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A novel amperometric algae-based biosensor was developed for the detection of photosynthetic herbicides in river water. The green photosynthetic algae Chlamydomonas reinhardtii was immobilized on carbon black modified screen-printed electrodes, exploiting carbon black as smart nanomaterial to monitor changes in algae oxygen evolution during the photosynthetic process. The decrease of oxygen evolution, occurring in the presence of herbicides, results in a decrease of current signals by means of amperometric measurements, in an analyte concentration dependent manner. Atrazine as case study herbicide was detected in a concentration range of 0.1 and 50 μM, with a linear range from 0.1 to 5 μM and a detection limit of 1 nM. No interference was observed in presence of 100 ppb arsenic, 20 ppb copper, 5 ppb cadmium, 10 ppb lead, 10 ppb bisphenol A, and 1 ppb paraoxon, tested as safety limits. A ~25% matrix effect and satisfactory recovery values of 107 ± 10% and 96 ± 8% were obtained in river water for 3 and 5 μM of atrazine, respectively. Stability studies were also performed obtaining a high working stability up to 10 h and repeatability with an RSD of 1.1% (n = 12), as well as a good storage stability up to 3 weeks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this study we reported the development of a paper-based algal biosensor for the optical detection of nanoencapsulated-atrazine, a forefront nanoformulated herbicide with a high effective post-emergence herbicidal activity. In particular, the unicellular green photosynthetic algae Chlamydomonas reinhardtii was immobilised on a paper substrate soaked with an agar thin film and placed in a glass optical measurement cell, obtaining a totally environmental-friendly device. Nanoencapsulated-atrazine was detected by following the variable fluorescence (1-V J ) parameter, which decreased inversely proportional to the herbicide concentrations, in a range between 0.5 and 200 nM, indicating a linear relationship in the measured dose-response curves and a detection limit of 4 pM. Interference studies resulted in a very slight interference in presence of 2 ppm copper and 10 ppb arsenic at safety limits, as well as a slight matrix effect and a satisfactory recovery value of 96 ± 5% for 75 nM nanoencapsulated-atrazine in tap water. Stability studies were also performed obtaining a good storage stability up to 3 weeks. Results demonstrated the suitability of the proposed paper-based optical biosensor as a valid support in smart agriculture for on site, environmental friendly, cost effective and sensitive nanoencapsulated-atrazine analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Polybrominated diphenyl ethers (PBDEs) are a type of flame retardants which are currently banned in EU and USA due their hazardousness for humans and mammals. However, these compounds were highly used during more than 30 years and still persist in the environment since they are resistant to degradation. Herein we present a biosensor for the detection of PBDEs using screen printed carbon electrodes (SPCEs) based on the electrochemical monitoring of water oxidation reaction (WOR) catalyzed by iridium oxide (IV) nanoparticles (IrO 2 NPs). Our assay shows a limit of detection of 21.5 ppb of PBDE in distilled water. We believe that such an IrO 2 NPs-based electrocatalytic sensing system can lead to a rapid, sensitive, low cost and miniaturizable device for the detection of PBDEs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Herein, we report an impedimetric DNA-based aptamer sensor for a single-step detection of B. anthracis spore simulant (B. cereus spore). Specifically, we designed a miniaturized label-free aptasensor for B. cereus spores based on a gold screen-printed electrode functionalized with B. cereus spores-binding aptamer (BAS-6R). Several parameters were optimized to fabricate the aptasensor such as the concentration of DNA aptamer solution (0.5 µM), the time (48 h), the temperature (4 °C), and the pH (7.5) for aptamer immobilization on the working electrode surface. Once the aptasensor was developed, it was tested against B. cereus spores 14579 evaluating the effect of incubation time and MgCl 2 concentration. Under the optimized conditions (incubation time equal to 3 h and absence of MgCl 2 ), B. cereus spores 14579 were detected with a linear range between 10 4 CFU/ml and 5 × 10 6 CFU/ml and a detection limit of 3 × 10 3 CFU/ml. Furthermore, the study of selectivity toward B. cereus 11778, B. subtilis, Legionella pneumophila, and Salmonella Typhimurium has demonstrated the capability of this sensor to detect B. cereus spores, proving the suitability of the DNA-based sensing element combined with a portable instrument for a label-free measurement on site of B. anthracis spore simulant., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Herein, we propose the first three-dimensional origami paper-based device for the detection of several classes of pesticides by combining different enzyme-inhibition biosensors. This device was developed by integrating two different office paper-based screen-printed electrodes and multiple filter paper-based pads to load enzymes and enzymatic substrates. The versatile analysis of different pesticides was carried by folding and unfolding the filter paper-based structure, without any addition of reagents and any sample treatment (i.e. dilution, filtration, pH adjustment). The paper-based platform was employed to detect paraoxon, 2,4-dichlorophenoxyacetic acid, and atrazine by exploiting the capability of these different types of pesticides (i.e. organophosphorus insecticides, phenoxy-acid herbicides, and triazine herbicide) to inhibit butyrylcholinesterase, alkaline phosphatase, and tyrosinase, respectively. The degree of inhibition correlating to the quantity of pesticides was evaluated by chronoamperometrically monitoring the enzymatic activity in the absence and in the presence of pesticides by using a portable potentiostat. To improve the sensitivity, the paper-based electrodes were modified with carbon black alone in the case of platforms for 2,4-dichlorophenoxyacetic acid and atrazine detection, or decorated with Prussian blue nanoparticles for the detection of paraoxon. The paper-based device was applied for the detection of paraoxon, 2,4-dichlorophenoxyacetic acid, and atrazine at ppb level in both standard solutions and river water sample. The accuracy of this origami multiple paper-based electrochemical biosensor was evaluated in river water by recovery studies, obtaining satisfactory values (e.g. for paraoxon 90 ± 1% and 88 ± 2%, for 10 and 20 ppb, respectively). The proposed three-dimensional origami paper device allows for rapid, cost-effective and accurate pesticide detection in surface water as a result of combining filter and office papers, screen-printing, wax-printing and nanomaterial technology., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bioassays based on enzyme inhibition are analytical tools widely employed for inhibitor analysis. Beside the conventional analytical techniques such as chromatography and mass spectrometry, these bioassays are cost-effective, easy to use, and suitable for in situ measurement but they are often characterised by a quite narrow linear range. Herein, we report a novel graphical method based on integrated Michaelis-Menten equation, valid for all types of reversible inhibition, which provides an extended linear range. The suitability of this innovative approach was demonstrated in the case of fluoride quantification using a colorimetric bioassay based on acetylcholinesterase inhibition. The "half time reaction", estimated by the progress curve of cholinesterase inhibition, was plotted versus the fluoride inhibitor concentration, observing an extended linear range up to 5 mM, instead of 0.6 mM using initial rate measurements. The applicability of this new concept was further demonstrated in the case of catalase enzyme inhibited by cyanide. Furthermore, it was demonstrated that fixed substrate conversion at level of 10-50% allows determination of inhibitor concentration in a wide linear range with high precision and in short time of analysis. This novel theoretical and practical approach allows for the extension of the linear range without any further experiments, with several advantages including low reagent consumption, reduced waste generation and time of measurement., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The development of rapid, cost-effective, and single-step methods for the detection of small molecules is crucial for improving the quality and efficiency of many applications ranging from life science to environmental analysis. Unfortunately, current methodologies still require multiple complex, time-consuming washing and incubation steps, which limit their applicability. In this work we present a competitive DNA-based platform that makes use of both programmable DNA-switches and antibodies to detect small target molecules. The strategy exploits both the advantages of proximity-based methods and structure-switching DNA-probes. The platform is modular and versatile and it can potentially be applied for the detection of any small target molecule that can be conjugated to a nucleic acid sequence. Here the rational design of programmable DNA-switches is discussed, and the sensitive, rapid, and single-step detection of different environmentally relevant small target molecules is demonstrated.

