| The overuse of antibiotics in animal husbandry leads to the increasingly serious problem of antibiotic resistance rates in foodborne bacterias.Effective detection methods can prevent and control the infection and spread of antibiotic resistance bacteria and ensure human health.The existing methods for the detection of pathogenic bacteria from food sources have many problems,such as long detection period,inability to distinguish live bacteria,huge instruments,expensive price,etc.Electrochemical sensor technology has good selectivity,high sensitivity,with advantages of low cost and rapid detection,it can provide technical support for the construction of effective detection methods.Based on the theory of electron transfer in the process of bacterial energy metabolism,this paper introduces resazurin as a redox probe.In this paper,the electrochemical characteristics of resazurin in E.coli were studied.The aim of this study was to obtain the electrochemical signals from the sensing system in vivo,so as to construct a rapid and portable method.Low-energy and low-cost electrochemical methods for rapid detection of antibiotic resistance of foodbome bacteria provide theoretical basis.The results are as follows:(1)In this paper,the electrochemical characteristics of resazurin in pH6.8 buffer solution were studied.The working electrode and reference electrode in three-electrode system were studied by cyclic voltammetry(CV)and control variable method.At the same time,the change of peak potential under different buffer solutions and different pH values of BR buffer solution was studied,and the sweep speed of subsequent test was optimized.On this basis,the electrochemical characteristics of resazurin with different concentrations were measured by three electrochemical methods:cyclic voltammetry(CV),differential pulse voltammetry(DPV)and square wave voltammetry(SWV).When the working electrode is glassy carbon electrode and the reference electrode is Ag/AgCl electrode,the optimal electrochemical signal can be obtained.Considering the effect of peak potential current and background noise,the scanning speed of 0.15 v/s is chosen as the subsequent scanning velocity parameter.The electrochemical signals measured by the three methods all increase with the increase of the concentration of resazurin.The electrochemical response signals of the SWV method is the most sensitive,which lays a foundation for the further study of the electrochemical characteristics of the edge resazurin in the presence of Escherichia coli in vivo.(2)The electrochemical properties of resazurin in BR buffer solution supplemented with E.coli ATCC 25922 were studied by means of SWV,DPV and CV methods.Spectrophotometric method,plate counting method and CV method were used to study the effects of different concentrations of resazurin on E.coli in vivo(OD600= 0.9).The effects of oxygen,concentration of E.coli and the reaction time of E.coli with resazurin were studied by using CV method.The effect of the presence of E.coli ATCC 25922 on the electrochemical characteristics of resazurin was studied by three different electrochemical methods.Results show that the presence of oxygen in the solution had an significant effect on the electrical signal,nitrogen treatment for 10 min could eliminate the effect of oxygen.The interaction time between E.coli and resazurin had a significant effect on the electric signal.The optimal action time was 60 mins.With the increase of E.coli concentration,the electrical signals of CV,DPV and SWV were negatively correlated with the varies of concentration.The linear equations are:the CV oxidation peak current equation y=0.026x-0.589,R2=0.908;CV reduction peak current equation y=0.045x+0.756,R2=0.918;DPV positive sweep equation y = 0.018x-1.427,R2=0.950;DPV negative sweep equation y= 0.02x+1.697,R2=0.966;SWV positive sweep equation y=0.197x+2.351,R2=0.971;SWV negative sweep equation y=0.203x-2.192,R2=0.977,respectively.the three methods can be used for quantitative analysis of E.coli in a certain range,but the signal sensitivity of SWV is the highest.The results show that the electrochemical method can detect the activity of E.coli ATCC 25922 in a sensitive and accurate manner.It provides a theoretical support for the further study of the electrochemical characteristics of resazurin in isolated strains E.coli.(3)SWV method was used to explore the electrochemical characteristics of resazurin in the foodborne of antibiotic resistance of isolated strains E.coli.Based on the advantage of SWV method in quantitative detection of E.coli in vivo,electrochemical signal changes of resazurin and isolated strains E.coli were obtained by different time;the change of electrochemical signal of isolated strains E.coli under the action of resazurin with different concentrations of antibiotics;Electrochemical signal changes of different sensitive strains and resistant strains added ampicillin were analysed.%RA value was measured,as bacterial activity on behalf of electrochemical methods.Reference to clinical and Laboratory Standards Institute(CLSI)standard antibiotics sensitivity test set,the method of electrochemical discrimination bacterial antibiotics sensitivity was established.Antibiotics susceptibility test of 5 isolated strains of foodborne E.coli with 3 antibiotics were detected rapidly using the established method,and at the same time,comparison with traditional t antibiotic sensitivity test was analysed.Definition of%RA values for antibiotics sensitive and resistant bacteria were proposed and then help to validate and improve the electrochemical rapid susceptibility method.The results show that,the determination results of antibiotics sensitivity using electrochemical method with 50%RA value as threshold to test the antibiotics sensitivity of 5 isolated foodborme E.coli strains with 3 kinds of antibiotics were consistent with the disk diffusion method.Compare to disk diffusion method which report the results of 18?24 h,this method is only 2.5 h,and provides a theoretical basis for the study of rapid antibiotics sensitivity test. |
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