Analytical and Bioanalytical Chemistry (v.405, #11)

Amperometric sensing — Bioelectroanalysis by Renato Seeber; Wolfgang Schuhmann; Fabio Terzi; Chiara Zanardi; Nicolas Plumere; Magdalena Gebala (3423-3426).
has been Full Professor of Analytical Chemistry since 1986 at the universities of Sassari, Bologna and Modena, where he now works. In the past, his research interests were in the fields of molecular electrochemistry, namely electrode mechanisms of organic and inorganic compounds in aprotic media, the development of novel methods for signal filtering and for development of digital simulation techniques, thermodynamic and kinetic aspects of mobilization of iron and other metal ions in soil, and electroanalytical and chemometric methods applied to food chemistry. For almost 15 years his main interest has been in the field of modification of electrodes with intrinsically and redox conducting polymers, metal nanoparticles, graphene and relevant composites. The analytical application, even to real matrices, has always been coupled to an as deep as possible electrochemical, spectroscopic, and microscopic characterization of the electrode system, aiming at giving a rationale to its behaviour and at optimizing its structure for the best analytical performance. A parallel activity has dealt with the development and testing of electronic tongues based on amperometric sensors—differently modified electrodes—applied to characterization of food matrices. obtained his diploma in chemistry from the University of Karlsruhe, Germany, and his PhD degree in 1986 from the Technical University of Munich. After finishing his habilitation thesis at the Technical University of Munich in 1994, he was appointed to be a professor of analytical chemistry at Ruhr-Universität Bochum in 1996. He is the co-author of more than 360 research articles spanning topics from the development of electrochemical biosensors to microelectrochemistry, scanning electrochemical microscopy, combinatorial microelectrochemistry, localized corrosion, electrocatalysis, photoelectrocatalysis and battery materials. He is a fellow of the Royal Society of Chemistry (2005) and the International Society of Electrochemistry (2012). He has received the Biosensors & Bioelectronics Award (2000), the Julius von Haast Fellowship Award (2008) and the Katsumi Niki Prize for Bioelectrochemistry (2012). is a postdoctoral researcher in analytical chemistry at the University of Modena and Reggio Emilia. His research activities are focused on the synthesis and characterization of nanomaterials, modification of electrode surfaces using conducting polymers, self-assembled monolayers and nanomaterials, spectroscopic and microscopic characterization of electrode coatings and development and testing of amperometric sensors based on the systems realized. He has published more than 40 articles in international peer-reviewed journals. is a researcher in analytical chemistry at the University of Modena and Reggio Emilia. Her research interests are in the field of electroanalysis, particularly dealing with the development of new materials as electrode coatings possessing electrocatalytic and antifouling properties. These materials are applied in electrochemical sensors and biosensors for the quantification of meaningful analytes in food and environmental matrices. She is the co-author of more than 50 articles in international journals and a book chapters. studied chemistry at the University of Strasbourg and the University of the West of Scotland, Glasgow, and obtained his PhD degree in 2009 from the University of Tübingen. He was awarded a DFG fellowship for his PhD work as well as a DAAD fellowship for a research visit to San Jose State University (CA, USA). After a postdoctoral stay at the laboratory of Wilbur H. Campbell (NECi, Lake Linden, MI, USA), he started as a junior group leader at the Center for Electrochemical Sciences (CES) at Ruhr-Universität Bochum in 2010. He is the co-author of 15 publications dealing with electrochemical biosensors, single-molecule methods and redox-active nanomaterials. The research activities of his group at CES focus on the development of new strategies for efficient electrical connectivity between redox enzymes and electrodes. studied biochemistry at Wroclaw University of Technology, Poland. After finishing her diploma thesis in 2006, she started her PhD work at Ruhr-Universität Bochum. In 2010, she successfully defended her thesis entitled “From impedimetric investigation of the surface architecture to label-free DNA assays”. She is the co-author of 14 research articles. Her research interests includes the in-depth understanding of the properties of biomolecules at electrified interfaces and their impact on the design of improved biosensors and bioassays. Presently she is a postdoctoral fellow at Stanford University working on ion–RNA interactions.

Many cathodic electrochemiluminescence (ECL) systems require very negative potentials; it is difficult to achieve stable cathodic ECL in aqueous solutions because of hydrogen evolution and instability of intermediates. In this study, tricresyl phosphate-based carbon paste electrode (CPE) was used to achieve cathodic ECL. It exhibits no obvious hydrogen evolution even at a potential up to −1.6 V and dramatically stabilizes electrogenerated [Ru(bpy)3]+. Therefore, a reversible wave of [Ru(bpy)3]2+/1+ in aqueous solutions at carbon electrode has been observed for the first time, and cathodic ECL of [Ru(bpy)3]2+/S2O 8 2− has been achieved. Under the optimum conditions, the plots of the ECL versus the concentration of S2O 8 2− are linear in the range of 10−6 to 10−2 M with the detection limit of 3.98 × 10−7 M. Common anions have no effect on the ECL intensity of the [Ru(bpy)3]2+/S2O 8 2− system. Since CPEs have been widely used, CPEs with high hydrogen evolution potential are versatile platforms for electrochemical study and cathodic ECL study.
Keywords: Electrochemistry; Luminescence; Carbon paste electrode; Hydrogen evolution potential

Nanomaterial-based functional scaffolds for amperometric sensing of bioanalytes by Ramendra Sundar Dey; Raj Kumar Bera; C. R. Raj (3431-3448).
Functional nanomaterials have emerged as promising candidates in the development of an amperometric sensing platform for the detection and quantification of bioanalytes. The remarkable characteristics of nanomaterials based on metal and metal oxide nanoparticles, carbon nanotubes, and graphene ensure enhanced performance of the sensors in terms of sensitivity, selectivity, detection limit, response time, and multiplexing capability. The electrocatalytic properties of these functional materials can be combined with the biocatalytic activity of redox enzymes to develop integrated biosensing platforms. Highly sensitive and stable miniaturized amperometric sensors have been developed by integrating the nanomaterials and biocatalyst with the transducers. This review provides an update on recent progress in the development of amperometric sensors/biosensors using functional nanomaterials for the sensing of clinically important metabolites such as glucose, cholesterol, lactate, and glutamate, immunosensing of cancer biomarkers, and genosensing.
Keywords: Amperometric biosensors; Functional nanomaterials; Graphene; Carbon nanotubes; Metal and metal oxide nanoparticles; Clinical metabolites; Immunosensors; Cancer biomarkers; DNA; Genosensors

In this critical review, new nanomaterials based on graphene (GN) are described, especially those used for the assembly of miniaturized electrochemical transducers. In particular, the physicochemical properties and mechanical features of few layers of graphene (FLGs) are described, as is their use for assembly of chemically modified sensors, biosensors, and immunosensors. The FLGs described here were functionalized by chemical treatment in solution, resulting in oxidized and/or reduced surfaces, edges, and sides. The presence of oxygenated functionality strongly affects the electrocatalysis and the electron-transfer properties of several molecular targets, not only in the solid phase (e.g. in field-effect transistors, FETs) but also in liquid matrices (chemically modified electrodes and biosensors). In addition, “green chemistry” reagents, for example ionic liquids (ILs) can be used for exfoliation and intercalation of graphene planes, to obtain stable and homogeneous nanodispersions. The assembled sensors, biosensors, and immunosensors are extremely useful for electrochemical detection of several electro-active targets of importance in food analysis, environmental monitoring, and clinical diagnosis. A detailed description of each analytical application has been given in this critical review and brief remarks on the emerging disciplines of nanomedicine and nanofoods are also discussed.
Keywords: Few layers of graphene; Oxidized graphene nanoribbons; Reduced graphene nanoribbons; Sensors; Biosensors; Immunosensors; Green chemistry and ionic liquids; Nanomedicine and nanofoods

