Preparation Of Poly Neutral Red Modified Glassy Carbon Electrode And Application In The Detection Of Antimicrobial Efficacy

Objective Neutral red(NR),a basic phenazine dye,have been widely used as an attractive mediator because of redox center in its structure.Poly(neutral red)modified glassy carbon electrode(PNR/GCE)was prepared by electropolymerization and used as an electrochemical probe to investigate the effectiveness of representative antimicrobial agents against E.coli.Subsequently,the feasibility of the bioelectrochemical system based on PNR/GCE for rapid detection of antimicrobial agents was evaluated.On this basis,GR/CS/E.coli/PNR/GCE was prepared by preparation and direct modification of biocomposite nanomaterial using chitosan(CS)as film-forming material and graphene(GR)as conductive nanomaterial.The prepared GR/CS/E.coli/PNR/GCE under the optimal conditions was applied to the detection of the effectiveness of antimicrobial agents,and the feasibility of the fabricated microbial electrochemical sensor for rapid detection of antimicrobial efficacy was evaluated.Methods PNR/GCE was prepared by electropolymerization with GCE as substrate and characterized by cyclic voltammetry(CV).The-0.45 V oxidation peak current in cyclic voltammogram as an indicator was used to investigate the effects of NR concentration,the number of scanning segments,the p H value of buffer and the Na NO3 concentration of supporting electrolyte on the polymerization process.Subsequently,the optimum conditions for polymerization were selected.Then a new method for rapid detection of antimicrobial efficacy in vitro was constructed,where NR as a electron mediator and E.coli as a model microorganism were employed in detecting the relationship between the activity of E.coli and the concentration of antibacterials.On this basis,GR/CS/E.coli/PNR/GCE was obtained by immobilization of E.coli on PNR/GCE with CS as film-forming material and GR as conductive nanomaterial.The immobilization conditions of E.coli were screened by orthogonal design.Then the results of orthogonal experiment were analyzed by F-test,and the effects of GR concentration,CS concentration and E.coli concentration on the experiment were obtained.The prepared GR/CS/E.coli/PNR/GCE under the optimal conditions was applied to detect the effectiveness of representative antimicrobial agents.Results(1)The optimum polymerization conditions of NR were as follows: 0.5 mmol/L of neutral red,30 scan segments,buffer solution p H of 7.0,0.5 mol/L of Na NO3 as supporting electrolyte.(2)The highest sensitivity in the detection of antibacterials efficacy was gotten when the final absorbance value of bacterial suspension was 0.5 and the standard glucose-glutamic acid solution(GGA,BOD220)was used as nutrition.The incubating time was set at 40 min.(3)Under the optimum experimental conditions,the 50% inhibiting concentration(IC50)of cefoperazone,amikacin,gentamycin and levofloxacin on E.coli cells were 34.15 mg/L,27.98 mg/L,8.83 mg/L,11.49 mg/L,respectively.However,vancomycin,as an antibiotic only affects gram-positive bacteria,showed a poor inhibition on E.coli cells.(4)Microbial immobilization conditions were investigated based on PNR/GCE.The results showed that the optimum final concentrations of GR,CS and E.coli were 0.50 mg/m L,0.75%,0.50 g/m L,respectively.(5)The prepared GR/CS/E.coli/PNR/GCE under the optimal conditions was applied to the detection of the effectiveness of gentamycin,levofloxacin,ceftriaxone sodium,piperacillin sodium and vancomycin.The 10% inhibiting concentration(IC10)of the first four antimicrobial agents against E.coli were 15.81mg/L ? 19.62mg/L ? 65.74mg/L ? 62.47mg/L,respectively.However,vancomycin,as an antibiotic against gram-positive bacteria,had little inhibitory effect on E.coli cells.Conclusions(1)The prepared PNR/GCE by electropolymerization has excellent stability,reproducibility and sensitivity.The bioelectrochemical system based on PNR/GCE as working electrode and E.coli as model microorganism can be used to detect the effectiveness of antimicrobial agents.Compared with traditional antimicrobial susceptibility tests,this method is simple and rapid,and can be used to determine the efficacy of antimicrobial agents accurately.so our method should be supposed as a supplementary method to detect the effectiveness of antimicrobial agents,and it shows that its promising application in the research and development of new antimicrobial agents and the detection of clinical bacterial resistance.(2)The prepared GR/CS/E.coli/PNR/GCE under the optimal conditions for immobilization of microorganisms can be used to detect the effectiveness of antimicrobial agents and retain original efficacy in several weeks.In addition,this method avoids the tedious operation of culturing microbes in advance before each test and effectively reduce the detection cycle.Therefore,it has a promising application potential in the detection of antimicrobial efficacy and bacterial resistance surveillance.