François Bessueille

A novel nitrite biosensor based on conductometric electrode modified with cytochrome c nitrite reductase composite membrane

A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples.

Development of a conductometric phosphate biosensor based on tri-layer maltose phosphorylase composite films.

A conductometric biosensor for phosphate detection was developed using maltose phosphorylase (MP) from recombinant Escherichia coli immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin (BSA). The process parameters for the fabrication of the mono-enzymatic sensor and various experimental variables such as the enzyme loading, time of immobilization in saturated GA vapour, working buffer solution and temperature were investigated with regard to their influence on sensitivity, detection limit, dynamic range, operational and storage stability. The biosensor can work well at the temperature between 20 degrees C and 50 degrees C, and reach 90% of steady-state conductance in about 10s. The sensor has two linear ranges, one is from 1.0 microM to 20 microM phosphate with a detection limit of 1.0 microM, and the other is between 20 microM and 400 microM phosphate. When stored in citrate buffer (0.1M, pH 6.0) at 4 degrees C, the biosensor showed good stability over two months. No obvious interference from other anionic species like SO(4)(2-), Cl(-), NO(3)(-), NO(2)(-) and HCO(3)(-) was detected. The biosensor is suitable for use in real water samples.

Impedimetric immunosensor based on SWCNT-COOH modified gold microelectrodes for label-free detection of deep venous thrombosis biomarker

Measurement of D-dimer has subsequently become an essential element in the diagnostics of deep vein thrombosis and pulmonary embolism; in this context microelectrodes with an area of 9×10(-4) cm(2) were used to develop impedimetric immunosensor for detecting deep venous thrombosis biomarker (D-dimer). The biosensor is based on functionalized carbon nanotubes (SWCNT-COOH) where the antibody (anti-D-dimer) was immobilized by covalent binding. The electrical properties and the morphology of the biolayer were characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry and atomic force spectroscopy (AFM). Impedimetric microimmunosensor allows to obtain sensitivity of 40.1 kΩ μM(-1) and detection limit of 0.1 pg/mL (0.53 fM) with linear range from 0.1 pg/mL to 2 μg/mL (0.53 fM to 0.01 μM). We demonstrate that using carbon nanotubes and microelectrodes, high sensitivity and dynamic range were obtained. The biosensor exhibited a short response time of 10 min. Moreover, the studied immunosensor exhibits good reproducibility (R.S.D. 8.2%, n=4).

Anticancer drug detection using a highly sensitive molecularly imprinted electrochemical sensor based on an electropolymerized microporous metal organic framework

A simple and highly sensitive approach for the detection of the anti-neoplastic drug gemcitabine is presented, based on a one-step electropolymerized molecularly imprinted microporous-metal-organic-framework. The sensitive layer was prepared by electropolymerization of the aniline moieties of p-aminothiophenol- gold nanoparticles on the surface of gold electrodes tethered with p-aminothiophenol, in the presence of gemcitabine as a template molecule. Experimental parameters that control the performance of the sensor were investigated and optimized. Under optimal conditions a calibration curve was obtained in the linear range from 3.8 fM to 38 nM with a limit of detection of 3 fM. The obtained imprinted sensor has the advantages of easy manufacture, high sensitivity and selectivity and good reproducibility. Furthermore the feasibility of the proposed technique has been investigated on spiked serum samples and infusion solution containing gemcitabine.

Molecularly imprinted polymer-based electrochemical sensor for the sensitive detection of glyphosate herbicide

ABSTRACT A sensitive electrochemical molecularly imprinted sensor was developed for the detection of glyphosate (Gly), by electropolymerisation of p-aminothiophenol-functionalised gold nanoparticles in the presence of Gly as template molecule. The extraction of the template leads to the formation of cavities that are able to specifically recognise and bind Gly through hydrogen bonds between Gly molecules and aniline moieties. The performance of the developed sensor for the detection of Gly was investigated by linear sweep voltammetry using a hexacyanoferrate/hexacyanoferrite solution as redox probe, the electron transfer rate increasing when concentration of Gly increases, due to a p-doping effect. The molecularly imprinted sensor exhibits a broad linear range, between 1 pg/L and 1 µg/L and a quantification limit of 0.8 pg/L. The selectivity of the proposed sensor was investigated towards the binding of Gly metabolite, aminomethylphosphonic acid, revealing excellent selectivity towards Gly. The developed sensor was successfully applied to detect Gly in tap water samples.

Selective Metal Pattern Fabrication Through Micro-Contact or Ink-Jet Printing and Electroless Plating onto Polymer Surfaces Chemically Modified by Plasma Treatments

Simple versatile processes combining plasma treatments, micro-contact printing (µCP) or ink-jet printing (IJP), and electroless deposition (ELD) have been developed to produce micrometer and sub-micrometer scale metal (Ni, Ag) patterns at the surface of polymer substrates. Plasma treatments were mainly used to graft the substrate surfaces with either nitrogen-containing functionalities on which a palladium-based catalyst can be subsequently chemisorbed (case of Ni deposition through a tin-free process in solution) or oxygen-containing functionalities on which a tin-based sensitization agent can be subsequently chemisorbed (case of Ag deposition through a redox reaction). µCP of the catalyst or of self-assembled monolayers (SAMs) as well as ink printing were used to obtain locally active or non-active areas at the polymer surfaces. The metal micro-patterns were characterized using optical microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Surface chemical characterization was carried out by X-ray photoelectron spectroscopy (XPS).

