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Rapid and sensitive detection of SARS-CoV-2 virus genetic material is of paramount importance to mitigate the COVID-19 pandemic outbreak and lower the death toll. Herein, we report the design of a magnetofluorescent bioplatform for the direct and specific detection of the viral RNA of SARS-CoV-2 in the total RNA extracted from nasopharyngeal swabs of COVID-19-positive patients. A higher fluorescence response was achieved using two capture probes tethered to magnetic beads using a biotin/streptavidin linkage, targeting two specific sites in the ORF1a and S genes. Two horseradish peroxidase (HRP)-conjugated reporter sequences, complementary to the loci of the S and N genes, were used to reveal the presence of the viral RNA through the oxidation of o-phenylenediamine to fluorescent 2,3-diaminophenazine. Under optimal conditions, the bioplatform showed high selectivity and sensitivity and was able to detect as low as 0.01 ng of viral RNA (1 × 103 copies/μL) with a linear dynamic range varying from 0.01 to 3.0 ng (1 × 103 to 9 × 107 copies/μL). The bioplatform was also able to discriminate the SARS-CoV-2 RNA from those of other related viruses such as hepatitis C, West Nile, measles, and non-polio viruses. Furthermore, the developed biosensor was validated in 46 clinical samples (36 COVID-19-positive patients and 10 COVID-19-negative subjects, as assessed with the gold standard RT-qPCR method). Both sensitivity and specificity of the developed method reached 100%. Finally, making such a simple and specific method available in the field, at a primary point of care, can better help the detection of SARS-CoV-2 infection in low-resource settings.

International audience; Ochratoxin A (OTA) is a carcinogenic mycotoxin that contaminates food such as cereals, wine and beer; therefore it represents a risk for human health. Consequently, the allowed concentration of OTA in food is regulated by governmental organizations and its detection is of major agronomical interest. In the current study we report the development of an electrochemical aptasensor able to directly detect trace OTA without any amplification procedure. This aptasensor was constructed by coating the surface of a gold electrode with a film layer of modified polypyrrole (PPy), which was thereafter covalently bound to polyamidoamine dendrimers of the fourth generation (PAMAM G4). Finally, DNA aptamers that specifically binds OTA were covalently bound to the PAMAM G4 providing the aptasensor, which was characterized by using both Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR) techniques. The study of OTA detection by the constructed electrochemical aptasensor was performed using Electrochemical Impedance Spectroscopy (EIS) and revealed that the presence of OTA led to the modification of the electrical properties of the PPy layer. These modifications could be assigned to conformational changes in the folding of the aptamers upon specific binding of OTA. The aptasensor had a dynamic range of up to 5 μg L−1 of OTA and a detection limit of 2 ng L−1 of OTA, which is below the OTA concentration allowed in food by the European regulations. The efficient detection of OTA by this electrochemical aptasensor provides an unforeseen platform that could be used for the detection of various small molecules through specific aptamer association.

The involvement of oxidative stress in the mechanism of antibiotics-meditated cell death is unclear and subject to debate. The kinetic profile and a quantitative relationship between the release of reactive oxygen species (ROS), bacteria and antibiotic type remain elusive. Here we report direct measurements and analytical quantification of the release of superoxide radicals (O2(·-)), a major contributor to ROS, in antibiotics-treated bacterial cultures using a cytochrome c electrochemical biosensor. The specificity of electrochemical measurements was established by the addition of superoxide dismutase (SOD) which decreased the O2(·-) signal. Measurements using a general ROS-specific fluorescence dye and colony forming units (CFU) assays were performed side-by-side to determine the total ROS and establish the relationship between ROS and the degree of lethality. Exposure of Escherichia coli and Listeria monocytogenes cultures to antibiotics increased the release of O2(·-) radicals in a dose-dependent manner, suggesting that the transmembrane generation of ROS may occur as part of the antibiotic action. The study provides a quantitative methodology and fundamental knowledge to further explore the role of oxidative stress in antibiotics-meditated bacterial death and to assess physiological changes associated with the complex metabolic events related to oxidative stress and bacterial resistance.

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.

One of the key challenges to both control foodstuff composition and detect chemical or biological contaminants in food is the availability of selective, sensitive, and reliable analytical methods. In this context, electrochemical biosensors are particularly attractive due to their low-cost, high sensitivity, and selectivity. They can be easily miniaturized and integrated in high density arrays for multianalyte and high throughput analysis. To improve their sensitivity and robustness, different strategies have been developed, including the incorporation of nanomaterials such as gold or magnetic nanoparticles, carbon-based nanostructures (eg, nanotubes, graphene). These nano-objects offer high surface areas and can be used as platforms to enhance bioreceptors loading and stability, also serving as relays for electron transfer between the biomolecules and the electrode. In this chapter, new trends in nanomaterial-based electrochemical biosensors for food analysis are reviewed, focusing on the determination of major contaminants, that is, foodborne pathogens, toxins, veterinary drug residues, and pesticides.

