [Fano (1956)]) formally equivalent to those that appear in dielectric waveguides, but in this case in conductive materials. From the electromagnetic perspective, an electronic vibration wave propagates across the surface. These waves are supported by free electrons, and for this reason plasmons appear only in highly conductive materials. For a metallic layer of thickness d, parallel to the y-z plane, the TE and TM modes admit the general solution provided in eq. 1.1.12: − T E : Ey (x) = a+ i exp(iki x) + ai exp(−iki x)

Ex = Ez = 0

(1.3.1)

− T M : Hy (x) = b+ i exp(iki x) + bi exp(−iki x)

Hx = Hz = 0

(1.3.2)

where i is the layer index, and the modal index has been supressed. After applying the boundary conditions of continuity, derivability and extinction away the interface, it can be demonstrated that any non-trivial solution must verify the following expressions [Homola (2006)]:

T E : tan(kd) =

T M : tan(kd) =

γ1 /k + γ3 /k 1 − (γ1 /k)(γ3 /k)

γ1 2 /k1 + γ3 2 /k3 1 − (γ1 2 /k1 )(γ3 2 /k3 )

(1.3.3)

where k 2 = ω 2 2 0 µ0 − β 2 and γi2 = β 2 − ω 2 i 0 µ0 are the propagation constants at each medium, along the direction perpendicular to the interface, and β is the propagation constant of the guided mode. The permittivities i are relative values. The dierence between both expressions results from the application of the boundary conditions at the interface between two media. TE polarized modes have no electrical eld components perpendicular to the interface, while the parallel components are equal [Reitz (1992)]. In the case of TM modes and in the absence of surface charges, the perpendicular components of the electric displacement are continuous:

Ex(2 /Ex(1 = 1 /2

(1.3.4)

The dierence between the eigenvalue equations 1.3.3 for each polarization mode is exactly this term. 37

CHAPTER 1. 1.3.



BIOSENSING

A metal-dielectric interface is the limit case, with d approaching zero. If the lower medium is conductive, the eigenvalue equations become:

T E : γm = −γd TM :

γd γm =− m d

(1.3.5)

where the indices m and d refer to metal and dielectric. Again, γd2 = β 2 − ω 2 d 0 µ0 and k 2 = ω 2 i 0 µ0 − β 2 , are the propagation constants, dened as real numbers at each medium. For TM modes, the solution is the surface plasmon wave number: r m d ω βSP W = (1.3.6) c m + d while TE modes do not admit propagating solutions. As the real part of the permittivity of metals and dielectrics have opposite signs, the term m d is negative. In order to get a real value for β , it is then needed that m < −d , i.e., any material able to hold surface plasmons must have a negative real part of its permittivity, larger in modulus to that of the dielectric with which is in contact. Metals usually verify this condition for certain ranges of frequencies. The permittivity of a material that follows the Drude's free electron model is

m = 0 [1 −

ωp2 ] ω 2 − iων

(1.3.7)

p N e2 /0 me is the plasma where ν is the collision frequency and ωp = frequency, at which the free electrons naturally oscillate. N is the free electron concentration, e the electron charge, and me is the electron mass. It is enough to substitute eq. 1.3.7 in the condition m < −d to nd the allowed frequency range. For gold, silver and platinum this range comprise the visible and near infrared wavelengths. The eld solutions are strongly conned at the interface: Hy (x > 0) ∝ exp(−γd x) ; Hy (x < 0) ∝ exp(γm x) At the penetration depth eld amplitudes decay by a factor e with respect to −1 and γ −1 their maxima. From the above expressions, these depths are γm d into metal and dielectric, respectively. Typical penetration depths inside the dielectric are between 100 and 200nm. For clarity, the discussion from above was limited to interfaces, not thin layers. In actual SPR biosensors the modes are supported by a thin metallic 38

CHAPTER 1.


BIOSENSING

1.3.


2.6

Effective index

2.4

2.2

2

(km ks)/(km + ks)

1.8

(km kc)/(km + kc)

1.6

1.4 20

40

60

80

100

120

140

120

140

film thickness (nm) 5

3.5

x 10

Attenuation [dB/cm]

3

2.5

2

1.5

1

0.5

0 20

40

60

80

100

film thickness (nm)

Figure 1.3.1.: Eective refractive index (up), and loss constant (down) of a surface plasmon wave, as a function of the layer thickness.

39

CHAPTER 1. 1.3.



BIOSENSING

lm, and eq. 1.3.6 is just an approximation for some cases. In gure 1.3.1 the eective index of a conductive waveguide is presented as a function of its thickness. This results were obtained using the general method described in sec. 1.1.4, and coincide with [Ctyroký (1999)]. The plots were calculated for a stack of three layers, the lower (substrate) with a RI of 1.47 , the upper (cover) with a RI of 1.40 and the middle (gold lm layer) with a complex refractive index of 0.197+j3.466, working at 633nm wavelength. The loss constant in db/cm is dened as 2·105 log(e)Im{β} if lengths are given in microns. As can be seen in this example, a symmetric solution always extends towards the substrate and, above an average cuto thickness of 50nm an anti-symmetrical solution appears towards the cover. When layer thickness is increased these solutions asymptotically tend to those given by eq. 1.3.6 for their corresponding interfaces.

1.3.1. Excitation and detection of Surface Plasmons Surface Plasmon Resonance (SPR) is the excitation of surface polaritons by means of a wave-number matching condition. Because of their analogue nature, the coupling schemes may be equal to those presented in sec. 1.1.6, except with respect to one dierence: the evanescent eld is generated in the substrate-lm interface instead of inside the prism. For accomplishing this, an oil (index-matching uid) with a high refractive index lls the gap between prism and waveguide (g 1.3.2). Grating coupling is, in contrast, completely analogue in both systems. Another dierence exists between plasmons and dielectric guided modes; the typical attenuation constants of surface plasmons (see g. 1.3.1) are of the order of 105 dB/cm, and hence no signicant propagation distances may be expected. For this reason, the coupling analysis cannot be based on the detection of the coupled energy directly, as explained in sec. 1.2.1. Instead of it, as metallic surfaces are highly reective, the resonances are usually studied indirectly: the reected light exhibits a sharp dip upon excitation (either in the angular or wavelength domains) because of the energy absorption.

40




Figure 1.3.2.: Surface Plasmon excitation by means of a prism coupler: In contrast with respect to the case of dielectrics (sec. 1.1.6) total internal reection takes place in the substrate of the waveguide instead of the prism, and the air gap is lled with a high refractive oil.

1.3.2. Surface Plasmon Resonance (SPR) biosensors The market leader company is Biacore, currently a division of General Electric [www.gehealthcare.com]. Most Biacore systems, like Biacore®3000 employ angular interrogation with prism coupling. Instead of moving components, Biacore instruments perform angular interrogation by means of a fan-shaped monochromatic source. An exception was the Biacore FlexChip® system, which used diractive coupling in order to excite up to 400 channels at the same time. Texas Instruments commercialize the Spreeta® instrument, a single channel compact SPR system. Ibis technologies [www.ibis-spr.nl] also assembles the Ibis iSPR®, a multi-channel (SPR-imaging) instrument based on prism coupling and angular interrogation. All these devices promise optical sensitivities around 10−6 RIU (Refractive Index Units), quantity that can be translated into 0.1ng/cm2 for typical molecular systems. The currently available systems were designed for general purpose, which, although not being a theoretical drawback, increase 41

CHAPTER 1. 1.4.


WAVEGUIDE FLUORESCENCE EXCITATION

BIOSENSING

their cost and the cost of the disposable sensors. There also exist many small companies selling SPR devices, and it is common to recycle or selffabricate the sensors in scientic laboratories. It is not only the (lack of) software and support what makes them a cheaper alternative, but also less valuable, turning maintenance and measurement tasks for an skilled technician instead of a regular user.

1.4. Waveguide Fluorescence Excitation Fluorescence and phosphorescence [Taitt (2005)] are quantum processes in which excited molecules relax to thier ground states through intermediate energy levels. The transitions between upper and intermediate levels do not necessarily involve the emission of photons and, in most cases, take place between molecular vibrational levels. Subsequently, the excess of energy is dissipated as heat. Because of the dierent energy gaps the photon nally emitted has a longer wavelength than that of the adsorbed one (g. 1.4.1). Fluorescence is the result of the relaxation of a non-degenerated (singlet) state, in which the total electronic spin is 0. In contrast, phosphorescence arises from the relaxation of a 3-fold degenerated (triplet) state, in which the electronic spin sums 1. Phenomenologically, because of the time an electron needs to recover its original spin, the lifetime of the triplet states (around 10−3 s) is several orders of magnitude larger than the lifetime of a singlet (10−9 s). This implies that, in contrast to phosphorescence, uorescence can be observed only while the excitation radiation is being applied. Both excitation and ground levels are splitted into a number of vibrational levels which are very close to each other. For this reason both processes take place in spectral bands instead of single wavelengths. Fluorescent tags are a family of molecular markers that become uorescent upon the linkage with a complementary target substance. Because of their security, these labels are the preferred alternative to radiative tags in microarray studies [Goldys (2009)]. In uorescent micro-arrays molecular targets are adsorbed onto an immobilized array of labels from which the recovered uorescence emission allows the estimation of the amount of target. The emission intensity is proportional to the excitation energy, the concentration of active tags and their quantum yield φ. As the concentration of active tags is the fraction of labels linked with target molecules, if cf and cx represent both surface concentrations:

If luorescent = φf (cf , cx )Iincident 42

(1.4.1)




Figure 1.4.1.: Waveguide excitation uorescence device. The uorescence emission leaves the surface of the waveguide, and then can be collected at whatever angle, preferably dierent to the reection or out-coupling directions. Conventionally, the labels are excited by a focused beam. In this conguration, the uorescence of other compounds along the optical path of excitation results in the increase of the background noise and a worse overall sensitivity. An elegant alternative is the use of an evanescent eld for the excitation of labels in the close vicinity of the adsorbing surface of interest [Duveneck et al. (2002)]. This concept has another intrinsic advantage: while the excitation radiation is conned into the waveguide, uorescence is isotropically emitted. In this way, a large aperture optical arrangement may recover uorescence from a broad angular range without the disturbance of the excitation source. The representative waveguide-uorescence instrument is the ZeptoREADER system [www.zeptosens.com], which claims a picomolar limit of detection of detection (LOD), equivalent to a zeptomol of target molecules in a nanoliter volume droplet [Pawlak (2002)]. In the context of the present research, the dual grating coupler conguration was proposed not only to carry out surface uorescence excitation, but also 43

CHAPTER 1. 1.5.


MOLECULAR ADLAYERS

BIOSENSING

for combining it with the complementary study of the coupled modes. In the applications chapter a system that can be congured for both techniques will be presented.

1.5. Molecular adlayers In the above sections it was assumed that the existence of small concentrations of particles on the surface of a waveguide can be mimicked by its mean eect: a change in the thickness and refractive index of an hypothetic, ideally homogeneous and continuous adhered layer (adlayer ). This assumption is nothing else than the continuum approximation [Landau (1987)]. In this context, some requisites guarantee its validity:

Particles are small : The processes takes place at large scales compared with the size of the involved elements (particles). The coupling areas range between square microns and square millimeters, while molecular diameters are of tens of nanometers. In addition, wavelengths in the visible range are also an order of magnitude larger than molecular diameters. The number of particles involved in the process is very high : Using 30KDa and 5ng/mm2 as reference values for the molecular weight and surface mass density, a square micron would still contain 1011 molecules. Particles must be homogeneously distributed : Along a characteristic length (e.g. wavelength) the particle concentration must be homogeneous. If this was not true their contribution would not be clear and it would not be possible to dene a density function [Schasfoort (2008)]. It is accepted that the cases of interest are described by the random sequential adsorption model [Ramsden (1993)]. The random nature of this process, together with the law of large numbers, lead to the acceptance of this third assumption.

As a conclusion, molecular layers can be considered continuous, and hence may be characterized using a thermodynamic (i.e. macroscopic) description. Accordingly, a molecular layer may be described with its thickness (an extensive variable) and density (an intensive variable). These parameters, even not representing the actual molecules, are unique at any coverage degree, so can be used for recovering the real data. In the optical point of view, density is replaced by refractive index. In 1978 De Feijter found that the refractive index of a colloidal system varies 44

CHAPTER 1.


BIOSENSING

1.5.

MOLECULAR ADLAYERS

linearly on the attached mass density for a wide range of concentrations [De Feijter (1978)]:

n(σm ) = nσ=0 +

dn σm dσm

(1.5.1)

where n(σm ) and nσ=0 are the refractive indices with and without attached particles, respectively, and σm is the adsorbed mass density. Equation 1.5.1 seems just an Euler expansion, but the derivative dn/dσm is constant in most cases. Typical values are 0.18ml/g for proteins and 0.16ml/g for nucleic acids [Wen (2000)]. Equation 1.5.1 is a key relation between particle concentration and refractive index and, without it, evanescent eld sensors would never be anything else than mere refractometers. The evanescent eld sensors measure eective indices, but no indices of the sample directly. Furthermore, the concentration of the considered species varies with distance, as these species get deposited onto the surface, while owing above at very weak concentrations. As seen in sec. 1.1.5, it can be assumed that the recovered signal of an evanescent eld sensor depends on the interaction between analite and evanescent eld, which is evaluated by means of its energy density. Once it was accepted that a dependence between concentration and refractive index exists, it is possible to formulate the shift of the eective index as [De Fornel (2001)]:

nef f − nef f (σm

dn = 0) = dσm

ˆ∞

dσm exp(−y/dp )dy dy

(1.5.2)

0

As eld energies behave as exponential functions with a characteristic depth dp , the above equation states a proportionality between the amount of interaction and the energy density, according with the above discussion. As said before, the term dn/dσm may be considered a constant, and the integral in general varies linearly on the attached mass. As an example, if it is supposed that mass is attached homogeneously within certain adlayer thickness wad and non adsorbed above it, σm is a step function, and eq. 1.5.2 turns into nef f = nef f (σm = 0) + (dn/dσm )exp(−wad /dp ), which expresses the dependence of the response on the ratio between adlayer thickness and penetration depth. It is important to notice that the fundamental questions of why and how density and refractive index are related remain unanswered. 45

CHAPTER 1. 1.6.


MOLECULAR RECOGNITION

BIOSENSING

1.5.1. The refractive index of an adlayer For clarity the following discussion will be limited to a system composed by a single type of molecules. This is enough to answer the above questions, but also can be easily generalized. It is well known that the wave solutions of the Maxwell equations propagate at the speed of light c2 = (µ)−1/2 . The refractive index is dened as the quotient between speeds of light in vacuum and in certain medium: c √ n = = r µr (1.5.3) v where the relative permittivity (also known as dielectric constant ) r and permeability µr appear explicitly. The materials considered here are non magnetic, so always µr = 1. Then, the problem gets reduced to understand the dependence of the dielectric constant on density and molecular structure. The molecular polarizability α, which describes the ability of a molecule to get polarized is an intrinsic property of the particles and depends on their morphology. The collective eect of molecular dipoles is reected by the electrical susceptibility, and nally in the permittivity of a material. The explicit dependence between molecular polarizability and dielectric constant is known as the Clausius-Mossotti relation [Feynman (1964)]: 30 (r − 1) α= (1.5.4) N (r + 2)

N is the number of molecular dipoles per unit volume. Finally, after combining 1.5.3 and 1.5.4, for the non-magnetic case (µr = 1) : n2 = r =

2ψ+1 1−ψ

; ψ=

αN 30

(1.5.5)

It can be seen that for the limits of non- polarizability or no presence of particles (i.e. ψ → 0), the medium behaves like vacuum. Under similar polarizabilities, the De Feijter equation (eq. 1.5.1) appears naturally from eq. 1.5.5. The conclusion from this result is:

Lemma 4. The refractive index of a material grows with its density and with the polarizability of its molecules. The overall parameter that covers both contributions is called optical density.

1.6. Molecular recognition As it happens to other physical sensors (e.g. spectrometers), the merely quantitative data obtained from waveguide sensors -thickness and refrac46




tive index- has no meaning without an interpretation. Biosensors need a transduction block as those already described, but also a molecular recognition block that guarantees that the observed stimuli are specic. For instance, the surface of a waveguide covered by antibodies has the ability to anchor their specic antigen molecules from a ow stream and to reject the linkage of other species. This conguration, on one hand, guarantees the specicity of the measurement and, on the other, monitors the reaction while being produced, which provides information about its kinetics.

Denition. Biorecognition is an interaction process between biomolecules

favored by an anity between each other signicantly higher than that of any of both with respect to whatever other composite of the same medium. This phenomenon is relevant in the case of macromolecules, which according their unique topographies and local compositions may exhibit specic anities with concrete locations (epitopes) of other molecules. The composition of biomolecules is determined by its sequence of amino acids [Stryer (1975)], but the same sequence may lead to dierent topographies, depending on ambient conditions, such as temperature and pH. These topographies range from full functionality to complete denaturation. For this reason whatever instrument designed for monitoring molecular recognition must be able to control these environmental variables. Although the molecular recognition elements are extracted or synthesized from living organisms using biological or chemical techniques, the creation of stable active surfaces using these antibodies is known as functionalization, and is considered a matter of bioengineering, rather than of chemistry.

1.6.1. Functionalization and specicity Functionalization is the coating of a surface with some specic (bio)molecular recognition system, in order to promote specic adsorption and to inhibit inespecic adhesion. This system usually consists on an antibody or complementary DNA strand layer on its upper face, and linkage molecules like silanes [Trummer (2001)] or thiols [Ulman (1998)] to anchor the system to the supporting surface. Ideally, species of only one kind will be deposited on the surface, and hence the measured thickness and refractive indices would be uniquely determined by the amount of specically attached mass. The actual picture involves some nonspecic attachment and shifts due to other eects (specially thermal) which worsen the selectivity. Taking this into account, selectivity is considered acceptable if biorecognition leads to 47

CHAPTER 1. 1.6.



BIOSENSING

signicant dierences when compared to the other sources of signal. Generally, a negative control experiment performed under the same conditions allows to solve this situation. Along section 2.6 a more ecient alternative is proposed.

1.6.2. Information from the adsorption kinetics Adsorption events can be divided in two dierent stages: a) the transport (either diusive or convective) of the species from the bulk liquid to the close vicinity of the surface and b) the attachment of these species. According to [Gray (2004)] the same substance may be solute, intimate solute or adsorbate depending on its role. In each case, the concentration of this substance is Ci , {Ci } and (Ci )ads , respectively (g. 1.6.1). Each of both subprocesses has its own rate parameters: ka

k10

kb

k2

Ci {Ci } (Ci )ads

(1.6.1)

ka and kb are phenomenological transport coecients, k10 is the rate function and k2 is the reverse rate constant (with units of s−1 ). The need of a forward rate function arises because dierent surfaces accept molecules dierently. The easiest approach: k10 = (Cx )tot k1 f (φ) (1.6.2) i )ads φ = n(C (Cx )tot Here k1 is an intrinsic, second order, rate constant, (Cx )tot is the total concentration of matrix sites (available locations for binding) and f is a function of the fraction of the still available matrix sites, φ. For instance, as the number of free matrix sites decreases along adsorption, the forward reaction gets slowed down. From eq.1.6.1 it can be written a general expression for the rate of formation of intimate solute:

d{Ci } = ka Ci − kb {Ci } − k10 {Ci } + k2 (Ci )ads dt

(1.6.3)

Two dierent limit cases can be distinguished, the transport limited and the reaction limited regimes. In the rst one, the reaction consumes intimate 48

CHAPTER 1.


BIOSENSING

1.6.


Figure 1.6.1.: The adsorption process: the solute, after being transported to the close vicinity of the surface is called intimate solute. After adsorption, this specie is called adsorbate. The dierent concentrations and reaction rates govern the dependence of each concentration on the others. solute at a higher rate than that of at which transport resets it; the second case is the oposite. Although in most cases adsorption cannot be classied as a limit case, these examples provide a way to get valuable information from the kinetic curves.

Transport-limited regime: transport:

In this case, adsorption events dominate over

[k10 {Ci } − K2 (Ci )ads ] >> [ka Ci − kb {Ci }]

(1.6.4)

As intimate solute is rapidly consumed, d{Ci }/dt → 0. This, combined with eq.1.6.4 provides the rate of formation of adsorbate in the transport-limited regime. d(Ci )ads ka Ci + k2 (Ci )ads = k10 − k2 (Ci )ads (1.6.5) dt kb + k10 Upon the startup of an adsorption experiment the adsorbate concentration 49

CHAPTER 1. 1.6.



BIOSENSING

is usually zero and, using that kb [k10 {Ci } − k2 (Ci )ads ]

(1.6.8)

Using again eq.1.6.3 combined with eq.1.6.8 and d{Ci }/dt → 0, it is found that: ka def {Ci } w Ci = kT Ci (1.6.9) kb The number kT is known as equilibrium partition coecient for the formation of intimate solute. The corresponding rate of formation of adsorbate, in this case is d(Ci )ads = k10 kT Ci − k2 (Ci )ads (1.6.10) dt Considering the limit cases of zero adsorbate or solute concentrations, the equations 1.6.6 and 1.6.7 are obtained again, with the only need to identify the eective association and dissociation rate parameters as:

fi = k10 kT bi = −k2 50

(1.6.11)

CHAPTER 1.


BIOSENSING

1.6.

Partition coecient:

same description:


The above described regimes equally lead to the

d(Ci )ads = fi Ci − bi (Ci )ads dt

(1.6.12)

The eective adsorption partition coecient characterizes either the concentrations of solute and adsorbate at equilibrium or the forward and background rates during the adsorption.

(Ki )ef f =

fi (Ci )ads k 0 ka = 1 = bi k2 kb Ci

(1.6.13)

It is important to notice that, if (Ki )ef f is known, the actual adsorbate and the bulk solute concentrations can be obtained from each other. It is also important to remark that the eective forward and backward rates can be obtained from a set of adsorption and desorption experiments.

1.6.3. Hydrodynamics and random sequential adsorption The random arrival and adsorption of species from certain stream, where the bulk concentration is kept constant is called random sequential adsorption [Ramsden (1993)]. In this situation, the rate of encounter is only limited by diusion, and the rate of linkage is only limited by the existence of vacancies. As the second limitation was described above, it just rests to mention how the rst may behave in a typical system. Generally, the experiments are carried out inside channels, in which ow is laminar and the Poiseulle equation (eq. 1.6.14) is a good approximation [Landau (1987)]. If the axis OZ relies on the cross section and OX points towards the ow direction:

vX (z) = 4vx |zmax /2 (

z zmax

−

z2 2 zmax

)

(1.6.14)

This solution has a zero speed limit on the sidewalls, which is contradictory with the fact that analite is actually transported towards the surface by the stream. The compartment model is a renement of the uidic model in which a stagnant layer of thickness δ exists, in which the speed of the uid can be considered zero. Above this layer, the bulk liquid is moving at a constant speed vbulk , with a constant concentration cbulk . Inside the stagnant layer species are transported towards the surface only by means of diusion. According to it,

d{Ci } Di w 2 [Cbulk − {Ci }] − k10 {Ci } dt δ 51

(1.6.15)

CHAPTER 1. 1.7.


AIMS OF THIS THESIS

BIOSENSING

Di is the diusion constant of the i-th specie. The rst term of the right side represents the transport, and decreases as the stagnant thickness increases, which happens at smaller ow rates. The second term represents an (ideally irreversible) adsorption process, (see eq. 1.6.2), and for this reason is proportional to the matrix site concentration. In the stationary regime, using d{Ci }/dt = 0, the rate of concentrations at the surface and the bulk is {Ci } Di /δ 2 w (1.6.16) Cbulk (Di /δ 2 ) + k10 This equation links the denitions of stagnant layer and intimate solute. The ratio between concentrations of intimate solute and solute in bulk depends on (D2 /δ 2 ) and the forward rate constant, that can be obtained from a series of kinetic experimets.

1.7. Aims of this thesis From eq.1.2.1 it may be seen that coupling resonances in planar grating couplers can be found in either the angular or spectral domains. Along section 1.1.5 it was shown that, by following the evolution of these resonances it is possible to monitor biochemical reactions, leading to a family of tools called evanescent eld biosensors. These devices may be used in two dierent ways:

From a previous knowledge of the system features (anities, ow rate, diusion constants) biosensors may be employed to quantify tiny amounts of specic biomolecules. In most cases, the complete knowledge of the system is actually not needed, and the sensor outcome may be related with some other variables after an initial calibration cycle. Starting from a prepared system (i.e. with known concentrations) the sensor readouts may be used for the obtainment of the reaction parameters. For instance, the direct measure of anities has an obvious interest in pharmacology.

The main core of this project is the development and demonstration of an instrument of this kind, a double grating coupler. A rst grating couples a light beam into a planar waveguide, while a second grating couples it out, using the angle as a measure of the eective index. The energy coupled into a dielectric waveguide experiments a very small attenuation along distances of centimeters, so there is no technical challenge in doing this, provided that 52

CHAPTER 1.


BIOSENSING

1.7.

AIMS OF THIS THESIS

coupling eciencies are high enough. This idea was originally proposed by Tiefenthaler and Lukosz [Lukosz (1990)], but commercial systems based on it were never fabricated. For this reason, this development is not strictly new and it will not be presented as a goal by itself. Nevertheless, this work revises all the development stages, form the concept to the instrument, and demonstrates several improvements. This research intends to answer the following questions:

The cost per assay is too high compared with traditional techniques: Solutions based on low cost technologies like polymers and/or design strategies will be proposed. The samples frequently need to be puried in advance: On-line referencing, assisted by a multichannel detection scheme would be helpful for distinguishing crossed sensitivities, specially if samples are heterogeneous.

Not always the obtained resolutions or dynamic ranges satisfy the requirements for an application : It is desired to get adaptable sensing ranges. Sampling rates may be too slow depending on the reaction kinetics : It is a goal to design faster devices, lowering idle times and creating smarter ways to handle data. Often, the validity of the results rely on models instead of evidences : The strength of grating couplers with respect to other biosensing techniques like SPRs is that thickness and refractive indices are obtained independently, with no model assumptions.

The rst question leads to the fabrication cost reduction, the second and third suggest the improvement of the techniques, and the last two suggest the improvement of the instrumentation. Another point was added, about the ideas on which the rest of the work will rely. Finally, this is a brief summary of the goals of this research:

Model unication

The techniques that will be studied along the present work are conceptually very close to each other, as all of them are evanescent eld techniques. Nevertheless, along this chapter these techniques were presented separately, and a global prospective about the systems would be an interesting contribution. In chapter 2 a unied model is developed, in order to describe, from the TE mode propagation in a at 53

CHAPTER 1. 1.7.


AIMS OF THIS THESIS

BIOSENSING

dielectric waveguide to the adlayer growth on a corrugated surface of an SPR, using a few ideas and a simple mathematical formulation.

Fabrication cost reduction

At rst, it was proposed to fabricate the sensors using materials and techniques available as elements of the CMOS fabrication chain. On one hand this decreases the costs by allowing the fabrication without the purchase of additional equipment and, on the other, this makes possible the integration with sources and detectors in monolithic systems. It was found that, apart from standard techniques (PECVD, LPCVD, photolitography, RIE, and HF-Etch) only a nanopatterning technique was needed. This method may be as expensive as electron beam lithography or as cheap as hot embossing molding. With the cost eectiveness in mind, the soft lithography fabrication was explored, and conclusions about what is feasible and what is not were found. In this case the nal result was a fourth instrument, a Surface Plasmon Resonance sensor, with diractive coupling and spectral interrogation operation mode. As in the above case, this instrument is a demonstration of the feasibility of the proposal.

Improving sensing techniques

The use of a multisensing scheme is common in other elds. Apart from the parallelization of measurements, this idea exhibits some other strengths, and for that reason it was decided to bring it to Grating Coupler Biosensing. At the time of writing this report, this group still could not nd any other reference about it applied to these sensors. One of the strengths of the multisensing approach is the integrated on-chip reference, which may be regarded as an additional goal. Taking advantage of the exibility introduced by a complete re-design of the sensors, it was proposed to extend some congurations in order to perform complementary analyses. In this way it was introduced the waveguide-uorescence excitation system.

Improving instrumentation

An aim of this work was to propose a fully scalable multichannelling approach. The underlying idea consists on implementing the channels 54

CHAPTER 1.


BIOSENSING

1.7.

AIMS OF THIS THESIS

depending on the application rather than on the instrument. For this purpose local functionalization was combined with a specic instrument interface.

Unlike xed OGCBs or spectral SPR systems, rotary grating couplers have an intrinsic performance limitation because of the way the motors search the angular resonances (sec. 1.2.2). Nevertheless, these instruments are unique in the sense that they provide two independent data sets and subsequently their readouts do not rely on any model. It was hence desired to improve the eciency of such systems, and this led to some other proposals.

As sensing is the core subject of the work, dierent sensors, together with the instrumentation needed for their use, were developed (chapter 4). The list of systems is: 1)A rotary OGCB, 2) a multi-sensing double pad OGCB, 3) a surface uorescence excitation device and 4) a spectral interrogation SPR-G sensor. These instruments summarize the dierent proposals and explore their actual applicability. Several common features like thermal control or sample delivery are also translated into components, after a systematic study. As a summary, from these four optical biosensors based on nanostructured surfaces, new ideas were introduced at the model, design, fabrication and instrumentation levels respect the current systems.

55

2. Design of a congurable sensing platform Along this thesis, several sensors were completely developed from the concept. The big advantage, but also the challenge, is the chance to decide every feature from the beginning. Along this chapter the steps of the sensor design are presented, in a bottom-up approach. Although the design parameters are a result from optimization studies, in most cases the chosen values were a compromise between performance and technological or cost restrictions. Some special features were also added to the developed sensors (e.g. multisensing and on-chip reference) which involved additional requirements. To the knowledge of the author, at their date of publication, these features only had been applied in other kinds of sensor. The fabrication of the dierent sensors and the development of their corresponding instrumentation, described along the next chapters, is determined by the following results.

