Jean-Louis Marty

Aptamer-based colorimetric biosensing of Ochratoxin A using unmodified gold nanoparticles indicator

This work presents an aptasensor for Ochratoxin A (OTA) using unmodified gold nanoparticles (AuNPs) indicator. The assay method is based on the conformation change of OTAs aptamer in phosphate buffered saline (PBS) containing Mg(2+) and OTA, and the phenomenon of salt-induced AuNPs aggregation. A single measurement took only five minutes. Circular dichroism spectroscopic experiments revealed for the first time that upon the addition of OTA, the conformation of OTAs aptamer in PBS buffer changed from random coil structure to compact rigid antiparallel G-quadruplex structure. This compact rigid G-quadruplex structure could not protect AuNPs against salt-induced aggregation, and thus the color change from red to blue could be observed by the naked eye. The linear range of the colorimetric aptasensor covered a large variation of OTA concentration from 20 to 625 nM and the detection limit of 20 nM (3σ) was obtained.

Improved multianalyte detection of organophosphates and carbamates with disposable multielectrode biosensors using recombinant mutants of Drosophila acetylcholinesterase and artificial neural networks

Engineered variants of Drosophila melanogaster acetylcholinesterase (AChE) were used as biological receptors of AChE-multisensors for the simultaneous detection and discrimination of binary mixtures of cholinesterase-inhibiting insecticides. The system was based on a combination of amperometric multielectrode biosensors with chemometric data analysis of sensor outputs using artificial neural networks (ANN). The multisensors were fully manufactured by screen-printing, including enzyme immobilisation. Two types of multisensors were produced that consisted of four AChE variants each. The AChE mutants were selected in order to obtain high resolution, enhanced sensitivity and minimal assay time. This task was successfully achieved using multisensor I equipped with wild-type Drosophila AChE and mutants Y408F, F368L, and F368H. Each of the AChE variants was selected on the basis of displaying an individual sensitivity pattern towards the target analytes. For multisensor II, the inclusion of F368W, which had an extremely diminished paraoxon sensitivity, increased the sensors capacity even further. Multisensors I and II were both used for inhibition analysis of binary paraoxon and carbofuran mixtures in a concentration range 0-5 microg/l, followed by data analysis using feed-forward ANN. The two analytes were determined with prediction errors of 0.4 microg/l for paraoxon and 0.5 microg/l for carbofuran. A complete biosensor assay and subsequent ANN evaluation was completed within 40 min. In addition, multisensor II was also investigated for analyte discrimination in real water samples. Finally, the properties of the multisensors were confirmed by simultaneous detection of binary organophosphate mixtures. Malaoxon and paraoxon in composite solutions of 0-5 microg/l were discriminated with predication errors of 0.9 and 1.6 microg/l, respectively.

Acetylcholinesterase in organic solvents for the detection of pesticides: Biosensor application

Abstract Acetylcholinesterase (AChE) from electric eel, in a free or immobilized state, can be used for the detection of insecticides. This system is convenient because of the selectivity and specificity of the inhibition of AChE by organophosphorus and carbamate insecticides. However, these pesticides are highly soluble only in organic solvents. This article deals with the determination of the activity of AChE in organic solvents. Firstly, the effect of different organic solvents on activity was determined using the free enzyme. The AChE activity was maintained using the free enzyme. The AChE activity was maintained depending on the nature of the solvent. The results were applied to the biosensing system and a new method for the detection of organophosphorus and carbamate insecticides was developed. A correlation between the AChE activity and a physico-chemical parameter, was found in order to predict the effect of the solvent on enzyme activity. Upon comparison of the correlation curve obtained with free and immobilized enzyme, it appeared that immobilization enhanced the stability of the enzyme and increased the number of usable organic solvents. The inhibition of AChE by organophosphorus and carbamate insecticides was tested in organic solvents and the limit of detection determined. The inhibitory capacity of AChE was maintained in most organic solvents. The reactivation of immobilized enzyme with 2-pyridine aldoxime methiodide (2-PAM) allowed the repeated use of the same enzyme electrode. The application of the biosensor for the detection of organophosphorus and carbamate insecticides in organic solvents using chemical knowledge will be useful for the detection of pesticide residues present in water and food at very low levels.

