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(-)(8) M) and a linear calibration range starting exactly from the detection limit and extending over 6 orders of magnitude of H(2)O(2) concentrations (1 x 10(-)(8) to 1 x 10(-)(2) M), which are the most advantageous analytical performances in hydrogen peroxide electroanalysis.

This work examines the fabrication regime and the properties of microgel and microgel/enzyme thin films adsorbed onto conductive substrates (graphite or gold). The films were formed via two sequential steps: the adsorption of a temperature- and pH-sensitive microgel synthesized by precipitation copolymerization of N-isopropylacrylamide (NIPAM) and 3-(N,N-dimethylamino)propylmethacrylamide (DMAPMA) (poly(NIPAM-co-DMAPMA) at the pH-condition corresponding to its noncharged state (first step of adsorption), followed by the enzyme, tyrosinase, adsorption at the pH-condition when the microgel and the enzyme are oppositely charged (second step of adsorption). The stimuli-sensitive properties of poly(NIPAM-co-DMAPMA) microgel were characterized by potentiometric titration and dynamic light scattering (DLS) in solution as well as by atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) at solid interface. Enhanced deposition of poly(NIPAM-co-DMAPMA) microgel particles was shown at elevated temperatures exceeding the volume phase transition temperature (VPTT). The subsequent electrostatic interaction of the poly(NIPAM-co-DMAPMA) microgel matrix with tyrosinase was examined at different adsorption regimes. A considerable increase in the amount of the adsorbed enzyme was detected when the microgel film is first brought into a collapsed state but then was allowed to interact with the enzyme at T < VPTT. Spongelike approach to enzyme adsorption was applied for modification of screen-printed graphite electrodes by poly(NIPAM-co-DMAPMA)/tyrosinase films and the resultant biosensors for phenol were tested amperometrically. By temperature-induced stimulating both (i) poly(NIPAM-co-DMAPMA) microgel adsorption at T > VPTT and (ii) following spongelike tyrosinase loading at T < VPTT, we can achieve more than 3.5-fold increase in biosensor sensitivity for phenol assay. Thus, a very simple, novel, and fast strategy for physical entrapment of biomolecules by the polymeric matrix was proposed and tested. Being based on this unique stimuli-sensitive behavior of the microgel, this stimulated spongelike adsorption provides polymer films comprising concentrated biomaterial.

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This work examines the adsorption regime and the properties of microgel/enzyme thin films deposited onto conductive graphite-based substrates. The films were formed via two-step sequential adsorption. A temperature- and pH-sensitive poly­(N-isopropylacrylamide)-co-(3-(N,N-dimethylamino)­propylmethacrylamide) microgel (poly­(NIPAM-co-DMAPMA microgel) was adsorbed first, followed by its interaction with the enzymes, choline oxidase (ChO), butyrylcholinesterase (BChE), or mixtures thereof. By temperature-induced stimulating both (i) poly­(NIPAM-co-DMAPMA) microgel adsorption at T > VPTT followed by short washing and drying and then (ii) enzyme loading at T < VPTT, we can effectively control the amount of the microgel adsorbed on a hydrophobic interface as well as the amount and the spatial localization of the enzyme interacted with the microgel film. Depending on the biomolecule size, enzyme molecules can (in the case for ChO) or cannot (in the case for BChE) penetrate into the microgel interior and be localized inside/outside the microgel particles. Different spatial localization, however, does not affect the specific enzymatic responses of ChO or BChE and does not prevent cascade enzymatic reaction involving both BChE and ChO as well. This was shown by the methods of electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and amperometric analysis of enzymatic responses of immobilized enzymes. Thus, a novel simple and fast strategy for physical entrapment of biomolecules by the polymeric matrix was proposed, which can be used for engineering systems with spatially separated enzymes of different types.

A versatile guest matrix was fabricated from a temperature- and pH-sensitive poly(N-isopropylacrylamide)-co-(3-(N,N-dimethylamino)propylmethacrylamide) microgel (poly(NIPAM-co-DMAPMA), MG) for the gentle incorporation of butyrylcholinesterase (BChE). The microgel/BChE films were built up on a surface of graphite-based screen-printed electrodes (SPEs) premodified with MnO nanoparticles via a two-step sequential adsorption under careful temperature and pH control. On this basis, a rather simple amperometric biosensor construct was formed, which uses butyrylthiocholine as BChE substrate with subsequent MnO-mediated thiocholine oxidation at a graphite-based SPE. The complexation of BChE with the microgel was found to be safe and effective, as confirmed by a high operational and rather good long-term storage stability of the resultant SPE-MnO/MG/BChE biosensors. The small mesh size of the microgel with respect to the size of BChE results in a predominant outer complexation of BChE within the dangling chains of the microgel rather than a deep penetration of the enzyme into the microgels. Given such surface localization, BChE is easily accessible both for the substrate and for cholinesterase inhibitors. This was supported by the analytical characteristics of the SPE-MnO/MG/BChE biosensor that were examined and optimized both for the substrate and for the enzyme detection. The SPE-MnO/MG/BChE biosensor enabled precision detection of organophosphorus pesticides (diazinon(oxon), chlorpyrifos(oxon)) in aqueous samples with minimized interference from extraneous (nonanalyte) substances (e.g., ions of heavy metals). The detection limits for diazinon(oxon) and chlorpyrifos(oxon) were estimated to be as low as 6 × 10 M and 8 × 10 M, respectively, after 20 min of preincubation with these irreversible inhibitors of BChE.

We highlight microgel/enzyme thin films that were deposited onto solid interfaces via two sequential steps, the adsorption of temperature- and pH-sensitive microgels, followed by their complexation with the enzyme choline oxidase, ChO. Two kinds of functional (ionic) microgels were compared in this work in regard to their adsorptive behavior and interaction with ChO, that is, poly(N-isopropylacrylamide-co-N-(3-aminopropyl)methacrylamide), P(NIPAM-co-APMA), bearing primary amino groups, and poly(N-isopropylacrylamide-co-N-[3-(dimethylamino) propyl]methacrylamide), P(NIPAM-co-DMAPMA), bearing tertiary amino groups. The stimuli-sensitive properties of the microgels in the solution were characterized by potentiometric titration, dynamic light scattering (DLS), and laser microelectrophoresis. The peculiarities of the adsorptive behavior of both the microgels and the specific character of their interaction with ChO were revealed by a combination of surface characterization techniques. The surface charge was characterized by electrokinetic analysis (EKA) for the initial graphite surface and the same one after the subsequent deposition of the microgels and the enzyme under different adsorption regimes. The masses of wet microgel and microgel/enzyme films were determined by quartz crystal microbalance with dissipation monitoring (QCM-D) upon the subsequent deposition of the components under the same adsorption conditions, on a surface of gold-coated quartz crystals. Finally, the enzymatic responses of the microgel/enzyme films deposited on graphite electrodes to choline were tested amperometrically. The presence of functional primary amino groups in the P(NIPAM-co-APMA) microgel enables a covalent enzyme-to-microgel coupling via glutar aldehyde cross-linking, thereby resulting in a considerable improvement of the biosensor operational stability.

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