The recent goal of sustainability in analytical chemistry has boosted the development of eco-designed analytical tools to deliver fast and cost-effective analysis with low economic and environmental impact. Due to the recent focus in sustainability, we report the use of low-cost filter paper as a sustainable material to print silver electrodes and to load reagents for a reagent-free electrochemical detection of chloride in biological samples, namely serum and sweat. The electrochemical detection of chloride ions was carried out by exploiting the reaction of the analyte (i.e. chloride) with the silver working electrode. During the oxidation wave in cyclic voltammetry the silver ions are produced, thus they react with chloride ions to form AgCl, while in the reduction wave, the following reaction occurs: AgCl + e - -->Ag + Cl - . These reactions at the electrode surface resulted in anodic/cathodic peaks directly proportional to the chloride ions in solution. Chloride ions were detected with the addition of only 10μL of the sample on the paper-based electrochemical cell, obtaining linearity up to 200mM with a detection limit equal to 1mM and relative standard deviation lower than 10%. The accuracy of the sensor was evaluated in serum and sweat samples, with percentage recoveries between 93 ± 10 and 108 ± 8%. Moreover, the results achieved with the paper-based device were positively compared with those obtained by using the gold standard method (Ion Selective Electrode) adopted in routine clinical analyses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Abstract : Salmonella has represented the most common and primary cause of food poisoning in many countries for at least over 100 years. Its detection is still primarily based on traditional microbiological culture methods which are labor-intensive, extremely time consuming, and not suitable for testing a large number of samples. Accordingly, great efforts to develop rapid, sensitive and specific methods, easy to use, and suitable for multi-sample analysis, have been made and continue. Biosensor-based technology has all the potentialities to meet these requirements. In this paper, we review the features of the electrochemical immunosensors, genosensors, aptasensors and phagosensors developed in the last five years for Salmonella detection, focusing on the critical aspects of their application in food analysis.

Paper-based microfluidic devices are gaining large popularity because of their uncontested advantages of simplicity, cost-effectiveness, limited necessity of laboratory infrastructure and skilled personnel. Moreover, these devices require only small volumes of reagents and samples, provide rapid analysis, and are portable and disposable. Their combination with electrochemical detection offers additional benefits of high sensitivity, selectivity, simplicity of instrumentation, portability, and low cost of the total system. Herein, we present the first example of an integrated paper-based screen-printed electrochemical biosensor device able to quantify nerve agents. The principle of this approach is based on dual electrochemical measurements, in parallel, of butyrylcholinesterase (BChE) enzyme activity towards butyrylthiocholine with and without exposure to contaminated samples. The sensitivity of this device is largely improved using a carbon black/Prussian Blue nanocomposite as a working electrode modifier. The proposed device allows an entirely reagent-free analysis. A strip of a nitrocellulose membrane, that contains the substrate, is integrated with a paper-based test area that holds a screen-printed electrode and BChE. Paraoxon, chosen as nerve agent simulant, is linearly detected down to 3µg/L. The use of extremely affordable manufacturing techniques provides a rapid, sensitive, reproducible, and inexpensive tool for in situ assessment of nerve agent contamination. This represents a powerful approach for use by non-specialists, that can be easily broadened to other (bio)systems., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Through the years, scientists have developed cutting-edge technologies to make (bio)sensors more convenient for environmental analytical purposes. Technological advancements in the fields of material science, rational design, microfluidics, and sensor printing, have radically shaped biosensor technology, which is even more evident in the continuous development of sensing systems for the monitoring of hazardous chemicals. These efforts will be crucial in solving some of the problems constraining biosensors to reach real environmental applications, such as continuous analyses in field by means of multi-analyte portable devices. This review (with 203 refs.) covers the progress between 2010 and 2015 in the field of technologies enabling biosensor applications in environmental analysis, including i) printing technology, ii) nanomaterial technology, iii) nanomotors, iv) biomimetic design, and (v) microfluidics. Next section describes futuristic cutting-edge technologies that are gaining momentum in recent years, which furnish highly innovative aspects to biosensing devices., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report a screen-printed electrode (SPE) modified with a dispersion of carbon black (CB) and chitosan by drop casting. A cyclic voltammetry technique towards ferricyanide, caffeic acid, hydroquinone, and thiocholine was performed and an improvement of the electrochemical response with respect to bare SPE as well as SPE modified only with chitosan was observed. The possibility to detect thiocholine at a low applied potential with high sensitivity was exploited and an acetylcholinesterase (AChE) biosensor was developed. A dispersion of CB, chitosan, and AChE was used to fabricate this biosensor in one step by drop casting. The enzymatic activity of the immobilized AChE was determined measuring the enzymatic product thiocholine at +300 mV. Owing to the capability of organophosphorus pesticides to inhibit AChE, this biosensor was used to detect these pollutants, and paraoxon was taken as model compound. The enzyme inhibition was linearly related to the concentration of paraoxon up to 0.5 μg L(-1), and a low detection limit equal to 0.05 μg L(-1) (calculated as 10% of inhibition) was achieved. This biosensor was challenged for paraoxon detection in drinking waters with satisfactory recovery values. The use of AChE embedded in a dispersion of CB and chitosan allowed an easy and fast production of a sensitive biosensor suitable for paraoxon detection in drinking waters at legal limit levels. Graphical Abstract Biosensors based on screen-printed electrodes modified with Acetylcholinesterase, Carbon Black, and Chitosan for organophosphorus pesticide detection.

