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The immobilization of enzymes onto transducer support is a mature technology and has been successfully implemented to improve biocatalytic processes for diverse applications. However, there exists still need to design more sophisticated and specialized strategies to enhance the functional properties of the biosensors. In this work, a biosensor platform based on innovative fabrication strategy was designed, and employed for the detection of organophosphate (OP) in natural waters. The biosensor was prepared by incorporating acetylcholinesterase enzyme (AChE) to the graphite paste modified with tetracyanoquinodimethane (TCNQ) mediator, along with the use of a macroalgae (Cladaphropsis membranous) as a functional immobilization support. The novel immobilization design resulted in a synergic effect, and led to enhanced stability and sensitivity of the biosensor. The designed biosensor was used to analyze methyl parathion OP insecticide in water samples collected from a demonstrably contaminated lake of São Luis Island, Maranhão, Northeast of Brazil. Water analysis revealed that the aquatic ecosystem was polluted by sub-ppm concentrations of the OP insecticide, and a good correlation was found between values obtained through biosensor and GC-MS techniques. Our results demonstrated that macroalgae-biosensor could be used as a low-cost and sensitive screening method to detect target analyte. [ABSTRACT FROM AUTHOR]

Abstract: The glycated hemoglobin (HbA1c) level in blood is a measure of long-term glycemic status in patients with diabetes mellitus. Current clinical methods for determination of the HbA1c level include electrophoresis/electroendosmosis, ion exchange chromatography, high-performance liquid chromatography, boronate affinity chromatography, immunoassay, and liquid chromatography–tandem mass spectroscopy in addition to fluorometry and colorimetry. These methods have certain drawbacks such as being complex, time-consuming, and requiring expensive apparatus and trained persons to operate. These drawbacks were overcome by biosensing methods. We review these biosensors, which are based on (i) measurement of electrons, that is, current generated from splitting of hydrogen peroxide released during oxidation of fructosyl valine by immobilized fructosyl amino acid oxidase, which is directly proportional to HbA1c concentration, and (ii) direct measurement of HbA1c by some specific reaction. HbA1c biosensors work optimally within 4 to 1800s, between pH 7.0 and 9.0 and between 25 and 45°C, and in the range of 1 to 10,000μM, with a detection limit between 20 and 500μM and sensitivity between 4.6nA and 21.5μAmM−1 cm−2 and stable over a period of 5 to 90days. We suggest the ways to modify existing HbA1c biosensors, leading to simple, reliable, and economical sensors ideally suited for point-of-care treatment. [Copyright &y& Elsevier]

The direct electrochemical behaviour of bilirubin in the physio-pathological concentration range and at physiological pH was investigated by cyclic voltammetry. Nanostructured electrodes with a thin film of multi-walled carbon nanotubes exhibited a higher sensing performance than bare electrodes. The detection limit obtained with nanostructured electrodes (4.2±0.1 μM) allows the detection of both normal and pathological levels of bilirubin. Due to its sparse solubility in aqueous solvents, in human fluids bilirubin is found in the form of soluble complex with albumin. Therefore, the nanostructured-sensor response was studied in presence of different concentrations of this protein. A signal weakening was observed with increasing concentrations of albumin due to the decrease of free bilirubin. Finally, bilirubin detection was tested at concentrations typical of newborn jaundice (200–500 μM) and in the presence of normal albumin levels. A detection limit of 9.4±0.3 μM was identified. Since this value is below the minimum critical bilirubin concentration for newborns, our sensor, modified with a thin film of carbon nanotubes, could potentially be used for bilirubin detection in cases of newborn jaundice. [ABSTRACT FROM AUTHOR]

Abstract: Different enzyme immobilization approaches of Trametes versicolor laccase (TvL) onto gold surfaces and their influence on the performance of the final bioanalytical platforms are described. The laccase immobilization methods include: (i) direct adsorption onto gold electrodes (TvL/Au), (ii) covalent attachment to a gold surface modified with a bifunctional reagent, 3,3'-Dithiodipropionic acid di (N-succinimidyl ester) (DTSP), and (iii) integration of the enzyme into a sol–gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface (TvL/MPTS/Au). The characterization and applicability of these biosensors are described. Characterization is performed in aqueous acetate buffer solutions using atomic force microscopy (AFM), providing valuable information concerning morphological data at the nanoscale level. The response of the three biosensing platforms developed, TvL/Au, TvL/DTSP/Au and TvL/MPTS/Au, is evaluated in the presence of hydroquinone (HQ), used as a phenolic enzymatic substrate. All systems exhibit a clear electrocatalytic activity and HQ can be amperometrically determined at −0.10V versus Ag/AgCl. However, the performance of biosensors – evaluated in terms of sensitivity, detection limit, linear response range, reproducibility and stability – depends clearly on the enzyme immobilization strategy, which allows establishing its influence on the enzyme catalytic activity. [Copyright &y& Elsevier]

