No abstract

A novel hybrid system based on NAD-dependent glucose dehydrogenase immobilized on gold nanoparticles (AuNPs) covered with C70fullerene has been developed for effective biosensing and quantitative detection of glucose.

A novel bioelectrocatalytic system was prepared by immobilizing alcohol oxidase (AOx) onto multiwalled carbon nanotubes (MWCNT) modified with 4‐(pyrrole‐1‐yl) benzoic acid (PyBA). Functional carboxylic groups from PyBA create covalent amide linkages with amine groups from the enzyme molecule and provide an anchor for the effective immobilization of AOx improving the stability of the whole system. The immobilized enzyme displayed a pair of reversible redox peaks of flavin adenine dinucleotide (FAD) cofactor with the formal potential E0’=−0.451 V. The response showed a surface‐controlled electrode process with the heterogeneous electron transfer rate constant ks=2.7 s−1. Under aerobic conditions AOx(FADH2) can be oxidized to AOx(FAD) by oxygen, which then reacts with ethanol decreasing the cathodic response, which could be used for ethanol detection with a high sensitivity 13.1 μA mM−1 cm−2. The lack of bioactivity towards ethanol in anaerobic conditions suggests the presence of two types of AOx molecules in the system: active with oxygen maintaining the direct electron transfer feature and not active without a redox mediator, due to the deeply embedded FAD cofactor. The polarization curve showed that the electrooxidation current of ethanol appears at −410 mV and reaches 2.0 µA cm−1 at −300 mV. In this case, the bioactivity of AOx to ethanol can be observed offering promising solution for the development of mediatorless systems for application to biosensors and biofuel cells.

In recent years, carbon nanotubes (CNT) have gained considerable attention, because of their remarkable electronic and mechanical properties, which have made them extremely attractive for a wide range of sensing applications from structural materials to nanoelectronic components. The ability of CNT-modified electrodes to promote electron transfer reactions has been documented in connection with important biomolecules [1]. Our goal is to explore new applications of CNT as an electrode material in facilitating the electron transfer between enzyme molecule and electrode in the bioelectrocatalytic system for glucose oxidation that is of potential utility for bioelectronic devices such as biosensors and biofuel cells. Our research focuses on the direct electrochemical performance of glucose oxidase (GOx) immobilized on 4-(pyrrole-1-yl) benzoic acid (PyBA) modified multi-walled carbon nanotubes (CNTs). GOx is a homodimer containing two tightly bound flavin adenine dinucleotide (FAD) redox centers embedded deeply in the enzyme [2]. Modification of carbon nanostructures allow to obtain thin and organized films, causing a rapid and effective transfer of electrons between the active center of the biocatalyst (GOx) and the electrode surface. The presence of PyBA in our composite film significantly improves their stability and introduces new functional groups that have great importance in the enzyme immobilization process onto the CNTs-modified electrode [3]. In this work stable immobilization and direct electron transfer of glucose oxidase were achieved on the composite film modified glassy carbon electrode. The resulting electrode gave a well-defined redox peaks with a formal potential of about −440 mV (vs. Ag/AgCl) in 0.1 phosphate buffer solution pH=7.0. The electron transfer rate constant was estimated to be 3.15 s-1, due to the combined contribution of CNTs/PyBA and GOx. Furthermore, the method for detecting of glucose was proposed based on the decrease of oxygen caused by the enzyme-catalyzed reaction between GOx and glucose. The low calculated apparent Michaelis–Menten constant (KM app) was 10.2 mM, implying the high enzymatic activity and affinity of immobilized enzyme for glucose. It can reasonably be expected that this observation might hold true for other noble carbon nanostructure-electroactive protein systems, providing a promising platform for the development of biosensors and biofuel cells. References [1] J.J.Gooding, R.Wibowo, J. Liu, W. Yang, D. Losic, S. Orbons, F.J. Mearns, J.G. Shapter, D.B. Ilibbert, J. Am. Chem. Soc. 125 (2003) 9006. [2] H.J. Hecht, H.M. Kalisz, J. Hendle, R.D. Shmid, D.Schomburg, J. Mol. Biol. 229 (1993) 153. [3] B. Kowalewska, M. Skunik, K. Karnicka, K. Miecznikowski, M. Chojak, G. Ginalska, A. Belcarz, P.J. Kulesza, Electrochim. Acta 53 (2008) 2408.

