|Figure 1: A sketch of the biosensor "EVA" based on angle interrogation of a PC SW. The typical reflection profile is shown near the CMOS matrix. Note that this setup also may be used as a critical-angle refractometer, if only q TIR angle is measured.|
Photonic Crystal Surface Wave (PC SW)-based biosensor "EVA" is label-free biosensor with an independent registration of the critical angle of total internal reflection (TIR) from the liquid.
In Fig. 1 a sketch of the biosensor and typical signals from the photodiode array are shown. A laser beam from fiber-coupled diode laser is sent to the sensor surface through a polarization-maintaining fiber cable (to improve the quality of a beam profile). The beam is focused by a cylindrical lens so that the excitation angle of one s -polarized PC SW (existing in this 1D PC) structure and TIR angle (in p -polarization) are contained in the convergence angle of the beam.
After reflection from the sensor surface, the reflection profile contains information about the TIR angle (transferred by the p -polarized part of the beam) and about the angle of the PC SW excitation (transferred by the s -polarized part of the beam). Moreover, the sharpness of the reflection near the critical angle, and the measurement precision of the liquid RI herein are much higher than those used in standard critical-angle refractometers on uncoated prisms that enhance the RI sensitivity of the biosensor [1,2].
Such types of PC SW sensors also possess one-dimensional spatial selectivity in a direction perpendicular to the plane of the Figure 1 (i.e., along the focus line of the cylindrical lens). This fact permits recording of several reactions with an analyte simultaneously if different ligands are deposited on the PC in several linear target bands. In this way several tests can be performed at once that increases the throughput of the sensor.
The flow cells are made from a glass slide with two or four holes through which glass tubes are fitted to serve as inlet and outlet, respectively. The inlet tube is connected to a small tank filled with the solution under investigation. Flow velocity is controlled by a peristaltic pump "Ismatec Reglo Digital". A typical depth is from 35 mm to 50 mm, and corresponding flow cell volume is from 3.5 mL to 5 mL. The dead volume of the flow cell system was approximately 25 mL.
|Figure 2: A flow cell of the biosensor with two inlet tubes and two outlet tubes. Such flow cell design permits movement of the border between two liquids by simply changing the pressure in the inlets.|
The construction of the cell with two inlets and two outlets presented in Figure 2 permits movement of the border between two liquids by changing the pressure in the inlets. It may be useful if the surface of the PC chip is covered by one target ligand and several analytes should be tested with this ligand. If the inert reference liquid (e.g., PBS) is sent in one inlet and several analytes under study are sequentially sent to another inlet with sequentially increasing pressure, then all needed information about analytes-ligand interaction may be obtained without chip changing. This is one more way to increase the sensitivity and throughput of the sensor.
|Figure 4: Adlayer thickness change during free biotin (m=244 Da) binding to the streptavidin monolayer (top) and corresponding changes of RI of the buffer during this injection (bottom). The measurement time was 1 s per point (no posterior data averaging and smoothing). In the color inset a corresponding process is illustrated.|
|Figure 3: Immobilization of streptavidin on a biotinylated surface (top) and corresponding changes of RI of the buffer during this injection (bottom). The measurement time was 1 s per point (no posterior data averaging and smoothing). In the color inset a corresponding process is illustrated.|
In the first step, the free carboxyl groups on the surface were chemically activated using EDC / NHS mixture. Then the ligand (Rabbit IgG) is immobilized following by ethanolamine deactivation of the remaining activated carboxyl groups and detachment of not covalently bound proteins. Finally a receptor binding to the surface modified with a ligand is observed following by IgG complex dissociation in PBS buffer and regeneration procedure.
The RI of the liquid was derived as in that for classical critical-angle Abbe refractometers, through the angle of total internal reflection qTIR. The liquid RI is then given byne=n0sin(qTIR) ,
where n0 is the RI of the prism in which the critical angle qTIR is measured.To derive the changes of the adlayer thickness from the changes of the resonance angle DqSW and Dne (known from (1)), we used the dispersion relation for optical surface waves.