A New Technology For Rapid Detection Of E.coli O157:H7 And Staphylococcus Aureus Based On Biocathodes Reducing Oxygen

Electroactive bacteria（EAB）are a series of microorganisms that transfer or capture generated electrons to or from the cell outside through metabolic reactions.While metabolizing,reduction/oxidation of extracellular dissolved and solid electron acceptors/donors occurred,and differences in redox potential between the bacteria and the extracellular electron acceptors/donors can lead to redox reactions.Bioelectrochemical systems could simplify complex bioelectrochemical processes into a single redox reaction in the cathode/anode chamber by constructing an electrical potential difference between the cathode and the anode,and the interaction between microbial-electrode interface is the key to the realization of bioelectrochemical systems.At present,most researches have focused microorganisms working on the anode,while little researches have been done for microorganisms on the cathode due to the limitations of high oxygen reduction potentials.In recent years,various types of microbial cathodes have received widespread attention due to the leapfrogging in conjunction with emerging sensing and other technologies.In this paper,a novel bioelectrochemical system based on screen-printed bipolar electrode（BPE）and foodborne pathogenic bacteria was constructed,investigating the mechanism of interaction between foodborne pathogenic bacteria and extracellular electron acceptors,resolving the electron transfer between foodborne pathogenic bacteria and the electrode interface,and discussing its catalytic ability for oxygen reduction.We combined the catalytic oxygen reduction performance of foodborne pathogenic bacteria on the cathode with the technique of bipolar electrode-electrochemiluminescence（BPE-ECL）to develop a new method for the rapid detection of foodborne pathogenic bacteria.The research contents of this paper are as follows:1.Investigation of the mechanism for cathodic oxygen reduction catalyzed by foodborne pathogenic bacteriaCurrently,most known electrically active bacteria tend to oxidize at the anode,using the electrode as an electron acceptor,as in microbial fuel cells（MFC）,where the EAB oxidize the organic fuel in the anode chamber and release electrons and protons.At the cathode,EAB usually catalyse oxygen reduction,reducing the overpotential for oxygen reduction,but the phenomenon of solid electrodes acting as electron donors has not been extensively studied.In this study,we demonstrated that foodborne pathogenic bacteria was electrochemically active and can catalyse oxygen reduction at the cathode.The oxygen reduction capacity of foodborne pathogenic bacteria was characterised using cyclic voltammetry（CV）.Comparing living foodborne pathogenic bacteria,Lactobacillus rhamnosus,dead foodborne pathogenic bacteria and a range of single food substrates and real sample substrates,revealing that under aerobic conditions,only living foodborne pathogenic bacteria could catalyse oxygen reduction at the cathode of the BPE.Also,a novel bioelectrochemical system based on screen-printed BPE foodborne pathogenic bacteria was constructed.Applying a constant potential to the BPE,foodborne pathogenic bacteria catalyzed electrochemical reduction of O₂,and decreased the overpotential of O₂reduction on the cathode.The specific mechanism of catalytic oxygen reduction may be related to the high concentration of Co Q presented in foodborne pathogenic bacteria.As an electron transmitter,Co Q can accept the electrons at the cathode of BPE and transfer the electrons to O₂through complexⅣ,then eventually converted to water.2.Biocathodes reducing oxygen in BPE-ECL system for rapid screening of E.coli O157:H7After discovery of electron transfer from bacteria,most bacteria known to be electrochemically active are utilized as a self-regenerable catalyst at the anode of microbial fuel cells（MFCs）.However,the reverse phenomenon,cathodic catalysts is not so widely researched.This present study demonstrated that E.coli O157:H7 was electrochemically active,and it was able to catalyze oxygen reduction at the cathode of bipolar electrode（BPE）.Applying a constant potential to the BPE,E.coli O157:H7can catalyze electrochemical reduction of O₂,decrease the overpotential of O₂reduction at the cathode,which in turn generates an electrochemiluminescence（ECL）reporting intensity change at the anode.Significantly,a majority of food matrix does not exhibit catalytic activity for electrochemical reduction of O₂.Meanwhile,due to the physically separation of two poles of closed BPE,complex food matrix at the cathode