Electrochemical biosensor array for the identification of microorganisms based on lectin-lipopolysaccharide recognition.

Rapid identification of bacterial strains remains a well-known problem in applied medicine and, for viable pathogens, is an important diagnostic goal. We have investigated an electrochemical biosensor array, in which transduction is based on respiratory cycle activity measurements, where the microorganism's native respiratory chain is interrupted with non-native external oxidants. The selective biochemical recognition agents employed in this study are lectins that, once immobilized, recognize and bind to cell surface lipopolysaccharides. Porous membranes with different surface properties were examined as potential immobilization supports for these lectins. Optimizations performed using concanavalin A and E. coli JM105 show that immobilization methods involving pre-activated membranes significantly reduce the time required to create a functional lectin layer on the membrane surface. Overall, we found general agreement between agglutination test results and the electrochemical assessment of lectin-cell binding. Chronocoulometric measurements were made for cells captured on lectin-modified Immunodyne ABC membranes physically affixed to Pt working electrodes. This lectin-based sensor array was exposed to viable cells of gram-negative and gram-positive bacteria as well as yeast, and chronocoulometric measurements were used to generate a pattern of responses for each organism toward each lectin. Principal component analysis was used to classify the chronocoulometric results for the different microbial strains. With this new method, six microbial species (Baccilus cereus, Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Enterobacter aerogenes, Saccharomyces cerevisiae) were readily distinguished.

Ferricyanide reduction by Escherichia coli: kinetics, mechanism, and application to the optimization of recombinant fermentations.

Ferricyanide reduction was studied by flow injection analysis (FIA) and chronoamperometry (CA) using two host strains and one recombinant strain of E. coli. Samples taken from batch cultures of E. coli JM105 and HB101 showed maximal specific ferricyanide reduction rates in the late exponential phase of growth, with values (micromol/min x g) of 24 (FIA) and 17 (CA) for JM105, and 36 (FIA) for HB101, when shake-flask cultures were sampled, and 70 for HB101, when a chemostat was used to control pH and dissolved oxygen concentration throughout the cultivation. Remarkably higher ferricyanide reduction rates were obtained with HB101 cells cultivated continuously at very slow growth rate, when chilled, resuspended cell samples were incubated for 5 min in solutions containing 10 mM succinate or formate. These compounds are substrates for primary, membrane-bound dehydrogenases that transfer electrons via ubiquinone to the cytochrome oxidase complexes. Apparent Michaelis-Menten kinetics were observed with respect to ferricyanide concentration when 10 mM succinate was included in the assay buffer; apparent Km values of 10.1+/-0.6 mM and 14.4+/-1.2 mM ferricyanide were obtained for exponential- and stationary-phase E. coli JM105, respectively. Cyanide inhibition studies show that ferricyanide is reduced mainly by cytochrome o oxidase in exponentially growing cells. The large difference in ferricyanide reduction rates observed in the absence and presence of succinate and formate were used to signal stationary-phase entry 5 h after induction of recombinant human Cu/Zn superoxide dismutase expression in a batch fermentation of E. coli HMS174(DE3)(pET3ahSOD). This new method can be used as an adjunct to the quantitation of medium components for the optimization of recombinant fermentations.

Rapid antibiotic susceptibility testing via electrochemical measurement of ferricyanide reduction by Escherichia coli and Clostridium sporogenes.

Electrochemical measurement of respiratory chain activity allows rapid and reliable screening for antibiotic susceptibility in microorganisms. Chronoamperometry and chronocoulometry of suspensions of aerobically cultivated E. coli combined with the non-native oxidant potassium hexacyanoferrate(III) (ferricyanide) yield signals for reoxidation of the reduction product ferrocyanide that are much smaller if the E. coli has been incubated briefly with an effective antibiotic compound. Chronocoulometric results, obtained following 20-min incubation with antibiotic and 2-min measurement in assay buffer containing 50 mM ferricyanide and 10 mM succinate, at +0.50 V vs Ag/AgCl at a Pt working electrode, were compared with traditional disk diffusion susceptibility testing, which requires overnight incubation on agar plates; the results show significantly lower accumulation of ferrocyanide in all cases in which growth inhibition was observed in the disk diffusion assay. A range of antibiotic compounds (13) were examined that possess different mechanisms of action. Quantitative determination of IC50 values for penicillin G and chloramphenicol yielded values that were 100-fold higher than those obtained by standard turbidity methods after 10-h incubation; this is likely a result of the very brief (10 min) exposure time to the antibiotics. Addition of 5 microM 2,6-dichlorophenolindophenol, a hydrophobic electron-transfer mediator, to the assay mixture allowed susceptibility testing of a Gram-positive obligate anaerobe, Clostridium sporogenes. This rapid new assay will facilitate clinical susceptibility testing, allowing appropriate treatment virtually as soon as a clinical isolate can be obtained.

