Sensitive And Rapid Analysis Of Escherichia Ocli Based On Electrochemical Sensing Platform

Escherichia coli is a common bacteria in the intestines of humans and other warm-blooded animals.After being excreted with feces,it is very likely to cause water pollution,food pollution and environment pollution.Pathogenic E.coli is prone to cause a variety of human diseases.Although traditional E.coli detection methods are very accurate,they are complex and take a long time(≥24 hours).In this study,a portable intelligent electrochemical sensor was established through electrochemical analysis technology to achieve rapid and sensitive analysis and detection of E.coli in the environment.The theoretical reference for rapid analysis and detection of pathogens in the environment was provided.The specific research contents are as follows:(1)Electrochemical sensing analysis method for rapid detection of E.coliE.coli is induced to produce β-galactosidase in LB medium containing isopropylthio-β-D-galactosidase(IPTG).The β-galactosidase released after breaking the wall can hydrolyze 4-Aminobenzene-β-D-galactoside(PAPG)produces p-aminophenol(PAP),and the concentration of E.coli can be determined indirectly based on the response of PAP on the modified electrode.The modified electrode is a bimetallic Pt-Pd nanocomposite electrode(Pt-Pd/BP/GCE)made by self-made high-stability black phosphorene.The morphology,structure,specific surface area of electrochemical activity and electrochemical stability of the material were investigated.The preparation process of the material,the optimal enzyme activity temperature of β-galactosidase,the optimal detection pH of PAP and other parameters were explored and optimized,and successfully applied to the detection of E.coli.The results showed that the prepared nanomaterial BP/Pt-Pd has good electrochemical stability and electrocatalytic performance,and the modified Pt-Pd/BP/GCE electrode has a good linear response to the indirect detection of E.coli(6 × 106-1.6 × 108 cfu·mL-1),the limit of detection(LOD)was 6.65 × 105 cfu·mL-1,and the sensitivity was 2.44μA.(cfu·mL-1)-1·cm-2.In addition,the method is also suitable for the determination of PAP concentration and β-Gal enzyme activity,corresponding to the linear range of PAP is 2 × 10-8 to 5 × 10-6 M,LOD is 1.41 × 10-1 M,and the sensitivity is 115.81 μA·μM-1·cm-2,linear range corresponding to β-Gal is 0.20.8 mU·L-1,LOD is 9.35 nU·L-1,sensitivity is 2.77 μA(mU·L-1)-1·cm-2.(2)Optimization of the conditions for the activity of β-galactosidase produced by E.coliAfter the initial establishment of a rapid electrochemical detection method for E.coli,the method for electrochemical determination of β-galactosidase enzyme activity previously established was used to optimize the conditions for enzyme production during the pretreatment of E.coli for electrochemical detection.After optimizing the five conditions of carbon source,nitrogen source,inducer,pH and temperature,Plackett-Burman test design was carried out on the enzyme-producing medium,the significant factors of fermentation process conditions were screened,three significant factors of sucrose,IPTG and pH were determined.The response surface analysis of three factors and five levels was carried out to establish the optimal enzyme production conditions,which were 3%sucrose,2%yeast extract,0.5%sodium chloride,1.5 mM IPTG inducer,pH 6.5 and induction at a temperature of 30℃,the optimized enzyme-producing activity is 182.63%of the original.(3)The preparation method of intelligent electrochemical sensor for rapid detection of E.coliBased on the establishment of a rapid detection method for E.coli,the feasibility of applying this method to an intelligent portable electrochemical workstation was explored.A new modified material nitrogen-doped graphene oxide-carboxymethylcellulose sodium dispersion(NGR-CMC)was prepared,and the effect of pH on β-galactosidase enzyme activity was studied.CMC’s morphology,structure,electrochemical active specific surface area,stability and other parameters were investigated.The results showed that the prepared material has good oxygenated water stability,large specific surface area and high electrocatalytic activity.The optimized modified electrode and portable sensor showed a good linear response to the detection of E.coli.The linear range is 2.5 × 103-2.5 × 105 cfu·mL-1,and the detection limit is 1.68 × 103 cfu·mL-1.The sensitivity is 0.589 μA·(cfu·mL-1)-1·cm-2.It only taked 3.5 hours from the pretreatment of E.coli to be tested to the final result analysis.