Ultramicroelectrode sensor arrays in which each electrode, or groups of electrodes, are individually addressable are of particular interest for detection of several species concomitantly, by using specific sensing chemistry for each analyte, or for mapping of one analyte to achieve spatio–temporal analysis. Microfabrication technology, for example photolitography, is usually used for fabrication of these arrays. The most widespread geometries produced by photolithography are thin-film microdisc electrode arrays with different electrode distributions (square, hexagonal, or random). In this paper we review different electrochemical sensor arrays developed to monitor, in vivo, NO levels produced by cultured cells or sliced tissues. Simultaneous detection of NO and analytes interacting with or released at the same time as NO is also discussed.
Keywords: Nitric oxide; Electrode array; Sensor array; Electrochemical sensors; Cell culture analysis; Peroxynitrite

In this topical review, progress achieved in amperometric sensing of different analytes over conducting polymer-based hybrid electrocatalysts is summarized. We report a variety of synthetic methods and the resulting hybrid assemblies, with the effectiveness of such strategies, for designing conjugated polymer-based hybrids as robust sensors for amperometric detection. Beyond incorporation of metal nanoparticles, metal-oxide and non-oxide semiconductors, carbon-based nanomaterials (nanotubes, graphene, and graphene oxide), and special dopant ions are also discussed. Moreover, some particularly interesting miscellaneous approaches, for example photo-amperometric sensing or use of overoxidized polymers, are also emphasized. Determination of dissolved gases (for example O2, NO, and NO2), ions (sulfite, nitrite, nitrate, chlorate, bromate, and iodate) and smaller and larger molecules (for example H2O2, ascorbic acid (AA), dopamine (DA), urea (UA), amino acids, hydrazine, NADH, serotonin, and epinephrine) is discussed. These achievements are reviewed from the materials perspective, addressing both synthetic and electrocatalytic aspects of the polymer-based modified electrodes. Beyond simple or more sophisticated mixing, a wide range of methods of preparation is presented, including chemical (one-pot polymerization, impregnation), electrochemical (co-deposition, doping type inclusion, etc.) and combined strategies. Classification of such synthetic routes is also included. However, it is important to note that we omit studies in which conducting polymers alone were used for determination of different species. Furthermore, because excellent reviews—cited in this work also—are available on immobilization of biomolecules (for example enzymes) for biosensing purposes, this topic, also, is excluded.
Keywords: Amperometric sensing; Conducting polymer; Modified electrode; Composite; Electrodeposition; Sensor

Hybrid and biohybrid layered double hydroxides for electrochemical analysis by Christine Mousty; Vanessa Prévot (3513-3523).
Layered double hydroxides (LDH) are lamellar materials that have been extensively used as electrode modifiers. Nanostructured organic–inorganic materials can be designed by intercalation of organic or metallic complexes within the interlayer space of these materials or by the formation of composite materials based on biopolymers (alginate or chitosan) or biomolecules, such as enzymes. These hybrid or biohybrid materials have interesting properties applicable in electroanalytical devices. From an exhaustive review of the literature, the relevance of these hybrid and biohybrid LDH materials as electrode materials for electrochemical detection of species with an environmental or health impact is evaluated. The analytical characteristics (sensitivity and detection limit) of LDH-based amperometric sensors or biosensors are scrutinized. Figure (Bio) Hybrid LDH based modified electrodes
Keywords: Layered double hydroxides; Hybrid; Biohybrids; Electrodes; Sensors; Biosensors

Recent advances in graphite powder-based electrodes by Dolores Bellido-Milla; Laura Ma Cubillana-Aguilera; Mohammed El Kaoutit; Ma Purificación Hernández-Artiga; José Luis Hidalgo-Hidalgo de Cisneros; Ignacio Naranjo-Rodríguez; José Ma Palacios-Santander (3525-3539).
Graphite powder-based electrodes have the electrochemical performance of quasi-noble metal electrodes with intrinsic advantages related to the possibility of modification to enhance selectivity and their easily renewable surface, with no need for hazardous acids or bases for their cleaning. In contrast with commercial electrodes, for example screen-printed or sputtered-chip electrodes, graphite powder-based electrodes can also be fabricated in any laboratory with the form and characteristics desired. They are also readily modified with advanced materials, with relatively high reproducibility. All these characteristics make them a very interesting option for obtaining a large variety of electrodes to resolve different kinds of analytical problems. This review summarizes the state-of-the-art, advantages, and disadvantages of graphite powder-based electrodes in electrochemical analysis in the 21st century. It includes recent trends in carbon paste electrodes, devoting special attention to the use of emergent materials as new binders and to the development of other composite electrodes. The most recent advances in the use of graphite powder-modified sol–gel electrodes are also described. The development of sonogel–carbon electrodes and their use in electrochemical sensors and biosensors is included. These materials extend the possibilities of applications, especially for industrial technology-transfer purposes, and their development could affect not only electroanalytical green chemistry but other interesting areas also, for example catalysis and energy conversion and storage.
Keywords: Graphite powder; Composite materials; Carbon paste; New binders; Sol–gel; Sonogel–carbon; Modified electrodes; Applications

Amperometric homogeneous competitive immunoassay in a perfluorocarbon emulsion oxygen therapeutic (PEOT) by Rebecca E. Barlag; H. Brian Halsall; William R. Heineman (3541-3547).
The effect of a perfluorocarbon emulsion oxygen therapeutic (PEOT) on the detection of the drugs theophylline and phenytoin was explored using a commercial enzyme multiplied immunoassay technique (EMIT®). The EMIT technique is based on the enzymatic production of NADH, which is typically detected in serum samples spectrophotometrically. Here, amperometry using the rotating disk electrode on a single drop of solution is demonstrated to detect theophylline and phenytoin in the presence of PEOT. In the study, 2,6-dichloroindophenol (DCIP) added to the immunoassay mixture is reduced by the NADH to DCIPH2. Oxidation of DCIPH2 is monitored electrochemically at +200 mV using a glassy carbon rotating disk electrode. Slopes of amperograms are proportional to the concentration of drug in the immunoassay sample. This technique yields excellent quantitative data in the therapeutic range for both drugs in 2–20 % PEOT.
Keywords: Immunoassay; Perfluorocarbon; Electrochemistry; Theophylline; Phenytoin

