Integrated dual-slab waveguide interferometer for

Integrated dual-slab waveguide interferometer for glucose concentration detection in the physiological range Meng Wang*a, Sanna Uusitalob, Miia Määttäläa, Risto Myllyläa, Markku Känsäkoskib Optoelectronics and Measurement Techniques Laboratory, University of Oulu, Finland; b VTT Technical Research Center of Finland, Oulu, Finland

a

ABSTRACT This paper presents a label-free optical biosensor based on a Young’s interferometer configuration that uses a vertically integrated dual-slab waveguide interferometer as sensing element. In this element, linearly polarized light is coupled into a dual-slab waveguide chip from the input end-face, and the in-coupled zeroth order mode propagates in separated upper and lower waveguides. At the output end-face, the two closely spaced coherent beams diffract out and produce an interference fringe pattern. An evanescent wave field, generated on the surface of the upper waveguide, probes changes in the refractive index of the studied sample, causing a phase shift in the fringe pattern. Compared to a conventional integrated Young’s interferometer utilizing a Y-junction as the beam splitter, the dual-slab waveguide Young’s interferometer has the advantage of easy fabrication and large tolerance to the input-coupling beam. This paper builds a measurement system to investigate sensor performance using glucose solutions with various concentrations. These glucose concentration measurements are performed within the physiological range of 30mg/dl ~ 500mg/dl. The results indicate that a dual-slab waveguide interferometer yields an average phase resolution of 0.002 rad, which corresponds to an effective refractive index change of 4×10-8 with an interaction path length of 15 mm. Keywords: Interferometric biosensor, dual-slab waveguide, evanescent wave field, glucose concentration, effective refractive index

1. INTRODUCTION In recent years, there has been a rapidly growing interest in integrated optical (IO) sensors, because they allow sensitive, real-time, label-free on-site measurements of the concentration of (bio-) chemical species1. Most IO sensors are based on a planar multilayer waveguide structure deposited on top of a silicon or glass substrate. A multilayer waveguide structure is composed of a very thin higher refractive index layer, consisting of a network of optical waveguide channels, and adjacent lower refractive index layers. As a result of total internal reflection (TIR) of light propagating in the layer with the higher refractive index, an evanescent wave field penetrates the boundaries of the layer and reaches the top of the waveguide surface with a typical penetration depth of a few hundred nanometres. IO sensors utilize this evanescent wave field to excite attached fluorescent labels 2 or, in label-free detection schemes, to probe the refractive index change of the sample area and the absorption of molecules 3. A direct label-free detection method does not require the preparation steps needed for label tagging. This simplifies the sensing process and avoids the risk of contamination, since there is less need for auxiliary chemicals during sample preparation procedures. Interferometry is a well-known direct sensing technique with high sensitivity and resolution. The sensing mechanism transduces a change in the concentration of chemical species on the waveguide surface into a change in the real part of the effective refractive index of a guided mode propagating in the waveguide. This change in the effective refractive index also changes the phase of the propagating light, which can be measured as a shift in the interference fringe pattern. Interference pattern modulation can be used with many sensing platforms, such as the MachZehnder, Michelson and difference interferometer. However, unlike these methods, Young’s interferometer (YI) offers the advantage of determining the absolute sign of the induced phase change during sensing. Furthermore, there is a linear correlation between the change in the position of the interference pattern and the induced phase change4. Originally, an integrated YI has a two-branch structure (constituting a sensing and a reference arm) with the Y-junction functioning as *

[email protected]; phone +358 8 5532776; fax +358 8 553 2774; www.ee.oulu.fi Optical Sensors 2008, edited by Francis Berghmans, Anna Grazia Mignani, Antonello Cutolo, Patrick P. Meyrueis, Thomas P. Pearsall, Proc. of SPIE Vol. 7003, 70031N, (2008) · 0277-786X/08/$18 · doi: 10.1117/12.780184

