Instrumental improvements in optical waveguide light mode spectroscopy for the study of biomolecule adsorption R. Kurrata) and M. Textor Laboratory for Surface Science and Technology, Department of Materials, Swiss Federal Institute of Technology, CH-8092 Zu¨rich, Switzerland
J. J. Ramsden Department of Biophysical Chemistry, Biocenter of the University, CH-4056 Basel, Switzerland
P. Bo¨ni Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland
N. D. Spencer Laboratory for Surface Science and Technology, Department of Materials, Swiss Federal Institute of Technology, CH-8092 Zu¨rich, Switzerland
~Received 1 October 1996; accepted for publication 4 February 1997! Optical waveguide light mode spectroscopy ~OWLS! is a new technique that is particularly well suited to the in situ study of biomolecule adsorption kinetics on surfaces. Here we describe improvements to a commercial OWLS instrument in order to allow for easy combination with other ex situ surface-characterization methods, such as x-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and atomic force microscopy. Further, the problem of contamination of the waveguide surface arising from the use of silicone in the flow-through cuvette with which biomolecules are brought into contact with the adsorbing surface had to be resolved, as it greatly altered the wetting and adsorption properties of the waveguide. Finally, through physical vapor deposition of thin, nanosized layers of titanium oxide onto the waveguide layer, it is possible to simulate the surface properties of oxide-covered titanium implant surfaces. However, scanning angle constraints set by the mechanics of the commercial instrument must be borne in mind. © 1997 American Institute of Physics. @S0034-6748~97!03105-5#
I. INTRODUCTION
The interaction of biomaterials ~such as those used as surgical implants! with their surroundings is strongly influenced by the adsorption of biomolecules. Knowledge of the adsorption kinetics of such biomolecules is therefore essential to the understanding of biocompatibility, i.e., of the interface between an artificial biomaterial and its biological environment.1 The integrated optics technique, optical waveguide light mode spectroscopy ~OWLS!, is an excellent tool for the in situ study of the kinetics and equilibrium constants of relevant surface processes, e.g., adsorption of proteins,2,3 binding of analyte molecules to immobilized receptors,4 or the adsorption, spreading, and growth of cells.5 The basic principle of the technique ~Fig. 1! consists of measuring the mode spectrum of an optical waveguide,6 the surface of which is exposed to a solution of biomolecules or cells. The parameters characterizing adlayers of adsorbed molecules or cells can be derived from the spectrum.7,8 The present article covers improvements to a commercial OWLS instrument with the aim of eliminating some drawbacks and artifacts as well as extending its application to surfaces with chemical composition and properties that are closer to those characteristic of real implant materials. The first improvement is a modification of the optical arrangement that detects the incoupled light, in order to ena!
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able small, approximately square waveguides to be used. This provides the advantage of direct introduction of the waveguides ~without the need to cut them! into surface characterization instruments, such as the photoelectron spectrometer ~XPS!, secondary ion mass spectrometer ~SIMS!, and atomic force microscope ~AFM!. These techniques can therefore be used to characterize the waveguide surface before introduction into the OWLS instrument, and provide complimentary information about the structure of the adsorbed biomolecule adlayer following the OWLS adsorption experiment. Second, we have found that the silicone polymers, out of which part of the flow-through cuvette is currently fabricated, are a source of severe contamination of the waveguide surface, altering the wettability and hence the adsorption properties of the original, clean sample surface. New polymeric materials to replace silicone were evaluated in order to eliminate contamination of the waveguide surface with surface active contaminants. Third, we describe the surface modification of currently available commercial planar waveguide/grating couplers via physical vapor deposition ~PVD! of titanium dioxide (TiO2), in order to simulate the surface of titanium surgical implants. These are known to be covered by a natural thin oxide film, which renders the titanium implant chemically inert and provides long-term corrosion resistance. Although, in principle, the entire waveguide layer could be fabricated out of TiO2, it is more straightforward to use a commercially available standard mixed SiO2-TiO2 waveguide, which can
0034-6748/97/68(5)/2172/5/$10.00
© 1997 American Institute of Physics
thin layers ~here ;180 nm!, only discrete modes exist, which means that the incoupled power of the light reaches a maximum for discrete values of a determining the corresponding N. By solving the mode equations simultaneously, the optogeometric parameters of the layer system can be calculated from the effective refractive indices and the quantity of adsorbed molecules determined.6,8 B. XPS and ToF-SIMS surface analysis
FIG. 1. The measuring principle of the OWLS method.