Highly porous magnetite/graphene nanocomposites for a solid-state electrochemiluminescence sensor on paper-based chips by Yuanhong Xu; Zhaozi Lv; Yong Xia; Yanchao Han; Baohua Lou; Erkang Wang (3549-3558).
Graphene-nanosheet-based highly porous magnetite nanocomposites (GN-HPMNs) have been prepared using a simple solvothermal method and used as an immobilization matrix for the fabrication of a solid-state electrochemiluminescence (ECL) sensor on paper-based chips. Highly porous Fe3O4 nanocrystal clusters were coated with acrylate and wrapped tightly on the skeleton of graphene nanosheets. The structures and sizes of the GN-HPMNs could be tuned by varying the proportions of the solvents ethylene glycol and diethylene glycol. Then, the relatively highly porous ones with an average diameter of about 65 nm were combined with Nafion to form composite films on an electrode surface for immobilization of Ru(bpy)3 2+ (bpy is 2,2′-bipyridine). Because of their porosity, negatively charged surface, and cooperative characteristics of magnetic nanomaterials and graphene, under an external magnetic field, the GN-HPMNs ensured effective immobilization, excellent electron transfer, and long-term stability of Ru(bpy)3 2+ in the composite film. The sensor developed exhibited excellent reproducibility with a relative standard deviation of 0.65 % for 30 continuous cycles. It was found to be much more favorable for detecting compounds containing tertiary amino groups and DNAs with guanine and adenine. A detection limit (signal-to-noise ratio of 3) of 5.0 nM was obtained for tripropylamine. As an application example, 0.5 nM single-nucleotide mismatch could be detected. This was the first attempt to introduce magnetic nanomaterials and an external magnetic field into paper-based chips. The sensor developed has the advantages of high sensitivity, good stability, and wide potential applicability as well as simplicity, low cost, and good disposability. Figure Schematic diagram of using graphene-nanosheet-based highly porous magnetite nanocomposites for fabrication of a solid-state electrochemiluminescence sensor on paper-based chips and application example of the developed sensor for single-nucleotide mismatch discrimination
Keywords: Magnetite nanocomposite; Graphene; Electrochemiluminescence; Immobilization; Paper-based chips

Voltammetric platform for detection of 2,4,6-trinitrotoluene based on a molecularly imprinted polymer by M. Pesavento; G. D’Agostino; G. Alberti; R. Biesuz; D. Merli (3559-3570).
New methods for determination of explosive substances as, for example, 2,4,6-trinitrotoluene (TNT), in a rapid way and at low cost are highly required. An electrochemical platform has been here developed with good characteristics of low dimension, fast response, low cost, and high selectivity. It is based on a commercially available screen printed cell with graphite ink working and auxiliary electrodes and a silver ink quasi-reference electrode. The whole cell is covered with a thick layer of cation exchanging acrylic polymer molecularly imprinted with 2,4,6-trinitrotoluene. The polymeric layer acts at the same time as electrolytic medium and selective receptor. It has been demonstrated that, in this medium, 2,4,6-trinitrotoluene is electroactive at graphite electrode, being reduced by a non-reversible reaction. The peak current (differential pulse voltammogram) is proportional to TNT concentration with limit of detection for TNT around 5 × 10−7 M and linearity range up to 2 × 10−5 M. The selectivity for TNT relative to other reducible compounds as, for example, nitroaromatic derivatives, and to other possible interfering substances, as negatively charged ions, is good. Measurements can be performed in not de-aerated solution and in small volumes (20 μl), so that the proposed platform is very promising for in situ determinations. Figure Molecularly imprinted polymer for TNT as selective artificial receptor and ionic medium of the electrochemical cell
Keywords: Electrochemical sensor; Molecularly imprinted polymers; Cation exchangers as electrolytic media; Trinitrotoluene

Electrochemical gas sensors based on paper-supported room-temperature ionic liquids for improved analysis of acid vapours by Rosanna Toniolo; Nicolò Dossi; Andrea Pizzariello; Alice Casagrande; Gino Bontempelli (3571-3577).
A prototype of a fast-response task-specific amperometric gas sensor based on paper-supported room-temperature ionic liquids (RTILs) is proposed here for improved analysis of volatile acid species. It consists of a small filter paper foil soaked with a RTIL mixture containing an ionic liquid whose anion (acetate) displays a basic character, upon which three electrodes are screen printed by carbon ink profiting from a suitable mask. It takes advantage of the high electrical conductivity and negligible vapour pressure of RTILs and of their easy immobilization into a porous and inexpensive supporting material such as paper. The performance of this device, used as a wall-jet amperometric detector for flow injection analyses of headspace samples in equilibrium with aqueous solutions at controlled concentrations, was evaluated for phenol and 1-butanethiol vapours which were adopted as model acid gaseous analytes. The results obtained showed that the quite high potentials required for the detection of these analytes are lowered significantly, thanks to the addition of the basic acetate RTIL. In such a way, overlap with the medium discharge is avoided, and the possible adverse effect of interfering species is minimised. The sensor performance was quite satisfactory (detection limits, ca. 0.3 μM; dynamic range, ca. 1–200 μM, both referred to solution concentrations; correlation coefficients in the range 0.993–0.997; repeatability, ± 6 % RSD; long-term stability, 9 %); thus suggesting the possible use of this device for manifold applications. Figure Layout and cross-section of the RTIL-PED sensor adopted in flow injection analyses. R pseudo-reference electrode, W working electrode, C counter electrode
Keywords: Room-temperature ionic liquids (RTILs); Paper-based electrochemical detectors (PEDs); Electrochemical gas sensors for acid vapours; Flow injection analysis (FIA); Phenol analysis; 1-Butanethiol analysis

Graphene-modified electrode. Determination of hydrogen peroxide at high concentrations by Fabio Terzi; Jonathan Pelliciari; Chiara Zanardi; Laura Pigani; Antti Viinikanoja; Jukka Lukkari; Renato Seeber (3579-3586).
A gold electrode partially coated by graphene multilayer is developed and tested with respect to high concentrations of hydrogen peroxide. The effective use of conventional electrode materials for the determination of such an analyte by anodic oxidation or cathodic reduction is prevented by the occurrence of adsorptions fouling the electrode surface. This prevents reliable and repeatable voltammetric curves for being recorded and serious problems arise in quantitative analysis via amperometry. The gold–graphene electrode is shown to be effective in quantitative evaluation, by cathodic reduction, of hydrogen peroxide at concentration levels that are of interest in an industrial. Acid, neutral, and basic pH values have been tested through correct adjustment of a Britton Robinson buffer. The experiments have been performed both by cyclic voltammetry and with amperometry at constant potential in unstirred solution. The latter technique has been employed in drawing a calibration linear plot. In particular, the performances of the developed electrode system have been compared with those of both pure gold and pure graphene electrode materials. The bi-component electrode was more sensitive; co-catalytic action by the combination of the two components is hypothesised. The system is stable over many potential cycles, as checked by surface-enhanced Raman spectra recorded over time.
Keywords: Graphene; Gold; Nanostructured electrode; Hydrogen peroxide amperometry; High analyte concentrations

Direct electrochemical detection of bisphenol A at PEDOT-modified glassy carbon electrodes by Elisabetta Mazzotta; Cosimino Malitesta; Eleonora Margapoti (3587-3592).
The electrochemical behavior of bisphenol A (BPA) was studied on poly(3,4-ethylenedioxythiophene) (PEDOT)-modified glassy carbon electrodes by cyclic voltammetry. It was observed that BPA oxidation on PEDOT film produced a BPA polymer (pBPA) showing excellent redox activity with anodic and cathodic peaks at 0.15 and 0.01 V, respectively; the former being evaluated for BPA electrochemical sensing. The amount of deposited pBPA has been estimated by electrochemical and spectroscopic analysis by X-ray photoelectron spectroscopy. The effect of scan rate and pH on the oxidation of pBPA film has been studied. The oxidation current was found to vary linearly with BPA concentration in the range 90–410 μM, and a detection limit of 55 μM was evaluated. Results of BPA amperometric detection have also been collected by using a repetitive potential step program to give a linear response to BPA in the concentration range 40–410 μM with a detection limit of 22 μM and a sensitivity of 1.57 μAμM−1 cm−2. The developed sensor showed satisfactory reproducibility and anti-interference properties and was successfully applied to BPA determination in mineral water samples.
Keywords: Bisphenol A; Poly(3,4-ethylenedioxythiophene) (PEDOT); Modified electrodes; Electrochemical sensors