A synthetic cysteine oxidase based on a ferrocene-cyclodextrin conjugate.

We report a novel synthetic cysteine oxidase consisting of a ferrocene-beta-cyclodextrin conjugate in which the ferrocene moiety is bound to the secondary hydroxyl side of the cyclodextrin cavity through an ethylenediamine linker. Cysteine oxidation occurs after the ferrocene group is electrochemically oxidized to the ferricinium form, and this generates a voltammetric electrocatalytic wave, the magnitude of which is related to the rate constant for cysteine oxidation. Comparison of cysteine oxidation rates for the primary and secondary beta-cyclodextrin derivatives (105 and 1470 M-1 s-1, respectively) shows that the secondary derivatives are more effective synthetic enzymes. Substrate selectivity of the secondary derivative is demonstrated by comparison of oxidation rates for cysteine (1470 M-1 s-1) and glutathione (260 M-1 s-1) at pH 7.0. The rate constant for cysteine oxidation was 3-fold higher at pH 8.0. With a constant synthetic enzyme concentration, electrocatalytic limiting currents increased linearly with increasing cysteine concentration to a maximum at 6 mM cysteine; above this concentration, the current decreased significantly. These and other results suggest that product inhibition of the catalytic cycle occurs as a result of cystine binding more strongly to the cyclodextrin than cysteine.

Comparison of an intercalating dye and an intercalant-enzyme conjugate for DNA detection in a microtiter-based assay.

Two methods have been developed for the detection of DNA immobilized on the surface of microtiter wells. An intercalating dye, 3,6-diaminoacridine, is used in stain and rinse solutions, so that measured absorbance values (450 nm) reflect the sum of DNA-bound and free dye. With diaminoacridine, signal increases of 0.056 +/- 0.010 were achieved on immobilizing double-stranded calf thymus DNA. An intercalant-enzyme conjugate, consisting of an average of four daunomycin moieties covalently bound to each glucose oxidase, was shown to provide a 10-fold signal enhancement (optimum 0.25 microM, with rinsing and peroxidase-o-dianisidine detection) compared to diaminoacridine, due to catalytic amplification; signals of 0.50 +/- 0.05 were obtained. This conjugate possesses 56% of the activity of native glucose oxidase and was prepared using water-soluble carbodiimide and N-hydroxysuccinimide reagents. Single-stranded DNA was immobilized onto avidin-coated polystyrene plates and commercially available (Covalink) plates possessing secondary amine groups. Following hybridization with complementary DNA, detection was performed with the daunomycin-glucose oxidase conjugate. Both immobilization methods showed optimum DNA concentrations of 0.10 microgram/mL, and maximum signal intensities were obtained when > 0.5 microgram/mL complementary DNA was present in the hybridization solution. Some nonspecific binding of the intercalant-enzyme conjugate was suggested by results obtained with avidin-coated polystyrene plates, but not with Covalink plates.

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis.

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-beta-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2 micrograms/ml. The calibration curves are linear from 0.4 to 16 micrograms/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.

Targeting glucose oxidase at aspartate and glutamate residues with organic two-electron redox mediators.

The bimolecular rate constants for the reactions of five organic two-electron redox mediators with reduced glucose oxidase (GOx) were determined by measuring voltammetric electrocatalytic currents at glassy carbon electrodes in the presence of excess glucose under anaerobic conditions. The mediators studied were thionine, brilliant cresyl blue, azure A, daunomycin, and dopamine, and the bimolecular rate constants for electron transfer between GOx and the oxidized mediator (M-1 s-1) are 1.6 x 10(4), 4.0 x 10(2), 9.8 x 10(2), 9.0 x 10(3), and 1.2 x 10(6), respectively. GOx was covalently derivatized using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysulfosuccinimide to form amide bonds between the aliphatic primary amine groups on daunomycin and dopamine and carboxylate side chains of aspartate and glutamate residues. Derivatives with 2.5 +/- 0.1 daunomycin groups and 4 +/- 1 dopamine groups were obtained, with activities of 50% and 75%, respectively, relative to native GOx in a dye-peroxidase assay. Although the daunomycin derivative did not show measurable intramolecular electron-transfer rates, the dopamine derivative rapidly transfers electrons from active-site FADH2 groups to the oxidized (quinone) form of dopamine. Because the heterogeneous oxidation of dopamine is relatively slow, the currents measured at +0.75 V vs Ag/AgCl were not at their limiting (plateau) values, and only a minimum value of the intramolecular rate constant (4.5 s-1) could be determined. This value is > 20 times larger than values obtained for GOx-ferrocene derivatives in which surface lysine residues were covalently modified using identical coupling reagents and similar reaction conditions. This work shows that targeting GOx carboxylate groups with electron-transfer mediators may represent a promising approach to the design of reagentless glucose biosensors.

