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We report on the novel protocol for enzyme immobilization into gel of siloxanes using water--organic mixtures with the high content of organic solvent as a reaction medium. Hydrolysis of alkoxysilanes carried out without excessive dilution with water resulted in more active and stable enzyme containing membranes. Immobilization of an inherently labile lactate oxidase according to the proposed sol-gel protocol over Prussian Blue modified electrode resulted in an advanced lactate biosensor characterized with a sensitivity of 0.18 A[M.sup.1] [cm.sup.-2] in the flow injection analysis (FIA) mode over a wide dynamic range. A comparison with the known sensors has shown that analytical performances of the elaborated lactate biosensor are advantageous over both published systems and commercialized devices. The biosensor shows an appropriate stability and is suitable for clinical analysis (including noninvasive diagnostics) and food quality control. 10.1021/ac9027615

Electropolymerizing the prosthetic group (flavin adenine dinucleotide, FAD) responsible in the active sites of dehydrogenases for [NAD.sup.+]|NADH regeneration, we succeeded in mimicking enzyme activity. Poly(FAD) characterized by an additional polymer-type redox reaction has been discovered as a highly effective electrocatalyst for NADH oxidation: operating at the lowest potentials reported for NADH transducers (0.00 V, pH 7.4), poly-(FAD) is characterized by the electrochemical rate constant of 1.8 [+ or -] 0.6 x [10.sup.-3] cm [s.sup.-1], which is at the level of the NADH mass-transfer constant. Flow injection analysis of NADH with the poly(FAD)-modified wall-jet electrode as a detector has been characterized by a linear calibration range prolonged down to 5 x [10.sup.-7] M and a sensitivity of 0.08 A [M.sup.-1] [cm.sup.-2], which taking into account the dispersion coefficient (~3), is at the diffusion-limiting value. In contrast to the low molecular weight mediators able to exhibit similar electrocatalytic properties, poly(FAD)-modified electrodes are characterized by the dramatically improved stability and, thus, can be considered as the most advantageous NADH transducers for analytical chemistry.

We propose to form nanoelectrode arrays by deposition of the electrocatalyst through lyotropic liquid crystalline templates onto inert electrode support. Whereas Prussian Blue is known to be a superior electrocatalyst in hydrogen peroxide reduction, carbon materials used as electrode support demonstrate only a minor activity. We report on the possibility for nanostructuring of Prussian Blue by its electrochemical deposition through lyotropic liquid crystalline templates, which is noticed from atomic force microscopy images of the resulting surfaces. The resulting Prussian Blue based nanoelectrode arrays in flow injection analysis mode demonstrate a sub-part-per-billion detection limit (1 x [10.sup.-8] M) and a linear calibration range starting exactly from the detection limit and extending over 6 orders of magnitude of [H.sub.2][O.sub.2] concentrations (1 x [10.sup.-8] to 1 x [10.sup.-2] M), which are the most advantageous analytical performances in hydrogen peroxide electroanalysis.

An optimal environment for glucose oxidase (GOx) in Nation membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water--organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Nation is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx--Nafion membranes. A glucose biosensor was prepared by casting the Gox--Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 [micro]M level, along with high sensitivity (0.05 A [M.sup.-1] [cm.sup.-2]), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar [H.sub.2][O.sub.2] transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.

The specially deposited Prussian Blue denoted as 'artificial peroxidase' was used as a transducer for hydrogen peroxide. The electrocatalyst was stable, highly active, and selective to hydrogen peroxide reduction in the presence of oxygen, which allowed sensing of H(sub 2)O(sub 2) around 0.0 V (Ag/AgCl). Glutamate oxidase was immobilized on the surface of the Prussian Blue-modified electrode in a Nafion layer using a nonaqueous enzymology approach. The calibration range for glutamate in flow injection system was 1 x 10(super -7)-1 x 10(super -4) M. The lowest concentration of glutamate detected (1 x 10(super -7) M) and the highest sensitivity in the linear range of 0.21 A M(super -1) cm(super -2) were achieved. The influence of reductants was practically avoided using the low potential of an indicator electrode (0.0 V Ag/AgCl). The attractive performance characteristics of the glutamate biosensor illustrate the advantages of Prussian Blue-based 'artificial peroxidase' as transducer for hydrogen peroxide detection.