Proc. of SPIE Vol. 7003 70031N-1 2008 SPIE Digital Library -- Subscriber Archive Copy

the beam splitter5-6. Recently, new integrated YI structures have been developed, including multichannel integrated YI7, planar channel-type YI8-10 and vertically integrated dual-slab YI4,11,12. At the present stage, the detection resolution of chemically induced changes in the effective refractive index of the different YI structures is in the magnitude of 10-6 ~ 10-8. Compared to all the other YI structures, the vertically integrated YI requires less manufacturing procedures, less complicated manufacturing methods and has a higher tolerance to light in-coupling and system alignment. That brings the benefits of lower manufacturing costs and the possibility for mass production. In this paper, we describe a set of refractive index measurements with a commercially available vertically integrated dual-slab waveguide Young’s interferometer13. We have analyzed the effect of glucose solutions on sensor performance using a set of glucose distilled-water samples with a concentration range of 30 mg/dl to 1500 mg/dl. Of most interest for the present purpose are concentrations within the physiological range of 30 mg/dl ~ 500 mg/dl, while samples with a higher glucose concentration are used for better measurement system calibration. Measurements were performed in both transverse electric (TE) and transverse magnetic (TM) modes using a He-Ne laser as the light source.

2. MATERIALS AND METHODS 2.1. Dual-slab waveguide Young’s interferometer In a conventional Young’s interferometer, a Y-junction beam splitter is used to produce coherent sensing and reference beams, as can be seen in Fig. 1 a). However, in a dual-slab waveguide Young’s interferometer, the coherent sensing and reference beams are produced by splitting the incident light through a structure of vertically spaced parallel slab waveguides, Fig. 1 b). This simplifies the in-coupling of light and the fabrication of the chip.

Etched sensing channels Reference arm

N

Cladding

Sensing arm a)

Fig. 1. Schematic illustration of a) a conventional Young’s interferometer with a Y-junction and b) a dual-slab waveguide Young’s interferometer.

The sensor structure used here has a five-layer dielectric stack structure of silicon oxynitride (SiOxNy), which is fabricated on a silicon wafer substrate with silicon oxynitride dielectric layers deposited by PECVD 11. Of the layers, the second and fourth, forming the reference and sensing waveguide layers, have higher refractive indices than the adjacent cladding and cover layers. The first and third layers are cladding layers, while the fifth layer is the cover layer. With its etched sensing windows, the cover layer functions as a chemo-optical interface. The cladding layer between the sensing and the reference waveguide is thick enough to separate the light propagating in both waveguides. Further, it enables the two light beams to diffract at the output end-face of the sensor and to interfere with each other. An evanescent wave field, generated on the surface of the sensing waveguide, probes the refractive index change of the sample solution and the density and thickness of the formed adlayer in the sensing window. This type of five-layer stack structure makes a Yjunction beam splitter unnecessary and greatly simplifies the manufacturing process. Conventional Y-junction interferometers often have such small waveguide channels that light in-coupling requires the use of an optical fiber or a high magnification lens. In a dual-slab waveguide interferometer, the light of a collimated laser beam can be coupled directly into the waveguide sensor at its end-face to excite both upper and lower mode without any additional optics. At

Proc. of SPIE Vol. 7003 70031N-2

the other end of the sensor, the two modes diffract in free space and generate a Young’s interference fringe pattern on the detector. The diameter of the input laser beam must be sufficiently large, relative to the dimension of the waveguide stack, to ensure almost equal coupling efficiency to both the upper and lower waveguides, even if the source and the waveguides are slightly misaligned from device to device4. With an optimal laser choice, the optical tolerance of the input-coupling beam can be hundreds of microns and, thus, a small misalignment of the laser does not cause any change in the interference fringe pattern. Theoretically the generated interference fringe pattern can be written as

I (x ) = W ( x )[1 + cos(2πfx − ϕ )]

