Sensitive Detection of Organophosphorus Pesticides Using a Needle Type Amperometric Acetylcholinesterase-based Bioelectrode. Thiocholine Electrochemistry and Immobilised Enzyme Inhibition

International audienceAn acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concen¬ trations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of + 0.410 V vs. Ag/AgCl, KClSat was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthio-choline detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm~" of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 jxM for dipterex and 0.4 jaIVI for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots

Amperometric Biosensor For Ethanol Based On A Phenothiazine Derivative Modified Carbon Paste Electrode

A new amperometric biosensor for ethanol, based on carbon paste electrode modified with alcohol dehydrogenase (ADH), polyethylenimine (PEI) and using a phenothiazine derivative (DDDP; 16H,18H-dibenzo[c,1]7,9-dithia-16,18-diazapentacene) as redox mediator for NADH recycling, was developed. The biosensor response is the result of mediated oxidation of NADH, generated in the enzymatic reaction between ADH and ethanol (in the presence of NAD(+)). The biosensor sensitivity (calculated as the ratio I-max/K-M(app)) was 0.035 mA M-1 and the detection limit was 0.26 mM, while the linear response range was from 0.1 to 20 mM ethanol

Influence of the Electrode Material on the Electrochemical Behavior of Carbon Paste Electrodes Modified with Meldola Blue and Methylene Green Adsorbed on a Synthetic Zeolite

Carbon paste electrodes modified with a phenoxazine derivative, Meldola blue, and a phenothiazine derivative, methylene green, both strongly adsorbed on a synthetic zeolite were investigated using either glassy carbon powder (Sigradur K, SK) or single-walled carbon nanotubes (SWCNT) as conductive electrode material. In the case of SWCNT based electrodes, the formal potential of both mediators was pH dependent, as expected for a redox process involving proton transfer. In contrast, the formal potential of both mediators of SK based modified electrodes was practically insensitive to pH. This behavior is discussed in terms of interactions existing in the heterogeneous system mediator-zeolite-electrode material

Poly-phenothiazine derivative-modified glassy carbon electrode for NADH electrocatalytic oxidation

Electropolymerization of a new phenothiazine derivative (bis-phenothiazin-3-yl methane; BPhM) on glassy carbon (GC) electrode generates a conducting film of poly-BPhM. in stable contact with the electrode surface. The heterogeneous electron-transfer process corresponding to the modified electrode is characterized by a high rate constant (50.4 s(-1), pH 7). The CC/poly-BPhM electrode shows excellent electrocatalytic activity toward NADH oxidation. The rate constant for catalytic NADH oxidation, estimated from rotating disk electrode (RDE) measurements and extrapolated to zero concentration of NADH, was found to be 9.4 x 10(4) M-1 s(-1) (pH 7). The amperometric detection of NADH, at +200 mV vs. SCE, is described by the following electroanalytical parameters: a sensitivity of 1.82 mA M-1, a detection limit of 2 mu M and a linear domain up to 0.1 mM NADH. (c) 2008 Elsevier Ltd. All rights reserved

NADH electrocatalytic oxidation at glassy carbon paste electrodes modified with Meldola blue adsorbed on alpha-titanium phosphate

Basic electrochemistry of glassy carbon paste electrodes (GCPEs) modified with Meldola Blue adsorbed on crystalline a-titanium phosphate (MB-a-TP) electrodes and their ability to catalyze the NADH oxidation have been investigated. Various ratios of glassy carbon (Sigradur K) to MB-alpha-TP, were used to obtain GCPEs. Cyclic voltammetry (CV) and rotating disc electrode (RDE) measurements, performed in Tris buffer solution, showed that: (1) the MB formal potential vs. pH dependence is linear; (2) the addition of polyethyleneimine (PEI) to the paste electrode has a significant beneficial effect on the electrooxidation of NADH. As a general conclusion, it can be stated that GCPEs modified with MB-alpha-TP and incorporating 2.44% (w/w) PEI exhibits the best behavior for NADH electrocatalytic oxidation