Optical fiber spectroelectrochemical device for detection of catechol at press-transferred single-walled carbon nanotubes electrodes by Jesus Garoz-Ruiz; Daniel Izquierdo; Alvaro Colina; Susana Palmero; Aranzazu Heras (3593-3602).
A new long-optical-pathway spectroelectrochemical cell for absorptometric measurements in the UV–Vis region was developed. This cell consists of two optical fibers brought face to face and fixed on the working electrode support. As a proof of concept, the spectroelectrochemical cell was applied to the determination of catechol using a press-transferred single-walled carbon nanotube film as the working electrode. Voltabsorptometry was demonstrated to be very helpful in understanding the mechanism of catechol oxidation. The experiments showed that the main oxidation product is o-benzoquinone, but other soluble side products are also observed. Multivariate calibration explains the selection of 390 nm as the best wavelength for the univariate absorptometric determination of catechol, avoiding the interference of oxidation side products. Catechol was quantified using both the electrochemical and the spectroscopic signal, demonstrating that this hybrid technique is an autovalidated analytical method. Dual detection of catechol was also carried out using amperometric spectroelectrochemistry. Finally, spectroelectrochemistry was used to quantify catechol in the presence of hydroquinone.
Keywords: Single-walled carbon nanotubes; Electrochemistry; Spectroscopy; UV–Vis spectroelectrochemistry; Catechol

Voltammetry of microparticles is applied to characterise and to identify solid analytes of interest in the field of cultural heritage. Nafion® is used for the immobilisation of solid microparticles onto the surface of a glassy carbon electrode by exploiting the deposition onto the electrode surface of a micro-volume of a suspension of the microsample in polymeric solution. Cyclic voltammetry and square wave voltammetry are applied to characterise and to identify the microparticles immobilised in the Nafion® coating. The analyte studied in this work is Prussian Blue as a typical inorganic pigment, with a relatively simple electrochemical behaviour. The proposed method is applied to a sample of Venetian marmorino plaster. The performance of Nafion® for this analysis is compared with that of the polymer Paraloid B72. Figure From sampling the pigment in the work of art to recording the voltammetric signal with Nafion coated electrodes
Keywords: Voltammetry; Microparticles; Nafion®; Cultural heritage; Prussian Blue

Low-cost reduced graphene oxide-based conductometric nitrogen dioxide-sensitive sensor on paper by Jukka Hassinen; Jussi Kauppila; Jarkko Leiro; Anni Määttänen; Petri Ihalainen; Jouko Peltonen; Jukka Lukkari (3611-3617).
The fabrication concept for a low-cost sensor device using reduced graphene oxide (rGO) as the sensing material on a porous paper substrate is presented. The sensors were characterized using conductivity and capacitance measurements, atomic force microscopy and X-ray photoelectron spectroscopy. The effects of different reducing agents, graphene oxide (GO) flake size and film thickness were studied. The sensor was sensitive to NO2, and devices based on a thin (10-nm) hydrazine-reduced GO layer had the best sensitivity, reaching a 70 % reduction in resistance after 10 min of exposure to 10 ppm NO2. The sensitivity was high enough for the detection of sub-parts per million levels of NO2. Desorption of gas molecules, i.e. the recovery of the sensor, could be accelerated by UV irradiation. The structure and preparation of the sensor are simple and up-scalable, allowing their fabrication in bulk quantities, and the fabrication concept can be applied to other materials, too. Figure Low‐cost reduced graphene oxide based conductometric nitrogen dioxide sensitive sensor on paper
Keywords: Graphene; Gas sensor; Sensor fabrication; Nitrogen dioxide; Printed electronics; Paper substrate

Small electron-transfer proteins as mediators in enzymatic electrochemical biosensors by Célia M. Silveira; M. Gabriela Almeida (3619-3635).
Electrochemical mediators transfer redox equivalents between the active sites of enzymes and electrodes and, in this way, initiate bioelectrocatalytic redox processes. This has been very useful in the development of the so-called second-generation biosensors, in which they transduce a catalyzed reaction into an electrical signal. Among other pre-requisites, redox mediators must be readily oxidized and/or reduced at the electrode surface and readily interact with the biorecognition component. Small chemical compounds (e.g. ferrocene derivatives, ruthenium, or osmium complexes and viologens) are frequently used for this purpose but, lately, small redox proteins (e.g. horse heart cytochrome c) have also been used as redox partners in biosensing applications. In general, docking between two complementary proteins introduces a second level of selectivity to the biosensor and enlarges the list of compounds analyzed. Moreover, electrochemical interferences are frequently minimized owing to the small overpotentials achieved. This paper provides an overview of enzyme biosensors that are mediated by electron-transfer proteins. The paper begins with a brief discussion of mediated electrochemistry in biosensing systems and proceeds with a detailed description of relevant work on the cooperative use of redox enzymes and biological electron donors and/or acceptors.
Keywords: Electrochemical biosensors; Redox partner; Electron-transfer protein; Mediated electrochemistry

Cellobiose dehydrogenase modified electrodes: advances by materials science and biochemical engineering by Roland Ludwig; Roberto Ortiz; Christopher Schulz; Wolfgang Harreither; Christoph Sygmund; Lo Gorton (3637-3658).
The flavocytochrome cellobiose dehydrogenase (CDH) is a versatile biorecognition element capable of detecting carbohydrates as well as quinones and catecholamines. In addition, it can be used as an anode biocatalyst for enzymatic biofuel cells to power miniaturised sensor–transmitter systems. Various electrode materials and designs have been tested in the past decade to utilize and enhance the direct electron transfer (DET) from the enzyme to the electrode. Additionally, mediated electron transfer (MET) approaches via soluble redox mediators and redox polymers have been pursued. Biosensors for cellobiose, lactose and glucose determination are based on CDH from different fungal producers, which show differences with respect to substrate specificity, pH optima, DET efficiency and surface binding affinity. Biosensors for the detection of quinones and catecholamines can use carbohydrates for analyte regeneration and signal amplification. This review discusses different approaches to enhance the sensitivity and selectivity of CDH-based biosensors, which focus on (1) more efficient DET on chemically modified or nanostructured electrodes, (2) the synthesis of custom-made redox polymers for higher MET currents and (3) the engineering of enzymes and reaction pathways. Combination of these strategies will enable the design of sensitive and selective CDH-based biosensors with reduced electrode size for the detection of analytes in continuous on-site and point-of-care applications.
Keywords: Biosensors; Carbohydrates; Catecholamines; Cellobiose dehydrogenase; Electron transfer; Nanomaterials

Electrode interfaces switchable by physical and chemical signals for biosensing, biofuel, and biocomputing applications by Evgeny Katz; Segiy Minko; Jan Halámek; Kevin MacVittie; Kenneth Yancey (3659-3672).
This review outlines advances in designing modified electrodes with switchable properties controlled by various physical and chemical signals. Irradiation of the modified electrode surfaces with various light signals, changing the temperature of the electrolyte solution, application of a magnetic field or electrical potentials, changing the pH of the solutions, and addition of chemical/biochemical substrates were used to change reversibly the electrode activity. The increasing complexity in the signal processing was achieved by integration of the switchable electrode interfaces with biomolecular information processing systems mimicking Boolean logic operations, thus allowing activation and inhibition of electrochemical processes on demand by complex combinations of biochemical signals. The systems reviewed range from simple chemical compositions to complex mixtures modeling biological fluids, where the signal substrates were added at normal physiological and elevated pathological concentrations. The switchable electrode interfaces are considered for future biomedical applications where the electrode properties will be modulated by the biomarker concentrations reflecting physiological conditions. Figure Modified electrodes were reversibly switched between active and inactive states by various physical and chemical signals.
Keywords: Modified electrode; Signal-responsive material; Switchable electrode; Biocomputing; Logic gate; Biosensor