The potentiometric E-tongue system was employed for water toxicity estimation in terms of cyanobacterial microcystin toxins (MCs) detection. The data obtained from E-tongue were correlated to the MCs content detected by the standard chromatographic technique UHPLC-DAD (Ultra High Performance Liquid Chromatography with Diode Array Detector), as far as by the colorimetric enzymatic approach. The prediction of MCs released by toxic Microcystis aeruginosa strains was possible with Root Mean Squared Error of Validation (RMSEV) lower or very close to 1µg/L, the provisional guideline value of WHO for MCs content in potable waters. The application of E-tongue system opens up a new perspective offset for fast and inexpensive analysis in the field of environmental monitoring, offering also the possibility to distinguish toxin producing and non-toxic M. aeruginosa strains present in potable water., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Enzyme inhibitors like drugs and pollutants are closely correlated to human and environmental health, thus their monitoring is of paramount importance in analytical chemistry. Enzymatic biosensors represent cost-effective, miniaturized and easy to use devices; particularly biosensors based on enzyme inhibition are useful analytical tools for fast screening and monitoring of inhibitors. The present review will highlight the research carried out in the last 9 years (2006-2014) on biosensors based on enzyme inhibition. We underpin the recent advances focused on the investigation in new theoretical approachs and in the evaluation of biosensor performances for reversible and irreversible inhibitors. The use of nanomaterials and microfluidic systems as well as the applications of the various biosensors in real samples is critically reviewed, demonstrating that such biosensors allow the development of useful devices for a fast and reliable alarm system., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Oxidized Single-Wall Carbon Nanohorns (o-SWCNHs) were used, for the first time, to assemble chemically modified Screen Printed Electrodes (SPEs) selective towards the electrochemical detection of Epinephrine (Ep), in the presence of Serotonine-5-HT (S-5HT), Dopamine (DA), Nor-Epineprhine (Nor-Ep), Ascorbic Acid (AA), Acetaminophen (Ac) and Uric Acid (UA). The Ep neurotransmitter was detected by using Differential Pulse Voltammetry (DPV), in a wide linear range of concentration (2-2500 μM) with high sensitivity (55.77 A M(-1) cm(-2)), very good reproducibility (RSD% ranging from 2 to 10 for different SPEs), short response time for each measurement (only 2s) and low detection of limit (LOD=0.1 μM). o-SWCNHs resulted in higher analytical performances when compared with other nanomaterials used in literature for electrochemical sensors assembly., (Copyright © 2014 Elsevier B.V. All rights reserved.)

An electrochemical sensor for palytoxin (PlTX) detection, based on a strip of eight screen-printed electrodes connected to a cost-effective and portable apparatus, is reported. Sheep erythrocytes were used to test the palytoxin detector and degree of haemolysis was evaluated by measuring release of the cytosolic lactate dehydrogenase (LDH). Percentage haemolysis and, therefore, the amount of LDH measured, by use of NADH/pyruvate and appropriate electrochemical mediators, was correlated with the concentration of the toxin. Two different electrochemical approaches were investigated for evaluation of LDH release, but only one based on the use of a binary redox mediator sequence (phenazine methosulfate in conjugation with hexacyanoferrate(III)) proved useful for our purpose. After analytical and biochemical characterization, the sensor strip was used to measure palytoxin. Sheep blood and standard solutions of PlTX were left to react for two different incubation times (24 h or 4 h), resulting in working ranges of 7 × 10(-3)-0.02 ng mL(-1) and 0.16-1.3 ng mL(-1), respectively. The specificity of the test for palytoxin was evaluated by use of ouabain, which acts in the same way as PlTX on the Na(+)/K(+)-ATPase pump. A cross-reactivity study, using high concentrations of other marine biotoxins was also conducted. Experiments to evaluate the matrix effect and recovery from mussels are discussed.

A simple graphical method for the determination of reversible inhibition type, inhibition constant (Ki) and estimation of fifty percent of inhibition I₅₀ of an enzyme reaction is described. The method consists of plotting experimental data as "degree of inhibition" versus the inhibitor concentration at two or more concentrations of substrate. Diagnosis of inhibition type is based on determination of and the observation of the shift of the inhibition curves. Relationship between I50 and inhibition constant Ki was discussed. A simplified hyperbolae equation of degree of inhibition showing kinetic orders of 1 and zero at low and high concentrations of inhibitors respectively is proposed. The relative error of inhibitor concentration increased drastically when degree of inhibition reached values of 90%. Examples of published inhibition reports as well as an experimental example of amperometric biosensor based on tyrosinase inhibition by benzoic acid were in agreement with the proposed theoretical approach., (© 2013 Published by Elsevier B.V.)

