Preparation Of The Polyclonal Antibody Against Leucomalachite Green And The Assembly Of The Electrochemical Impedence Immunosensor

Leucomalachite Green(LMG) is the main metabolite and existence form of malachite green(MG) with a long resiue time in vivo. It may cause carcinogenesis, mutagenesis, teratogenesis, and respiratory toxicity because of the triphenylmethane functional group. Thus, LMG and MG have been prohibited to be used in aquatic products for human consumption in many countries including China. However, illegal use occurs frequently in aquaculture due to its low cost and ready availability. Given this, an efficient and low-cost method for monitoring LMG and MG would be highly appreciated to realize the effective supervisor. The specifis details in this study were as follows.Four haptens of LMG were synthesized according to the principles of the chemosynthesis and the design requirements of the hapten. In addition, a new systhetic route was proposed in the paper which could be expanded to produce a series of haptens with alkyl chains as spacer arms. Morever, the targets were characterized by MS and NMR to identify their molecular weight and structure, respectively.The four haptens were coupled with BSA and OVA, respectively via carbodiimide method. The complete antigens were characterized by UV-VIS and the coupling ratios were calculated by MALDI-TOF-MS. It turned out that the antigens were sucefully synthesized and the coupling ratios were between 10:1 and 20:1. Prepare the polyclonal antibodies by immunizing the rabbits and terminate the immune process by the ELISA detection method. The ELISA detection method was finally established by choosing the best antibody and optimizing the working conditions. The ELISA assay was further used to analyze the LMG residue in fish tissue with the sensitivity of 2.21 ng/mL with a linear range of 0.15~31.7 ng/mL and the limit of detection(LOD) around 0.06 ng/mL. Recoveries ranged from 79.8~92.4%, indicating that the ELISA could be used for effective and low-cost analysis of LMG in fish samples.The electrochemical impedence sensor was assembled using the antibody with the highest sensitivity and electrochemical impedence spectroscopy(EIS) was used to do the qualitative and quantitive analysis. In order to improve the analytical performance, glassy carbon electrode(GCE) was modified with 1, 4-phenylenediamine to form a stable layer, and then BSA-AuNCs were covalently bound to the GCE. An adequate quantity of the polyclonal antibody of LMG was immobilized onto the surface of the BSA-AuNCs by the chemical reaction of EDC/NHS. The surface of the electrode was finally blocked by the BSA to eliminate the influence of the nonspecific absorption. The calibration curve for LMG was linear in the range of 0.1~10 ng/mL with the limit of detection(LOD) 0.03 ng/mL. Furthermore, the sum of MG and LMG was detected in fish farm water by the MG reduction. The recovery was between 89.7~99.2% in spiked samples.