Imaging ellipsometry combined with surface plasmon resonance for real-time biospecific interaction analysis
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1 Imaging ellipsometry combined with surface plasmon resonance for real-time biospecific interaction analysis Won Chegal *a, Hyun Mo Cho a, Yong Jai Cho a, Young Pil Kim b, Hak Sung Kim b a Division of Optical Metrology, Korea Research Institute of Standards and Science, Daejeon , Republic of Korea b Department of Biological Science, Korea Advanced Institute of Science and Technology, Daejeon , Republic of Korea *wchegal@kriss.re.kr ABSTRACT We present imaging ellipsometry technique for kinetic measurement of bimolecular interactions with high sensitivity. When combined with surface plasmon resonance (SPR) effects, the ellipsometry becomes powerful technique for analyzing adsorption and desorption of biomolecules on gold layer based sensor chip surfaces. Because ellipsometric measurement gives ellipsometric parameters, namely, that is very sensitive to surface layer changes. The SPR combined ellipsometry is realized by Kretschmann configuration SPR cell comprising with about 30-nm-thick gold film deposited on top of glass slides, SF10 glass prism, and flow injection system. We used nulling type of imagining ellipsometer to acquire two dimensional ellipsometric parameters with spatial resolution down to one micrometer. We present results of kinetic measurements of biotin-streptavidin interactions for custom-built sensor chip. Keywords: imaging ellipsometry, surface plasmon resonance, kinetic measurements, biospecific interaction 1. INTRODUCTION Optical methods for measuring thickness of thin films are widely used in many applications fields. Ellipsometry is known as very highly sensitive techniques for characterization and measurement of optical properties of both surfaces and absorbing thin films. This technique is based on measurement of change for polarization state of light fields. The ellipsometry measures two parameters, ψ and, defined as the ratio of relative amplitude and phase difference for p- and s-polarized light before and after reflecting on sample surfaces 1-2. The optical and geometrical properties of thin film layers are analyzed using model fitting procedure with these measured ellipsometric parameters. Among two ellipsometric parameters, is extremely sensitive to surface properties, one can investigate distinct changes in parameter of, before growing up to mono-layer coverage. That is, the dynamic range of this metrology is ranging form sub angstroms to several hundreds nanometers. The surface plasmon is collective oscillation of surface charge density of free electrons at a metal surface. Because dispersion relation of the surface plasmon is different from that of the light in the ambient air, it is necessary some coupling mechanism to match the dispersion relation to excite the surface plasmon. The most widely used configuration is Kretschmann s attenuated total reflection coupling methods 3, which is comprised with high refractive index dielectric prism coated with thin metallic layer of approximately 30 ~ 50 nm thickness. The optimal thickness of the metallic layer is varied with refractive index of dielectric prism 4. Only p-polarized light is able to excite surface plasmon mode. S- polarized light does not excite surface plasmon mode. In the case of measuring reflectance of p-polarized light with the attenuated total reflection coupling scheme, one can get sharp absorption dip around surface plasmon resonance (SPR) condition. And this dip can be used to detect surface sensitive changes in vicinity of metallic layer surfaces. Since dispersion relation of surface plasmon is very sensitive to the change of bottom side of metallic layer, surface plasmon resonance sensor can be used for diagnostic tools 5 for antibody-antigen, biocompatible binding kinetics, gas adsorption, and etc. Recently, several modulation techniques have been introduced to analysis for excitation of surface plasmon resonance by using reflected light beam. These techniques include angular modulation 6, wavelength modulation 7, and polarization modulation 8. The polarization modulation technique combines the ellipsometry and surface plasmon resonance Nanosensing: Materials and Devices II, edited by M. Saif Islam, Achyut K. Dutta, Proc. of SPIE Vol. 6008, 60081G, (2005) X/05/$15 doi: / Proc. of SPIE Vol G-1