Accumulation of cholesterol in human blood can cause several health problems such as heart disease, coronary artery disease, arteriosclerosis, hypertension, cerebral thrombosis, etc. Therefore, simple and fast cholesterol determination in blood is clinically important. In this study, two types of amperometric cholesterol biosensors were designed by physically entrapping cholesterol oxidase in conducting polymers; thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemically and cholesterol oxidase was immobilized by entrapment during electropolymerization. The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H2O2at +0.7 V in the absence of a mediator. Kinetic parameters, such as Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. Km values were found as 1.47 and 5.16 mM for PEO-co-PPy and CP-co-PPy enzyme electrodes, respectively. By using these Km values, it can be observed that ChOx immobilized in PEO-co-PPy shows higher affinity towards the substrate. [ABSTRACT FROM AUTHOR]

A modified glassy carbon (GC) electrode was developed for the amperometric detection of biogenic amines, particularly histamine. The electrode was modified with the co-enzyme pyrroloquinoline quinone (PQQ) by entrapment during electropolymerziation of pyrrole to form polypyrrole (PPy). This method formed a thin film on the electrode surface possessing very good stability with a shelf-life exceeding one month without loss of signal. Optimal conditions for the PQQ/PPy electrode were determined and a linear response was found for histamine in phosphate buffer (pH 6) at +550 mV from 40 to 170 mg L−1 with a limit of detection ( S/N≥3) of 38 mg L−1. The practical linear range offered by this method suggests ideal use for spoilage detection in fermented foods. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: This paper presents the construction of amperometric biosensors for the detection of organophosphorus insecticides widely used in the treatment of olive trees. The systems are based on the immobilisation of acetylcholinesterase on screen-printed electrodes by bioencapsulation in a sol–gel composite. The enzyme activity was estimated by measuring the thiocholine produced by the enzymatic hydrolysis of the acetylthiocholine chloride using cobalt phtalocyanine as mediator. The developed devices have been used to carry out inhibition studies with three pesticides: malathion, methidathion and dimethoate (in their oxidized form), and tested using standard solutions and real samples of olive oil. These biosensors showed good operational stability as they maintained their initial analytical signal response during 10 successive measurements, and a good reproducibility with a relative standard deviation of 3%. The limits of detection of the developed devices were very compatible with the maximum residue limit tolerated by international regulations, they were as low as 10−9 M for the widely used pesticide malaoxon(oxidized malathion). The developed sensors were successfully used for the determinations of insecticides in real samples of olive oil. [Copyright &y& Elsevier]

Abstract: The electrochemical characterization of a class II cellobiose dehydrogenase (CDH), from the ascomycete fungus Neurospora crassa, adsorbed on graphite (G), was performed in regard to direct (DET) and mediated electron transfer (MET). The effects of the applied potential, mediator (1,4 benzoquinone) concentration and flow carrier pH on the amperometric response of the G/CDH modified electrodes were investigated under flow conditions. From the calibration curves, recorded at two pH values (5.2 and 7.0) for nine different sugars, the kinetic and the analytical parameters were evaluated under DET and MET operation modes. These results together with those obtained from long term operational stability measurements showed that: (i) for all nine investigated sugars the sensitivity was higher for MET than for DET and for pH 5.2 compared to pH 7.0; (ii) irrespective of DET or MET operation mode, the sensitivity of the new enzyme towards the investigated sugars decreased in the following sequence: cellobiose>lactose>(cellotriose≈cellopentaose) >>(maltotriose≈maltotetraose≈maltopentaose)>(maltose≈glucose); (iii) for all tested substrates, the apparent CDH affinity was roughly higher in DET than in MET operation mode. [Copyright &y& Elsevier]

Reaction-diffusion in porous enzyme-doped carbon paste electrode has been analyzed. The plate-gap model of such electrode has been proposed. The steady state current was calculated for the wide range of given parameters. The apparent parameters (apparent maximal currents and apparent Michaelis constants) of the modelled electrode were calculated. Relationships between apparent and given parameters were derived.

