Burkhard Schulz’s research while affiliated with Universität Potsdam and other places

This study introduces a thermally responsive, polymer-based electrode system. The key component is a surface-attached, temperature-responsive poly(oligoethylene glycol) methacrylate (poly(OEGMA)) type polymer bearing photoreactive benzophenone and carboxy groups containing side chains. The responsive behavior of the polymer in aqueous media has been investigated by turbidimetry measurements. Polymer films are formed on gold substrates by means of the photoreactive 2-(dicyclohexylphosphino)benzophenone (DPBP) through photocrosslinking. The electrochemical behavior of the resulting polymer-substrate interface has been investigated in buffered [Fe(CN)6]3-/[Fe(CN)6]⁴⁻solutions at room temperature and under temperature variation by cyclic voltammetry (CV). The CV experiments show that with increasing temperature structural changes of the polymer layer occur, which alter the output of the electrochemical signal. Repeated heating/cooling cycles analyzed by CV measurements and pH changes analyzed by quartz crystal microbalance with dissipation monitoring (QCM-D) reveal the reversible nature of the restructuring process. The immobilized films are further modified by covalent coupling of two small biomolecules - a hydrophobic peptide and a more hydrophilic one. These attached components influence the hydrophobicity of the layer in a different way - the resulting change of the temperature-caused behavior has been studied by CV indicating a different state of the polymer after coupling of the hydrophobic peptide.

We report on the pH-dependent bioelectrocatalytic activity of the redox enzyme xanthine dehydrogenase (XDH) in the presence of sulfonated polyaniline PMSA1 (poly(2-methoxyaniline-5-sulfonic acid)-co-aniline). UV-vis spectroscopic measurements with both components in solution reveal electron transfer from the hypoxanthine (HX) reduced enzyme to the polymer. The enzyme shows bioelectrocatalytic activity on ITO electrodes, when the polymer is present. Depending on solution pH different processes can be identified. It can be demonstrated that not only product-based communication with the electrode but also efficient polymer-supported bioelectrocatalysis occur. Interestingly, substrate-dependent catalytic currents can be obtained in acidic and neutral solutions although the highest activity of XDH with natural reaction partners is in the alkaline region. Furthermore, opera-tion of the enzyme electrode without addition of the natural co-factor of XDH is feasible. Finally, macroporous ITO electrodes have been used as immobilization platform for the fabrication of HX-sensitive electrodes. The study shows that the efficient poly-mer/enzyme interaction can be advantageously combined with the open structure of an electrode material of controlled pore size, resulting in a good processability, stability and defined signal transfer in the presence of substrate.

Es wurde ein dreidimensionales Polymerfasernetzwerk aufgebaut, charakterisiert und anschließend daran das Enzym Pyrrolochinolinchinon-abhängige Glukosedehydrogenase (PQQ)GDH gebunden. Das Polymerfasernetzwerk wurde durch Elektrospinnen einer Mischung des Polymers Polyacrylnitril und verschiedener leitfähiger Polymere der Polyanilin-Familie auf Indium-Zinn-Oxid-Elektroden aufgebracht. Die so hergestellten Fasermatten erwiesen sich bei mikroskopischen Untersuchungen gleichförmig präpariert und die Faserdurchmesser bewegten sich im Bereich weniger hundert Nanometer. Das Redoxpaar Kaliumhexacyanoferrat (II/III) zeigte an diesen Polymer-Elektrodenstrukturen eine quasi-reversible Elektrochemie. Bei weitergehenden Untersuchungen an den enzymmodifizierten Fasern ((PQQ)GDH) konnten unter Substratzugabe (Glukose) bioelektrokatalytische Ströme nachgewiesen werden. Das Fasernetzwerk fungiert hier nicht nur als Immobilisierungsmatrix, sondern als auch als Teil des Signalwandlers.

