Amperometric Immunosensor Based On Gold Nanoparticle/thionine Modified Gold Electrode For The Detection Of Abscisic Acid

Abscisic acid(ABA) is an important phytohormones. There are many traditional detection methods, such as HPLC, gas chromatography, gas chromatography mass spectrometry, liquid chromatography-mass spectrometry ,enzyme-linked immunosorbent assay, and radioimmunoassay. The electrochemical amperometric immunosensors, which combine their simple pretreatment procedure, fast analytical time, precise and sensitive current measurement with inexpensive and miniaturizable instrument have gained considerable attention, for phytohormonal analysis . Besides, gold nanoparticles(nano-Au) have been successfully applied to electrochemical immunoassay and DNA assay for its strong intrinsic adsorption property and biocompatibility.In this paper, a highly sensitivie, stabile, convenient and simple amperometric ABA immunosensor based on gold nanoparticle and the catalytic wave system of thionine, H₂O₂ and HRP is proposed. The optimized assay conditions, calibration curve, analysis of plant material samples, and comparison with HPLC were studied . The main contents in the thesis are presented as follows:(1) Procedure and principle of the sensor were introduced, feasibility and electrochemical characterization of this immunosensor were confirmed .(2) Several important aspects which effect performance of this new sensor, includeing the incubation time of the mixture of thionine and ABA-antibody conjugated HRP, the proportionality of thionine and ABA-antibody conjugated HRP, the optimal pH value of buffer solution and the concentration of H₂O₂ in buffer solution were systematically studied . The optimized detection condition of this new sensor was obtained . Research showed that the optimal incubation time was 1h , the optimal thionine percentage was 75 % for adsorption. The optimal pH value was 7.0 and the optimal concentration of H₂O₂ was 1.2mol·L^-1 in buffer for ABA determination.(3) The calibration curve of the amperometric immunosensor based on gold nanoparticle (nano-Au)/thionine modified gold electrode for the determination of abscisic acid was obtained under optimal conditions. Ampere response of the immunosensor decreased as increasing of ABA concentration. Depressed percentage has linear relationship with ABA concentration ranges from 0.5ng·mL^-1 to 1000ng/mL, the linear regression equation was y = 0.0209x + 17.071, the correlation coefficient was 0.9922. The detection limit was 0.2 ng·mL^-1.(4) ABA concentration of cotton leaf samples was detected by this under optimal conditions and was compared with HPLC. The result showed the accuracy and feasibility of using the proposed amperometric immunosensor method for the analysis of plant material samples.