Local control of protein binding and cell adhesion by patterned organic thin films by Frank Meiners; Inka Plettenberg; Julia Witt; Britta Vaske; Andreas Lesch; Izabella Brand; Gunther Wittstock (3673-3691).
Control of the cell adhesion and growth on chemically patterned surfaces is important in an increasing number of applications in biotechnology and medicine, for example implants, in-vitro cellular assays, and biochips. This review covers patterning techniques for organic thin films suitable for site-directed guidance of cell adhesion to surfaces. Available surface patterning techniques are critically evaluated, with special emphasis on surface chemistry that can be switched in time and space during cultivation of cells. Examples from the authors’ laboratory include the use of cell-repellent self-assembled monolayers (SAM) terminated by oligoethylene glycol (OEG) units and the lifting of the cell repellent properties by use of electrogenerated Br2/HOBr which can be performed with positionable microelectrodes. Structural changes of the SAM were analyzed by polarization-modulated infrared reflection absorption spectroscopy (PM IRRAS). Use of a soft array system of individually addressable microelectrodes enables formation of flexible and complex patterns in a short time and has the potential for further acceleration of probe-induced local manipulation of cell adhesion.
Keywords: Cell adhesion; Scanning electrochemical microscopy; Surface modification; Self assembled monolayers; Multielectrode probes

Electroanalysis of single-nucleotide polymorphism by hairpin DNA architectures by Alireza Abi; Elena E. Ferapontova (3693-3703).
Genetic analysis of infectious and genetic diseases and cancer diagnostics require the development of efficient tools for fast and reliable analysis of single-nucleotide polymorphism (SNP) in targeted DNA and RNA sequences often responsible for signalling disease onset. Here, we highlight the main trends in the development of electrochemical genosensors for sensitive and selective detection of SNP that are based on hairpin DNA architectures exhibiting better SNP recognition properties compared with linear DNA probes. SNP detection by electrochemical hairpin DNA beacons is discussed, and comparative analysis of the existing SNP sensing strategies based on enzymatic and nanoparticle signal amplification schemes is presented.
Keywords: Genosensors; DNA hairpin beacons; Single-nucleotide polymorphism; Electroanalysis

In this paper we critically review detection limits of electrochemical DNA biosensors enabling DNA detection without target labelling. The review includes transduction principles and latest breakthroughs. To compare the efficiency of each type of electrochemical DNA biosensor, a simple DNA biosensors classification is established on the basis of the nature of the bio-electrochemical transduction.
Keywords: Biosensor; DNA; Electrochemical; Label-free

Bioelectroanalysis with nanoelectrode ensembles and arrays by Michael Ongaro; Paolo Ugo (3715-3729).
This review deals with recent advances in bioelectroanalytical applications of nanostructured electrodes, in particular nanoelectrode ensembles (NEEs) and arrays (NEAs). First, nanofabrication techniques, principles of function, and specific advantages and limits of NEEs and NEAs are critically discussed. In the second part, some recent examples of bioelectroanalytical applications are presented. These include use of nanoelectrode arrays and/or ensembles for direct electrochemical analysis of pharmacologically active organic compounds or redox proteins, and the development of functionalized nanoelectrode systems and their use as catalytic or affinity electrochemical biosensors.
Keywords: Nanoelectrode; Ensemble; Array; Voltammetry; Biosensor; Mediated electrochemistry

Bioelectroanalytical procedures based on cathodic processes are often subject to interference from dissolved oxygen. At the potentials applied for analyte detection, oxygen reduction may occur directly at the electrode or may be catalyzed by the electron mediators or the sensing enzyme of the biosensor. These processes affect the background current and may thus result in erroneous analyte quantification. In this review, current strategies to circumvent these oxygen interferences are presented and critically assessed with respect to their compatibility for on-site monitoring with amperometric biosensing devices operating at low potential. The main strategies consist in (1) use of oxygen scavenging systems to remove dissolved oxygen from the sample, (2) design of bioelectroanalytical approaches to shift the applied potential for analyte detection to more positive values, and (3) development of electrode materials to increase the overpotential for the oxygen reduction reaction. The latest developments in these approaches have recently led to the first biosensing devices based on reductases fully compatible with on-site monitoring requirements and this opens up possibilities for their widespread application.
Keywords: Oxygen reduction reaction; Oxygen scavengers; Overpotential; Amperometric biosensors; Reductase; Cathodic processes; Electron mediator

Quantum dots on electrodes—new tools for bioelectroanalysis by F. Lisdat; D. Schäfer; A. Kapp (3739-3752).
The review covers recent developments in which quantum dots (QDs) are combined with electrodes for detection of analytes. Special focus will be on the generation of photocurrents and the possibility of spatially resolved, light-directed analysis. Different modes for combining biochemical reactions with QDs will be discussed. Other applications involve the use of QDs as labels in binding analysis. Different methods have been developed for read-out. In addition to photocurrent analysis, voltammetric detection of metals and electrochemiluminescence (ECL) can be used. In the latter, light is the sensor signal. ECL-based systems combine the advantage of very sensitive analytical detection with rather simple instrumentation. Figure Scheme of an enzymatic signal chain on a quantum dot electrode. Here the detection of glucose is achieved by the conversion of the enzymatically generated NADH at the illuminated QDs
Keywords: Quantum dot; Photocurrent; Electrochemiluminescence; Catalysis

New trends in the electrochemical sensing of dopamine by Krystyna Jackowska; Pawel Krysinski (3753-3771).
Since the early 70s electrochemistry has been used as a powerful analytical technique for monitoring electroactive species in living organisms. In particular, after extremely rapid evolution of new micro and nanotechnology it has been established as an invaluable technique ranging from experiments in vivo to measurement of exocytosis during communication between cells under in vitro conditions. This review highlights recent advances in the development of electrochemical sensors for selective sensing of one of the most important neurotransmitters—dopamine. Dopamine is an electroactive catecholamine neurotransmitter, abundant in the mammalian central nervous system, affecting both cognitive and behavioral functions of living organisms. We have not attempted to cover a large time-span nor to be comprehensive in presenting the vast literature devoted to electrochemical dopamine sensing. Instead, we have focused on the last five years, describing recent progress as well as showing some problems and directions for future development.
Keywords: Dopamine; Biosensors; Sensors; In vivo detection; Implantable sensors