2.1. Architectural levels The design of the dierent instruments of the present thesis consisted on series of steps, each of them based on the results of the previous and on the requirements of the next. This sequence can be summarized in three main blocks:

At sensor level:

The selection of the materials and the corresponding techniques for the sensor fabrication, with the nal goal of getting utility, performance and cost eectiveness.

57

CHAPTER 2. 2.1.

DESIGN OF A CONFIGURABLE SENSING

ARCHITECTURAL LEVELS

PLATFORM

Figure 2.1.1.: One of the proposed sensors. Several coupled light beams propagate across a planar waveguide, between two grating coupling pads. The device is passivated, except at the areas used for sensing.

The search of the optimal parameters for waveguides based on

these materials, and the design of the diractive elements that will be added to them. Once the previous step decided the intrinsic properties of the involved materials, the optical behavior of the structures is a result of purely geometrical parameters, such as layer thicknesses and grating periods.

The overall sensor conguration, which encompasses waveguides and diractive elements. Questions like the size and distribution of the diractive components play a critical role in the success of the devices.

At instrument level:

The design and fabrication of a sample delivery system and the

full optical setup, including light sources and imaging devices. The requirement of sensor disposability had consequences on the ow cell and the sensors, as will be described later. 58

CHAPTER 2. PLATFORM

DESIGN OF A CONFIGURABLE SENSING 2.2.

MATERIALS AND WORKING CONDITIONS

The control and acquisition instrumentation, from the hardware

to the programmed interfaces. The limitations here were cost and computer performance. Although computers are better every day, ecient programming provides better results at the time of application, promises improvements in the future and gets more prot from the same equipment. It seems a contradiction that these questions, which are not controversial while talking about hardware are forgotten so often in the case of software.

About applications:

The selection of a functionalization procedure, ecient for sev-

eral applications and compatible with the sensing surfaces. Part of this decision depends directly on the chosen materials, while the rest may vary depending on the application.

Ideally, another level in which readouts would be automatically

interpreted should be added. In this way, the best commercial instruments provide relevant biological information with ease to non specialized users.

In g 2.1.1 one of the developed sensors is shown, with a set of coupled beams, each one devoted for sensing a chemical channel. For their operation several intrinsic requirements must be met, about materials, layer structure, grating periods and other optogeometrical parameters. In addition, some other features are relevant, as the areas and distances between gratings and the placement of the passivation windows. These questions are the subject of the following sections.

2.2. Materials and working conditions About materials and processes, the four dierent criteria that were considered are knowledge, properties, cost, and compatibility. The word knowledge here refers to the control of the right parameters in order to get the desired results. Two dierent fabrication approaches may be chosen, either based on lithography or on molding. Lithography (either optical, electronic or whatever other) applied on silicon or its derivatives and the complementary thin lm fabrication techniques require clean room ambient conditions. In contrast, molding (e.g. imprinting) is applied to polymers and require a not so controlled environment [O'Shea (2003)]. The need to get an accurate control of the fabrication results suggested the 59

CHAPTER 2. 2.2.


MATERIALS AND WORKING CONDITIONS

PLATFORM

use of materials that, within either option, were standards of application. In addition, the compatibility of non standard materials with processes and infrastructures must be studied in each case. Finally, the search of alternative materials or processes could cover many material science projects by itself, but it can not be discarded as a way for improving evanescent eld sensors in the future. The desired properties are a high refractive index contrast and a high chemical stability. In the case of hard material techniques, silicon nitride (Si3 N4 , with a refractive above 2.02) and fused silica (SiO2 , with an index around 1.46) were chosen because of a good ratio between refractive indices and their common use in CMOS technological processes. Unfortunately, although polymers are in general chemically stable, the most protable alternatives are quite limited [Wang (1991)], and refractive indices above 1.5 are not common. While considering molding and hard fabrication techniques separately, the term cost has dierent meanings. In the case of hard materials, the mere fact of processing the samples inside a clean room represents a large part of the expenses. The use of dierent materials would just vary the costs if the needed steps were very dierent from one case to another. In contrast, in the case of polymers, even without a great improvement in the properties, large dierences between the material costs exist, although processes are very similar. In the case of polymeric waveguides the spin-coating [Madou (2002)] of Poly-methyl Methacrylate (PMMA) was tested as a cost-eective fabrication alternative. Its refractive index (1.492) is close to the average value for polymers [vor£ík (2007)]. Along this work it was evaluated the possibility of making sensors based on this or other polymers, with the conclusion that their refractive indices are too limited for their use as lm layers. Nevertheless, it is still possible to get prot from the molding easiness of these materials, giving them the role of a substrate. Then, using thin lm technology, a lm layer of whatever other material may be deposited, keeping the shape of the structured substrate. In this thesis, this idea led to low cost Surface Plasmon Resonance sensors. Finally, compatibility is considered both with respect to the samples and to the surface modication procedures. Neither the above mentioned polymers, silicon nitride or silica produce modications to biological samples because of contact. Gold is also a commonly used material in biosensing applications, because of its extreme stability and high conductivity, which makes it specially suitable for electrochemical and optical (e.g. SPR) ap60

CHAPTER 2. PLATFORM


WAVEGUIDE OPTIMIZATION

plications. The surface modication techniques depend on the composition of the surfaces. The inability of antibodies to be anchored directly onto the sensor surface gets solved by the use of intermediate linkers [Davies (2005)], usually thiols in the case of gold surfaces and silanes in the case of Si3 N4 . The knowledge of the working conditions is important because the properties of the materials depend on them. In concrete, the activity of biomolecules depends on temperature and pH. For this reason the sensors are expected to operate at certain xed temperatures. Later (chapter 4) the thermal dependence of the system responses will be experimentally exemplied. Although the selection of the light sources will be discussed along chapter 4, each material presents a dierent refractive index which depends on wavelength (chromatic dispersion). For this reason the working wavelengths must be chosen before designing the sensors. Theoretically whatever wavelength is suitable, but, in order to observe collective eects as a well dened polarization, grating coupling or wave guiding, light sources must be coherent. For this reason, illumination was performed by lasers, and hence wavelengths were nally restricted accordingly. In most cases, the He-Ne laser wavelength (632.8nm) was used, while in some others a wavelength of 635nm from a laser diode was chosen. These wavelengths are so close to each other that the dierence between predictions were not signicant, and only the results for the rst one were presented in this document. For another conguration, in which a coupled beam was used to excite uorescent labels, a solid state laser of 488nm wavelength was used. The table 2.1 [Palik (1985)] summarizes the properties of the dierent materials at the mentioned wavelengths at commonly applied temperatures. It must be mentioned that fused silica was the type of SiO2 that was used. Although the numbers presented in the table were used along the optimization process, actual values depend on fabrication parameters, concentration of impurities and temperature, in a manner not fully described here. This problem becomes less important with a fault-tolerant waveguide design.

2.3. Waveguide optimization Once that materials and working conditions have been dened, their intensive properties, like refractive index and absorption, become specied. The subsequent optimization process nds the geometry (layer thicknesses, grating periods, etching depths, etc.) that improve the sensitivity of the projected sensors. At rst, the waveguides will be studied alone but, as 61

CHAPTER 2. 2.3.



Si PMMA Au SiO2 (quartz) (fused silica) Si3 N4 H2 O

PLATFORM

488nm 633nm 25o C 37o C 25o C 37o C n = 4.422, k = 0.0163 n = 3.918, k = 0.0122 between 1.491 and 1.489 (see eq. 2.3.1) n = 0.97, k = 1.85 n = 0.20, k = 3.32 1.549 1.543 1.465 2.043 2.021 1.338 1.337 1.333 1.332

Table 2.1.: Standard refractive indices of the materials used in the devices, at application temperatures.

Figure 2.2.1.: Waveguide structures subject of this thesis. The lm and substrate media are supposed to extend innitely above and below the lm layers, for which optimal thicknesses around the shown numbers were found.

62

CHAPTER 2.


PLATFORM

2.3.


the presence of gratings modies their behavior, the resulting predictions will be rened with an improved method. In this way the results and their meanings can be presented with more clarity. In gure 2.2.1 the simulated structures are presented. The rst one is a dielectric grating coupler, the second is an analogous system based on polymers and the last one is an SPR-G device.

2.3.1. Waveguide thickness A target waveguide fabrication thickness has to be chosen in order to get the highest feasible application sensitivity. The following analysis was restricted to the proposed structures, which are shown in g. 2.2.1 (see also table 2.1). Although these sensors were designed for the detection of adlayer parameters, the optimization process was carried out with respect to buer refractive indices (refractometric operation). In concrete, Milli-Q water was chosen because of its refractive index (which is similar to that of common buers) and its ubiquitous availability. This decision was taken because a unique parameter was needed for comparing dierent structures. On one hand waveguides will be actually optimized, and on the other it will be seen that the provided estimations can be translated to biolayers. As liquids extend homogeneously across and beyond the eld penetration depths, a unique characteristic sensitivity can only be obtained if all the systems are characterized as refractometers. In the case of material layers, dierent thicknesses or penetration depths give rise to dierent sensitivities, which cannot be given before the selection of an application. Rigorously, the last statement is not complete, because refractometric sensitivities depend also on the refractive index of the buer itself (cover medium). Nevertheless, the eect of buer1 may be neglected with respect to that of solid layers. Along sec. 2.5 these statements are justied, and some experimental proofs are given along sec. 4.3. Using the matrix method (see section 1.1.4) the eective indices of a set of wave guiding structures were obtained. Then, sensitivities were calculated by numerical derivation2 and represented vs. waveguide thickness (gures 1 2

On the sensitivity, not on the eective index. This process calculates dierences between close data points instead of innitely narrow intervals. As an example, if ∆ is an increment below the resolution limit of the instrument for nc : nef f (nc + ∆) − nef f (nc ) dnef f ' dnc ∆

63

CHAPTER 2. 2.3.



PLATFORM

Figure 2.3.1.: Predicted eective indices (blue) and coupling angles (red) for a 0.5µm period grating. Waveguide materials are: Si3 N4 (up) and PMMA (down), and the wavelength is 633nm.

64

CHAPTER 2. PLATFORM



Figure 2.3.2.: Analogous gures, at a wavelength of 488nm.

65

CHAPTER 2. 2.3.



PLATFORM

Figure 2.3.3.: Dependence of the eective index (blue) and coupling angle (red) sensitivities on the lm layer thickness, for the same structures described above and a wavelength of 633nm. Si3 N4 (up) and PMMA (down).

66

CHAPTER 2. PLATFORM



Figure 2.3.4.: Analogous gures, at a wavelength of 488nm.

67

CHAPTER 2.



488nm, TM0

0.2 0.18

633nm, TM0

488nm, TE0

0.16 0.14 0.12 0.1 0.08

633nm, TE0

0.06

Index sensitivity dneff/dnc (RIU/RIU)

eff

PLATFORM

0.22

c

Index sensitivity dn /dn (RIU/RIU)

2.3.

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0.11

0.12

Film thickness (µm)

−3

7.5

x 10

7

488nm, TM0

6.5

633nm, TM0

6 5.5

488nm, TE0

5 4.5

633nm, TE

4

0

3.5 3 2.5 0.4

0.5

0.6

0.7

0.8

0.9

1

Film thickness (µm)

Figure 2.3.5.: Predicted sensitivities for Si3 N4 (up) and PMMA-based waveguides (down) at two dierent wavelengths.

68

CHAPTER 2. PLATFORM



2.3.1/2.3.2 and 2.3.3/2.3.4). The method is analogue to that presented by Tiefenthaler and Lukosz [Tiefenthaler (1989)]. As stated before, the cover medium was water and the studied wavelengths were 633nm and 488nm. (m

Checking the grating coupler equation (nef f = next sinθm + m Λλ ; eq. 1.2.1), it can be observed that a high angular sensitivity and hence a good resolutive limit need a high refractive sensitivity. In these simulations the effective indices (left axes) were translated into angles (right axes) under the assumption of a 0.5µm grating period. A slight shift between indices and angles appear due to a variable proportionality. Checking the grating equation (eq. 1.2.1) a one-to-one correspondence between eective indices and angles is found in case of a xed wavelength, but, for the derivatives the relation between scales depends on the eective index itself3 . As the relation between scales also depends explicitly on the wavelength, the eective index and coupling angle curves were presented separately while comparing behaviors at two dierent wavelengths. Depending on the propagation mode, a unique value of the thickness optimizes the sensitivity. It is remarkable that, as the material properties are xed, the obtained sensitivity is an upper limit. Figure 2.3.5 compares results for dierent wavelengths. Several conclusions can be obtained from the simulation results:

3

The most evident fact is the dependence of the sensitivity on thickness and waveguide refractive index. In the case of a waveguide made of silicon nitride, 65nm is the most sensitive lm thickness at 633nm wavelength and T E0 propagation mode. For the same wavelength, 850nm is the optimal thickness for PMMA. Although these results depend on the buer and the temperature, their validity will be assumed for other buers, provided that their indices are not signicantly dierent (e.g. PBS has an index of 1.339, a 0.6% above that of water). These dependencies, together with a not so sharp peak prole (large thickness range without signicant sensitivity loss) suggested to consider a tolerance around a 10% above the mode cuto thickness. h

i

1 λ nef f − m Λ From the grating equation, the coupling angle θ(nef f , λ) = asin next is a function of the eective index. In contrast, dierentiating the same equation the result is:

dθ dnef f λ dθ dnef f = = [n2ext − (nef f − m )2 ]−1/2 dnext dnef f dnext Λ dnext where the ratio between scales dθ/dnef f depends explicitly on the eective index

and, consequently, on the layer thickness.

69

CHAPTER 2. 2.3.



PLATFORM

Because of the dierent contrasts between the refractive indices of their layers, the sensitivities exhibit very dierent values for both materials: 0.15 (RIU/RIU)4 for the nitride-based waveguides and 0.006 (RIU/RIU) for the PMMA ones. The third conclusion is that the optimal sensitivities, obtained for different thicknesses, are not signicantly dierent from one wavelength to another, due to the refractive index homogeneity along the penetration depth. The last important thing is the appearance of higher order propagation modes (T E0 , T M0 , T E1 , T M1 ... and so on) as the lm thickness is increased above their respective cuto values. Depending on the lm thickness, each of these modes may be suitable for sensing or not. For instance, with a wavelength of 633nm and a lm thickness of 65nm, although getting a good sensitivity for the T E0 mode, no TM modes will propagate. In contrast, a wavelength of 488nm allows both the T E0 and T M0 propagation modes with the same thickness, as can be seen in g. 2.3.5.

2.3.2. Grating - SPR sensors It was seen that Surface Plasmon Polaritons can be explained in almost the same way that propagating solutions in dielectric waveguides (sec. 1.3). For this reason, in order to guarantee the existence and the appropriate sensitivity of SPR modes it is relevant again to calculate the optimal thickness of the supporting lm layers. A generalized version of the simulation algorithm described in 1.1.4 was programmed in order to explore resonances in the complex refractive index space. In this way, the same method becomes useful for the study of both dielectric waveguides and SPR systems. An example of the eigenfuntion with two modes can be seen in g. 1.1.3. The results (g. 1.3.1) exhibit the well known fact that a symmetric solution always exists, while an antisymmetric solution appears only above a certain cuto lm thickness (50nm in the case of the proposed devices). The symmetric solution is not suitable for sensing, because this mode is mostly spread into the substrate, while the desired behavior is the detection of the cover refractive index. In contrast, the antisymmetric mode solution pushes a signicant fraction of the energy 4

The RIU/RIU units of some gures are actually non-dimensional, but have an important qualitative meaning: Eective Index shift vs. Refractive Index shift of the cover medium. The units involved are the same, but correspond to dierent systems.

70

CHAPTER 2.


Normalized electric field

PLATFORM

2.3.


0.8

0.6

PC substrate

H2O cover

0.4

0.2

0

Au film

−0.2

100nm −0.4 −600

−400

−200

0

200

400

Cross section [nm] Figure 2.3.6.: Antisymmetric Surface Plasmon wave, supported by a 50nm thickness Au lm layer, resting on a PC substrate. A eld depth of 100nm extends into the cover (positive OX axis).

0

Sensitivity [RIU/RIU]

−0.1 −0.2

TMsym

−0.3 −0.4

Cutoff thickness

−0.5 −0.6 −0.7 −0.8

TMasym

−0.9 −1 40

50

60

70

80

90

100

film thickness (nm) Figure 2.3.7.: Sensitivity of the wave number of a Surface Plasmon vs. lm thickness. Materials: Au lm layer, polycarbonate (PC) substrate and an aqueous cover. 71

CHAPTER 2. 2.3.



PLATFORM

on the cover (g. 2.3.6) and hence it is the usual choice for these kind of devices. In g. 2.3.6 an evanescent eld penetration depth of about 100nm is obtained for an Au thickness of 50nm onto a polycarbonate substrate. As a consequence, symmetric and antisymmetric propagation modes exhibit very dierent sensitivities with respect cover refractive index (g. 2.3.7).

2.3.3. Thickness and fault-tolerance From gure 2.3.3 it can be seen that the sensitivity maxima with respect to thickness of dielectric lms are not sharp, and hence at thicknesses close to the optimal values the loss of sensitivity is not very important. For instance, a PMMA waveguide has (for 37o C , 488nm wavelength and T M0 mode) an optimal thickness at 650nm. At this value, a sensitivity of 7.40·10−3 RIU/RIU is obtained. A 10% larger thickness leads to roughly the same sensitivity. Obviously, these numbers get worse further above the maximum, but it can also be observed that this worsening is greater towards thicknesses below it. In the same example, a thickness of 800nm (a 25% above 650nm) leads to a sensitivity around 7.0·10−3 RIU/RIU , while a thickness of 500nm (a 25% below the same value) does not even reach the cuto point, so no guidance is allowed at all. The same behavior can be observed in all the obtained plots, and hence it may be assumed that thicker waveguides are safer than thinner ones. Deposition techniques as Chemical Vapor Deposition or Sputtering guarantee thicknesses within less than a 5% around the target value, but others like Spin Coating may lead to much larger errors. Even assuming an exact value for the fabricated thickness, refractive indices may not be completely guaranteed, specially when dispersion is important. This means that the same estimations may not be as accurate as expected, and the optimal thicknesses may eventually be higher or lower than estimated. From this it may be understood that a slight overestimation of the target thicknesses may be a safe choice for device fabrication in most cases. In the case of metallic layers devoted for the support of antisymmetric surface plasmons, it was seen that the layer thickness must be kept above a cuto value. Above this cuto point the sensitivity of the antisymmetric mode remains almost unchanged (g. 2.3.7). Although techniques as sputtering provide accuracies below 1nm, less accurate techniques like thermal evaporation are often used, specially in mass production. 72

CHAPTER 2.


PLATFORM

2.3.


2.3.4. Optical similarity Along sec. 1.1.5 it was mentioned that wavelength is an scaling q factor for the eld penetration depth, which can be written as dp = λ0 /[2π n2c − n2ef f ]. As the term aected by the square root is bounded between n2c −n2s and n2c − n2f (with ns and nf the indices of the substrate and the lm, respectively) the penetration depth depends mainly on wavelength. Something similar happens with the appearance of modal solutions. Remembering eq. 1.1.4 ( −2π λ nf (2dcosθ) + 2∆ψf c + 2∆ψf s = 2mπ ) it can be seen that, if phase shifts are equal, the ratio d/λ determines the propagation angle (θ in g. 1.1.1). This means that the characteristic lengths in planar waveguides are the wavelength and layer thickness. As an example, in g. 2.3.5 the quotient between the rst cuto thickness and its corresponding wavelength is 0.0717 (35nm/488nm) for 488nm wavelength and 0.0758 (48nm/633nm), for 633nm. The same conclusion may be obtained using the scaling method for waveguides [Kogelnik & Ramaswamy (1974)], which is based on the fact that the guidance condition (eq. 1.1.2) may be written in terms of non-dimensional parameters as the normalized guide index b = b(V, a), where V is the normalized and a is the index of asymmetry. For TE solutions, these parameters are dened as: q 2 2 n2 −n2 c V = λd (2π) n2f − n2s ; b = nef2f−n2s ; a = nn2s −n −n2 f

s

f

s

In the above expressions the waveguide thickness d only appears in the rst equation, divided by the wavelength. This suggests the same solutions nef f for whatever system with the same refractive indices and an equal value of (d/λ). Finally, for the same eective index the penetration depth, eq. 1.1.14 is proportional to the wavelength. With gratings it happens something similar. The coupling equation (eq. 1.2.1) may be written as nef f − next sinθc = m(λ/Λ) where, if indices are the same, the coupling angles result determined by the ratio λ/Λ. Again, for coupling angles, wavelength scales the grating pitch.

2.3.5. Polymers or dielectrics There were several reasons for rejecting the use of polymers in waveguide lm layers. The following considerations refer to PMMA, but are applicable to other polymers, because of their similar characteristics. 73

CHAPTER 2. 2.3.



PLATFORM

A rst problem is the diculty to get a reproducible PMMA thickness below 1µm, because manufacturers do not document these range of parameters. Nevertheless, the biggest problem is sensitivity. For instance (see g. 2.3.5) at 633nm wavelength the dierence between sensitivities at thicknesses of 900nm and 1µm is an 8%, while the sensitivity of a silicon nitride based waveguide is about a 2500% higher (0.006RIU/RIU for PMMA, vs. 0.15RIU/RIU for Si3 N4 ). There exists an additional reason for discarding PMMA as a wave guiding material. Due to a large thermal volume expansion coecient, polymers exhibit strong changes in their refractive indices. Using the data provided in [Cariou (1986)] for the refractive index of PMMA, it was found, for temperatures around 25o C , the next calibration:

n = 1.493715 − (1.05·10−4 )·T [o C]

(2.3.1)

Refractive indices of 1.49109 and 1.48984 were obtained for PMMA at 25o C and 37o C , respectively. In contrast, most dielectrics, like Si3 N4 vary their refractive index at about 10−5 RIU ·o C −1 , which leads to non signicant dierences within the same temperature range. As can be expected, the refractive index gets lowered upon expansion (higher specic volumes lead to lower optical densities). In gure 2.3.8, the coupling angles depend strongly on temperature. Even if a thermal control system is added, a thermal ripple of 0.2o C around 37o C keeps the resolution limited above 4·10−3 RIU (g. 2.3.9).

Figure 2.3.8.: Predicted thermal shift of the coupling angles for a PMMA waveguide (left) compared with a silicon nitride system (right). The working wavelength is 633nm and the grating pitch is 0.5µm. 74

CHAPTER 2. PLATFORM


DESIGN OF GRATING COUPLERS

Figure 2.3.9.: Eective index uncertainty due to thermal ripple in PMMA waveguides: The same coupling angle may correspond to signicantly dierent external indices. Finally, it was not mentioned that the same thermal expansion responsible of the shift of the refractive index is expected to vary the lm thickness, which complicates the analysis even more. Silicon nitride, as a covalent amorphous solid, exhibit no signicant expansion or dispersion within the studied range of temperatures. Although the use of PMMA as a wave guiding material was denitely discarded, it is still desirable to get gratings at the cost of a molding technique. An alternative is to use PMMA as an structured substrate, above which another lm material can be used. Any material that can be deposited at temperatures below the melting point of the polymer (i.e., at room temperature) may be appropriate [Kunz (1005)]. Here it will be studied the use of gold, with which a Surface Plasmon Resonance sensor may be developed. In this manner, not only another sort of optical biosensor is analyzed, but also the model unication can be tested.

2.4. Design of Grating couplers Once waveguides are properly designed, the next components to design are the diractive coupling elements. Intrinsically, as shown in g(2.4.1), square proled gratings have three parameters that can be chosen: i) grating 75

CHAPTER 2. 2.4.



PLATFORM

Figure 2.4.1.: Confocal microscopy image of a 2µm period square proled grating, with its design parameters highlighted. period, ii) etching depth, and iii) duty cycle (i.e. the fraction of the period occupied by the grooves). Then, the grating area must be optimized in order to get a good ratio between coupling eciency and cost. Finally, the placement of the dierent coupling elements must be chosen, regarding the sensor operation mode and the waveguide parameters.

2.4.1. Grating period: angular interrogation and dielectric OGCBs It has been shown that the coupling angle for each eective index depends on the grating period (see eq. 1.2.1). Now that waveguides have been optimized the following task is the selection of a grating period which couples the working light beam at an appropriate angular range. It is true that the presence of gratings modies the eective index of the waveguide and, consequently, the coupling angles, but this is a second order eect [Darwish (2007)], which just leads to slight shifts with respect the rst predictions. The proof of this statement requires the obtainment of the raw predictions. Figure 2.4.2 shows these predictions against the grating period in polar coordinates, for a Si3 N4 waveguide of 65nm thickness. In the plot, the coupling angles appear as if the physical system was sketched. It may be 76

CHAPTER 2.


PLATFORM

2.4.


90 120

Λ = 0.6µm

60

θ = Coupling angle (deg.)

Λ = 0.5µm Λ = 0.4µm

150

30

Λ = 0.2µm

180

0

330

210

240

300 270

R = Λ (µm) 90 120

Λ = 0.6µm

60 TE0

θ = Coupling angle (deg.)

Λ = 0.5µm

TM0 30

Λ = 0.4µm

150

Λ = 0.2µm

180

0

210

330 TM0 TE 240

300

0

270

R = Λ (µm)

Figure 2.4.2.: Up: Coupling angles for a 65nm Si3 N4 waveguide working at wavelengths of 488nm (blue) and 633nm (red), in the T E0 propagation mode. The in-coupling angles correspond to the passivated areas, while the out-coupling angles were obtained with water as a cover medium. Down:Coupling angles for T E0 and T M0 propagation modes in the same waveguide, working at 488nm. Both gures were obtained using the Thin Grating Approximation (TGA) 77

CHAPTER 2. 2.4.



PLATFORM

easier to understand the meaning of the gures considering that the small rectangles at the centers represent the cross section of the waveguides. The in-coupling angles (represented with arrows pointing inwards) correspond to gratings passivated with SiO2 (n = 1.46), and the out-coupling angles (arrows pointing outwards) correspond to an aqueous cover.

Figure 2.4.3.: Upwards and downwards light coupling. The Grating Coupler equation provides an invariant next sinθext , leading to the same measured angles in air.

The coupling angles appear at the intersections between the solutions for the waveguide (color lines) and the circle that represents a 0.5µm grating period. The symmetries observed in the plots are a consequence of the exchangeability of the grating pads and the symmetry with respect ips of the devices (see g. 2.4.3). This symmetry exists because, although frequently not specied, the grating coupler equation determines the value of next sinθext instead of the coupling angle θext directly. According to the Snell's law, this quantity is preserved in every media, and hence it becomes an invariant of the coupling process. For instance, out-coupling through out , which rethe liquid (upwards) is described with the term nsamp sinθsamp out ceives another another meaning (nair sinθair ) while leaving the ow cell. 78

CHAPTER 2. PLATFORM



While coupling light out through the substrate (downwards), the invariout (entering the substrate), and then ant is rst translated into nSiO2 sinθSiO 2 out again, while leaving the chip. As in both cases the last into nair sinθair medium is the same, the refractive index nair and the measured coupling out are equal. The same happens to the in-coupling process, with its angle θair corresponding eective index. Finally, this structure does not allow transverse magnetic propagation at a wavelength of 633nm, but the T M0 propagation mode may be excited at 488nm. Typically, rotary congurations like the OWLS system scan both polarizations in order to get two independent sources of information.

2.4.2. Grating period: spectral interrogation and SPR It was mentioned that coupling resonances exist either in the angular or spectral domains. These choices are known as angular or spectral interrogation, respectively. Although angular interrogation is the most common option, spectral interrogation has also been reported for both dielectric Grating Couplers [Jenq Nan (2006)] and Surface Plasmon Resonance Sensors [Homola (2002)]. Here the spectral interrogation mode was chosen, for one reason: new complementary instrumentation blocks would be developed. If a series of almost exchangeable instrumentation blocks are provided together with a modular software interface many other systems could be rapidly assembled. Two examples of such systems are spectral interrogation OGCBs and angular interrogation SPR-G systems. The description of the coupling process is quite similar to that of dielectric waveguides, for which the grating coupler equation (eq. 1.2.1) was introduced. In this context the incident radiation excites a surface plasmon polariton by means of the fulllment of a wave number matching condition. The eective index associated with the SPP replaces that of a guided mode in dielectric waveguides, but it is more common to speak about Surface Plasmon Wave Number instead of eective index. These quantities are related with each other by kspw = nef f k0 , where k0 is the propagation constant in vacuum. The grating coupler equation, in this case:

kSP W /k0 (λ) = next sinθinc + m

λ Λ

(2.4.1)

From the above equation it can be seen that resonance may be accomplished either by varying the incidence angle θinc while keeping the wavelength constant, or by varying the wavelength λ, at a xed incidence angle. In 79

CHAPTER 2. 2.4.



PLATFORM

both cases the symptom, instead of the appearance of a peak at some outcoupling direction, is somehow the opposite; the energy absorption by the Surface Plasmon Polariton produces a dip in the reected intensity. If spectral interrogation is chosen, the incidence and reection angles can be kept constant, which has been done here at normal incidence. Under these conditions eq. 2.4.1 with m = 1 can be written as:

kspw (λ) = k0 (λ)

λ Λ

This equation was numerically solved for a 50nm Au layer onto a polycarbonate substrate (g. 2.4.4). The permittivities of gold and polycarbonate exhibit a signicant dependence on the wavelength that had to be taken into account [Johnson (1972); Kasarova (2007)]. 0.1 0 −0.1

RTM/ RTE

−0.2 −0.3 −0.4 −0.5

λ+:739nm

λ−:698nm

−0.6 −0.7 −0.8

λc:703nm

−0.9 −1 650

700

750

800

Wavelength [nm]

Figure 2.4.4.: Surface plasmon reectance, with water as an external medium. The resulting spectrum is modulated by that of the excitation source. The red curve corresponds to the thin layer approximation (TGA), which neglects the inuence of the gratings on the eective propagation constants. On nanostructured surfaces the appearance of standing Bloch waves results in the split of the resonances, as reported in [Gérard (2004)]. The blue curve corresponds to the application of the expressions provided in [Darmanyan (2003)] for the frequencies of resonance, with respect that of a at surface. 80

CHAPTER 2.


PLATFORM

2.4.