It was suggested that reaction-diffusion conditions in pores of bulk enzymatic electrode resemble particular conditions in thin enzyme filled gap between parallel conducting plates. The plate-gap model of porous enzymatic electrode is based on the diffusion equations containing a nonlinear term related to the Michaelis-Menten kinetics of the enzymatic reaction inside the gap. Steady state current was calculated for the wide range of given values of substrate diffusion coefficient, depth of the gap and substrate concentrations. Simple approximate relationships between “apparent” parameters of amperometric biosensor (maximal currents and apparent Michaelis constants) and given values of diffusion characterising parameters were derived. Association of these dependences with previously reported relationships led to derive approximate formulae that bind apparent parameters with the complete set of given parameters of the plate-gap enzymatic electrode. The limit case of slow diffusion into deep gap was also characterised. In this specific case, the highest numerical values of the apparent parameters were obtained. However, this gain is achievable at the expense of biosensor response time.

Abstract: Nanorods and nanospheres of FeS with different sizes have been synthesized from a single-source precursor Fe(ACDA)3 [HACDA=2-aminocyclopentene-1-dithiocarboxylic acid] complex by decomposing it in structure-directing solvents such as ethylene glycol (EG), ethylenediamine (EN) and ammonia (NH3). The phase purity, morphology, structure and properties of the FeS nanoparticles (NPs) were studied by powder X-ray diffraction, transmission electron microscopy, UV–vis absorption and photoluminescence spectroscopic techniques. The photocatalytic activity of FeS NPs for the degradation of Methylene Blue (MB) has been found to be much better compared to that exhibited by commercial TiO2. The synthesized FeS NPs showed catalytic activity towards peroxidase substrates 3,3′,5,5′-tetramethyl benzidine (TMB) and H2O2, which follows typical Michaelis–Menten kinetics and good catalytic efficiency. Furthermore, the FeS NPs immobilized on glassy carbon (GC) electrode performs as excellent amperometric sensor for the detection of H2O2. The FeSNPs/GC electrode showed a linear range for the detection of H2O2 from 10 to 130μM with a sensitivity of 26μA/mM and detection limit of 4.03μM. [Copyright &y& Elsevier]

The amperometric responses of G/CDH and Au-4-mercaptophenol/CDH modified electrodes to lactose were comparatively recorded under flow injection and cyclic voltammetry operating modes, respectively. The differences noticed between the characteristic parameters of the two investigated bioelectrodes were explained in terms of the influences exerted by the given experimental conditions on direct electron transfer process, existing between cellobiose dehydrogenase (CDH) and the investigated electrode materials. [ABSTRACT FROM AUTHOR]

Abstract: In the present paper, we continue to utilize the sequential action of sialic acid aldolase and pyruvate oxidase to construct improved sialic acid (SA) amperometric biosensors and immobilized enzyme reactors (IERs). The enzymes were co-immobilized using glutaraldehyde crosslinking at the surface of a platinum disc electrode and by covalent attachment to the modified controlled pore glass beads packed in stainless steel columns to prepare the SA biosensors and IERs, respectively. Two different configurations were adapted in the present work to produce the first report on flow-injection amperometric determination of SA. In the first configuration, the SA biosensor was used as a detector in a flow injection setup. In the second configuration, an IER was used in conjunction with a flow-through amperometric cell equipped with a Pt disc electrode polarized at 0.7V vs Ag/AgCl for the anodic detection of the produced hydrogen peroxide. We also report a simple way to control the temperature in the flow injection setups. The FI determination of SA based on IER was found to be more superior and showed linear dependence of the FI peak heights on SA concentration in the range of 0.002–5.0mM (r 2 =0.9997), RSD of 1.1% at 250μM SA and a sample throughput of 35 sampleh−1. [Copyright &y& Elsevier]

Abstract: A direct electrochemical method to reduce graphene oxide nanoribbons was proposed. The reduced graphene nanoribbons (RGNRs) could be functionalized with water-soluble iron(III) meso-tetrakis(N-methylpyridinum-4-yl) porphyrin (FeTMPyP) via π–π noncovalent interaction on electrode surface. The resulting FeTMPyP/RGNRs film showed excellent electrocatalysis toward the reduction of dissolved oxygen at peak potential of −0.28V. Using glucose oxidase as model enzyme, a biosensor based on the consumption of O2 was developed for amperometric detection of glucose ranging from 0.5mM to 10mM. This biosensor could be successfully applied in the detection of glucose in human serum. The FeTMPyP functionalized RGNRs provided a platform for electrocatalysis and biosensing of oxidase substrates. [Copyright &y& Elsevier]