An advanced potentiometric pH transducer based on processible polyaniline (PCPAn) is reported on. Both glassy carbon and screen-printed carbon electrodes modified with PCPAn by dip-coating exhibited a fully reversible potentiometric response of approximately 90 mV/pH unit over the range from pH 3 to 9. Such a significantly higher potentiometric response of PCPAn-modified electrodes as compared with those of existing devices is explained on the basis of the thermodynamics of polyaniline redox reactions. The PCPAn-based pH transducers exhibit both good operational stability and prolonged shelf life and display a negligible response toward singly charged cations. The new thick-film pH transducer was employed for designing a potentiometric biosensor for urea. In the model solution which mimics blood serum, the urea-sensitive electrode has a detection limit of [10.sup.-5] M urea and a maximum response of [approximately]120 mV. The attractive performance characteristics are advantageous over those of existing pH sensors and offer great promise for sensing and biosensing applications.

We propose immobilizing enzymes into Nation membranes by suspending the enzyme in a water-ethanol mixture with a high (>90%) ethanol content, followed by mixing with the dissolved polyelectrolyte, and finally allowing the enzyme-polyelectrolyte solution to dry at the target surface (electrode surface). Since Nation solution was deposited from a solution where it is truly dissolved and without excessive dilution with water, the resulting membranes were more uniform and stable than those otherwise obtained. Enzyme suspensions in concentrated ethanol solutions were prepared without any prior modifications of the protein. The remaining activity after 30 min exposure to such solutions under optimal conditions was up to 100%. The stability of the enzymes in these suspensions was higher than that in aqueous solution. Electrochemical biosensors made accordingly showed a several times increased response compared with those of enzyme electrodes based on the traditional way of using excessive dilution of Nation with water. The remaining activity, after the drying-washing cycle of the enzyme electrode made by enzyme immobilization from concentrated organic solvent, was at least 10 times higher than that of the traditional one.

A first-generation amperometric glucose biosensor based on a Prussian Blue-modified electrode was developed. Prussian Blue was found to be a better electrocatalyst for hydrogen peroxide reduction than platinum. [H.sub.2][O.sub.2] was detected at the Prussian Blue-modified electrode in the presence of oxygen by both electrooxidation and electroreduction. The glucose amperometric biosensor was made by glucose oxidase immobilization onto a Prussian Blue-modified electrode with a Nation layer. The biosensor response exhibited a linear dependence on analyte concentration in the range 1 x [10.sup.-6]-5 x [10.sup.-3] M. The cathodic current density after addition of [10.sup.-6] M glucose was 0.18 [micro]A/[cm.sup.2]. When hydrogen peroxide produced via the enzyme reaction was detected by electroreduction, the biosensor response was independent of reductants. This amperometric biosensor is expected to obey the requirements for noninvasive diagnostics.

Abstract: We report on Prussian Blue based (bio)sensors displaying advantageous analytical performance characteristics when operated both in conventional three‐electrode regime powered by potentiostat and in power generation mode. ‘Self‐powered’ sensors are commonly aimed to produce sufficient energy for signalling and, thus, being energetically autonomous. In order to generate maximum energy output, the electrodes with high potential difference are preferable. In contrast, we propose the systems with the lowest potential difference between the working and the counter electrodes. Short‐circuiting the Prussian Blue modified working and the Ag|AgCl reference electrode, working electrode potential is adjusted to 0.00 V without potentiostat. The efficiency of the (bio)sensors in power generation mode is achieved due to high catalytic activity of Prussian Blue at 0.00 V versus Ag|AgCl reference. Here we discuss the benefits of the Prussian Blue based (bio)sensors operated through power generation in a short‐circuit regime without potentiostat, including an order of magnitude decreased noise and simplified readout. The multimodality of the sensors would provide their versatile functionality for the electroanalysis and elaboration of miniature highly sensitive wearable devices. [ABSTRACT FROM AUTHOR]