The origin of these studies is a paper by Ebbesen [Ebbesen (1998)], in which the nanostructuration of their surface leads to the enhanced transmission of light through metallic layers.

2.4.3. Etching depth and duty cycle The previous study of planar waveguides did not actually take the gratings into account. The approach that ignores the presence of the gratings is called Thin Grating Approximation (TGA). Here this model comes into contradiction with its application. First, adsorbed molecules, as gratings exist on the lm layer. Moreover, typical etching depths of tens of nanometers used in this application cannot be ignored with respect to lm thicknesses in the same order of magnitude. For these reasons, it was proposed to model the grating in the same manner as any other layer (g. 2.4.5). Among other calculation methods (e.g. coupled wave analysis or modal expansion [Botten (1985); Hutley (1982); Li (1993); Petit (1980)]) the Equivalent Layer Approximation (ELA) provides good computational performance, integrability and a clear meaning of the dierent involved terms. This approximation, proposed in [Kunz (1996)] represents the gratings as equivalent layers, with a thickness equal to their etching depth and a refractive index modulated by their duty cycle. Along the rst chapter it was described a method for solving planar waveguides with an arbitrary number of layers (see sec. 1.1.4). This method, combined with the ELA approximation, may be used to describe multilayer waveguides, grating couplers (g. 2.4.5), molecular adlayers (see sec. 2.5) and screening layers (sec. 2.5.4). In all the aforementioned cases the incorporated substructures are described by averaged refractive indices. The proposed expressions that give the eective index of an equivalent layer depend on whether a TE of TM propagation mode is being considered:

neq =

q τ n2f + (1 − τ )n2c (2.4.2)

neq =

q

τ n2f

+

1−τ n2c

and (T E

neq

(T M

≡ neq ; neq

81

≡ 21 (neq + neq )

CHAPTER 2. 2.4.



PLATFORM

Figure 2.4.5.: Equivalent Layer Approximation for the description of a nite depth square-proled grating. As usually, nc , ns and nf are, respectively, the refractive indices of cover, substrate and lm layer, and τ is the duty cycle of the grating. The eective indices were obtained using the ELA approximation for different gratings and the coupling angles were determined using the grating equation (eq. 1.2.1). Then, the sensitivities were obtained by numerical derivation [Darwish (2007)], analogously as in sec. 2.3.1. The duty cycles of 40%, 50% 60% and 80%, together with the etching depths of 10nm, 20nm, 30nm and 40nm were compared to the ideal case of grating absence (TGA), with the results presented in g. 2.4.6. In contrast with the coupling angles, nor the eective indices nor the sensitivities can be measured directly. A good agreement between measured coupling angles and theoretical predictions is a proof of the validity of the ELA approximation. This comparison was left to chapter 3. It was previously shown that larger eective indices produce higher sensitivities and shorter penetration depths. The lm layer has a larger refractive index than its surroundings, but, because of the grooves, a fraction of the volume that otherwise would be occupied by the lm is lled by the buer. This results in the lowering of the eective indices and explains why sensitivities worsen when this fraction is increased. The cases in which this 82

CHAPTER 2.


PLATFORM

2.4.


10

80% of Si3N4 8

Fabricated thickness: 65nm

Angular sensitivity: d θ /dnc

Thin grating (TGA) 9

7

6

5

4

0.04

0.06

60% of Si3N4

50% of Si3N4 40% of Si N

3 4

0.08

0.1

0.12

0.14

Waveguide thickness (µm) 10

9

dg = 10nm dg = 20nm

8

Fabricated thickness: 65nm

Angular sensitivity: d θ/dnc

dg = 0 (TGA)

7

6

5

4

0.04

0.06

d = 30nm g

dg = 40nm 0.08

0.1

0.12

0.14

Waveguide thickness (µm)

Figure 2.4.6.: Eects of the nite size of the grating features on the system sensitivity. The upper gure shows dierent duty cycles for a xed etching depth (20nm) and the lower one shows dierent etching depths for a xed duty cycle (50%).

83

CHAPTER 2. 2.4.



PLATFORM

happens are the lower duty cycles and the higher etching depths, as can be seen in the above curves. Finally, it may be noticed that the limit cases of 100% duty cycle and zero etching depth lead to the predictions given by the TGA approximation, as could be expected.

2.4.4. Size and distribution of the gratings Although a rigorous approach would provide estimations of the coupling lengths and eciencies, the actual parameters depend on non-ideal features, like scattering due to surface corrugations or non-ideal etching proles. Because of these reasons, a mixed study was performed, based on theoretical predictions and parameters measured from a set of testing devices.

Figure 2.4.7.: Coupling geometry: the same grating starts to de-couple energy immediately.

Length of the gratings Unlike the out-coupling gratings, for which the amount of de-coupled energy always grows with length, the in-coupling gratings not only recover the radiant energy from free space, but also couple the modal energy out, as 84

CHAPTER 2.


PLATFORM

2.4.


shown in g. 2.4.7. This means that the same grating plays two dierent roles; rst as a coupler for an incoming beam of constant power (provided by the illumination source) and then as a de-coupler for the vanishing propagating mode. It can be understood that the best coupling eciency will be obtained if the incoming spot covers the entire length of the grating. According with [Pascal (1997); Lyndin (1997)], this eciency is proportional to:

η(L) ∝ [1 + erf (

L ω 2 ω 2 L − )] exp[2[( ) − ]] ω 4Lc 4Lc 2Lc

(2.4.3)

where η is the coupling eciency, dened as the ratio of the coupled to the incident powers. L is the distance between the center of the spot and the border of the grating, Lc is the de-coupling length (distance along which a 60% power loss is obtained), and ω is the beam waist on the surface. As can be expected, depending on the attenuation distance, the extinction may be considered complete. In order to optimize the length L of the gratings, the rest of the parameters of eq. 2.4.3 were measured. First, from the de-coupling patterns (g. 2.4.8) a value of 100µm was estimated for the coupling distance Lc . Then, the beam waist was estimated from cross section images of the coupled beam (g. 2.4.9). From the shown images 270µm can be estimated for an He-Ne laser. For a laser diode 500µm is a good approximation. Using equation 2.4.3 and the obtained parameters, the normalized coupling eciency vs. the distance between impingement point and grating edge was plotted (g. 2.4.11). For the beam waists considered ecient grating lengths range between 100µm and 200µm. This keeps the beam onto the optimal location, but also fully covered by the grating. As rotary systems (see chapter. 4) couple incident beams symmetrically at certain coupling angles ±θc , the in-coupling beams impinge the center meridian of these gratings, which are twice as long as this optimal distance. Finally, from these considerations a grating length of 500µm (overestimated for slightly broader beams) was chosen for a simplied rotary setup.

Width of the gratings If the cost was the same, the best option would be the etch of the gratings across the entire width of the sensors chips, as in some commercial systems like OWLS or ZeptoREADER. Although the rst example is a single 85

CHAPTER 2. 2.4.



PLATFORM

0.95

Intensity counts(arbitray)

0.9 0.85 0.8 0.75 0.7 0.65 0.6

150µm

0.55 0.5 0.45 0

50

100

150

200

250

propagation direction (µm) Figure 2.4.8.: Image analysis of a de-coupling area, under the standard conditions; 20nm etching depth, 0.5µm grating period, square prole. A typical de-coupling length was 100µm.

86

CHAPTER 2.


PLATFORM

2.4.


Intensity counts (arbitrary)

0.035

0.03

0.025

0.02

0.015

0.01

0.005 0

100

200

300

400

500

600

Cross section (µm) Figure 2.4.9.: Cross section of a coupled beam, from which a spot diameter of 270µm was estimated.

87

CHAPTER 2. 2.4.



PLATFORM

Intensity counts (arbitrary)

0.017

0.016

0.015

0.014

0.013

0.012

0.011

0

100

200

300

400

500

600

700

800

Propagation direction (µm)

Figure 2.4.10.: Propagation prole outside the grating area. A typical extinction distance was 6.5mm, which corresponds to 6.7dB/cm.

88

CHAPTER 2.


PLATFORM

2.4.


Normalized coupling efficiency

1 0.9 0.8

ω:1000µm

0.7 0.6

ω:500µm

0.5 0.4

ω:270µm

0.3 0.2 0.1 0 0

100

200

300

400

500

600

700

800

900

1000

Distance between spot and edge (µm) Figure 2.4.11.: Normalized coupling eciency vs. distance between spot center and grating edge, for three dierent beam waists.

channel instrument, the multichannel capabilities of the second are in part a result of this feature. Nevertheless, in this project a sequential lithography procedure was used (sec. 3.2), for which costs and process times grow linearly with grating area, and an option had to be chosen between larger grating areas and a larger number of sensors.

The split of the sensing area into several coupling pads leads to the question of their width. In this regard, if limited beams impinge the coupling areas their waists may be considered, but the coupling elements have not necessarily to be broader than these beams. This, on one hand is due to the need of the system (including source powers, coupling eciencies and scattering losses) to redirect a signicant amount of energy onto the detector. It was observed that this is clearly fullled with grating widths clearly below that of the beams. In fact, it is possible to split a single beam into several channels (see sec. 4.3). On the other hand, the outgoing beams must be narrow enough to avoid exchange between channels on the surface of the detector (more details in chap. 4). A grating width of 200µm was selected according both requirements. 89

CHAPTER 2. 2.5.


RESPONSE TO MOLECULAR ADSORPTION

PLATFORM

Placement of the gratings The attenuation of a guided mode is a limiting factor either for the distance between the grating and the border of the sensor in single grating coupler systems or between in- and out-coupling pads in the dual-grating conguration (see sec. 1.2.1). Subsequently, this limits the size of the chips. An attenuation length between 6mm and 7mm was found (g. 2.4.10), from the analysis of microscopy images. This corresponds to an attenuation about 7dB/cm in the worst case. Because of symmetry, in rotary devices gratings should be placed across the center meridian of the sensors. In xed angle congurations some other features should be taken into account. The dual pad OGCB places a constant evanescent power on the surface of the waveguide, suitable for the excitation of uorescent labels attached on it [Taitt (2005)]. This is an excellent chance for testing a complementary technique with the same device. For this reason, and considering that longer propagation distances lead to higher excitation areas it was proposed to place both gratings as far from each other as possible. Once after the estimation of the propagation losses, it was decided that these were small enough for not taking them into account, and the only constraints arose from handling considerations.

2.5. Response to molecular adsorption As an adsorption analysis tool, evanescent eld biosensors are intended to monitor the growth of an adsorbed (in general proteic or DNA) layer. The assumption that getting a good refractometer guarantees a good biosensor is still not justied. The evanescent eld probes the media adjacent to the lm layer, and it was seen that this allows the measurement of its refractive index. Nevertheless, this resolution has not been translated into surface mass concentrations yet. For this purpose, in this work a model based extensively on the Equivalent Layer Approximation was proposed. As along sec. 3.2.3 some experimental proofs of the accuracy of this approach are given, from now on the validity of this model will be assumed.

2.5.1. Interpretation of the sensor outcome Restricting this analysis to the case in which an adsorbed layer thickness can be dened, the sensor outcome (eq. 1.5.2) may be expressed as a function 90

CHAPTER 2. PLATFORM



of two variables; the adlayer thickness wad and its refractive index nad : (no adlayer

nef f (wad , nad ) = nef f

+ (dn/dσm )exp(−wad /dp (nad ))

(2.5.1)

The other parameters are the eld penetration depth dp , which is a function of both (see eq. 1.1.14) and the coecient dn/dσm , which may be considered constant (see sec. 1.5). It is clear that the adlayer thickness depends on the way that molecules are adsorbed, and it was seen that the adlayer refractive index depends not only on the attached mass concentration but also on the molecular polarizability of the adsorbed species, which varies from one substance to another. If adlayer density and thickness are known, it is trivial to estimate the surface density of attached mass. In the preface it was said that very often results rely on assumptions instead of evidences. In this context, a goal of the proposed sensors is to avoid the need of assumptions about how or which substance is being adsorbed. In terms of variables, it is desired to recover adlayer thickness and refractive index directly from the sensor outcome. A key dierence between dielectric grating couplers and surface plasmon sensors arises here. The ability of dielectric waveguides to support several propagation modes (e.g. T E0 and T M0 ) allows the obtainment of two independent variables. In contrast, surface plasmons provide a single data source, and adlayer indices are generally modeled in advance.

2.5.2. Modeling sensor response In order to understand how sensors behave in real applications the way how parameters are recovered from readouts is not interesting. Instead of it, the attachment process will be modeled and the expected sensor outcome will be obtained. In both ways the sensor readout and the adsorbed mass may be related but, while the rst choice provides a mere algebraic algorithm, the second will show the expected response depending on the type of adsorption. The application of the ELA model to the adsorption of thin molecular layers is sketched in g. 2.5.1. Once assumed that the refractive index of a protein layer follows eq. 1.5.1 (sec. 1.5) this model was programmed to simulate the adsorption of a series of adlayers. It was considered that grating etching depths are not negligible when compared to adlayer thicknesses, specially in the rst stages of the process. For this reason, the molecular adsorption process was divided in two main stages: 91

CHAPTER 2. 2.5.



PLATFORM

Figure 2.5.1.: Adlayer growth model: Depending on whether the adsorbed layer is or not thicker than the grating etching depth two dierent situations are considered. The transition from one regime to another must be continuous. For simplicity the substrate was not shown in the gures.

Adsorbed thickness below the grating etching depth (g. 2.5.1, bottom). The system may be described using six layers: 1) the substrate (not shown), 2) the unetched fraction of the lm layer, 3) the etched and covered volume, comprising a fraction covered by the lm material and another lled by the adsorbate, 4) the etched and uncovered volume, in which the adsorbate volume is replaced by the liquid buer, 5) the volume above the grating in which some adsorbate can be found, and 6) the buer cover. Adsorbed thickness above grating etching depth (g. 2.5.1, top). Again, six layers describe the situation, but not the same: 1) the substrate (not shown), 2) the unetched part of the lm layer, 3) the full etching depth of the grating, with the same structure than layer (3) in the previous situation, 4) a pure adsorbate layer, which thickness is the dierence between that of the adsorbed layer and etching depth, 5) the top of the adlayer, following the corrugations of the surface of the 92

CHAPTER 2. PLATFORM



waveguide, and 6) the buer cover. Obviously, both cases should converge in the limit case of equal adsorbed thickness and grating depth (g. 2.5.1, middle). First, it was supposed that molecules are adsorbed onto a single layer, and only after an eventual saturation a new layer would appear. This leads to the top plot in g 2.5.2. Then, layers were supposed optically identical and the adsorption of a single homogeneous virtual layer was modeled. This layer had the refractive index of saturation. As can be seen, the results are clearly equivalent. In these simulations a proteic adlayer is adsorbed onto a Si3 N4 waveguide of 65nm. The refractive index of a monolayer was set to 1.34327, which corresponds to 90ng/cm2 of adsorbed protein mass [Vörös (2004)], according to a De Feijter coecient of 0.18g/ml. It should be mentioned that the translation between surface and volume concentrations takes the thickness of the adlayer into account (here set to 30nm). In practice, except for very low concentrations, not all the particles are adsorbed on a single layer, and a mixture of both approaches must be considered [Schasfoort (2008)]. The combination of the provided results cover all the possible adsorption modes. For the determination of the minimum detectable mass the adsorption of a single layer was considered. After this simulation the external indices at which the grating coupler produce the same outcome were found (g. 2.5.3). Under similar situations an index sensitivity of 8·10−2 RIU/deg corresponds to a mass sensitivity of 40µg ·cm−2 deg −1 . The ratio between both is 50µg ·cm−2 deg −1 RIU −1 . Using standard instrumentation it is possible to quickly scan by steps of 10−2 degrees and, at lower speeds, one degree can be sampled in thousands of steps. With a resolution of 10−3 degrees for the coupling angles, the preceding estimations can be translated into 8·10−5 RIU and 4ng/cm2 for the refractive index and attached mass resolutive limits, respectively. The OWLS instrument, with about 2000 samples per degree oers resolutions up to 1ng/cm2 [Vörös (2002)]. It must be mentioned that all evanescent eld sensors are equally represented by eq. 1.5.2. For convenience, it will be written here again: ˆ∞ dn dσm −y/dp o nef f = nef f + e dy dσm dy o

The term dn/dσm represents the dependence of the refractive index on the surface concentration (represented by the De Feijter model) and the term 93

CHAPTER 2. 2.5.



PLATFORM

13.96 1.5068

3rd adlayer

13.95

1.5066

13.945

nd

2

1.5065

adlayer 13.94 13.935

1.5064

1st adlayer

1.5063

13.93

Coupling angle (deg)

Effective index (RIU)

13.955 1.5067

13.925 1.5062 13.92

10

20

30

40

50

σad (ng/cm2)

60

1.5069

70

80

3rd adlayer

1.5068


13.9641 13.9593

1.5068

13.9545

2nd adlayer

1.5067

13.9497

1.5066

13.9449

1.5065 1.5064

90

13.9401

st

1 adlayer

13.9353

1.5063

13.9305

1.5063

13.9257

1.5062

13.9209

1.5061 0

0.5

1

1.5

2

2.5

Coupling angle (deg.)

1.5061 0

13.9161 3

Number of adlayers

Figure 2.5.2.: Eective index and coupling angle vs. adsorbed mass. Upper gure: Adsorption of three adlayers, according to the De Feijter model (eq. 1.5.1), onto a Si3 N4 waveguide of 65nm. The chosen value for dn/dσm was (0.18ml/g) and the thickness of each adlayer was set to 30nm. Lower Figure: Another point of view leads to equivalent results. With a xed refractive index (n = 1.34327 corresponding to 90ng/cm2 ), the adlayer thickness was continuously increased (each unit in the X axis represents 30nm). 94

CHAPTER 2. PLATFORM



1.506135

13.9181

1.506118

13.9171

1e−4deg. 1.506109

13.9166

1.5061

13.9161

1.506092 0

1

2

3

4

5

6

σad (ng/cm2)

7

8

1.50622

13.9156 10

13.923

1.5062


9

13.922

1.6e−4 RIU

13.921 1.50618 13.92 1.50616

2e−3 deg.

13.919

1.50614 13.918 1.50612

1.5061 1.3379


13.9176



4ng/cm2 1.506126

13.917

1.338

1.3381

1.3382

1.3383

1.3384

13.916 1.3385

Buffer refractive index (RIU)

Figure 2.5.3.: Up: Adsorption of 10ng/cm2 mass on a single adlayer, Si3 N4 system. With a grating period of 0.5µm the coupling angle shifts 2.5 ∗ 10−3 deg. Down: The same system working as a refractometer, where a dierence of 6 ∗ 10−4 RIU leads to a shift of 7.5 ∗ 10−3 degrees.

95

CHAPTER 2. 2.5.



PLATFORM

dσm /dy represents how mass is being adsorbed (single layer, islands, etc.). If this last term is a step function eq. 2.5.1 is obtained. None of both depend on which sensor is being used, and hence the dierence between sensors must arise mainly from the penetration depth dp . This is important here because the proposed dielectric grating couplers and SPR sensors have eld penetration depths of 100nm.

2.5.3. Sensing beyond Regarding scaling considerations, eld penetration depths are increased with wavelength (sec. 2.3.4). This fact suggests that the better sensitivities found for 488nm in the above gures are due to a stronger evanescent eld connement in the adsorption volume, which is, in any case, very small in this example. In g. 2.5.4 these sensitivities are evaluated. Figure 2.5.5 shows how sensor response become saturated above certain thickness which depends on the system, because of the limited eld penetration depth. As a conclusion, longer wavelengths scan further distances away the lm layer, but with less sensitivity.

Sensitivity dneff/dwad

0.02

0.015

λ: 488nm

0.01

λ: 633nm 0.005

0 0

60

120

180

240

300

360

Adlayer thickness [nm]

Figure 2.5.4.: Sensitivities of the eective indices under the same conditions.

96

CHAPTER 2.



1.5137

14.3672

Sensor saturation

1.5136


1.5135

full range / e

14.3603 14.3533

1.5134

14.3464

1.5133

14.3394

1.5131

14.3325

1.513 1.5129

14.3255

Sensing range

λ: 488nm

14.3186

1.5128 1.5127 1.5126 0


PLATFORM

14.3116 14.3047

No adlayer 60

120

180

240

300

14.2977 360

Adlayer thichness [nm] 1.5072

13.9832


full range / e

13.9697

1.5068

13.9563

1.5066

13.9429

Sensing range

λ: 633nm

1.5063

1.5061 0


Sensor saturation 1.507

13.9295

No adlayer 60

120

180

240

300

13.9161 360

Adlayer thickness [nm]

Figure 2.5.5.: Eective indices for T E0 propagation modes upon adlayer adsorption from an aqueous medium. The waveguide (Si3 N4 , 65nm thickness) operates at 488nm (up) and 633nm (down). Because of the limited eld penetration depth, and regardless the adsorption process itself, above certain thickness the sensors become insensitive. The coupling angles correspond to a grating period of 500nm.

97

CHAPTER 2. 2.5.



PLATFORM

2.5.4. Field distribution and chemical passivation Passivation is a common practice in micro-technology [Madou (2002)]. This process consists on protecting parts of a functional device from the contact with the surroundings by means of a deposited layer. Reasons may be the ambient pollution or the possibility of short circuits between electrical contacts. Here chemical passivation will be ned as the process by which the sensitivity of a sensor to the chemistry of its surroundings is inhibited. Two good reasons for its incorporation are the need to couple light into the sensors at a xed angle in the double grating scheme and the possibility to select the sensitive areas. Although this concept was demonstrated for SPR sensors [Homola (2001)] at the time of its publication [Darwish (2010)] the authors could not nd any previous reference about its application in the context of grating couplers.

Figure 2.5.6.: Eect of passivation on evanescent elds. With SiO2 at both sides, the eld extends symmetrically towards substrate and cover, but away the sample. Figure 2.5.6 compares the eld distributions of screened and unscreened devices at 633nm wavelength. A convenient screening layer must cover the evanescent eld suciently, causing a dramatic reduction of the local sensitivity. With the assistance of such an insensitive channel, analyses can be performed in a dierential conguration, with the out-coupling spot 98

CHAPTER 2.


PLATFORM

2.6.

MULTISENSING AND ON-CHIP REFERENCE

always referenced. For this reason, chemical passivation becomes a tool for the design of an on-chip referenced sensor system. Roles of blocked reference channels include angular referencing and light source monitoring.

Coupling angle respect 0 (deg.)

3.5

3

λ: 488nm; TM0

2.5

λ: 633nm; TM

0

2

λ: 488nm; TE0

1.5

λ: 633nm; TE0

1

0.5

0 0

50

100

150

200

250

300

350

400

450

500

Passivation thickness [nm]

Figure 2.5.7.: Coupling angles vs. passivation thickness, with respect to a non passivated Si3 N4 waveguide of 100nm, at two dierent wavelengths, using water as a cover medium. Figure 2.5.7 shows how, as the thickness of a SiO2 passivation layer is increased, the sensors get inhibited. Accordingly, in refractometric mode, with no passivation layer (zero thickness) the coupling angle corresponds to the water buer, while for larger thicknesses this angle tends to that of an innitely thick screen medium. From the obtained estimations, a passivation thickness of 500nm was proposed.

2.6. Multisensing and on-chip reference Traditionally, separate negative control experiments were needed to distinguish between specic and unspecic responses. This is denitely a drawback for at least two reasons. First, it is generally needed to use a dierent sensor chip, and hence an additional calibration should be performed. Second, there is no reason for getting the same random eects at equivalent stages of the measurement. These eects involve wavelength or intensity drifts at the light sources and the mere, but unavoidable, impossibility of 99

CHAPTER 2. 2.7.


GEOMETRICAL DESIGN CONSTRAINTS

PLATFORM

having exactly the same temperatures at the same exact instants. The third question is the need to know whether the attachment is specic or not, which cannot be deduced only from the fact of getting signal from the functionalized pad, specially if the sample is heterogeneous. The evident solution of these problems is to perform the negative control experiment at the same time and on the same chip, for which a multiple sensing scheme is a requirement. A multiple sensing scheme is not only useful for simultaneous negativecontrol [Darwish (2010)], but also for heterogeneous or multiple sample analysis. In the rst case, apart from the detection of more than one substance, it is possible to nd additional information from the correlation between measurements [Soper (2005)]. On the other hand, if each channel is employed to analyze a dierent sample, a noticeable reduction in the cost per assay is also obtained. As an example, in the current devices, the placement of nine channels (assays) instead of one at each sensor produced a cost increment of about a 4%, leading to a reduction by a factor 8 in the cost per assay.

2.7. Geometrical design constraints At the time of designing the wafer the problems of optical losses, handling comfort, torsional resistance, distribution of uidic elements and number of sensor devices within the same cost were taken into account.

Chip size and torsional stress A 4 wafer can be cut into a number of square devices which depends on their size. It is evident that smaller nite elements approximate any area with more accuracy, and hence smaller devices make wafers more protable. Furthermore, the endurance of the resulting sensors depends on their area. As an example, a few insertions broke many 20mm x 20mm test devices, but the nal sensors (16mm x 16mm) remained always intact. The torque caused by the same force with an arm of 2mm is 1.25 times higher than that of an arm of 16mm. At the same time, the section to length ratio of the smaller chips is 1.25 times larger than that of the bigger ones. It is hence preferred to design the smallest chips possible, within some other restrictions. 100

CHAPTER 2.


PLATFORM

2.7.


Other constraints There are two sensor types; single and dual OGCBs. In the rst, the coupling symmetry requires the placement of the diractive gratings across the meridian of the devices. If the dual pad sensors are going to be used for the excitation of Surface Fluorescence the separation between gratings must be as large as possible, in order to improve the excitation area (g. 2.7.1). Biosensors for in-vitro applications use uidic channels or chambers. The rst requirement for these structures is watertightness. Poly( dimethyl Siloxane) (PDMS) is an elastometer employed for this purpose [McDonald (2000)]. In most cases PDMS gaskets are sticked by plasma activation in order to prevent sample leakages [Katzenberg (2005)]. Here it was preferred to develop an eective way for the insertion and removal of both sensors and uidic gaskets. In the applications chapter (chap. 4) a ow cell is presented in which gaskets are sealed by means of pressure. In this case, depending on their aspect ratio and the applied force, channels may eventually collapse. It is commonly accepted that an aspect ratio up to 2:1 prevents this. Because of the fabrication procedure, channels typically have depths between 50µm and 100µm. A distance between paralel channels of 1mm was set in order to reserve space for 200µm width gratings (sec. 2.4.4) and uidic sidewalls. The length of the sensing elements was set to 500µm, according to the coupling distances. The inlets for the uidic channels will be drilled through the PDMS gasket. Because of availability of tools it is recommended to assign 1mm for the diameter of the inlet and outlet tubbing connections, and for this reason a 3mm width border was reserved around the patterned area. Additionally, this space makes handling - and hence insertion - easier.

Wafer mask description The mask set shown in g. 2.7.2 was generated using a the Lasi freeware package [www.lasihomesite.com]. This le had four layers, the rst one for the placement of alignment marks on the wafer, the second one with the gratings, the third one with the sensing windows of the passivation layer and the fourth one with the dicing lines which dene the chips.

101

CHAPTER 2. 2.7.



PLATFORM

Figure 2.7.1.: Design of single (up) and dual (down) grating couplers. A fraction of the area of both kinds of chips was reserved for the uidic inlet and outlet ports (see insets).

102

CHAPTER 2. PLATFORM



Figure 2.7.2.: Up: Zoom view of a 50% duty cycle grating (250nm paths). Down: The complete wafer. On the left it can be seen 12 dual-grating chips, each with six channels. On the right, 12 single grating couplers appear, each with nine channels. 103

3. Cost-ecient sensor fabrication and test Although dierent sensing schemes have been proposed according to expected performances, the previous analysis may be still considered as purely speculative. For this reason, the present chapter at rst describes the fabrication of these devices, and then compares their experimental responses with the theoretical predictions given along chapter 2.

3.1. The selection of a technology Along the design process it was stated the need to fabricate a single-mode optical waveguide, as much sensitive as possible with respect to the external refractive index. The guidance medium should have a refractive index above its surroundings, with clear consequences on the list of compatible materials. Then, once after materials have been chosen, their refractive indices become xed parameters of the structure. The proposed transduction elements are diractive gratings, which must be placed in such a way that coupling and de-coupling of light beams propagating in free space can be achieved at appropriate angles and in the rst diractive order, in order to prevent energy loss in spurious outgoing beams. As it happens with whatever nanoscaled device (e.g. transistors [Millman (1979), pp. 210]), the functionality of these elements is a result of material intrinsic properties and geometry. The very rst requirement of the proposed devices is that substrates should have lower refractive indices than lm layers (see chapter 1). The same applies to passivation layers if they exist. There are also compatibility issues between materials that cannot be ignored. For instance, polymeric substrates may be damaged by organic solvents, and CVD dielectric deposition is performed at temperatures far above the melting point of polymers. Furthermore, unlike silicon or silicon nitride, polymeric surfaces oer the molding option as a cheap alternative to photolitography for being structured. The conclusion is that fabrication must be conceived as a single 105

CHAPTER 3. 3.1.

COST-EFFICIENT SENSOR FABRICATION AND

THE SELECTION OF A TECHNOLOGY

TEST

process, restricted by the chosen materials, and it is desired to restrict it to standard or semi-standard choices. Because of their operation principles, the optimal design parameters of the proposed sensors are scaled by the wavelength of the working light. It was seen that the visible spectrum is convenient because of alignment easiness and a better average cost of the detection systems. The visible range of wavelengths pushes these sizes towards the nanometric scale (see chapter 2). From this fact thin lm deposition and nanopatterning techniques become unavoidable for the present project. In all cases the fabrication sequences have analogue steps: a standard lm material is proposed, for which a compatible substrate is selected. The properties of the involved materials determine the ratio of thicknesses of the layers, but also restrict the applicable fabrication technologies. The range of options is deliberately restricted to standard materials and well controlled fabrication processes. Doing so, fabrication parameters (temperatures, exposure or etching times, etc.) can be obtained from previous studies. Of course new materials may improve the performance of the nal devices, but the development of new fabrication protocols represents a topic by itself.

