Peptide-modified electrolyte-gated organic field effect transistor. Application to Cu2+ detection.

This work proposes an approach for Cu2+ sensing in water which combines the selectivity of the Gly-Gly-His (GGH) peptide probe with the sensitivity of the electrolyte-gated organic field-effect transistor (EGOFET). The oligopeptide probe was immobilized onto the gate electrode of the transistor by electrooxidation of the primary amine of the glycine moiety. Cu2+ complexation by the grafted GGH was at first electrochemically evidenced, using cyclic and square wave voltammetries, then it was demonstrated that GGH-functionalized EGOFETs can transduce Cu2+ complexation through a significant threshold voltage shift and therefore a change in drain current. The limit of detection is ca. 10-12 M and the sensitivity in the linear range (10-12 - 10-8 M) is 1 mA dec-1 (drain current variations).

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.

Enzyme-less electrochemical displacement heterogeneous immunosensor for diclofenac detection.

We describe an electrochemical immunosensor based on functionalization of a working electrode by electrografting two functional diazonium salts. The first one is a molecular probe, diclofenac, coupled with an arylamine onto which a specific antibody is immobilized by affinity interactions; the second is a redox probe (a quinone) also coupled with an arylamine, able to transduce the hapten-antibody association into a change in electroactivity. The steric hindrance induced by the antibody leads to a current decrease upon binding of the antibody on the grafted molecular probe; conversely, when diclofenac is present in solution, a displacement equilibrium occurs between the target diffusing into the solution and the grafted probe. This leads to dissociation of the antibody from the electrode surface, event which is transduced into a current increase ("signal-on" detection). The detection limit is ca. 20 fM, corresponding to 6pgL-1 diclofenac, which is competitive compared to other label-free immunosensors. We demonstrate that the sensor is selective and is able to quantify diclofenac in tap water.

Grafting of a peptide probe for Prostate-Specific Antigen detection using diazonium electroreduction and click chemistry.

The main objective of this work was to validate a label-free electrochemical method of protein detection using peptides as capture probes. As a proof-of-concept, we used a 7 amino acids sequence (HSSKLQL) specific for Prostate Specific Antigen. We investigated various electrografting conditions of two anilines (2-[(4-aminophenyl)sulfanyl]-8-hydroxy-1,4-naphthoquinone and 4-azidoaniline) further converted in situ into their corresponding diazonium salts on glassy carbon electrodes. It was demonstrated that the best method to obtain a mixed layer is the simultaneous electroreduction of the two diazonium salts. 4-azidoaniline was used to covalently immobilize the ethynyl-functionalized peptide probe by click coupling, and the hydroxynaphthoquinone derivative plays the role of electrochemical transducer of the peptide-protein recognition. The proteolytic activity of PSA towards a small peptide substrate carrying streptavidin at its distal end was also investigated to design an original sensing architecture leading to a reagentless, label free, and "signal-on" PSA sensor. Without optimization, the limit of quantification can be estimated in the nM to pM range.

General approach for electrochemical detection of persistent pharmaceutical micropollutants: Application to acetaminophen.

We propose in this work a general and versatile methodology for electrochemical monitoring of persistent pharmaceutical micropollutants. The system presented is based on an electroactive and electropolymerized hapten (mimetic molecule of the pollutant to be detected) and a specific antibody that competitively binds either the hapten or the pollutant. The current delivered by the device depends on this competitive equilibrium and therefore on the pollutant's concentration. The determination of the pharmaceutical product operates within minutes, using square wave voltammetry without labeling or addition of a reactant in solution; the competitive hapten/antibody transduction produces a "signal-on" (a current increase). Applied to acetaminophen, this electrochemical immunosensor presents a very low detection limit of ca. 10 pM, (S/N=3) and a very high selectivity towards structural analogs (aspirin, BPA, and piperazine) even in a mixture.

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.

An electrochemical ELISA-like immunosensor for miRNAs detection based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes.

We design an electrochemical immunosensor for miRNA detection, based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes. An original immunological approach is followed, using antibodies directed to DNA.RNA hybrids. An electrochemical ELISA-like amplification strategy was set up using a secondary antibody conjugated to horseradish peroxidase (HRP). Hydroquinone is oxidized into benzoquinone by the HRP/H2O2 catalytic system. In turn, benzoquinone is electroreduced into hydroquinone at the electrode. The catalytic reduction current is related to HRP amount immobilized on the surface, which itself is related to miRNA.DNA surface density on the electrode. This architecture, compared to classical optical detection, lowers the detection limit down to 10 fM. Two miRNAs were studied: miR-141 (a prostate biomarker) and miR-29b-1 (a lung cancer biomarker).