Abstract: The chrono-impedance technique (CIT) for real time determination of glucose concentration in a first generation glucose oxidase/carbon paste electrode was implemented. The biosensor was polarized with a signal composed of 900mV DC potential and 50mVRMS AC signal at 0.4Hz. A frequency response analyzer was used to measure the complex impedance (magnitude |Z| and phase (Φ)) of the biosensor-bulk interface. Real time measurements were performed while glucose was added to the bulk within a concentration range of 0–40mM. The cumulative impedance dose–response curves were used to construct calibration curves, both for magnitude and phase. The best fitting was obtained with a hyperbolic equation. Four biosensors were built obtaining five calibration curves for each of them. A single test measurement (unknown glucose concentration) was also obtained after each calibration procedure. Glucose concentrations were estimated with the calibration curves and also measured by colorimetry, the latter being the reference method. Besides, one-way ANOVA test evaluated repeatability. Difference between means was not statistically significant (p >0.01) for both magnitudes (|Z| and Φ). The Student''s t-test assessed the differences significance, which produced in all cases p levels lower or equal than 0.44. Thus, CIT was proved to be a reliable method to measure glucose concentration in real time. Moreover, it showed high repeatability and compared well against colorimetry (r 2 =0.98). [Copyright &y& Elsevier]

Abstract: The development of enzyme-mediated amperometric biosensors on the indium–tin oxide (ITO) glass electrode via surface-initiated atom-transfer radical polymerization (ATRP) was investigated. A trichlorosilane coupling agent, containing the sulfonyl halide ATRP initiator, was immobilized initially on the ITO electrode surface for consecutive surface-initiated ATRP of ferrocenylmethyl methacrylate (FMMA) and glycidyl methacrylate (GMA). Glucose oxidase (GOD) was subsequently immobilized on the modified ITO electrode surface via coupling reactions between the epoxide groups of GMA and the amine groups of GOD. The surface composition after each functionalization step was ascertained by X-ray photoelectron spectroscopy (XPS). With the introduction of redox-P(FMMA) block as the electron-transfer mediator, the enzyme-mediated ITO electrode exhibits high sensitivity, as revealed by cyclic voltammetry measurement. The sensitivities of the ITO-g-P(GMA–GOD)-b-P(FMMA) and ITO-g-P(FMMA)-b-P(GMA–GOD) electrodes are about 3.6μA/(mMcm2) (in the linear concentration range 0–5mM of glucose) and 10.9μA/(mMcm2) (in the linear concentration range of 0–17mM of glucose), respectively. For both biosensors, the steady-state response time and the detection limits are estimated to be less than 20s and 0.4±0.1mM of glucose concentration, respectively. Furthermore, the spatial effect of the redox mediator on the electrode surface is revealed by the fact that the block copolymer brush-functionalized ITO electrode with P(FMMA) as the inner (first) block is more sensitive to glucose than that with P(GMA) as the inner block. [Copyright &y& Elsevier]

Abstract: Boron-doped diamond (BDD) electrodes outperform conventional electrodes in terms of high stability, chemical inertness, wide potential window and low background current. Combining the superior properties of BDD electrodes with the merits of biosensors, such as specificity, sensitivity, and fast response, amperometric biosensors based on BDD electrodes have attracted the interests of many researchers. In this review, the latest advances of BDD electrodes with different surfaces including hydrogen-terminated, oxygen-terminated, metal nanoparticles-modified, amine-terminated, and carboxyl-terminated thin films, and microelectrodes, for the construction of various biosensors or the direct detection of biomolecules were demonstrated. The future trends of BDD electrodes in biosensing were also discussed. [Copyright &y& Elsevier]

Abstract: The majority of the amperometric glucose biosensors employs stabilization membranes to prevent enzyme release and to improve selectivity. Here, we describe a comparative study between some of the most common membranes and two more innovative systems based on titania and Pd hexacyanoferrate hydrogels. The study has been carried out on electrodes modified by a Ni/Al hydrotalcite electrochemically deposited on Pt surfaces together with the enzyme glucose oxidase. The effect of the different membranes on the selectivity, stability and sensitivity of the biosensors has been described and the best ones have been successfully applied to the analysis of real matrices. To further improve biosensor selectivity a pre-oxidizing enzyme membrane containing also horseradish peroxidase was employed. [Copyright &y& Elsevier]

Abstract: Biomimetic condensation of silicic acid onto a polyethyleneimine (PEI) coated surface was investigated using quartz crystal microbalance measurements (QCM). It was shown that precipitation of silicic acid proceeds in two sequential stages. Very fast PEI-assisted silica formation is followed by slow non-specific gel deposition. Electrostatic adsorption of enzyme and precipitation of silicic acid on the surface of PEI-modified screen-printed carbon electrodes (SPCE) were found to be competing and therefore silica formation does not increase enzyme load. However, incubation of the PEI-modified surface in silicic after immobilization of enzyme can be used to solidify and reinforce the PEI backbone via non-covalent concreting by silica. This simple and quick procedure ultimately stabilizes electrostatically immobilized enzymes against washing-off in low pH and/or ionic strength environments. [Copyright &y& Elsevier]