Availability and cost Two of the most important restrictions are process availability and cost. For instance, holographic patterning is known as the most cost eective alternative for the translation of regular nanometric patterns onto a surface [Chen (I977)]. It is true that the purchase of the needed equipment would be, in this case, similar to that of the fabrication of several wafers, employing other methods. Nevertheless, this and other options were rejected and a process based on electron beam (e-beam) lithography was planned, for a two reasons. First, the set-up and tuning of a technique would involve costs and delays far above the restrictions of the project and second, a sequential lithography method provides a higher exibility, suitable for the study of etching depth or duty cycle eects on the performance, between others. In addition, the Nanofabrication Laboratory at MC2, in the Chalmers University of Technology, through the MC2ACCESS program1 gave this group the option of a fully founded fabrication study under the European Research 1

European contract no. 026029, Program "Structuring ERA", "Research Infrastructures" Action. Call identier "FP6-2004-Infrastructures-5".

106

CHAPTER 3. TEST

COST-EFFICIENT SENSOR FABRICATION AND 3.2.

HARD SUBSTRATE GRATING COUPLERS

Infrastructures Program, using their fabrication facilities, which included ebeam lithography, but no holography.

3.2. Hard substrate Grating Couplers Because of the need to get a substrate with a refractive index below that of the wave guiding layer, two options were evaluated. In the rst one, the surface of a silicon wafer is oxidized up to one micron thickness. The other alternative is the use of wafers of fused silica (SiO2 ). Although being equivalent structures, the last alternative generates a fully transparent device, with the desired option of substrate coupling (see g. 2.4.3). The chosen substrates were a) 0.5mm thickness, 4 silicon wafer and b) 1mm thickness, 4 fused silica wafer. The fabrication sequence was the following:

Cleaning

The rst step was the standard RCA clean (RCA1 and RCA2) of the wafers, which consists on three steps: 1) Organic clean, under a 1:1:5 solution of N H4 OH , H2 O2 and H2 O, at 75o C , for 10 minutes, 2) Oxide strip: in order to remove the thin oxide layer resulting from the last step, the wafers were immersed in a 50:1 solution of HF and H2 O at 25o C and nally 3) Ionic clean; using a 1:1:6 solution of HCl, H2 O2 and H2 O at 80o C for 15 minutes in order to remove the rests of metallic pollutants. A Stangl 1109 wet process bench was employed along this process.

Waveguide fabrication

Thermal Oxidation of a buer layer. The silicon wafers were placed in a dedicated Centrotherm CESAR furnace, kept at 1100o C for 8h. The target thickness was 1000nm for the oxide layer. This step, as the oxide strip in the RCA clean, was skipped for the fused silica wafers. The resulting layer not only is again composed of fused silica, but also it is thick enough to be considered innite with respect to evanescent eld penetration depths rarely above 100nm. The thickness of the SiO2 layer was measured using a Woollam M2000 Spectroscopic ellipsometer, with a result of 1100nm. 107

CHAPTER 3. 3.2.



TEST

The silicon nitride (Si3 N4 ) of the waveguiding lm layer was deposited by Low Pressure Chemical Vapor Deposition (LPCVD). According to [Tonnberg (2006)] for a target thickness of 65nm the samples were processed in another dedicated Centrotherm CESAR furnace, for 18min at 770ºC. The chemical reactions that take place inside the chamber are: 3SiH4 + 4N H3 → Si3 N 4 + 12H2

3SiCl2 H2 + 4N H3 → Si3 N4 + 6HCl + 6H2

A thickness of 64.1nm was measured using the Woollam ellipsometer. The distance between this value and the target produces no signicant dierences with respect to the optimal behavior. This dierence, below a 0.4% demonstrates the ne control of the fabrication process, and represents a reason for the use of this technique instead of others [Vörös (2002)].

Alignment marks The nanometric diractive patterns must be aligned with a set of passivation windows that will be added later (g. 3.2.1, upper image). These features will be patterned on dierent layers, and hence these layers will need alignment marks at their corresponding masks. The rst option is the etch of these alignment marks together with the gratings and then to align them with the marks of the window mask. Unfortunately, as the gratings would be planned to be a few nanometers thick, the alignment marks would be too shallow for their observation under the microscope. The solution was the etch of a deeper version of the same marks (1µm etch) with an additional photolithography step. The corresponding mask (mask#1), contained only four small marks on the corners. In gure 3.2.1 it can be seen how their design preserves the orientation against rotations and ips. In g. 2.7.2 these marks can be seen as tiny crosses outside the sensors.

For the alignment marks, 1µm photoresist was spin coated, using a Headway resist spinner. The marks of the mask#1 were transferred to the resist by UV exposure, employing a Suss MicroTec MA6 mask aligner. The working wavelength of this aligner is 400nm, and the power per surface unit is 6mW/cm2 . Photoresist was developed and patterns were inspected using a metallographic microscope. 108

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Figure 3.2.1.: Devices and alignment marks: placed at the corners of the design (up), alignment marks appear at each mask. Each layer color corresponds to a mask (down). Red corresponds to mask#2 (gratings) and blue corresponds to mask#3 (passivation windows).

109

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The marks were dry etched onto the unprotected areas of the wafer using an Oxford Plasmalab® system. Finally, the photoresist was wet stripped with an organic photoresist remover, leaving the clean Si3 N4 surface with four easy-to-nd marks.

Diractive gratings

The electron-beam lithography (e-beam, EBL) for the grating etching uses a high denition electron sensitive photoresist (HSQ) which was spin coated using the above mentioned spinner. The resist thickness was 50nm, still above the 20nm depth of the nal structures. This is important because later the protected and unprotected areas will be etched at the same rate, leading to no dierences above the thickness of a resist layer. The gratings were dened by EBL. In this process a beam of electrons sequentially impinges the surface of an electron sensitive resist, like HSQ. Under the presence of free charges dielectric surfaces would also get charged, and the electrons from the beam would nd an increasing potential against their way towards the target surface. In order to overcome this problem a thin Cr layer was sputtered onto the resist. This step is mandatory for dielectrics, as the silicon nitride and the resist. An electron beam sequentially inhibits cross-linkage (if a positive resist is used) on the impinged areas. The mask (mask#2) is purely virtual; the CAD le that denes the gratings is translated into a software sequence of instructions, and never actually fabricated. The employed instrument was a JEOL model JBX-9300FS. With it, feasible spot diameters range between 4nm and 100nm, far below the narrowest paths fabricated (200nm, for the 40% duty cycle of a 0.5µm period grating). During the development of the resist the Cr deposited on it was lifted-o. The patterns of the resist were transferred onto the silicon nitride layer by means of Reactive Ion Etching (RIE) in an Oxford Plasmalab® system, at an etching rate of 10nm/min. A reactive plasma etches anisotropically the surface of the entire wafer, without distinguishing the photoresist protection. For this reason the grating depth cannot exceed the thickness of the electron-sensitive resist. The resist was nally stripped under an organic solvent solution, unveiling the nanostructured surface of the silicon nitride layer. The chromium sputtered onto the resist gets lifted o along this step. 110

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3.2.


Passivation Every light beam that propagates through an unprotected waveguide will experiment scattering due to corrugations or pollutants on its surface [Payne (1994)]. In addition, it is desired to make some coupling gratings insensitive to the external conditions, in order to get a constant coupling angle. The proposed solution for both problems consisted on the deposit of a SiO2 passivation layer onto the entire surface of the wafer, and then to etch windows on the areas devoted to sensing. Before doing so, it was thought convenient to reserve some unprotected devices in order to use them as embossing masters for their use with polymeric substrates.

The passivation SiO2 layer needed to be thick enough to cause a signicant shielding of the evanescent eld. According with previous simulations (sec. 2.5.4) 500nm was chosen as the target value. The Plasma Enhanced Chemical Vapor Deposition (PECVD) is a convenient technique for this range of thicknesses, one order of magnitude above those typically obtained by LPCVD. A Centrotherm furnace chamber was kept at 710ºC for 60min under a TEOS (tetraethylorthosilicate; Si(OC2 H5 )4 ) atmosphere. The chemical process was the following: Si(OC2 H5 )4 → SiO2 + residues The Woolam ellipsometer measured a passivation layer thickness of 510nm. Further tests (sec. 4.3.4) conrmed the adequacy of this thickness.

Window opening

The removal of the passivation oxide from the sensing areas requires a last etching step, with a third lithography sequence. A new photoresist layer was spin coated and exposed to UV radiation through the mask#3 (window denition). The conditions were equivalent to the rst lithography sequence, and the alignment marks placed along previous steps played here their critical role. The windows were etched under a 50% solution of HF. This process is characterized by automatic stop, due to a dierence of about three orders of magnitude between the etching rates for SiO2 and Si3 N4 . After a few minutes the oxide from the unprotected areas was completely etched, leaving the nitride surface beneath intact. The protec111

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Figure 3.2.2.: Scanning Electron Microscopy (SEM) image of a fabricated device. tive resist was nally stripped with an organic remover, leaving the nished devices ready for being diced from the wafer.

Sawing

Sticked on a plastic strip, the wafers were diced into 16mm x 16mm chips, using a Loadpoint Microace 3+ dicing saw facility. Because light coupling takes place through gratings, no subsequent polishment of the borders was needed.

Results inspection About the accuracy of the fabrication parameters, a Woollam ellipsometer measured a 64.1nm thickness Si3 N4 layer, a 1.4% error with respect to the target thickness of 65nm. This dierence has no a noticeable translation in the sensitivity of the sensors (see g. 2.3.3). About the etched grating sidewalls, they were perfectly vertical and the etching depth predicted in each case was accurately obtained (g. 3.2.2). 112

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3.2.1. Summary of the technological process The fabrication sequence consisted on 26 steps, divided in nine blocks: 1. Wafer RCA clean: 1.1) Organic clean, 1.2) oxide removal and 1.3) ionic clean. 2. Buer layer: 2.1) Thermal oxidation of the wafer surface, if needed (skipped for fused silica wafers) and 2.2) ellipsometric measurement of the oxide thickness. 3. Waveguiding layer: 3.1) Silicon nitride (Si3 N4 ) deposition by LPCVD, and 3.2) ellipsometric measurement of the nitride thickness. 4. Lithography of the rst alignment marks: 4.1) Spin coating of the photoresist, 4.2) UV exposure though mask#1, 4.3) resist development, 4.4) dry silicon etching and, 4.5) wet strip of the undeveloped resist. 5. Grating denition: 5.1) Sputtering of a thin Cr layer, 5.2) spin coating of an electron sensitive resist, 5.3) Electron Beam Lithography (e-beam) etching of the resist, using a virtual mask (mask#2). 5.4) Resist development with lift o of the Cr layer. 6. Grating etching: 6.1) Reactive Ion Etching (RIE) of the full surface, and 6.2) wet strip of the remaining resist. 7. Passivation: 7.1) PECVD deposition of silicon oxide onto the entire wafer and 7.2) ellipsometric measurement of the resulting thickness. 8. Window etching: 8.1) Spin coating of a photoresist, 8.2) UV exposure through mask#3, 8.3) development of the openings on the protective layer, 8.4) HF etching of the SiO2 and 8.5) wet strip of the protective undeveloped resist, using an organic solvent. 9. Dicing: 9.1) Dicing of the wafer and 9.2) device packaging under a clean atmosphere. 113

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Figure 3.2.3.: Sketch of the fabrication sequence, from the point of view of a single sensor.

3.2.2. Characterization Along sec. 2.3.1 (g. 2.3.3) it was predicted that the sensitivity of an optimized Si3 N4 waveguide of 65nm thickness with a grating period of 0.5µm would be about 9 degrees per refractive index unit. This value also corresponds to the experimental results obtained by a developed rotary instrument, which is described in the applications chapter. As this number 114

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represents the shift in the coupling angle due to buer refractive index, it also provides the angular sensitivity of a rotary conguration. In the dual-pad case, (see g. 1.2.1 for more details about both congurations) the out-coupling angles are translated into linear shifts on the surface of an optical detector (like a Position Sensitive Detector, PSD), and hence the sensitivity depends also on the distance between coupler and detector. As an example, gure 3.2.4 shows the linear shift for several concentrations of glycerol, measured by a 7µm pixel width camera (Thorlabs LC1). Between the extreme cases a 50 pixel (350µm) distance can be observed, corresponding to a dierence of 0.002RIU (from 1.333 to 1.335). An optical path of about 90cm was prepared in this case, using a couple of mirrors between sensor and detector.

Figure 3.2.4.: Readouts on the surface of a LC1 detector corresponding to dierent aqueous solutions of glycerol.

In any case, the instrument resolution depends on the ability of the instrumentation for resolving either the coupling angles or the linear shifts, and the use of additional processing tools. As, depending on the distance between sensor and detector dierent shifts may be obtained for the same stimulus, not only the sensitivity, but also the sensing range may be adjusted by choosing the appropriate distance. 115

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Water

Water passivated 15.5

17



17.5

10nm 20nm

16.5

16

30nm 40nm

15.5

15

Etching depth * Experimental data − Simulated model

14.5

14 0.5

0.55

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0.8

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14

30nm 40nm

13.5

13

Etching depth

* Experimental data − Simulated model

12.5

12 0.5

0.85

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14.5

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0.6

Ethanol passivated

17

10nm



0.75

0.8

0.85

15.5

20nm

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16

30nm 15.5

40nm

Etching depth o Experimental data − Simulated model

14.5

14 0.5

0.7

Ethanol

17.5

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0.65

Duty Cycle

Duty Cycle

0.55

0.6

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0.8

20nm 14.5

30nm

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12.5 0.5

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10nm

15

Etching depth

0.55

o Experimental data − Simulated model 0.6

0.65

0.7

0.75

0.8

0.85

Duty Cycle

Figure 3.2.5.: Predicted and measured coupling angles for a Si3 N4 waveguide of 65nm thickness, for dierent grating duty cycles and etching depths, working at 633nm wavelength.

3.2.3. Validity of the Equivalent Layer Approximation The experimental results shown in this section are a proof of the validity of the estimations given in sec. 2.4.3. The measurements were performed on a set of devices specically fabricated for testing purposes. These devices were also a source of information used later along the optimization of the nal devices (see sec. 2.4.4). The Equivalent Layer Approximation was used for the determination of coupling angles and system sensitivities, as a function of grating etching depth and duty cycle. These are the relevant parameters of the squared proled gratings that result from the applied technology. Three dierent duty cycles were prepared at mask level: 50%, 60% and 80%. At a known RIE etching rate of 10nm/min dierent depths were also etched by controlling the processing times. These depths were 10nm, 20nm, 30nm and 40nm. In gure 3.2.5 it can be observed a good match between model and predictions [Darwish (2007)]. Two additional facts will be mentioned here: a) Coupling angles coincide with the expected values in the equivalent layer approximation, even if the 116


TEST

3.3.

EMBOSSED BIOSENSING DEVICES

1.56

17

ethanol 1.55

TGA

16

1.54

15.5

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1.52

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1.51

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30nm 13.5

1.5

40nm

SiO2

eff

Coupling angle θ

16.5

H2O

Effective index n

CHAPTER 3.

1.49

13 12.5

1.48 1.34

1.36

1.38

1.4

1.42

1.44

1.46

External refractive index n

ext

Figure 3.2.6.: Expected values for the eective indices of the same structure, restricted to a 50% duty cycle. external medium was completely composed of silicon dioxide (see g. 3.2.6) and b) the curves for dierent etching depths converge if the duty cycle tends to 100%, and the limit is the prediction of the thin grating approximation. It may be mentioned that the substrate refractive index that best tted the measured angles (1.465) corresponds to fused silica. This material is often called fused quartz. This, from the point of view of the author, is confusing. Unlike quartz, thermal SiO2 is not crystalline, and both refractive indices are signicantly dierent (see table 2.1). As expected, the cover layer inhibits the coupling sensitivity to the external medium, and for this reason the gures that correspond to passivated systems coincide with each other.

3.3. Embossed biosensing devices One of the goals of the present project was to reduce the fabrication costs of the sensors. Unlike other disposable elements, the life cycle of grating couplers is very short, and it is preferred to replace the sensors immediately after each experiment or, at most, after a short series of them. This gives to any cost reduction a strong impact on the protability of the technique. It must be noticed that prototypes are not intended to achieve this goal directly. The purpose of the project is the demonstration of a low cost 117

CHAPTER 3. 3.4.


POLYMER WAVEGUIDES AND GRATING COUPLERS

TEST

technology, applied into the optical biosensing context and with mass production volumes in mind. Several solutions have been proposed by other groups [Vörös (2002); Horváth (2002b)]. The present proposal tries to take advantage of pre-existent processes, and discards the use of any custom fabrication equipment. Here it is shown that optical biosensors based on embossing techniques are as useful as any other, and hence this protable fabrication alternative exists. Embossing techniques are known as a low cost alternative for the fabrication of nanometric structures [Shen (2002)]. Analogously to the case of a Compact Disc, from a single embossing master thousands of replicas may be obtained. The fabrication of this master is a xed cost, which is rapidly redeemed during its subsequent use. In Nano Imprint Lithography (NIL), a nanostructured mold is pressed against a thermoplastic polymer at its glass transition temperature [Gottschalch (1999)]. In this state the polymer still remains solid, but preserves its new shape after been cooled again. Two options were considered, leading to the results of the next sections. The rst (sec. 3.4) consists on patterning the surface of the lm layer. In this case, the waveguide is constructed using a soft material and then the gratings are embossed on its surface. The second option (sec. 3.5) is to emboss the gratings on a substrate and then to deposit the lm layer on it. Then, the lm layer gets patterned because of the shape of the surface on which relies.

3.4. Polymer waveguides and grating couplers In the case of polymeric materials, the most common thin lm deposition technique is the spin-coating process [Madou (2002)], which consists on the centrifugation of a liquid polymer solution and the subsequent solvent evaporation and cross-linking with heat. As a lm material the Poly-methyl Methacrylate (PMMA) [vor£ík (2007)] was chosen, because of its low cost. Its refractive index (1.492) produce single mode waveguides for lm thicknesses below one micron. Cleanroom processing is very restrictive about the allowed materials that can be involved in a fabrication sequence. For instance, non-pure substrates are considered a source of contamination and hence are not allowed. One of these substrates is PYREX. As an ordinary glass, its cost per surface unit is far below that of a fused silica wafer. Fortunately, it is possible to integrate PYREX substrates in soft fabrication processes instead of fused silica. The fabrication sequence is in this case much shorter to that of dielectric devices: 118

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3.4.

Substrates were already supplied as 20mm x 20mm slices, which eliminates the need of a further dicing step, but forces to fabricate sensors one by one. In case of mass production this may be improved, but this limitation is actually an advantage for parametric studies about these sensors.

Organic clean of substrates: The dierent PYREX substrates were left in an ultrasonic bath lled with acetone, for 5 minutes, then rinsed under DI water and nally dried with nitrogen.

Each substrate was spin-coated with a lm layer of PolyMethyl Metacrilate (PMMA) with a thicknesses between 1.5µm and 4µm, following the datasheet provided by MicroChem [www.microchem.com].

Embossing of the surfaces: The grating structures were transferred

onto the PMMA surface of the devices by NanoImprint Lithography (NIL), using an Obducat Eitre® system [Diéguez (2007)]. The process parameters were 30Bar pressure, 150o C and 5min. The embossing masters were the previously fabricated dielectric sensor chips, without passivation. In order to recover the same structures, the original devices were prepared with a 50% duty cycle.

3.4.1. Characterization Several unembossed waveguides were characterized using a prism coupler (g. 3.4.1, top) and compared with the theoretical predictions calculated according to [Ulrich (1973)]. The coincidence is quite accurate (see g. 3.4.1, bottom) except for the lowest thickness, which was slightly underestimated with respect to the supplier calibration curve. Grating coupling was also tested (see g. 1.1.1, in the theory chapter), but results were considered redundant, specially after the decision of discarding the use of these waveguides for biosensing. These were the reasons:

Passivation problem: The dual-pad conguration is not feasible

for embossed systems, because of the need of passivation of the incoupling pad. Although the entire surface may be easily passivated at room temperature (e.g. by spin coating or sputtering) there exist no easy ways for etching sensing windows without damaging the underlaying structures.

Scattering: The contact of the sidewalls of uidic channels or other obstacles with the unprotected surface of the waveguide, along an optical path results in scattering and strong energy losses. This does not 119

CHAPTER 3. 3.4.



TEST

Figure 3.4.1.: Up: Prism coupler with a spin-coated PMMA (n=1.492) waveguide. Down: Prediction and (two) measurements of the eective indices vs. waveguide thickness, for several PMMA waveguides with air as a cover medium.

120

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EMBOSSED GRATING SPR SENSORS

prevent the system to operate, but is clearly disadvantageous, when compared with the passivated area of a dielectric chip, completely inert to this eect.

Sensitivities: The main problem is obviously sensitivity. As the

direct measure of this parameter is not possible, the theoretical predictions, based on the well proven ELA model and the accurate characterization of the waveguides, were accepted. According to these predictions, polymers could be up to ten times less sensitive than dielectrics (see g. 2.3.3 in the design chapter).

Although it may seem that embossing techniques are not a good choice for grating couplers they have been used, for single grating congurations, from the beginning [Lukosz & Tiefenthaler (1983)]. The proposal of alternative solutions for the aforementioned problems in the framework of dual-pad systems is a matter of technology and material science, which are not the subject of this research. Nevertheless, it is possible to develop ecient Surface Plasmon Sensors based on embossed polymeric substrates, as described along the next section.

3.5. Embossed Grating SPR sensors An alternative to the use of polymers for the waveguiding lm is to assign them the role of a substrate. The main advantages are that in this way the possibility of embossing gratings already exists and that there is no need to rise their refractive indices. The new problem is the low temperature2 deposition of any material devoted to work as a lm layer. An easy answer for this question is the use of sputtered metals, leading to Grating Surface Plasmon Resonance (SPR-G) devices [Darwish (2008a)]. It should be noticed that SPR-Gs are actually grating couplers. Although frequently described as electrical charge waves, surface plasmons are also associated to strongly conned electromagnetic waves, and the same equations describe well both kinds of sensors, if parameters are handled and interpreted correctly, as shown in chapter 2. 2

At least below the polymer melting point.

121

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Figure 3.5.1.: AFM image of the embossed surface of the SPR sensors.

Again using dielectric chips as embossing masters, the fabrication sequence was even shorter than that for embossed OGCBs:

Several 250µm thickness Polycarbonate sheets were purchased from Goodfellow [www.goodfellow.com], for their use as substrate layers. No PYREX or other supporting layers were needed. These sheets were shipped free of pollutants and covered by a protective lm, and can be cut using scissors.

Gratings were embossed on the surface of the PC sheets using a Nano

Imprint facility (the aforementioned Obducat Eitre® 6 system), again working at 60bar pressure for 3 minutes, at dierent temperatures around 130o C , without noticeable dierences. A 50nm layer of gold was sputtered using a Von Ardenne 730s system, controlling the process with a Quartz Crystal Microbalance.

In g. 3.5.1 the result of the entire process can be observed. This case corresponds to the use of an embossing master with gratings of 10nm depth. The defects on the embossed surface appeared due to the use of recycled dielectric samples as embossing masters, and, because of their average sizes, gave problems only when embossing features had thicknesses below 20nm. 122

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3.5.1. Spectral response of the Surface Plasmon Waves Plasmon waves are strongly attenuated, which prevents their use in the same congurations that were studied for grating couplers. For these kind of devices it is generally preferred to study the reectance spectrum, either in the angular or spectral domains. This is also an option for dielectric grating couplers [Jenq Nan (2006)], but propagating beam congurations, when feasible, are easier to handle and provide higher exibilities, as will be shown along the applications chapter. In the proposed measuring conguration the reectance spectrum was studied (see sec. 2.4.2). A plasmon wave produces a dip in this spectrum because of a strong energy absorption around a single resonance wavelength. A broadband light source was focused normally onto the surface of a grating, and the reectance spectrum was recovered using a spectrophotometer (Ocean Optics SD2000). If a dip in the spectrum corresponds to the excitation of a surface polariton it should only appear if the incidence polarization is TM.

Figure 3.5.2.: Dip generated at the reection spectrum by an excited surface plasmon wave. It can be observed a coincidence between g. 3.5.1 and g. 2.4.4. A few conclusions can be obtained from this coincidence: 123

CHAPTER 3. 3.5.



TEST

From the agreement between theoretical predictions and measurements, the validity of the presented model can be conrmed. Nevertheless, as a result of the fabrication process slight dierences between devices may appear, and each sensor must be calibrated. The aforementioned split in the frequencies of absorption is a symptom of the apparition of standing Surface Plasmon Polaritons, and suggest the study of these structures in a transmission conguration [Ebbesen (1998)].

124

4. Developed instruments and application results The sensors described along previous chapters, although under dierent situations, were conceived to carry out a generic task: the specic detection and quantication of species along their adsorption onto a surface. Of course, these devices need additional instrumentation for the complementary tasks involved in their use: the recreation of a processes of interest, the transduction of the signicant parameters, the translation of the obtained signals into whatever kind of information suitable for analysis and nally, the storage of this information. The requirements for these tasks and their solutions will be studied along this chapter (g. 4.0.1).

The complete list of components can be divided in the following blocks:

The sample delivery block, which ends at a uidic cell where the sample comes into contact with the sensor, includes the tubing and connections from and to the external reservoirs, and nally a pump and its control electronics for the establishment of a controlled ow rate. The thermal control system, which encompasses whatever kind of system heater, a thermal sensor and a closed-loop electronic control system. A well designed ow cell may be also considered a part of this system, as it combines the other elements, the sample and the sensor. The optical module consists on the illumination source, the discreet optical components (lenses, polarizers, beam splitters, etc.) that dene the beam characteristics, the optomechanics that controls angles and distances and, if the setup includes moving components, the corresponding control electronics. The imaging or acquisition block, which may include, depending on the conguration, photodetectors, cameras or spectrometers, and the corresponding control electronics and informatics. 125

CHAPTER 4. 4.1.

DEVELOPED INSTRUMENTS AND APPLICATION

GENERAL INSTRUMENTATION BLOCKS

RESULTS

Figure 4.0.1.: An optical biosensing instrument comprise at least a sensor in a ow cell, sample delivery, thermal stabilization, optics and signal acquisition.

The sensor itself may be considered a block apart, but transductors are obviously optical, and their design have unavoidable consequences on the entire optical module. Finally, ow cells were conceived as common blocks, with well dened uidic, optical and thermodynamic features.

These blocks are described at the beginning of this chapter. Then, the rest of sections describe the instruments developed for each kind of sensor, and the -often preliminary- results from their application. The four instruments that will be described are a rotary OGCB system, a xed angle dual-pad OGCB, a surface uorescence excitation device (OGCB-F) and an spectral mode SPR-G instrument.

4.1. General instrumentation blocks In all cases, the developed sensors are designed for in vitro applications, and hence the rst requirement that arises is the ability to recreate adsorp126

CHAPTER 4. RESULTS

DEVELOPED INSTRUMENTS AND APPLICATION 4.1.


tion events in a reproducible manner. It was seen along the theory chapter (see sec. 1.5) that adsorption dynamics depends on the rates of encounter between molecules and their linkage eciencies. The rates of encounter depend on the design of the uidic system and the injected ow rates, and for this reason ow channels must be designed and ow rates must be controlled. The linkage eciency of an active surface depends on an intrinsic factor (chemical anity) and on an extrinsic one, its rate of occupation. As a linkage event occurs, the surface becomes occupied by target particles, reducing the surface concentration of vacancies. When an experiment departs from a known state (either absence or saturation of adsorbed species) the surface occupation is the most relevant factor, which produces the characteristic adsorption or desorption curves. This behavior is predictable, and from its kinetics quantitative information can be obtained (see sec.1.5). Finally, chemical anity depends on molecular structure, which depends on the environment, mainly temperature and pH. The extreme cases are a living organism, in which these molecules exhibit their highest anity, and the conditions in which they become denatured. While pH may be controlled with the use of the appropriate buers, temperature must be controlled electronically.

4.1.1. Sample delivery system The way to keep a constant rate of encounter consists in the control of the ow rate, combined with a regular shape for the exposure volume. In practice, this is a uidic circuit fed by a high precision pump and with a custom microuidic cell. A desired requirement of biosensors is a low sample consumption. Both injection and peristaltic pumps handle extremely small ow rates, but the last ones have the option of closed-loop recirculation. In this manner, higher ow rates would not mean larger sample consumptions. A Gilson MINIPULS® 3 [www.gilson.com] peristaltic pump was chosen. This pump is able to perform forward and backward injection at a controlled rate, and provides a simple remote control interface [Jaramillo (2009)]. This interface consists on a six-pin connector, placed on the rear side of the pump (g. 4.1.1):

The two upper pins control the head rotation speed. With the supplied voltage, the actual speed may be selected between zero (0V) and the maximum speed (5V). This maximum achievable speed can be selected in the front panel of the pump. 127

CHAPTER 4. 4.1.



RESULTS

The middle connectors act as a switch, stopping the injection if a voltage dierence of 5V is supplied. The two bottom pads decide the rotation direction, which depends on the polarity.

The ow rate of the system depends not only on the rotation speed, but also on the diameter of the tubes of the circuit and the geometry of the ow cell. After calibrating this pump, it was obtained a ow rate of 5ml·hr−1 ·rpm−1 , using 1/16 internal diameter pipes from Upchurch® Scientic. A computer interface was programmed in LabVIEW® for the injection control, assisted by an USB data acquisition (DAC) board, model NI USB6250, from National Instruments [www.ni.com]. With this system, a constant ow rate could be accurately stablished along an entire experiment.

4.1.2. Thermal module A thermal control subsystem was included in the presented instrumentation, for the establishment of realistic experimental conditions. For instance, human proteins act in the human body at a single temperature of 37o C , and studies carried out at 25o C would not be representative of this situation. The thermal module consists on the following parts (g. 4.1.1):

A Peltier cell, which transfers heat towards the sample while a current is applied between its terminals. A PT100 thermal resistor, which measures the temperature of the sample. A Jumo eTRON microstat, which acts as a switch for the current supplied to the Peltier cell. Depending on the voltage given by the thermal resistor, this microstat opens or closes the circuit that feeds the Peltier cell. A 12V DC source, for feeding the Peltier cell and the microstat.