Direct, reagentless electrochemical detection of the BIR3 domain of X-linked inhibitor of apoptosis protein using a peptide-based conducting polymer sensor.

In this work, we report a reagentless electrochemical peptide (AVPFAQKG) sensor to directly detect the BIR3 domain of X-linked inhibitor of apoptosis protein (XIAP-BIR3). The bioreceptor was based on a conducting copolymer film electrosynthesized from juglone and a juglone-peptide conjugate (JP) newly designed. The peptide-protein interactions generated an important increase of steric hindrance at the interface and a current decrease (signal off) of the redox reaction from quinone embedded in the polymer backbone as evidenced by Square Wave Voltammetry. This allowed a specific and sensitive detection of XIAP-BIR3 with a detection limit of 1 nM (13 ng mL(-1)). The peptide-protein complex could be then dissociated by adding the free precursor peptide (AVPFAQKG) into solution, causing a shift-back on the signal, i.e. an increase in the current intensity (signal-on). This "off-on" detection sequence was used in this work as a double verification of the specificity and this approach can be employed as a general way to increase the reliability of the results. In general, the approach described in this work may be inspired to develop other direct and reagentless electrochemical protein assays with high specificity and sensitivity.

E-assay concept: detection of bisphenol A with a label-free electrochemical competitive immunoassay.

A label-free electrochemical immunosensor was developed by electropolymerization of N-(3-(4-(2-(4-hydroxyphenyl)propan-2-yl)phenoxy)propyl) 3-(5-hydroxy-1,4-dihydro-1,4-dioxonaphthalen-2(3)-yl)propionamide (JugBPA). By combination with an antibody directed to bisphenol A (αBPA), this conducting polymer-based biosensor can detect BPA directly with a limit of detection of 2pgmL(-1). Square wave voltammetry shows that the polymer film presents a current decrease upon anti-BPA binding and an opposite current increase upon BPA addition in solution. This electrochemical immunosensor (E-assay) also shows high selectivity towards closely related compounds (bisphenol A dimethacrylate, and dibutyl phthalate). The E-assay concept described here could be a promising tool for simple, low-cost and reagentless on-site environmental monitoring.

An innovative strategy for direct electrochemical detection of microRNA biomarkers.

We report an electrochemical method for direct, reagentless, and label-free detection of microRNA, based on a conjugated copolymer, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone), acting as hybridization transducer. Hybridization between the oligonucleotide capture probe and a microRNA target of 22 base pairs generates an increase in the redox current ("signal-on"), which is evidenced by square wave voltammetry. Selectivity is good, with little hybridization for non-complementary targets, and the limit of detection reaches 650 fM. It is also evidenced that this sensitivity benefits from the high affinity of DNA for RNA.

Design of interpenetrated network MWCNT/poly(1,5-DAN) on interdigital electrode: toward NO2 gas sensing.

In this paper, poly(1,5-diaminonaphthalene) was interpenetrated into the network made of multiwalled carbon nanotubes (MWCNT) on platinum interdigital electrode (IDE) by electro-polymerization of 1,5-diaminonaphthalene (1,5-DAN). The electro-polymerization process of 1,5-DAN on MWCNT was controlled by scanning the cyclic voltage at 50 mV s(-1) scan rate between -0.1 V and +0.95 V vs. saturated calomel electrode (SCE). The results of voltammetric responses and Raman spectroscopy represented that the films MWCNT/poly(1,5-DAN) were successfully created by this polymerization process. The films MWCNT/poly(1,5-DAN) were investigated for gas-sensing to NO2 at low concentration level. The gas-sensing results showed that the response-recovery times were long and strongly affected by thickness of the film MWCNT/poly(1,5-DAN). Nevertheless, these films represented auspicious results for gas sensors operating at room temperature.

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.

Label-free and reagentless electrochemical detection of microRNAs using a conducting polymer nanostructured by carbon nanotubes: application to prostate cancer biomarker miR-141.

