聚中性红修饰玻碳电极的制备及其在抗菌药物有效性检测中的应用

采用电聚合法制备了聚中性红修饰玻碳电极(PNR/GCE),运用循环伏安法(CV)对修饰电极进行表征,并以CV图中的峰电流为指标,研究了中性红浓度、扫描段数、缓冲液pH值及支持电解质NaNO_3浓度对聚合过程的影响,筛选了最佳聚合条件。继而借助中性红可作为电子媒介体的特性,以E.coli为模型微生物,通过考察E.coli活性与抗菌药物浓度的关系,构建了一种新型体外药效检测的方法,用于抗菌药物有效性的快速检测。以中性红浓度0.5 mmol/L,扫描段数Segment 30,缓冲液pH 7.0,NaNO_3浓度0.5 mol/L为最佳电极修饰条件。选择菌悬液吸光度(OD₆₀₀) 0.5,反应时间40 min,以标准葡萄糖-谷氨酸溶液(GGA,BOD²²⁰)为营养物质,此时检测抗菌药物有效性的灵敏度最高。在最佳实验条件下,头孢哌酮、阿米卡星、庆大霉素、左氧氟沙星4种抗菌药物对E.coli的半数抑菌浓度分别为34.15、27.98、8.83、11.49 mg/L;作为抗革兰氏阳性菌的万古霉素对E.coli的抑菌效果较差。上述结果与经典纸片扩散法具有良好一致性。

Preparation of a Poly Neutral Red Modified Glassy Carbon Electrode and Its Application in Efficacy Detection of Antimicrobials

A poly neutral red modified glassy carbon electrode(PNR/GCE) was prepared by electropolymerization,and characterized by cyclic voltammetry(CV).The peak current in cyclic voltammogram as an indicator was used to investigate the effects of neutral red(NR) concentration,number of scanning segments,pH value of buffer and concentration of supporting electrolyte NaNO3 on the polymerization process.Subsequently,the optimum conditions for polymerization were selected.Then a new method for the rapid detection of antimicrobial efficacy in vitro was developed with NR as an electron mediator and E-coli as a model microorganism to investigate the relationship between activity of E-coli and concentration of antibacterials.The optimum conditions for electrode modification were as follows:concentration of neutral red:0.5 mmol/L,scan segment:30,buffer:pH 7.0,supporting electrolyte:0.5 mol/L NaNO3.The highest sensitivity for the detection of antibacterials efficacy was obtained when a final absorbance value(OD600 ) for bacterial suspension of 0.5,a standard glucose-glutamic acid solution(GGA,BOD220) as nutrition and an incubating time of 40 min were selected.Under the optimum experimental conditions,the half-inhibitory concentrations of cefoperazone,amikacin,gentamycin and levofloxacin on E-coli cells were 34.15,27.98,8.83 and 11.49 mg/L,respectively.However,vancomycin,as only an antibiotic affecting gram positive bacteria,showed a poor inhibition on E-coli cells.The results obtained above were in good agreement with those obtained by classical disk diffusion method.