Leonard Stoica

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

Development of a cellobiose dehydrogenase modified electrode for amperometric detection of diphenols

A new amperometric biosensor based on cellobiose dehydrogenase (CDH) was created for the detection of ortho- and para-diphenolic compounds. The developed electrode efficiently discriminates between diphenolic and monophenolic compounds. The analyte, a diphenolic compound, is oxidised on the surface of a graphite electrode at an applied potential of +300 mV vs. Ag/AgCl. The diphenol is then regenerated by the adsorbed CDH in the presence of cellobiose, thus allowing an amplified response signal. Different parameters of the CDH–electrode system were optimised, e.g., applied potential, immobilisation time, flow rate, substrate concentration and storage conditions. Using the optimised parameters the sensitivity and detection limits for various diphenolic compounds were evaluated, resulting in detection limits below 5 nM for most of the compounds tested. The highest sensitivity recorded was obtained for dopamine, 3.6 A l mol–1 cm–2. The operational stability of the electrodes was high: during 2 h of continuous operation only a 1–2% decrease in response signal was observed.

Direct Electrochemistry of Proteins and Enzymes

Publisher Summary This chapter summarizes the achievements on direct electron transfer (DET) between redox enzymes and electrodes, with a special focus on haem and copper-containing redox enzymes. Haem enzymes involve peroxidases, catalases, cytochromes of the P450 group, and a variety of multi-cofactor complex enzymes that contain haem(s), along with other cofactors such as flavin(s), copper, and iron–sulphur cluster(s). There are a variety of electron tunneling pathways within the enzyme molecule between the redox active centre and the protein surface. The chapter presents two groups of redox enzymes: the intrinsic and extrinsic ones. To realize efficient DET between redox enzymes and electrodes, a proper orientation of the redox enzyme onto the electrode, through the site of the electron-tunneling pathways, where the partner protein commonly binds or through the domain of the active site exposed to the protein surface, becomes important. The copper sites in the redox proteins have been divided into three classes based on their spectroscopic features that reflect the geometric and electronic structure of the active site: type 1 (T1) or blue copper, type 2 (T2) or normal copper, and type 3 (T3) or coupled binuclear copper centers. The chapter describes all copper-containing proteins that are divided into four groups according to the structure of their active sites: (1) type-1 copper proteins, (2) type-2 copper enzymes, (3) type-3 copper proteins, and (4)‘‘blue’’ multi-copper oxidases.

Mass transport controlled oxygen reduction at anthraquinone modified 3D-CNT electrodes with immobilized Trametes hirsuta laccase

Carbon nanotubes covalently modified with anthraquinone were used as an electrode for the immobilization of Trametes hirsuta laccase. The adsorbed laccase is capable of oxygen reduction at a mass transport controlled rate (up to 3.5 mA cm(-2)) in the absence of a soluble mediator. The storage and operational stability of the electrode are excellent.

In-field monitoring of cleaning efficiency in waste water treatment plants using two phenol-sensitive biosensors

Abstract Two amperometric biosensors based on the enzymes cellobiose dehydrogenase (CDH) and quinoprotein-dependent glucose dehydrogenase (GDH), have been applied for monitoring the phenolic content in water samples, collected at different stages of a waste water treatment process, thus representing different cleaning levels of two waste water treatment plants (WWTPs). The biosensor measurements were performed in-field, compared with the results obtained by liquid chromatography-mass spectrometry and were further correlated with the cleaning efficiencies of the WWTPs. The effect of several potentially interfering compounds on the sensor response was also studied. The general purpose of the study was to evaluate the potential use of biosensors, not as quantitative tools for phenol analysis, but rather as screening tools indicating a certain trend, i.e. compounds present or not present, and potential correlation with sample toxicity. It was found that the biosensors and LC-MS results were not quantitatively comparable, however, both sensors could follow the decrease of the phenol content from the influent, primary treated and effluent waters. In addition, the correlation between biosensor inhibition and sample toxicity is discussed.

Glucose Oxidase/Horseradish Peroxidase Co‐immobilized at a CNT‐Modified Graphite Electrode: Towards Potentially Implantable Biocathodes