In this paper, a label-free and reagentless microRNA sensor based on an interpenetrated network of carbon nanotubes and electroactive polymer is described. The nanostructured polymer film presents very well-defined electroactivity in neutral aqueous medium in the cathodic potential domain from the quinone group embedded in the polymer backbone. Addition of microRNA miR-141 target (prostate cancer biomarker) gives a "signal-on" response, i.e. a current increase due to enhancement of the polymer electroactivity. On the contrary, non-complementary miRNAs such as miR-103 and miR-29b-1 do not lead to any significant current change. A very low detection limit of ca. 8 fM is achieved with this sensor.

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 and reagentless electrochemical detection of PCR fragments using self-assembled quinone derivative monolayer: application to Mycobacterium tuberculosis.

We report a signal-on, label-free and reagentless electrochemical DNA biosensor, based on a mixed self-assembled monolayer of thiolated hydroxynaphthoquinone and thiolated oligonucleotide. Electrochemical changes resulting from hybridization were evidenced with oligonucleotide targets (as models), as well as with polymerase chain reaction (PCR) products related to different lineages of Mycobacterium tuberculosis strains. With pure oligonucleotides, this system achieves high sensitivity (∼300 pM of DNA target, i.e. 30 fmol in a 100 µL sample) and excellent selectivity, allowing to detect a single mismatch on a sequence of 20 bases. With PCR products, current changes are specific to the bacterial strain from which the PCR fragment is produced. In addition, the sensor response is of the signal-on type, giving a positive signal change upon hybridization, and therefore does not suffer from false positive responses due to non-specific adsorption of DNA.

Direct and rapid electrochemical immunosensing system based on a conducting polymer.

A system device using multifunctional conjugated copolymer poly(5-hydroxy-1,4-naphthoquinone-co-hydroxy-2-thioacetic acid-1,4-naphthoquinone) acting both as immobilizing and transducing element for reagentless immunosensor has been constructed. Its functionality was evaluated in an antigen-antibody interaction model using ovalbumin-anti-ovalbumin. It was shown that the system specifically detects via electrochemical signal the antigen-antibody immune interaction in a reagentless context. Comparison to the conventional ELISA technique relevant to performance and sensitivity was presented.

Design of a new electrogenerated polyquinone film substituted with glutathione. Towards direct electrochemical biosensors.

We developed a method to graft a tripeptide (glutathione) onto 5-hydroxy-1,4-naphthoquinone, an electropolymerizable molecule. The resulting thin conducting polymer presents a well-defined and stable electroactivity in neutral buffered solution, due to the embedded quinone group, and is able to covalently graft amino-modified DNA probe strands. It is shown that the bioelectrode presents positive current change following DNA hybridization. This makes a "signal-on" direct electrochemical DNA sensor. The results were obtained with low target concentration (50nM) and the selectivity is excellent as a single-mismatch sequence can be discriminated from the full-complementary target.

Label-free DNA electrochemical sensor based on a PNA-functionalized conductive polymer.

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probe is presented. PNA were attached covalently onto a quinone-based electroactive polymer. Changes in flexibility of the PNA probe strand upon hybridization generates electrochemical changes at the polymer-solution interface. A reagentless and direct electrochemical detection was obtained by detection of the electrochemical changes using square wave voltammetry (SWV). An increase in the peak current of quinone was observed upon hybridization of probe on the target, whereas no change is observed with non-complementary sequence. In addition, the biosensor is highly selective to effectively discriminate a single mismatch on the target sequence. The sensitivity is also presented and discussed.

Investigations of the steric effect on electrochemical transduction in a quinone-based DNA sensor.

Electrochemical biosensors for DNA hybridization are receiving increasing interest. A key point for their efficiency is to obtain a high signal level for low DNA concentration. This implies the design of an efficient transducing surface. Conducting polymers are interesting for this purpose but the great majority of conducting polymer-based electrodes present a signal decrease upon hybridization (a "signal-off" behavior), which impedes their response and makes them sensitive to false positive ones. The sensor described here presents a "signal-on" behavior, due to the use of a quinone group as the transducing agent. The specific aim of this work is to study the steric effect on transduction. To this end, the electrochemical response was monitored versus the DNA target length, for a constant DNA probe length. The results indicate that the current depends on the length of the double strand. A model which can explain the electrochemical behavior takes into account the steric hindrance of the ODN strands.