Development of an electrochemical biosensor for the rapid identification and quantitation of microorganisms.

摘要

We have investigated a new type of biosensor that is based on respiratory cycle activity measurements, by interrupting the microorganism's native respiratory chain with non-native external oxidants.;The respiratory cycle activity of E. coli JM105 is studied electrochemically by flow injection analysis (FIA) and chronoamperometry (CA) using ferricyanide and other electron-transfer mediators. Apparent Michaelis-Menten kinetics are observed when 10 mM succinate is included in the assay buffer, with obtained Km values of 10.1 +/- 0.6 mM and 14 mM ferricyanide for exponential and stationary phase E. coli JM105, respectively. In addition, cyanide inhibition studies with exponential phase E. coli show that ferricyanide is reduced mainly by cytochrome o oxidase (86%).;A range of antibiotic compounds (13) were examined by chronocoulometry and compared to standard agar disk-diffusion testing. Chronocoulometric results, obtained following 20 min incubation with antibiotic and 2 min measurement (at +0.5 V vs Ag/AgCl at a Pt working electrode) in assay buffer (300 muL) containing 50 mM ferricyanide and 10 mM succinate, yield 100% efficiency, specificity and sensitivity. Quantitative determination of IC50 values for penicillin G and chloramphenicol yield values that are 100-fold higher than those obtained by standard turbidity methods (10 h). Further, the addition of 5 muM 2,6-dichlorophenolindophenol, a hydrophobic electron-transfer mediator, to the assay mixture allows susceptibility testing of a Gram-positive obligate anaerobe, Clostridium sporogenes. The rapid, new low-volume assay will facilitate clinical susceptibility testing, allowing appropriate treatment as soon as a clinical isolate can be obtained.;The application of membranes that feature different surfaces was examined as potential immobilization matrices for lectins, where immobilized lectins recognize and bind to cell-surface lipopolysaccharides in a sensor array. The application of a variety of lectins as recognition agents was then used to assess their usefulness in a sensor array. A biosensor that rapidly identifies pathogens, along with the assessment of antibiotic susceptibilities, will yield a useful and complete tool for medical diagnostics. (Abstract shortened by UMI.)