Biosensors and Bioelectronics 25 (2010) 2633–2638
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Surface plasmon resonance ellipsometry based sensor for studying biomolecular interaction Rakesh Singh Moirangthem a,b,c , Yia-Chung Chang b,∗ , Shih-Hsin Hsu b , Pei-Kuen Wei b a b c
Department of Engineering and System Science, National Tsing Hua University, Hsinchu 300, Taiwan Research Centre for Applied Sciences, Academia Sinica, Taipei 115, Taiwan Nano Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan
a r t i c l e
i n f o
Article history: Received 6 March 2010 Received in revised form 22 April 2010 Accepted 22 April 2010 Available online 4 May 2010 Keywords: Spectroscopic ellipsometry Kretschmann configuration Dove prism Surface plasmon resonance Biosensor
a b s t r a c t A simple surface plasmon resonance (SPR) ellipsometry equipped with a dove prism and micro-fluidic flow cell is adopted to investigate and study basic properties of biomolecular interaction. Using a dove prism greatly simplifies the optical alignment and the use of micro-fluidic cell helps reduce significantly the volume of the biological sample required in the experiment. By recording the ellipsometry data in terms of relative changes in the ellipsometric parameters, and as sensor signals we can understand the biomolecular interaction. Spectroscopic measurements were performed to check the bulk sensitivity, which further helps determine the corresponding wavelength for maximum sensitivity. Furthermore, dynamic measurements at a fixed wavelength were also done and allow the observation of real-time response to the changes in surface properties on a metallic film. Such a simple technique gives an index resolution around 1.64 × 10−6 which is better than the conventional SPR method based on the resonance angular detection. This technique yields sensitivity sufficient enough to detect changes in the effective thickness of biomolecular layer. Biological processes such as adsorption of protein to metal and protein–protein interaction can be understood from the optical response of the sample surface. Such technique is a promising candidate in developing profitable and user-friendly biosensors. Furthermore, this kind of characterization technique is non-destructive, label free, and sensitive with a sub-nanometer resolution in thickness. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The analysis of biomolecular interaction is a key area of research in the healthcare, pharmaceutical and biotechnology fields. Various techniques have been employed to study the biomolecular interaction in the past few decades. These include radioimmunoassay (RIA), capillary electrophoresis, fluorescence, surface plasmon resonance, etc. (Benesch et al., 2000; He et al., 2004; Dostalek and Knoll, 2008; Homola, 2003). Many optical biosensors have very high sensitivity but problems arise by using biomolecular labels such as radioactive isotopes or fluorophores. These factors have motivated the research in developing label free optical detection techniques for biosensing. Most of label free optical biosensors are based on affinity-sensor detection of small changes in refractive index near the interface. Surface plasmon resonance (SPR) sensors can be classified according to the way light interacts with the
∗ Corresponding author at: Research Center for Applies Sciences, Academia Sinica, 128 Sec. 2, Academia Road, Nankang, Taipei 115, Taiwan. Tel.: +886 2 2652 5183; fax: +886 2 2652 5184. E-mail address:
[email protected] (Y.-C. Chang). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.04.037
surface plasmon such as angular, intensity, wavelength, phase, or polarization modulation. Reviews on SPR biosensor can be found in (Homola et al., 1999; Homola, 2003). Ellipsometry refers to a class of optical experiments which is self-referencing and it measures the polarization states (ellipsis of the polarization) after and before reflection from the sample. The simultaneous measurement of the ellipsometry parameters, and provides rich information about the sample under investigation, which allows quantitative analysis based on fitting theoretical predictions to experimental results. Numerous applications in a variety of fields such as semiconductor structure inspection, in situ monitoring of nucleation and growth, surface biology, measuring adsorption on metal surfaces in opaque liquids, biosensing have been reported (Aspnes, 1993; Basa et al., 1998; Arwin, 2000; Poksinski and Arwin, 2003; Nabok and Tsargorodskaya, 2008). When the SPR effect is combined with ellipsometry in total internal reflection mode, one obtains a technique with very high sensitivity (Arwin et al., 2004). In recent years, there are increasing numbers of reports on the SPR ellipsometry (Kretschmann configuration) for detecting biomolecules (Poksinski and Arwin, 2007; Hsu et al., 2009; Hooper et al., 2009). It has been reported that the protein adsorption on the semi-transparent gold film monitored by total internal reflection ellipsometry shows a
