Thierry Noguer

Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents

A screen-printed biosensor for the detection of pesticides in water miscible organic solvents is described based on the use of p-aminophenyl acetate as acetylcholinesterase substrate. The oxidation of p-aminophenol, product of the enzymatic reaction was monitored at 100 mV vs. Ag/AgCl screen-printed reference electrode. Miscible organic solvents as ethanol and acetonitrile were tested. The acetylcholinesterase (AChE) was immobilised on a screen-printed electrode surface by entrapment in a PVA-SbQ polymer and the catalytic activity of immobilised AChE was studied in the presence of different percentages of organic solvents in buffer solution. The sensor shows good characteristics when experiments were performed in concentrations of organic solvents below 10%. No significant differences were observed when working with 1 and 5% acetonitrile in the reaction media. Detection limits as low as 1.91x10(-8) M paraoxon and 1.24x10(-9) M chlorpyrifos ethyl oxon were obtained when experiments are carried out in 5% acetonitrile.

Enzyme sensors for the detection of pesticides

Abstract Various families of pesticides (insecticides, fungicides and herbicides) can be detected using enzymes. Organophosphorus and carbamate insecticides (e.g. paraoxon, methyl-parathion, aldicarb and carbofuran) inhibit acetylcholinesterase; dithiocarbamate fungicide (e.g. maneb) inhibits aldehyde dehydrogenase; and sulfonylurea herbicides (e.g. sulfomethuron methyl and thifensulfuron methyl) inhibit acetolactate synthase. When these pesticides are in the presence of these enzymes, their activity decreases. We have monitored this decrease with a thiocholine sensor for acetylcholinesterase, a NADH sensor for aldehyde dehydrogenase and spectrophotometrically for acetolactate synthase. In all cases detection is better than those obtained by CPG (gas phase chromatography), colorimetric or HPLC (high performance liquid chromatography) methods. We can detect 0·02 ppb equivalent paraoxon, 0·05 ppm maneb and 19 ppb thifensulfuronmethyl.

Screen-printed poly(3,4-ethylenedioxythiophene) (PEDOT): A new electrochemical mediator for acetylcholinesterase-based biosensors

This work describes the use of a PEDOT:PSS-based conductive polymer for designing AChE-based biosensors. The transducers were obtained directly by screen-printing a PEDOT:PSS suspension on the surface of thick film carbon electrodes. The obtained working electrodes showed a high conductivity when compared with electrodes modified with conventional mediators like cobalt phthalocyanine or tetracyanoquinodimethane. The PEDOT:PSS polymer was shown to be suitable for thiocholine oxidation, allowing the measurement of AChE activity at 100 mV vs Ag/AgCl. The high conductivity of PEDOT:PSS allowed the accurate detection of the organophosphate insecticide chlorpyrifos-oxon at concentrations as low as 4x10(-9)M, corresponding to an inhibition ratio of 5%.

Screen-printed electrodes with electropolymerized Meldola Blue as versatile detectors in biosensors

Electropolymerization of Meldola Blue was carried out by cyclic voltammetry in the range from -0.6 to +1.4 V vs. Ag/AgCl, thus defining a new immobilization procedure of the phenoxazine mediator on screen-printed graphite electrodes. Evidence of polymer formation was provided by electrochemical and Fourier transform infrared spectroscopy (FTIR) data. Following polymerization, Meldola Blue preserved the ability to catalyze NADH oxidation allowing to achieve a detection limit of 2.5 x 10(-6) mol l(-1) and a sensitivity of 3713 microA l mol(-1) in amperometric determinations at 0 V vs. Ag/AgCl. In addition, the polymeric mediator was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase. Typical calibration at -0.1 V vs. Ag/AgCl shows a detection limit of 8.5 x 10(-5) mol l(-1), a sensitivity of 494 microA l mol(-1) and a linear range from 2.5 x 10(-4) to 5 x 10(-3) mol l(-1) hydrogen peroxide.

Site-specific immobilization of a (His)6-tagged acetylcholinesterase on nickel nanoparticles for highly sensitive toxicity biosensors

This paper reports site-specific affinity immobilization of (His)6-tagged acetylcholinesterase (AChE) onto Ni/NiO nanoparticles for the development of an electrochemical screen-printed biosensor for the detection of organophosphate pesticides. The method is based on the specific affinity binding of the His-tagged enzyme to oxidized nickel nanoparticle surfaces in the absence of metal chelators. This approach allows stable and oriented attachment of the enzyme onto the oxidized nickel through the external His residue in one-step procedure, allowing for fast and sensitive detection of paraoxon in the concentration range from 10(-8) to 10(-13) M. A detection limit of 10(-12) M for paraoxon was obtained after 20 min incubation. This method can be used as a generic approach for the immobilization of other His-tagged enzymes for the development of biosensors.

