A microfluidic electrochemical biosensor based on multiwall carbon nanotube/ferrocene for genomic DNA detection of Mycobacterium tuberculosis in clinical isolates.

Herein we present a microfluidic-multiplexed platform that integrates electrochemical sensors based on carbon nanotubes associated with ferrocene as redox marker (carbon nanotube (CNT)/ferrocene) for direct detection of pathogenic viral DNA from Hepatitis C and genomic DNA from Mycobacterium tuberculosis in clinical isolates. By operating the fluidic device under high flow (150 µl/min), the formation of a very thin depletion layer at the sensor surface (δS = 230 nm) enhances the capture rate up to one DNA strand per second. By comparison, this capture rate is only 0.02 molecule/s in a static regime without flow. This fluidic protocol allows thus enhancing the limit of detection of the electrochemical biosensor from picomolar in bulk solution to femtomolar with a large dynamic range from 0.1 fM to 1 pM. Kinetics analysis also demonstrates an enhancement of the rate constant of electron transfer (kS) of the electrochemical process from 1 s(-1) up to 6 s(-1) thanks to the geometry of the miniaturized fluidic electrochemical cell. This microfluidic device working under high flow allows selective direct detection of a Mycobacterium tuberculosis (H37Rv) rpoB allele from clinical isolate extracted DNA. We envision that a microfluidic approach under high flow associated with a multiwall CNT/ferrocene sensor could find useful applications as the point-of-care for multi-target diagnostics of biomarkers in real samples.

Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%.

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole.

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP(C)) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP(C) interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP(C) with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP(C) and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

Electrochemical detection of D-dimer as deep vein thrombosis marker using single-chain D-dimer antibody immobilized on functionalized polypyrrole.

We describe a rapid and sensitive method for detection and quantification of d-dimer which is a biomarker present at elevated concentrations in patients with deep vein thrombosis (DVT) disorders. The method uses an immunosensor based on a single-chain antibody (ScAb) immobilized on a transducer surface and with a densely packed receptor layer. Detection is based on the redox activity of a N-alpha bis(carboxymethyl)-L-lysine (ANTA)/Cu2+ complex attached to a polypyrrole backbone. The resulting hybrid material: polypyrrole ANTA/metal complex/His-tag ScAb was characterized by AFM, surface plasmon resonance (SPR) and differential pulse voltammetry (DPV) for the optimization of the biosensor formation. The biosensor offers a promising template for antibody immobilization and for immunodetection of a specific D-dimer. The biosensor shows a remarkable variation in redox activity of the ANTA/Cu2+ complex after the D-dimer association with a binding constant Kd of 1 ng mL(-1). Electrochemical impedance spectroscopy (EIS) allows monitoring D-dimer association with a linear response between 0.1 ng mL(-1) and 500 ng mL(-1) and a detection limit of 100 pg mL(-1) in PBS is obtained. The biolayer exhibits the same sensitivity for the detection of d-dimer in human patient plasma samples. This assay method is versatile, offers enhanced performance for the evaluation of proteins association and could easily be extended to the detection of other proteins, present in serum human sample.

Investigation of SPR and electrochemical detection of antigen with polypyrrole functionalized by biotinylated single-chain antibody: a review.

An electrochemical label-free immunosensor based on a biotinylated single-chain variable fragment (Sc-Fv) antibody immobilized on copolypyrrole film is described. An efficient immunosensor device formed by immobilization of a biotinylated single-chain antibody on an electropolymerized copolymer film of polypyrrole using biotin/streptavidin system has been demonstrated for the first time. The response of the biosensor toward antigen detection was monitored by surface plasmon resonance (SPR) and electrochemical analysis of the polypyrrole response by differential pulse voltammetry (DPV). The composition of the copolymer formed from a mixture of pyrrole (py) as spacer and a pyrrole bearing a N-hydroxyphthalimidyl ester group on its 3-position (pyNHP), acting as agent linker for biomolecule immobilization, was optimized for an efficient immunosensor device. The ratio of py:pyNHP for copolymer formation was studied with respect to the antibody immobilization and antigen detection. SPR was employed to monitor in real time the electropolymerization process as well as the step-by-step construction of the biosensor. FT-IR demonstrates the chemical copolymer composition and the efficiency of the covalent attachment of biomolecules. The film morphology was analyzed by electron scanning microscopy (SEM). Results show that a well organized layer is obtained after Sc-Fv antibody immobilization thanks to the copolymer composition defined with optimized pyrrole and functionalized pyrrole leading to high and intense redox signal of the polypyrrole layer obtained by the DPV method. Detection of specific antigen was demonstrated by both SPR and DPV, and a low concentration of 1 pg mL(-1) was detected by measuring the variation of the redox signal of polypyrrole.

Effect of the size of electrode on electrochemical properties of ferrocene-functionalized polypyrrole towards DNA sensing.

A simple and highly sensitive electrochemical DNA sensor based on a ferrocene-functionalized polypyrrole has been prepared on a microelectrode array substrate for a multi-DNA detection chip format. A copolymer formed with 1-(phthalimidylbutanoate)-1'-(N-(3-butylpyrrole)butanamide)ferrocene (Py-Fe-NHP) and pyrrole was electrocopolymerized on the gold surface of both macroelectrode and biochip formats. DNA probes bearing an amino group were covalently grafted by substitution of NHP groups and the hybridization reaction was followed by monitoring the redox signal of the ferrocenyl group acting as the probe. The integration of the polymers into chip format produces high-density arrays of individually addressable oligonucleotide microelectrodes. Results show that reducing the size of the electrodes from a macroelectrode to the chip format allows a variation of the nucleation and the growth process during electropolymerization of modified pyrrole monomers. These modifications enable an increase in the sensitivity and selectivity of DNA hybridization.

Electrochemical probing of DNA based on oligonucleotide-functionalized polypyrrole.

We have developed a new type of electrochemical biosensor based on oligonucleotide-functionalized polypyrrole. At first, we analyzed the experimental conditions necessary for building such modified electrodes that show a high electroactivity in aqueous media. We developed a precursor polymer bearing an easy leaving ester group on which amino-labeled oligonucleotides with various sequence lengths were directly substituted. The electrochemical response of the modified electrode was analyzed in various aqueous media containing either complementary or noncomplementary oligonucleotides. Results show that the cyclic voltammogram of oligonucleotide-functionalized polypyrrole is not modified when in the presence of a noncomplementary oligonucleotide in solution. On the other hand, a significant modification of the voltammogram is observed upon addition of a complementary oligonucleotide "target" to the electrolytic medium, which can be quantitatively determined by amperometric methods. The detection limit of this electrochemical biosensor is about 10(-11) mol, without any signal processing.