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large enhancement in sensitivity in comparison to the traditional ellipsometry (Poksinski and Arwin, 2004). Also, kinetic measurements of immobilized antibody layers and of DNA hybridization with a commercially available ellipsometer have been reported with a resolution limit of 10 pm/mm2 (Westphal and Bornmann, 2002). This resolution corresponds to the refractive index (RI) resolution of about 10−5 . Another study on the phase measurements based on the rotating analyzer ellipsometry achieved a RI resolution of 10−7 (Naraoka and Kajikawa, 2005), comparable to that of the heterodyne dependent detection (Nelson et al., 1996). Most of these studies mainly focused on improving the sensitivity. However, a detailed study on the optical properties of biomolecues (in solution) is lacking. In this report, we present systematic investigation on the biomolecular interaction and find out the effective thickness, effective refractive index and surface mass density of the protein adsorbed on the metal film for various interaction times. In addition, it has been shown that the use of a dove prism can circumvent complicated optical set-up or high precision alignment. A beam of light entering one of the slanted faces of the dove prism undergoes total internal reflection from the inside of the longest face and emerges from the opposite slanted face. It inverts the image of a collimated light beam impinging parallel to the long edge of the dove prism. The total internal reflection angle of propagation in a dove prism (BK7) is around 72.8◦ (with a weak wavelength dependence), which can activate the surface plasmon resonance when coated with a proper thickness of metal film (say silver or gold) on the longest face of the prism with excitation wavelength between 600 nm and 1000 nm depending on the refractive index of the surrounding aqueous medium (Bolduc et al., 2009). In this study, we use a similar technique, which can be performed in spectroscopic as well as dynamic (with single-wavelength) modes without changing set-up by using ellipsometry equipped with a dove prism (BK7) and a custom built micro-fluidic flow cell.
2. Experimental details 2.1. Materials Chemicals were obtained from commercial suppliers and used without further purification. Bovine serum albumin (BSA) and antiBSA were purchased from Sigma–Aldrich (USA). 10× phosphate buffer saline (PBS) solutions were obtained from UniRegion BioTech (Taiwan). Glycerol (99%) was purchased from Acros Organics, USA. Dove prism (BK7) was purchased from Edmund Optics (Singapore). Glass slide used in all the experiments were purchased from Gold Seal (USA). Ultra-pure water (Milli-Q Element, Millipore) was used for all experiments.
2.2. Apparatus The SPR ellipsometry set-up used in our experiment is based on the commercially available variable angle spectroscopic ellipsometry (VASE) from J.A. Woollam Company (USA) equipped with a dove prism and custom built micro-fluidic flow cell (see Fig. 1). The use of dove prism in this optical configuration and the readymade mounting of all the optical components such as source, polarizer, sample stage, compensator, and analyzer in the VASE instrument further simplifies the single-axis optical alignment and in finding the optimum angle for SPR excitation which provides a user-friendly environment. Though the present experimental setup is done with a commercial instrument, the idea can be used when developing a standalone optical instrument for sensing application.
Fig. 1. (a) Schematic diagram showing the polarization of incident and reflected light for a sample attached parallel to the VASE sample stage in the instrument frame and (b) illustration of polarization of incident and reflected light (within the prism) for a sample attached perpendicular to the sample stage. Note the exchange of s- and p-polarization from the instrument frame to the prism frame.