Supramolecular immobilization of glucose oxidase on gold coated with cyclodextrin-modified cysteamine core PAMAM G-4 dendron/Pt nanoparticles for mediatorless biosensor design by Paula Díez; Ciprian-George Piuleac; Paloma Martínez-Ruiz; Santiago Romano; María Gamella; Reynaldo Villalonga; José M. Pingarrón (3773-3781).
Cysteamine core polyamidoamine G-4 dendron branched with β-cyclodextrins was chemisorbed on the surface of Au electrodes and further coated with Pt nanoparticles. Adamantane-modified glucose oxidase was subsequently immobilized on the nanostructured electrode surface by supramolecular association. This enzyme electrode was used to construct a reagentless amperometric biosensor for glucose, making use of the electrochemical oxidation of H2O2 generated in the enzyme reaction. The amperometric response of the biosensor was rapid (6 s) and a linear function of glucose concentration between 5 and 705 μmol L−1. The biosensor had a low detection limit of 2.0 μmol L−1, sensitivity of 197 mA mol−1 L cm−2, and retained 94 % of its initial response after storage for nine days at 4 °C.
Keywords: Biosensor; β-cyclodextrin; Dendrimer; Glucose oxidase; Platinum nanoparticles; Supramolecular complex

In situ electrochemical evaluation of anticancer drug temozolomide and its metabolites–DNA interaction by Ilanna C. Lopes; S. Carlos B. Oliveira; Ana Maria Oliveira-Brett (3783-3790).
Temozolomide (TMZ) is an antineoplastic alkylating agent with activity against serious and aggressive types of brain tumours. It has been postulated that TMZ exerts its antitumor activity via its spontaneous degradation at physiological pH. The in vitro evaluation of the interaction of TMZ and its final metabolites, 5-aminoimidazole-4-carboxamide (AIC) and methyldiazonium ion, with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine residues. The results obtained revealed the decrease of the dsDNA oxidation peaks with incubation time, showing that TMZ and AIC/methyldiazonium ion interact with dsDNA causing its condensation. Furthermore, the experiments of the in situ TMZ and AIC/methyldiazonium ion–dsDNA interaction using the multilayer dsDNA-electrochemical biosensor confirmed the condensation of dsDNA caused by these species and showed evidence for a specific interaction between the guanosine residues and TMZ metabolites, since free guanine oxidation peak was detected. The oxidative damage caused to DNA bases by TMZ metabolites was also detected electrochemically by monitoring the appearance of the 8-oxoguanine/2,8-dyhydroxyadenine oxidation peaks. Nondenaturing agarose gel electrophoresis of AIC/methyldiazonium ion–dsDNA samples confirmed the occurrence of dsDNA condensation and oxidative damage observed in the electrochemical results. The importance of the dsDNA-electrochemical biosensor in the in situ evaluation of TMZ–dsDNA interactions is clearly demonstrated.
Keywords: Temozolomide; Spontaneous chemical degradation; Oxidation; Multilayer dsDNA-electrochemical biosensor; DNA oxidative damage

At-line measurement of lactose in dairy-processing plants by Nick Glithero; Claire Clark; Lo Gorton; Wolfgang Schuhmann; Neil Pasco (3791-3799).
Environmental and process control applications have needs for sensors that operate continuously or repeatedly, making them applicable to batch measurement and flowing product stream measurement. Additionally, for lactose monitoring in dairy-processing plants, the sensors must have sufficient flexibility to handle a wide range of substrate concentration and be resilient to withstand wide pH excursions brought about by frequent exposure to clean-in-place chemicals that happen without any warning. This paper describes the development and trialling of an at-line lactose biosensor that meets the needs of the dairy industry for loss monitoring of lactose in dairy-processing plants by the combination of a third-generation enzyme biosensor with a sequential injection analyser. Results, both from grab sample analysis and an at-line factory prototype, are shown from their operation when installed at a Fonterra dairy factory (New Zealand) during the 2011–2012 season. Previous sensor fabrication methods were converted to a single-step process, and the flow-through cell was adapted to bubble-free operation. The lactose concentration in wastewater-processing streams was successfully monitored by taking and analysing samples every 2–3 min, semi-continuously, for 3 months by an unskilled operator. The Fonterra site flushes approximately 100–300,000 L of wastewater per hour from its lactose plant. In the 2011–2012 season, the daily mean lactose content of this wastewater varied significantly, from 0.0 to 8.0 % w/v (0–233,712 μM) and equated to substantial total losses of lactose over a 6-month period. These lactose losses represent lost saleable or useable product.
Keywords: At-line process analysis; Lactose measurement; Screen-printed electrodes; CDH-based biosensors; Flow injection analysis

Detection of haemoglobin using an adsorption approach at a liquid–liquid microinterface array by Eva Alvarez de Eulate; Lauren Serls; Damien W. M. Arrigan (3801-3806).
The behaviour of haemoglobin (Hb) at the interface between two immiscible electrolyte solutions (ITIES) has been examined for analytical purposes. When Hb is fully protonated under acidic conditions (pH I p = 7.46 C − 0.109, R = 0.996, where I p is the peak current (in nanoampere) and C is haemoglobin concentration (in micromolar). The calculated detection limit (3σ) was 48 nM for a 60 s preconcentration period, while the relative standard deviation was 13.3 % for six successive measurements at 0.1 μM Hb. These results illustrate the prospects for simple, portable and rapid label-free detection of biomacromolecules offered by electrochemistry at arrays of liquid–liquid microinterfaces.
Keywords: Haemoglobin; Adsorption; ITIES; Liquid–liquid interface; Adsorptive stripping voltammetry

Mediated glucose enzyme electrodes by cross-linking films of osmium redox complexes and glucose oxidase on electrodes by Peter Ó Conghaile; Sirisha Kamireddy; Domhnall MacAodha; Paul Kavanagh; Dónal Leech (3807-3812).
Here, we report on a novel, versatile approach for the preparation of mediated enzyme electrodes, demonstrated using cross-linked films of glucose oxidase and a range of functionalised osmium complexes on graphite electrodes. Response of enzyme electrodes are optimised by evaluation of glucose response as a function of variation in ratios of [Os(2,2′-bipyridine)2(4-aminomethyl pyridine)Cl]+ redox mediator, polyallylamine support and glucose oxidase enzyme cross-linked using a di-epoxide reagent in films on graphite. Lowering of the redox potential required to mediate glucose oxidation is achieved by synthesis of complexes using (4,4′-dimethyl-2,2′-bipyridine) or (4,4′-dimethoxy-2,2′-bipyridine) as a ligand instead of (2,2′-bipyridine). Enzyme electrodes prepared using the complexes based on dimethoxy- or dimethyl-substituted bipyridines provide glucose oxidation current densities of 30 and 70 μA cm−2 at 0.2 and 0.35 V applied potential compared to 120 μA cm−2 at 0.45 V for the initial enzyme electrode, under pseudo-physiological conditions in 5 mM glucose, with stability of signals proving inadequate for long-term operation. Current output and stability may be improved by selection of alternate anchoring and cross-linking methodology, to provide enzyme electrodes capable for application to long-term glucose biosensors and anodes in enzymatic fuel cells. Figure Glucose enzyme electrodes for application as biosensors or anodes in enzymatic fuel cells prepared by crosslinking films of osmium complex, glucose oxidase and polymer support on graphite electrodes.
Keywords: Biosensors; Electrochemical sensor; Enzymes; Glucose oxidase; Osmium; Enzymatic fuel cell