Abstract: Three phenoloxidases based biosensors were successfully developed using as electrochemical transducer a new type of electrode recently developed by our group: the “Sonogel–Carbon electrode”. The employed enzymes were Trametes versicolor laccase (Lac), Mushroom tyrosinase (Tyr), and Horseradish peroxidase (HRP). Immobilization step was accomplished by doping the electrode surface with a mixture of the individual enzyme and Nafion ion exchanger as additive-protective. The biosensor responses, optimized in beer real samples, were evaluated for five individual polyphenols. It was found that the developed biosensors were sensitive to nanomolar concentrations of the tested polyphenols. As example, the limit of detection, sensitivity, and response linear range for caffeic acid for Nafion-Lac/Sonogel–Carbon biosensor were 0.06μmolL−1, 99.6nAμmol−1 L, and 0.04–2μmolL−1, respectively. The stability and reproducibility of the biosensors were evaluated by applying them directly to beer real samples. It has been demonstrated that the Nafion-Lac/Sonogel–Carbon system is the more stable with a relative standard deviation of 3.3% (n =10), maintaining 84% of its stable response for at least three weeks. Estimation of polyphenol index in eight lager beers and a comparison of the results with those obtained by a classical method was carried out. [Copyright &y& Elsevier]

A selective and sensitive method is presented for biogenic amines (BA) determination. The novelty consists in coupling a highly selective electrochemical biosensor to a weak acid cation-exchange column for online detection of amines. A bienzyme design, based on a recently isolated amine oxidase from grass pea and commercial horseradish peroxidase, was used for the biosensor construction. The enzymes were co-immobilized on the surface of a graphite electrode together with the electrochemical mediator (Os-redox polymer). The electrochemical detection was performed at a low applied potential (−50 mV vs . Ag/AgCl, KCl0.1 M), where biases from interferences are minimal. The separation and determination of six BA, with relevance in food analysis (tyramine, putrescine, cadaverine, histamine, agmatine and spermidine), were investigated. Irrespective of the BA nature, the amine oxidase-based biosensor showed a linear response up to 5 mM, and its sensitivity decreases in the following order: cadaverine, putrescine, spermidine, agmatine, histamine and tyramine. The approach was used to estimate the BA content in fish samples, after their extraction with methanesulfonic acid. [ABSTRACT FROM AUTHOR]

The responses of a PQQ-GDH entrapped in a polymer structure to mixtures of glucose and maltose were evaluated. Each compound was considered in the concentration range of 0–0.2 mM. Imaging was performed at constant height in the enzymatic feedback mode of scanning electrochemical microscopy (SECM). The enzyme-polymer spot was discretized into 15 × 15 μm2 substructures which were treated as independent individual microsensors. The response surfaces of the individual microsensors were approximated with a linear regression model. The coefficients in the derived equations represent contributions from topography, glucose concentration, maltose concentration, and the competition of glucose and maltose for the same active site of PQQ-GDH to the measured signal. The ratio of glucose and maltose contributions to the current at the SECM tip was constant for all microsensors and it was predominantly determined by the ratio of the turnover rates of both analytes in the PQQ-GDH catalyzed reaction. Using the difference between these coefficients, it was possible to select the microsensors within the overall enzyme-polymer spot that provided the best data for quantifying glucose and maltose by the artificial neural network used. The quantification of glucose and maltose was successful, except when the contributions from the components of the mixture were n g = k n units of glucose and simultaneously n m = 1.86(1− k) n units of maltose, for each constant n > 0 and k∈<0,1>. [ABSTRACT FROM AUTHOR]

Abstract: A conductive nanocage composed of Prussian blue (PB) nanoparticles doped mesocellular silica–carbon foam was prepared. This nanocage possessed ink-bottlelike structure with narrow and controllable pore-size distribution, good biocompatibility and favorable conductivity. The Prussian blue nanoparticles dispersed homogeneously on the mesowalls and provided the nanocage with highly catalytic ability toward the reduction of hydrogen peroxide. The nanocage could be used for volume-selective entrapment of enzyme to prepare a biosensor. The use of the Prussian blue nanoparticles doped nanocage would open new horizons for fabrication of biosensors and biocatalysts. [Copyright &y& Elsevier]