(1)

where x is the spatial coordinate, f and φ are the spatial frequency and the intrinsic phase of the fringes and W(x) is the envelope function of the fringe pattern. In a dual-slab waveguide structure, the upper slab is the sensing waveguide, which detects changes in the refractive indices of the applied sample solutions and reacts to the accumulated adlayer on top of the sensor surface. The lower slab is the reference waveguide, which is unaffected by changes occurring at the surface, since the evanescent wave field in the reference waveguide rapidly decays in the cladding between the two guiding layers. 2.2. Theoretical background On the sensor surface, the concentration change ∆C is measured as a phase-shift ∆φ of the interference fringe pattern. The transduction relationship can be described with the following chain: ∆C → ∆X → ∆neff → ∆ϕ , where ∆neff denotes the change in the effective refractive index of the waveguide sensor. ∆neff is a function of the following parameters

neff = neff (λ , ns , n f , d f , nad , d ad , nc , pol.) ,

(2)

where λ is the wavelength of the incident light; ns, nf, nad, and nc are the refractive indices of the substrate, the waveguide, the adlayer and the covering sample, respectively; df and dad are the thicknesses of the waveguide and the adlayer; and pol indicates polarization. ∆X denotes a change of each of the above mentioned parameter X which affects neff. In the case of glucose concentration measurements, where no adlayer is formed on the surface of the sensing window, the only parameter affected by ∆C is the change in the refractive index of the sample’s cover layer ∆nc. Since a change in the effective refractive index ∆neff produces a phase change ∆φ in the interference fringe pattern, this change can be quantified by the expression

∆ϕ =

2π

λ0

L ⋅ ∆neff ,

(3)

where L indicates interaction length (i.e., the length of the sensing window) and λ0 is the vacuum wavelength of the monochromatic light source. In IO sensors designed for measuring refractive index changes of liquid samples covering a waveguide, the change in the effective index is, to first order 14

∆neff =

∂neff ∂nc

∆nc ,

(4)

where ∆nc is the change in the refractive index of the liquid sample covering the waveguide, and the derivative ∂neff/∂nc is the corresponding sensitivity of the waveguide, obtainable for a given wavelength and waveguide structure by the following equation

n P ⎡ ⎛n = c ⋅ c ⎢2 ⋅ ⎜⎜ eff neff P ⎢ ⎝ nc ∂nc ⎣

∂neff

ρ

2 ⎤ ⎞ ⎟⎟ − 1⎥ , ⎥⎦ ⎠

(5)

where Pc/P is the proportion of the power in the cover layer compared to the total power of the chip, and ρ is 0 for the TE mode and 1 for the TM mode. In reality, we first measure the phase change ∆φ and then, using Equation (3), we calculate the change in the effective refractive index ∆neff from ∆φ. If we know the physical parameters of the chip, such as the refractive indices of the stack layers and the distance between the sensing and reference waveguide layers, we can


apply Equation (4) to calculate the change in the refractive index of the sample ∆nc from ∆neff. Finally, ∆C can be obtained by using the known relationship between a particular glucose concentration and its refractive index, which can be found in the literature 15. We have calculated theoretical phase shift values using the Marcatili method with known glucose sample concentrations. Marcatili’s is an analytical method, designed to model three-dimensional optical waveguides, as shown in Fig. 2.

2

p

UC -p

2

9

-9

Fig. 2. Waveguide mode of Marcatili’s method.

The method assumes that electric and magnetic fields are confined to the core and do not exist in the four hatched regions. As a result, electric fields in these regions decay exponentially in the x and y directions. Moreover, wave propagation for TE and TM mode coupling can be characterized by the following equations 16

β = k02 n 2f − k x2 − k y2

TE & TM:

(

(6)

)

⎡ k 2n2 − k 2 k 2 n2 − n2 − k 2 ⎤ π 0 f c x y ⎥ + ( p − 1) , k x a = arctan ⎢ 02 2f ⋅ 2 kx ⎢ k0 nc − k y ⎥ 2 ⎣ ⎦

TE:

(

)

⎛ k 2 n2 − n2 − k 2 1 0 f c y k y b = arctan⎜ ⎜ 2 ky ⎝ TM:

,

(

)

2 2 ⎛ 2 2 ⎞ 1 ⎟ + arctan⎜ k0 n f − ns − k y ⎜ ⎟ 2 ky ⎝ ⎠

⎛ n2 k02 (n 2f − nc2 ) − k y2 ⎞⎟ 1 + arctan⎜ 2f ⎟ ⎜ nc ky 2 ⎠ ⎝

(7)