2 measurement techniques. And some other researchers called as surface plasmon resonance enhanced ellipsometry 8 or total internal reflection ellipsometry 9. All of above mentioned techniques are theoretically identical in the aspect of ellipsometric parameters are measured with attenuated total reflection coupling of surface plasmon waves. Since ellipsometric measurements offers both of intensity and phase information of reflection coefficients, the measurement resolution can be greatly improved compared with conventional SPR reflectance measurement techniques. In this paper, we present imaging ellipsometry technique for kinetic measurement of bimolecular interactions with high sensitivity. The SPR combined ellipsometry is realized by Kretschmann s configuration SPR cell comprising with about 30-nm-thick gold film deposited on top of glass slides, SF10 glass prism, and flow injection system. We used nulling type of imagining ellipsometer (EP3, Nanofilm, German) to acquire ellipsometric parameter. We present results of kinetic measurements of biotin-streptavidin interactions with custom-built sensor chip. 2. THEORY AND EXPERIMENTAL SETUP The quantities measured by an ellipsometer are ψ and, and that is defined as complex Fresnel reflection coefficients of p- and s-polarized light 1. The amplitude ratio of reflection coefficients represents ψ. is the phase difference between p- and s-polarized light. The total reflection coefficients are evaluated with n-phase thin film modeling. In the simplest case for two semi infinite medium two phase model, the total reflection coefficients are substituted by Fresnel reflection coefficients with R p = r p and R s = r s. R p ρ = = tanψ exp( i ) (1) R s There are various types ellipsometer for measuring two ellipsometric parameters. Those include rotating components types, phase modulating types, and classical nulling types. In this study an ellipsometer which is based on classical nulling scheme is used. This ellipsometer is commercial equipment, EP3 (Nanofilm, German), and the optical structure is shown in figure 1. In this system the 532 nm laser is used for light source. A polarizer and a compensator are arranged in light source part of the ellipsometer. A microscopic objective lens and an analyzer are arranged in detector arm with CCD camera as a detector. When linearly polarized light different from p- or s-polarized light is incident on the sample, the reflected light will be elliptically polarized light in general. The other way, if the same elliptically polarized light incident on the surface will generate a linearly polarized light after reflection. So, using adequate azimuth angle combination for polarizer and compensator one can always find an elliptically polarized light that produces an exactly linearly polarized light after reflection. So in the nulling type ellipsometer, iterative rotation for polarizer, compensator, and analyzer are scanned to find exact nulling of measurement light incident on detector surface. Consequently the azimuth angles of polarizer, compensator, and analyzer can be used to calculate ellipsometric parameters. Sa.. laser COD camera polarizer compensator analyzev 0 bjedive thin film Figure 1 Schematic diagram for nulling type imaging ellipsometer (EP3, Nanofilm, German) Figure 2 shows the setup used for experiments. The attenuated total reflection coupler is composed with equilateral prism made by SF10 glass and its bottom side is covered with thin gold film of 30 nm thickness. The reaction chamber is made Proc. of SPIE Vol G-2
3 by silicon rubber seals and its dimension is 500 µm (width) 15 mm (length) 200 µm (height). Liquid inlent and outlet ports are connected with standard 1/16" peek tubing and fittings (Upchurch). A syringe pump (PHD22/2000, Harvard) and two positional injection valve (MX9900, Rheodyne) are used for liquid delivery. During fluid injection and bio molecules growing ellipsometric parameters are measured with every 15 seconds intervals. H. syriiige Piflul) laser polarizer coin!) ens ator ATIt coupler detector waste injection valve buffer reservoir Figure 2 Experimental setup with attenuated total reflection coupler for ellipsometric measurements 3. RESULTS Some basic experiments are conducted before starting bio molecules reaction experiments. Solution mixture of distilled water and ethanol is injected in reaction chamber and the real-time ellipsometric parameters are measured under various flow rates. Figure 3 shows the measured ellipsometric parameters with 100 µl/min and 50 µl/min flow rates. The noisy line is for ψ and clear line is for. 0) C) Time (mmn) Figure 3 Ellipsometric parameters plot for injecting distilled water and its mixture with ethanol. At startup the plot rate is 100 µl/min and at about 80 min the flow rate is downed to 50 µl/min. Proc. of SPIE Vol G-3
