Bienzymatic-based electrochemical DNA biosensors: a way to lower the detection limit of hybridization assays

5 pagesInternational audienceThe use of the alkaline phosphatase (AP) as enzyme label and the amplification of its analytical response with a diaphorase (DI) secondary enzyme were investigated in an electrochemical hybridization assay involving arrays of carbon screen-printed DNA biosensors for the sensitive quantification of an amplified 406-base pair human cytomegalovirus DNA sequence (HCMV DNA). For this purpose, PCR-amplified biotinylated HCMV DNA targets were simultaneously bound to a monolayer of neutravidin irreversibly adsorbed on the surface of the electrodes and hybridized to complementary digoxigenin-labeled detection probes. The amount of hybrids immobilized on the electrode surface was labeled with an anti-digoxigenin AP conjugate and quantified electrochemically by measuring the activity of the AP label through the hydrolysis of the electroinactive p-aminophenylphosphate (PAPP) substrate into the p-aminophenol (PAP) product. The intensity of the cyclic voltammetric anodic peak current resulting from the oxidation of PAP into p-quinoneimine (PQI) was related to the number of viral amplified DNA targets present in the sample, and a detection limit of 10 pM was thus achieved. The electrochemical response of the AP label product was further enhanced by adding the diaphorase enzymatic amplifier in the solution. In the presence of the auxiliary enzyme DI, the PQI was reduced back to PAP and the resulting oxidized form of DI was finally regenerated in its reduced native state by its natural substrate, NADH. Such bienzymatic amplification scheme enabled a 100-fold lowering of the HCMV DNA detection limit obtained with the monoenzymatic system

Noble Metal Nanoparticles for Biosensing Applications

In the last decade the use of nanomaterials has been having a great impact in biosensing. In particular, the unique properties of noble metal nanoparticles have allowed for the development of new biosensing platforms with enhanced capabilities in the specific detection of bioanalytes. Noble metal nanoparticles show unique physicochemical properties (such as ease of functionalization via simple chemistry and high surface-to-volume ratios) that allied with their unique spectral and optical properties have prompted the development of a plethora of biosensing platforms. Additionally, they also provide an additional or enhanced layer of application for commonly used techniques, such as fluorescence, infrared and Raman spectroscopy. Herein we review the use of noble metal nanoparticles for biosensing strategies—from synthesis and functionalization to integration in molecular diagnostics platforms, with special focus on those that have made their way into the diagnostics laboratory

Subfemtomolar electrochemical detection of target DNA by catalytic enlargement of the hybridized gold nanoparticle labels

7 pagesInternational audienceAfter showing the failure of conventional gold-enhancement procedures to amplify the gold nanoparticle-based electrochemical transduction of DNA hybridization in polystyrene microwells, a new efficient protocol was developed and evaluated for the sensitive quantification of a 35 base-pair human cytomegalovirus nucleic acid target (tDNA). In this assay, the hybridization of the target adsorbed on the bottom of microwells with an oligonucleotide-modified Au nanoparticle detection probe (pDNA-Au) was monitored by the anodic stripping detection of the chemically oxidized gold label at a screen-printed microband electrode (SPMBE). Thanks to the combination of the sensitive AuIII determination at a SPMBE with the large number of AuIII released from each pDNA-Au, picomolar detection limits of tDNA can be achieved. Further enhancement of the hybridization signal based on the autocatalytic reductive deposition of ionic gold (AuIII) on the surface of the gold nanoparticle labels anchored on the hybrids was first envisaged by incubating the commonly used mixture of AuIII and hydroxylamine (NH2OH). However, due to a considerable nonspecific current response of poor reproducibility it was not possible to significantly improve the analytical performances of the method under these conditions. Complementary transmission electronic microscopy experiments indicated the loss of most of the grown gold labels during the post-enlargement rinsing step. To circumvent this drawback, a polymeric solute containing polyethyleneglycol and sodium chloride was introduced in the growth media to act as aggregating agent during the catalytic process and thus retain the enlarged labels on the bottom of the microwell. This strategy, which led to an efficient increase of the hybridization response, allowed detection of tDNA concentrations as low as 600 aM (i.e., 104 lower than without amplification) and thus offers great promise for ultrasensitive detection of other hybridization events

MÉTHODE DE DÉTECTION ÉLECTROCHIMIQUE DE SÉQUENCES CIBLES D'ACIDE NUCLÉIQUE

L'invention concerne une méthode et un ensemble de détection électrochimique de séquences cibles d'acide nucléique. Selon la méthode, on fournit un échantillon biologique susceptible de renfermer un acide nucléique, ledit acide nucléique étant susceptible de contenir une séquence cible, ledit échantillon biologique étant mélangé à un agent oxydant, ladite séquence cible comportant au moins une base nucléotidique oxydable par ledit agent oxydant ; on fournit des moyens complémentaires susceptibles de se coupler avec ladite séquence cible ; selon l'invention lesdits moyens complémentaires comprennent des moyens d'amplification activables adaptés à répliquer ladite séquence cible, lesdits moyens d'amplification comprenant au moins des nucléotides incluant ladite base nucléotidique, lesdits nucléotides étant aptes à être consommés durant la réplication pour constituer des acides nucléiques répliqués, et ; on détermine la présence de ladite séquence cible en appliquant un champ électrique audit échantillon et en enregistrant la décroissance du courant électrique

Détection ampérométrique des <em>Escherichia coli</em> productrices de beta-lactamases à spectre étendu dans les effluents de station d’épuration