A new and simple-to-prepare hypoxanthine biosensor has been developed using xanthine oxidase (XOD) immobilised on carbon electrode surfaces. XOD was immobilised by glutaraldehyde cross-linking on carbon film (CF) electrodes and on carbon nanotube (CNT) modified CF (CNT/CF). A comparison of the performance of the two configurations was carried out by the current response using amperometry at fixed potential; the best characteristics being exhibited by XOD/CNT/CF modified electrodes. The effects of electrolyte pH and applied potential were evaluated, and a proposal is made for the enzyme mechanism of action involving competition between regeneration of flavin adenine dinucleotide and reduction of hydrogen peroxide. Under optimised conditions, the determination of hypoxanthine was carried out at −0.2 V vs. a saturated calomel electrode (SCE) with a detection limit of 0.75 μM on electrodes with CNT and at −0.3 V vs. SCE with a detection limit of 0.77 μM on electrodes without CNT. The applicability of the biosensor was verified by performing an interference study, reproducibility and stability were investigated, and hypoxanthine was successfully determined in sardine and shrimp samples.
Keywords: Hypoxanthine biosensor; Xanthine oxidase; Carbon film electrodes; Carbon nanotubes; Flavin adenine dinucleotide

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer-modified nanoporous gold electrodes by Urszula Salaj-Kosla; Micheál D. Scanlon; Tobias Baumeister; Kawah Zahma; Roland Ludwig; Peter Ó Conghaile; Domhnall MacAodha; Dónal Leech; Edmond Magner (3823-3830).
Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (±0.4), 16.0 (±0.1) and 2.0 (±0.1) μM were obtained for CtCDH-modified lactose and glucose biosensors and GOx-modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells composed of GOx- and CtCDH-modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase as cathodes, in biofuel cells. A maximum power density of 41 μW/cm2 was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O2-saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).
Keywords: Nanoporous gold electrodes; Mediated electron transfer; Biosensor; Bioanode; Biofuel cell

A microfluidic electrochemiluminescent device for detecting cancer biomarker proteins by Naimish P. Sardesai; Karteek Kadimisetty; Ronaldo Faria; James F. Rusling (3831-3838).
We describe an electrochemiluminescence (ECL) immunoarray incorporated into a prototype microfluidic device for highly sensitive protein detection and apply this system to accurate, sensitive measurements of prostate-specific antigen (PSA) and interleukin-6 (IL-6) in serum. The microfluidic system employed three molded polydimethylsiloxane (PDMS) channels on a conductive pyrolytic graphite chip (2.5 × 2.5 cm) inserted into a machined chamber and interfaced with a pump, switching valve, and sample injector. Each of the three PDMS channels encompasses three 3 μL analytical wells. Capture-antibody-decorated single-wall carbon nanotube forests are fabricated in the bottom of the wells. The antigen is captured by these antibodies on the well bottoms. Then, a RuBPY-silica-secondary antibody (Ab2) label is injected to bind to antigen on the array, followed by injection of sacrificial reductant tripropylamine (TPrA) to produce ECL. For detection, the chip is placed into an open-top ECL measuring cell, and the channels are in contact with electrolyte in the chamber. Potential applied at 0.95 V versus Ag/AgCl oxidizes TPrA to produce ECL by redox cycling the RuBPY species in the particles, and ECL light is measured by a charge-coupled device camera. This approach achieved ultralow detection limits of 100 fg mL−1 for PSA (9 zeptomole) and 10 fg mL−1 (1 zeptomole) for IL-6 in calf serum, a 10–25-fold improvement of a similar non-microfluidic array. PSA and IL-6 in synthetic cancer patient serum samples were detected in 1.1 h and results correlated well with single-protein enzyme-linked immunosorbent assays.
Keywords: Microfluidics; Electrochemiluminescence; Immunoarray; Single-wall carbon nanotube forest; RuBPY-silica nanoparticles

Interaction of antitumor flavonoids with dsDNA in the absence and presence of Cu(II) by Yassin M. Temerk; Mohamed S. Ibrahim; Mohamed Kotb; Wolfgang Schuhmann (3839-3846).
The binding of antitumor flavonoids, namely 3-hydroxyflavone (3HF) and hesperidin (Hesp) with dsDNA was investigated in the absence and presence of Cu(II) using cyclic voltammetry and square wave voltammetry at the hanging mercury drop electrode. The reduction currents of 3HF, 3HF-Cu complex, and the 3HF-β-cyclodextrin inclusion complex decreased after intercalation into dsDNA. The intercalation of Hesp into dsDNA is weak. dsDNA is reduced at a potential of −1.48 V overlaying the reduction of Hesp. In contrast, in the presence of Cu(II), the interaction of Hesp with dsDNA leads to a much stronger intercalation. The binding constants of the flavonoid-Cu complex with dsDNA were evaluated and calibration graphs for the determination of dsDNA were obtained from the decrease in the peak current in the cyclic voltammograms of 3HF in the presence of dsDNA. The proposed method exhibited good recovery and reproducibility for indirect determination of dsDNA.
Keywords: 3-hydroxyflavone; Hesperidin; dsDNA; Intercalation; Flavonoid-Cu complex; Voltammetry

Bioelectrochemical probing of intracellular redox processes in living yeast cells—application of redox polymer wiring in a microfluidic environment by Arto Heiskanen; Vasile Coman; Natalie Kostesha; David Sabourin; Nick Haslett; Keith Baronian; Lo Gorton; Martin Dufva; Jenny Emnéus (3847-3858).
Conventionally, microbial bioelectrochemical assays have been conducted using immobilized cells on an electrode that is placed in an electrochemical batch cell. In this paper, we describe a developed microfluidic platform with integrated microelectrode arrays for automated bioelectrochemical assays utilizing a new double mediator system to map redox metabolism and screen for genetic modifications in Saccharomyces cerevisiae cells. The function of this new double mediator system based on menadione and osmium redox polymer (PVI-Os) is demonstrated. “Wiring” of S. cerevisiae cells using PVI-Os shows a significant improvement of bioelectrochemical monitoring in a microfluidic environment and functions as an effective immobilization matrix for cells that are not strongly adherent. The function of the developed microfluidic platform is demonstrated using two strains of S. cerevisiae, ENY.WA and its deletion mutant EBY44, which lacks the enzyme phosphoglucose isomerase. The cellular responses to introduced glucose and fructose were recorded for the two S. cerevisiae strains, and the obtained results are compared with previously published work when using an electrochemical batch cell, indicating that microfluidic bioelectrochemical assays employing the menadione–PVI-Os double mediator system provides an effective means to conduct automated microbial assays. Figure Microfluidic platform for bioelectrochemical assays using osmium redox polymer “wired” living yeast cells
Keywords: Cellular redox activity; Microbial bioelectrochemistry; Osmium redox polymer; Double mediator system; Saccharomyces cerevisiae ; Microfluidic system

The electrochemical behavior of dopamine was examined under redox cycling conditions in the presence and absence of a high concentration of the interferent ascorbic acid at a coplanar, microelectrode array where the area of the generator electrodes was larger than that of the collector electrodes. Redox cycling converts a redox species between its oxidized and reduced forms by application of suitable potentials on a set of closely located generator and collector electrodes. It allows signal amplification and discrimination between species that undergo reversible and irreversible electron transfer. Microfabrication was used to produce 18 individually addressable, 4-μm-wide gold band electrodes, 2 mm long, contained in an array having an interelectrode spacing of 4 μm. Because the array electrodes are individually addressable, each can be selectively biased to produce an overall optimal electrochemical response. Four adjacent microbands were shorted together to serve as the collector, and were flanked on each side by seven microbands shorted as the generator (a ratio of 1:3.5 of electroactive area, respectively). This configuration achieved a detection limit of 0.454 ± 0.026 μM dopamine at the collector in the presence of 100 μM ascorbic acid in artificial cerebrospinal fluid buffer, concentrations that are consistent with physiological levels. Enhancement by surface modification of the microelectrode array to achieve this detection limit was unnecessary. The results suggest that the redox cycling method may be suitable for in vivo quantification of transients and basal levels of dopamine in the brain without background subtraction. Figure 1 Microelectrode array chip design and assignment of electrodes used for determination of dopamine (DA) in the presence of large excess of ascorbic acid (AA) by redox cycling. Analytes (DA and AA) are oxidized at the generator electrodes to form dopamine-o-quinone (DAQ) and dehydroascorbic acid (AAo) which diffuse to the nearest collector electrodes. DA is selectively detected at the collector electrodes, because DAQ can be reduced there, but AAo hydrolyzes to a nonelectroactive form prior to arrival
Keywords: Dopamine; Ascorbic acid; Electrochemistry; Redox cycling; Microelectrode array