Voltammetric DNA biosensor for cystic fibrosis based on a modified carbon paste electrode.

Carbon paste electrodes modified by the inclusion of either octadecylamine or stearic acid were used as solid phases to which DNA was covalently bound. Immobilized DNA was detected by voltammetry of solutions containing submillimolar quantities of Co(bpy)3(ClO4)3, Co(phen)3(ClO4)3, and Os(bpy)3-Cl2 (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline), all of which associate reversibly with immobilized DNA and yield increased peak currents at DNA-modified electrodes. Immobilization onto octadecylamine-modified electrodes was performed using a water-soluble carbodiimide, and at high DNA concentrations in the reaction mixture, it resulted in visible polymerization of DNA on the surface. Optimization of the deoxyguanosine- (dG-) selective immobilization reaction for stearic acid-modified electrodes, using water-soluble carbodiimide and N-hydroxysulfosuccinimide reagents to activate carboxylate groups on the surface, yielded conditions of 4.5% (w/w) stearic acid and 10 micrograms/mL DNA. Polythymidylic acid of 4000-base average length (poly(dT)4000) was immobilized at stearic acid-modified electrodes following enzymatic elongation with dG residues at the 3'-end. These DNA-modified electrodes were used to study hybridization with analyte poly(dA)4000 by in situ voltammetry of 60 microM Co(bpy)3(ClO4)3 at low ionic strength (20 mM NaCl), and by voltammetry of the same complex, following exposure of the electrode to poly(dA)4000 in a separate hybridization step conducted at high ionic strength (0.5 M NaCl). Results indicate slow (> or = 1 h) hybridization at low ionic strength and fast (< or = 10 min) hybridization at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence-selective biosensor for DNA based on electroactive hybridization indicators.

Deoxyribonucleic acid was covalently immobilized onto oxidized glassy carbon electrode surfaces that had been activated using 1-[3-(dimethylamino)-propyl]-3-ethylcarbodimide hydrochloride and N-hydroxysulfosuccinimide. This reaction is selective for immobilization through deoxyguanosine (dG) residues. Immobilized DNA was detected voltammetrically, using tris (2,2'-bipyridyl)cobalt(III) perchlorate and tris (1,10-phenanthroline)cobalt(III) perchlorate (Co(bpy)3(3+) and Co(phen)3(3+). These complexes are reversibly electroactive (1e-) and preconcentrate at the electrode surface through association with double-stranded DNA. Voltammetric peak currents obtained with a poly(dG)poly(dC)-modified electrode depend on [Co(bpy)3(3+)] and [Co(phen)3(3+)] in a nonlinear fashion and indicate saturation binding with immobilized DNA. Voltammetric peak currents for Co(phen)3(3+) reduction were used to estimate the (constant) local DNA concentration at the modified electrode surface; a binding site size of 5 base pairs and an association constant of 1.74 x 10(3) M(-1) yield 8.6 +/- 0.2 mM base pairs. Cyclic voltammetric peak separations indicate that heterogeneous electron transfer is slower at DNA-modified electrodes than at unmodified glassy carbon electrodes. A prototype sequence-selective DNA sensor was constructed by immobilizing a 20-mer oligo (deoxythymidylic acid) (oligo(dT)20), following its enzymatic elongation with dG residues, which yielded the species oligo(dT)20(dG)98. Cyclic voltammograms of 0.12 mM Co(bpy)3(3+) obtained before and after hybridization with poly-(dA) and oligo(dA)20 show increased cathodic peaks after hybridization. The single-stranded form is regenerated on the electrode surface by rinsing with hot deionized water. These results demonstrate the use of electroactive hybridization indicators in a reusable sequence-selective biosensor for DNA.

Rotating disc electrode characterization of immobilized glucose oxidase.

The kinetic properties of glucose oxidase (EC 1.1.3.4) which has been covalently immobilized to a rotating glassy carbon electrode surface have been investigated. Analysis of the rotation rate dependence of the hydrogen peroxide-derived current suggests that oxygen mass transport to the enzyme-electrode surface is rate controlling at low rotation rates. Only as the diffusion layer approaches zero thickness (i.e., infinitely fast rotation rate) does mass transport become unimportant. A diffusion-free glucose Km for air-saturated buffer is found to be 66 mM using this methodology. The importance of mass transport restrictions in two-substrate enzymes such as glucose oxidase is discussed in the context of biosensor design.

Conductometric transducers for enzyme-based biosensors.

The use of alternating current conductometric transducers in biosensing devices has been investigated for urea and D-amino acid sensors using the enzyme systems urease and D-amino acid oxidase/catalase. Transducers with copper and platinum electrodes were constructed and characterized, and two enzyme immobilization methods were tested. Detection limits of 1 x 10(-6)M and linear ranges of 2 orders of magnitude were routinely achieved for these model sensors with enzymes covalently immobilized on collagen films.