⎞ π ⎟ + (q − 1) , (q=1, 2, …) (8) ⎟ 2 ⎠

⎡ n 2 (k 2 n 2 − k 2 ) k 2 (n 2 − n 2 ) − k 2 ⎤ π 0 f c x x ⎥ + ( p − 1) , k x a = arctan ⎢ c2 02 f2 ⋅ 2 kx ⎢ n f (k0 nc − k x ) ⎥ 2 ⎣ ⎦

⎛ n2 1 k y b = arctan⎜ 2f ⎜ nc 2 ⎝

(p=1, 2, …)

(p=1, 2, …)

(9)

k02 (n 2f − ns2 ) − k y2 ⎞⎟ π + (q − 1) , (q=1, 2, …) (10) ⎟ 2 ky ⎠

where β is the wave propagation constant in the waveguide, k0 = 2π/λ0 is the wave number in vacuum and kx and ky are wave numbers in the x and y directions. As shown in Figure 2, a and b are half the width and length of the waveguide and nf, nc, and ns are the refractive indices of the waveguide, the covering sample and the substrate, respectively. In addition, p and q are wave modes in the x and y directions, equalling 1 in the case of zeroth TE and TM mode. Table 1 summarizes the input physical parameters for theoretical model calculations.


Table 1. Waveguide parameters for theoretical model calculations.

Parameter Substrate index (cladding index) Waveguide index Cover index (H2O) Width of the waveguide (2a) Height of the waveguide (2b)

Value ns = 1.475 nf = 1.520 nc = 1.333 2a = 0.01 m 2b = 1×10-6 m

The propagation constant β provided by the model can be used to determine the effective refractive index neff:

neff =

β

,

k0

(11)

Equation (3) allows us to calculate the theoretical phase shift of known samples. Section 3 compares the measured fringe pattern phase shifts with theoretical values calculated by the Marcatili method. 2.3. Fringe pattern analysis Direct measurement of the phase change ∆φ is possible by continuously monitoring the relative phase that is also the intensity distribution of the fringe pattern. In our case, this is achieved by recording the fringe pattern with a 1Hz frequency during the entire measurement period. The resulting interferogram picture measures 640×512 pixels, with each pixel digitized to 12-bit information. To analyze the fringe patterns, we use a two-dimensional fast-Fourier-transform (FFT) algorithm, which calculates the amplitude and phase spectrum of the fringe pattern f(m,n) 2D-FFT: F ( p, q ) =

M −1 N −1

∑ ∑ f (m, n)W

pm M

m=0 n=0

where p=0,1, …, M-1; q=0,1, …, N-1; WM

=e

⎛ 2π ⎞ − j⎜ ⎟ ⎝M ⎠

WNqn ,

; WN = e

Amplitude: A( p, q ) = F ( p, q ) , Phase:

ϕ ( p, q ) = arctan

imag [F ( p, q )] , real [F ( p, q )]

(12) ⎛ 2π ⎞ − j⎜ ⎟ ⎝ N ⎠

, (13) (14)

The phase of each recorded interference fringe pattern corresponds to the peak of its amplitude spectrum. It is then extracted at the given spatial frequency from the phase spectrum, and phase discontinuities for successive fringe patterns are corrected by a phase unwrapping method. A change in the phase ∆φ is seen as a phase difference between two successively recorded fringe patterns.

3. MEASUREMENT AND RESULTS 3.1. Measurement system setup Shown in Fig. 3, the measurement system presented here is a Young’s interferometer with a dual-slab waveguide chip as the sensing element. As light source, the system employs a 2mW He-Ne laser (Melles Griot 05-LHP-121), which is linearly polarized and has an emission wavelength of 632.8nm. Measuring 0.59mm in diameter, the laser beam is capable of covering both waveguides. As described before, the dual-slab waveguide sensor chip has the advantage of easy light coupling and a diminished probability for laser miss-alignment. Moreover, the input laser beam is simply endfire coupled into the waveguide to obtain a satisfied visibility of the interference pattern. Relative displacement smaller than the diameter of the laser beam between the laser and the dual-slab waveguide chip does not affect considerably the visibility of the interference pattern.


