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International audience; Herein, carboxypeptidase Y (CPY) and thermolysin (TLN) were compared as sensing elements to develop an original biosensor for the direct detection of ochratoxin A (OTA) in olive oil. Electrochemical detection of OTA was performed using biosensors containing either CPY or TLN immobilized through glutaraldehyde vapor cross-linking onto gold interdigitated microelectrodes. Different parameters affecting biosensor sensitivity towards OTA were first optimized. TLN and CPY exhibited an optimal activity under the same conditions: 35 min cross-linking time, working pH of 7 and temperature of 25 degrees C. TLN and CPY biosensors exhibited comparable analytical performances. The conductometric response of both modified electrodes was linear up to 75 mu M. The limits of detection were also very similar (1 mu M for the CPY biosensor and 0.7 mu M for the TLN biosensor). For both enzymes, initial rate vs. OTA concentration plots could be described by hyperbolic curves consistent with the Michaelis-Menten model. K-M(app) and V-max(app), deduced from these curves, were 26 mu M and 3.3 mu S min(-1) respectively for the TLN biosensor. The biosensors signal was reproducible, relative standard deviations calculated from three consecutive measurements of standards in the 1-75 mu M range, being lower than 5%. The signals were also very stable over a 30 days period when biosensors were stored at 4 degrees C in 20 mM phosphate buffer between two measurements. The TLN biosensor was further evaluated for OTA determination in spiked olive oil samples. Recovery values were close to 100% demonstrating the suitability of this method for OTA screening in olive oil. Interestingly, OTA concentrations could be determined without pretreatment of the sample and no matrix effect was observed.

This paper investigates the development of a biosensor associating two distinct enzymatic activities, that of the beta-galactosidase and that of the glucose oxidase, in order to apply it for the quantitative detection of lactose in milk. To eliminate interferences with glucose, a differential mode of measurement was used. Results show a linear calibration curve for lactose concentration between 60 and 800 μM (0.03 to 0.3 g/L). Tests with real commercial milk samples were carried out to validate the conductometric biosensor.

International audience; An original biosensor was developed for the direct conductometric detection of ochratoxin A (OTA) in olive oil samples. The biosensor is based on thermolysin (TLN) immobilization into a polyvinyl alcohol (PVA)/polyethylenimine (PEI) matrix containing gold nanoparticles (AuNPs) and cross-linked at the surface of gold interdigitated microelectrodes using glutaraldehyde. Under optimal conditions (35 min cross-linking time, working pH of 7 and temperature of 25 degrees C), the biosensor response was linear up to 60 nM OTA, with a sensitivity of 597 mu S mu M-1 and a limit of detection of 1 nM. This value was 700 times lower than the detection limit obtained using the more classical method based on enzyme cross-linking in the presence of bovine serum albumin (BSA). PVA/PEI hydrogel creates a very favorable aqueous environment for the enzyme. In addition, interactions between protonated amino groups of PEI and negative charges of both citrated AuNPs and thermolysin improve their dispersion in the polymer blend, favoring enzyme stabilization and accessibility of the substrate. No conductometric signal was observed after OTA injection in the absence of AuNPs, in agreement with the insulating properties of the cross-linked PVA/PEI hydrogel film. Incorporation of AuNPs into the TLN/BSA biomembrane helped improving the sensitivity by 5.3 but this latter remained 140 times lower than the sensitivity of TLN/AuNPs/(PVA/PEI) biosensor. The study of enzyme kinetics showed that V-i vs [OTA] plot exhibited a non-hyperbolic trend, indicating that kinetics does not display a Michaelis-Menten behavior. Biosensor response times are longer (7-48 min) comparatively to TLN/BSA biosensor with a maximal value of 25 min. This difference is due to the diffusion phenomenon through the pores of the polymer membrane. The proposed OTA biosensor was very reproducible with a relative standard deviations (RSDs) in the 3-15% range and stable over a 30-days period when stored at 4 degrees C in 20 mM phosphate buffer between two measurements. The method was further evaluated using commercial doped olive oil samples. No pretreatment of the sample was needed for testing and no matrix effect was observed. Recovery values were close to 100% demonstrating the suitability of the proposed method for OTA screening in olive oil.

International audience; A urea biosensor has been prepared by covalent binding of urease directly to the surface of an ammonium-sensitive field effect transistor (FET). The NH4+-sensitive membrane is based on a zeolite-incorporated polymeric membrane biosensor (clinoptilolite). The sensitivity of ammonium detection is sub-nernstian (32 mV/pNH4+) but the ISFET presents a high selectivity, which is interesting for measurements in biological media. The grafting of urease to the NH4+-sensitive membrane was performed by cross-linking with glutaraldehyde. The sensitivity of the urea ENFET is 15 mV/purea and this remains stable over 15 days with a detection limit of 3×10−5 M. Finally, in order to test feasibility of the urea biosensor for environmental applications, the remaining activity of the urease was determined after exposure to enzyme inhibiting heavy metals ions such as Hg(II). Using these urea biosensors, a detection limit of less than 5×10−8 M was obtained for Hg(II).