Electrospinning is known as a fabrication technique for electrode architectures that serve as immobilization matrices for biomolecules. The current work demonstrates a novel approach to construct a conductive polymeric platform, capable not only of immobilization, but also of electrical connection of the biomolecule with the electrode. It is produced upon electrospinning from mixtures of three different highly conductive sulfonated polyanilines and polyacrylonitrile on ITO electrodes. The resulting fiber mats are with a well-retained conductivity. After coupling the enzyme pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) to polymeric structures and addition of the substrate glucose an efficient bioelectrocatalysis is demonstrated. Depending on the choice of the sulfonated polyanilline mediatorless bioelectrocatalysis starts at low potentials; no large overpotential is needed to drive the reaction. Thus, the electrospun conductive immobilization matrix acts here as a transducing element, representing a promising strategy to use 3D polymeric scaffolds as wiring agents for active enzymes. In addition, the mild and well reproducible fabrication process and the active role of the polymer film in withdrawing electrons from the reduced PQQ-GDH lead to a system with high stability. This could provide access to a larger group of enzymes for bioelectrochemical applications including biosensors and biofuel cells.

On page 5178, R. E. Gyurcsányi, F. W. Scheller, and co-workers demonstrate specific recognition of acetylcholinesterase, a potential marker of Alzheimer's disease, by a hybrid material which shows threefold selectivity gain by the “shape-specific” electrosynthesized molecularly imprinted polymer, oriented binding to the propidium layer and signal generation exclusively by the enzyme bound to the nanofilm. Acetylcholinesterase is detected in the nanomolar range, whilst interfering proteins are effectively suppressed.

Effective recognition of enzymatically active tetrameric acetylcholinesterase (AChE) is accomplished by a hybrid nanofilm composed of a propidium-terminated self-assembled monolayer (Prop-SAM) which binds AChE via its peripheral anionic site (PAS) and an ultrathin electrosynthesized molecularly imprinted polymer (MIP) cover layer of a novel carboxylate-modified derivative of 3,4-propylenedioxythiophene. The rebinding of the AChE to the MIP/Prop-SAM nanofilm covered electrode is detected by measuring in situ the enzymatic activity. The oxidative current of the released thiocholine is dependent on the AChE concentration from ≈0.04 × 10−6 to 0.4 × 10−6m. An imprinting factor of 9.9 is obtained for the hybrid MIP, which is among the best values reported for protein imprinting. The dissociation constant characterizing the strength of the MIP-AChE binding is 4.2 × 10−7m indicating the dominant role of the PAS-Prop-SAM interaction, while the benefit of the MIP nanofilm covering the Prop-SAM layer is the effective suppression of the cross-reactivity toward competing proteins as compared with the Prop-SAM. The threefold selectivity gain provided by i) the “shape-specific” MIP filter, ii) the propidium-SAM, iii) signal generation only by the AChE bound to the nanofilm shows promise for assessing AChE activity levels in cerebrospinal fluid.

A nanohybrid consisting of poly(3-aminobenzenesulfonic acid-co-aniline) and multiwalled carbon nanotubes [MWCNT-P(ABS-A)]) on a gold electrode was used to immobilize the hexameric tyrosine-coordinated heme protein (HTHP). The enzyme showed direct electron transfer between the heme group of the protein and the nanostructured surface. Desorption of the noncovalently bound heme from the protein could be excluded by control measurements with adsorbed hemin on aminohexanthiol-modified electrodes. The nanostructuring and the optimised charge characteristics resulted in a higher protein coverage as compared with MUA/MU modified electrodes. The adsorbed enzyme shows catalytic activity for the cathodic H2O2 reduction and oxidation of NADH.

