Detection Method Based On Electrochemical Immunosensing Technology Of BmNPV

Bombyx mori nuclear polyhedrosis,also called silkworm blood-type abscess,always are the most serious virus disease in the sericulture production.Taking effective prevention measures is an effective way to avoid economic losses caused by the outbreak of silkworm disease.The traditional detection methods of BmNPV basically have defects.In order to reduce economic losses caused by Bombyx mori nuclear polyhedrosis,studying and establishing a rapid,specific,simple and high-sensitivity BmNPV detection technology are very important,which plays an important role for the sustainable and stable development in the sericulture production.Electrochemical immunosensors have the advantages of simple operation,low detection limit,high precision,low raw materials,short reaction time,and automatic operation.It has been successfully used in clinical medical diagnosis,food industry quality inspection,environmental composition analysis,animal pathogenic microorganism detection and other fields.In present study,electrochemical immunosensor for detecting Bombyx mori nuclear polyhedrosis virus was constructed by mix self-assembled membrane modified the gold electrode,the electrochemical immunosensor rapid detection technology of Bombyx mori polyhedrosis virus was established and applied to the actual sample detection.The main results are as follows:1.The electrochemical characterization of two hybrid self-assembled membranes and their effects on detecting antigens were studied,the result showed that 80%MUA/ME hybrid self-assembled membrane modified electrode was the best.The pre-processed working electrode was placed in 80%MUA/ME mixed solution for 24 h for self-assembly and in EDC/NHS mixed solution for 1 h for activation,then gradually added BmNPV antibody,BSA,antigen for performing layers of modification.The electrochemical behavior of the immunosensor is characterized by EIS and AFM.The results showed that the impedance value of the immunosensor and roughness of electrode increased with the electrodes gradual modification,and its impedance value has a linear relationship with the antigen concentration captured on the electrode.All those showed that the immunosensor of Bombyx mori nuclear polyhedrosis virus was successfully constructed.2.The detection conditions of the constructed BmNPV electrochemical immunosensor were optimized.The results showed that when the bound antibody was diluted 500 times,the electrochemical signal change value reached the maximum,and then tended to be smooth.When the temperature for antibody and antigen incubation was 37?,the reaction activity of antibody antigen reached the highest and the change of its impedance value was the most obvious.When the reaction time of antibody and antigen was 30 min,the electrochemical signal reached the maximum,and then tended to be flat.3.The detection performance of the electrochemical immunosensor was studied.It was found that the polyhedrin concentration was in the range of 0.0001-100 ng/mL,the linear equation was Y=4619.63 LgC+2108.36,and the detection limit reached 14.54 fg/mL,the linear correlation coefficient squared(R~2)was 0.9922,which spanned seven orders of magnitude and had high sensitivity.The results of specificity determination test showed that the impedance values of interfering substances,such as silkworm blood and silkworm cytoplasm polyhedrosis virus,were far lower than the response values of the detection antigen,it was indicated that the sensor had good specificity and strong anti-interference ability.Repeatability measurement results showed that the RSD of this method was less than5%,and it had good accuracy and precision.The regenerative test indicated that the immunosensor could be regenerated about 5 times in the dissociation solution of 0.2 mol/L glycine-0.2 mol/L HCl(pH=2.4),which reduced the actual application cost.4.The electrochemical immunosensor was used for actual sample detection.The experimental results of the standard serum addition method showed that the RSD value of each concentration was less than 10%,and the recovery rate was 92.31%-100.61%,which indicated that the detection method had high precision and practical application value.The silkworm was added BmNPV by feeding,and the blood of silkworm infected different periods was detected.The results showed that the silkworm could amplify the specificity of hemolymph when fed for 12 h.The impedance value of silkworm blood is significantly different from that of fed at 0 h(P<0.05),which is basically consistent with the PCR reaction detection results.But the detection period of the EIS method is significantly shortened,and the detection steps are simple,which provides a new detection technology for the early detection of silkworm nuclear polyhedrosis.