does not interfere with the ECL reaction at the anode.Therefore,the effect of food matrix is negligible when measuring E.coli O157:H7 levels in food.A low detection limit of 10 CFU/m L E.coli O157:H7 could be identified within 1 hour.Thus,biocathodes reducing oxygen in BPE-ECL system has shown excellent characteristics in the field of rapid detection of electroactive bacteria in food.3.Temporal sensing detection of staphylococcus aureus based on anodic dissolution of Ag and cathodic biocatalysis of oxygen reductionA Ag@C hybrid bipolar electrode（BPE）sensing platform has been established for the temporal detection of Staphylococcus aureus（S.aureus）in food.Combining the advantages of anodic dissolution of Ag and cathodic biocatalysis of oxygen（O₂）reduction,this strategy showed an ultralow detection limit down to 10 CFU/m L.The Ag@C hybrid BPE was fabricated by Ag nanoparticles（NPs）electrochemically deposition on the carbon film of anode,as the anodic dissolution probe.The duration of Ag layer dissolution was positively correlated with the amount of Ag but negatively related to the controlled potential and the conductivity of the circuit.Therefore,it was possible to amplify a slight conductivity change through tuning the other two factors.As the formation of Ag@C completely quenched the ECL emission of luminol,the ECL emission recovery reflected the extent of anodic dissolution.Meanwhile,S.aureus catalyzed the electrochemical reduction of O₂at the cathode,reducing the overpotential for cathodic O₂reduction and thus increasing the rate of anodic electron loss,facilitating Ag dissolution and restoring the ECL emission of luminol.When a constant potential was applied to the BPE,through monitoring the ECL recovery time before and after the incubation of S.aureus on the cathode,a few number of S.aureus could be quantified due to slight difference of the conductivity.This strategy has several merits:（i）Anodic dissolution was introduced to improve sensitivity and extend the detection pattern to the time dimension;（ii）The interference of food matrix can be ignored because most food matrix has no catalytic activity on electrochemical reduction of O₂;（iii）This strategy expands the application of BPE in the ultra-sensitive detection of food-borne pathogens.4.Double bipolar electrode electrochemiluminescence color switch for food-borne pathogens detectionColor-switch electrochemiluminescence（ECL）sensing platform based on a dual-bipolar electrode（D-BPE）is reported in this work.The D-BPE was composed of a cathode filled with buffer and two anodes filled with[Ru（bpy）₃]²⁺-TPr A and luminol-H₂O₂solutions,respectively.Both anodes were modified with capture DNA and served as ECL reporting platforms.After introducing ferrocene-labeled aptamer（Fc-aptamer）on both anodes,the ECL emission signal of the[Ru（bpy）₃]²⁺was difficult to be observed（anode 1）,while luminol emitted a strong and visible ECL signal（anode 2）.Ferrocene（Fc）did not only prevent the oxidation of[Ru（bpy）₃]²⁺due to its lower oxidation potential,its oxidation product Fc⁺also quenched the[Ru（bpy）₃]²⁺ECL through efficient energy transfer.For luminol,Fc⁺catalyzes the accelerated formation of the excited-state of the luminol anion radical,which leads to the enhancement of the luminol ECL.In the presence of food-borne pathogens,the aptamer was assembled with them,leading to the leaving of Fc from the surface of the D-BPE anodes.The ECL intensity of[Ru（bpy）₃]²⁺was enlarged,meanwhile,the blue emission signal of luminol became weakened.By self-calibrating the ratio of the two signals,1 to 10⁶CFU/m L food-borne pathogenic bacteria can be sensitively detected with a detection limit of 1 CFU/m L.Ingeniously,the color-switch biosensor can be used to detect S.aureus,E.coli and S.typhimurium by assembling the corresponding aptamers onto the D-BPE anodes.The new bioelectrochemical system of electrochemically active foodborne pathogenic bacteria constructed in this project can effectively inhibit the influence of dead bacteria and food complex substrates and other substances without catalytic oxygen reduction performance on signal acquisition,and has strong anti-interference ability.1)we find the capabiility of biocathodes reducing oxygen on E.coli O157:H7and S.aureus.2)The bipolar electrode electrochemiluminescence technology is used to convert the weak electrical signal in the aerobic respiration process of microorganisms into a light signal that can be sensitively detected,so as to realize the electrooptic signal conversion.A new sensitive detection method for E.coli O157:H7 and S.aureus is established by using the microbial cathode catalytic oxygen reduction.