Computational and experimental investigation of molecular imprinted polymers for selective extraction of dimethoate and its metabolite omethoate from olive oil.

This work presents the development of molecularly imprinted polymers (MIPs) for the selective extraction of dimethoate from olive oil. Computational simulations allowed selecting itaconic acid as the monomer showing the highest affinity towards dimethoate. Experimental validation confirmed modelling predictions and showed that the polymer based on IA as functional monomer and omethoate as template molecule displays the highest selectivity for the structurally similar pesticides dimethoate, omethoate and monocrotophos. Molecularly imprinted solid phase extraction (MISPE) method was developed and applied to the clean-up of olive oil extracts. It was found that the most suitable solvents for loading, washing and elution step were respectively hexane, hexane-dichloromethane (85:15%) and methanol. The developed MIPSE was successfully applied to extraction of dimethoate from olive oil, with recovery rates up to 94%. The limits of detection and quantification of the described method were respectively 0.012 and 0.05 μg g(-1).

High sensitive bienzymic sensor for the detection of dithiocarbamate fungicides

Abstract A bienzymic sensor for the determination of dithiocarbamate fungicides was developed based on aldehyde dehydrogenase inhibition. The NADH formed by the oxidation of propionaldehyde by aldehyde dehydrogenase was reoxidised by diaphorase using hexacyanoferrate(III) as electron acceptor. The hexacyanoferrate(II) produced was electrochemically oxidised at a potential of 250 mV vs. SCE. As dithiocarbamate fungicides inhibit aldehyde dehydrogenase, a decrease of the induced current was correlated to their concentration in the working medium. Aldehyde dehydrogenase and diaphorase were used in solution or entrapped in a photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups. The best results were achieved using entrapped enzymes and the sensitivity of the sensor was improved by lowering the amount of enzyme and by increasing the contact time between the pesticide and the enzyme. Using entrapped enzymes, the detection of 1.48 ppb of maneb was achieved whereas the commonly used spectrophotometric method allows to detect only 400 ppb of dithiocarbamate.

Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk

An aptasensor was designed for the determination of aflatoxin M1 (AFM1) in milk based on DNA-aptamer recognition and electrochemical impedance spectroscopy detection. A hexaethyleneglycol-modified 21-mer oligonucleotide was immobilized on a carbon screen-printed electrode through carbodiimide immobilization, after diazonium activation of the sensing surface. Cyclic voltammetry and electrochemical impedance spectroscopy in the presence of ferri/ferrocyanide redox probe were used to characterize each step of the aptasensor development. Aptamer-AFM1 interaction induced an increase in electron-transfer resistance, allowing the determination of AFM1 in buffer in the range 2-150 ng/L (LOD=1.15 ng/L). Application to milk analysis showed that a preliminary treatment was mandatory. A simple filtration through a 0.2 µm PTFE membrane allowed determination of AFM1 in milk for concentrations ranging from 20 to 1000 ng/kg. These performances are compatible with the AFM1 levels set in European Union for milk and dairy products for adults (50 ng/kg) and infants (25 ng/kg).

Biosensors based on enzyme inhibition: Detection of organophosphorus and carbamate insecticides and dithiocarbamate fungicides

The dithiocarbamate fungicides and the organophosphorus and carbamate insecticides are two families of pesticides of environmental concern used on a very large scale worldwide. The need for increasing numbers of analyses has created a demand for the development of rapid, simple, and low cost methods. Enzyme sensors based on the abilities of these compounds to inhibit enzymes are described. An enzyme sensor with immobilized aldehyde dehydrogenase is described allowing the detection of 9 ppb of zineb, whereas the classical method allows the determination of 400 ppb. Using screen-printed electrodes with acetylcholinesterase, it is possible to detect 0.5 ppb of paraoxon in aqueous buffer and 1.4 ppb of paraoxon in a mixture of hexane and water (80/20).

Phosphotriesterase: A complementary tool for the selective detection of two organophosphate insecticides : Chlorpyrifos and chlorfenvinfos

This work shows the possibility of combining the high sensitivity of genetically-modified Drosophila melanogaster acetylcholinesterase (B394) with the ability of phosphotriesterase (PTE) to hydrolyse organophosphate compounds, in the aim of developing a biosensor selective to two insecticides of interest: chlorpyrifos and chlorfenvinfos. The studies clearly demonstrate that chlorfenvinfos is a substrate that acts as competitive inhibitor of PTE, therefore preventing the efficient hydrolysis of other pesticides, including chlorpyrifos. A bi-enzymatic sensor was designed by immobilizing both B394 and PTE in a polyvinylalcohol matrix. The sensor was shown to be able to discriminate between chlorpyrifos and chlorfenvinfos inhibitions.