Here, we employ a model electrochemical DNA sensor to demonstrate that the detection limit and specificity of surface-based sensors often are not dependent on the true affinity of the probe for its target but are simply dependent on the effective probe concentration. Under these circumstances, the observed affinity (and thus the sensor's detection limit and specificity) will depend on the density with which the probes are packed on the surface of the sensor, the surface area, and even the volume of sample employed.

A glucose biosensor was assembled using gold microelectrodes (diameter of 250 μm) coated by Single-Walled Carbon Nanotubes (SWCNTs), via the Electrophoresis Deposition Process (EPD). This nanostructured platform was successfully used to deposit the poly(pyrrole)/glucose oxidase film (PPy/GOx). The most important result of this biosensors was the wide linear range of concentration, ranging from 4 to100 mM (covering the hypo- and hyper-glycemia range, useful in diabetes). This extended linearity offered the possibility to measure glucose from 0.560 to 12.0 mM, with a detection limit of 50 μM (useful for hypo-glycemia disease)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Air-filled lysozyme microbubbles (LSMBs) were engineered as a support for the immobilization of gold nanoparticles and an enzyme, alkaline phosphatase, in order to develop micro-antimicrobial and biosensing devices. Gold nanoparticles immobilized on LSMBs significantly improved the antimicrobial efficacy of the microbubbles against M. lysodeikticus. The surface functionalization of the microbubbles with gold nanoparticles did not affect their echogenicity when exposed to an ultrasound imaging probe. Alkaline phosphatase was conjugated on the surface of microbubbles without compromising its enzymatic activity. The functionalized microbubbles were used for the detection of paraoxon in aqueous solutions.

This work investigates the behaviour of two alternative systems that model the crucial event involved in any ELISA test, i.e. the molecular recognition between an antigen and its specific antibody on a solid phase, and its measurement. Each approach is devised with the goal of making possible a single-step, separation and wash-free amperometric magneto-immunosensor. Magnetic particles (MBs) are used as support for the immobilization of rabbit IgGs that are recognized by the specific anti-rabbit IgG-HRP. The assay protocol is based on the use of a series of small "reservoirs" containing phosphate buffer, hydroquinone, anti-rabbit IgG-HRP and an appropriate amount of MB-rabbit IgG. After a brief incubation, the content of each "reservoir" is transferred to one of the wells of a 8-well magnetized-screen-printed electrode strip. The resulting MB-IgG-anti-IgG-HRP chain, is then concentrated on the working electrode surface for electrochemical measurement. Two different approaches to monitor this immunological reaction are investigated. The first one is based on the enzyme-channeling principle (ECP) and involves the use of a second enzyme, glucose oxidase (GOD), immobilized on the working electrode previously modified with Prussian Blue. Since the H(2)O(2) produced by GOD is the co-substrate of the HRP enzyme, glucose is added into the well and the current, generated by the residual H(2)O(2), is measured. The second, more direct, approach is performed without exploiting ECP (no GOD enzyme), by adding H(2)O(2) into the well and measuring the current generated by the HRP product on a pristine screen-printed electrode. Both approaches yielded a typical sigmoidal binding curve, illustrating the discrimination between the signal produced by the immuno-bound HRP concentrated on the electrode surface, and the background signal due to HRP in the bulk solution.

This review covers applications of pristine and functionalized single-wall carbon nanotubes (SWCNTs) in nano-medicine, cultural heritage, and biosensors. The physicochemical properties of these engineered nanoparticles are similar to those of ultrafine components of airborne pollution (UF) and might have similar adverse effects. UF may impair cardiovascular autonomic control (inducing a high-risk condition for adverse cardiovascular effects), cause mammalian embryo toxicity, and increase geno-cytotoxic risk. SWCNTs coated with a biopolymer, for example polyethylenimine (PEI), become extremely biocompatible, hence are useful for in-vivo and in-vitro drug delivery and gene transfection. It is also possible to successfully immobilize a human enteric virus on PEI/SWCNT composites, suggesting application as a carrier in non-permissive media. The effectiveness of carbon nanostructured materials in the cleaning, restoration, and consolidation of deteriorated historical surfaces has been widely shown by the use of carbon nanomicelles to remove black dendritic crust from stone surfaces. The nanomicelles, here, have the twofold role of delivery and controlled release of the cleaning agents. The high biocompatibility of functionalized SWCNTs with enzymes and proteins is a fundamental feature used in the assembly of electrochemical biosensors. In particular, a third-generation protoporphyrin IX-based biosensor has been assembled for amperometric detection of nitrite, an environmental pollutant involved in the biodeterioration and black encrustation of historical surfaces.

In this paper we propose the construction and application of a portable multi-purpose biosensor array for the simultaneous detection of a wide range of endocrine disruptor chemicals (EDCs), based on the recognition operated by various enzymes and microorganisms. The developed biosensor combines both electrochemical and optical transduction systems, in order to increase the number of chemical species which can be monitored. Considering to the maximum residue level (MRL) of contaminants established by the European Commission, the biosensor system was able to detect most of the chemicals analysed with very high sensitivity. In particular, atrazine and diuron were detected with a limit of detection of 0.5nM, with an RSD% less than 5%; paraoxon and chlorpyrifos were revealed with a detection of 5 μM and 4.5 μM, respectively, with an RSD% less than 6%; catechol and bisphenol A were identified with a limit of detection of 1 μM and 35 μM respectively, with an RSD% less than 5%., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Paper-based artworks are among the most valuable assets for transmission of knowledge. Historical paper is composed of different polysaccharides (e.g. cellulose), binders, and glues. During aging all of these components undergo several degradation processes, as a result of external and intrinsic causes, and these can compromise the state of conservation of the document. In this work, application of a new biotechnological strategy for paper artefact preservation is reported. By making use of innovative and non-invasive materials, for example appropriate hydrogels, in combination with selective electrochemical biosensors, it is possible to simultaneously verify the degradation condition of the paper artwork and then to efficiently clean it, while monitoring the process of removal of both pollution and degradation products. In this paper, we focus on specific examples in which such techniques have been applied to paper artworks and that illustrate the advantages and potential of this biotechnology compared with the traditional paper-cleaning methods currently in use.