Abstract: In this work is reported the development and application of an amperometric biosensor for the determination of total biogenic amines content by using the commercial diamino oxidase (DAO from Porcine kidney E.C. 1.4.3.6) as the biocomponent, entrapped by glutaraldehyde onto an electrosynthesized bilayer film. In order to minimize both the fouling and the interference caused by the direct electrochemical oxidation of both the analytes (i.e., biogenic amines) and the common interferents usually present in food products the performances of Pt and Au electrodes and of several electroproduced anti-interferents mono- and bi-layer films were tested. In spite of a very low activity of the commercial DAO, the biosensor displayed a high response sensitivity in flow experiments, short response time, a good linear response and low detection limits. The excellent anti-interference characteristics allowed the use of the biosensor in screening analysis of food products. [Copyright &y& Elsevier]

Abstract: Enzyme–polymer layers immobilized on an electrode surface often serve as basis for amperometric biosensors. Caused by the formation process they show spatial variability in the polymer thickness which corresponds to a variability of immobilized enzyme activity. The relationship between topography and localized enzymatic activity of enzyme–polymer spots was studied using scanning electrochemical microscopy (SECM) in the feedback mode and generator-collector mode. Discretisation with a grid size corresponding to the scanning parameters defined substructures which can be treated as individual microsensors with specific response characteristics. The local responses are mainly governed by the polymer thickness but also influenced by neighbouring sites. Thus, discretisation allowed us to treat an enzyme–polymer spot with dimensions of about 300μm diameter like an array of more than 400 individual microsensors. Using suitable selection criteria and multivariate calibration it was possible to identify sensing sites which are optimal for the determination of glucose. It was demonstrated that an artificial neural network which was trained with the data provided by SECM images well predicted glucose concentration in the presence of ascorbic acid. [Copyright &y& Elsevier]

Carbon paste based biosensors for the determination of creatine and creatinine have been integrated into a sequential injection system. Applying the multi‐enzyme sequence of creatininase (CA), and/or creatinase (CI) and sarcosine oxidase (SO), hydrogen peroxide has been detected amperometrically. The linear concentration ranges are of pmol/L to nmol/L magnitude, with very low limits of detection. The proposed SIA system can be utilized reliably for the on‐line simultaneous detection of creatine and creatinine in pharmaceutical products, as well as in serum samples, with a rate of 34 samples per hour and RSD values better than 0.16% (n=10). [ABSTRACT FROM AUTHOR]

An amperometric biosensor has been developed for measurement of Umami, or the taste based on the amount of l-glutamate, in tomato foods. The biosensor is based on an enzyme-mediator system in which l-glutamate oxidase is used for biochemical oxidation of l-glutamate and a tetrafulvalene-tetracyanoquinodimethane (TTF-TCNQ) paste, prepared from the mixture of TTF-TCNQ salt, graphite powder, and silicone oil, serves as the mediator. The limit of detection, calculated by use of a four-parameter logistic model, was 0.05 mmol L−1, and the limit of quantification was 0.15 mmol L−1. The correlation coefficient ( R 2) was 0.990 and the relative standard deviation was no more than 1% ( n=5). The response time ( τ 95) was 20–50 s, depending on concentration. The repeatability of the sensor was better than 5% ( n=10). The sensor developed was stable for more than ten days. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

In order to determine creatine and creatinine, amperometric diamond paste biosensors were proposed. A bienzymatic biosensor based on creatinase and sarcosine oxidase was used for the assay of creatine and a trienzymatic biosensor based on creatinase, sarcosine oxidase, and creatininase was proposed for the assay of creatinine. The linear concentration ranges are of fmol/L magnitude order, with very low limits of detection. The biosensors proved to be highly reliable for determination of creatine and creatinine as raw materials in pharmaceutical formulations as well as in serum samples. [ABSTRACT FROM AUTHOR]

Abstract: Silver–DNA hybrid nanoparticles with controlled dimension were electrodeposited on a glassy carbon electrode by the reduction of silver in the aid of DNA. The hybrid nanoparticles showed a narrow size distribution and a favorable catalytic ability to reduction of hydrogen peroxide and dissolved oxygen, which were related to the DNA concentration in deposition solution and the deposition time. The presence of DNA avoided the further aggregation of silver nanoparticles and improved the catalytic ability of the nanoparticles. The modified electrode obtained showed a linear response to hydrogen peroxide concentration ranging from 2.0μM to 2.5mM with a limit of detection of 0.6μM and a sensitivity of 773μAmM−1 cm−2. Based on the dissolved oxygen consumption during the oxidation process of glucose catalyzed by the immobilized glucose oxidase, a glucose biosensor with a limit of detection of 9.0μM and a linear range from 50μM to 1.2mM was fabricated. The biosensor excluded the interference from ascorbic acid, acetaminophen, and uric acid that always coexist with glucose in samples. [Copyright &y& Elsevier]

