D. Centonze

Simultaneous monitoring of glucose and lactate by an interference and cross-talk free dual electrode amperometric biosensor based on electropolymerized thin films

An interference and cross-talk free dual electrode amperometric biosensor integrated with a microdialysis sampling system is described, for simultaneous monitoring of glucose and lactate by flow injection analysis. The biosensor is based on a conventional thin layer flow-through cell equipped with a Pt dual electrode (parallel configuration). Each Pt disk was modified by a composite bilayer consisting of an electrosynthesised overoxidized polypyrrole (PPYox) anti-interference membrane covered by an enzyme entrapping gel, obtained by glutaraldehyde co-crosslinking of glucose oxidase or lactate oxidase with bovine serum albumin. The advantages of covalent immobilization techniques were coupled with the excellent interference-rejection capabilities of PPYox. Ascorbate, cysteine, urate and paracetamol produced lactate or glucose bias in the low micromolar range; their responses were, however, completely suppressed when the sample was injected through the microdialysis unit. Under these operational conditions the flow injection responses for glucose and lactate were linear up to 100 and 20 mM with typical sensitivities of 9.9 (+/- 0.1) and 7.2 (+/- 0.1) nA/mM. respectively. The shelf-lifetime of the biosensor was at least 2 months. The potential of the described biosensor was demonstrated by the simultaneous determination of lactate and glucose in untreated tomato juice samples; results were in good agreement with those of a reference method.

Interference-free glucose sensor based on glucose-oxidase immobilized in an overoxidized non-conducting polypyrrole film

SummaryAn amperometric glucose sensor is described; it is based on glucose oxidase immobilized in an overoxidized non-conducting polypyrrole membrane. The overoxidation process of polypyrrole produces a permselective, antifouling membrane capable of rejecting ascorbate, urate, acetaminophen and cysteine, as well as proteins and other surface active components typically present in serum. The amperometric assay of glucose in serum correlates well with an established routine procedure based on an enzymatic-colorimetric method.

An interference-free biosensor based on glucose oxidase electrochemically immobilized in a non-conducting poly(pyrrole) film for continuous subcutaneous monitoring of glucose through microdialysis sampling

A glucose biosensor, based on glucose oxidase immobilized in a non-conducting (overoxidised) polypyrrole film, is described which proved practically immune from faradaic interference arising from endogeneous (ascorbate, urate, cysteine) and exogeneous (acetaminophen) electroactive interferents. The bias introduced in the measurement of 5 mM glucose by the given interferents at their maximum physiological levels never exceeded 2% which is, by far, the lowest value ever reported. The biosensor has been used for continuous subcutaneous monitoring of glucose in a rabbit implanted with a microdialysis probe. The potential and limits of this approach are discussed.

An in situ electrosynthesized amperometric biosensor based on lactate oxidase immobilized in a poly-o-phenylenediamine film: determination of lactate in serum by flow injection analysis

The electrochemical immobilization of lactate oxidase in a poly-o-phenylenediamine film permits the one-step and all-chemical construction of a lactate amperometric biosensor. The sensor was prepared in situ i.e. in the flow injection analysis (FIA) system by simply injecting a plug of a solution containing the monomer and the enzyme. At a flow rate of 50 microL/min linearity was observed up to 0.2 mM lactate and detection limits of about 2 microM could be easily achieved. Faradaic interferences caused by ascorbate, urate, cysteine and acetaminophen were sufficiently minimized to permit lactate determination in diluted serum by FIA. Results obtained by FIA-amperometric detection compared well (according to a proper t-test at a 95% confidence level) with those obtained by a standard enzymatic colorimetric assay. At a flow rate of 1 ml/min a sample throughput higher than 70 sample h-1 was achieved. After one week of continuous use in the FIA system a 75% decrease in biosensor sensitivity was observed.