2.3. Sample preparation and measurements The glass substrates used in the experiment were cleaned by piranha solution (70% H2 SO4 :30% H2 O2 ) followed by rinsing with ultra-pure water. Thereafter, the glass substrates were further cleaned in ultrasound bath with acetone solution for 20 min and with isopropyl solution for another 20 min, and finally rinsed with water. The clean glass substrates was stored in water and dried in oven prior to metallization. Then, 3 nm thick adhesion layer (titanium) followed by 30 nm gold film were deposited on the glass substrate using e-gun evaporator (AST, Taiwan). The deposited thicknesses of the gold and titanium layers were calibrated by using VASE in the standard mode. The modeling fitting result gives thicknesses of the gold and titanium thin film around 28 nm and 2 nm, respectively, close to the anticipated values. To carry out the experiment in aqueous medium a micro-fluidic flow cell is prepared with a central chamber. The micro-fluidic flow cell consists of an acrylic plastic sandwiched between two microscopic glass slides with inlet and outlet valves. The bottom glass slide attached to the micro-fluidic flow cell is replaced by a freshly prepared gold deposited glass substrate and directly mounts in optical contact with the prism where the light is internally reflected from the gold film. It should be noted that the glass substrate used for the experiment has very close optical properties compared with the prism. To suppress the unnecessary prism–slide interference due to the air gap, an index matching liquid was used. Thereafter, the prepared solutions were injected into the micro-fluidic cell through syringe pump and optical measurements were carried out. The use of a micro-fluidic cell (with a total volume of approximately 32 l) significantly reduces the volume of the solutions required in the experiment. To determine the bulk sensitivity, various concentrations of glycerol–water mixture having different refractive indices were prepared. Subsequent measurements were done by changing the mixture solutions in the micro-fluidic cell, and the resulting optical signals were used to calculate the bulk sensitivity. In the biomolecular interaction study, BSA and anti-BSA were diluted in 1× PBS buffer (pH 7.4) with concentration of 50 M and 1 M, respectively. Firstly, PBS buffer solution was injected into
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the microchip and measurements were done to obtain optical response from the buffer. Then, 50 M BSA was injected onto the gold surface and due to the physisorption of BSA on gold surface, the BSA will form a coating on the gold thin film surface. We kept the BSA solution in the micro-fluidic cell for 1 h in order to obtain sufficient BSA coverage on the gold surface, followed by washing with PBS buffer to remove unbound proteins. Finally, 1 M anti-BSA was injected into the micro-fluidic cell and kept for 3–4 h to undergo the protein–protein interaction, followed by washing with PBS buffer to wash away the unbound anti-BSA. The investigation was done in both spectroscopic and dynamic (for single-wavelength) modes with our prism-assisted SPR ellipsometry. Furthermore, all theoretical calculations used to fit experimental results were done using VASE-equipped software (WVASE32).
(tan · ei )corrected =
= where,
t2s t2p t2s t2p
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(tan
t2s = 2k2n /(k1n + k2n ),
t2p = (2k2n /ε2 )/(k2n /ε2 + k1n /ε2 ),
Rs Rp
t1s t1p
−1 · ei )prism
t1s t1p
(2)
t1p = (2k1n /ε1 )/(k2n /ε2 + k1n /ε1 ), k1n =
ε1 − ε1 sin2 1
and
2
k2n = ε2 − ε1 sin 1 . 1 is the angle of incidence. ε1 and ε2 are the dielectric constants for the ambient (air) and prism media, respectively. We transform all our experimental results by using Eq. (2) in order to analyze it with the VASE software. 4. Results and discussion
3. Theoretical analysis The ellipsometry parameters are measured by applying a probe beam with a known polarization state onto a sample and then investigating the change in the polarization state of the reflected light beam. In the reflection mode, ellipsometry parameters , are defined by the ratio of the complex-valued reflection coefficients Rp and Rs for the polarization parallel (p) and perpendicular (s) to the plane of incidence, respectively (Azzam and Bashara, 1987; Fujiwara, 2007):
=
Rp Rp = tan · exp(i) or tan = and = ıp − ıs Rs Rs
(1)