Here, we demonstrate a strategy to convert the graded Michaelis-Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the "branch point effect", a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter's response. Above an arbitrarily selected "threshold" substrate concentration, the nonsignaling HK enzyme saturates leading to a "sudden" activation of the first signaling GOx enzyme and a far steeper dose-response curve than that observed for simple Michaelis-Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications.

Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets.

A simple and reliable technique has been developed for the construction of an amperometric acetylcholinesterase biosensor based on screen-printed carbon electrodes. For the first time, one-step modification using single-walled carbon nanotubes and Co phtalocyanine has been proposed to decrease the working potential and to increase the signal of thiocholine oxidation. The biosensor developed made it possible to detect 5-50 ppb of paraoxon and 2-50 ppb of malaoxon with detection limits of 3 and 2 ppb, respectively (incubation 15 min). The biosensor showed high reproducibility when measurements of the substrate and inhibitor were performed (R.S.D. about 1% and 2.5%, respectively). The reliability of the inhibition measurements was confirmed by testing spiked samples of sparkling and tape waters., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An efficient electrochemical protocol to monitor hydrogen peroxide consumption during metal-catalyzed oxidation by using screen-printed electrodes modified with Prussian blue is presented. In particular, cyclooctene oxidation to cyclooctene oxide, catalyzed by a vanadium(V)-salophen complex (H(2)salophen=N,N'-o-phenylenebis(salicylideneimine)), in molecular and ionic media was tested. Initially, a protocol for batch analysis was developed for a monophasic system in acetonitrile, and subsequently, an in situ protocol was developed for a biphasic system of 1-butyl-3-methylimidazolium hexafluorophosphate/phosphate buffer. Calibration curves were performed in amperometric mode by applying -50 mV versus an Ag pseudo-reference. The calibration curve of hydrogen peroxide showed a linear correlation from 1 × 10(-6) up to 5 × 10(-3) mol L(-1) with satisfactory inter- and intra-electrode reproducibility (relative standard deviation (RSD) values of 5 and 13%, respectively, for the monophasic system and 11 and 13%, respectively, for the biphasic system). Kinetic studies to investigate the oxidation reaction for both the mono- and biphasic systems have been carried out in amperometric mode as well. Firstly, the decomposition of hydrogen peroxide was examined, which showed that, in 1-butyl-3-methylimidazolium hexafluorophosphate(,) it completely decomposed in 300 min, whereas in acetonitrile, in the same time frame, 20% of the initial amount was still active. In the presence of 1% of the catalyst the decomposition rate increased in both solvents. Finally, the complete oxidation of cyclooctene was followed and the effective conversion was determined. The developed protocols showed high reproducibility, with the advantage that the environmentally friendly biphasic system could also be recycled. The good analytical performance obtained, coupled with a short analysis time, the possibility of in-line automation and the use of ionic liquids instead of molecular solvents, made this system a very attractive choice for monitoring oxidative reactions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A reliable and cost-effective electrochemical method for the detection of deoxynivalenol (DON) in cereals and cereal-based food samples based on the use of a novel anti-DON Fab fragment is presented. The analytical system employed, Enzyme-Linked-Immunomagnetic-Electrochemical (ELIME) assay, is based on the use of immunomagnetic beads (IMBs) coupled with eight magnetized screen-printed electrodes (8-mScPEs) as electrochemical transducers. Using standard solutions of DON, a working range between 100 and 4500 ng/ml was obtained with an EC(50) of 380 ng/ml. The ELIME assay was employed to evaluate the cross-reactivity of the Fab fragment towards different trichothecenes revealing a good selectivity towards DON over other trichothecenes with the exception of 3-Ac-DON. The sensor was then applied to cereals and cereal-based food samples (wheat, breakfast cereal and baby-food) and a wide range of sample treatment procedures was tested. Within-laboratory precision (9-24% repeatability for breakfast cereals and 10-33% for baby-food) and recovery data (82-110% for breakfast cereals and 97-108% for baby-food) were calculated by analyzing blank breakfast cereals and baby-foods fortified with DON, demonstrating that the proposed method has the capability for use as a screening assay for DON in such products., (Copyright 2010 Elsevier B.V. All rights reserved.)

A novel biosensor assay format for aflatoxin based on acetylcholinesterase (AChE) inhibition by aflatoxin B(1) (AFB(1)) is proposed. The AChE was present in solution and an amperometric choline oxidase biosensor was used for monitoring its residual activity. To create the biosensor, the choline oxidase was immobilized by cross-linking onto screen-printed electrodes modified with Prussian Blue (PB) and these were used to detect the H(2)O(2) at low potential (-0.05V versus a screen-printed internal silver pseudoreference electrode). For the development of the AFB(1) assay, several parameters such as AChE and substrate concentration, the methanol effect, and pH were evaluated and optimized. The linear working range was assessed to be 10-60ppb. Concentrations as low as 2ppb, which correspond to the legal limit of AFB(1) in food for humans, were detected after a pre-concentration step. The suitability of the method was evaluated using commercial olive oil samples. A recovery equal to 78+/-9% for 10ppb of AFB(1) in olive oil samples was obtained.

A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen+phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml(-1). The total analysis time is the sum of 50 min for the off-line enzymatic incubations and 4 min for the biosensor response.

The present review deals with novel developments in immunosensors destined for final application in food analysis. In this perspective particular emphasis will be given to the most important approaches which recently have been used for immunosensor construction and assembling. For this reason, electrochemical, surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) techniques will be explored in detail and recent and practical examples on food matrices will be reviewed. Objective of this survey is to give a general overview of the possible application of immunosensors to the food analysis field.