To maximise the power output of enzymatic biofuel cells (BFC) it is necessary to maintain a large difference between the onset potentials of the anodic and cathodic reactions, represented by the cell voltage (Vcell) [2] at a concomitantly high current density (Icell). Aside from the power output characteristics, a BFC requires compatibility with the operational medium, that is, for a potentially implantable BFC a buffered solution at pH 7.4, in the presence of high concentrations of chloride ions. Presently, state-of-the-art cathodes for BFC rely on oxygen reduction catalysed by laccase or bilirubin oxidase, which have onset potentials at around +635 or +465 mV, respectively (all potentials are expressed vs. Ag jAgCl (3 m KCl)). While these biocathodes have acceptable onset potentials, their use is currently limited by the necessary acidic optimal value of the pH and the efficient inhibition of the enzymatic reaction by Cl ions at physiological concentration (140 mm Cl ) in the case of laccase. BiliACHTUNGTRENNUNGrubin oxidase-based cathodes exhibit a 200 mV lower onset potential and a substantially less efficient direct electron transfer communication with the electrode as compared with laccases. Molecular or polymer-bound mediators, which facilitate electron transfer between enzymes and electrodes, for example, 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonate) or Os-complex containing polymers, are usually introduced to improve electrochemical communication. However, these mediators introduce an additional drop of potential and in certain cases create a diffusional barrier for the substrate/ products within the film. Recently, we have described a novel electrode material based on two-generations carbon nanotubes (CNTs) and the surprisingly high onset potential for the reduction of H2O2 by direct electron transfer with horseradish peroxidase (HRP) of +650 mV. The composite structured carbon material was obtained through a sequential two-step chemical vapour deposition process (CVD) in which carbon microfibers (CMF) are generated first at 1150 8C, followed by the second electrodeposition of Fe catalyst on CMF/GR and carbon nanotube (CNT) growth by CVD at 750 8C, as branches of the CMF as well as onto GR (see related SEM images in ref. [10]). Thus, the overall increased surface is in good electrical contact with the underlying graphite rod (GR). In contrast to other bioelectrochemical studies involving an extensive use of CNTs, the proposed synthesis of the composite structured electrode material avoids an uncontrolled electric connection between the CNTs and the electrode. Moreover, an enhanced enzyme loading with minimum potential drop from thermodynamic potential of iron– oxo complex at HRP and an optimum connectivity of even the most remote enzyme molecules is envisaged. The concept of biocathodes running with H2O2 reduction by using its in situ generation by glucose oxidase (GOx) either immobilized at an anodic site or present in solution at the cathode compartment was demonstrated recently and in the early 1990s, respectively. In the present work, we extended the application of the efficient reduction of H2O2 at CNT/CMF/GR electrodes modified with pyrenehexanoic acid (PHA) and HRP by co-immobilization of GOx. Herein, the novelty consists of: 1) major shift of onset of cathodic reaction up to +600 mV, which is limited for reaching the thermodynamic limits of +750 mV only by competitive direct H2O2 oxidation at the electrode surface; 2) demonstration of its full operation at physiological pH and in the presence of Cl ions; and 3) a new concept in bioelectrochemistry for efficient enhancement of enzyme loading by two-generations enlargement [a] Dr. W. Jia, Prof. Dr. W. Schuhmann, Dr. L. Stoica Analytische Chemie, Elektroanalytik & Sensorik Ruhr-Universit t Bochum, Universit tsstr. 150 44780 Bochum (Germany) Fax: (+49) 234-3214683 E-mail : wolfgang.schuhmann@rub.de leonard.stoica@rub.de [b] Dr. C. Jin, Dr. W. Xia, Prof. Dr. M. Muhler Lehrstuhl f r Technische Chemie, Ruhr-Universit t Bochum Universit tsstr. 150, 44780 Bochum (Germany) Scheme 1. Representation of CMF/CNT composite carbon structure onto GR and involved enzymatic/heterogeneous reactions responsible for reductive current at cathode.

Scanning electrochemical microscopy (SECM) as a tool in biosensor research.

Scanning electrochemical microscopy (SECM) is discussed as a versatile tool to provide localized (electro)chemical information in the context of biosensor research. Advantages of localized electrochemical measurements will be discussed and a brief introduction to SECM and its operation modes will be given. Experimental challenges of the different detection modes of SECM and its applicability for different fields in biosensor research are discussed. Among these are the evaluation of immobilization techniques by probing the local distribution of biological activity, the visualization of diffusion profiles of reactants, cofactors, mediators, and products, and the elucidation of (local) kinetic parameters. The combination of SECM with other scanning-probe techniques allows to maximize the information on a given biosensing system. The potential of SECM as a tool in micro-fabrication aiming for the fabrication of microstructured biosensors will be shortly discussed.

Bioelectrochemical detection of L-lactate respiration using genetically modified Hansenula polymorpha yeast cells overexpressing flavocytochrome b2.

In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, also known as flavocytochrome b(2), FC b(2)) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b(2) the O(2) reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b(2), cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN(-), lactate respiration could be proven for causing the decrease in the O(2) reduction.

Activation/Inhibition Effects during the Coelectrodeposition of PtAg Nanoparticles: Application for ORR in Alkaline Media†

PtAg bimetallic nanoparticles for oxygen reduction reaction (ORR) in alkaline media were prepared by pulse electrodeposition (PED). During PED the reduction of Ag(+) ions predominates, thus an increased Ag content in the co-deposit is accomplished. The mechanism for this anomalous co-deposition was elucidated by potential pulse experiments, which revealed that nuclei formation mainly occurs via the reduction of Pt(2+) ions. The growth of the particles is diffusion controlled leading to the formation of a Ag shell covering a PtAg alloyed region. However, the shell is not growing homogeneously on the PtAg alloy. Hence, regions of the PtAg alloy are exposed, which exhibit an enhanced ORR activity compared to a pure Ag surface.

Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 microM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e(-)/H(+) acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.