Study On The Enzyme Immunosensor For Rapid Detection Of Vibrio Parahaemolyticus Based On Screen-Printed Electrode

Electrochemical immunosensor,which is a new field based on multi-subject such as biology,medicine,physics,chemistry and electronics, has been widely applied to the reach of food safety rapid detection,clinical medicine,environmental monitoring,military affairs and other areas. Because of its potential advantages such as miniaturization,low cost,high specific,high sensitivity,simple＆direct detection and on-line analysis, immunosensor has become the research hotspot.The selection and modification of supporting electrode,antigen-antibody immobilization technique are the most important and critical parts of fabrication of immunosensor.Also,they are the key technologies in the development of immunosensor.Vibrio Parahaemolyticus,which was the primary pathogen that cause food poisoning in littoral during summer and autumn,was chosen as experimental model in this paper.The researches carried out in this paper would be described as follows:1 A amperometric electrochemical enzyme immunosensor for rapid detection of Vibrio parahaemolyticus based on Chitosan-SiO₂ hybrid-composite membraneFor improving the key technical problems of existing immunesensors, which were the method of immobilizing biosensitive element and keeping its bioactivity,a chitosan-SiO₂ hybrid-composite membrane was prepared by sol-gel method.The enzyme immunosensor was fabricated by coating the membrane and horeseradish peroxidase labeled Vibrio parahaemolyticus antibody on the surface of four-channels screen-printed carbon electrode.The immunosensor was characterized by cyclic voltammetry.Vibrio parahaemolyticus could be detected according to the decrease percentage(DP) of peak current before and after immune response,while cyclic voltammetry was used as an electrochemical mean to detect the products of the enzymatic reaction.Under the optimum conditions of immunoreaction and electrochemical detection,the decrease percentage(DP) of peak current before and after immune response had a linear relation with lgC in the range of 10⁴-10⁹ cfu/mL,the linear regression equation was:DP=6.51gC-3.319,the correlation coefficient was 0.9958 and the detection limite was 6.9×10³ cfu/mL(S/N=3).The immunosensor possessed acceptable specificity,reproducibility(RSD＜6%), stability(the amperometric response was 95%of the initial response after a week) and accuracy(96.7%of the results obtained by the immunosensor were in agreement with those obtained by GB/T 4789.7-2003).Therefore, the immunosensor gave a good performance in rapid detection of Vibrio parahaemolyticus.2 A amperometric electrochemical enzyme immunosensor for Vibrio parahaemolyticus based on electric deposition Nano-Au modified four-channel screen-printed carbon electrodeIn order to improve the biocompatibility and strength of responing signals of immunosensor,a nano-Au modified electrode was prepared by constant potential deposition which was obtained by direct reduction of HAuCLj onto the four-channels screen-printed carbon electrode.The preparation condition of electric deposition nano-Au modified four-channels screen-printed carbon electrode had been selected.The concentration of HAuCl₄ was 0.05 g/L and the electric deposition time was 30s.The enzyme immunosensor was fabricated by immobilizing horeseradish peroxidase labeled Vibrio parahaemolyticus antibody on the surface of nano-Au modified four-channels screen-printed carbon electrode through electrostatic adsorption.The immunosensor was characterized by cyclic voltammetry.Vibrio parahaemolyticus could be detected according to the decrease percentage(DP) of peak current before and after immune response,while cyclic voltammetry was used as an electrochemical mean to detect the products of the enzymatic reaction.Under the optimum conditions of immunoreaction and electrochemical detection,the decrease percentage(DP) of peak current before and after immune response had a linear relation with lgC in the range of 10⁴-10⁹ cfu/mL,the linear regression equation was:DP=7.71gC-12.63,the correlation coefficient was 0.9973(n=6) and the detection limite was 8.1×10⁴ cfu/mL(S/N=3).The immunosensor possessed acceptable specificity,reproducibility(RSD＜6%), stability(the amperometric response was 90%of the initial response after a week) and accuracy(93.3%of the results obtained by the immunosensor were in agreement with those obtained by GB/T 4789.7-2003).Therefore, the immunosensor gave a good performance in rapid detection of Vibrio parahaemolyticus.3 A amperometric electrochemical enzyme immunosensor for Vibrio parahaemolyticus based on glutaraldehyde-crosslinked four-channel screen-printed carbon electrodeIn the search for a simple and useful method of immobilize antibody to fabricate the immunosensor based on four-channel screen-printed carbon electrode,glutaraldehyde cross-linking method was used.Horeseradish peroxidase labeled Vibrio parahaemolyticus antibody was immobilized by crosslinking with glutaraldehyde onto the surface of four-channels screen-printed carbon electrode.The immunosensor was characterized by cyclic voltammetry.Vibrio parahaemolyticus could be detected according to the decrease percentage(DP) of peak current before and after immune response,while cyclic voltammetry was used as an electrochemical mean to detect the products of the enzymatic reaction.Under the optimum conditions of immunoreaction and electrochemical detection,the decrease percentage(DP) of peak current before and after immune response had a linear relation with igC in the range of 10³-10⁹ cfu/mL,the linear regression equation was:DP=6.51gC-3.595,the correlation coefficient was 0.9935 and the detection limite was 2.0×10⁵ cfu/mL(S/N=3) The immunosensor possessed acceptable specificity,reproducibility(RSD<6%), stability(the amperometric response was 92%of the initial response after a week) and accuracy(93.3%of the results obtained by the immunosensor were in agreement with those obtained by GB/T 4789.7-2003).Therefore, the immunosensor gave a good performance in rapid detection of Vibrio parahaemolyticus.