AMPEROMETRIC BIOSENSORS FOR GLUCOSE AND ETHANOL DETERMINATION IN WINE USING FLOW INJECTION ANALYSIS

Reagentless amperometric biosensors for glucose and ethanol were developed and successfully applied for monitoring glucose and ethanol concentrations in wine during the fermentation process. The glucose biosensor was based on commercially available glucose oxidase and horseradish peroxidase co-immobilized on solid graphite using Os(II)-redox hydrogel (RH) [1]. In the case of ethanol biosensor, the quinohemoprotein dependent alcohol dehydrogenase was immobilized on the graphite electrode surface using the same RH [2]. Both biosensors were operated at low applied potentials (-50 mV vs. Ag/AgCl, KCl0.1M for glucose biosensor, and +250 mV vs. Ag/AgCl, KCl0.1M for ethanol biosensor), where biases from interferences are minimal. The bioelectroanalytical parameters, estimated from flow injection analysis measurements, were found as follows: sensitivity, 0.73 +/- 0.01 mu A mM(-1) for glucose and 0.45 +/- 0.01 mu A mM(-1) for ethanol; linear range up to 1 mM in both cases; detection limit, 7.0 mu M for glucose and 8.9 mu M for ethanol. The results for real samples were found in good agreement with those reported by Barsan et al. [3]

NADH electrocatalytic oxidation at glassy carbon paste electrodes modified with meldola blue adsorbed on acidic alpha-zirconium phosphate

The electrochemical behavior and the electrocatalytic activity for the NADH oxidation at glassy carbon paste electrodes (GCPEs) modified with Meldola Blue (MB) adsorbed onto acidic cc-zirconium phosphate (ZP*) have been investigated. Cyclic voltammetry and rotating disc electrode measurements, performed in Tris buffer solution (pH 7) in absence and in presence of different NADH concentrations, showed that: (i) the formal standard potential (E degrees') of NIB was found pH dependent, according to the equation E = E degrees (c)-0.059pH + 0.029log(1 + 10(pH-pKa)), where the best fitting value for pK(a) Was estimated to be 4.3 +/- 0.4; (ii) the best electrocatalytic response was obtained for MB-ZP*-modified GCPEs having a ratio between the glassy carbon powder (Sigradur K) and MB-ZP* of 19:1; (iii) the presence of polyethyleneimine in the paste has a significant beneficial effect on the electrocatalytic activity of the investigated modified electrodes. As a general conclusion, it was stated that the particular features of the MB-ZP*-GCPEs are related to the ZP* crystallinity

EFFECT OF ENZYME DEGLYCOSYLATION ON THE AMPEROMETRIC DETECTION OF GLUCOSE AT PDH-MODIFIED ELECTRODE

The effect of deglycosylation of pyranose dehydrogenase (PDH), obtained from Agaricus meleagris and recombinantly expressed in Pichia pastoris, on the amperometric detection of glucose was investigated. Glycosylated (gPDH) and deglycosylated (dgPDH) PDH were immobilized on spectrographic graphite (G) simultaneously with an Os redox polymer (Os-RP). The amperometric response of G/Os-RP/gPDH and G/Os-RP/dgPDH to glucose was recorded using flow injection measurements and cyclic voltammetry. A significant increase in the maximum catalytic current density was observed for G/Os-RP/dgPDH [(148.7 +/- 0.14) mu A/cm(2)) compared with G/Os-RP/gPDH [(81.4 +/- 1.4) mu A/cm(2)]. Additionally, the deglycosylation of the enzyme resulted in a higher substrate-enzyme affinity (K-M (app) = 2.44 +/- 0.10 mM), compared with glycosylated PDH (K-M(app) = 7.52 +/- 0.34 mM)

Amine oxidase amperometric biosensor coupled to liquid chromatography for biogenic amines determination

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

Graphite electrodes modified with Neurospora crassa cellobiose dehydrogenase: Comparative electrochemical characterization under direct and mediated electron transfer

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 approximate to cellopentaose) > > (maltotriose approximate to maltotetraose approximate to maltopentaose) > (maltose approximate to glucose); (iii) for all tested substrates, the apparent CDH affinity was roughly higher in DET than in MET operation mode. (C) 2012 Elsevier B.V. All rights reserved