then be readily modified by a thin ~nanosized! surface layer of defined stoichiometry. We describe the constraints imposed by the mechanical scanning limits of the IOS-1 instrument. II. EXPERIMENT A. OWLS instrument
The optical waveguide light mode spectrometer used in this study is a commercial IOS-1 goniometer scanner manufactured by Artificial Sensing Instruments ASI, Zu¨rich, Switzerland.9 In this instrument, a flow-through cuvette, into which biomolecule solutions can be introduced, is fixed over a diffraction grating incorporated into the optical waveguide. The entire assembly ~waveguide plus cuvette! is then mounted on a precision goniometer, which measures the angles of incidence of the external laser beam required to couple light into the waveguide. Typical mode spectra are shown in Fig. 5. There are several types of waveguides that may be used with this instrument. Some of the experiments referred to in this work have been carried out with the older ASI2400 type, consisting of a 0.5-mm-thick glass plate and a 200-nm-thick pyrolyzed sol-gel SiO2 TiO2 layer with an embossed diffraction grating of 2400 lines/mm. The newer ASI1400 waveguides have a diffraction grating of 1400 lines/mm and a 180-nm-thick SiO2 TiO2 layer, produced by sputtering of TiO2 and SiO2. Both these waveguide types are monomode waveguides, i.e., only supporting the zeroth modes of TE and TM polarizations, for which the sensitivity to adlayer changes is the highest.8 The measuring principle of the OWLS method is shown in Fig. 1. The linearly polarized light beam ~He–Ne laser, l5632.8 nm! enters from below, through the supporting glass slide. At the diffraction grating, the light is coupled into the waveguiding layer ~high refractive index mixture of SiO2 and TiO2!, providing that the incoupling condition N 5n air sin a1l•l/L is fulfilled, where N is the effective refractive index of the whole layer system, a the angle of incidence, l the diffraction order, l the wavelength of the light, and L the grating period. From there, the light is guided by total internal reflection to the end of the waveguide, where it is detected by photodiodes ~not shown!. By varying a, the mode spectrum can thus be measured. In very Rev. Sci. Instrum., Vol. 68, No. 5, May 1997
The surface composition of the waveguides before and after use in the OWLS instrument as well as before and after coating of the waveguides with TiO2, was determined using XPS and TOF-SIMS. XPS was performed with a SAGE100 spectrometer ~SPECS, Berlin, Germany! using Mg K a radiation. Peak intensities were converted to atomic concentration using published10,11 sensitivity factors. TOF-SIMS measurements of the outer surface of the waveguides were obtained by means of a Perkin–Elmer PHI 7000 instrument. An 8 keV cesium ion primary beam was scanned over an 1003100 mm2 area of the waveguide surface. A TOF analyzer was used to detect the positive secondary ions with a mass range of 1–1000 amu. Pulsed lowenergy electrons were used to neutralize the surface charges that built up during ion bombardment. Mass resolution (m/Dm) was typically 4000. The mass range was calibrated 1 using well-defined hydrocarbon peaks (CH1 3 , C2H3 , 1 C3H5 ). C. PVD coating of waveguide
The TiO2 coatings were produced in a Leybold dcmagnetron Z600 sputtering plant. The dc-power supply was pulsed ~the frequency was 20 kHz, i.e., we applied a negative voltage to the cathode for 37.5 ms followed by a positive voltage for 12.5 ms! in order to reduce the poisoning of the Ti targets by O2. Moreover, arcs were suppressed. The magnetrons ~area 488 mm387.5 mm! were operated at a power of 2 kW. The gas flows were 12 sccm for Ar and 17 sccm for O2 resulting in partial pressures p Ar57.7310 and p O2 53.4 31024 mbar. By means of a Tencor P2 long scan profiler, the thickness, d, of a test coating was determined to be 73 Å for a sample that passed the sputter target n55 times with a drive speed n 50.75 m/min. Based on this calibration, the waveguides were coated with nominal thicknesses of 20, 40, and 100 Å TiO2 by varying n and n appropriately. III. RESULTS A. Small waveguides and glass fiber optics
The waveguides for which the IOS-1 is designed have a size of 1634830.5 mm, which is too large to be directly used in typical XPS, SIMS, or AFM instruments. Hence, the need for smaller waveguides. The basic idea was to build an insert for the original IOS-1 measuring head, to hold smaller waveguides (831230.5 mm) and to provide an optical connection to the photodiodes. Normally, the photodiodes are very close to the waveguide, in order to collect all the light that emerges as a highly divergent beam from the waveguide. For the connection beOptical waveguide light mode spectroscopy
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FIG. 2. Schematic drawing of the waveguide holder with glass fiber optics and waveguide. The distance from left to right is 48 mm, i.e., the length of the original waveguide.