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.

Rapid and sensitive detection of bacterial pathogens is critical for assessing public health, food and environmental safety. We report the use of modularly designed and site-specifically oriented synthetic antimicrobial peptides (sAMPs) as novel recognition agents enabling detection and quantification of bacterial pathogens. The oriented assembly of the synthetic peptides on electrode surfaces through an engineered cysteine residue coupled with impedimetric detection facilitated rapid and sensitive detection of bacterial pathogens with a detection limit of 10(2)CFU/mL for four bacterial strains including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). The approach enabled differentiation between live and dead bacteria. The fabrication of the sAMPs functionalized surface and the importance of the sAMPs orientation for providing optimum recognition and detection ability against pathogens are discussed. The proposed methodology provides a universal platform for the detection of bacterial pathogens based on engineered peptides, as alternative to the most commonly used immunological and gene based assays. The method can also be used to fabricate antimicrobial coatings and surfaces for inactivation and screening of viable bacteria.

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; This paper presents a first report on an ammonium ISFET (ion-sensitive field effect transistor) based on a polymeric membrane (siloprene) including a zeolite selective for ammonium exchange: clinoptilolite. The effect of zeolite content on sensitivity of detection is studied; an optimum is obtained for 43 w/w%. A sensitivity of detection of 32 mV/pNH4+ and a detection limit of 10−8 M are obtained. A highly selective ammonium ISFET is obtained.

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).

Despite the significant advances of modern medicine and the availability of a wide variety of antibiotics for the treatment of microbial infections, there is an alarming increase of multiresistant bacterial pathogens. This chapter discusses the status of bacterial resistance mechanisms and the relationship with oxidative stress and provides an overview of the methods used to assess oxidative conditions and their contribution to the antibiotic resistance.

Despite the significant advances of modern medicine and the availability of a wide variety of antibiotics for the treatment of microbial infections, there is an alarming increase of multiresistant bacterial pathogens. This chapter discusses the status of bacterial resistance mechanisms and the relationship with oxidative stress and provides an overview of the methods used to assess oxidative conditions and their contribution to the antibiotic resistance.

The use of a voltammetric electronic tongue as tool for the prediction of concentration levels of certain water quality parameters from influent and effluent wastewater from a Submerged Anaerobic Membrane Bioreactor pilot plant applied to domestic wastewater treatment is proposed here. The electronic tongue consists of a set of noble (Au, Pt, Rh, Ir, and Ag) and non-noble (Ni, Co and Cu) electrodes that were housed inside a stainless steel cylinder which was used as the body of the electronic tongue system. As a previous step an electrochemical study of the response of the ions sulphate, orthophosphate, acetate, bicarbonate and ammonium was carried out in water using the electrodes contained in the electronic tongue. The second part of the work was devoted to the application of the electronic tongue to the characterization of the influent and effluent waters from the wastewater treatment plant. Partial Least Squares analysis was used to obtain a correlation between the data from the tongue and the pollution parameters measured in the laboratory such as soluble chemical oxygen demand (CODs), soluble biological oxygen demand (BODs), ammonia (NH 4-N), orthophosphate (PO 4-P), Sulphate (SO 4-S), acetic acid (HAC) and alkalinity (Alk). A total of 28 and 11 samples were used in the training and the validation steps, respectively, for both influent and effluent water samples. The electronic tongue showed relatively good predictive power for the determination of BOD, COD, NH 4-N, PO 4-P, SO 4-S, and Alk. © 2012 Elsevier Ltd., Financial support from the Spanish Government (project MAT2009-14564-C04-01 and PCI-Mediterraneo A/024590/09/A/03044/10), the Generalitat Valenciana (project PROMETEO/2009/016), the MICINN (MAT2009-14564-C04-02) for a research fellowship and the UPV (project PAID-05-10) is gratefully acknowledged.

[EN] A sensitive, simple and label-free immunosensor for atrazine detection was developed. The immunosensor was based on antibody physisorption as immobilization technique. This technique was proposed as a cost-effective gold immuno-functionalization strategy alternative to SAM-based strategy for pesticide detection. Each step of immunosensor construction was characterized by electrochemical impedance spectroscopy, and the antibody-atrazine interaction was measured in a dynamic range from 10 to 150 ng/mL. This method was highly selective for atrazine and propazine, showing weak or no cross-reactivity to other similar compounds like terbutylazine. A limit detection of 10 ng/mL of atrazine was detected in PBS buffer with a good reproductibility. Lower limit detection in tap water was obtained., This work was financially supported by the PCI cooperation project between Spain and Tunisia. The authors thank Dr. F. Del Campo and Dr. E. Baldrich from CNM-Barcelona (Spain) for the interdigitated microelectrode fabrication. Pr. A. Abdelghani has to thanks the NATO (North Atlantic Treaty Organization) for the material donation.

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.