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.

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.

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.

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.

Synthesis of new fluoroquinolones and evaluation of their in vitro activity on Toxoplasma gondii and Plasmodium spp.

The synthesis of four new computer-designed fluoroquinolones which have been predicted by QSAR analysis to be active against the protozoa Toxoplasma gondii is described. These compounds are inhibitory in vitro for T. gondii. One of these compounds has a remarkably high activity comparable to that of trovafloxacin. It combines the basic cyclopropyl-quinoline structure of gatifloxacin or moxifloxacin with the C-7 6-amino-3-azabicyclo[3.1.0]hexyl side chain of trovafloxacin. The four compounds are also inhibitory for blood stages of Plasmodium falciparum though at high concentration. These results confirm the potential of quinolones as anti-T. gondii and antimalarial drugs but also show that the QSAR models for T. gondii cannot be reliably extended for screening antimalarial activity.