The Study Of Label-Free Impedimetric Immunosensor For AFB1

This thesis summarized the research status and application of immunoensors and modified electrodes, and the advantages and disadvantages of the methods on AFB1 detection were analyzed. The study had focused on preparation conditions of the linear poly o-phenylenediamine (PoPD) modified electrode and AFB1 immunosensor. Furthermore, the good result of quantitative determination was obtained with EIS method by the use of the preparated AFB1 immunosensor.First of all, poly o-phenylenediamine (PoPD) modified electrode was developed by means of cyclic voltammetry. The membrane of PoPD rich in amino groups (-NH2) with linear structure was obtained under the potential ranging from -600 mV to 1000 mV, 50 mV/s scan rate, and 30 cycles in 0.05 mol/L o-PD + 1 mol/L H2SO4 solution. The resulting membrane not only possesses good selected penetrating properties, which can let H+ penetrate through, but also catalyzing redox reactions of H+/H.Secondly, the preparation conditions of AFB1 immunosensor based on the PoPD modified electrode and AFB1 antigen fixed conditions were optimized. The results showed that the time of three operating should be finished within 45 min at 25℃, including glutaraldehyde cross-linked, purified anti-AFB1 antibodies fixed, and fetal calf serum blocked. The best immobilization condition of AFB1 antigen was got in the solution of 80% PBS + 20% methanol (pH 7.40) at 30℃.Thirdly, the reactions that occurred during the assembly process of AFB1 immunosensor were studied. Results indicated that during glutaraldehyde cross-linking with PoPD/Au electrode and antibody fixed in the electrode surface, the two aldehyde groups (-CHO) of glutaraldehyde occurred Schiff base reacted with the amino (-NH₂) of PoPD and the residual amino (-NH₂) of antibody immunoglobulin, respectively, forming a stable Ar-N=C(CH₂)3C=N-R structure. The antibody was fixed on the electrode surface forming AFB1 immunosensor which was capable of molecular recognition.Fourthly, the EIS method was used to characterize functional interface formed during immunosensor assembly, and the interface model of molecular recognition was constructed. It was found that in the assembling process, glutaraldehyde cross-linked, anti-AFB1 fixed, and bovine serum closed on the PoPD modified electrode, electronic transfer resistance increased step by step; in AFB1 antigen specific immune response, the electronic transfer resistance with the combination of immune AFB1 volume increased. The EIS spectra has a significant"dispersion effect"due to the non-specific adsorption of H+ and the non-uniform surface of the immunosensor.At last, the prepared AFB1 immunosensor was used to detect AFB1 with EIS method. The results showed that two good linear relations were presented in 0.03μg/mL 0.7μg/mL. When the AFB1 concentration was in the range of 0.03μg/mL 0.1μg/mL, the linear equation 1 wasΔRP = -5960Ω+ 2.75×10¹¹×cAFB1, R2 = 0.9946; when the AFB1 concentration was in the range of 0.1μg/mL 0.7μg/mL, the linear equation 2 wasΔRP = 19410Ω+ 2.49×10¹⁰×cAFB1, R² = 0.9807. The detection limit (LOD) was 2.88×10-8 g/mL.In addition, the dissociation and preservation for AFB1 immunosensor were optimized. It was found that sensor can be used up to 10 times when it was dissociated in 0.2 mol/L glycine -0.2 mol/L HCl (pH = 2.60) mixed solution for the dissociation; The sensor can be kept under wet condition for temporary purpose, and it also can be kept up to 11 days under dry condition.Compared with the reported similar label-free impedimetric AFB1 immunosensor, this sensor prepared by this method shows better linear relationship and the detection limit is much lower than the reported result of 1×10-7 g/mL. In addition the immunosensor can be reused, time saving in preparation, low cost, and has a lost of commercial potential and market value.