tween the small waveguides and the photodiodes ~spaced for the large waveguides!, a very high quality glass fiber bundle ~Fiberoptic, Dietikon, Switzerland! had to be used. Figure 2 is a schematic drawing of the waveguide holder with the glass fibers and the waveguide in place. Figures 3 and 4 show detailed construction drawings of the new holder. The glass fibers have polished ends, allowing a nearly perfect transmission of the signal from the waveguide to the photodiodes. When inserting the waveguide in the holder, its contours can be seen very sharply at the end of the glass fibers, showing the perfect image transmission. No difference could be seen in the mode spectra after cutting a standard size waveguide and measuring it in the new holder. Figure 5 shows such a mode spectrum of a small waveguide. It appears perfect, without the slightest distortion. In fact, the new system appears even more efficient at collecting the guided light than the original setup, probably due to the shorter pathway and reduced intensity loses in the small waveguide setup; a waveguide layer section has now been replaced by a bundle of fibers. B. Silicone polymer contamination
After the modification of the waveguide holder to accept the small waveguides, it was possible to perform XPS and TOF-SIMS measurements both before and after an OWLS experiment. At this point, another problem of the IOS-1 instrument became apparent. The flow-through cuvette, which forms the channel for the liquid and which is sealed to the waveguide, is made of silicone rubber. Using this flowcell,
FIG. 3. Detailed top view of the holder. Dimensions in mm. 2174
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FIG. 4. Detailed side view in the center position. Dimensions in mm.
problems showed up due to air bubbles forming in the flowcell during long experiments.12 We realized that the surface of the waveguides became more and more hydrophobic during an experiments. Figure 6 shows a typical TOF-SIMS spectrum of a waveguide that had been in contact with the silicone cuvette. It is clear from the intense peak at m/e573 (SiC3H9), as well as from larger silicone-related fragments at m/e 543 (SiCH1 147 (Si2C5H15O1), 207 (Si3C5H15O1 3 ), 3 ), 1 221 (Si3C7H21O2 ), that there is severe surface contamination, most likely related to polydimethylsiloxane ~PDMS!. Hence silicone polymers in contact with the test liquids must be avoided. Since the cuvette material self-seals to the waveguide, only elastomers can be used to replace silicone. We found that Kalrez® ~Dupont!, a perfluoronated elastomer, was suitable. The new flowcell is made of two plates of Kalrez® ~see Fig. 7!. The lower one is a rectangular ring, which forms the walls of the flowcell, and the upper part, forming the top is a plate with two holes for the tubing. Placing the two elements of the flow cell onto the waveguide and applying a certain amount of pressure completely seals
FIG. 5. Mode spectrum with an ASI IOS-1 instrument, where a is the measured angle of incidence ~according to Fig. 1! and Eta is the light intensity at the photodiodes. ~a! From small-sized ~831230.5 mm! uncoated waveguide ~type ASI2400! in the new setup ~Sec. III A!. ~b! From a waveguide ~type ASI1400! coated with a 10 nm TiO2 layer ~Sec. III C!. Optical waveguide light mode spectroscopy
FIG. 7. Schematic drawing of the Kalrez cuvette, made of two Kalrez plates. The upper one contains the holes for the tubes, the lower one forms the flow channel.
FIG. 6. TOF-SIMS spectrum ~positive ions! of a type ASI1400 waveguide after contact with the silicone rubber cuvette for 4 h. Note the intense peak at m/e573 due to silicone surface contamination.
