We present the construction of layer-by-layer (LbL) assemblies of polyethylenimine and urease onto reduced-graphene-oxide based field-effect transistors (rGO FETs) for the detection of urea. This versatile biosensor platform simultaneously exploits the pH dependency of liquid-gated graphene-based transistors and the change in the local pH produced by the catalyzed hydrolysis of urea. The use of an interdigitated microchannel resulted in transistors displaying low noise, high pH sensitivity (20.3µA/pH) and transconductance values up to 800 µS. The modification of rGO FETs with a weak polyelectrolyte improved the pH response because of its transducing properties by electrostatic gating effects. In the presence of urea, the urease-modified rGO FETs showed a shift in the Dirac point due to the change in the local pH close to the graphene surface. Markedly, these devices operated at very low voltages (less than 500mV) and were able to monitor urea in the range of 1-1000µm, with a limit of detection (LOD) down to 1µm, fast response and good long-term stability. The urea-response of the transistors was enhanced by increasing the number of bilayers due to the increment of the enzyme surface coverage onto the channel. Moreover, quantification of the heavy metal Cu(with a LOD down to 10nM) was performed in aqueous solution by taking advantage of the urease specific inhibition.

Glucose oxidase (GOx) was covalently modified at pH 7.2 with ferrocenecarboxylic acid (FCA), ferrocenedicarboxylic acid (FDA), and ferroceneacetic acid (FAA) using 1-ethyl-3-( 3-(dimethy1amino)propyl)-carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide to promote selective coupling to surface lysines.Reagent ratios were varied to obtain derivatives with 5-12 ferrocene groups per GOx dimer and 260% activity. For comparison, GOx was derivatized with FCA in the presence of 3 M urea using only EDC as a promoter. Varying reagent ratios yielded derivatives with 4-39 FCA groups per GOx and 530% activity; linear sweep voltammetry results showed a slow but readily detectable release of FCA upon storage of these derivatives. Tryptophan fluorescence quenching in two media, 0.1 M phosphate buffer (pH 7.0) and 8 M urea, confirmed that GOx was covalently modified and not merely associated with ferrocene. In all cases, the GOx derivatives exhibited significantly greater quenching than controls containing native GOx with free ferrocenes. The results of voltammetric dilution experiments (performed in oxygen-free solutions in the presence of excess glucose) were consistent with rate-limiting intramolecular electron transfer from the reduced flavin centers to bound ferricinium. Using an expression derived here, values between 0.16 and 0.90 s-1 were obtained for intramolecular electron transfer in the FCA derivatives, suggesting that the location (rather than the number) of bound ferrocene groups is rate-determining. Approximately lo3-fold slower intramolecular electron transfer was measured in an FDA derivative, consistent with fluorescence quenching data which indicated that bound FDA is more solvent-exposed than bound FCA. The results of lysine-targeted modification of GOx are interpreted in light of the recently published 3-D structure of GOx; since the critical flavin-lysine separations are all >23 A, an alternative approach is necessary to obtain GOx derivatives for use in a practical, reagentless glucose sensor.

Layer-by-layer supramolecular structures composed of alternate layers of negatively charged enzymes and cationic redox polyelectrolyte have been assembled. Glucose oxidase (GOx), lactate oxidase (LOx) and soybean peroxidase (SBP) have been electrically wired to the underlying electrode by means of poly(allylamine) with [Os(bpy)2 ClPyCOH]c ovalently attached (PAAÈOs) in organized structures with high spatial resolution. Biotinylated glucose oxidase has also been used to assemble step-by-step on antibiotin goat immunoglobulin (IgG) layers and the enzyme was electrically wired by PAAÈOs. These spatially organized multilayers with mono-and bienzymatic schemes can work efficiently in molecular recognition, redox mediation and generation of an electrical signal. The concentration of redox mediator integrated into the multilayers, obtained from the voltammetric charge and an estimation of the layer thickness, exceeds by 100-fold the amount of deposited enzyme assessed by quartz crystal microbalance. Di †erences in GOx electrical wiring efficiency have been detected with the di †erent assembling strategies. The surface concentration of electrically wired enzyme represents a small proportion of all the enzyme molecules present in the multilayers which can be oxidized by the soluble mediator PyCOOH]Cl. This proportion, as well as the rate of [Os(bpy) 2 Cl FADH 2 oxidation by PAAÈOs, increases with the number of electrically wired enzyme layers and with the spatial accessibility of the Os moiety to the enzyme active center.

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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.

Single‐layer and bilayer bienzyme electrodes based on the combination of a three‐dimensional (3‐D) redox epoxy network that electrically connects redox centers of bound horseradish peroxidase (HRP) to electrodes with a hydrogen peroxide generating enzyme, the redox centers of which are not connected to the redox‐epoxy network, are described. In the single‐layer electrodes, H2O2 generated by the first enzyme oxidizes the second enzyme HRP, which oxidizes the redox polymer network that is electrochemically reduced at 0 mV saturated calomel electrode (SCE). When the redox centers of the H2O2 generating enzyme are also electrically connected to the redox‐epoxy network, the substrate reduced redox centers are oxidized by the redox polymer network, thus lowering the cathodic current. Such attenuation is avoided in bilayer electrodes, where the H2O2 producing enzyme and the redox‐epoxy‐HRP network are not electrically connected. The single‐layer bienzyme electrodes extend the range of amperometric biosensors based on directly redox‐epoxy “wired” enzymes to enzymes that are difficult to electrically connect to redox polymer networks and whose preferred or only cosubstrate is oxygen. For the difficult to wire enzyme‐choline oxidase, the cathodic current density in the single‐layer peroxidase and choline oxidase containing electrode is 80 μA cm−2 at 10 mM choline concentration, while the anodic current density of the directly wired enzyme is only 5 μA cm−2. Alcohol oxidase is not electrically connected to the wiring 3‐D redox‐epoxy network. The anodic current density of its redox‐epoxy wired electrodes is close to nil, while the cathodic current density, observed in alcohol oxidase and wired peroxidase containing single‐layer electrodes at 10 mM ethanol, is 5 μA cm−2. When well‐wired enzymes, such as glucose oxidase or lactate oxidase, are utilized in single‐layer electrodes, limiting cathodic current densities of 60 μA cm−2 are observed for both enzymes. These currents are much lower than those observed in the directly wired enzyme anodes.

Pyridine-based osmium complexes bearing either a carboxylate or aldehyde group were covalently attached to glucose oxidase and were shown to work as mediators for the reoxidation of the enzyme. For the complex containing the carboxylate group, the binding was made through carbodiimide coupling to the amine residues in the protein. For the complex containing the aldehyde group, the reductive coupling was carried out by condensation with the amino groups on the protein in the presence of sodium cyanoborohydride. Electrochemical studies show evidence for both intramolecular and intermolecular redox mediation for the electrochemical reoxidation of the modified glucose oxidases in the presence of glucose. The modified enzymes adsorbed on glassy carbon and platinum show different electrochemical responses for the two electrode materials, suggesting that orientation of the adsorbed enzyme is induced due to the interaction of the osmium complex with the different surfaces. Construction of enzyme switches based on these modified enzymes was carried out, and their responses were compared with those obtained using native glucose oxidase and a soluble redox mediator.

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