Carbon nanostructures, such as carbon nanotubes (CNT) and graphene have gained considerable attention during the last years, especially due to their remarkable electronic and mechanical properties, which have made them extremely attractive for a wide range of sensing applications from structural materials to nanoelectronic components. The ability of carbon nanostructure-modified electrodes to promote electron transfer reactions has been documented in connection with important biomolecules [1]. Our goal was to study the influence of carbon nanostructured platform as an electrode material in facilitating the electron transfer between the enzyme molecule and the electrode surface in bioelectrocatalytic systems for oxidation reaction of glucose and lactate that is of potential utility for bioelectronic devices such as biosensors and biofuel cells. Our research focuses on the direct electrochemical performance of flavin-dependent oxidases such as glucose oxidase (GOx) and lactate oxidase (LOx) immobilized on 4-(pyrrole-1-yl) benzoic acid (PyBA) modified CNT (CNT/PyBA) and/or (for comparison) on hybrid material composed of electrochemically reduced graphene oxide (ERGO) and CNT/PyBA (ERGO/CNT/PyBA). GOx and LOx are enzymes containing tightly bound flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) redox centers, respectively, embedded deeply in the protein shell [2]. Modification of carbon nanotubes allows to obtain thin and organized films, causing a rapid and effective transfer of electrons between the active center of the biocatalyst (GOx or LOx) and the electrode surface. The presence of PyBA in our hybrid systems significantly improves their stability and introduces new functional groups that have great importance in the enzyme immobilization process onto the modified electrode [3]. Moreover, graphene oxide (GO) has a unique ability to form stable aqueous dispersions, highly bio-compatible and possesses good electrocatalytic properties. In addition, its reductive product graphene is a unique material with potential applications in diverse fields and one of the hottest materials of interest. It was demonstrated that it is possible to prepare a water dispersible GO/CNT hybrid material via non-covalent π-π stacking interactions [4]. Dispersing CNT via non-covalent approaches cannot depress its intrinsic electrical, mechanical, and optical properties. The incorporation of GO with CNT render this hybrid material as a versatile platform for the electrocatalytic applications. Conversely, the insulating property of GO caused by the aliphatic sp3 hybridized domain, limits its conductivity. Therefore for improving the conductance, it can be reduced to graphene either by chemical, thermal or electrochemical methods. Recent studies reveal that the electrochemical reduction of GO to graphene (ERGO) was appreciably improved after the incorporation of CNT. In this work stable immobilization and direct electron transfer of oxidase enzymes were achieved on the hybrid system modified glassy carbon electrode. The resulting electrode gave a well-defined redox peaks with a formal potential of about -458 mV for GOx and -446 mV for LOx (vs. Ag/AgCl) in 0.1 phosphate buffer solution pH=7.0. Furthermore, the method for detecting of glucose and/or lactate was proposed based on the decrease of oxygen caused by the enzyme-catalyzed reaction between enzyme and substrate. The low calculated apparent Michaelis-Menten constants (KM app) were 13.3 mM for GOx and 8.25 mM for LOx, implying high enzymatic activity and affinity of immobilized enzyme for glucose and lactate, respectively. The calculated heterogeneous electron transfer rate constants were twice higher due to the combined contribution of GO with CNT/PyBA. It can reasonably be expected that this observation might hold true for other noble carbon nanostructure-electroactive protein systems, providing a promising platform for the development of biosensors and biofuel cells. References [1] J.J.Gooding, R.Wibowo, J. Liu, W. Yang, D. Losic, S. Orbons, F.J. Mearns, J.G. Shapter, D.B. Ilibbert, J. Am. Chem. Soc. 125 (2003) 9006. [2] H.J. Hecht, H.M. Kalisz, J. Hendle, R.D. Shmid, D.Schomburg, J. Mol. Biol. 229 (1993) 153. [3] B. Kowalewska, M. Skunik, K. Karnicka, K. Miecznikowski, M. Chojak, G. Ginalska, A. Belcarz, P.J. Kulesza, Electrochim. Acta 53 (2008) 2408. [4] C. Zhang, L. Ren, X. Wang, T. Liu, J. Physical Chemistry C 114 (2010) 11435.