Fig. 3. Measurement system setup.

Supplied by Farfield Group Ltd, the dual-slab waveguide chip has a sensing and reference waveguide, both measuring 1µm in height, with a 4µm cladding layer between them. Etched on the cover layer are two sensing channels with an interaction length of 15mm. In affinity measurements, the second sensing channel can be used as a referencing channel to compensate for non-specific binding events and environmental noise. Because no adlayer forms on the sensor surface in glucose concentration measurements, there is no need for more than one sensing channel. The surface of the sensing channel consists of bare silicon oxynitride without any additional surface treatment, while the unused channel is covered by a piece of Teflon slide, which also serves as a gasket to prevent leakage of the liquid sample at the interface of the sensor chip and the flow cell. At the output end-face of the sensor chip, the diffracted light beams interfere to produce a fringe pattern, which is efficiently collected by a microscope objective. This interference fringe pattern is then forwarded into a CCD camera (PCO SensiCam 12 Bit Cooled Imaging) through the microscope objective. Fig. 4 shows a collected interference fringe image of 640×512 pixels. The colour bar on the right side indicates the intensity distribution of the fringe pattern. 4000 3500 100

Pixel number

3000 200

2500 2000

300 1500 1000

400

500 500 100

200

300 400 Pixel number

500

600

0

Fig. 4. Captured interference fringe pattern on a CCD camera.


The flow system of the measurement setup consists of a single-channel flow cell and a pulse-free continuous-flow syringe pump (OPAM Instruments). As shown by Fig. 3, the flow cell has an inlet and outlet, allowing the sample liquid to flow through the whole sensing channel, where approximately a volume of 5µl stays on the surface of the sensing waveguide in the Teflon window. The pumping speed of the syringe pump is typically set to 3ml/min. A PC is used to control flow speed and to collect the raw digital images captured by the CCD camera. System stability was tested using a continuous flow of distilled water. The standard deviation of the fringe pattern phase shift was around 2×10-3 rad, corresponding to an effective refractive index of 1.3×10-8 over ten independent tests. Applying the 3σ criterion 10, the obtained detection limit of ∆neff was 4×10-8, corresponding to a 5×10-6 refractive index change in the covering sample liquid. This indicates that the lowest achievable detection resolution is 3.5mg/dl (0.0035% w/v) for glucose water solutions. 3.2. Glucose concentration measurement In evanescent wave field sensing, cover fluid concentrations can be detected by their intrinsic refractive index. In this study, the cover fluid samples were glucose distilled-water solutions. Since the concentration of a glucose water solution has a linear relationship with its intrinsic refractive index, it can be used for the precise calibration of the measurement system. In room temperature, the refractive index of a glucose water solution has the following relationship with its concentration

nc = 0.0014713 * C + 1.333 ,

(13)

where C indicates the glucose concentration of a percentage solution (w/v). This relationship was obtained from the Dglucose lookup table 15 with linear interpolation and regression for concentrations smaller than 0.5% (w/v). Most interesting for the current purpose were glucose concentrations in the physiological range varying from 30mg/dl (0.03%) to 500mg/dl (0.5%), while samples with a higher glucose concentration were used to calibrate the measurement system. In order to make the etched sensing channel hydrophilic, the measurement started by pumping distilled water for 90 seconds. Next, each glucose sample was measured twice using a pumping time of 150 seconds, followed by a washing procedure with distilled water, taking 90 seconds. The lowest glucose concentration was applied first, and the washing procedure served as reference. Fig. 5 shows the sensor response that is the phase shift of the interference fringe pattern of two measurement sets: one with smaller concentration and the other with a larger one. In this figure, the washing procedure constitutes the baseline between each measurement. The difference between the baseline and the sensor response represents the phase shift caused by the refractive index change between the reference (distilled water) and the samples (glucose water solutions). 3

0.2

1.5%

0.15

2.5

0.07%

phase shift [rad]

phase shift [rad]

0.1%

0.05%

0.1 0.03%

2

1.0%

1.5 0.5%

1

0.05

0.3% 0.5

0 0

500

1000 time [s]

1500

2000

0

0

500

1000 time [s]

1500

2000

Fig. 5. Sensor response (fringe pattern phase shift) for two sets of glucose water solutions with TE polarization.

