Steeve Reisberg

Antibodies directed to RNA/DNA hybrids: an electrochemical immunosensor for microRNAs detection using graphene-composite electrodes.

We report a simple and sensitive label-free immunosensor for detection of microRNAs (miRNA) based on a conducting polymer/reduced graphene oxide-modified electrode to detect miR-29b-1 and miR-141. Square wave voltammetry is used to record the redox signal. Current increases upon hybridization (signal on) from 1 fM to 1 nM of target miRNA. The limit of quantification is ca. 5 fM. The sensor exhibits high selectivity as it distinguishes mismatch. To double-check its selectivity, two specific RNA-DNA antibodies recognizing miRNA-DNA heteroduplexes, antipoly(A)-poly(dT) and anti-S9.6, were used. The antibody complexation with the hybrid leads to a current decrease that confirms the presence of miRNA, down to a concentration of 8 fM. The antibody-hybrid complex can be then dissociated by adding miRNA-DNA hybrids in solution, causing a shift-back on the signal, i.e., an increase in the current density (signal-on). This On-Off-On detection sequence was used as a triple verification to increase the reliability of the results.

Nanometric layers for direct, signal-on, selective, and sensitive electrochemical detection of oligonucleotides hybridization.

We report a signal-on, reagentless electrochemical DNA biosensor, based on an electroactive self-assembled naphthoquinone derivative (JUG(thio)) monolayer. This system achieves highly sensitive (approximately 300 pM) and selective signal-on detection. Before hybridization, the single strand can interact with JUG(thio) and slow down the redox reaction. When the complementary target is added, the formation of the double helix eliminates the single strand/JUG(thio) interactions and the JUG(thio) redox rate, and hence the current increase.

A label-free electrochemical immunosensor for direct, signal-on and sensitive pesticide detection

A new electropolymerizable monomer, [N-(6-(4-hydroxy-6-isopropylamino-1,3,5-triazin-2-ylamino)hexyl) 5-hydroxy-1,4-naphthoquinone-3-propionamide], has been designed for use in a label-free electrochemical immunosensor when polymerized on an electrode and coupled with a monoclonal anti-atrazine antibody. This monomer contains three functional groups: hydroxyl group for electropolymerization, quinone group for its transduction capability, and hydroxyatrazine as bioreceptor element. Square wave voltammetry shows that the polymer film, poly[N-(6-(4-hydroxy-6-isopropylamino-1,3,5-triazin-2-ylamino)hexyl) 5-hydroxy-1,4-naphthoquinone-3-propionamide], presents negative current change following anti-atrazine antibody complexation and positive current change after atrazine addition in solution. This constitutes a direct, label-free and signal-on electrochemical immunosensor, with a very low detection limit of ca. 1 pM, i.e. 0.2 ng L(-1), one of the lowest reported for such immunosensors. This is far lower than the detection limit required by the European Union directives for drinkable water and food samples (0.1 μg L(-1)). The strategy described has great promise for the development of simple, cost-effective and reagentless on-site environmental monitors.

Label-free electrochemical detection of prostate-specific antigen based on nucleic acid aptamer.

We report a label-free aptasensor to make direct detection of prostate specific antigen (PSA, a biomarker of prostate cancer) using a quinone-containing conducting copolymer acting as redox transducer and grafting matrix for immobilization of the short aptamer strands. It is shown that capture of PSA generates a current decrease (signal-off) measured by Square Wave Voltammetry. This current decrease is specific for PSA above a limit of quantification in the ng mL(-1) range. The change in current is used to determine the PSA-aptamer dissociation constant K(D), of ca. 2.6 nM. To consolidate the proof of concept, a heterogeneous competitive exchange with a complementary DNA strand which breaks PSA-aptamer interactions is studied. This double-check followed by a current increase provides full assurance of a perfectly specific recognition.

Hydroxynaphthoquinone ultrathin films obtained by diazonium electroreduction: toward design of biosensitive electroactive interfaces.

Electroactive 2-(phenylsulfanyl)-8-hydroxy-1,4-naphthoquinone has been electrodeposited via the reduction of the corresponding diazonium salt on Au electrodes. Surface characterizations by X-ray photoelectron spectroscopy (XPS) and infrared reflection-absorption spectroscopy (IRRAS) reveal that the mechanism of film deposition follows an aryl radical formation and its immobilization on the electrode surface. Electrochemical study shows that the surface coverage can be finely tuned (thickness between one and four layers) by adjusting the potential and the deposition time. By managing the potential applied when reducing diazonium in potentiostatic mode, the formed layer could mediate or not charge transfer. This is the first time that the films obtained by diazonium process are demonstrated to act as mediators in the growth process. Hence, with potentials higher than the formal potential of quinone group, very thin and homogeneous layers are obtained, whereas thicker films are formed when more cathodic potentials than that of quinone are applied. The possibility to manage the charge-transfer kinetics, the thickness, and the homogeneity of electroactive deposits is interesting in the scope of designing electrochemical transducers.