The microstat is internally calibrated by the manufacturer, and gets the temperature directly from the thermal resistor. By controlling the temperature tolerance the accuracy of the heat transfer may be improved, but this instrument has a resolution of ±0.1o C around the set point. 128

CHAPTER 4. RESULTS



Figure 4.1.1.: Scheme of the sample conditioning block. A Jumo eTRON microstat acts as a switch controlled by temperature between the Peltier cell and the current source. The control temperature is provided by a PT100 thermal resistor, inserted into the ow cell. Simultaneously, a computer controlled Gilson MINIPULS® peristaltic pump injects the sample into the same cell. Acquisition and control is managed by a DAQ system from National Instruments.

4.1.3. Fluidic cell and gasket The full custom part of the uidic system comprises two elements; an gasket based on an elastomer (PDMS) and a metallic ow cell.

Fluidic gasket Depending on the experiment, it may be desired to dispense the same sample among several channels, to dispense dierent samples among dierent groups of channels or to select a dierent sample for 129

CHAPTER 4. 4.1.



RESULTS

each channel. The consequence is that, even for identical sensors, a dierent gasket may be needed for each application. This strongly suggests the use of photolithography of SU-8 and PDMS casting [McDonald (2000)]. The uidic channels or chambers were designed to be in direct contact with the sensing zones of the chips, letting the dierent samples ow into and out from the cell through a standard microuidic tubing system. Figure 4.1.2 summarizes the fabrication process:

A single layer CAD le denes the uidic paths, according to the locations of the sensing pads and the space left for uidic connections. The Q-CAD [http://www.qcad.org/] Linux software was used for this purpose. A photo-mask is printed on a Polycarbonate Film using a high resolution printer. This method, aordable for features above 10µm, is widely used for uidics, here with channel sidewalls of 100µm width. An SU-8 (SU-8 50, purchased from Microresist.de) negative photoresist layer of 100µm thickness was spin-coated onto a microscopy glass slide, and then lithographically patterned giving rise to a complementary mold for the gasket. The surface of the molds were covered by PDMS in liquid phase and then cross linking was promoted in an oven along 4hr at 80o C . Finally, gaskets were detached from the SU-8 molds and cut appropriately.

Flow cell

Because of cost and exibility, a unique ow cell design was shared between all the developed instruments. Its development was a challenging task because of its shared role as an optical, uidic and thermal component. Its list of the requirements and the proposed solutions is the following:

Compatibility with the use of the aforementioned PDMS gaskets.

Easy insertion and removal of the sensors. Usually the microuidic

problem is solved by sticking PDMS gaskets to the hard supporting surfaces. This is done by surface activation, using plasma reactor [Katzenberg (2005)]. Although sensors were conceived as disposable devices, it is possible to give them several uses if they are correctly cleaned. This is a bad clinical practice, but a strongly desirable option in research applications. Under this situation, the watertightness is preferably guaranteed by pressing both parts (sensor and gasket) instead of sticking them. For this purpose the ow cell had a self-aligned housing block (see g. 4.1.3-(a)). 130

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Figure 4.1.2.: An example of uidic gasket devoted for its use into the uidic cell assembly. A photo-mask designed with the Q-CAD software was printed on a Polycarbonate lm, using a high resolution printer. The SU-8 photoresist was used for the fabrication of a mold, which nally led to the PDMS gasket.

Fault tolerance with respect to thickness of the gaskets: PDMS

gaskets are moulded from liquid phase, which cause some diculties. First, their thickness exhibit uncertainties up to 0.5mm due to the accuracy of the amount of PDMS used and second, depending on the oven in which they are prepared, gaskets may be slightly tilted. In order to solve this problem the thickness of the housing block can be adjusted using two screws, one on the top an one on the bottom (see g. 4.1.3-(b)). This is the minimum number that distributes pressure homogeneously, which results in an easy and reliable insertion.

Light coupling: although transparent, PDMS surfaces may be rough (specially those which were in contact with the mold) and hence produce scattering. In addition, PDMS gaskets are above 500µm of dispersive thickness. As shown before, light coupling may be accomplished by impinging either the top or the bottom surfaces of the sensors, and thanks to the Snell's invariance (see g. 2.4.3) the measured angles are the same. It is obvious that the top surface will be 131

CHAPTER 4. 4.1.



RESULTS

protected by the uidic gasket. In contrast, the bottom surface (the substrate is a SiO2 wafer) is perfectly at. It is hence preferable to place the gasket and the inlet/outlet ports in the rear of the housing block, and to leave the sensor bottom facing the coupling window of the cell. In addition, this keeps the tubing connections hidden, which is an additional advantage (see g. 4.1.3-(c)).

Safe placement of the sensor chips: Firsts tests demonstrated that tiny pressures easily break sensor chips if cells were made of plastic, but strong pressures didn't brake any sensor if cells were metallic. The reason is the dierence in the rigidness of both elements, being the torsion of the plastic pieces a source of stress that sensors cannot absorb. For this reason the ow cell was made of aluminum.

Thermal control integration: The need of temperature control combined with the high thermal conductivity of the aluminum suggested the use of a Peltier Cell for thermal stabilization. These devices have a 'cold' and a 'warm' faces, which can be switched and controlled by an applied current. In this case the cold size was left in contact with the bigger thermal mass of the rear block (see g. 4.1.3-(d)), which is also attached to the rest of the system. The warm side faces the housing block, which has a thermal resistor in order to monitor the temperature of the sensor. In order to prevent heat transfer between the cold and warm parts of the cell, these are attached with plastic screws. Spurious reections: In order to prevent them the entire cell was anodized.

Flexibility: For convenience it was proposed a common cell for dual

and single pad congurations. In the rst, the coupling pads are placed by the border of the cell window (square hole in block (b), g. 4.1.3). This window was small in order to increase the contact surface between sensor and supporting block, which reduces the stress while pressing sensors against gaskets, under the risk of getting shaded gratings. For this reason the housing cover sidewalls have a 45o inclination. In a rotary conguration the sensing pads are placed across the center meridian of the sensor, and sidewall tilt does not improve neither disturbs the incoming beams. In this case the coupled beams are observed through the border sections of the sensors and hence the sides of the cell are opened. Finally, in order to keep the impingement beam always onto the coupling pads, the rotation axis of the cell relies on the surface of the chip. 132

CHAPTER 4. RESULTS



Figure 4.1.3.: System ow cell: Sensors and uidic gaskets are placed in the housing block (a), with the chip substrate looking towards the window of the cover piece (b). Inlet and outlet pipes are connected through the rear block (c). A Peltier cell is placed in the thermal housing of the rear block (d), with its warm side facing the housing block.

133

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RESULTS

4.1.4. Illumination block The presented sensors exhibit resonances in both the angular and spectral domains, which can be exploited in a series of congurations. Unfortunately, this fact also leads to crossed sensitivities, which may worsen the overall resolution. Thus, resonances in the angular interrogation mode become broader because the system actually uses a narrow spectral band instead of a single wavelength. It can be understood that this turns a single coupling angle into a narrow angular coupling range. In g. 4.1.4 it can be seen the translation of a wavelength uncertainty of 0.01nm into estimations of attached mass based on coupling angles. Equivalently, the resolution of the spectral interrogation mode is limited by the collimation of the working beam. 13.918

∆ σ : 5ng/cm2. ad


13.9175

13.917

13.9165

∆ λ: 0.01nm 13.916

∆ θ: 1e−3deg. 13.9155

13.915

13.9145 0

1

2

3

4

5

6

7

Adsorbed mass σad [ng/cm2]

8

9

10

Figure 4.1.4.: Coupling angle shift due to variations in the working wavelength. With a De Fejter coecient of 0.18, a 65nm thick silicon nitride waveguide (n=2.01) and a working wavelength of 633nm, a shift of 0.01nm in the working wavelength fakes the adsorption of a mass of 5ng/cm2 . In order to prevent crossed sensitivities in the angular interrogation mode the light source must be spectrally as narrow as possible. Beam collimation must be guaranteed also, not only for spectral interrogation but also for the angular mode, as its inaccuracy would introduce an additional uncertainty. Lasers fulll these requirements, but their beam waists are broader than the 134

CHAPTER 4. RESULTS


ROTARY GRATING COUPLERS

grating areas. This problem was addressed by the use of telescope assemblies and pinholes. For a dual grating coupler it was found that, using a 5mW energy source the recovered energy was enough to clearly distinguish the out-coupled beam, regardless whether the incoming beam was limited or not. Lasers are also coherent light sources, which is a requirement for the appearance of collective eects like interference -required for guidance- and diraction -required for coupling-. Lasers also provide well dened polarization states, although for the accurate ltering of TE or TM polarization modes additional polarizers were employed. Although not mentioned, the dierent setups share these elements, rigidly assembled to optomechanical components.

4.2. Rotary grating couplers A rotary OGCB system, similar to the OWLS instrument (sec. 1.2.2) was developed as a biosensor and a test bench for the samples (g. 4.2.1). The ow cell was mounted on a Newport [www.newport.com] SR50 automatic goniometer, managed by a Newport SMC100CC controller. A Hamamatsu S2386 (320nm - 1100nm) photodetector was placed at each side of the ow cell, in order to scan the coupling resonances. This is done through the photocurrents generated by the coupled beams when they reach the borders of the sensors. A Thorlabs CPS196 laser-diode source of 635nm wavelength was chosen as a very compact illumination source.

4.2.1. Resonance tracking algorithm A modular software was developed for the management of the instrument, using the LabVIEW instrument control environment [Jaramillo (2009)]. These modules were organized as a series of layers:

Interface: The interface provides results from a series of angular scans along a period of time dened by the user. These results, automatically stored in a spreadsheet le format, consist on a series angular locations at which resonances were located and their corresponding time labels. The start button (g. 4.2.2) also connects the pump in order to start the injection automatically. 135

CHAPTER 4. 4.2.


ROTARY GRATING COUPLERS

RESULTS

Figure 4.2.1.: Rotary Grating Coupler instrument: A step motor rotates the ow clock- and counterclockwise, alternatively. Two photodiodes transform coupling beam intensities into photocurrents, and a DAC board digitalizes and sends these data to a computer.

Full scan: Each scan is completed after the location of two symmet-

rical peaks (or two couples of peaks, if TE and TM modes are being scanned together). Then, the corresponding peak locations are identied and centered, and the corresponding data are added to a pile. A center-of-mass peak identication algorithm was used, together with a special interval restriction block. In a rst scanning cycle the a full pre-dened angular range is scanned. Once the rst location of the resonance is found, the subsequent scans cover only their vicinity, nding the new locations and dening a new scan interval for the next cycle. This strategy improves the acquisition sample rate by a factor proportional to the ratio between the full and the limited intervals. In the lower image of g. 4.2.3 the software scans 3 degrees instead of 15, leading to a 5-fold improvement in this ratio.

Scan step: Scan cycles are organized as an ordered sequence of rotations and photocurrent acquisitions, according with an angular step 136

Rotary instrument interface:

Figure 4.2.2.: Under the LabVIEW® environment (1) the interface includes a starting angle selector for up to four resonances (2), a general control block (3), injection control (4), and several outputs, consisting on the current data (5), the last scan (6), and the sensogram (7).

137

ROTARY GRATING COUPLERS 4.2. RESULTS

DEVELOPED INSTRUMENTS AND APPLICATION CHAPTER 4.

CHAPTER 4. 4.3.


FIXED ANGLE GRATING COUPLER

RESULTS

dened by the user. Each of these steps is as a single instruction with respect to upper software levels.

Instrument level: The bottom software layer generates the instruc-

tions that are sent to the SMC100CC controller, and recovers the already amplied photocurrents acquired by a National Instruments NIUSB 6251 board.

4.2.2. Preliminary results The result from a calibration experiment performed with several liquids is shown in gure 4.2.3. The larger resonances, close to ±14o correspond to the sensing area, while the shorter ones, placed at ±9.5o correspond reections and are not sensitive, as can be seen. From the bottom image in the same gure of from g. 4.2.4 an angular sensitivity of 9deg/RIU is obtained, according with the theoretical predictions given along chap. 2. This sensitivity is high enough for biosensing applications, as discussed along sec. 2.5. The time evolution of the resonances is shown in g. 4.2.4. A sample rate of 1Hz was obtained, using the algorithm described above. Unfortunately, the interpreted language and the general purpose motion controller and step motor represent a bottleneck for the eciency. This makes the system suitable for low abundance sample detection, but quite limited as a kinetic analysis tool. The resolution of the grating coupler instrument depends on its ability to resolve angles. The SR50 rotation stage guarantees a 0.001deg precision, which, combined with a 9deg/RIU sensitivity of the sensors, gives a raw resolutive limit of 10−4 RIU . According to the predictions given in sec. 2.5 this optical resolution corresponds to 4.4ng/cm2 of specically attached mass.

4.3. Fixed angle grating coupler The OGCB conguration proposed by Lukosz [Lukosz (1990)] consists on a dual grating coupler assembly (see sec. 1.2.1): an incoming laser beam is coupled into a planar waveguide at a grating and then coupled out by 138

CHAPTER 4. RESULTS



Figure 4.2.3.: Angular spectrum prole obtained for dierent liquids using the rotary instrument.

139

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RESULTS

Figure 4.2.4.: Evolution of angular resonance when dierent buers are injected. another one (g 4.3.1). The out-coupling angle, which may be measured using whatever linear photodetector, depends on the amount of mass adsorbed onto the waveguide. Position Sensitive Detectors (PSD) (for instance [www.hamamatsu.com]) provide sub-micron precision at a very low cost. For this reason PSDs have been the preferred option in most cases for detection [Lukosz (1995)]. Nevertheless, PSD devices obtain the coordinates of impingement from the measurement of overall photocurrents, which allows the detection of only one spot per sensor. Then, if more than a single spot is being measured, the cost rises linearly with their number, while the cost of camera-based systems does not vary. In addition, the cost of the detection elements is still far below that of other components (e.g. the illumination source) and hence it has a little repercussion on the overall cost. The option of simultaneous monitoring of several spots carries the dualgrating sensors to a world of potential applications, starting by the selfreferenced detection and the multiple sensing scheme. Just as an example, multi-molecular detection, assisted by a PCA analysis may be used as a diagnose tool, in cases where a disease cannot be identied by a unique label. 140

CHAPTER 4. RESULTS



Figure 4.3.1.: Dual-pad Grating Coupler instrument, with a surface uorescence module (further details in sec. 4.4). This represents a second level of abstraction, in the same sense that functionalization transformed physical quantitative data into qualitative specic concentrations.

4.3.1. Multiple self-referenced detection The conditions that promote the adhesion of a new adlayer involve a change of the chemical composition of the buer, which by itself produces a shift in the detected signal. In addition, there exist global eects like the dependence of the refractive index of the buer on temperature, that cannot be stabilized completely [Diéguez (2009b)]. Multichannelling (g. 4.3.2) is a solution for the buer problem: a channel in which adsorption is inhibited may serve as a reference for the others. In this way, not only dierent adsorption processes may be studied together, but also the eect of the buer refractive index may be distinguished from adsorption. Thanks to this conguration, active channels could be acquired in a dierential operation mode, and then compared with each other. 141

CHAPTER 4. 4.3.



RESULTS

Figure 4.3.2.: Several spot pattern from a multichannel sensor, taken with an Allied Marlin SXGA camera of 1392x1040pixels.

Figure 4.3.3.: Interface for the dual grating instrument: On an image recovered by the camera a set of bands may be selected (1), the out-coupling spots are tracked along these bands (2) and their evolution is plotted vs. time (3). 142

CHAPTER 4. RESULTS



4.3.2. Channel denition by software The use of cameras instead of PSDs provides an almost-unlimited scalability, at the price of a higher software complexity, because of the need to resolve resonances from dierent channels. It is desired to use the same instrument in several dierent scenarios, form a single channel to a high number of locally functionalized channels in dierential mode with respect passivated and blocked areas. An instrument interface and analysis system was programmed using the Matlab® environment in order to track and store the readouts from an arbitrary number of resonances in real time (g. 4.3.3). The evolution of each angular resonance can be tracked as a movement of its corresponding out-coupling spot along a single axis on the imaging device. These axes are parallel, and hence there is no need to explore more than a few narrow bands (cross sections, one per channel) of these images. The computational speedup due to processing vectors instead of matrices results in the recovery of several samples per second.

4.3.3. Improving resolution The acquired signal may be aected by several sources of disturbance, including pressure or thermal ripple and interferences between neighbor channels. Furthermore, the overall resolution may be limited by the size of the detection elements and other constraints. Figure 4.3.4 corresponds to the use of a Thorlabs LC1 linear CMOS array. This detector, with a full width of 21mm, divided in 3000 pixels, gave a raw resolutive limit of 7.5·10−5 RIU when placed 60cm away the sensor, which corresponds to the assignment of the outgoing beam to a single pixel. This distance was chosen in order to keep a reference (passivated) and a sensing spot within the same image (g. 4.3.5). The corresponding out-coupling angles dier by 3deg. (see g. 3.2.6 in chapter 3), while realistic sensing ranges are around 1deg. (see g. 4.2.4). From this it may be understood that this on-chip referencing scheme sacrices 2/3 of the overall resolution. The alternative to this is the use of a chemically blocked channel as a reference, which certainly remain in the range of measurement. Another improvement was the sub-pixel processing (g. 4.3.5): In its denitive form, a 2D image (g. 4.3.2) is cut into a series of slices, each one corresponding to a single channel. The cross sections of these slices can be averaged, and nally the result was tted using a two shifted Gaussian x−xc2 2 1 2 model (A1 exp( x−xc w1 ) + A2 exp( w2 ) ). With a band width of 50 pixels 143

CHAPTER 4. 4.3.



RESULTS

Figure 4.3.4.: Dual Grating Coupler calibration curve, from several solutions of NaCl an Glycerol. The refractometric response is linear in the sensing range, with a negative slope of 7.5·10−5 RIU/pix.

Figure 4.3.5.: Sub-pixel peak determination in dierential-mode conguration (Thorlabs LC1 camera). The pattern was tted using a double gaussian model. 144

CHAPTER 4. RESULTS



measurements became independent of the shape of the peaks, but crosstalk between neighbor channels was also avoided. Finally, the combination of both strategies, together with an appropriate placement of the detectors may easily improve resolution up to 10−5 RIU.

4.3.4. Chemical passivation and local functionalization Two dierent passivation alternatives were tested, the rst implemented in the sensors and the second as a part of the functionalization process. Chip passivation inhibits the sensor response completely, while channel blocking along functionalization prevents the adsorption of biomolecules. In this way, a blocked channel may be used for the analysis of the dierent buers, while passivated channels act as a common reference. According to the results provided in sec. 2.5.4 and the fabrication sequence described in sec. 3.2, the entire surface of the sensors was covered by a 500µm SiO2 layer. Then, several sensing windows were etched on certain gratings. For instance, the in-coupling gratings remained passivated, which allowed beam coupling at a xed angle without the disturbance of the chemical composition of the buer or the deposit of whatever analite or pollutant. Some of the out-coupling gratings also remained passivated, in order to get xed reference spots on the optical detector. The eectiveness of this proposal was tested with a series of liquids (g. 4.3.6). For the analysis of dierent samples with the same sensor, local functionalization was proposed. Each out-coupling grating is functionalized with respect to a dierent target and then each spot corresponds to a dierent process, which can be studied simultaneously. Once after combining this with the strategies of channel denition by software and on-chip referencing, the number of channels is limited only by the size of the reactant droplets left on the sensing areas.

4.3.5. Application results Along the following experiment the response of two dierent channels was compared with respect to the injection of dierent concentrations of Human Serum Albumin (HSA). Further details can be found in [Darwish (2010)]. The functionalization process consisted in the following steps:

In order to covalently attach the amine-terminated composites, a Self Assembled Monolayer (SAM) of an organosilane crosslinker (triethoxysilane aldehide, TEA) was deposited onto the entire surface 145

CHAPTER 4. 4.3.



RESULTS

Figure 4.3.6.: Referenced buer detection: The sensing spot (right) measures the buer index, while the reference spot (left) remains xed. The buers were water (n=1.33) ethanol (n=1.36) and 2-propanol (n=1.38). of the chip by vapor phase exposure for one hour. The sample was heated at 90o C for another hour, and nally rinsed and dried with N2 .

Functionalized channel: A 10−7 M solution of IgG monoclonal

antibodies (Anti-human serum albumin (anti-HSA), purchased from Roche Diagnostics [http://www.roche.com/diagnostics/]) was left on the functional channel, using a pipette.

Blocked channel: For preventing unspecic adsorption, the surface

of the blocked channel was saturated with amino poly(ethylene glycol) (PEG-N H2 ). The grating coupler chip was placed in the ow cell described before, kept at a constant temperature of 37o C . Reactions took place in a pH-controlled Phosphate Buered Saline (PBS) medium. Then, HSA solutions with molarities of 10−15 M , 10−13 M ,10−11 M and10−9 M , were injected at a ow rate of 15ml/hr. Between these injections, a 0.1M solution of HCl was injected 146

CHAPTER 4. RESULTS



Figure 4.3.7.: Adsorption of dierent concentrations of Human Serum Albumin (HSA) onto an IgG monoclonal antibody surface (top). The red curve corresponds to a channel blocked with BSA (Bovin Serum Albumin). The rate curves are shown for both channels (bottom). 147

CHAPTER 4. 4.3.



RESULTS

in order to regenerate the surface. The illumination source was a TE polarized 5mW He-Ne laser from SPECTRA PHYSICS, of 633nm wavelength. The camera used for the detection of the out-coupling resonances was a 1392x1040 pixel Allied Marlin SXGA camera, with a 7µm x 7µm pixel size. A cylindrical lens spread the laser beam onto several in-coupling gratings. The results of the experiment can be seen in g. 4.3.7. In the upper image the sensogram for the entire process can be observed. Saturation was achieved before 1min in all cases, except for the lowest concentration. The blocked channel measures changes of the refractive index, but no adlayer formation can be observed. The middle and bottom images of the same gure show the rate of adsorption, expressed as the derivative dnef f /dt of the change in the eective refractive index along time, for both channels. According to the theory, higher concentrations led to higher adsorption rates. The saturation level is always the same, because adlayer formation is limited by the hydrodynamic conditions and the available matrix sites.

Figure 4.3.8.: Dynamic range estimation: from the adsorption kinetic curves the ability for resolving dierent concentrations was estimated. 148

CHAPTER 4. RESULTS



The height of each positive peak in the rate curve (g. 4.3.7-down) is proportional to the injected sample concentration at the beginning of an adsorption event (see sec. 1.6.2). The negative peaks exhibit roughly equal amplitudes, suggesting that the surface departs from saturation in the desorption cycles. These heights were related with the injected concentration in order to estimate a dynamic range curve. In g. 4.3.8 concentrations below 1f emtomolar provide adsorption rates within the noise level, and concentrations above 1nM saturate the dynamic response and cannot be related with actual concentrations.

4.4. Waveguide uorescence excitation Fixed angle OGCB systems allow the waveguide surface uorescence excitation (WG-F), for two reasons:

The waveguide is neither moving nor rotating, and hence light collection systems may focus the emission area without problems and the coupled energy remains unchanged. The low attenuations allow the excitation along large areas. The obtained attenuation length for a Si3 N4 waveguide was 7mm (see g. 2.7.1 in chapter 2). In order to take advantage of this option, sensing windows were opened along the full length between gratings.

As mentioned above, labeling is considered a drawback because of the requirement of sample conditioning, but uorescence still has a sensitivity potentially higher than that of other techniques [Grandin (2006)]. For instance, an extremely low attached mass of analite may still not fulll the assumptions required to be considered a layer (see sec. 1.5). A raw limit may be 10 adsorbed molecules on an area of 500nm x 500nm. For molecules of 75kDa weight, this means 0.5ng/cm2 . In such situation, although collective eects may not be clearly observed, the emission of uorescent labels may be still detected by optical means, with the only restriction of the background light.

4.4.1. Modications of the OGCB and preliminary tests Based on the dual OGCB instrument, several modications were proposed in order to carry out analyses of surface uorescence excitation (see g. 4.3.1). 149

CHAPTER 4. 4.4.



RESULTS

Figure 4.4.1.: Optical setup for the selection between an He-Ne laser (633nm) and a laser diode (488nm).

Selectable laser source and collection systems In order to excite the uorescent labels a Cyan Scientic 488 nm CW Laser from Spectra Physics was used. As the same sensors were used as grating couplers it was proposed to base the new arrangement on the previous experiment setup. A periscope assembly let the user switch between the 633 He-Ne laser and the 488nm Cyan laser by rotating a single mirror. The uorescence emission was quantied by several dierent means. The rst one was the Allied Marlin camera used before, assisted by a wavelength lter, spatially resolved the area of the device, in order to provide high throughput capabilities. The intensity proles of the images can be related with the amount of the active uorophores (g. 4.4.3).

Preliminary tests As a viability test, dierent concentrations of uorescein between 10−4 M and 10−9 M were injected. Fluorescein is a uorophore molecule of 332.306g/Mol weight, which can be excited by wavelengths around 495nm, and then reemits photons in a wavelength band around 519nm. A spectrometer probe 150

CHAPTER 4. RESULTS



Figure 4.4.2.: Sensor operation at two dierent wavelengths. A cylindrical lens is used for the excitation of several channels. This can be done using two dierent laser sources.

151

CHAPTER 4. 4.4.



RESULTS

Figure 4.4.3.: Filtered images of the uorescent-excited areas (top), and their corresponding intensity proles (bottom).

152

CHAPTER 4. RESULTS



was also focused onto a uorescent area in order to get the ngertip of the uorophores (g. 4.4.4).

Figure 4.4.4.: Surface emission spectrum: The wavelength of maximum emission is 519nm.

This arrangement was limited by the sensitivity of the pixels of the detector, the eciency of the λ-lter and the background noise, to uorescein concentrations above 1nM . The performance of the system may be improved, either with the use of a special camera with thermal stabilization or with the use of a dierent excitation geometry [Duveneck et al. (2002)]. To demonstrate the capability of the system to improve this limit of detection a photomultiplier was used, again assisted by an spectral lter. Further improvements of the detection limits may be achieved with the assistance of additional strategies. For instance, the background noise may be strongly canceled if the excitation source is synchronized (chopped) at certain frequency with any of the mentioned detectors. Figure 4.4.5 shows the results from the application of this strategy with a photomultiplier device. 153

CHAPTER 4. 4.4.



RESULTS

Figure 4.4.5.: Photomultiplier readouts: Concentrations up to 10−12 M can be distinguished above a base voltage level of 0.4V, due to an improvable light isolation.

From the measurements, bulk uorescein concentrations of 1nM can be easily distinguished. A bulk uorescein (332.32Da weight) concentration of 1nM , corresponds to 0.33ng/ml. Using the ALOGPS on-line software [www.vcclab.org/lab/alogps] an octanol/water partition constant of log(K) w 3.35 was estimated for uorescein. This is considered a good approximation for the absorption of biomolecules into the cell membrane. If these numbers are used in eq. 1.6.13 (Cadsorbed /Cf lowing = K ) in order to get a raw estimation of the ratio between the adsorbed and owing uorophore concentrations, and a layer of 100nm thickness is considered, a surface concentration of 7.4pg/cm2 would be obtained. This number, similar to that of the Zeptosensor system, is far below the typical detection limits of waveguide techniques of about 5ng/cm2 [Grandin (2006)]. It may be understood that these concentrations correspond to less than a molecule per square micron, so the continuum approximation is not applicable in this case. Nevertheless, uorescence techniques just estimate the amount of attached mass from the emission intensity, with no assumptions about the formation of adlayers. 154

CHAPTER 4.


RESULTS

4.5.

LOW-COST EMBOSSED SPR-G SYSTEMS

4.5. Low-cost embossed SPR-G systems For the embossed SPR-G systems described in sec. 3.5 a wavelength interrogation device was assembled (g. 4.5.1). Instead a monochromatic source, a LS-1 Tungsten Halogen Light Source from Ocean Optics was used as a broadband ber optic illumination lamp. A TM polarized beam from this lamp is focused perpendicularly onto the surface of an SPR-G sensor, placed in a custom thermally-controlled ow chamber. A beam splitter sends the reected light to a SD2000 optical spectrometer, also purchased from Ocean Optics. As plasmon waves may only be excited by TM polarized radiation, a TE reectance spectrum is recorded at rst as a reference signal, exhibiting the lamp continuous spectrum and no adsorption. Then, this reference vector is subtracted from the TM reection spectrum. The obtained curves show a characteristic narrow dip (see sec. 3.5.1) as a symptom of the energy adsorption carried out by a surface plasmon polariton wave. An algorithm for the identication and time tracking of these resonances was programmed, under the Matlab® software package.

4.5.1. SPR-G tests Due to the dependence of the permittivity of gold on the temperature, the acquired data are not reliable unless temperature is stabilized (g. 4.5.2). As in the above cases, the thermal control module consisted on a Peltier cell and a PT100 thermal resistor, connected in a loop controlled by a Jumo eTRON microstat. A clear dierence between SPR sensors and grating couplers is the strong thermal dependence of the rsts, in which a ripple in the recorded signal tracks the story of the power switches of the microstat. Using this setup it could also be observed that the placement of the thermal resistor in the middle of the ow cell reduced the amplitude of the thermal ripple. An explanation for the rst fact, regardless the features of the gold itself, is the small thermic mass of a system supported by a 0.25mm thick plastic layer instead of a wafer. For the second fact, it can be understood that heat transmission from the Peltier cell is more ecient at the center of its surface, and thermal gradients close to 1o C may exist across the employed ow cell. Several concentrations of glycerol were nally injected, in order to characterize the sensitivity of the sensors and the resolutive limit of the instrument. 155

CHAPTER 4. 4.5.



RESULTS

Figure 4.5.1.: Wavelength interrogation SPR-G device setup (up) and scheme (down). Readouts from the optical ber spectrometer can be seen in the upper left inset. 156

CHAPTER 4. RESULTS



Figure 4.5.2.: Time evolution and thermal sensitivity of SPR-G devices: When glycerol concentration was increased by a 5% a shift was obtained, equal to that caused by an increase of 5o C .