The nerve agents are chemical warfare agents known to be used during terrorist attacks. An inexpensive and portable system to be used by first responders and military personnel is of interest owing to the continuing threat of possible terrorist attacks. Amperometric biosensors based on cholinesterase inhibition show such potentialities. In this work butyrylcholinesterase was immobilized onto screen-printed electrodes modified with Prussian blue and the nerve agent detection was performed by measuring the residual activity of enzyme. The optimized biosensor was tested with sarin and VX standard solutions, showing detection limits of 12 and 14 ppb (10% of inhibition), respectively. The enzymatic inhibition was also obtained by exposing the biosensors to sarin in gas phase. Two different concentrations of sarin gas (0.1 and 0.5 mg m(-3)) at different incubation times (from 30 s up to 10 min) were tested. It is possible to detect sarin at a concentration of 0.1 mg m(-3) with 30-s incubation time, with a degree of inhibition of 34%, which match the legal limits (immediate danger to life and health).

Lipase activity against triacylglycerols has been measured using an amperometric enzyme biosensor based on glycerol dehydrogenase/NADH oxidase. A Prussian Blue modified screen-printed electrode was selected as substrate for the two immobilised-enzyme systems due to their higher operative stability reported in previous works. Various parameters such as cofactor (flavin mononucleotide FMN) concentration (1 mM), NAD+ coenzyme concentration (2 mM), pH effect (phosphate buffer pH 6 to 8, Tris buffer pH 8-10) response time and storage stability were evaluated and optimised. The glycerol biosensor was then investigated for lipase activity. The system was challenged against an olive or sunflower oil real samples in order to detect fatty acids and the results were compared with those provided either by the manufacture or reference methods with good agreement.

Glucose biosensors based on the use of planar screen-printed electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring performed with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 degrees C) with an operative stability of ca. 72 h, thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system.

Ochratoxin A (OTA) is a fungal metabolite that occurs in foods, beverages, animal tissues, human blood and presents carcinogenic, teratogenic and nephrotoxic properties. This study concerns the redox properties of OTA using electrochemical techniques which have the potential for providing insights into the biological redox reactions of this molecule. The in situ evaluation of the OTA interaction with DNA using a DNA-electrochemical biosensor is also reported. The oxidation of OTA is an irreversible process proceeds with the transfer of one electron and one proton in a diffusion-controlled mechanism. The diffusion coefficient of OTA was calculated in pH 7 phosphate buffer to be D(O) = 3.65x10(-6) cm2 s(-1). The oxidation of OTA is also pH dependent for electrolytes with pH<7 and involves the formation of a main oxidation product which adsorbs strongly at the GCE surface undergoing reversible oxidation. In alkaline electrolytes OTA undergoes chemical deprotonation, the oxidation involving only the transfer of one electron. The electrochemical dsDNA-biosensor was also used to evaluate the possible interaction between OTA and DNA. The experiments have clearly proven that OTA interacts and binds to dsDNA strands immobilized onto a GCE surface, but no evidence of DNA-damage caused by OTA was obtained.

Bulk screen-printed electrodes (bSPEs) modified with zirconium phosphate (ZrP) and Meldola blue (MB) and by electrochemical deposition of a Reineckate film (bMBZrPRs-SPEs) have been constructed and used as NADH sensors. Cyclic voltammetric investigation of these bulk electrochemically modified screen-printed electrodes revealed stable catalytic activity in oxidation of the reduced form of the coenzyme nicotinamide adenine dinucleotide (NADH). Flow-injection analysis (FIA) coupled with amperometric detection confirmed the improved stability of the bMBZrPRs-SPEs (10(-4) mol L(-1) NADH, %RSD = 4.2, n = 90, pH 7.0). Other conditions, for example applied working potential (+50 mV relative to Ag|AgCl), flow rate (0.30 mL min(-1)) and pH-dependence (range 4.0-10.0) were evaluated and optimized. A glycerol biosensor, prepared by immobilizing glycerol dehydrogenase (GDH) on the working electrode area of a bMBZrPRs-SPE, was also assembled. The biosensor was most stable at pH 8.5 (%RSD = 5.6, n = 70, 0.25 mmol L(-1) glycerol). The detection and quantification limits were 2.8 x 10(-6) and 9.4 x 10(-6) mol L(-1), respectively, and the linear working range was between 1.0 x 10(-5) and 1.0 x 10(-4) mol L(-1). To assess the effect of interferences, and recovery by the probe we analyzed samples taken during fermentation of chemically defined grape juice medium and compared the results with those obtained by HPLC.

Modified screen-printed electrodes for amperometric detection of H(2)O(2) and nicotinamide adenine dinucleotide (NADH) at low applied potential are presented in this paper. The sensors are obtained by modifying the working electrode surface with Prussian Blue, a well known electrochemical mediator for H(2)O(2) reduction. The coupling of this sensor with phenazine methosulfate (PMS) in the working solution gives the possibility of measuring both NAD(P)H and H(2)O(2). PMS reacts with NADH producing PMSH, which in the presence of oxygen, gives an equimolar amount of H(2)O(2). This allows the measurement of both analytes with similar sensitivity (357 mA mol(-1)L cm(-2) for H(2)O(2) and 336 mA mol(-1)L cm(-2) for NADH) and LOD (5x10(-7)mol L(-1) for H(2)O(2) and NADH) and opens the possibility of a whole series of biosensor applications. In this paper, results obtained with a variety of dehydrogenase enzymes (alcohol, malic, lactate, glucose, glycerol and glutamate) for the detection of enzymatic substrates or enzymatic activity are presented demonstrating the suitability of the proposed method for future biosensor applications.

Analytical technology based on sensors is an extremely broad field which impacts on many major industrial sectors such as the pharmaceutical, healthcare, food, and agriculture industries as well as environmental monitoring. This review will highlight the research carried out during the last 5 years on biosensors that are based on enzyme inhibition for determination of pollutants and toxic compounds in a wide range of samples. Here the different enzymes implicated in the inhibition, different transducers forming the sensing devices, and the different contaminants analyzed are considered. The general application of the various biosensors developed, with emphasis on food and environmental applications, is reviewed as well as the general approaches that have been used for enzyme immobilization, the enzyme catalysis, and the inhibition mechanism.