where ıp and ıs are the phases of Rp and Rs . The amplitude ratio of is thus given by tan and is the phase difference between the reflection coefficients for the p and s polarizations. The detailed theory about the ellipsometry working under SPR condition can be found elsewhere (Arwin et al., 2008). For VASE in the prism-assisted SPR ellipsometry configuration, the plane of incidence needs to be redefined. In normal ellipsometry measurements working on reflection mode (called instrument frame), the sample plane is parallel to the vertical sample stage frame as shown in Fig. 1(a). If we treat the plane of the page as the sample stage frame of VASE, then the plane of incidence is out of the page. Hence, the plane of incidence is perpendicular to the page while the s-polarized wave (Es ) is in the page. But when measurement is done in internal reflection mode using a dove prism (called prism frame), the sample plane is actually perpendicular to the sample stage frame (see Fig. 1(b)). Thus, the plane of incidence and the p-polarized electrical field (Ep ) in the prism frame are in the page, and the s-polarized electrical field (Es ) is perpendicular to the page. Hence, it is necessary to interchange the s- and p-polarized reflection coefficients defined in the instrument frame and the prism frame when we interpret the VASE data. There is also imperfect transmission taking place at the interfaces between air and the prism at an oblique angle, which needs to be considered, as shown in red circles in Fig. 1(b) (For interpretation of the references to color in text, the reader is referred to the web version of the article). Again the difference between measurements and model calculations has to be corrected by taking these changes due to transmission (twice) into account because the VASE-equipped software (WVASE32) can only handle the reflection from a multilayered structure, which in this case is prism/gold/medium. Including the polarization interchange and oblique transmission factors we obtain:
The SPR ellipsometry equipped with dove prism and custom built micro-fluidic flow cell was used to determine the refractive index resolution and further used for investigating biomolecular interaction spectroscopically and dynamically. Under the resonance condition the surface plasmon wave generated on the gold surface is very sensitive to the change in the refractive index of the surrounding medium; hence it can monitor the changes of the surface properties. This forms the basis of the principle behind the SPR and SPR ellipsometry. Spectroscopic measurements show that and spectra are red-shifted when the refractive index of the surrounding aqueous medium or the thickness of the biomolecular layer adsorbed on the metal surface increases. There is an advantage for spectroscopic measurement over single-wavelength measurement in terms of sensitivity when the change in thickness is very small. As mentioned in a previous report, for measurements at fixed wavelength to monitor small thickness changes on the gold surface, the proper choice of the wavelength is important (Arwin et al., 2004). However, the spectroscopic measurements allow us to determine the highest resolution wavelength. Therefore, it will be advantageous to have an instrument which can perform both spectroscopic and dynamic measurements (in singlewavelength mode). Our SPR ellipsometry set-up based on VASE provides such flexibility and helps overcome the above issue. The highest resolution wavelength is found via the spectroscopic measurement and subsequent dynamic measurement is done at that wavelength. Fig. 2 shows the ellipsometry spectra for (a) and (b) of a 30 nm gold thin film in surrounding aqueous medium for the glycerol–water mixture with various refractive indices. We use a three-layer model with titanium as the adhesive layer between gold layer and prism (treated as the ambient) with different refractive index medium of glycerol–water mixture near the gold surface to fit the experimental data. The refractive index of the glycerol–water mixture is modeled using the Cauchy dispersion with the following relation: n() = An +
Bn 2
(3)
where An and Bn are two constants. The calculated results (solid curves) can fit the corrected experimental data very well as seen in Fig. 2(a and b) for most wavelengths, and the fitting parameters An , Bn and mean square error (MSE) are given in Supplementary Information (Table S1). Here we have demonstrated that this optical set-up can be used to determine the dielectric constant of aqueous medium conveniently. In this work, we are mainly focusing on how the surface plasmon resonance responds to the change in the surrounding media with varying refractive index. The optical spectra for ellipsometry parameters and resemble typical SPR results with the reflection amplitude reaching a local minimum near the resonance wavelength (as marked by an arrow),
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Fig. 2. Spectral response of the ellipsometry parameters for (a) , and (b) for the glycerol–water mixture with varying refractive index. The model fitted results (solid curves) are also shown for comparison. (c) Wavelength shift (ı) of the SPR dips in () versus the change in refractive index (ın) of the surrounding aqueous medium on the sample surface. (d) Dynamic response of and measured at fixed wavelength (633 nm) with varying refractive index of glycerol–water mixture.