Flexible micro(bio)sensors for quantitative analysis of bioanalytes in a nanovolume of human lachrymal liquid by Viktor Andoralov; Sergey Shleev; Thomas Arnebrant; Tautgirdas Ruzgas (3871-3879).
A flexible electrochemical micro(bio)sensor has been designed for determination of several biological compounds, specifically, ascorbate, dopamine, and glucose, in human lachrymal liquid (tears). The microsensor for simultaneous determination of ascorbate and dopamine concentrations was based on a gold microwire modified with the tetrathiafulvalen–7,7,8,8-tetracyanoquinodimethane complex as a catalyst. To monitor glucose concentration in tears, glucose dehydrogenase was immobilized on a gold microwire modified with carbon nanotubes and an osmium redox polymer. A capillary microcell was constructed for sampling tears. The cell had a working volume of 60–100 nL with a sampling deviation of 6.7 %. To check if the microcell was properly filled with buffer or tear sample, a control electrode was introduced into the construction. The electrode was used to measure the electrical resistance of a fully filled nanovolume cell. The mechanical flexibility is one of the most important features of the prototype and allowed direct collection of tears with minimized risk of damage to the eye. Figure Tear sampling and electrochemical analysis
Keywords: Flexible micro(bio)sensor; Nanovolume; Tears; Ascorbate; Dopamine; Glucose

Online rapid sampling microdialysis (rsMD) using enzyme-based electroanalysis for dynamic detection of ischaemia during free flap reconstructive surgery by M. L. Rogers; P. A. Brennan; C. L. Leong; S. A. N. Gowers; T. Aldridge; T. K. Mellor; M. G. Boutelle (3881-3888).
We describe an enzyme-based electroanalysis system for real-time analysis of a clinical microdialysis sampling stream during surgery. Free flap tissue transfer is used widely in reconstructive surgery after resection of tumours or in other situations such as following major trauma. However, there is a risk of flap failure, due to thrombosis in the flap pedicle, leading to tissue ischaemia. Conventional clinical assessment is particularly difficult in such ‘buried’ flaps where access to the tissue is limited. Rapid sampling microdialysis (rsMD) is an enzyme-based electrochemical detection method, which is particularly suited to monitoring metabolism. This online flow injection system analyses a dialysate flow stream from an implanted microdialysis probe every 30 s for levels of glucose and lactate. Here, we report its first use in the monitoring of free flap reconstructive surgery, from flap detachment to re-vascularisation and overnight in the intensive care unit. The on-set of ischaemia by both arterial clamping and failure of venous drainage was seen as an increase in lactate and decrease in glucose levels. Glucose levels returned to normal within 10 min of successful arterial anastomosis, whilst lactate took longer to clear. The use of the lactate/glucose ratio provides a clear predictor of ischaemia on-set and subsequent recovery, as it is insensitive to changes in blood flow such as those caused by topical vasodilators, like papaverine. The use of storage tubing to preserve the time course of dialysate, when technical difficulties arise, until offline analysis can occur, is also shown. The potential use of rsMD in free flap surgery and tissue monitoring is highly promising. Figure Free flap surgery timeline: The flap is raised and MD probe inserted. Glucose and lactate levels were monitored at 1 minute intervals throughout flap removal and the reconstruction of the tongue. Grey lines indicate key events as communicated by the surgeons in real time.
Keywords: Microdialysis; Free tissue transfer; Glucose; Lactate; Real time; Ischaemia

A displacement immunoassay involves having a labelled analogue of the analyte (the epitope) already bound to the antibody. The presence of the analyte causes a competition for antibodies, and some of the antibodies dissociates from the epitope so that it can bind with the analyte. Herein, the influence of the affinity of the surface-bound epitope for the antibody on the sensitivity and selectivity of a displacement immunosensor is explored both theoretically and experimentally. An electrochemical immunosensor described previously [1], where the dissociation of antibodies from an electrode surface causes an increase in current from surface-bound ferrocene species, is used for this purpose. As expected, the ease and effectiveness of the bound antibody being displaced is inversely related to the affinity of the antibody to the surface-bound epitope relative to the analyte in solution as expected. However, if the affinity constant is too low, selectivity and/or sensitivity are compromised. Experimental results are qualitatively compared with a simple mass-action model. Figure The important parameters in displacement immunoassays are investigated theoretically via a simple mass action model and compared with experimental data generated using a novel electrochemical immunosensor, as shown, where antibody on the surface suppresses electrochemistry and, hence, displacement of the antibody increases the current
Keywords: Immunosensor; Displacement assay; Theory; Electrochemistry

Reagentless d-sorbitol biosensor based on d-sorbitol dehydrogenase immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite by Zhijie Wang; Mathieu Etienne; Veronika Urbanova; Gert-Wieland Kohring; Alain Walcarius (3899-3906).
A reagentless d-sorbitol biosensor based on NAD-dependent d-sorbitol dehydrogenase (DSDH) immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite has been developed. It was prepared by durably immobilizing the NAD+ cofactor with DSDH in a sol–gel thin film on the surface of carbon nanotubes functionalized with poly(methylene green). This device enables selective determination of d-sorbitol at 0.2 V with a sensitivity of 8.7 μA mmol−1 L cm−2 and a detection limit of 0.11 mmol L−1. Moreover, this biosensor has excellent operational stability upon continuous use in hydrodynamic conditions. Figure Reagentless D-sorbitol biosensor based on NAD-dependent D-sorbitol dehydrogenase (DSDH) immobilized in sol-gel/carbon nanotubes/poly(methylene green) composite
Keywords: Carbon nanotubes; Sol–gel; Poly(methylene green); Cofactor; Dehydrogenase; Reagentless biosensor

Hybridization detection of enzyme-labeled DNA at electrically heated electrodes by Anne Walter; Annette-Enrica Surkus; Gerd-Uwe Flechsig (3907-3911).
In this report we describe an electrochemical DNA hybridization sensor approach, in which signal amplification is achieved using heated electrodes together with an enzyme as DNA-label. On the surface of the heatable low temperature co-fired ceramic (LTCC) gold electrode, an immobilized thiolated capture probe was hybridized with a biotinylated target using alkaline phosphatase (SA-ALP) as reporter molecule. The enzyme label converted the redox-inactive substrate 1-naphthyl phosphate (NAP) into the redox-active 1-naphthol voltammetrically determined at the modified gold LTCC electrode. During the measurement only the electrode was heated leaving the bulk solution at ambient temperature. Elevated temperature during detection led to increased enzyme activity and enhanced analytical signals for DNA hybridization detection. The limit of detection at 53 °C electrode temperature was 1.2 nmol/L.
Keywords: DNA hybridization; Heated biosensor; Enzyme label; Electrochemical detection; Alkaline phosphatase