This work shows the feasibility of an olfactory biosensor based on the immobilization of Saccharomyces cerevisiae yeast cells genetically modified to express the human olfactory receptor OR17-40 onto interdigitated microconductometric electrodes. This olfactory biosensor has been applied to the detection of its specific odorant (helional) with a high sensitivity (threshold 10(-14) M). In contrast, no significant response was observed using a non-specific odorant (heptanal), which suggests a good selectivity. Thus, this work may represent a first step towards a new kind of bioelectronic noses based on whole yeast cells and allowing a real time monitoring of olfactory receptor activation.

International audience; A conductometric proteinase K biosensor for organic matter monitoring in rivers has been developed. In fact, with approximately 30% of the total Chemical Oxygen Demand (COD), proteins were chosen to be used as indicators of urban pollution. Proteinase K hydrolyzes proteins into different ionic amino acids which results in local conductivity changes. In this work, we began with the optimization of biosensor response using bovine serum albumin (BSA) as standard protein. A stable biosensor with a constant repeatability and a detection limit about 0.5 mg/ml BSA were obtained. Then, response biosensor was tested with samples of rivers water. Good correlations between conductance changes and values given by standard methods (chemical oxygen demand and protein concentration evaluated by microBCA protein assay) have been shown.; Développement d'un biocapteur conductimétrique à proteinas K pour le suivi de la matière organique dans les rivières. Les proteines sont utilisées comme estimateur de la quantité de matière organique polluante. La protéinase K hydrolyse les protéines en différents amino acides ionisés qui induisent une changement de conductivité locale. Dans ce travail nous optimisons le biocapteur avec de la BSA. Le biocapteur est stable avec une bonne répétabilité et un LD de 0,5µg/mL de BAS; l'évaluation du biocapteur avec des eaux de rivières donne un bonne corrélation avec le COD et la teneur en azote organique des eaux.

International audience; This new biosensor for protein determination is based on a possibility of translation of either a proton or hydroxyl ion arising during protein hydrolysis in the presence of trypsin by means of ion selective field effect transistor (ISFET). The conditions of trypsin immobilization and main biosensor characteristics were optimized using hydrolysis reaction of nalpha-benzoyl-L-arginine ethyl ester hydrochloride (BAEE). The trypsin was immobilized on the ISFET surface using a co-reticulation process between the enzyme and BSA (4% trypsin. 6% BSA) in saturated glutaraldehyde vapour. The limit of detection of BAEE with this biosensor is 0.5 mM with a linear dynamics range from 0.5 to 5 mM, and sensitivity is about 6 mV/mM. The time of biosensor response was 5-7 min. The possibility of the application of developed biosensor for detection of small penta-peptide, which is used in some cosmetic products, has been demonstrated.

International audience; The traditional systems of analysis are not any more in adequacy with the new needs, such as in situ control of pollution (industrial wastes, heavy metals, quality of water) or in vivo measurements in the biomedical field. With approximately 30% of the total chemical oxygen demand (COD), the proteins are the major fraction of the organic matter of the effluents. For this reason, a conductometric enzyme biosensor based on a proteinase K was developed and then tested with real samples of water from rivers and sewers. The biosensor was optimized with the bovine serum albumin (BSA) as a standard protein. A good reproducibility and high storage stability (more than 1 month) are observed. A large linear range of response for BSA determination from 0.4 to 8 mg/l was obtained. The results given by the biosensor for water analysis are, qualitatively, in adequacy with those given by the colorimetric method using the bicinchroninic acid.

The potential toxicity of pesticide residues in drinking water has meant a rigid regulation for the appearance of these pollutants. Thus, in this work, we developed a new immunosensor for atrazine detection. We focused on the optimisation of the antibody immobilisation method on sensor surface for the enhancement of the biosensor sensitivity. First, with site-directed immobilisation of rabbit anti-atrazine antibodies using goat anti-rabbit immunoglobulin, a detection limit of 0.5 ng/mL was obtained. This value is 20 times lower than the detection limit obtained with non-oriented antibodies. The second way to improve immunosensor sensitivity consisted of the addition of carbon nanotubes (CNT). As result of using these CNT, detection limit has been improved again from 0.5 ng/mL to 100 pg/mL., This work was financially supported by the PCI cooperation project between Spain and Tunisia.