while the spectra in Fig. 2(b) show a quick rise near the resonance. It is also seen in Fig. 2(a) that the SPR dip shifts to longer wavelength when the refractive index of the surrounding aqueous medium increases. Fig. 2(c) shows the wavelength shift (ı)dip of the SPR dip in spectra versus the change in refractive index ın of the surrounding aqueous medium. Inverse of this slope in Fig. 2(c) gives the refractive index (RI) detection sensitivity which can be written as ın/(ı)dip . The slope obtained from a linear fitting of experimental data is 6084 nm/RIU. The RI resolution can be defined as RI = (ın/(ı)dip ) , where is the wavelength resolution of the instrument (Nelson et al., 1996; Xinglong et al., 2003). With a wavelength resolution of 0.01 nm we obtain a RI resolution of 1.64 × 10−6 for the () spectra. A comparison of the refractive index resolution of the various techniques is shown in Table 1. However, experimental results for () spectra indicate a fairly broad resonance which may be due to roughness at all surfaces. Care must be exercised when interpreting the behavior of the () spectra because there is a sudden phase shift in the value of whenever it crosses the boundary defined within the VASE
software, which is completely different from the resonance behavior. This feature can lead to misinterpretation of the experimental results. We also observed a sudden drop or phase shift in the () spectra due to the value of crossing the border defined as −180 ≤ ≤ 180 or 0 ≤ ≤ 360 (to simplify the plotting). If we add 360◦ to all the values beyond the crossing point, then we can obtain smooth curves for as shown in Fig. 2(b). Since the () spectra in Fig. 2(b) have a broad resonance feature, it is difficult to judge the wavelength shift of the surface plasmon resonance based on (). Therefore, the () spectra are more useful for monitoring the spectral shift in terms of chemical or biological reaction. It also helps us to determine the wavelength where the optical response is optimal. Fig. 2(d) shows the dynamic measurement at a fixed wavelength (633 nm) for detecting glycerol–water mixture with various refractive indices. The refractive index values for different glycerol–water mixture were measured using a refractometer. The standard deviation of and measurement at this wavelength within certain time interval was approximately 0.005 and 0.09, respectively. Here, we choose a wavelength near the optimum
Table 1 Comparison of the refractive index resolution obtained from various SPR experimental techniques. Methods
Refractive index resolution
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1.6 × 10−6 RIU 1.3 × 10−7 RIU 3 × 10−6 RIU 3 × 10−5 RIU ∼10−5 RIU
Our system Hooper et al. (2009) SPR-Navi (BioNavis Ltd., Finland) Xinglong et al. (2008) Westphal and Bornmann (2002)
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Fig. 4. The surface mass density of BSA adsorbed on the gold thin film after 1 h, 3 h, 6 h and 12 h on incubation in 50 M BSA solution. Data are plotted for singlewavelength at 580 nm versus incubation time. Error bars were determined by doing three independent measurements.
Fig. 3. Spectral response of the ellipsometry parameters for (a) and (b) for various configurations with respect to the addition of BSA and anti-BSA. The model fitted results (solid curves) are also shown for comparison.
response. However, one can choose other wavelengths depending upon their specific application. It also shows that the changed of is much more significant than in the resonance region, which is consistent with the previously reported results (Cho et al., 2005). Thus, spectroscopic measurement of () helps us to determine the optimum wavelength for sensing of biomolecules or other sensing applications and through a proper theoretical analysis we can also determine the effective thickness of the biomolecules, while () provides better sensitivity. Next, we perform label free bio detection of protein–protein interaction by measuring the interaction between BSA and anti-BSA. First, the PBS buffer solution was injected into the microchip and measurements were done to obtain optical response from the buffer. Next, the BSA molecules were immobilized on the metal surface followed by washing with buffer solution to remove unbound proteins. Subsequently antiBSA molecules were injected into the chip to undergo biomolecular interaction. Spectroscopic measurements were done at various configurations with respect to the addition of BSA and anti-BSA and we analyze the spectral shift due to different surface properties on the gold thin film surface using a four-layer model with titanium as the adhesive layer and treating the biomolecule layer (such as BSA or BSA + anti-BSA) as a layer following the above Eq. (3) (see inset figure in Fig. 3(a)). In this model, we fix the value of Bn and only fit the An and thickness. Fig. 3 shows the ellipsometry spectra for (a) , and (b) of a 30 nm gold thin film in surrounding aqueous medium with the addition of BSA and then anti-BSA. The model fitting results for various configurations are also shown as solid curves. The fit for is almost perfect, so the experimental and theoretical curves overlap completely. Note that the addition of anti-BSA leads to