Each measurement was performed both in the TE and TM polarization mode. Fig. 6 compares the measured phase shift values with theoretical values calculated by the Marcatili method. Due to fabrication tolerances, the dual-slab waveguide chips used in the TE and TM polarization measurements showed some small variations in their physical parameters. This has been taken into account with the theoretical calculations.


3.0

TM

experimental theoretical

2.5

phase shift [rad]

TE 2.0

1.5

1.0

0.5

0.0 0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

glucose concentration [%]

Fig. 6. Theoretical and experimental phase shift as a function of glucose concentration in both TE and TM polarization.

As shown in Fig.6, the sensor response increases nearly linearly within the examined concentration range and is in good accordance with the theoretical calculations. This is particularly true for the physiological range, where the mean measurement error between experimental and theoretical phase shifts never exceeds 4% of the expected theoretical value. Fig 7 shows a calibration curve, where the change in ∆neff, obtained from Equation (3) for each concentration, is plotted against the refractive index ∆nc obtained from Equation (13). A linear regression is then fitted to the data to determine the slope of the curve, which indicates the sensitivity constant ∂neff/∂nc of the dual-slab waveguide interferometer sensor. Finally, the obtained sensitivity constants for TE and TM polarization are compared to theoretical sensitivity constants calculated by Equation (5). 0.000022 0.000020 0.000018 0.000016

TM

TM: Linear Regression Y = A + B * X A B

4.00834E-8 ± 4.68813E-8 0.00913 ± 4.74452E-5

Theoretical value for B:

TE

0.009172

0.000014

∆neff

0.000012 0.000010 0.000008 TE: Linear Regression Y = A + B * X

0.000006

A B

0.000004 0.000002 0.000000 0.0000

-2.60453E-8 ± 4.03458E-8 0.00778 ± 4.08311E-5

Theoretical value for B:

0.0005

0.0010

0.0015

0.0020

0.007859

0.0025

∆nc

Fig. 7. Change in the refractive index of the sample ∆nc versus change in the effective refractive index ∆neff.


As shown in Fig. 7, the sensitivity constants obtained from the linear regression for TE and TM mode are 0.00778±4.08311×10-5 and 0.00913±4.74452×10-5, respectively. They agree nicely with the theoretical values of 0.007859 and 0.009172. This confirms that the dual-slab waveguide interferometer sensor can be used for very accurate refractive index measurements.

4. CONCLUSIONS This paper applied a dual-slab waveguide interferometer sensor to concentration detection in glucose water solutions. The vertically integrated structure of Young’s interferometer greatly simplifies the manufacturing process and has a good tolerance for small misalignments in adjusting the laser beam for light in-coupling. Tests to establish the stability of the system configuration yielded a detection limit of 4×10-8 for ∆neff, corresponding to a refractive index change of 5×10-6 in the covering sample liquid ∆nc at a 1 Hz sampling rate. Used in refractive index measurements to determine glucose concentrations, the waveguide sensor demonstrated high reproducibility and sensitivity. Moreover, the results were in good agreement with theoretical phase shift values calculated by the Marcatili method for the TE and TM coupling modes. Within the physiological range, the largest mean measurement error deviated from the theoretical value only by 4%. Also the sensitivity constants of the TE and TM mode, obtained with the linear regression method, were in good accordance with theoretical calculations. In future, our research will focus on affinity measurements on the binding processes of biomolecules. Potential research objects include Mouse-IgG and Anti Mouse-IgG. In these measurements, the other sensing channel will be utilized for subtraction of non-specific adsorption.

ACKNOWLEDGEMENTS The authors would like to thank the Infotech Oulu Graduate School and Finnish Funding Agency for Technology and Innovations (TEKES) for supporting this research project. In addition, the authors wish to extend their warmest thanks to Ms. Inka Mäkelä for technical support.

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