Surface Plasmon Resonance (SPR) Based Biosensor Immunoassays For Drug Residues

In this dissertation, sensitive and fast assays were developed to determinesulfonamides in PBS with surface plasmon resonance (SPR)-based biosensor. TheSPR biosensor-2002 and the multi-analyte SPR biosensor were home-made and thesensor chip had 3×5 cells. The change of temperature and non-specific adsorptioncould be canceled by using reference surface to improve the quality of the data.The Sulfonamide-bovine serum albumin (BSA) conjugate (antigen) wascovalently immobilized on a modified gold film. The antibody against sulfonamidewas used to develop an inhibitive assay in which the antibody was mixed withsulfonamide of different concentrations prepared with PBS to construct a standardcurve.The SPR biosensor-2002 was used to study the detection conditions ofSulfamethazine(SMZ),Sulfamethoxazole(SMOZ)and sulfadiazine(SDZ). Theconditions of the immobilization of the antigen were studied and the workingconcentration of antibody was optimized. The scope of the detection was 0-10ng/mL.The limit of detection for sulfamethazine and sulfamethoxazole was less than 5ng/mL,and for sulfadiazine 0.1ng/mL. The specificity and stability of the antibody were alsostudied. A sensitive and fast continuous SPR-based biosensor assay in which adetection cycle consists of five detection steps followed by one regeneration step wasdeveloped and evaluated according to the kinetics of antigen-antibody interaction.This method can effectively decrease the times of regeneration. The total time span ofa detection step was five minutes.The multi-analyte SPR biosensor was used to determine sulfamethoxazole andsulfamethazine in PBS buffer. The immobilization of the conjugations ofsulfonamide-BSA was carried out in the instrument to monitor the quantity of theantigen immobilized. The antibody mixed with the sulfonamide in the buffer wasinjected over the surface of the chip to get a response curve with a slope inverselyproportional to the concentration of the sulfonamide in buffer. Two calibration curves were constructed and the detection scopes were in the range of 1-20ng/mL. The limitof detection for sulfamethazine was 0.6ng/mL, and for sulfamethoxazole 3.5 ng/mL.The effect of matrix in milk was also studied. Fat in milk was a great hinderanceto the interaction between the antibody and the antigen. Antibody in the milk with fatcan't bind with the antigen immobilized.The chip without antigen immobilized could be stored in 4℃for a long time.The chip immobilized with antigen could be stored in 4℃for more than one monthwithout distinctive changes.The kinetics of the binding between the sulfamethazine antibody and the antigen(SMT-BSA) were analyzed. The kinetic constants were calculated: The binding rateconstant (K_a) was 1.38×10~4M~-s~-, the dissociate constant (K_d)was 0.0136s~-, theaffinity constant was 1.0×10~6M~-。...