Disposable Screen Printed Electrochemical Sensors: Tools for Environmental Monitoring

Screen printing technology is a widely used technique for the fabrication of electrochemical sensors. This methodology is likely to underpin the progressive drive towards miniaturized, sensitive and portable devices, and has already established its route from “lab-to-market” for a plethora of sensors. The application of these sensors for analysis of environmental samples has been the major focus of research in this field. As a consequence, this work will focus on recent important advances in the design and fabrication of disposable screen printed sensors for the electrochemical detection of environmental contaminants. Special emphasis is given on sensor fabrication methodology, operating details and performance characteristics for environmental applications.

Label-free impedimetric immunosensor for sensitive detection of ochratoxin A

A novel label-free electrochemical impedimetric immunosensor for sensitive detection of ochratoxin A (OTA) was reported. A two-step reaction protocol was elaborated to modify the gold electrode. The electrode was first derivatized by electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt (4-CPDS) in acidic aqueous solution yielded stable 4-carboxyphenyl (4-CP) monolayer. The ochratoxin A antibody was then immobilized making use of the carbodiimide chemistry. The steps of the immunosensor elaboration and the immunochemical reaction between ochratoxin A and the surface-bound antibody were interrogated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect ochratoxin A. The increase in electron-transfer resistance (DeltaR(et)) values was linearly proportional to the concentration of OTA in the range of 1-20ngmL(-1), with a detection limit of 0.5ngmL(-1).

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.

Aptamer-DNAzyme hairpins for biosensing of Ochratoxin A

We report an aptasensor for biosensing of Ochratoxin A (OTA) using aptamer-DNAzyme hairpin as biorecognition element. The structure of this engineered nucleic acid includes the horseradish peroxidase (HRP)-mimicking DNAzyme and the OTA specific aptamer sequences. A blocking tail captures a part of these sequences in the stem region of the hairpin. In the presence of OTA, the hairpin is opened due to the formation of the aptamer-analyte complex. As a result, self-assembly of the active HRP-mimicking DNAzyme occurs. The activity of this DNAzyme is linearly correlated with OTA concentration up to 10 nM, showing a limit of detection of 2.5 nM.

Amperometric biosensors based on nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors

Screen-printed electrodes coated with the nafion layer have been investigated for cholinesterase biosensor design. The butyrylcholinesterase (ChE) from horse serum was immobilised onto the nafion layer by cross-linking with glutaraldehyde vapours. The biosensors obtained showed better long-term stability and lower working potential in comparison to those obtained with no nafion coating. The sensitivity of a biosensor toward organophosphate pesticides is not affected by the nafion coating. The detection limits were found to be 3.5x10(-7) M for trichlorfon and 1.5x10(-7) M for coumaphos.

Biosensors to detect marine toxins: Assessing seafood safety

This article describes the different types of marine toxins and their toxic effects, and reviews the bio/analytical techniques for their detection, putting special emphasis to biosensors. Important health concerns have recently appeared around shellfish (diarrheic, paralytic, amnesic, neurologic and azaspiracid) and fish (ciguatera and puffer) poisonings produced by different types of phycotoxins, making evident the urgent necessity of counting on appropriate detection technologies. With this purpose, several analysis methods (bioassays, chromatographic techniques, immunoassays and enzyme inhibition-based assays) have been developed. However, easy-to-use, fast and low-cost devices, able to deal with complicated matrices, are still required. Biosensors offer themselves as promising biotools, alternative and/or complementary to conventional analysis techniques, for fast, simple, cheap and reliable toxicity screening. Nevertheless, despite the wide range of seafood toxins and the already rooted biosensing systems, the literature on biosensors for phycotoxins is scarce. This article discusses the existing biosensor-based strategies and their advantages and limitations. Finally, the article gives a general overlook about the regulation toxin levels and monitoring programmes currently established around the world concerning seafood safety.

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.