Acetylcholinesterase (ChE) sensor based on Prussian blue (PB) modified electrode was developed and tested for the detection of organophosphorus and carbamic pesticides. The signal of the sensor was generated in PB mediated oxidation of thiocholine recorded at +200 mv in DC mode. ChE from electric eel was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) on the surface of screen-printed carbon electrode covered with PB and Nafion. The content of the surface layer (specific enzyme activity, Nafion and BSA amounts) was optimized to establish high and reliable response toward the substrate and ChE inhibitors. The ChE/PB sensor makes it possible to detect Aldicarb, Paraoxon and Parathion-Methyl with limits of detection 30, 10 and 5 ppb, respectively (incubation 10 min). The feasibility of practical application of the ChE/PB sensor developed for the monitoring of degradation of the pesticides in wine fermentation was shown. To diminish matrix interferences, the electrolysis of the grape juice with Al anode and evaporation of ethanol were suggested, however the procedures decrease the sensitivity of pesticide detection and stability of the sample tested.

Being one of the most commonly used electrochemical mediators for analytical applications, Prussian Blue has found a wide use in the biosensor field during the last years. Its particular characteristic of catalysing hydrogen peroxide reduction has been applied in the construction of a large number of oxidase enzyme-based biosensors for clinical, environmental and food analysis. By modifying an electrode surface with Prussian Blue, it is in fact possible to easily detect hydrogen peroxide at an applied potential around 0.0 V versus Ag/AgCl, thus making possible coupling with oxidase enzymes while also avoiding or reducing electrochemical interferences. Papers dealing with glucose, lactate, cholesterol and galactose biosensors that are based on the use of Prussian Blue have recently appeared in the most important analytical chemistry journals. Another recent trend is the use of a choline probe based on choline oxidase for pesticide determination to exploit the inhibition of acetylcholinesterase by these compounds. In addition, the use of Prussian Blue in the development of biosensors for food analysis has captured the interest of many research groups and led to improved methods for the detection of glutamate, galactose, alcohol, fructosyl amine, formate, lysine and oxalate. This review will focus on the biosensing aspects of Prussian Blue-based sensors giving a general overview of the advantages provided by such mediator as well as its drawbacks. A comprehensive bibliographic reference list is presented together with the most up to date research findings in this field and possible future applications. The commercial potential of sensors based on this mediator will also be discussed.

Novel planar glucose biosensors to be used for continuous monitoring have been developed. The electrodes are produced with the "screen printing" technique, and present a high degree of reproducibility together with a low cost and the possibility of mass production. Prior to enzyme immobilisation, electrodes are chemically modified with ferric hexacyanoferrate (Prussian Blue). This allows the detection of the hydrogen peroxide produced by the enzymatic reaction catalysed by GOD, at low applied potential (ca. 0.0 V versus Ag/AgCl), highly limiting any electrochemical interferences. The layer of Prussian Blue (PB) showed a high stability at the working conditions (pH 7.4) and also after 1 year of storage dry at RT, no loss of activity was observed. The assembled glucose biosensors, showed high sensitivity towards glucose together with a long-term operational and storage stability. In a continuous flow system, with all the analytical parameters optimised, the glucose biosensors detected glucose concentration as low as 0.025 mM with a linear range up to 1.0mM. These probes were also tested over 50-60 h in a continuous flow mode to evaluate their operational stability. A 0.5 mM concentration of glucose was continuously fluxed into a biosensor wall-jet cell and the current due to the hydrogen peroxide reduction was continuously monitored. After 50-60 h, the drift of the signal observed was around 30%. Because of their high stability, these sensors suggest the possibility of using such biosensors, in conjunction with a microdialysis probe, for a continuous monitoring of glucose for clinical purposes.

Conductive polymer nanotubules of 1,2-diaminobenzene (1,2-DAB) were prepared using a porous polycarbonate membrane template, placed on a Pt foil and used to support the polymer, then, the electropolymerisation was performed by chronocoulometry. The obtained conductive polymer nanostructures were then placed on Pt electrode and used to support highly dispersed prussian blue (PB), which acts as the active component for H2O2 detection. The observed good stability of PB as catalyst of H2O2 was related to the presence of organic non-conventional conducting polymers in a composite nanostructured film. These nanostructured polymer/PB composite films were also characterised by scanning electron microscopy (SEM) and Raman spectroscopy. The non-conventional conducting polymer nanotubules/PB modified Pt electrodes were tested by cyclic voltammeter for stability at different pH values, then, by amperometry, for hydrogen peroxide, ascorbic acid, acetaminophen, uric acid and acetylcholine. Glucose oxidase (GOD), lactate oxidase (LOD), L-amino acid oxidase (L-AAOD), alcohol oxidase (AOD), glycerol-3-phosphate oxidase (GPO), lysine oxidase (LyOx), and choline oxidase (ChOx) were immobilised on PB layer supported on 1,2-diaminobenzene (1,2-DAB) nanotubules onto the Pt electrodes. Different strategies for enzyme immobilisation were performed and used. Analytical parameters such as reproducibility, interference rejection, response time, storage and operational stability of the sensors have been studied and optimised. Results provide a guide to design high sensitive, stable and interference-free biosensors. The glucose biosensors assembled with nanostructured poly(1,2-DAB) showed a detection limit of 5 x 10(-5) mol l(-1), a wide linearity range (5 x 10(-5) to 5 x 10(-3) mol l(-1)), a high selectivity, a stability of 3 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical parameters and stability were also studied for L-(+)-lactic acid, L-leucine, ethanol, glycerol-3-phosphate, lysine, and choline biosensors.

The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples.

Studies of the oxidation of beta-nicotinamide adenine dinucleotide (NADH) at glassy carbon (GCEs) electrode surfaces, modified with nonconventional conducting polymer nanotubules, are reported. In contrast to the situation with conventional carbon electrodes, chemical reversibility of the NADH oxidation reaction was achieved by means of poly(1,2-diaminobenzene) conducting nanotubule coatings. A Delta E(p) of 425 mV (vs Ag/AgCl; pH 7.0) was observed. The NADH amperometric response of the conducting nanotubule modified GCEs was shown to be extremely stable, with 98% of the initial response remaining after 48 h of stirring in the presence of 1 x 10(-4) M NADH solutions (compared to 14% at the poly(1,2-diaminobenzene) modified GCEs). The nonconventional conducting polymer nanotubule-coated electrodes, when tested in amperometric mode for NADH electrochemical oxidation at an applied potential of 450 mV, showed a sensitivity of 99 nA/mM, an operational stability for 2 days, a storage stability of 2 weeks at 4 degrees C, a linearity from 5 x 10(-5) to 1 x 10(-3) M, and good NADH chemical reversibility, all of which make them useful tools for dehydrogenase enzyme probe assembly.