National audienceLes Escherichia coli productrices de β-lactamases à spectre étendu (E. coli BLSE) sont résistantes à la plupart des β-lactamines et deviennent un problème majeur de santé publique en médecine humaine. Leur présence dans le tube digestif des humains fait que grandes quantités d’E. coli BLSE sont présentes dans les eaux usées qui arrivent dans les stations d’épuration (STEP). Malgré les traitements réalisés dans les STEP, des quantités non négligeables d’E. coli BLSE sont encore présentes dans les effluents traités directement rejetés dans l’environnement. Ces rejets peuvent être à l’origine d’une contamination des eaux récréatives et constituer un risque sanitaire pour les populations exposées. La mise au point de tests rapides et pratiques est donc nécessaire pour détecter et quantifier ces E. coli BLSE dans les réseaux d’eaux usées et les environnements naturels. Cette étude à eu pour objectif de développer une méthode ampérométrique rapide pour quantifier les E. coli BLSE dans les effluents de STEP. La première étape consiste en une double filtration sur membranes d’un échantillon d’eau de STEP, suivie d’une analyse ampérométrique en deux étapes : (1) une mise en culture (4-5h) de chaque membrane dans un milieu liquide contenant du céfotaxime ± acide clavulanique (inhibiteur de BLSE) et (2) une incubation (15min) de chaque culture filtrée sur membrane en présence de Nitrocéfine, dont l’hydrolyse par les β-lactamases est suivie par ampérométrie. Deux intensités sont ainsi mesurées : iCef et iClav et la valeur i = iCef - iClav est utilisée pour quantifier les E. coli BLSE présentes dans l’échantillon en se basant sur des courbes de calibration. Une bonne corrélation de cette estimation ampérométrique (5-6h) avec un dénombrement classique sur milieu gélosé (24h) est obtenue après avoir analysé une quarantaine d’échantillons. Cette méthode est très prometteuse dans le domaine de l’analyse des eaux usées et des eaux récréatives

Evaluation of the analytical performances of avidin-modified carbon sensors based on mediated horseradish peroxidase enzyme label and their application to the amperometric detection of nucleic acids

8 pagesInternational audienceIn this study, neutravidin-coated screen-printed carbon sensors were fully characterized and further used for the amperometric detection of specific DNA sequences of human cytomegalovirus (HCMV DNA). For this purpose, we took advantage of an earlier established relationship between the amount of HRP affinity immobilized on the surface of the electrode and the steady-state current recorded in the presence of H2O2 as substrate and the single electron donor [OsIII(bpy)2pyCl]2+ as cosubstrate. After incubating a saturating concentration of biotinylated horseradish peroxidase (Bio-HRP) onto the neutravidin-modified sensors, a surface concentration of active HRP of 3.6 pmol.cm-2 was calculated from the measurement of the electrocatalytic plateau current value. This result indicates that monolayers of neutravidin were adsorbed on the screen-printed carbon sensors. These neutravidin-covered platforms were then used to immobilize biotinylated nucleic acid targets. After hybridization with a complementary digoxigenin-labeled detection probe, the extent of hybrids formed was determined with an anti-digoxigenin HRP conjugate. The biosensor assay was applied to the detection of a synthetic oligonucleotide target, and then to the determination of an amplified viral DNA sequence. Monolayers of HRP-labeled oligonucleotide hybrids were immobilized onto the sensing surface whereas one third of the surface was covered with HCMV DNA hybrids. On the other hand, detection limits of 200 pM and 1 nM were obtained for the short oligonucleotide and the longer DNA targets, respectively. Finally, we demonstrated that the sensitivity of the electrochemical assay could be significantly improved by using high concentrations of the reduced form of the mediator [OsII(bpy)2pyCl]+, thus allowing one to detect as low as 30 pM of amplified HCMV DNA fragment

Evaluation de la qualité microbiologique des eaux de baignade via la mesure ampérométrique d’activités enzymatiques

BIOMEEAUBEvaluation de la qualité microbiologique des eaux de baignade via la mesure ampérométrique d’activités enzymatiques. XV. Colloque du Groupe Français de Bioélectrochimi

Amperometric detection of extended-spectrum β-lactamase activity : application to the characterization of resistant <em>E.coli</em> strains

EA MERS CT3International audienceThe amperometric detection of extended-spectrum β-lactamase (ESBL) with carbon screen-printed sensors was investigated in the presence of the Nitrocefin, a commercially-available β-lactamase chromogenic cephalosporin substrate. Using an ESBL isolated from a clinical sample, it was shown for the first time that the intensity of a specific anodic pic current (EP = [similar]+0.3 V vs. Ag/AgCl) resulting from the catalytic hydrolysis of the β-lactam ring was proportional to the amount of ESBL. The proof-of-principle of a novel susceptibility assay for the rapid and accurate identification of ESBL- producing bacteria was then demonstrated. The detection scheme relied on (i) the culture of the sample in a medium containing the cefotaxime supplemented or not with the clavulanic acid inhibitor to allow the specific determination of ESBL producers (ii) followed by the incubation of the bacteria with the Nitrocefin and (iii) the measurement of the enzyme product by cyclic voltammetry. The amperometric assay was further applied to the characterization of E. coli strains and to the quantification of the ESBL producers. A detection limit of 5 × 104 cfu mL−1 ESBL-producing E. coli was achieved after a 10 min incubation time. In contrast to the approved routine assays, the electrochemical approach, which did not require isolated colonies to be performed, provided quantified results regarding ESBL activity within a few hours. Finally, owing to its cost-effectiveness, portability and simplicity, this test holds great promise for clinical and environmental applications

Une méthode électrochimique pour la détection rapide des bactéries résistantes aux antibiotiques

BIOMEEAUBUne méthode électrochimique pour la détection rapide des bactéries résistantes aux antibiotiques. XV. Colloque du Groupe Français de Bioélectrochimi