much more significant changes in and than the addition of BSA alone. This is because the molecular weight of anti-BSA (150 kDa) is larger than that of BSA (66 kDa) and the spectra are red-shifted as the thickness/refractive index of the molecular layer on the surface of the gold film increases, which shows very similar response to the conventional spectroscopic SPR biosensor. The effective thickness of BSA obtained from model fitting is around 6.9 nm and that of the BSA/anti-BSA combined layer is 22.2 nm, which is close to the value obtained from AFM measurement as shown in Supplementary Information (Fig. S2). However, the thickness of BSA molecular layer may depend on the preferred molecular orientation after its adsorption on the sensing surface. For example, two different adsorption areas were suggested for the “side on” mode (9 nm × 5.5 nm) and “end on” mode (5.5 nm × 5.5 nm) by Rezwan et al. (2004). The effective refractive indices of BSA and the combined layer (marked as anti-BSA) are shown as solid and dashed lines in Fig. S1 of Supplementary Information, and the corresponding fitting parameters and thickness obtained are given in Table S1 of Supplementary Information. To determine the surface mass density (ng/mm2 ) of BSA adsorbed on the gold thin film, we separately performed spectroscopic measurements after 1 h, 3 h, 6 h, 12 h of incubation in 50 M BSA solution. To determine the error bars, the experiment was repeated three times under similar experimental condition. The calculation was done using the de Feijter formula (de Feijter et al., 1978). Fig. 4 shows the amount of BSA adsorbed on gold surface (surface mass density) as a function of time for a wavelength fixed at 580 nm. As it can be seen in Fig. 4, the adsorbed amount of BSA protein on gold surface after incubating for 1 h is about 0.60 ng/mm2 which is close to the result from neutron reflectivity data (Su et al., 1998). The minor difference in the obtained value could be due to the different adsorbing surface. The spectroscopic measurements on PBS buffer and adsorbed BSA on gold thin film after 1 h of incubation give a wavelength shift of 5 nm in the SPR dip. Thus, a wavelength resolution of 0.01 nm giving a RI resolution of 1.64 × 10−6 will correspond to the surface mass density of 1.2 pg/mm2 of BSA protein adsorbed on the sensor surface, which is close to the detection limit 3 pg/mm2 of commercial SPR instrument such as the SPR-Navi (BioNavis Ltd., Finland). Since most of the biomolecular interactions are dynamic processes, it is important to know the real-time response of the biological process. Therefore, we extend our study to measure the dynamic response of BSA and anti-BSA interaction.
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length shift of SPR dip in spectra gives an RI resolution of 1.64 × 10−6 , while dynamic measurement done at fixed wavelength shows that the phase () is much more sensitive than the amplitude ( ). Through studies of BSA and anti-BSA molecular interactions, we have demonstrated both spectroscopic and dynamic measurement capabilities to sense high-affinity bimolecular interactions by using SPR ellipsometry without any labeling. Through a theoretical modeling, we are able to determine the effective thickness and effective refractive index of the biomolecules absorbed on the gold thin film at various configurations with respect to the addition of BSA and anti-BSA. Additionally, we are able to correlate RI resolution to the surface concentration of the protein adsorbed on the sensor surface. Furthermore, real-time dynamic responses of biological processes such as adsorption of protein and protein–protein interaction have been presented and information such as adsorption time and interaction time can be clearly understood from the optical response of the sample surface. Hence, such a technique is promising for developing user-friendly, high-affinity bio detection at low cost. Acknowledgements The authors would like to thank Dr. Chau-Hwang Lee and Shu Han Wu for their helpful discussion. This work was supported by Academia Sinica, Taiwan and by the National Science Council of the Republic of China under Contract No. NSC 98-2112-M-001-022MY3. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2010.04.037. Fig. 5. Dynamic response of the ellipsometry parameters (a) and (b) as a function of interaction time for BSA anti-BSA interaction at fixed wavelength.
Fig. 5 shows the real-time response of our measurement at various interaction times for a fixed wavelength (580 nm). It is confirmed from the graph that the signal for is more significant than that of , which is consistent with our result on bulk sensitivity measurement at fixed wavelength. In other words, for single-wavelength detection the phase response is much more sensitive than the amplitude response. From the dynamical study, it is seen clearly that the adsorption of BSA molecules on the gold surface occurs and further interaction between BSA and anti-BSA molecules exits. The binding of the anti-BSA molecules with BSA shows more pronounced optical response due to its larger molecular weight as compared to BSA molecules. However, the decrease in the optical signal after washing with PBS buffer is due to the removal of the non-specific or unbound protein from the surface which reduces the thickness of the biomolecular layer. Thus, the dynamical study shows a clear understanding of the biomolecular interaction evolving with time, which helps the understanding of the complex biological processes. 5. Conclusion A dove prism-assisted SPR ellipsometry for investigating the bimolecular interactions in solution is presented. A single-axis alignment of optical components simplifies the complicated and high precision optical alignment procedure in developing an optical tool for biosensing. Spectral sensitivity obtained from the wave-
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