Polymers can provide a suitable chemical environment for the immobilization of biomolecules in an active form on surfaces. Furthermore they can be used to wire redox proteins and enzymes with electrodes. For this purpose mainly redox polymers with well-defined redox centres and conductive polymers with intrinsic electron conductivity are applied. Polyanillines are belonging the latter class of polymers. Their properties can be easily tuned by the substitution pattern on the monomeric units. Recently we have shown that sulfonated polyanillines are suitable partners in the interaction with the enzyme PQQ-dependent glucose dehydrogenase [1]. On the basis of efficient electron exchange different enzyme electrodes can be constructed [2-4]. In this study we have investigated whether it is possible to connect the multi-domain enzyme fructose dehydrogenase FDH with electrodes using the same group of polymers. The interaction of a sulfonated and methoxy-substituted polyanilline with FDH is studied in solution using UV/Vis spectroscopy. It can be demonstrated that electron transfer from the substrate reduced enzyme to the polymer is feasible, but also that this interaction is enhanced by the presence of calcium ions. Subsequently the reaction has been investigated by electrochemical means with all partners in solution verifying the spectroscopic results. In a next step of development the enzyme has been fixed on planar ITO electrodes by means of a polymer layer. Such an enzyme electrode exhibits effective, substrate-induced bioelectrocatalysis starting at rather low potential (~0mV vs Ag/AgCl). The efficiency can be significantly enhanced when macroporous ITO electrodes are used. They have pore size diameters of ~ 300nm and allow incorporation of higher amounts of polymer and enzyme. A current enhancement of ~ 35 is found compared to the flat ITO. The study demonstrates that sulfonated polyanillines can be considered as valuable matrices in connecting redox enzymes with electrodes. [1] D. Sarauli; C.G. Xu; B. Dietzel; B. Schulz; F. Lisdat, Acta Biomater. 2013 , 9 , 8290 [2] I. Schubart; G. Göbel; F. Lisdat, Electrochimica Acta 2012 , 82 , 224 [3] D. Sarauli, C.G. Xu, B. Dietzel, B. Schulz, F. Lisdat, Journal of Materials Chemistry B 2 (21) 2014 3151-3404 [4] D. Sarauli, K. Peters, Xu, B. Schulz, D. Fattakhova-Rohlfing, F. Lisdat, ACS Appl. Mater. Interfaces 2014 , 6 , 17887

We report on efficient bioelectrocatalysis of the redox enzyme fructose dehydrogenase (FDH) upon its interaction with a sulfonated polyaniline PMSA1 [poly(2-methoxyaniline-5-sulfonic acid)-co-aniline]. This interaction has been monitored in solution and on the surface of planar and macroporous indium tin oxide (ITO) electrodes by UV-vis and cyclic voltammetric measurements. Moreover, an enhancement of the catalytic activity for fructose conversion induced by a structural change of sulfonated polyaniline PMSA1 caused by the presence of Ca2+ ions is observed. An entrapment of the Ca2+-bound polymer and enzyme inside the pores of macroporous ITO electrodes leads to a significantly increased (~35-fold) bioelectrocatalytic signal compared to a flat ITO and allows the fabrication of highly efficient electrodes with good stability.

Polymer-multiwalled carbon nanotube (MWCNT) nanohybrids, which differ in surface charge have been synthesized to study the bioelectrocatalysis of adsorbed cellobiose dehydrogenase (CDH) from Phanerochaete sordida on gold electrodes. To obtain negatively charged nanohybrids, poly(3-amino-4-methoxybenzoic acid-co-aniline) (P(AMB-A)) was covalently linked to the surface of MWCNTs while modification with p-phenylenediamine (PDA) converted the COOH-groups to positively charged amino groups. Fourier transform infrared spectroscopy (FTIR) measurements verified the p-phenylenediamine (PDA) modification of the polymer-CNT nanohybrids. The positively charged nanohybrid MWCNT-P(AMB-A)-PDA promoted direct electron transfer (DET) of CDH to the electrode and bioelectrocatalysis of lactose was observed. Amperometric measurements gave an electrochemical response with KMapp = 8.89 mM and a current density of 410 nA/cm(2) (15 mM lactose). The catalytic response was tested at pH 3.5 and 4.5. Interference by ascorbic acid was not observed. The study proves that DET between the MWCNT-P(AMB-A)-PDA nanohybrids and CDH is efficient and allows the sensorial detection of lactose.