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

Abstract

The diagnostics field is undergoing rapid changes and biosensors are a key technology because of their speed and simplicity. Rapid identification of bacterial strains remains a well-known problem in applied medicine; thus, the rapid detection of viable pathogens is an important diagnostic goal. 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 kinetic 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%).

The rapid assessment of the respiratory cycle activity allows rapid and reliable screening for antibiotic susceptibility in microorganisms. Chronoamperometry and chronocoulometry of aerobically cultivated E. coli yield signals for reoxidation of the reduction product ferrocyanide that are much smaller if E. coli has been briefly incubated with an effective antibiotic compound. 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 uL) 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 uM 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.

In the development of biosensors, biochemically selective recognition agents must be associated intimately with a transducer. 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. Optimizations performed using the model lectin Concanavalin A show that immobilization methods involving preactivated membranes significantly reduce the time required to create a functional lectin layer on the membrane surface. Completeness and homogeneity of the applied lectin layer were confirmed by atomic force microscopy.

The application of a variety of lectins as recognition agents was the used to assess their usefulness in a sensor array. Chronocoulometric measurements of cells captured on the lectin modified membranes were performed in an assay buffer (200 uL) containing 50 mM ferricyanide, 0.1 mM menadione, 10 mM succinate and 10 mM formate. The implementation of factor analysis (pattern recognition) allowed the discrimination of six microbial strains (B. cerfeus, S. aureus, P. vulgaris, E. coli, E. aerogenes and S. cerevisia). A biosensor that rapidly identifies pathogens, along with the assessment of antibiotic susceptibilities, will yield a useful and complete tool for medical diagnostics.