Monitoring the cholesterol level is of great importance, especially for people with high risk of developing heart disease. Here we report on reagentless cholesterol detection in human plasma with a novel single-enzyme, membrane-free, self-powered biosensor, in which both cathodic and anodic bioelectrocatalytic reactions are powered by the same substrate. Cholesterol oxidase was immobilised in a sol-gel matrix on both the cathode and the anode. Hydrogen peroxide, a product of the enzymatic conversion of cholesterol, was electrocatalytically reduced, by the use of Prussian blue, at the cathode. In parallel, cholesterol oxidation catalysed by mediated cholesterol oxidase occurred at the anode. The analytical performance was assessed for both electrode systems separately. The combination of the two electrodes, formed on high surface-area carbon cloth electrodes, resulted in a self-powered biosensor with enhanced sensitivity (26.0 mA M -1 cm -2 ), compared to either of the two individual electrodes, and a dynamic range up to 4.1 mM cholesterol.Reagentless cholesterol detection with both electrochemical systems and with the self-powered biosensor was performed and the results were compared with the standard method of colorimetric cholesterol quantification.

Single molecule enzymology provides an opportunity to examine details of enzyme mechanisms that are not distinguishable in biomolecule ensemble studies. Here we report, for the first time, detection of the current produced in an electrocatalytic reaction by a single redox enzyme molecule when it collides with an ultramicroelectrode. The catalytic process provides amplification of the current from electron-transfer events at the catalyst leading to a measurable current. This new methodology monitors turnover of a single enzyme molecule. The methodology might complement existing single molecule techniques, giving further insights into enzymatic mechanisms and filling the gap between fundamental understanding of biocatalytic processes and their potential for bioenergy production.

High potential multicopper oxidases (MCOs) have T1 reduction potentials >600 mV (vs normal hydrogen electrode), making them important catalysts for O 2 reduction in various biotechnological applications. The oxygen reduction mechanism for the low-potential MCOs is well-characterized; however, O 2 reactivity of high potential MCOs is not well understood. In this study, we have shown that in the laccase from Trametes versicolor, the T1 redox potential is increased by ~350 mV over that of the low-potential MCOs corresponding to an 8 kcal/mol decrease in the driving force, and thus exhibits a slower intramolecular electron transfer (IET) rate to the trinuclear Cu cluster (TNC) in the native intermediate (NI), relative to the low potential MCO from Rhus vernicifera laccase. This IET rate is, however, >10 2 times faster than the decay rate of the NI, demonstrating that this intermediate form of the enzyme is catalytically relevant enabling fast turnover. However, in contrast to the low potential MCOs where T1 reduction by substrate is rate limiting, the rate limiting step in turnover of high potential MCOs is the first IET to NI. Part of the reduction potential difference of the T1 sites in high vs. low potential MCOs is balanced by an ~100 mV higher reduction potential of NI due to the more positive protein environment in the vicinity of the TNC.

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Anodization of a graphene monolayer on silicon carbide was monitored with electrochemical impedance spectroscopy. Structural and functional changes of the material were observed by Raman spectroscopy and voltammetry. A 21 fold increase of the specific capacitance of graphene was observed during the anodization. An electrochemical kinetic study of the Fe(CN)6 3-/4redox couple showed a slow irreversible redox process at the pristine graphene, but after anodization the reaction rate increased by several orders of magnitude. On the other hand, the Ru(NH3)6 3+/2+ redox couple proved to be insensitive to the activation process. The results of the electron transfer kinetics correlate well with capacitance measurements. The Raman mapping results suggest that the increased specific capacitance of the anodized sample is likely due to a substantial increase of electron doping, induced by defect formation, in the monolayer upon anodization. The doping concentration increased from less than 1×10 13 of the pristine graphene to 4-8×10 13 of the anodized graphene.

Wiring glucose oxidase in the membrane with an immobilized mediator is possible due to the diffusion ability of the latter, if the enzyme containing membrane is formed according to the proposed protocol, including exposing proteins to water-organic mixtures with the high content of organic solvent. In the course of the study, the new glucose oxidase mediator, unsubstituted phenothiazine, was discovered. The diffusion coefficient of the mediator in the resulting membrane is independent of the presence of enzyme. The cyclic voltammograms of the enzyme electrode after appearance of the only glucose in solution obtain a well-defined catalytic shape, which is normally observed for both the enzyme and the mediator in solution. Analytical performances of the resulting biosensor are comparable to the advanced second generation ones, which, however, require covalent linking of the mediator either to the membrane forming polymer or to the enzyme. Even without such covalent linking, the reported biosensor is characterized by an appropriate long-term operational stability allowing reagentless sensing.

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The mediation of oxidases: glucose oxidase (GOx), lactate oxidase (LOx) and cholesterol oxidase (ChOx) by a new electron shuttling mediator, unsubstituted phenothiazine (PTZ), was studied. Cyclic voltammetry and rotating-disk electrode measurements in nonaqueous media were used to determine the diffusion characteristics of the mediator and the kinetics of its reaction with GOx, giving a second-order rate constant of 7.6 × 10 3 M -1 s -1 to 2.1 × 10 4 M -1 s -1 for water -acetonitrile solutions containing 5 % to 15 % water. These values are in the range reported for commonly used azine-type mediators, indicating that PTZ is able to function as an efficient mediator. PTZ and GOx, LOx and ChOx were successfully co-immobilized in sol-gel membrane on a screen-printed electrode to construct glucose, lactate and cholesterol biosensors, respectively, which were then optimised in terms of stability and sensitivity. The electrocatalytic oxidation responses showed a dependence on substrate concentration ranging from 0.6 to 32 mM for glucose, from 19 to 565 mM for lactate and from 0.015 to 1.0 mM for cholesterol detection.Oxidation of substrates on the surface of electrodes modified with PTZ and enzyme membrane was investigated with double-step chronoamperometry and the results showed that the PTZ 2 displays excellent electrochemical catalytic activities even when immobilised on the surface of the electrode. Highlights: The kinetics of the electrocatalytic oxidation of glucose with GOx mediated by a new electron shuttling mediator, PTZ, was studied. PTZ delivers superior potential performance among azine-type redox mediators for GOx. PTZ and GOx, LOx and ChOx were successfully co-immobilized in sol-gel membrane on a screen-printed electrode to construct glucose, lactate and cholesterol biosensors, respectively. Second-order rate constants for the reaction between the mediator and variety of oxidases co-immobilised inside a sol-gel membrane were obtained by chronoamperometry.