Study Of Electrochemical Behavior Of Methylene Blue In The Viable Food-Borne Escherichia Coli

The abuse of antibiotics in clinical,animal husbandry and aquaculture industry has led to a growing problem of food-borne bacterial resistance in China.Rapid and portable detection method is one of the important measures to control the wide spread of food-borne resistant bacteria from source and ensure human health.Electrochemical methods are characterized by rapidness,sensitivity,low cost and miniaturization,which provide technical support for the development of effective detection methods.Therefore,based on the theory of electron transfer in the process of bacterial energy metabolism,this article introduces methylene blue as a redox probe to study the electrochemical properties of methylene blue in viable food-borne Escherichia coli(E.coli).Through the acquisition of biosensor electrochemical signals,the aim was to provide a theoretical basis for developing a rapid,portable,low energy consumption and low cost electrochemical detection systems of food-borne bacterial.First,the measurement conditions for the electrochemical characteristics of methylene blue were optimized.We used both Cyclic voltammetry(CV)and square wave voltammetry to investigated the scan curves of MB at different working electrodes(Au electrode and glassy carbon electrode)and different scan rates(0.025?0.05 and 0.1 V/s).Effect of dissolved oxygen on the electrochemistry of MB was discussed and then the correlation curve between MB concentration and CV/SWV peak current was constructed.The results show that MB is an irreversible redox reaction on glassy carbon electrode.In order to obtain better CV/SWV signal,the optimum test parameters would be 0.05 V/s scan rate of CV with glassy carbon electrode as working electrode following N2 purge.In the range of 0.1?0.5 mmol/L,the peak currents of CV/SWV are positively correlated with MB concentration.The regression equations are Y =-15.447X-0.641,R2=0.9996 and Y =-101.159-1.035,R2=0.9986 respectively.Second,the electrochemical properties of methylene blue in viable E.coli ATCC 25922 were explored.We analyzed the electrochemical signal changes of MB after addition of E.coli ATCC 25922 were by CV and SWV,respectively.In the presence of E.coli ATCC 25922,different cultivation times with MB and effect of dissolved oxygen were further explored.The correlation curve between E.coli ATCC 25922 concentration and CV/SWV peak current were constructed.The results showed that:the respiration of bacteria reduced MB in the solution,resulting in the continuous decrease of CV/SWV peak current with the prolongation of the interaction time,which reached the steady state at 8 min and the removal of dissolved oxygen could accelerated this process.In the range of 1.7?8.7×108 CFU/mL,the concentration of E.coli ATCC 25922 was linearly correlated with the peak current of CV/SWV with regression equations of Y = 0.256X-3.773,R2=0.9945 and Y = 1.909X-23.915,R2=0.9942,respectively.SWV were chosen for subsequent experiments because it is more sensitive and stable than CV.Electrochemical method can sensitively and accurately detect the change of methylene blue concentration in the solution caused by the respiration of E.coli ATCC 25922,providing a theoretical basis for the subsequent research.Third,the electrochemical properties of methylene blue in food-resistant E.coli isolates were studied,and the feasibility of electrochemical rapid susceptibility testing was explored.We studied the electrochemical signal changes of MB after different cultivation time with isolates by SWV.After interacting with ciprofloxacin,the difference in signal change between sensitive and resistant strains were analyzed,and%RA value were introduced as an index of respiratory activity.With reference to the drug susceptibility test standards established by the Clinical and Laboratory Standards Institute(CLSI),an electrochemical rapid drug sensitivity test system was initially established.The drug sensitivity test of 3 antibiotics for 5 strains of food-borne E.coli were carried out using both electrochemical method and paper diffusion method.Results show that with%RA value of 75 as the threshold for judging the susceptibility and drug resistance of bacterial,electrochemical susceptibility test can receive a report that consistent with paper diffusion method results within only 2 h while paper diffusion method need 18 h or longer.It will provide a theoretical basis for future study of rapid antibiotic sensitivity test.