APPLICATION OF A BIOSENSOR BASED ON SPR USING SPECTROSCOPIC ELLIPSOMETER

. The work is focused on the possibility to use spectroscopic ellipsometer in biosensor application. Operation of the ellipsometer is enhanced by the feature of TIRE (Total Internal Reflection Ellipsometry). The principal goal is to detect analytes in aqueous solutions at low concentrations below 0.1 nmol/l.


Introduction
Surface Plasmon Resonance (SPR) is an effect occurring at the metal/dielectric interface. This phenomenon allows toobserve nano-changes in thickness, density fluctuation or molecular adsorption on a metal surface [4]. Surface Plasmon Resonance (SPR) sensors are widely used in biochemistry and pharmacology due to their high sensitivity. They are based on the angular measurement of reflectance of p-polarized light reflected from a metal-dielectric surface.
For a specific wavelength of light and an angle of incidence when the condition of surface plasmon generation is fulfiled, the light energy leaks to the surface plasmon polariton and a rapid decrease in R p is observed. The resonance condition is highly sensitive to material properties at the metal-dielectrics interface. If any change occurs near the metal surface, e.g. organic molecules are adsorbed onto the metal, the minima in reflectance are shifted to different angles of incidence or wavelengths [5].
Total Internal Reflection Ellipsometry (TIRE) combines two techniques, ellipsometry and SPR, in order to reach better sensitivity. By TIRE, higher sensitivity can be achieved allowing monitoring small molecules binding to the metal substrate. The method can be used to detect affinity interactions on the surface in optical biosensors.
The presented work shows possibility of using ellipsometer Horiba Jobin-Yvon MM-16 enhanced by TIRE as a biosensor based on SPR [3]. Spectroscopic ellipsometer is a device set to investigate properties of thin layers on a solid substrate. The measurement time of spectroscopic ellipsometer is sufficient enough for monitoring slow surface chemical processes. Moreover, SPR enhancement improves sensitivity of the ellipsometer below 1 nm in layer thickness.

Ellipsometry and TIRE
Ellipsometry is a nondestrustive optical method used in a wide range of thin layers applications. The basic principle is observation of polarization state changes in the reflected light. After the reflection of light from a layered surface, both the amplitude and the phase of p and s polarization components are changed. Analyzing the data measured, the ellipsometric parameters Δ (phase difference) and ψ (ratio of Fresnel's reflection amplitudes) are extracted using the ellipsometric equation [2]   i s p e r r  tan / (1) where r p , r s are complex Fresnel's reflection amplitudes [3].
When specific conditions (angle of incidence AOI, wavelength) for SPR generation are fulfilled, the energy of p component of incident light is transformed into the surface plasmon polariton. This resonant process results in light intensity extinction, which could be observed as a considerable minimum in reflectance R p spectra ( Fig. 2 left). TIRE can be applied only when the light passes through optical boundary with the lower index of refraction on the other side and under the total internal reflection condition. If the incident angle is greater than the critical (cut off) angle, the beam does not penetrate into the second medium and the evanescent wave occurs.

Results and discussion
Kretschmann's configuration was used in the TIRE experiment. Light beam emitted from the ellipsometer output head passes the optical right-angle BK-7 prism and impinges under the total reflection condition on the glass plate covered with 50 nm gold layer.
The glass plate was optically coupled to the prism via immersion oil. The plate with the gold layer was attached to the Teflon trough of 3 cm 3 (Fig. 1). The spectroscopic polarimeter Horiba Jobin MM-16 with a spectral range of 430  850 nm and 2 nm resolution was used. The device is equipped with a motorized goniometer providing the external angle of incidence between 45° and 90° with 0.01° step [1].
Taking into account 2 nm spectral resolution of MM-16 and considering SPR generation as a resonant process, Δ parameter measurement in the vicinity of the SPR point is more sensitive than R p . Comparing the standard deviation   = 0.62° of Δ as given by a long-term measurement and the temperature gradient of water near the room temperature ∂n/∂T = -8.0×10 -5 K -1 , the standard deviation of the refractive index of water  n = 3.3×10 -6 was obtained.
This number indicates that variations of material parameters can be determined at the level of order of magnitude 10 −5 using this method. The gradient of rapidly falling Δ in the vicinity of the resonance wavelength (λ R = 682 nm) was -1.52° per the limiting AOI step (Fig. 2) [2].

. Shift by the change of different angle of incident
We carried out several measurements to prove how the SPR effect depends on the angle of incidence (Fig. 3). The varying conditions also have an influence on the dependence of Δ on wavelength, as it is obvious from Fig. 4. The slopes of the linear parts are as follows -115, -59.7, -25.9, -15.2, and -8.5 in unit's°/nm (Fig. 4).   Some thermal measurements are shown in Fig 7a and 7b. The water was cooled from temperature 35° to room temperature (19°). The temperature of water was measurement by a digital thermometer. The angle of incidence was set to 83° and Δ was measured at 677 nm. Fig. 7a shows a comparison of the measurement and the theoretical model. The reason of the difference between the measurement and the calculation at higher temperature is caused by the fact that the theoretical model includes only temperature and refractive index of water. In the real experiment the increasing temperature is relevant also for the glass substrate and the prism. In our theoretical calculations we were able to estimate changes in R P and Δ attributed to subtle variations in thickness of the organic layer. In Fig. 8 a simulation of increasing thickness of the layer is presented and its effect on the wavelength. The SPR resonant wavelengths are 717.97 nm and 718 nm, for thickness of 2 nm and 2.01 nm, respectively. It means that the increase in thickness of the organic layer with the index of refraction of 1.45 between 2.00 nm and 2.01 nm caused a SPR resonant wavelength shift by 0.03 nm, which is beyond the MM-16 spectral resolution and this effect, cannot be detected at all. On the other hand, as to the parameter Δ, it changes by 0.59° and this shift is measurable. The change of the Δ parameter with increasing thickness of the layer on the metal surface with step 0.01 nm is visible from Fig. 9. The working point was chosen at 718 nm. As biosensor application a monolayer of an aptamer molecule for human thrombin deposited on 50 nm gold layer was used. The exponentially shaped saturation curve of the thrombin adsorption on the aptamer receptor is shown in Fig. 10. The detection limit of thrombin concentration in the buffer solution was less than 0.3 nmol/l.

Remarks and conclusion
The possibility of using spectroscopic ellipsometer Horiba Jobin-Yvon MM-16 enhanced by TIRE as an SPR biosensor was shown. In the vicinity of the SPR resonance λ R the rapid linear change of Δ was exploited to detect the limiting index of refraction of water change σ n = 3.3×10 -6 and to reveal the exponentially saturated adsorption process of 0.3 nmol/l concentration of the human thrombin on aptamer biosensor. TIRE approach serves as a sufficient step to achieve considerably enhanced sensitivity of the device in monitoring biomolecules adsorption processes.