Abstract: An interference-free first generation alcohol biosensor based on alcohol oxidase immobilized, by glutaraldehyde co-crosslinking with bovine serum albumin, on a perm-selective electropolymerized film modified gold electrode is described. The use, in place of platinum, of a gold electrode, properly conditioned by an electrochemical pre-treatment, allowed the elimination of the electrode fouling and of the interference caused by the direct alcohol oxidation at the electrode surface. The efficiency of overoxidized polypyrrole and poly-ortho-phenylendiamine as anti-interferent perm-selective films towards the most common electroactive interferents was also investigated. The low response time and the fabrication procedure employed in this study allowed the use of the biosensor in a flow injection system equipped with a conventional thin-layer electrochemical cell. The biosensor showed a high sensitivity, a good stability and a complete suppression of electroactive interferences in operational conditions. Biosensor performances were tested by the rapid determination of the alcohol content in just diluted wine samples and the results compared well with those obtained with the classical method of distillation. [Copyright &y& Elsevier]

Abstract: The incorporation of fungal laccases into novel applications has been delayed mainly due to their intrinsic sensitivity towards halides and alkaline conditions. In order to explore new sources of enzymes we evaluated the multipotent polyphenol oxidase PPO1 from the marine bacterium Marinomonas mediterranea. Here we report that, in contrast to its fungal counterparts, PPO1 remained functional above neutral pH presenting high specificity for phenolic compounds, in particular for methoxyl-substituted mono-phenols and catechols. These properties, in addition to its tolerance towards chloride (up to 1M) and its elevated redox potential at neutral pH (0.9V), suggest this enzyme may be an interesting candidate for specific applications such as the Amperometric determination of phenolic compounds and bio-fuel cells. [Copyright &y& Elsevier]

A sequential injection analysis/amperometric biosensor system is proposed for the enantioselective analysis of the S-enantiomer of enalapril, ramipril and pentopril. The amperometric biosensor used as detector in the sequential injection analysis was designed by immobilization of l-amino acid oxidase in carbon paste. The proposed SIA system can be utilized reliably for the enantioanalysis of the S-enantiomer from the raw materials as well as from their pharmaceutical formulations, with a rate of 75 samples per hour and R.S.D. values better than 0.1% (n = 10). [Copyright &y& Elsevier]

Sensitive amperometric biosensors for phenols compounds, based on tyrosinase (polyphenoloxidase, PPO) immobilized on a Pt electrode in an electropolymerized poly-amphiphilic pyrrole matrix or cross-linked with glutaraldehyde, were constructed and compared. Steady-state amperometric measurements, performed at −50 mV vs. SCE in aqueous phosphate buffer containing LiClO4 0.1 M (pH 7) as well as in a chloroform solution containing 0.1 M C6H5CH2N(CH3)3Cl, were used in order to compare the electroanalytical and kinetic parameters of the investigated amperometric biosensors in aqueous and nonaqueous media. It was established that the polypyrrole matrix has a higher efficiency for enzyme retention resulting in higher bioelectrode sensitivity, both in aqueous buffer (690 μA M−1) and in chloroform (149 μA M−1). [Copyright &y& Elsevier]

A mathematical model of a sandwich membrane biosensor with an enzyme layer between two membranes is considered. The electrochemically active product of the enzymatic reaction is detected by a catalytic electrode on one side of the sandwich membrane. A finite but different mass transfer resistance across the solution-membrane interface was assumed few the substrate and product. The steady state solution depended on the Blot numbers and the diffusivities of the substrate and electrochemically active product. At low Blot numbers the signal depended on the mass transfer coefficient and was independent of the membrane thickness. At high Blot numbers the signal depended on the position of the enzyme layer and the relative diffusivity of the electrochemically active product in the two membranes. [ABSTRACT FROM AUTHOR]

