Design Of Electrochemical Impedance Biological Sensing System Based On Insect Sex Pheromone-binding Proteins And Living Validation

Insect sex pheromone is a trace level chemical substance that is secreted in vitro by individual one sex and can be accepted by the opposite sex of the same,and can lead to the opposite sex produce some behavior and physiological reaction.It plays an important role in maintaining the insect sex relation between the male and female individuals and species reproduction.The sex pheromone binding proteins(sex pheromone-binding proteins,PBPs)are water-soluble protein which widely exist in the ensillum lymph of insects male tentacles,which can produce specificity recognition with sex pheromones of females release.Therefore,using the specificity binding of the both,which is very suitable to develop related biosensor and can be used to insect scouting and pests prevention and control.In order to achieve the detection of important chemical pheromones in a complex environment,this paper puts forward a set of sex pheromone-binding proteins of electrochemical impedance sensor system,after studying the common biosensors,the paper puts forward a set of electrochemical impedance sensor system based on sex pheromone-binding proteins,by simulating the specificity binding of insect PBPs with corresponding pheromone ligands to achieve the goal of specific detection.First using the Altium-design software designs and draws a interdigitated electrodes connection PCB layout,and producing the PCB layout by corrosion;next Using AutoCAD software designs and draws the electrode base,using aluminum material processing the finished product;then fixing the prepared PBPs protein in the nitrocellulose membrane which is adsorbed on the interdigitated electrodes.Finally,the electrochemical workstation was used to measure the impedance change of the electrode,and the impedance spectrum was used to record the ligands response from protein with chemical synthesis pheromones.In the concrete experiment,we do four groups of protein ligand binding impedance measurement experiment,respectively,the bee queen pheromone vanillyl alcohol(HVA)and methyl-p-hydroxy benzoate(HOB),plant volatile?-ionone and Ethyl oleate(control group).The results show that impedance decreases with the increase of concentration,and the charge transfer resistance(charge transfer resistance,Rct)relative changes is proportional to the logarithm of ligands concentration.The biggest change of Rct in combination test can achieve 60~70%,the detection limit is 10~-77 M,the degree of specificity binding of Am-ASP1 with HVA,HOB are significantly higher than the other two.It shows that the sensor has retained the Am-ASP1 function and can be used for selective and quantitative detection of ligands of bee pheromones.To test the detection ability of the sensing system in vivo,selecting the Ectropis obliqua female moth as the research object.First building electrochemical impedance sensing system based on E.obliqua sex pheromone-binding proteins PBP2,choosing two kinds of E.obliqua sex pheromone to do the ligands combination experiment,respectively(Z,Z,Z)-3,6,9-18 carbon triene and 6,7-epoxy(Z,Z)-3,9-18 carbon diene,getting the corresponding normalized impedance-concentration logarithm figure.And then designing a set of collection system,to used to collect the smell of E.obliqua female moth release and for impedance detection.Results show that the PBP2 fixed on the electrode can combine sex pheromones in the mixed scent of E.obliqua female moth release,when E.obliqua female moth reach a certain amount,the smell of its release is enough to be detected,it shows that the sensor system has live detection ability,which can be used in tea garden for E.obliqua plant prediction.This research will provide some reference of the development of biosensor based on specific insect olfactory function protein,which has important theoretical significance and practical value.