Quinone-Based Polymers for Label-Free and Reagentless Electrochemical Immunosensors: Application to Proteins, Antibodies and Pesticides Detection

Polyquinone derivatives are widely recognized in the literature for their remarkable properties, their biocompatibility, simple synthesis, and easy bio-functionalization. We have shown that polyquinones present very stable electroactivity in neutral aqueous medium within the cathodic potential domain avoiding side oxidation of interfering species. Besides, they can act as immobilized redox transducers for probing biomolecular interactions in sensors. Our group has been working on devices based on such modified electrodes with a view to applications for proteins, antibodies and organic pollutants using a reagentless label-free electrochemical immunosensor format. Herein, these developments are briefly reviewed and put into perspective.

Recent Advances in Electrochemical Immunosensors

Immunosensors have experienced a very significant growth in recent years, driven by the need for fast, sensitive, portable and easy-to-use devices to detect biomarkers for clinical diagnosis or to monitor organic pollutants in natural or industrial environments. Advances in the field of signal amplification using enzymatic reactions, nanomaterials such as carbon nanotubes, graphene and graphene derivatives, metallic nanoparticles (gold, silver, various oxides or metal complexes), or magnetic beads show how it is possible to improve collection, binding or transduction performances and reach the requirements for realistic clinical diagnostic or environmental control. This review presents these most recent advances; it focuses first on classical electrode substrates, then moves to carbon-based nanostructured ones including carbon nanotubes, graphene and other carbon materials, metal or metal-oxide nanoparticles, magnetic nanoparticles, dendrimers and, to finish, explore the use of ionic liquids. Analytical performances are systematically covered and compared, depending on the detection principle, but also from a chronological perspective, from 2012 to 2016 and early 2017.

Versatile transduction scheme based on electrolyte-gated organic field-effect transistor used as immunoassay readout system

We report on an innovative heterogeneous bisphenol A (BPA) immunoassay based on an electrolyte-gated organic field-effect transistor whose organic semiconductor is poly(2,5-bis(3-tetradecylthiophen-2-yl)thieno[3,2-b]thiophene) co-crystallized with an alkyl derivative of bisphenol A. A decrease of the transistor output current is first observed upon antibody specific binding onto the organic semiconductor. Upon bisphenol A addition, the competitive dissociation of the antibody from the semiconductor surface leads to an opposite increase of the output current. We present here a proof-of-concept for bisphenol A detection; the device could be readily adapted to other small organic molecules of interest and is a promising tool for simple, low-cost, portable and easy-to-use biosensors.

Triggering the Electrolyte-Gated Organic Field-Effect Transistor output characteristics through gate functionalization using diazonium chemistry: Application to biodetection of 2,4-dichlorophenoxyacetic acid

We investigated an Electrolyte-Gated Organic Field-Effect transistor based on poly(N-alkyldiketopyrrolo-pyrrole dithienylthieno[3,2-b]thiophene) as organic semiconductor whose gate electrode was functionalized by electrografting a functional diazonium salt capable to bind an antibody specific to 2,4-dichlorophenoxyacetic acid (2,4-D), an herbicide well-known to be a soil and water pollutant. Molecular docking computations were performed to design the functional diazonium salt to rationalize the antibody capture on the gate surface. Sensing of 2,4-D was performed through a displacement immunoassay. The limit of detection was estimated at around 2.5 fM.

Transistors for Chemical Monitoring of Living Cells

Featured Application Animal testing will be soon replaced by better accepted and less expensive in-vitro cell culture models, which explains the recent demand for real-time cell culture monitoring systems. They can bring high throughput screening and could be used not only for biomedical purposes (drug discovery, toxicology, protein expression, cancer diagnostic, etc.), but also for environmental ones (qualification of pollutants cocktails, for example). Beyond this, in-situ monitoring also participates in strengthening the fundamental knowledge about cells metabolism. Abstract We review here the chemical sensors for pH, glucose, lactate, and neurotransmitters, such as acetylcholine or glutamate, made of organic thin-film transistors (OTFTs), including organic electrochemical transistors (OECTs) and electrolyte-gated OFETs (EGOFETs), for the monitoring of cell activity. First, the various chemicals that are produced by living cells and are susceptible to be sensed in-situ in a cell culture medium are reviewed. Then, we discuss the various materials used to make the substrate onto which cells can be grown, as well as the materials used for making the transistors. The main part of this review discusses the up-to-date transistor architectures that have been described for cell monitoring to date.