Conducting electroactive polymers (CPs) are materials discovered just over 20 years ago which have aroused considerable interest on account of their electronic conducting properties and unique chemical and biochemical properties. Consequently, they have numerous (bio)analytical and technological applications. CPs are easily synthesized and deposited onto the conductive surface of a given substrate from monomer solutions by electrochemical polymerization with precise electrochemical control of their formation rate and thickness. Coating electrodes with CPs under mild conditions opens up enormous possibilities for the immobilization of biomolecules and bioaffinity or biorecognizing reagents, the improvement of their electrocatalytic properties, rapid electron transfer and direct communication to produce a range of analytical signals and new analytical applications. Co-immobilization of other molecules (enzymatic co-factors or charge-transfer mediators) by entrapment within electropolymerized films or by covalent binding on these films permits straightforward fabrication of reagentless biosensors. The characteristics of CPs and their uses, mainly in amperometric biosensors, are reviewed. The most recent applications and lines of research related to CP films are summarized in the different sections of the paper, and probable future trends are discussed. [ABSTRACT FROM AUTHOR]

A sequential injection analysis (SIA) system is described for the simultaneous determination of l-and d-carnitine using amperometric biosensors as detectors. The SIA system was used, because of its high precision, accuracy and low sample and buffer consumption. The biosensors were designed using physical and chemical immobilization of l-amino acid oxidase and horseradish peroxidase (HRP) for the assay of l- carnitine, and d-amino acid oxidase and HRP for the assay of d-carnitine. The linear concentration ranges are in the pmol/l to nmol/l magnitude order, with very low limits of detection. The biosensors/SIA system was used reliably for on-line process control of the enantiopurity of carnitine with a frequency of 34 samples per hour. [Copyright &y& Elsevier]

In order to determine creatine and creatinine amperometric biosensors were proposed. A bienzymatic biosensor based on creatinase (CI) and sarcosine oxidase (SO) was used for the assay of creatine and a trienzymatic biosensor based on CI, SO and creatininase (CA) for the assay of creatinine. The linear concentration ranges are of pmol l−1 to nmol l−1 magnitude order, with very low limits of detection. The biosensors proved high reliability for determination of creatine and creatinine as raw material, and in the pharmaceutical formulation. [Copyright &y& Elsevier]

In order to determine ortho-acetyl-L-carnitine, two biosensors were proposed. The biosensors were designed using physical immobilization of L-amino acid oxidase (L-AAOD) and horseradish peroxidase (HRP). Electrode characteristics were obtained and compared for the two carbon paste (graphite powder and paraffin oil) biosensors. The linear concentration ranges for the proposed biosensors were in the ranges of fmol/L to nmol/L, magnitude order with low limits of detection. Due to their reliability, the biosensors were used as detectors in a sequential injection analysis system, and gave reliable results for on-line assay of ortho-acetyl-L-carnitine in synthesis process control with a frequency of 75 samples per hour. [ABSTRACT FROM AUTHOR]

Libraries consisting of acrylic acid-based polymer resins were synthesized by copolymerizing acrylic acid with di4erent alkyl acrylates and 1-vinyl imidazole concomitantly varying the relative concentrations and the number of monomers present in the reaction mixture. The obtained polymer resins are suitable for electrochemically-induced precipitation on electrode surfaces caused by the protonation of acidic side groups at the polymer chains induced by a local electrochemical formation of protons in the di4usion zone in front of the electrode. Addition of suitable biological recognition elements such as glucose oxidase to suspensions of these electrodeposition paints (EDP) led to the entrapment of the enzyme within the slowly precipitating polymer film, and hence to the nonmanual fabrication of amperometric biosensors. The suitability of the obtained copolymers was screened with respect to the performance of the resulting glucose sensors. It could be demonstrated that the chemical composition of the used EDP significantly influences the properties of the sensors in terms of sensitivity and linear range of their response to glucose. [ABSTRACT FROM AUTHOR]

A simple and reliable method for rapid evaluation of mixtures of phenolic compounds (phenol/chlorophenol, cathecol/phenol, cresol/chlorocresol and phenol/cresol) using a dual amperometric device is described. This new approach is based on the difference between the sensitivity of laccase and tyrosinase for different phenolic compounds. A multichannel potentiostat was used to monitor simultaneously laccase- and tyrosinase-based biosensors, and the data were treated using the partial least squares (PLS) chemometric algorithm. This system showed an excellent efficiency for the resolution of the phenolic mixtures. For example, in the phenol/chlorophenol mixture it was studied the determination of individual species in a concentration range from 1.0×10−6 to 10.0×10−6 mol l−1 obtaining relative standard deviations of 3.5 and 3.1% for phenol and chlorophenol, respectively. The excellent correlation between the estimated and the real concentrations can also be observed by the correlation coefficients (0.9958 and 0.9981 for phenol and chlorophenol, respectively). These results show that proposed methodology can be successfully employed to the simultaneous determination of phenolic compounds in mixtures, even in more diluted solutions. [Copyright &y& Elsevier]