Figure 4.5.3.: Refractometric calibration of the SPR-G sensors: From the slope of the tting line a sensitivity of 611nm/RIU was estimated. 157

CHAPTER 4. 4.5.



RESULTS

The refractive index of these solutions (tted with respect the glycerol volume fraction as n = 1.333+1.4·10−3 ·[conc(%)]), ranged between 1.333 (0%) and 1.351 (16%). From the calibration data (g. 4.5.3), a wavelength sensitivity of 611nm/RIU could be observed. With an spectrometer resolution of 0.01nm, a resulting resolutive limit of 1.6·10−5 RIU may be obtained, similar to that of a OGCB system. In sec. 2.5 of chapter 2 it was justied that the performance of an evanescent eld biosensor depends on penetration depth, while a refractometer operates with an homogeneous medium. Working as refractometers, the proposed dielectric waveguides and SPR sensors obtain similar (although not equal) resolutions. In addition, for the optimized geometries similar eld penetration depths of 100nm were obtained in both cases. With similar refractometric resolutions and penetration depths similar mass sensitivities are expected. The ratio between both sensitivities, obtained for OGCB biosensors (50µg ·cm−2 ·RIU −1 ) will hence be used here for the estimation of the mass resolution of SPR-G sensors. With a refractometric resolution of 1.6·10−5 RIU a mass resolutive limit of 1ng/cm2 may be expected for these sensors.

158

5. Conclusions The aim of this work (sec. 1.7) was to answer some relevant questions about understanding, reliability, protability, and performance of optical biosensors. In order to do it, a series of devices were developed, following the unavoidable sequence of modeling, design, fabrication and test, which is reected along this document.

5.1. Scientic contributions The development of a series of sensors based on well known concepts and technologies may not seem a good starting point for the generation of relevant scientic contributions. Nevertheless, this work, subjected to the philosophy of basic academic research, intended to be original and to obtain as general conclusions as possible. Although at the purely theoretical level there was not room for doing this, a generalized representation tool for all the evanescent eld sensors was presented. With respect to design and fabrication, an implementation of on-line reference based on chemical passivation was proposed, for a grating coupler structure of a conceptually unlimited scalability. These systems also allowed the realization of simultaneous complementary analyses of the same sample. Finally, a low cost fabrication process was proposed for the SPR-G sensors.

5.1.1. Theory Some numerical models were developed and combined for the study of devices and processes.

Equivalent Layer Approximation (ELA) The application of the Equivalent Layer Approximation was combined with an implementation of the transfer matrix method in order to study waveguides with an arbitrary number of layers and dierent surface structures. 159

5.1.

SCIENTIFIC CONTRIBUTIONS

CHAPTER 5.

CONCLUSIONS

This approximation demonstrated a good applicability when comparing theoretical predictions and measurement results (see 3.2.3). These results were published in [Darwish (2007)].

Model of adlayer formation An adlayer formation model was developed based on the same ELA approximation, which helps to estimate the amount of attached mass, specially in those cases in which optical density cannot be obtained directly from the sensor outcome (e.g. while using an SPR system). This model allowed the establishment of a correspondence between the resolution of a technique while measuring refractive indices or adsorbed mass, and led to results similar to those presented elsewhere for the same techniques (see sec. 2.5).

Unied representation for OGCB and SPR The described method was implemented in such a way that not only dielectric, but also adsorbing and conductive waveguides could be analyzed (sec. 1.1.4). The predictions were accurate for both grating couplers and SPR-G sensors (see sec. 3.5.1). Although physically equivalent, both sorts of sensors are not usually represented using the same model. Here this model was also applied for the study of adsorption events in both kinds of systems.

5.1.2. Design and fabrication The contributions at the engineering level were basically the application of concepts developed for other systems in the context of grating couplers. Standard technologies were used for the fabrication of the sensors. Althought it is true that new materials would probably improve these devices, the conceptual developments presented here are not incompatible with this option.

Multiple sensing Multichannel grating coupler devices were designed, fabricated and tested, based on the dual-grating pad sensor conguration. 160

CHAPTER 5.

CONCLUSIONS

5.1.


Chemical passivation Passivation was demonstrated for grating coupler devices, initially as a way for keeping a constant in-coupling angle, but later demonstrating to be a good on-chip reference tool (g. 4.3.6).

On-chip reference The presence of chemically passivated sensing elements in multichannel devices allowed the establishment of on-chip reference channels (4.3). In this way, refractometric and thermal drifts can be subtracted from measurements in real time [Darwish (2010)].

Complementary techniques Capabilities of surface uorescence excitation were implemented in grating coupler devices, in a manner similar to that presented in [Duveneck et al. (1997)], but with two essential dierences. The system is actually devoted to perform guided mode spectroscopy and surface uorescence together, and on samples that ow independently in dierent channels.

Representation tools Along the design stage it was found that, if coupling angles were represented in polar coordinates vs. whatever other parameter, the working operation conguration could be observed as if it was actually sketched (see g. 2.4.2).

Low cost SPR-Gs Some low cost polymers (as PMMA) that can be embossed are not suitable as waveguide lms because of their low refractive indices. Their use as a substrate with lm layers made of dierent materials give rise to a cost eective fabrication alternative. Here this was demonstrated for Grating Surface Plasmon Resonance sensors (sec. 3.5). The obtained devices exhibited a performance at least as good as that of gating couplers [Darwish (2008a)]. 161

5.1.


CHAPTER 5.

CONCLUSIONS

5.1.3. Instrumentation and application The created modular instrumentation blocks can be shared or exchanged between sensors and sensing modes. These blocks were based on commercial optomechanics and interpreted programming languages in order to lower development times and hence to broaden the range of studied techniques. As many instruments (the OWLS among others) explore angular resonances, the related instrumentation has been developed along years, and no signicant improvements with respect to commercial alternatives have been presented. For instance, the resonance tracking algorithm described in sec. 4.2.1 is necessary for the presented rotary instrument, but it also existed in the market. The most relevant feature is the analysis of regions of interest that is performed in the dual grating coupler systems.

Channel denition by software The developed dual-pad couplers allow an scalability only restricted by the camera resolution and the size of the droplets used for surface preparation. The associated instrumentation software was developed accordingly, with a user interface that allows the operator to select an arbitrary number of channels (sec. 4.3.2). These channels correspond to regions of interest (ROI) on the acquired images, which are not necessarily dened on the chip except by local functionalization (e.g. using gratings etched across the full width of the sensors). As a result, three advantages were obtained: rst, a huge exibility, not only with respect to the number of channels but also with respect their placement, second, a signicant improvement in the sample rate because of the image analysis of smaller subsets and third, the avoidance of crosstalk between channels.

Application In all cases the systems were calibrated in the refractometric operation mode and, by means of the developed model of adlayer adsorption, estimations of their applicability for biosensing were given. According to it, both OGCB and SPR-G sensors are expected to resolve a few nanograms per square centimeter of adsorbed mass (sec. 4.3). In the cocrete case of the dual grating coupler biosensor this was already demonstrated in an application experiment [Darwish (2010)]. 162

CHAPTER 5.

CONCLUSIONS

5.2.

SENSOR COMPARISON

5.2. Sensor comparison Dual-pad grating couplers are better than rotary systems, not only because of their multichannel capabilities, but also because of the harmful inuence of the mechanical components on the sample rates. Nevertheless, rotation may be an excellent way to calibrate and align the sensors automatically. The three techniques proposed are not incompatible with each other. It is true that their limits of resolution are dierent, but the rest of their features determine dierent ranges of application. In table 5.1 the main features the devices fabricated in this work are summarized.

Technique Sensor Application

Dielectric waveguides

OGCB

Adsorption, dielectric surfaces.

Surface Plasmon Resonance

SPR-G

Adsorption, metallic surfaces.

Fluorescence WG-F

Measurement of low abundance samples.

Resolution Other features Simultaneous measurement of thickness and refractive index, 8·10−5 RIU adjustable sensing 4ng/cm2 range, multichannel capabilities, on-chip reference. Low cost 1.6·10−5 RIU technology, high 1ng/cm2 sensitivity, high sample rate. Non refractometric, need of sample preparation, zeptomoles unmatched limit of detection. Research in progress.

Table 5.1.: Comparative summary of the studied optical biosensing devices: guided mode spectroscopy (OGCB), surface plasmon resonance (SPR-G) and waveguide-excited uorescence (WG-F).

163

5.3.

FUTURE TRENDS.

CHAPTER 5.

CONCLUSIONS

Briey, OGCB biosensors are probably the best tool for the characterization of biolayers, specially if the adsorbate itself is being characterized, SPR-G may be more protable for the detection of well known antigens (e.g. in medical diagnostics) and, in cases of extremely low abundance, label techniques as WG-F are unavoidable.

5.3. Future trends. After this study there still exist ways for the improvement of the described techniques. Two challenges, for instance, are the detection of large and small analite particles. Because of the eld penetration depths, analites like cells cannot actually be identied, and their presence is just assumed if certain membrane proteins are detected. In the other extreme, whatever target molecule with a low molecular mass represents a sensitivity challenge, as it cannot produce a change in the optical density of a signicantly thick layer.

Waveguide improvements These limitations have nothing to do with gratings. As shown along the theory chapter, there exists an upper limit for the period -where a grating starts to generate a second diractive order- but also a lower limit, below which light cannot be coupled. For the above reason, it may be assumed that improvements should be found for waveguides instead of gratings. In particular, the improvement of penetration depths or sensitivities is just a matter of eld connement and hence depends on the layer structure of the waveguides. Of course, as these systems are scaled by the working wavelengths, the option of dierent illumination sources exist, but it seems much more reasonable to look for new materials, with dierent properties.

Anti-reective coatings Some minor improvements could also be included, as the deposit of a antireective coatings (e.g. M gF ) onto the rear face of the devices, with which spurious reections like those observed in g. 4.3.2 would disappear. The use of carbon black in the uidic system [Johnson White (2005)] would also improve the contrast in surface uorescence detection systems. 164

CHAPTER 5.

CONCLUSIONS

5.3.

FUTURE TRENDS.

Diractive multiplexing The simultaneous stimulus of several grating channels done by a single cylindrical can be considered a waste of energy, as areas not devoted for sensing are also illuminated. The possibility of splitting the in-coupling beam by means of diraction into an appropriate number of channels could be explored, although very precise alignment would be required and some exibility would be lost.

Monolithic implementation Convenience, disposability and reliability are well known features of monolithic devices like integrated circuits (ICs). Translated to optical biosensors this means the integration of light sources, focusing elements, biological transductors, uidics and detection elements in a single chip. Although the obvious diculties, problems like alignments, thermal stabilization or vibrations would automatically disappear. Moreover, the actual implementation of a lab-on-a-chip device would allow the jump from in vitro to in vivo applications, which would represent an obvious improvement of the eciency of some analyses and drug delivery. As the demonstrated integration of negative control makes these tasks more reliable and these sensors more autonomous, the author thinks that this work represents an step towards this paradigm.

165

Bibliography Abdiche, Y. N.; Malashock, D. S.; Pinkerton A; Pons J. 2009. Exploring Blocking Assays Using Octet, ProteOn, and Biacore Biosensors. Analytical Biochemistry, 386(2), 172180. Adányi, N.; Levkovets, I. A.; Rodriguez-Gil S.; Ronald A.; Váradi M.; Szendro I. 2007. Development of immunosensor based on OWLS technique for determining Aatoxin B1 and Ochratoxin A. Biosensors and Bioelectronics, 22(6), 797 802. Adányi, N.; Váradi, M.; Kim N.; Szendro I. 2006. Development of new immunosensors for determination of contaminants in food. Current Applied Physics, 6, 279286. Ahel, I.; Ahel, D.; Matsusaka T.; Clark AJ.; Pines J.; Boulton SJ.; West SC. 2008. Poly(ADP-ribose)-binding zinc nger motifs in DNA repair/checkpoint proteins. Nature, 451, 8185. Alessandrini, A.; Facci, P. 2005. AFM: a versatile tool in biophisics. Meas. Sci. Tech., 16, R65R92. Aulin, C.; Varga, I.; Claesson PM.; Wågberg L.; Lindström T. 2008. Buildup of polyelectrolyte multilayers of polyethyleneimine and microbrillated cellulose studied by in situ dual-polarization interferometry and quartz crystal microbalance with dissipation. Langmuir, 24, 25092518. Aung, KMM.; Ho X, Su X. 2008. DNA assembly on streptavidin modied surface: a study using quartz crystal microbalance with dissipation or resistance measurements. Sensors and Actuators B: Chemical, 131, 371 378. Bakker, E.; Pretsch, E. 2005. Potentiometric sensors for trace-level analysis. Trac-Trends in Analytical Chemistry, 24(3), 199207. Bard, A. J.; Fox, M.A. 1995. Articial Photosynthesis: Solar Splitting of Water to Hydrogen and Oxygen. Accounts of Chemical Research, 28(3), 141145. Beusink, JB.; Lokate, AM.; Besselink GA.; Pruijn GJ.; Schasfoort RB. 2008. Angle-scanning SPR imaging for detection of biomolecular interactions on microarrays. Biosens. Bioelectron., 23, 839844. 167

Bibliography

Bibliography

Bilitewski, U. 2000. Peer Reviewed: Can Anity Sensors Be Used To Detect Food Contaminants? Analytical Chemistry, 72(21), 692 A701 A. Born, M.; Wolf, E. 1997. Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diraction of Light. 6th edn. Cambridge University Press. Botten, L.C.; McPhedran, R.C. 1985. Completeness and Modal Expansion Methods in Diraction Theory. Journal of Modern Optics, 32(12), 1479 1488. Byres, E.; Paton, AW.; Paton JC.; Löing JC.; Smith DF.; Wilce MCJ.; Talbot UM.; Chong DC.; Yu H.; Huang S.; Chen X.; Varki NM.; VarkiA.; Rossjohn J.; Beddoe T. 2008. Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature, 456, 648652. Cariou, J.M.; Dugas, J.; Martin L.; Michel P. 1986. Refractive-index variations with temperature of PMMA and polycarbonate. Appl. Opt., 25(3), 334336. Chan, LL.; Pineda, M.; Heeres JT.; Hergenrother PJ.; Cunningham BT. 2008a. A general method for discovering inhibitors of protein-DNA interactions using photonic crystal biosensors. ACS Chem. Biol., 3, 437448. Chan, LL.; Gosangari, SL.; Watkin KL.; Cunningham BT. 2008b. Labelfree imaging of cancer cells using photonic crystal biosensors and application to cytotoxicity screening of a natural compound library. Sensors and Actuators B: Chemical, 132, 418425. Chaubey, A.; Malhotra, B. D. 2002. Mediated Biosensors. Biosensors & Bioelectronics, 17(6-7), 441456. Chen, T.T. ; Hawang, Da H-Min. I977. Grating Fabrication by Interference of Laser Beams. Chinese Journal of Physics, 15, NO. 1, 69. Chorley, BN.; Wang, X.; Campbell MR.; Pittman GS.; Noureddine MA.; Bell DA. 2008. Discovery and verication of functional single nucleotide polymorphisms in regulatory genomic regions: current and developing technologies. Mutat. Res., 659, 147157. Chouteau, C.; Dzyadevych, S.; Chovelon J. M.; Durrieu C. 2004. Development of novel conductometric biosensors based on immobilised whole cell chlorella vulgaris microalgae. Biosensors & Bioelectronics, 19(9), 10891096. 168

Bibliography

Bibliography

Clark, L.C.; Lyons, C. 1962. Electrode systems for continuous monitoring in cardiovascular surgery. Annals of the New York Academy of Sciences, 102(1), 29. Clerc, D., & Lukosz, W. 1997. Direct immunosensing with an integratedoptical output grating coupler. Sensors and Actuators B: Chemical, 40(1), 53 58. Corne, C.; Fiche, J-B.; Gasparutto D.; Cunin V.; Suraniti E.; Buhot A.; Fuchs J.; Calemczuk R.; Livache T.; Favier A. 2008. SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes. Analyst, 133, 10361045. Cunningham, Brian, Li, Peter, Lin, Bo, & Pepper, Jane. 2002. Colorimetric resonant reection as a direct biochemical assay technique. Sensors and Actuators B: Chemical, 81(2-3), 316 328. Darmanyan, S.A.; Zayats, A.V. 2003. Light tunneling via resonant surface plasmon polariton states and the enhanced transmission of periodically nanostructured metal lms: An analytical study. Phys. Rev. B, 67, 035424. Darwish, N.; Caballero, D.; Moreno M.; Errachid A.; Samitier J. 2010. Multi-analytic grating coupler biosensor for dierential binding analysis. Sensors and Actuators B: Chemical, 144, Issue 2, 413417. Darwish, N.; Diéguez, L.; Moreno M.; Muñoz F.; Mas R.; Mas J.; Samitier J.; Nilsson B.; Petersson G. 2007. Second order eects of aspect ratio variations in high sensitivity grating couplers. Microelectronic Engineering, 84(5-8), 1775 1778. Proceedings of the 32nd International Conference on Micro- and Nano-Engineering. Darwish, N.; Moreno, M.; Karlsson M.; Nikolaje F.; Samitier J. 2008a. Low cost Surface Plasmon Resonance sensor based on diraction gratings. In: Proceedings of the eurosensors xxii conference, dresden, germany. Darwish, N.; Moreno, M.; Viguès N.; Muñoz F. J.; Mas J.; Samitier J. 2008b. Multichannel high resolution Grating Coupler Biosensor. In: Proceedings of the eurosensors xxii conference, dresden, germany. Davies, C. 2005. The Immunoassay Handbook Third edition. ELSEVIER. Chap. Introduction to Immunoassay Principles, page p8. De Feijter, J.A.; Benjamins, J.; Veer F.A. 1978. Ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the air-water interface. Biopolymers, 17(7), 17591772. 169

Bibliography

Bibliography

De Fornel, F. (ed). 2001. Evanescent Waves: From Newtonian Optics to Atomic Optics. Springer-Verlag, Berlin. Diéguez, L.; Darwish, N.; Graf N.; Vörös J.; Zambelli T. 2009a. Electrochemical tuning of the stability of pll/dna multilayers. Soft Matter, 5, 2415 2421. Diéguez, L.; Darwish, N.; Mir M.; Martínez E.; Moreno M.; Samitier J. 2009b. Eect of the Refractive Index of Buer Solutions in Evanescent Optical Biosensors. Sensors Letters, 7, 851855. Diéguez, L.; Darwish, N.; Moreno M.; López-Bosque M.J.; Samitier J. 2007. Optical Structures Patterned in Polymeric Waveguides. In: 5ª reunión española de optoelectrónica, optoel'07. Do, T.; Ho, F.; Heidecker B.; Witte-K.; Chang L.; Lerner L. 2008. A rapid method for determining dynamic binding capacity of resins for the purication of proteins. Protein Expr. Purif., 60, 147150. Dostalek, J.; Homola, J.; Miler M. 2005. Rich information format surface plasmon resonance biosensor based on array of diraction gratings. Sensors and Actuators B: Chemical, 107(1), 154 161. Proceedings of the 7th European Conference on Optical Chemical Sensors and Biosensors EUROPT(R)ODE VII. Drapp, B., Piehler, J., Brecht, A., Gauglitz, G., Lu, B. J., Wilkinson, J. S., & Ingenho, J. 1997. Integrated optical Mach-Zehnder interferometers as simazine immunoprobes. Sensors and Actuators B: Chemical, 39(1-3), 277 282. 3rd European Conference on Optical Chemical Sensors and Biosensors. Duveneck, G. L., Pawlak, M., Neuschäfer, D., Bär, E., Budach, W., Pieles, U., & Ehrat, M. 1997. Novel bioanity sensors for trace analysis based on luminescence excitation by planar waveguides. Sensors and Actuators B: Chemical, 38(1-3), 88 95. 3rd European Conference on Optical Chemical Sensors and Biosensors. Duveneck, Gert L., Abel, Andreas P., Bopp, Martin A., Kresbach, Gerhard M., & Ehrat, Markus. 2002. Planar waveguides for ultra-high sensitivity of the analysis of nucleic acids. Analytica Chimica Acta, 469(1), 49 61. Ebbesen, T.W.; Lezec, H.J.; Ghaemi H.F.; Thio-T.; Wol P.A. 1998. Extraordinary optical transmission through sub-wavelength hole arrays. Nature, 391, 667669. 170

Bibliography

Bibliography

Eggleston, CM.; Vörös, J.; Shi L.; Lower-BH.; Droubay TC.; Colberg PJS. 2008. Binding and direct electrochemistry of OmcA, an outer-membrane cytochrome from an iron reducing bacterium, with oxide electrodes: a candidate biofuel cell system. Inorg. Chim. Acta, 361, 769777. Engvall, E.; Perlman, P. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry, 8 (9), 8714. Fan, X.; White, I.M.; Shopova S.I.; Zhu-H.; Suter J.D.; Sun Y. 2008. Sensitive optical biosensors for unlabeled targets: A review. Analytica Chimica Acta, 620, 826. Fang, Y.; Frutos, AG.; Verklereen R. 2008a. Label-free cell-based assays for GPCR screening. Comb. Chem. High Throughput Screen., 11, 357369. Fang, Y.; Ferrie, AM. 2008b. Label-free optical biosensor for ligand-directed functional selectivity acting on β 2 adrenoceptor in living cells. FEBS Lett., 582, 558564. Fang, Y.; Ferrie, A.M.; Fontaine N.H.; Mauro-J.; Balakrishnan J. 2006a. Resonant Waveguide Grating Biosensor for Living Cell Sensing. Biophysical Journal, 91, 19251940. Fang, Y. 2006b. Label-Free Cell-Based Assays with Optical Biosensors in Drug Discovery. ASSAY and Drug Development Technologies, 4, n.5, 583595. Fano, U. 1956. Atomic Theory of Electromagnetic Interactions in Dense Materials. Phys. Rev., 103, 1202. Feynman, R.P.; Leighton, R.B.; Sands M. 1964. Feynman Lectures on Physics, Volume 2. Addison-Wesley. Chap. Refractive Index of Dense Materials, pages 113. Fiche, JB.; Fuchs, J.; Buhot A.; Calemczuk-R.; Livache T. 2008. Point mutation detection by surface plasmon resonance imaging coupled with a temperature scan method in a model system. Anal. Chem., 80, 1049 1057. García Aljaro, C.; Muñoz-Berbel, X.; Jenkins-ATA.; Blanch AR.; Muñoz FX. 2008. Surface plasmon resonance assay for real-time monitoring of somatic coliphages in wastewaters. Appl. Environ. Microbiol., 74, 40544058. Ghatak, A.K.; Thyagarajan, K.; Shenoy M.R. 1987. Numerical Analysis of Planar Optical Waveguides Using Matrix Approach. Journal of Lightwave Technology, LT-5, NO. 5,, 660668. 171

Bibliography

Bibliography

Goldys, E.M. (ed). 2009. Fluorescence applications in Biotechnology and Life Sciences. Wiley-Blackwell. Gottschalch, F.; Homann, T.; Sotomayor Torres C.M.; Schulz H.; Scheer H.C. 1999. Polymer issues in nanoimprinting technique. SolidState Electronics, 43(6), 1079 1083. Grandin, H.M.; Städler, B.; Textor M.; Vörös J. 2006. Waveguide excitation uorescence microscopy: A new tool for sensing and imaging the biointerface. Biosensors and Bioelectronics, 21(8), 1476 1482. Gray, J.J. 2004. The interaction of proteins with solid surfaces. Current Opinion in Structural Biology, 14(1), 110 115. Grieshaber, Dorothee, MacKenzie, Robert, Vörös, Janos, & Reimhult, Erik. 2008. Electrochemical Biosensors - Sensor Principles and Architectures. Sensors, 8(3), 14001458. Gérard, D.; Salomon, L.; De Fornel F.; Zayats A.V. 2004. Ridge-enhanced optical transmission through a continuous metal lm. Phys. Rev. B, 69, 113405. Homola, J.; Lu, H.B.; Nenninger G.G.; Dostalek J.; Yee S.S. 2001. A novel multichannel surface plasmon resonance biosensor. Sensors and Actuators B: Chemical, 76(1-3), 403 410. Homola, J. 2006. Frontiers in Planar Lightwave Circuit Technology. Springer. Chap. Surface Plasmon Resonance (SPR) biosensors and their applications in food safety and security, pages 101118. Homola, J.; Dostalek, J.; Chen S.; Rasooly A.; Jiang S.; Yee S.S. 2002. Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk. International Journal of Food Microbiology, 75, 61 69. Homola, Jiri. 1997. On the sensitivity of surface plasmon resonance sensors with spectral interrogation. Sensors and Actuators B: Chemical, 41(1-3), 207 211. Homola, Jiri (ed). 2006. Surface Plasmon Resonance Based Sensors. 1 edition edn. Springer Series on Chemical Sensors and Biosensors, vol. Vol. 4. Springer. Homola, Jiri, Yee, Sinclair S., & Myszka, David. 2008. Surface plasmon resonance biosensors. Second edition edn. Amsterdam: Elsevier. Horvath, R.; McColl, J.; Yakubov G.; Ramsden J. 2008. Structural hysteresis and hierachy in adsorbed glycoproteins. J. Chem. Phys., 129, 07110210711024. 172

Bibliography

Bibliography

Horváth, R.; Pedersen, H.C.; Larsen N.B. 2002a. Demonstration of reverse symmetry waveguide sensing in aqueous solutions. Appl. Phys. Lett., 81(12), 21662168. Horváth, R.; Lindvold, L.R.; Larsen N.B. 2002b. Reverse-symmetry waveguides: theory and fabrication. Applied Physics B-Lasers and Optics, 74(4-5), 383393. Hutley, M.C. 1982. Diraction Gratings (Techniques of Physics). Academic Press. Huygens, Ch. 1690. Traité de la Lumière. Jaramillo, J.J. 2009. Automatización de un sistema de control para biosensores ópticos resonantes. M.Phil. thesis, Faculty of Physics, Universitat de Barcelona. Jenq Nan, Y.; Yi-Ming, C.; Yen-Chieh M.; Wei-Han W.; Fan-Ching C.; Chun-Yu L.; Kuang-Li L.; Pei-Kuen W.; Shean-Jen C. 2006. Optical waveguide biosensors constructed with subwavelength gratings. Applied Optics, 45(9), 193842. Jessel, N.; Oulad-Abdelghani, M.; Meyer F.; Lavalle P.; Haîkel-Y.; Schaaf P.; Voegel J.-C. 2006. Multiple and time-scheduled in situ DNA delivery mediated by B-cyclodextrin embedded in a polyelectrolyte multilayer. PNAS, 103 no. 23, 86188621. Johnson, P. B.; Christy, R. W. 1972. Optical Constants of the Noble Metals. Phys. Rev. B, 6, 43704379. Johnson White, B.;Golden, J. 2005. Reduction of background signal in automated array biosensors. Meas. Sci. Technol., 16, N29N31. Jönsson, U.; Fägerstam, L.; Ivarsson B.; Johnsson B.; Karlsson R.; Lundh K.; Löfås S.; Persson-B.; Roos H.; Rönnberg-I.; et al. 1991. Real-time biospecic interaction analysis using surface plasmon resonance and a sensor chip technology. BioTechniques, 11, 620622. Karamanska, R.; Clarke, J.; Blixt O.; Macrae JI.; Zhang JQ.; Crocker PR.; Laurent N.; Wright-A.; Flitsch SL.; Russell-DA.; Field RA. 2008. Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays. Glycoconj. J., 25, 6974. Kasarova, S.N.; Sultanova, N.G.; Ivanov C.D.; Nikolov I.D. 2007. Analysis of the dispersion of optical plastic materials. Optical Materials, 29, 1481 1490. 173

Bibliography

Bibliography

Kasemo, B. 1998. Biological surface science. Current Opinion in Solid State and Materials Science, 3(5), 451459. Katzenberg, F. 2005. Plasma-bonding of poly(dimethylsiloxane) to glass. e-Polymers, 60. Khan, TR.; Grandin, HM.; Mashaghi A.; Textor M.; Reimhult E.; Raviakine I. 2008. Lipid redistribution in phosphatidylserine-containing vesicles adsorbing on titania. Biointerphases, 3, FA90FA95. Kogelnik, H., & Ramaswamy, V. 1974. Scaling rules for thin-lm optical waveguides. Applied Optics, 13, No. 8, 18561862. Kueng, A.;Kranz, C.; Mizaiko B. 2004. Amperometric ATP biosensor based on polymer entrapped enzymes. Biosensors & Bioelectronics, 19(10), 13011307. Kunz, R.E.; Dübendorfer, J.; Morf R.H. 1996. Finite grating depth effects for integrated optical sensors with high sensitivity. Biosensors and Bioelectronics, 11(6-7), 653 667. Kunz, R.E.; Edliger, J.; Sixt P.; Gale M.T. 1005. Replicated chirped waveguide gratings for optical sensing applications. Sensors and Actuators A: Physical, 46-47, 482486. Lan, Y-b.; Wang, S-z.; Yin Y-g.; Homann WC.; Zheng X-z. 2008. Using a surface plasmon resonance biosensor for rapid detection of Salmonella Typhimurium in chicken carcass. J. Bionic Eng., 5, 239246. Landau, L.D. 1987. Fluid mechanics / by L.D. Landau and E.M. Lifshitz; translated from the Russian by J.B. Sykes and W.H. Reid. Pergamon Press. Lane, TJ.; Fletcher, WR.; Gormally-MV.; Johal MS. 2008. Dual-beam polarization interferometry resolves mechanistic aspects of polyelectrolyte adsorption. Langmuir, 24, 1063310636. Lausted, CG.; Hu, Z.; Hood-LE. 2008. Quantitative serum proteomics from surface plasmon resonance imaging. Mol. Cell. Proteomics, 7, 24642474. Lee, SJ.; Youn, B-S.; Park JW.; Niazi-JH.; Kim YS.; Gu MB. 2008a. ssDNA aptamer-based surface plasmon resonance biosensor for the detection of retinol binding protein 4 for the early diagnosis of type 2 diabetes. Anal. Chem., 80, 28672873. Lee, S-K.; Kim, H-C.; Cho S-J.; Jeong SW.; Jeon WB. 2008b. Binding behavior of CRP and anti-CRP antibody analyzed with SPR and AFM measurement. Ultramicroscopy, 108, 13741378. 174