4 The ellipsometric parameter of is about phase information for reflection coefficients, and then these results show that phase measurement has great advantages compared with conventional SPR-reflectance measurement. The position around = is when the distilled water is flowing and two major dips approaching = are sensor output in the case of mixture solution is flowing. The different dip width is due to varied flow rate as mentioned above. Thereafter the flow rate is fixed to 50 µl/min. e V ci 45-1 Figure 4 Ellipsometric parameters plot for flowing mixture solution of distilled water ethanol. Four different solutions in refractive index are used, and numbers printed on upper part of the plot represent specific zone. The detailed data of each zone are summarized in table 1. To evaluate sensitivity of ellipsometric measurements with attenuated total reflection coupler, various concentrations of distilled water and ethanol solution is injected into SPR cell. Each time before injecting solution the refractive index is measured by refractometer (DR-A1, Atago). In this experiment difference of refractive index of injected solution with that of distilled water is ~ and corresponding ellipsometric parameter difference is 1.85 ~ The sensitivity is defined as how much refractive index changes are occurred in the unit degree change in ellipsometric parameter,. The sensitivity values are varied in the range from ~ , these non-uniform sensitivity values are due to nonlinear response of and refractive index measurement error in using refractometer. Table 1 Average values of ellipsometer parameters correspond to each zone in figure 1. Refractive index (RI) difference is evaluated with those of mixture solution and distilled water. Ellipsometric parameter (deg) Zone δ( ) RI difference ( 10-3 ) Sensitivity ( 10-3 RI/deg) Proc. of SPIE Vol G-4
5 Since in stationary measurement of ellipsometric parameter of, the standard deviation value is calculated less than 0.01, so one can evaluate the measurement resolution about Finally ellipsometric parameters measurement is conducted while binding biotin and streptavidin. Figure 5 shows measured plot during experiments. Three different concentration of streptavidin solutions are injected fluidics channel successively. The sensorchip used in SPR experiments are prepared as follows. First, 30 nm gold film is deposited on top surface of glass slides (25 mm 25 mm 1 mm, SF10 glass). MUAM (11-Amino-1-undecanethiol, Dojindo) monolayer is self-assembled onto the gold surface. Then MUAM-coated surface is biotinlayted with Sulfo-NHS-LC-biotin (EZ- Link Sulfo-NHS-LC-Biotin, Pierce). And index matching oil is used for mating the prism and biotinlayted glass slide. PBST solution is used for running buffer throughout experiments. Three different concentration of streptavidin (10 µg/ml, 20 µg/ml, 50 µg/ml) solutions are injected to form biotin-streptavidin binding. Figure 5 shows the results of biotin-streptavidin binding experiments. The distinct response of ellipsometric parameter of is shown at each injection of streptavidin solutions buffer < 260 buffer 255 SA gmL SA 20.uL buffer SA buffer I 120 Time (mm) 150 Figure 5 Ellipsometric parameters plot for biotin-streptavidin binding experiments. Three stage of measurement is conducted with streptavidin concentration as 10 µg/ml, 20 µg/ml and 50 µg/ml. 4. SUMMARY AND CONCLUSIONS Imaging ellipsometry technique for kinetic measurement of bimolecular interactions has been presented. When the ellipsometry combined with surface plasmon resonance (SPR) effects, the ellipsometry becomes powerful technique for analyzing adsorption and desorption of biomolecules on gold layer based sensor chip surfaces. We achieved detection limit in the refractive index in the order of This detection limit can be improved by signal averaging technique and sensor chip optimization by gold thin film thickness tuning. It is showed kinetic measurement of ellipsometric parameters of during biotin and streptavidin. At present only single fluidic channel system is used in experiments. And in the near future the microfluidics system with four or more reaction channel will be fabricated to get various concentration experiments simultaneously. ACKNOWLEDGEMENT The authors acknowledge the support of this research by the NT-BT Measurement and Manipulation Project. REFERENCES 1. R. M. A. Azzam and N. M. Basara, Ellipsometry and polarized light, Elsevier, Amsterdam, Proc. of SPIE Vol G-5
6 2. H. G. Tompkins, Spectroscopic ellipsometry and reflectometry: a user s guide, Willey, New York, E. Kretschmann, Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflächenplasmaschwingugnen, Z. Phys., 241, , Z. Salamon, H. A. Macleod and G. Tollin, Surface plasmon resonance spectroscopy as a tool for investigating the biochemical and biophysical properties of membrane proteins systems. I: Theoretical principles, Biochimica et Biophysica Acta, 1331, , J. Davies, Surface analytical techniques for probing biomaterial processes, CRC, K. Matsubara, S. Kawata and S. Minami, Optical chemical sensor based on surface plasmon measurement, Applied Optics, 27, , K. S. Johnston, S. R. Karlson, C. Jung and S. S. Yee, New analytical technique for characterization of thin films using surface plasmon resonance, Mater. Chem. Phys., 42, , P. Westphal and A. Bornmann, Biomolecular detection by surface plasmon enhanced ellipsometry, Sensors and Actuators B, 84, , H. Arwin, M. Poksinski and Knut Johansen, Total internal reflection ellipsometry: principles and applications, Applied optics, 43, , Proc. of SPIE Vol G-6
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