The Research On SPR Biosensor And Its Combination With Electrochemical Technique

Surface Plasmon Resonance (SPR) biosensing has been widely applied in many fields, and its combination with other techniques has become the research hot spot. The thesis is aimed at enhancing the performance of our home-built SPR biosensor instrument. The bimetallic chip with gold and silver was designed to improve the performance of the SPR sensor. Combined with different biological molecular recognition systems, the biosensor was used to detect H7N9 influenze virus and the antibiotics of ciprofloxacin (CIP). Combined with electrochemical technique, a two-electrode electrochemical SPR (ESPR) biosensor has been proposed. Choosing cyclic voltammetry, the ESPR biosensor was used to detect the concertation of toluidine blue (TB), and to monitor the polymerization of polyaniline (PAn). Main contents and achievements of this thesis include:1. Novel metal film systems are designed to improve the performance of the SPR sensor. The optimal thickness of the single gold, silver, and the bimetallic with gold and silver chips are determined according to the theoretical analysis and simulation. Experiments were carried out to obtain the performance of the sensor with the bimetallic chip (40 nm Ag+10 nm Au) and single gold (50 nm) chip, respectively. For the refractometry experiment of NaCl, the noise of the bimetallic chip is 0.004 pixel, and the refractive index resolution is 5.3×10-7 RIU, which shows an improvement for the performance of the SPR sensor compared with the single gold chip. For the adsorption experiment of BSA molecules, the response of the SPR resonance angle is lager than the single gold chip.2. A SPR biosensor was developed for the detection of H7N9 influenza viruses based on the bimetallic chip. The monoclonal antibodies to the hemagglutinin (HA) of H7N9 viruses was immobilized on the surface of the chip, then the anti-HA chip was ready for rapid identification and quantitative detection for H7N9 viruses. Results showed that the response of SPR was linearly related to the concentration of HA in the range of 1～100 ng/mL, and the limit of detection for HA was 1 ng/mL. The equilibrium dissociation constant, KD is 2.28×10-11 M, which suggest that the anti-HA chip is recognized efficiently by the H7N9 virus.3. A SPR biosensor was proposed based on the metallic chip combined with molecularly imprinted polymers (MIP) for the rapid detection of CIP. The MIP chip was prepared by photo-polymerization with CIP as template molecule and itaconic acid as functional monomer on the surface of the SPR chip. The MIP chip exhibited high selectivity for CEP, the limit of detection for CIP is 1×10-13 mol/L, and the recovery of CIP samples is 93.5%～108.8%. This MIP chip could be regeneration for the physical and chemical stability.4. A two-electrode ESPR biosensor has been proposed combined SPR with electrochemical technique. Active carbon (AC) is employed as the counter electrode for its potential stability. The ESPR system with cyclic voltammetry was used for detecting the concentration of toluidine blue (TB) from its redox reaction. The peak potential of oxidation and reduction could be obtained from the cyclic voltammogram. The time differential of the resonance angle dθ/dt was correlated with the current of the cyclic voltammogram. Two peaks were also obtained from the curves of E-dθ/dt, and the corresponding potential of the two peaks were similar to the peak potential of oxidation and reduction. The concertation of TB was fitting with the value of current and the value of dθ/dt at oxidation potential, respectively, which could be used to detection the concentration of TB.5. The two-electrode ESPR setup was also used for investigating the electropolymerization of polyaniline (PAn). The information of the redox reaction could be obtained from the cyclic voltammograms. The resonance angle shift curves can provide more detailed information about the polymerization process, from which we can obtain the transition processes of PAn to different oxidation states. Then the growth kinetics of PAn could be analyzed, which provided a new way for the study of the polymerization mechanism of PAn.