A disposable electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed using a screen printed electrode (SPE) system as a differential pulse voltammetry (DPV) transducer with mouse anti-erythromycin (and anti-tylosin) monoclonal antibodies (MAb) serving as molecular recognition elements. The immunochemical system makes use of the competition assay principle, and employs an erythromycin (or tylosin)-BSA conjugate as coating molecule. After competition between free and coated analyte for the antibodies, the activity of the alkaline phosphatase labelled antiglobulins was measured electrochemically using 1-naphthylphosphate as substrate. Using standard solutions of erythromycin and tylosin, the detection limit of the assay was 0.2 ng mL(-1) determined to be for erythromycin and 2.0 ng mL(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.0 ng mL(-1) for erythromycin and 3.0 ng mL(-1) for tylosin. The suitability of the assay for quantification of erythromycin and tylosin in bovine muscle was also studied. Spiked and real samples were analysed using the immunosensor system developed here. The ELISA showed precision values (relative standard deviation, RSD%) ranging from 4 to 9% for erythromycin and from 8 to 15% for tylosin; the accuracy (relative error, RE%) ranged from -11 to 6% and from -4 to 12% for erythromycin and tylosin, respectively. Results obtained on real samples were confirmed by micro-liquid chromatography coupled on line with tandem mass spectrometry (micro-LC-MS-MS), using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest.

Cholinesterase sensors based on screen-printed electrodes modified with polyaniline, 7,7',8,8'-tetracyanoquinodimethane (TCNQ), and Prussian blue have been developed and tested for detection of anticholinesterase pesticides in aqueous solution and in spiked grape juice. The influence of enzyme source and detection mode on biosensor performance was explored. It was shown that modification of the electrodes results in significant improvement of their analytical characteristics for pesticide determination. Thus, the slopes of the calibration curves obtained with modified electrodes were increased twofold and the detection limits of the pesticides were reduced by factors of 1.6 to 1.8 in comparison with the use of unmodified transducers. The biosensors developed make it possible to detect down to 2 x 10(-8) mol L(-1) chloropyrifos-methyl, 5 x 10(-8) mol L(-1) coumaphos, and 8 x 10(-9) mol L(-1) carbofuran in aqueous solution and grape juice. The optimal conditions for grape juice pretreatment were determined to diminish interference from the sample matrix.

Glucose oxidase, lactate oxidase, L-aminoacid oxidase and alcohol oxidase were immobilised on new films based on 2,6-dihydroxynaphthalene (2,6-DHN) copolymerised with 2-(4-aminophenyl)-ethylamine (AP-EA) onto the Pt electrodes. The electropolymerisation was performed by cyclic voltammetry. Different scan rates and scan potential ranges were investigated and selected according to the monomers used. These sensors were tested for hydrogen peroxide, ascorbic acid and acetaminophen by cyclic voltammetry and amperometry. The amperometric studies were carried out in batch as well as in a flow injection analysis (FIA) system. Analytical parameters such as reproducibility, interference rejection, response time, buffer, storage and operational time of the sensors have been studied. These films were also characterised by X-ray photoelectron spectroscopy (XPS). Different strategies for enzyme immobilisation were performed and discussed: enzyme entrapment in the film during the electropolymerisation and covalent attachment of the enzyme to the film via a carbodiimide (1-ethtl-3-(3-dimethylaminopropyl)carbodiimide, EDC) or glutaraldehyde. Different parameters were considered in order to optimise the immobilisation procedures. Results provide a guide to design high sensitive, stable and interference-free biosensors. In addition, studies were performed using these probes in an original FIA based on solenoidal valves. Sensor stability, life time and dynamic range were also optimised in these conditions.

The promising advantages of Prussian Blue (PB) as catalyst and of the thick film screen printing technology have been combined to assemble sensors with improved characteristics for the amperometric determination of H(2)O(2). PB-modified screen printed electrodes were applied to detect H(2)O(2) at an applied potential of -0.05 V versus the internal screen printed Ag pseudoreference electrode, showing a detection limit of 10(-7) mol l(-1), a linearity range from 10(-7) to 5x10(-5) mol l(-1), a sensitivity of 234 microA mmol l(-1) cm(-2), and a high selectivity. Improved stability at alkaline pH values was also observed, which made possible their use with enzymes having an optimum basic pH. Then, the immobilisation of a single enzyme (glucose oxidase (GOD) or choline oxidase (ChOX)) or of two enzymes, acetylcholinesterase (AchE) coimmobilised with ChOX, has been performed on the surface of PB modified screen-printed electrodes (SPEs) using glutaraldehyde and Nafion. ChOX has been selected as an example of enzyme working at alkaline pH. The choline biosensors showed a detection limit of 5x10(-7) mol l(-1), a wide linearity range (5x10(-7)-10(-4) mol l(-1)), a high selectivity and a remarkable long term stability of 9 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical characteristics and stability were observed with the acetylcholine biosensors.

A fast procedure for the determination of choline in food was developed by coupling a microwave hydrolysis procedure with an O(2)/choline oxidase-based electrochemical biosensor. Time and temperature were varied to select the best conditions for the microwave hydrolysis. Results have been compared with those found by the traditional method, constituted by hydrolysis at 70 degrees C followed by enzymatic-colorimetric assay. Data obtained by the biosensor method correlated well with the enzymatic-colorimetric assay (R(2) = 0.998). Microwave versus traditional hydrolysis gave a good correlation both with the colorimetric and with biosensor procedures with a relative error below 6%. The method is sensitive and selective enough to be used for a wide variety of food items reducing remarkably the analysis time.