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.

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.

This work examines the fabrication regime and the properties of polymer-enzyme thin-films adsorbed onto conductive substrates (graphite or gold). The films are formed via two-steps, sequential adsorption of poly(n-butylmethacrylate)-block-poly(N,N-dimethylaminoethyl methacrylate) (PnBMA-b-PDMAEMA) diblock copolymer micelles (1st step of adsorption), followed by the enzyme choline oxidase (ChO) (2nd step of adsorption). The solution properties of both adsorbed components are studied and the pH-dependent step-by-step fabrication of polymer-enzyme biosensor coatings reveals rather drastic differences in their enzymatic activities in dependence on the pH of both adsorption steps. The resulting hybrid thin-films represent highly active biosensors for choline with a low detection limit of 30 nM and a good linearity in a range between 30 nM and 100 μM. The sensitivity is found to be 175 μA mM(-1) cm(-2) and the operational stability of the polymer-enzyme thin-films can be additionally improved via enzyme-to-enzyme crosslinking with glutaraldehyde.

The benefits of stimuli‐responsiveness of microgels for surface modification and further engineering of microgel‐enzyme biosensor constructs are highlighted. Accordingly, the adsorption behavior of the pH‐ and temperature‐sensitive poly(N‐isopropylacrylamide‐co‐N‐(3‐dimethylaminopropyl)methacrylamide) microgel (P(NIPAM‐co‐DMAPMA) microgel) is examined by means of atomic force microscopy (AFM) and quartz‐crystal microbalance with dissipation monitoring (QCM‐D). An enhanced adsorption of the microgel onto relatively hydrophobic surfaces is observed at elevated pH and temperature as manifested by a high surface coverage and a large adsorbed mass. This is a consequence of pronounced hydrophobization of the microgel as revealed by means of dynamic light scattering (DLS) and turbidimetry (cloud points). The subsequent electrostatic loading of the surface‐bound microgel with enzymes is examined by means of QCM‐D and demonstrates a highly‐capacious integration of biomolecules into the microgel films. Finally, the biosensor responses of the microgel‐enzyme films fabricated onto screen‐printed graphite electrodes are assessed to demonstrate a biorelated application. Thus, a comprehensive conceptual understanding is attained on the key points: (1) how the stimuli‐responsive properties of the microgel correlate with its amount deposited onto the surface, (2) what determines the highly‐capacious loading of the surface‐bound microgel with the enzymes, and ultimately (3) how the tandem of the stimuli‐sensitive microgels and enzymes can be used for easy engineering of biosensor systems.