Bibliography

Bibliography

Li, L.; Haggans, C.W. 1993. Convergence of the coupled-wave method for metallic lamellar diraction gratings. J. Opt. Soc. Am. A, 10(6), 1184 1189. Lukosz, W.; Stamm, Ch. 1990. Integrated optical interferometer as relative humidity sensor and dierential refractometer. Sensors and Actuators A: Physical, 25(1-3), 185 188. Lukosz, W. 1995. Integrated optical chemical and direct biochemical sensors. Sensors and Actuators B: Chemical, 29(1-3), 37 50. Proceedings of the 2nd European Conference on Optical Chemical Sensors and Biosensors. Lukosz, W., & Tiefenthaler, K. 1983. Embossing technique for fabricating integrated optical components in hard inorganic waveguiding materials. Opt. Lett., 8(10), 537539. Lukosz, W., Nellen, Ph. M., Stamm, Ch., & Weiss, P. 1990. Output grating couplers on planar waveguides as integrated optical chemical sensors. Sensors and Actuators B: Chemical, 1(1-6), 585 588. Lyndin, N.M.; Parriaux, O.; Sychugov-V.A. 1997. Waveguide excitation by a Gaussian beam and a nite size grating. Sensors and Actuators B: Chemical, 41(1-3), 23 29. Madou, M.J. 2002. Fundamentals of microfabrication: The science of miniaturization. 2nd edn. Boca Raton, FL: CRC Press. Mashaghi, A.; Swann, M.; Popplewell-J.; Textor M.; Reimhult E. 2008. Optical anisotropy of supported lipid structures probed by waveguide spectroscopy and its application to study of supported lipid bilayer formation kinetics. Anal. Chem., 80, 36663676. McDonald, J.C.; Duy, D.C.; Anderson-J.R.; Chiu D.T.; Wu H.; Schueller O.J.; Whitesides-G.M. 2000. Fabrication of microuidic systems in poly(dimethylsiloxane). Electrophoresis, 21, 2740. Merz, C.; Knoll, W.; Textor-M.; Reimhult E. 2008. Formation of supported bacterial lipid membrane mimics. Biointerphases, 3, FA41FA50. Millman, J. 1979. Microelectronics Digital and Analog Circuits and Systems. Mc Graw-Hill Book Company. Mir, M.; Jenkins, ATA.; Katakis-I. 2008a. Ultrasensitive detection based on an aptamer beacon electron transfer chain. Electrochem. Commun., 10, 15331536. 175

Bibliography

Bibliography

Mir, M.; Katakis, I. 2008b. Target label-free, reagentless electrochemical DNA biosensor based on sub-optimum displacement. Talanta, 75, 432 441. Moraes, ML.; Baptista, MS.; Itri-R.; Zucolotto V.; Oliveira ON Jr. 2008. Immobilization of liposomes in nanostructured layer-by-layer lms containing dendrimers. Mater. Sci. Eng. C, 28, 467471. Moreira, CS.; Lima, AMN.; Ne-H.; Thirstrup C. 2008. Temperaturedependent sensitivity of surface plasmon resonance sensors at the goldwater interface. Sensors and Actuators B: Chemical, 134, 854862. Muñoz Berbel, X.; Vigués, N.; Jenkins ATA.; Mas J.; Muñoz FJ. 2008. Impedimetric approach for quantifying low bacteria concentrations based on the changes produced in the electrode-solution interface during the pre-attachment stage. Biosens. Bioelectron., 23, 15401546. Myers, FB.; Lee, LP. 2008. Innovations in optical microuidic technologies for point-of-care diagnostics. Lab Chip, 8, 20152031. Nepal, D.; Balasubramanian, S.; Simonian AL.; Davis VA. 2008. Strong antimicrobial coatings: single-walled carbon nanotubes armored with biopolymers. Nanoletters, 8, 18961901. O'Shea, D.C.; Suleski, T.J.; Kathman A.D.; Prather D.W. 2003. Diractive Optics: Design, Fabrication, and Test. SPIE Publications; illustrated edition edition. O'Sullivan, C.K.; Guilbault G.G. 1999. Commercial quartz crystal microbalances - theory and applications. Biosensors & Bioelectronics, 14, 663?670. Otero, J.; Puig-Vidal, M. ; Frigola M.; Casals A. 2009a. Micro-to-nano optical resolution in a multirobot nanobiocharacterization station. Pages 53575362 of: Proceedings of the 2009 ieee/rsj international conference on intelligent robots and systems. IROS'09. Piscataway, NJ, USA: IEEE Press. Otero, J.; Puig-Vidal, M.; Samitier J. 2009b. Implementation of a multirobot system using self-sensing probes for cell and molecular biology experimental research. In: Proceedings of the 9th ifac symposium on robot control. Palik, E.D. (ed). 1985. Handbook of optical constants of solids. Academic Press. 176

Bibliography

Bibliography

Pascal, D.; Orobtchouk, R.; Layadi A.; Koster A.; Laval S. 1997. Optimized coupling of a Gaussian beam into an optical waveguide with a grating coupler: comparison of experimental and theoretical results. Applied Optics, 36, No. 12 y, 24432447. Pawlak, M.; Schick, E.; Bopp M. A.; Schneider M.J.; Oroszlan P.; Ehrat M. 2002. Zeptosens' protein microarrays: A novel high performance microarray platform for low abundance protein analysis. Proteomics, 2, 383393. Payne, F.P.; Lacey, J.P.R. 1994. A theoretical analysis of scattering loss from planar optical waveguides. Optical and Quantum Electronics, 26-10, 977986. Petit, R.; Cadilhac, M.; Maystre D.; Vincent P.; Neviere M. 1980. Electromagnetic Theory of Gratings (Topics in Applied Physics). Springer. Prabhakar, N.; Arora, K.; Arya SK.; Solanki PR.; Iwamoto M.; Singh H.; Malhotra-BD. 2008. Nucleic acid sensor for M. tuberculosis detection based on surface plasmon resonance. Analyst, 133, 15871592. Raiteri, Roberto; Grattarola, Massimo; Butt Hans-Jürgen; Skládal Petr. 2001. Micromechanical cantilever-based biosensors. Sensors and Actuators B: Chemical, 79, 115126. Ralin, D.; Dultz, S.; Silver J.; Travis J.; Kullolli M.; Hancock W.; HincapieM. 2008. Kinetic analysis of glycoprotein-lectin interactions by label-free internal reection ellipsometry. Clin. Proteomics, 4, 3746. Ramadas, M.R.; Garmire, E.; Ghatak A.K.; Thyagarajan K.; Shenoy M.R. 1989. Analysis of absorbing and leaky planar waveguides: a novel method. Opt. Lett., 14(7), 376378. Ramsden, J.J. 1993. Review of new experimental techniques for investigating random sequential adsorption. Journal of Statistical Physics, 73, 853 877. Ramsden, J.J. 1997. Optical biosensors. Journal of Molecular Recognition, 10(3), 109120. Reitz, J.R.; Milford, F.J.; Christy R.W. 1992. Foundations of Electromagnetic Theory. 4th edn. Addison Wesley. Rich, R.L.; Myszka, D.G. 2009. Grading the commercial optical biosensor literature - Class of 2008: 'The Mighty Binders'. J. Mol. Recognit, 23, 164. Rich, RL.; Cannon, MJ.; Jenkins J.; Pandian P.; Sundaram S.; Magyar R.; Brockman-J.; Lambert-J.; Myszka DG. 2008. Extracting kinetic rate 177

Bibliography

Bibliography

constants from surface plasmon resonance array systems. Anal. Biochem., 373, 112120. Rich, R. L., & Myszka, D. G. 2005. Survey of the year 2004 commercial optical biosensor literature. J.Mol.Recognit., 18, 431478. Rich, R.L., et al. . 2009. A Global Benchmark Study Using Anity-based Biosensors. Analytical Biochemistry, 386 (2), 194216. Rosenblatt, D.; Sharon, A.; Friesem A.A. 1997. Resonant Grating Waveguide Structures. IEEE Journal of Quantum Electronics, 33, NO. 11, 20382059. Ruiz, A.; Buzanska, L.; Gilliland D.; Rauscher H.; Sirghi L.; Sobanski T.; Zychowicz-M.; Ceriotti-L.; Bretagnol F.; Coecke-S.; Colpo P.; Rossi F. 2008. Micro-stamped surfaces for the patterned growth of neural stem cells. Biomaterials, 29, 47664774. Ryu, J.; Joung, HA.; Kim MG.; Park CB. 2008. Surface plasmon resonance analysis of Alzheimer's beta-amyloid aggregation on a solid surface: from monomers to fully-grown brils. Anal. Chem., 80, 24002407. Saliterman, S.S. 2006. Fundamentals of BioMEMS and Medical Microdevices. Wiley. Schasfoort, R.B.M.; Tudos, A.J. (ed). 2008. Handbook of Surface Plasmon Resonance. Springer. Schmidt, C.E.; Baier Leach, J. 2003. NEURAL TISSUE ENGINEERING: Strategies for Repair and Regeneration. Annual Review of Biomedical Engineering, Vol. 5, 293347. Sepúlveda, B.; Sánchez del Río, J.; Moreno M.; Blanco F.J.; Mayora K.; Domínguez C.; Lechuga-L.M. 2006. Optical biosensor microsystems based on the integration of highly sensitive Mach-Zehnder interferometer devices. Journal of Optics A: Pure and Applied Optics, 8 - 7, S561. Shaklan, S.; Roddier, F. 1988. Coupling starlight into single-mode ber optics. Appl. Opt., 27, 23342338. Shen, X.J.; Pan, L.; Lin L. 2002. Microplastic embossing process: experimental and theoretical characterizations. Sensors and Actuators A: Physical, 97-98, 428 433. Shenoy, M.R.; Thyagarajan, K.; Ghatak A.K. 1988. Numerical analysis of optical bers using matrix approach. Journal of Lightwave Technology, 6(8), 12851291. 178

Bibliography

Bibliography

Solanki, PR.; Prabhakar, N.; Pandey MK.; Malhotra BD. 2008. Selfassembled monolayer for toxicant detection using nucleic acid sensor based on surface plasmon resonance technique. Biomed. Microdevices, 10, 757767. Soper, S.A.; Hashimoto, M.; Situma C.; Murphy M.C.; McCarley R.L.; Cheng Y.W.; Barany F. 2005. Fabrication of DNA microarrays onto polymer substrates using UV modication protocols with integration into microuidic platforms for the sensing of low-abundant DNA point mutations. Methods, 37, 103113. Srivastava, R.; Weiss, M.N.; Groger H.; Lo P.; Luo S.-F. 1996. A theoretical investigation of environmental monitoring using surface plasmon resonance waveguide sensors. Sensors and Actuators A: Physical, 51(2-3), 211 217. Stryer, L. 1975. Biochemistry. W. H. Freeman and Company, San Francisco. Szendro, I.; Erdélyi, K.; Fábián M.; Puskás Z.; Adányi N.; Somogyi K. 2008. Combination of the optical waveguide lightmode spectroscopy method with electrochemical measurements. Thin Solid Films, 516, 81658169. Székács, A.; Adányi, N.; Székács I.; Majer-Baranyi K.; Szendro I. 2009. Optical waveguide light-mode spectroscopy immunosensors for environmental monitoring. Appl. Opt., 48, No. 4, B151B158. Taitt, C.R.; Anderson, G.P.; Ligler F.S. 2005. Evanescent wave uorescence biosensors. Biosensors and Bioelectronics, 20(12), 2470 2487. 20th Anniversary of Biosensors and Bioelectronics. Tiefenthaler, K.; Lukosz, W. 1985. Grating couplers as integrated optical humidity and gas sensors. Thin Solid Films, 126, 205211. Tiefenthaler, K.; Lukosz, W. 1989. Sensitivity of grating couplers as integrated-optical chemical sensors. J. Opt. Soc. Am. B, 6(2), 209220. Trummer, N.; Adányi, M.V.; Szendro I. 2001. Modication of the surface of integrated optical wave-guide sensors for immunosensor applications. Fresenius J. of Anal. Chem., 371, 2124. 10.1007/s002160100929. Tonnberg, S. 2006. Optimisation and characterisation of LPCVD silicon nitride thin lm growth. M.Phil. thesis, Department of Microtechnology and Nanoscience - MC2, Chalmers University of Technology. Ctyroký, J.; Homola, J.; Lambeck P.V.; Musa-S.; Hoekstra H.J.W.M.; Harris R.D.; Wilkinson J.S.; Usievich-B.; Lyndin N.M. 1999. Theory and modelling of optical waveguide sensors utilising surface plasmon resonance. Sensors and Actuators B: Chemical, 54(1-2), 66 73. 179

Bibliography

Bibliography

Ulman, A. (ed). 1998. Self-assembled Monolayers Of Thiols. Academic Press, San Diego. Ulrich, R.; Torge, R. 1973. Measurement of Thin Film Parameters with a Prism Coupler. Appl. Opt., Vol. 12, No. 12, 29012908. Van Weemen, B.K.; Schuurs, A.H. 1971. Immunoassay using antigenenzyme conjugates. FEBS Letters, 15 (3), 2326. Vörös, J. 2004. The Density and Refractive Index of Adsorbing Protein Layers. Biophysical Journal, 87(1), 553 561. Vörös, J.; Ramsden, J.J.; Csúcs G.; Szendro-I.; De Paul S.M.; Textor M.; Spencer N.D. 2002. Optical grating coupler biosensors. Biomaterials, 23(17), 3699 3710. vor£ík, V.; Huttel, I.; Palá£ek P. 2007. Polymethylmethacrylate optical waveguides prepared in electrical eld. Materials Letters, Volume 61, Issues 4-5, Pages 953955. Wang, B.; Wilkes, G.L.; Smith C.D.; Mc Grath G.E. 1991. High refractiveIndex hybrid ceramer materials prepared from titanium tetraisopropoxide and poly(arylene ether phosphine oxide) through sol-gel processing. Polymer Communications, 32 (13), 400402. Wattendorf, U.; Coullerez, G.; Vörös J.; Textor M.; Merkle HP. 2008. Mannose-based molecular patterns on stealth microspheres for receptorspecic targeting of human antigen-presenting cells. Langmuir, 24, 1179011802. Wen, J.; Arakawa T. 2000. Refractive Index of Proteins in Aqueous Sodium Chloride. Analytical Biochemistry, 280, Issue 2, 327329. Wiki, M.; Kunz, R.E. 2000. Wavelength-interrogated optical sensor for biochemical applications. Opt. Lett., 25, No. 7, 463465. Yalow, R.; Berson S. 1960. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest., 39, 115775. Yu, X.; Tsibane, T.; McGraw PA.; House FS.; Keefer CJ.; Hicar MD.; Tumpey TM.; Pappas-C.; Perrone LA.; Martinez-O.; Stevens J.; Wilson IA.; Aguilar-PV.; Altschuler EL.; Basler CF.; Crowe JE Jr. 2008. Neutralizing antibodies derived from the B cells of 1918 inuenza pandemic survivors. Nature, 455, 532536. Ziegler, c. 2004. Cantilever-based biosensors. Anal. Bioanal. Chem., 379, 946959. 180

A. Overview of the current relevant biointeraction analysis tools Some good review papers exist about, among others, the most relevant biosensing techniques [Grieshaber et al. (2008); Fan (2008); Rich (2009)], so it was not intended to be exhaustive here. Instead of it, the biosensing scene will be sketched, with an emphasis in the comparison between the concepts developed in this thesis and their commercial counterparts. Biosensors are basically a scientic tool. For this reason it happens that the literature about the development of biosensors is rapidly overshadowed by application studies, either based on the same technique or on any other. The obvious need for manufacturers to have more customers than employees make application references much more numerous than those about any technique itself. Nevertheless, the sensors and their applications are different things and their comparison would not be fair, although sensors are aimed for sensing, and their development cannot be unbound from their use. Finally, the success, in terms of the number of publications based on a certain sensing technique is not uniquely determined by its sensitivity or reliability. For instance, the costs, use easiness and the mere previous existence of a community of users make certain techniques predominant.

Detection and biosensing There exist many techniques for the specic detection of molecules from biological samples, which are not intended for the same purposes. First, detection and measurement are dierent concepts. One of the most relevant detection techniques in biomedicine is the Enzymelinked ImmunoSorbent Assay (ELISA) [Van Weemen (1971); Engvall 181

APPENDIX A. OVERVIEW OF THE CURRENT RELEVANT BIOINTERACTION ANALYSIS TOOLS

(1971)]. Briey, this technique consists in at least two consecutive adsorption/wash cycles. Along the rst cycle, an antibody complementary to the antigen to be detected is specically linked to the sample, which consists on inespecically adsorbed molecules in the bottom of a microtitier plate. In the second cycle a labeled (e.g. chromogenically) enzyme, is specically attached to the antibody, so that the amount of attached target sample gives rise to a certain observable eect, such as a change of the color. Many other techniques are based on the same concept, either modifying the order in which reagents are adsorbed or the way of labeling them. All of these detection methods are hence quite similar and suer common limitations:

The way to evaluate the amount of the studied sample is not quantitative. The tests are performed manually.

These limitations are not decisive when the mere detection of a certain substance leads to a conclusive diagnosis. This is, for instance, the case of pregnancy tests. Of course it is desired to detect the target antigens as sensitively as possible, but the broad application of assay techniques show that this is not a problem. The situation is quite dierent when the questions are how much or at what rate a target is being adsorbed, for which the aforementioned methods give poor results [Rich & Myszka (2005) ]. Many of the most relevant problems involve the study of bioanities and hence require more specic tools. It should be noticed that every detection technique takes prot of some physical or chemical eect for the discovery or molecules, so this is not new in general. Automation and transduction, which are the answer for the listed problems are, in contrast, a result of the last decades and gave rise to the concept of biosensing. From the historical point of view, the glucose oxidase (GOx) biosensor [Clark (1962)] marked the start-point of this entire eld. A transduction block also takes prot from physical or chemical eects, but here replaces the operator in the task of judging whether a phenomenon is being happening or not. Moreover, quantitative data is continuously recorded along an automated realization of the experiment. Biosensors are classied depending on the transduction principle (e.g. waveguide vs. SPR), while automation distinguishes between instruments (e.g. xed of rotary grating couplers). As a conclusion, biosensors are a special subset of detection techniques, with unique real-time and quantication capabilities. As biosensors are the subject of the present research other detection methods will be excluded from the rest of the discussion. 182


Label and label-free biosensing With respect to the use of markers, biosensing techniques may be divided in label and label-free. The label-based biosensors are derived from detection techniques, to which resemble. Fluorescence analysis is probably the best example. The use of (uorescent) labels provides merely qualitative results unless a signal quantication (e.g. photometric) method is utilized for detection. This laboratory did not originally intended to develop label systems, but along the development of the grating coupler it was found that it was possible to incorporate this possibility, which permits extraordinary high sensitivities [Taitt (2005)] just by keeping some design restrictions in mind. This idea was proposed [Duveneck et al. (1997)] for high throughput arrays and by Gradin [Grandin (2006)] as an improvement of uorescence microscopy. Here a mid-size implementation with a congurable ow system was presented, with a selectable number of channels. Label-free techniques also rely on complementary chemistries for the obtainment of specicity, but skip the use of additional labels. This means that the specic antibodies are used as they are, without subsequent processes, and the transduction method involves the change of a property of the system directly caused by interaction, not a property of the interacting species.

Biosensing techniques With respect to their transduction principle, the basic types of biosensors are electrochemical, acoustic, calorimetric, based on scanning probe microscopy, and optical.

Electrochemical biosensing Electrochemical biosensing monitors some electrical parameter of the chemical reaction. Choices are the current exchanged and the variation of the voltage or the impedance of a sample [Chaubey (2002)]. An excellent review paper about these techniques is [Grieshaber et al. (2008)]. In most cases electrochemical biosensors have three electrodes; a reference electrode to keep a constant stable potential, a counter electrode that supplies certain voltage to the reaction volume, and a working electrode, which 183


performs the transduction by recovering the generated current. Depending on the measured magnitude, electrochemical biosensors may be:

Amperometric / voltametric: The catalytic eect of enzymes

permits a very accurate quantication of the concentration of the species through the current generated along the reaction. If current is measured under a constant voltage, the technique is referred as amperometry, and if current is measured under controlled variations of voltage the technique is called voltammetry. In [Kueng (2004)] it is reported the detection of concentrations of ATP below 10nmol/l.

Potentiometric. The voltage at which the red-ox process may be started is a ngerprint that can be used for the identication of many biological processes. The detection limits of these kind of devices range between 10nmol/l and 0.01nmol/l [Bakker (2005)].

Conductometric. Because of the exchange of ions between the reacting species, the conductivity of the solution suers changes that are monitored though the application of a periodic voltage [Chouteau (2004)].

Acoustic biosensors Acoustic sensors take prot of the dependence between mass and harmonic resonance in solid materials. Accordingly, as the mass of an adsorbing membrane varies, the frequency of the supported acoustic waves is measured. For instance, the Attana A100® [www.attana.com] Quartz Crystal Microbalance (QCM) [O'Sullivan (1999)] based system makes a quartz crystal oscillate at its resonance frequency by means of an AC voltage. Then, the frequency of resonance is tracked in order to calculate the amount of attached mass y binding experiments. Other systems are the TTP LabTec RAPid 4 Resonant Acoustic Proling (RAP) [Aung (2008)] and the Akubio [www.akubio.com] RAPuid Resonant Acoustic Proling system.

Calorimetry Calorimetry is the study of chemical processes by means of the analysis of the involved heat exchange. Two common alternatives are:

Isothermal Titration Calorimetry (ITC). Under isothermal conditions, two samples are injected. As these substances react, the release of heat is monitored. A feed-back system keeps a constant 184


temperature equal to that of a reference cell. The amount of heat exchanged is proportional to the amount of bound mass. This method does not distinguish the kind of reaction by itself, but if this is known, anities may be determined for concentrations between 10−2 M and 10−12 M .

Dierential Scanning Calorimetry (DSC). This method is of special interest in stability studies (e.g. protein unfolding). The temperature inside the reaction chamber is varied constantly and the heat generated by induced processes is monitored. For instance, in denaturation studies, at a temperature Tm (50% protein unfolding rate) a peak in the heat capacity (cp = (∂Q/∂T )P ) is observed. Away this temperature the reaction is less ecient, and the provided energy is spent mostly in varying temperature.

Representative instruments are the Ge Healthcare MicroCal [www.microcal.com] series, (e.g. MicroCal Auto-iTC200). The studied processes take place in the whole volume of a reaction chamber, not on a surface. These instruments are not suitable for the study of surface reactions, but for their range of applications these instruments do not require immobilization.

Scanning Probe Microscopy (SPM) and cantilevers Due to its resolution and the possibility to study samples under physiological conditions, Atomic Force Microscopy (AFM) is a suitable tool for a broad range of biological applications [Alessandrini (2005)]. The scan of the interaction forces between the sample and a proximal probe allows the structural study of individual entities, from cell topography to protein unfolding [Lee (2008b); Otero (2009a,b)]. It possible to functionalize the surface of a cantilever and to measure its frequency of resonance along the adsorption of some analite [Ziegler (2004); Raiteri (2001)]. This may be either considered a SPM or an acoustic technique.

Optical biosensing Because of its intrinsic sensitivity, optics gave rise to a large and fruitful series of biosensing techniques. The most relevant are interferometry, ellipsometry, SPR, guided mode spectroscopy and colorimetry. 185


Interferometry. The nanometric wavelength range of the visible spectrum makes interference a suitable mean for the study of differences in the optical paths caused by the adsorption of biolayers. In Mach-Zehnder interferometers [Drapp et al. (1997); Sepúlveda (2006)] a channel waveguide is spit into two dierent paths (arms), one of which is exposed to the analite. Then, an optical path difference arises due to the dierence between the eective index along each arm, which depends on the adsorbed molecules. Dual polarization interferometry (DPI) (see the Farled Scientic [www.fareldscientic.com] Analight® instrument) is an alternative, in which two polarizations are propagated together along the same waveguide, one of which acting as a reference for the other. As these polarizations are exchangeable, two independent sources of information are obtained, which makes the technique suitable for adlayer thickness and density measurements [Aulin (2008); Mashaghi (2008); Lane (2008); Khan (2008); Wattendorf (2008)]. Another possibility is the interferometry of light reected on a biolayer. In this case the adsorbed thickness has a direct translation into the optical path. The FortéBio Octet® [www.fortebio.com] instrument uses white light and a reference spot to carry out this analysis [Do (2008); Rich et al. (2009); Abdiche (2009)]. Ellipsometry. The eld components of a light beam may behave dif-

ferently, depending on their polarizations. The Maven biotechnologies LFIRE instrument [http://www.mavenbiotech.com/] uses total internal reection to generate an evanescent eld that probes the external medium, leading to dierent reectivities for each polarization [Ralin (2008)].

Surface Plasmon Resonance. The Surface Plasmon Resonance

is one of the most widespread biosensing techniques. In most cases a rotary prism coupling system is used to explore resonances in the angular domain. The Metrohm Autolab [http://www.ecochemie.nl/] family of systems is one of the most fruitful examples with respect literature [Lee (2008a); Prabhakar (2008); Solanki (2008); Ryu (2008); Mir (2008a,b); Muñoz Berbel (2008); García Aljaro (2008)]. The Nomadics SensiQ® instrument [www.discoversensiq.com/] is another popular example [Myers (2008); Nepal (2008); Moraes (2008); Lan (2008); Moreira (2008)]. Because of a large history of innovations since the rst commercial SPR instrument, Biacore [http://www.biacore.com/] is the market leader and this is reected in the quality of the derived publications 186


[Yu (2008); Ahel (2008); Byres (2008)]. Currently, the most remarkable SPR instrument is the Biacore®3000 system. Many of the features developed in the context of grating couplers along this work are already implemented in it: A single wavelength and a fan-shaped incoupling beam are combined to perform angular interrogation without the need of moving components, and consequently response times are in the order of 0.5ms. This system also studies up to 4 channels together, one of which may be used as a reference. Furthermore, local functionalization allow the study of up to 20 reactions inside each channel. This feature, limited to only two channels, appears also in the Reichert SR7000DC instrument [http://www.reichert.com/].

Spectroscopy of guided modes. Commercially, the spectroscopy

of guided modes is solely represented by the OWLS instrument from Microvacuum [www.owls-sensors.com/]. This is an angular interrogation grating coupler system, in the single grating pad conguration, assisted by a rotary stage for the scan of the coupling resonances. The dielectric surface of the waveguides make this system suitable for cell membrane mimics [Merz (2008)], but also for the combination with complementary techniques as electrochemistry [Szendro (2008); Diéguez (2009a)] among a broad variety of other applications [Eggleston (2008)]. In addition, the availability of aordable data sources for layer thickness and densities make this system suitable for structural studies [Horvath (2008)].

Colorimetric Resonant Reection. In the SRU Biosystems BIND®

[www.srubiosystems.com] uses the narrow range of wavelengths reected from a broadband light source, in a similar manner to that of an SPR-G system with spectral interrogation mode [Cunningham et al. (2002)]. The monitorization of this Peak Wavelength Value (PWV) allows the Real time study of molecular binding events [Chan (2008b,a)]. Finally, the Corning Epic® system [Fang (2008a,b)] uses the wavelength of the light reected as a symptom of the molecular adsorption.

Optical array systems Specially in the context of genetics, the need to quantify a large variety of DNA sequences from the same sample turned high throughput a common requisite. For this reason, array systems have been gaining a strong attention from the scientic community, and optical biosensing is one of the most 187


promising alternatives.

SPR imaging. Instead of a unique binding channel, SPR imag-

ing systems use broad beams, with which many dierent plasmons are locally excited. Then, the corresponding resonances are spatially resolved by means of an imaging system, usually a CCD camera. Historically, the IBIS MX96 instrument, which could analyze up to 500 regions of interest (ROI) [Beusink (2008) ] and the Biacore FlexChip (based on SPR-G instead of prism coupling) [Karamanska (2008); Rich (2008); Chorley (2008)] were important. Currently, the Genoptics [www.genoptics-spr.com] SPRi-Plex array system [Ruiz (2008); Fiche (2008); Corne (2008)] and the Plexera PlexArray HT system [www.plexera.com/] are able to analyze about 1000 spots [Lausted (2008) ], and the Granity Pharmaceuticals PlasmonImager [http://www.granity.com/] is able to analyze up to 9126 spots per array.

Surface uorescence. The ZeptoREADER system from Bayer AG [http://www.zeptosens.com/] apart from providing extremely low limit of detection thanks to the use of uorescent labels (picomolarities), is an array-based instrument with 1584 data points per array. Other techniques. Some of the aforementioned devices, like the Maven LFIRE, the SRU Biosystems BIND® and the Corning®Epic® systems are also array instruments.

Final remarks It must be recognized that some ideas applied in the prototypes of this thesis were mere implementations of features that already existed in commercial systems. This did not avoid the need to develop them again, because even when they were obviously present, their implementation was never intended to be public. This is the case of the Resonance tracking algorithm, which limited the angular scanning scope of the rotary systems to an interval around the expected resonance. In the developed system, this feature restricted the scan to two intervals of 1deg around ±14deg , instead of a full scan of an interval of 30deg. This signicantly improved the sample rate, and hence it is not surprising that other rotary instruments like the Microvacuum OWLS, or the AnalyticalµSystems / Mivitec [www.microsystems.de] BIOSUPLAR 6 already had the same feature incorporated. 188


Some other ideas were taken from certain instruments or sensors and applied to others. This is the example of multichannel sensing and Background subtraction, which are common in SPR systems. Dual grating couplers had not received enough interest since the apparition of the OWLS instrument, because it was an already excellent system, and the technique had already been demonstrated. Nevertheless, OWLS have no on-line referencing capabilities because of its single channel structure. For these reasons, the implementation of previous concepts as multichannelling, on-chip reference and SiO2 passivation (e.g. [Homola (2001); Dostalek (2005)]) can be considered new in the context of grating couplers. Finally, some of the most innovative ideas presented here were inspired in others, like the concept of the analysis of regions of interest of some array systems, which suggested a channel selection feature for the achievement of an unlimited scalability.