A microdialysis fibre based sampler for flow injection analysis: determination of L-lactate in biofluids by an electrochemically synthesised bilayer membrane based biosensor

A microdialysis fibre based, low volume sampler is described which can be used in flow injection analysis (FIA) when an on-line dilution of the sample and/or removal of high molecular weight interferents is required. This device used in combination with a lactate amperometric biosensor based on lactate oxidase electrochemically immobilised in a bilayer membrane of poly(o-phenylendiamine) and overoxidized poly(pyrrole) permits the extension of the linear range of response up to 10 mM lactate. Combining microdialysis sampling with FIA and amperometric detection at an interference-free and fast-response biosensor, lactate determination in complex media such as serum, milk and yoghurt can be easily achieved with a high sample throughput and no sample pre-treatment.

Permselective Behavior of an Electrosynthesized, Nonconducting Thin Film of Poly(2‐naphthol) and Its Application to Enzyme Immobilization

The electrooxidation of 2-naphthol in phosphate buffer at pH 7 leads to the formation of a nonconducting polymer of poly(2-naphthol) on a platinum electrode. Such a resulting thin film displays an interesting permselective behavior, which proved useful in minimizing the interference of ascorbate, acetaminophen, cysteine, and urate sample molecules. Electrochemical detection in flowing streams was used to investigate the relevance of permselectivity for sensor development. Nonconducting poly(2-naphthol) film demonstrated useful also as a novel permselective membrane for glucose oxidase immobilization. The glucose response time, t0.95, evaluated in batch addition experiments, was lower than 4u2005s. The calibration plot was linear up to 15u2005mM of glucose with a sensitivity of 2.2u2005nA/mM.

Permselective and enzyme-entrapping behaviours of an electropolymerized, non-conducting, poly(o-aminophenol) thin film-modified electrode: a critical study.

Non-conducting polymeric films synthesised by the electrooxidation of o-aminophenol on a platinum electrode in acetate or phosphate buffer displayed an interesting permselective behaviour, which proved valuable in minimising the electrochemical interferences from ascorbate, acetaminophen, cysteine and urate sample molecules in amperometric detection mode. The electrosynthesis of poly(o-aminophenol) (p(oAP)) film showed also useful as permselective membrane for enzyme immobilization as demonstrated by the production of an interference-free glucose oxidase biosensor. In this respect, the glucose response time, t(0.95), evaluated in batch addition experiments, was lower than 5s while the calibration curve was linear up to 10mM of glucose with a sensitivity of 69.7nA/mM. Both the permselective behaviour and the enzyme-entrapping property of the film were critically compared with the relevant studies until now reported. With respect to the sophisticated but complex approaches described elsewhere, this study shows that simply a proper optimization of p(oAP) electrosynthesis and its permselective behaviour is the key to improve significantly the selectivity of the resulting analytical devices.

Electrochemically prepared glucose biosensors : kinetic and faradaic processes involving ascorbic acid and role of the electropolymerized film in preventing electrode-fouling

The influence is discussed of ascorbic acid (AA) on the response of a glucose biosensor based on glucose oxidase immobilized in electropolymerized poly(ophenylenediamine) (PPD) or overoxidized poly(pyrrole) (oxPPy) films on a Pt electrode. The kinetics of the homogeneous reaction between AA and H2O2 has been investigated by two independent methods and found to be too slow to influence the response of typical glucose biosensors. Therefore, the decrease in the sensor response, observable when working in batch under typical experimental conditions, can in no way be ascribed to a depletion of H2O2, which is produced in the biocatalytic cycle via the homogeneous reaction with AA. While the purely additive Faradaic interference can practically be nullified by both entrapping membranes, electrode fouling by electro-oxidation products of AA (responsible for the observed decrease in glucose sensitivity) might still represent a problem when working with PPD based biosensors. In this respect the permeability characteristics of the film markedly influence the magnitude of the observed phenomena. The oxPPy film achieves the goal of completely eliminating ascorbate without blocking the access of glucose to the immobilized enzyme.