the flow cell. Further, the adhesion of the two Kalrez® plates to each other is much stronger than to the glass surface, so that the lower plate stays on the upper one after lifting the cell off the waveguide at the end of an experiment. Using the Kalrez cuvette solved the contamination problems. Even after a long time in contact with the fluoroelastomer, e.g., three days, the surface remained unchanged. Figure 8 shows a TOF-SIMS spectrum of a waveguide which has been in contact with the Kalrez® flow cell for one day. As can be seen, there is no contamination, except for hydrocarbons which are always found on the surface as natural contaminants. C. TiO2 coatings on waveguides
In order to be able to simulate the surfaces of titanium implants, the waveguide surface should be modified to achieve a surface composition as close to that of titanium implants as possible. TiO2 was deposited by PVD on conventional waveguides ~type ASI1400! as a thin ~few nanometers thick!, transparent coating, well suited for studies using the OWLS technique. However, it must be borne in mind
that TiO2 has a very high refractive index (;2.4), and even a thin adlayer significantly changes the effective refractive index of the waveguide. As the TiO2 layer thickness increases, the TM and TE peaks, shown in Fig. 5, quickly shift to higher angles. However, the IOS-1 instrument is limited mechanically to a 567°. Figure 5 also shows the peak positions for a 10-nmcoated waveguide and, as can be seen, this is about the maximum coating thickness for a standard waveguide and used with the IOS-1 instrument. Thicker coatings shift the TE0 peak out of the measuring range. Since an adlayer of adsorbed biomolecules is characterized by two optogeometrical parameters, n A and d A , two effective refractive indices ~i.e., of the TM0 and TE0 modes! must be measured. On the other hand, a minimum thickness of the TiO2 coating must be used to ensure that the coating completely covers the underlying material. Figure 9 shows the Si/Ti ratio measured by XPS at the surface for different coating thicknesses. As can be seen, 10 nm is an ideal value for the coating thickness. It is still within the constrains of the IOS-1 optics and, at the same time, the surface is completely covered by the TiO2 layer. The problem with the peaks shifting out of the measuring window can alternatively be solved by using waveguides with a thinner (SiO2•TiO2) waveguiding layer. The optogeometrical properties of the waveguide are linked by the mode
FIG. 8. TOF-SIMS spectrum ~positive ions! of an ASI1400 (SiO2•TiO2) waveguide after contact with the Kalrez cuvette for one day. There is no contamination except for the usual hydrocarbons. Rev. Sci. Instrum., Vol. 68, No. 5, May 1997
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FIG. 9. Atomic ratio Si/Ti at the surface vs TiO2 coating thickness, calculated from XPS data.
equations. For a four-layer system with support S, high refractive film F, additional adlayer A, and outer medium C, the following mode equation links the refractive indices n S , n F , n A , and n C to the thicknesses d F and d A :
p m5
2p l
3
F
An 2F 2N 2
H
S D S S D S FS D FS D N nC N nC
d F 1d A
2
N nA 2 N 1 nF 1
2arctan
nF nS
2arctan
nF nC
2r
D D
n 2A 2n 2C n 2F 2n 2C
GJ r
2
21 2
21
A A
N 2 2n 2S
n 2F 2N 2
2r
G G
N 2 2n 2C n 2F 2N 2
,
~1!
where r 50 and 1 are for the transverse electric and transverse magnetic modes,13 respectively, and m50,1,... is the mode number. Figure 10 shows the calculated optimum thickness d 1 of the waveguiding (SiO2•TiO2) layer for any given TiO2 layer thickness d 2 , where optimum thickness is defined as the thickness d 1 for which the additional TiO2 layer leads to the same incoupling angles as the uncoated standard waveguide. For the experiments, any data pair d 1 /d 2 from Fig. 10 can be used, but according to Fig. 9, a minimum thickness d 2 of 10 nm should be used. IV. DISCUSSION
A system for using small-sized waveguides in a commercial OWLS instrument has been devised. The necessary changes merely involve the replacement of a small part of the measuring head. This exchange is very simple and hence allows for the measurement of both the new small sized and also the conventional waveguides with the same instrument. The new system has the advantage that the waveguides can now be directly measured in surface-analytical instruments before and after the adsorption experiments. Second, a new flow-through cuvette based on a fluoropolymer (Kalrez® ) 2176
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FIG. 10. Calculated optimum thickness of the waveguiding layer d 1 as a function of TiO2 coating thicknesses d 2 on top of the waveguide layer between 0 and 40 nm. For the proposed 10 nm TiO2 coating, the optimum waveguide (SiO2•TiO2) thickness is 160 nm. For the coated waveguides, the waveguide thickness d F consist of the mixed SiO2•TiO2 layer with thickness d 1 and the pure TiO2 layer with thickness d 2 .
proved to be successful for contamination-free adsorption experiments, replacing conventional silicone polymers that always carry the risk of silicone surface contamination. Third, conventional waveguides have been coated by a PVD technique with thin ~10 nm! TiO2 layers, in order to simulate the surface ~oxide! of titanium implant material. The constraints that have to be observed regarding film thickness of the waveguiding layer and of the TiO2 top coat were calculated in a four-layer model and experimentally verified. The resulting improved instrument is believed to be very suitable for the measurement of biomolecule ~e.g., peptides, proteins, glycoproteins! adsorption of the interaction of preadsorbed biomolecules with the corresponding antibodies as well as of cell-surface interactions.
ACKNOWLEDGMENT
The authors would like to thank the Eidgeno¨ssische Stiftung zur Fo¨rderung Schweizerischer Volkswirtschaft durch Wissenschaftliche Forschung for financial support.
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Optical waveguide light mode spectroscopy