Sensitivity Improvement of an Impedimetric Immunosensor Using Functionalized Iron Oxide Nanoparticles

This work has explored the development of impedimetric immunosensors based on magnetic iron nanoparticles (IrNP) functionalized with streptavidin to which a biotinylated FAB part of the antibody has been bound using a biotin-streptavidin interaction. SPR analysis shows a deviation on the measured (angle) during antigen-antibody recognition whereas label free detection using by EIS allows us to monitor variation of polarization resistance. Before detection, layers were analyzed by FTIR and AFM. Compared to immobilization of antibody on bare gold surface using aminodecanethiol SAM, antibody immobilization on nanoparticles permitted to reach lower detection limit: 500 pg/ml instead of 1 ng/ml to in the case of EIS and 300 ng/ml instead of 4.5 g/ml in the case of SPR. Thus, it permitted to improve the sensitivity: from 257.3 to 1871 in the case of EIS and from to in the case of SPR.

Electrically addressable deposition of diazonium-functionalized antibodies on boron-doped diamond microcells for the detection of ochratoxin A

This work reports the manufacturing procedure for an impedimetric immunosensor for sensitive detection of the mycotoxin, ochratoxin A (OTA), through electroaddressing of diazonium functionalized antibodies on the working electrode of a planar Boron Doped Diamond (BDD) electrochemical microcell. The immunosensor elaboration and the immunochemical reaction between ochratoxin A and the surface-bound antibody were monitored using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The impedance variation due to the specific antibody–OTA interaction was correlated with the OTA concentration in the samples. The increase in electron-transfer resistance values presents a sigmoidal shape versus log concentration of OTA, with a dynamic range between 7 pg mL−1 and 25 ng mL−1. A limit of detection (LOD) of 7 pg mL−1 and a IC50 of 1.2 ng mL−1 were obtained. The immunosensor thus fabricated exhibited high sensitivity, good reproducibility, long-term stability, and was used for the detection of OTA in real coffee samples with a good recovery rate. The reported validated manufacturing procedure is compatible with the production of microarrays for multidetection.

Validation of a conductometric bienzyme biosensor for the detection of proteins as marker of organic matter in river samples.

This article describes a conductometric bi-layer based bienzyme biosensor for the detection of proteins as a marker of organic matter in rivers. Proteins were chosen to be used as indicators of urban pollution. The working mechanism of the bienzyme biosensor is based on the enzymatic hydrolysis of proteins into several fractions (peptides and amino acids), which results in a local conductivity change depending of the concentration of proteins. In this work, we began with the optimization of biosensor response using bovine serum albumin (BSA) as standard protein. For this objective seven enzymatic biosensors were prepared: four enzymatic sensors with only one layer of enzyme (proteinase K, trypsin, pronase or protease X) and three other enzymatic sensors with two layers (first layer: membrane containing proteinase K; second layer: one of the three other enzymes: trypsin, pronase or protease X). The biosensors were obtained through the deposition of enzymatic layers and the cross-linking process between enzymes and BSA in saturated glutaraldehyde vapour. The response of the various biosensors, described previously, were compared with the values of total organic carbon (TOC), and those of organic nitrogen (Norg), as determined by the laboratory accredits (CEMAGREF of Lyon) using the traditional method of analysis (NF EN 1484, infrared spectroscopy) and (NF EN 25663, mineralization/colorimetry assay) respectively for each water sample obtained from different sites in Lyon (France). The linear correlations obtained with the response of the seven biosensors showed the most important indices of correlations for the biosensor with two enzymatic layers: proteinase K + pronase (pkp). The optimum conditions for the preparation of the pkp biosensor increased the sensitivity and gave a limit of quantification of 0.583 microg/L for TOC and 0.218 microg/L for Norg in water samples. This sensor shows good reproducibility (2.28%), a capacity to be used at temperatures range 10-30 degrees C (depending on the season) and moreover a long lifetime (5 weeks).

Aflatoxin B1 Detection Using a Highly-Sensitive Molecularly-Imprinted Electrochemical Sensor Based on an Electropolymerized Metal Organic Framework

A sensitive electrochemical molecularly-imprinted sensor was developed for the detection of aflatoxin B1 (AFB1), by electropolymerization of p-aminothiophenol-functionalized gold nanoparticles in the presence of AFB1 as a template molecule. The extraction of the template leads to the formation of cavities that are able to specifically recognize and bind AFB1 through π-π interactions between AFB1 molecules and aniline moities. The performance of the developed sensor for the detection of AFB1 was investigated by linear sweep voltammetry using a hexacyanoferrate/hexacyanoferrite solution as a redox probe, the electron transfer rate increasing when the concentration of AFB1 increases, due to a p-doping effect. The molecularly-imprinted sensor exhibits a broad linear range, between 3.2 fM and 3.2 µM, and a quantification limit of 3 fM. Compared to the non-imprinted sensor, the imprinting factor was found to be 10. Selectivity studies were also performed towards the binding of other aflatoxins and ochratoxin A, proving good selectivity.