Régis Rouillon

Biosensors : potential in pesticide detection

Abstract Considerable advances are being made in detection and measurement using sensors based on enzymes, antibodies, whole cells, and organelles. This article attempts to identify the constraints of each system. Improvements are required in the design of biosensors before they become widely accepted in environmental control. Despite their limitations they are often able to deliver the information required.

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).

Acetylcholine biosensor involving entrapment of two enzymes. Optimization of operational and storage conditions

Abstract A bi-enzymatic sensor for the determination of acetylcholine was prepared by co-entrapment of acetyl-cholinesterase and choline oxidase in poly(vinyl alcohol) bearing styrylpyridinium groups. The measurements were based on the detection of enzymatically liberated hydrogen peroxide. The best conditions of use are pH 8, 30°C and a buffer concentration higher than 0.05 M. The storage stability in a dry state is excellent.

Development of a cytochrome c-based screen-printed biosensor for the determination of the antioxidant capacity of orange juices.

This paper describes the development of an amperometric cytochrome c (cyt c)-based biosensor and its later application to the quantification of the scavenging capacity of antioxidants. The enzymatic biosensor was constructed by covalently co-immobilizing both cyt c and XOD on a mercaptoundecanol/mercaptoundecanoic acid (MU/MUA) mixed self-assembled monolayer (SAM)-modified screen-printed gold electrode. The applicability of this method was shown by analyzing the antioxidant capacity of pure substances, such as ascorbic acid and Trolox, and natural sources of antioxidants, particularly 5 orange juices.

Entrapped Enzymes in Photocrosslinkable gel for Enzyme Electrodes

Abstract Enzyme sensors were prepared for the determination of glucose, ethanol, choline and acetylcholine by entrapment in photocrosslinkable PVA-SbQ gel. The determination is based on the electrochemical detection of enzymatically generated hydrogen peroxide. A linear response in a wide range of substrate concentrations and excellent storage stability were recorded for all enzymes tested.

Immobilization of thylakoids in polyvinylalcohol for the detection of herbicides

Abstract Thylakoid membranes isolated from spinach leaves were immobilized by entrapment in polyvinylalcohol bearing styrylpyridinium groups. The reduced species produced by photosynthetic membranes are oxidized at a platinum working electrode in a microphotoelectrochemical cell. Free and entrapped thylakoids were compared for their responses to inhibition by two herbicides: diuron and atrazine. The detection limits were respectively 2×10 −8 and 2×10 −7 M for diuron and atrazine.

Entrapment of chloroplasts and thylakoids in polyvinylalcohol-SbQ. Optimization of membrane preparation and storage conditions

Abstract Chloroplasts and thylakoids isolated from spinach were entrapped in six different polyvinylalcohol bearing styrylpyridinium groups (PVA-SbQ). Measurements of oxygen production were carried out in 0.01 M phosphate buffer (pH 6) at 25 °C. Immobilized preparations were compared for their electron transport activity measured as the initial rate of oxygen evolution with p -benzoquinone as artificial electron acceptor. The entrapped chloroplasts in PVA-SbQ 1700 and 3500 betaine retained 20% of their initial activity after 90-day storage in the dark at + 4 °C. A membrane made with PVA-SbQ 2300 kept 20% activity after 427-day storage at −18 °C in the dark whereas the same membrane retained only 10% activity after 90-day storage at + 4 °C in the dark. A 1% BSA concentration increased the stability of the membrane. The limits of detection (corresponding to a 10% inhibition) of atrazine were 10 μg/l and 12 μg/l for native and entrapped chloroplasts, respectively. The good stability of entrapped chloroplasts suggests that this system could be useful for the detection of pollutants in water.

Electrochemical impedimetric sensor based on molecularly imprinted polymers/sol-gel chemistry for methidathion organophosphorous insecticide recognition.

We report here a novel method to detect methidathion organophosphorous insecticides. The sensing platform was architected by the combination of molecularly imprinted polymers and sol-gel technique on inexpensive, portable and disposable screen printed carbon electrodes. Electrochemical impedimetric detection technique was employed to perform the label free detection of the target analyte on the designed MIP/sol-gel integrated platform. The selection of the target specific monomer by electrochemical impedimetric methods was consistent with the results obtained by the computational modelling method. The prepared electrochemical MIP/sol-gel based sensor exhibited a high recognition capability toward methidathion, as well as a broad linear range and a low detection limit under the optimized conditions. Satisfactory results were also obtained for the methidathion determination in waste water samples.

Potential for use of a cyanobacterium Synechocystis sp. immobilized in poly(vinylalcohol): Application to the detection of pollutants

A cyanobacterium, Synechocystis sp. PCC 6803, was immobilized by entrapment in poly(vinylalcohol) bearing styrylpyridinium groups. Its properties in a single-compartment micro-photoelectrochemical cell using platinum electrodes in potentiosatic mode were compared with the native material. The operational activity was measured in the presence of an electrolytic solution containing 20 mM sodium phosphate, 0.15 mM NaCl and 1 mM MgCl2. The best conditions of use are pH 7.0, 38 C and a 2,5-dichlorobenzoquinone concentration equal to 350 M with native cyanobacteria or pH 6.5, 25C and 500M 2,5-dichlorobenzoquinone after entrapment. Using this procedure, the photocurrent could be inhibited by pollutants such as Diuron or HgCl2. After entrapment, the detection limits (corresponding to a 10% inhibition) were respectively 0.5 M and 50M for Diuron and HgCl2 after five minutes of incubation. A permeabilization technique was used to increase sensitivity of the procedure to the detection of HgCl2 (25% inhibition with 50M after five minutes of incubation).

Comparative study of thylakoid membranes sensitivity for herbicide detection after physical or chemical immobilization.

A micro-test using immobilized thylakoid membranes as sensing element in a micro-electrochemical cell has been developed to assess impairment at the level of the light-driven transport of electrons. In this study, thylakoids isolated from spinach leaves were either immobilized by entrapment in poly(vinylalcohol) bearing styrylpyridinium groups or by chemical immobilization in an albumin-glutaraldehyde crosslinked matrix. The two immobilization procedures were compared upon the sensitivity of the immobilized materials to detect nine herbicides targetting photosystem II. Despite the largely differing mode of immobilization, the procedures led to strikingly similar detection capabilities for herbicides. Inherent characteristics of both immobilization procedures are also discussed.