189

B. Biosensado óptico mediante supercies nanoestructuradas Introducción El objetivo de la presente investigación es la profundización en el desarrollo de una serie de técnicas de medida, para el estudio de la adsorción especíca de biomoléculas procedentes de un ujo. Éste es un problema central en biología, pues cualquier sustancia llega a los diferentes tejidos a través de uidos, siendo el ujo sanguíneo sólo un ejemplo. Disponer de herramientas de este tipo es, pues, de gran utilidad para problemas como el desarrollo de fármacos, el estudio del rechazo a implantes y la medicina regenerativa [Kasemo (1998)].

Biosensores ópticos La naturaleza ondulatoria de la luz establece un vínculo entre el micromundo de los procesos en estudio y el mundo macroscópico de los observadores. Así, todos los fenómenos derivados de este carácter ondulatorio (desde la interferometría a la espectroscopia) son útiles para medidas de gran precisión de distintas magnitudes. Tradicionalmente los equipos ópticos requerían un alineamiento de gran precisión, que obligaba a la integración de elementos opto-mecánicos de elevado coste y volumen. Sin embargo, la óptica integrada proporciona una manera de autoalinear los haces luminosos sin necesidad de elementos adicionales, siempre que se haya provisto un modo ecaz de introducir la luz en las guías de onda. Para hacerlo se propone el acoplo difractivo [Hutley (1982)], como un modo de conseguir la inserción a lo largo de amplias áreas, cosa que puede resultar útil en aplicaciones de multisensado. A pesar de que a lo largo de las dos últimas décadas mucho se ha avanzado en las técnicas de biosensado óptico [Fan (2008)], aún quedan cuestiones sin una respuesta denitiva, y éstas serán el objeto del presente documento: 191

APÉNDICE B. BIOSENSADO ÓPTICO MEDIANTE SUPERFICIES NANOESTRUCTURADAS

No siempre la resolución o rango dinámico de una técnica satisface los requisitos de su aplicación. La frecuencia de muestreo puede ser insuciente en función de la cinética de reacción.

El coste por muestra es elevado en comparación con otras técnicas.

Las muestras se han de preparar por adelantado.

La interpretación de las medidas se apoya a menudo sobre modelos en lugar de sobre evidencias.

Técnicas empleadas Las técnicas sujeto de estudio en el presente trabajo se conocen como Biosensado Óptico de Campo Evanescente, y se basan en una serie de suposiciones. Para su justicación sugerimos consultar el documento original en inglés:

Bajo las condiciones de estudio las moléculas que se adsorben sobre una supercie pueden considerarse como una capa homogénea. Los parámetros optogeométricos de estas capas dependen de la cantidad de moléculas adheridas. A bajas concentraciones una única capa se forma, cuya densidad crece con la concentración, y con ella su índice de refracción. Cuando tal capa se satura, otras capas pueden crecer sobre ella. Mediante el uso de químicas complementarias una supercie puede prepararse para la adsorción especíca de biomoléculas. Bajo estas condiciones la monitorización de espesores e índices de capa otorga información sobre concentraciones especícas.

Todos los dispositivos propuestos tenían en común el uso de guías de onda planares como medio para poner en interacción la radiación con la materia. La energía propagada a través de estas estructuras no está completamente connada en su interior, sino que se atenúa exponencialmente más allá de su supercie. Sin entrar en la naturaleza de este fenómeno, se dene la profundidad de penetración del campo dentro de la cubierta dp como aquélla a lo largo de la que la intensidad de los campos disminuye en un factor e2 . El índice efectivo de una guía de ondas, según estas consideraciones se modeliza mediante las siguientes expresiones [De Fornel (2001); De Feijter (1978)]: 192


nef f − nef f (σm

dn = 0) = dσm

ˆ∞

dσm exp(−y/dp )dy dy

(B.0.1)

0

n(σm ) = nσ=0 +

dn σm dσm

(B.0.2)

La primera ecuación expresa la dependencia del índice efectivo de la guía con respecto a la distribución de masa adherida sobre su supercie. La segunda expresión, conocida como ecuación de De Fejter, modeliza el índice de refracción de una capa en función de su concentración de partículas. La cantidad dn/dσm es generalmente constante (0,18ml/g para proteínas y 0,16ml/g para cadenas de ADN [Wen (2000)]). Las profundidades de penetración dp típicas del campo evanescente rondan los 100nm, lo que hace éstas técnicas especialmente recomendables para la detección de (bio)moléculas. Tres tipos de sensores fueron estudiados: Los acopladores difractivos (Grating Couplers, OGCB) [Lukosz (1995)], los sensores difractivos de plasmón supercial (SPR-G ) [Homola et al. (2008)] y los sistemas de uorescencia excitada por luz guiada (WG-F) [Duveneck et al. (1997)].

Grating couplers Sobre una guía de onda óptica dieléctrica de estructura planar se hace circular un uido que contiene el analito que se desea detectar. Dado que el índice de refracción efectivo de una guía óptica depende tanto del espesor de las capas que la componen como de sus respectivos índices de refracción, la adsorción de moléculas sobre su supercie repercutirá sobre este índice efectivo [Vörös (2002)]. Para la inserción de la luz en la guía de ondas se propone el grabado de redes de difracción sobre la supercie de la guía. Dado que si un haz incide sobre la supercie de la red de difracción éste se difractará hacia el interior de la guía de onda, la condición de acoplamiento resonante será la coincidencia entre la constante de propagación de la guía y la del haz difractado. Si Λ es el periodo de la red de difracción, se tiene:

nef f = next sinθinc + m

λ Λ

(B.0.3)

Observando la ecuación B.0.3 se encuentra que existen dos posibilidades para el estudio del índice efectivo a través de las condiciones de acoplo: 193


La interrogación angular, que trabaja con una única longitud de onda λ y busca los ángulos a los que se produce resonancia. Para este tipo de interrogación es posible emplear tanto el esquema de grating de entrada como el de grating de salida. En el primero, un dispositivo encuentra las resonancias a medida que se hace rotar con respecto a la fuente, mientras que en el segundo un haz ya presente en la guía de onda se desacopla a través de un ángulo variable [Lukosz et al. (1990)]. La interrogación espectral [Homola (1997); Jenq Nan (2006)], que mantiene constante el ángulo de incidencia θinc y busca las longitudes de onda de resonancia. Dado que las condiciones de guiado en los sistemas en estudio son aplicables a una única longitud de onda, el esquema de grating de salida no es aplicable en interrogación espectral.

Resonancia de Plasmón Supercial (SPR) Los plasmones superciales pueden modelizarse de la misma manera que los modos guiados de una guía dieléctrica, aunque van asociados a vibración de cargas libres y están sometidos a una fuerte atenuación. Así, para una capa metálica lo sucientemente gruesa, el número de onda del plasmón supercial acepta la conocida expresión :

βSP W

ω = c

r

m d m + d

(B.0.4)

donde m y d representan, respectivamente, las permitividades del metal y del dieléctrico que conforman la interfaz. Sin embargo este es el caso límite hacia el que convergen los modos de propagación anteriormente descritos para espesores de capa mucho menores. Para estos sistemas se propone también el acoplo difractivo como vía para conseguir la condición de resonancia.

Fluorescencia excitada por luz guiada El desarrollo de biosensores basados en guías dieléctricas sugiere la posibilidad de emplear parte del campo evanescente para la excitación de marcadores uorescentes. Así, la guía de onda pone la energía de excitación directamente en contacto con la muestra, evitando los problemas de atravesar con ella un medio dispersivo o incluso uorescente. La intensidad de la 194


emisión uorescente dependerá de la energía suministrada y de la concentración de marcadores uorescentes activados por la interacción especíca. A cambio de la utilización de marcadores, los sistemas de uorescencia ofrecen límites resolutivos mucho mejores que otras técnicas ópticas [Duveneck et al. (1997); Grandin (2006)], y por este motivo se consideró adecuado implementar esta característica en los sensores propuestos. Se propuso una conguración exible de los sensores de tipo grating coupler que hiciese posible la excitación de uorescencia.

Diseño Tanto la adhesión de biomoléculas como la variación del líquido circundante implican un cambio estructural de las guías de onda en estudio. Este cambio varía el índice efectivo de propagación de la luz, y con él las condiciones de acoplamiento difractivo. Diferentes parámetros fueron optimizados para garantizar la fabricación de sistemas con sensibilidades adecuadas: el espesor de capa, el periodo de la red de difracción, la profundidad de grabado y el ciclo de trabajo de estas redes [Darwish (2007)] y nalmente su ubicación en cada sensor. La adecuada estimación de estos valores necesita la modelización de los sistemas en estudio, que se describirá a continuación.

Modelización de los sensores Los sistemas en estudio no pueden modelizarse únicamente mediante una capa plana, pues además de consistir en sustrato, capa de connamiento y medio cubierta, las guías de onda van recubriéndose de capas moleculares a medida que un experimento tiene lugar. Además, tampoco la supercie de las redes de difracción es plana, y por lo tanto no puede ser caracterizada con precisión mediante un modelo sencillo. Se propone como herramienta de modelización sistemática una combinación entre el método de matriz de transferencia generalizado [Shenoy (1988); Ghatak (1987)] y el modelo de capa equivalente [Kunz (1996)].

Método de matriz de transferencia en el plano complejo Como punto de partida se asume la existencia de una serie de constantes de propagación complejas {βm,r +iβm,z }, solución de la ecuación de autovalores que establece los requisitos de guiado (reexión total interna e interferencia 195


constructiva). Asumiremos que esta ecuación puede escribirse del siguiente modo:

F (βm ) = 0

(B.0.5)

donde m es el índice modal, que distingue unas soluciones de otras, y F () es una función, aún por determinar. Cuando la ecuación B.0.5 se verique, la función F −2 (β) pasará por un polo, que se asumirá lorentziano. Suponiendo inicialmente una parte imaginaria de β mucho menor que su parte real, y suponiendo una proporcionalidad entre el valor de F −2 y la distancia euclídea hasta sus polos:

k

1 A k2 = F (βγ ) [(β − βr )2 + βz2 ]

(B.0.6)

Si se desarrolla en serie de Taylor la función F alrededor de su cero βm el resultado es F (β) = F (βm ) + (β − βm )( dF dβ )βm = (β − βm )C , donde la constante compleja C puede identicarse con A−1/2 . Se propone una solución modal genérica como suma de ondas planas, Ψm,i = Am,i cos(km,i (y − di )) + Bm,i ξi sin(km,i (y − di )), donde, m es el índice modal y Am,i y Bm,i son las amplitudes que se propagan en cada sentido. Para obtener las amplitudes modales se propone una solución en forma de ondas planas, a la que se requiere las condiciones de contorno de continuidad y derivabilidad en las interfaces, y cancelación en el innito. El resultado es: q 2 km,i = k02 n2i − βm (B.0.7) A1 = 1 B1 = (−i/ξs ) Ac (β) + iξc Bc (β) = 0 La última de éstas ecuaciones es precisamente la ecuación B.0.5, y por tanto la obtención de Ac y Bc permite evaluar la función F , cosa que se hace con un rango de valores posibles de β en el espacio complejo, a n de encontrar los polos de kF −2 k. Las coordenadas de los polos resultantes podrán nalmente identicarse con las componentes real e imaginaria de las constantes de propagación de cada modo.

Modelo de capa equivalente Este modelo considera cada una de las secciones de una estructura como una capa diferente, caracterizada por un determinado espesor y un índice de 196


refracción modulado por su estructura. Así, una monocapa molecular puede entenderse que tiene el espesor de una molécula y un índice de refracción proporcional a su concentración en supercie, pero una red de difracción también puede caracterizarse, en función de su espesor de grabado y ciclo de trabajo. Siguiendo a [Kunz (1996)] se propone:

neq =

q q τ n2f + (1 − τ )n2c ; neq = nτ2 + f

y

(T E

neq

(T M

≡ neq ; neq

1−τ n2c

(B.0.8)

≡ 21 (neq + neq )

Este modelo se insertará en los algoritmos de matriz de transferencia para representar las guías de onda (incluidas las redes de difracción) en condiciones refractométricas y de adsorción.

Materiales y parámetros propuestos A partir del método anteriormente descrito para el cálculo de índices efectivos y la derivación numérica para la obtención de sensibilidades, se obtuvieron las siguientes conclusiones para cada sistema:

Guías dieléctricas Se propuso como estructura para el guiado de ondas una capa de alto índice de refracción (nitruro de silicio, n = 2,02) sobre óxido de silicio (n = 1,46). Esta combinación fue propuesta por su integrabilidad con la tecnología de fabricación CMOS. Trabajando en medio acuoso y a una longitud de onda de 633nm, se encontró que el espesor óptimo de la capa de guiado es de 65nm, para el que se consigue una sensibilidad refractiva de 0,15RIUnef f /RIUnc (ver gura 2.3.3). Si bien fueron también estudiadas las capas de guiado de polímero (en concreto Poly-methil metacrilate, PMMA), las sensibilidades obtenidas fueron un orden de magnitud inferiores.

Capas metálicas para SPR Con el n de aprovechar las posibilidades de nanoestampación de los polímeros, una vez descartados éstos como soporte para los modos guiados, se propuso su utilización como sustrato en otro tipo de sistemas. Si bien también 197


sería posible el depósito de una capa dieléctrica sobre sustrato polimérico, se prerió el ensayo de la técnica de acoplo difractivo de plasmón supercial (SPR-G), empleando para ello una capa de oro como guía. En los sensores de este tipo se necesita la creación de un modo antisimétrico de propagación, que se extiende principalmente hacia la cubierta en lugar de hacia el sustrato (véase la gura 2.3.6). Para la aparición de este modo existe un espesor de corte, que para la estructura propuesta es de 50nm (gura 2.3.7). La sensibilidad de este modo se mantiene en torno a 0,9RIUnef f /RIUnc , actuando como refractómetro.

Redes de difracción Por eciencia de acoplo era necesario que las redes de difracción actuasen dentro del primer orden de difracción. Observando la ecuación B.0.3 puede verse que esto implica que el periodo de red debe ser del orden de la longitud de onda empleada, si bien ésta podría variarse en función de la aplicación. Por geometría de acoplo se decidió emplear un periodo de red de 500nm, con las siguientes consecuencias para líquidos:

El acoplo de un haz de 635nm en la guía dieléctrica se produce entre 13 y 14 grados (gura 2.4.2). Actuando en interrogación espectral, las longitudes de onda de acoplo del plasmón supercial están entre 720 y 730nm (g. 4.5.3).

Se estudió además el efecto de la presencia de la red de difracción sobre el índice efectivo, mediante las técnicas descritas al principio. Las conclusiones fundamentales son las siguientes (g. 2.4.6):

La aproximación de capa delgada, según la cual la presencia del grating puede despreciarse, es un caso límite cuando la profundidad de grabado tiende a cero o cuando el ciclo de trabajo (fracción de cada periodo sin grabar) tiende a la unidad. La sensibilidad disminuye a medida que un mayor volumen de muestra penetra en la estructura, de modo que la aproximación de capa delgada conduce a las sensibilidades más elevadas. Esto es así debido a que las capas equivalentes tienen índices de refracción más bajos, lo que disminuye el índice efectivo y con él la concentración del campo evanescente.

Los resultados teóricos fueron avalados por medidas experimentales realizadas con unos sistemas de prueba fabricados expresamente para este propósito (g. 3.2.5). 198


Los dispositivos inicialmente fabricados sobre nitruro de silicio fueron propuestos también como máster para na nanoestampación de estructuras difractivas sobre polímero. Al hacer esto, la réplica obtenida es necesariamente la estructura complementaria del molde. Por este motivo se propuso un ciclo de trabajo del 50 %, que se reproduce en las estructuras complementarias. Además, una profundidad de grabado de 20nm fue propuesta, en parte para asegurar la reproducibilidad de las estructuras estampadas y en parte para mejorar la manejabilidad de los dispositivos, pues tales espesores son visibles a simple vista. Para la ubicación de las redes de difracción se tuvo en cuenta la atenuación en la propagación de los modos en guías dieléctricas, y para su tamaño se estimó su eciencia de acoplo. Dependiendo del ancho del haz incidente, se encontró que la longitud de acoplo apropiada variaba entre 100µm (para 270µm de ancho de haz) y 200µm para un ancho de haz de un milímetro. Para que estas redes de difracción operasen en ambos sentidos se propuso una longitud de 0,5mm, ligeramente superior, adecuada para haces ligeramente más anchos. A partir de una serie de medidas sobre los sistemas de test se encontró que la atenuación era de alrededor de 7dB/cm (g. 2.4.10), lo que conducía a intensidades perfectamente detectables al cabo de entre 6 y 7mm.

Validez como biosensores El modelo de capa equivalente fue empleado para simular la adsorción de partículas en la supercie de los sensores, a n de establecer una relación entre su sensibilidad como refractómetros con su respuesta biosensora (g. 2.5.1). Como valores de referencia se propuso 30nm de espesor y 30kDa para el peso molecular de las partículas, además de un coeciente de De Fejter (dn/dσm ) de 0.18ml/g. Los resultados fueron los siguientes:

Para un grating coupler optimizado de nitruro de silicio con una resolución angular de 10−3 grados, se obtienen los límites resolutivos de 8·10−5 RIU y 4ng/cm2 , como refractómetro y biosensor, respectivamente. Para un sistema SPR-G funcionando en interrogación espectral y con una resolución de 0.01nm se obtienen resoluciones de 1,6·10−5 RIU o 1ng/cm2 , respectivamente. 199


Doble red, pasivación y multisensado Existen dos posibilidades para la exploración de las resonancias de acoplo en el dominio angular (g. 1.2.2):

Esquema de red de entrada En este caso, a medida que el sensor se hace rotar, la orientación del haz incidente varía con respecto a la dirección normal a la red de difracción. Eventualmente puede alcanzarse un ángulo de incidencia que verique la condición de acoplo, ec. B.0.3. Si esta exploración se realiza continuamente, el ángulo de resonancia puede monitorizarse en función del tiempo, y así obtener información cinética en tiempo real [Vörös (2002)].

Esquema de red de salida Si, de algún modo, pudiese mantenerse un haz guiado constantemente, la presencia de una red de difracción sobre la supercie de la guía provocaría el desacoplamiento de este haz, a través de un ángulo dado nuevamente por la ecuación B.0.3. Este ángulo puede medirse con una cámara, y la no necesidad de explorar mecánicamente el rango de ángulos de acoplo acelera enormemente la tasa de muestreo del sensor. Para mantener la luz dentro de la guía de onda se propone la utilización de otra red de difracción [Lukosz et al. (1990)]. Esto presenta el problema de mantener las condiciones de acoplo invariables sobre la red de entrada, mientras la red de salida aún actúa como elemento de transducción.

Pasivación A n de afrontar esta dicultad se propuso para los biosensores basados en guías dieléctricas la incorporación de una capa de pasivación, de óxido de silicio, de tal manera que sólo las redes de difracción empleadas para la detección estuviesen expuestas al medio exterior [Darwish (2007)]. La idea es similar a la descrita en [Dostalek (2005)] para sistemas SPR.

Multisensado y autoreferencia En los sensores de doble red de difracción, se propuso la implementación de hasta un total de 8 parejas de redes (canales), a n de permitir la medida 200


paralela de las concentraciones de diferentes sustancias que puedan uir al mismo tiempo sobre el sensor. Adicionalmente, se mantuvo la pasivación sobre alguno de los elementos de desacoplo, lo que permitió la obtención de una medida de desacoplo invariable, con respecto a la cual los demás ángulos pudiesen medirse. Este sistema de referencia en el chip fue complementado con una estrategia de funcionalización y bloqueo selectivo de los canales, que permite distinguir entre la sensibilidad a la adsorción especíca y la respuesta sensibilidad al índice de refracción del buer en el que el analito está disuelto.

Fabricación y test Resumiremos aquí la secuencia de fabricación de los biosensores basados en guía de onda dieléctrica y de los sistemas SPR-G, además de la uídica diseñada para su aprovechamiento.

Fabricación de Grating Couplers Partiendo de una oblea de silicio de 4, las etapas de fabricación de estos sensores fueron las siguientes:

Limpieza RCA: Limpieza orgánica con una solución de hidróxido

amónico (N H4 OH ), agua oxigenada (H2 O2 ) y agua en proporciones 1:1:5, a 75o C durante 10min., seguida de la eliminación de óxido en una disolución 1:50 de HF en agua a 25o C y de la limpieza iónica, con una disolución en proporciones de 1:1:6 de ácido clorhídrico (HCl), agua oxigenada y agua, a 80o C durante 15min.

Creación de una capa buer como sustrato, de óxido de silicio:

Oxidación térmica de la oblea en un horno CESAR Centrotherm a 1100o C durante 8hr., seguida de la medición del espesor obtenido mediante elipsometría. El valor observado fue de 1,1µm. En algunos casos se partió directamente de una oblea de SiO2 (fused silica). En ellos, tanto el baño en HF de la limpieza RCA como esta etapa fueron suprimidos.

Depósito de una capa de guiado de nitruro de silicio por fase de

vapor a baja presión (LPCVD). El proceso tuvo lugar en otro horno Centrotherm, a 770o C durante 18min [Tonnberg (2006)]. El espesor deseado tras el proceso de optimización, de 65nm, fue conrmado por elipsometría. 201


Denición de las marcas de alineamiento de la oblea por fotolitografía y grabado seco, en un sistema Oxford Plasmalab®.

Denición de las redes de difracción por litografía por haz de

electrones (e-beam). Tras el depósito de una resina sensible a los electrones y el sputtering de una capa de Cr, la supercie de la muestra fue expuesta secuencialmente a un haz de electrones, empleando un sistema JEOL JBX-9300FS. A continuación se procedió al revelado de la resina, con el lift-o del cromo depositado sobre ella.

Grabado iónico (Reactive Ion Etchig) de las redes de difracción: Re-

cubierta con la resina recién revelada, la muestra fue grabada anisotrópicamente. Las redes de difracción resultaron del grabado de las áreas no protegidas por la resina. Finalmente toda la resina fue eliminada con un disolvente orgánico, quedando al descubierto la supercie de la capa de nitruro de silicio, ya nanoestructurada.

Pasivación de toda la oblea: depósito de SiO2 en fase vapor asistido

por plasma (PECVD). Este proceso tuvo lugar en un horno Centrotherm a 710o C durante 60min., con una atmósfera de tetraethylorthosilicate (TEOS).

Denición de las ventanas de sensado por fotolitografía, y grabado sobre la capa de pasivación con HF . Este grabado húmedo se caracteriza por la parada automática, pues una vez el grabado alcanzada la capa de nitruro de silicio la su velocidad disminuye tres órdenes de magnitud.

Fabricación de SPR-Gs A partir de una lámina sustrato de policarbonato de 250µm de espesor adquirida a Goofellow, se propuso el siguiente proceso de fabricación [Darwish (2008b)]:

Nanoestampación de las redes de difracción, empleando como moldes algunos de los sensores fabricados sobre dieléctrico, sin pasivar (es decir, con las redes de difracción expuestas). Los parámetros de estampación fueron 130o C , 3min y 60bar de presión, en un equipo Obducat Eitre® 6. Sputtering de 50nm de Au sobre la supercie de las muestras, empleando un equipo Von Ardenne 760, que controlaba el espesor depositado mediante balanza de cuarzo. 202


Inspección de los resultados mediante microscopía de fuerzas atómicas, AFM (g. 3.5.1).

Elementos uídicos Los canales uídicos que habían de denirse sobre la supercie de los sensores fueron trazados en juntas de polydimethyl-siloxane (PDMS). Este material se adapta a un molde estando en fase líquida, solidicándose a continuación. La fabricación del molde se realiza empleando SU-8, una fotoresina negativa especial para la denición de estructuras con elevadas relaciones de aspecto. Esta resina es iluminada a través de una máscara imprimida sobre una lámina de acetato. Una vez revelada la capa depositada de SU-8, de su relación de aspecto resulta denida una profundidad del canal entre 50 y 100µm. El curado del PDMS en contacto con los moldes se realiza en una estufa, a 80o C durante 4hr. Una vez nalizado, el resultado es la estructura complementaria a la denida en el molde (g. 4.1.2).

Instrumentación y medidas A partir de los diferentes sensores propuestos se desarrollaron los elementos de instrumentación necesarios para hacerlos funcionar. Se comenzará describiendo los bloques comunes para pasar a continuación a los especícos.

Bloques instrumentales En todos los equipos es necesaria la inyección controlada de muestras y la estabilización de la temperatura. Los elementos encargados de ello son:

Una bomba Gilson Minipuls, que gestionada a través de un puerto de control remoto mantiene un ujo de inyección constante, y el software creado para su operación. Una celda Peltier que suministra calor al sensor para mantener su temperatura constante, en función de la medición suministrada por una resistencia Pt100. La gestión de ambos elementos la realiza un microstato Jumo eTRON. La celda de ujo (g. 4.1.3): Diseñada con una cavidad para alojar el sensor y la junta uídica y otra para la celda Peltier, que bombea 203


calor entre sus caras. La celda de ujo fue diseñada para aislar las caras caliente y fría, manteniendo la muestra a temperatura constante y drenando el calor residual.

Iluminación Por otra parte, la iluminación ha de ser coherente y monocromática en los casos de interrogación angular, para evitar sensibilidades cruzadas. Además, debe tratarse de luz linealmente polarizada, para poder distinguir los casos transversal eléctrico y transversal magnético. Esto se consigue con iluminación Láser y elementos ópticos discretos. Se propuso un sistema de fuentes de luz intercambiables mediante un espejo rotatorio para aplicaciones en diferentes longitudes de onda.

Instrumentación especíca Los sistemas instrumentales dieren más entre sí por el tipo de interrogación que por la naturaleza del sensor. Así, tanto los bloques de instrumentación como las interfaces software diseñadas lo fueron con el propósito de ser intercambiables entre sí, mejorando la congurabilidad de los sistemas. Para la interrogación angular por ángulo de entrada se programó un sistema motorizado basado en un controlador y un motor de pasos de Newport, mientras que para la interrogación angular por ángulo de salida se programó una interfaz para una cámara Marlin SXGA de 1392x1040 píxeles. En este último caso se implementó un algoritmo de análisis de regiones de interés sobre la imagen que permite su uso para un número arbitrario de canales (g. 4.3.3). En el caso de los sistemas de excitación de uorescencia se empleó un ltro de longitudes de onda en combinación con un fotomultiplicador para medir la intensidad luminosa en la emitida en la dirección normal a la supercie, en la banda de emisión de los marcadores (g. 4.4.5). Los sensores de plasmón supercial funcionaban con interrogación espectral. La iluminación se realizaba con una fuente de luz blanca Ocean Optics LS1, y las resonancias de acoplo eran detectadas mediante un espectrómetro Ocean Optics SD-2000, sobre la luz reejada. En todos los sistemas las resonancias eran identicadas a partir del ajuste numérico de las curvas de respuesta. Esto se traduce en el análisis sub-píxel en el caso de cámaras o en la mejora de la resolución del espectrómetro empleado, según el caso. 204


Pruebas con sistemas biológicos Si bien en la mayoría de los casos la viabilidad de los dispositivos como biosensores se evaluó a partir de pruebas de calibración refractométrica, en el caso de los Grating Couplers de doble red se documenta un experimento de adhesión especíca [Darwish (2010)]. En este experimento se tienen en cuenta los ángulos de acoplo de entrada (pasivado), que se mantiene jo durante el experimento y los de salida a través de una red de difracción funcionalizada y otra bloqueada. Los resultados demuestran la capacidad del sistema para detectar concentraciones adsorbidas en ujos donde la proteína (Bovin Serum Albumin) está presente en concentraciones picomolares (g. 4.3.7).

Conclusiones Tres técnicas de biosensado óptico con supercies nanoestructuradas han sido presentadas: los acopladores dieléctricos (OGCB), los sensores SPR con acoplo difractivo (SPR-G) y los sensores de uorescencia excitada por guía de onda (WG-F). En relación a los problemas planteados al principio, este trabajo aporta las siguientes respuestas:

Sería deseable disponer de una herramienta potente y fácil de interpretar para representa r todos los sistemas : Se presenta un método de modelización unicado para sensores basados en guiado óptico con capas nanoestructuradas, capaz de representar efectos de segundo orden y procesos de adhesión de partículas en supercie. No siempre la resolución o el rango dinámico de una técnica satisfacen los requisitos de su aplicación: La implementación de sistemas OGCB de doble red, interrogados por cámara, además de una elevada sensibilidad, posee un rango dinámico ajustable en función de la distancia entre red de desacoplo y detector. La frecuencia de muestreo puede ser insuciente en función de la cinética de reacción : Si bien un proceso de barrido optimizado fue descrito para los equipos de acoplo resonante por red de entrada, las

velocidades de muestreo en los sistemas de doble red y SPRG mostraron tasas de muestreo notablemente superiores, debido a la supresión de piezas móviles.

La interpretación de las medidas se apoya a menudo sobre modelos en lugar de sobre evidencias : los equipos Grating Coupler permiten 205


la obtención simultánea de espesor e índice de refracción de las capas depositadas, sin necesidad de hacer suposiciones sobre cómo el proceso tiene lugar. Además, un diseño de sensor e instrumentación que habilita el análisis simultáneo mediante acoplo por red de difracción y excitación de marcadores uorescentes facilita la realización de pruebas complementarias.

El coste por muestra es elevado en comparación con otras técnicas : A nivel de aplicación, predomina la necesidad de detección de analito conocido, para el que hay disponibles modelos de depósito ables. Este es, por ejemplo, el caso clínico. En este contexto los sistemas SPRG propuestos proporcionan buenos límites de detección y requieren técnicas de fabricación de bajo coste. Las muestras se han de preparar por adelantado : La implementación de referencia en chip para sensores basados en guías de onda dieléctricas, combinada con sus características multicanal permite el análisis de muestras heterogéneas.

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