Real-time electrochemical PCR with a DNA intercalating redox probe.

The proof-of-principle of a nonoptical real-time PCR method based on the electrochemical monitoring of a DNA intercalating redox probe that becomes considerably less easily electrochemically detectable once intercalated to the amplified double-stranded DNA is demonstrated. This has been made possible thanks to the finding of a redox intercalator that (i) strongly and specifically binds to the amplified double-stranded DNA, (ii) does not significantly inhibit PCR, (iii) is chemically stable under PCR cycling, and (iv) is sensitively detected by square wave voltammetry during PCR cycling. Among the different DNA intercalating redox probes that we have investigated, namely, methylene blue, Os[(bpy)(2)phen](2+), Os[(bpy)(2)DPPZ](2+), Os[(4,4'-dimethyl-bpy)(2)DPPZ](2+) and Os[(4,4'-diamino-bpy)(2)DPPZ](2+) (with bpy = 2,2'-bipyridine, phen = phenanthroline, and DPPZ = dipyrido[3,2-a:2',3'-c]phenazine), the one and only compound with which it has been possible to demonstrate the proof-of-concept is the Os[(bpy)(2)DPPZ](2+). In terms of analytical performances, the methodology described here compares well with optical-based real-time PCRs, offering finally the same advantages than the popular and routinely used SYBR Green-based real-time fluorescent PCR, but with the additional incomes of being potentially much cheaper and easier to integrate in a hand-held miniaturized device.

Real-time electrochemical monitoring of the polymerase chain reaction by mediated redox catalysis.

We described the proof-of-principle of a nonoptical real-time PCR that uses cyclic voltammetry for indirectly monitoring the amplified DNA product generated in the PCR reaction solution after each PCR cycle. To enable indirect measurement of the amplicon produced throughout PCR, we monitor electrochemically the progressive consumption (i.e., the decrease of concentration) of free electroactive deoxynucleoside triphosphates (dNTPs) used for DNA synthesis. This is accomplished by exploiting the fast catalytic oxidation of native deoxyguanosine triphosphate (dGTP) or its unnatural analogue 7-deaza-dGTP by the one-electron redox catalysts Ru(bpy)(3)(3+) (with bpy = 2,2'-bipyridine) or Os(bpy)(3)(3+) generated at an electrode. To demonstrate the feasibility of the method, a disposable array of eight miniaturized self-contained electrochemical cells (working volume of 50 microL) has been developed and implemented in a classical programmable thermal cycler and then tested with the PCR amplification of two illustrated examples of real-world biological target DNA sequences (i.e., a relatively long 2300-bp sequence from the bacterial genome of multidrug-resistant Achromobacter xylosoxidans and a shorter 283-bp target from the human cytomegalovirus). Although the method works with both mediator/base couples, the catalytic peak current responses recorded with the Ru(bpy)(3)(3+)/dGTP couple under real-time PCR conditions are significantly affected by a continuous current drift and interference with the background solvent discharge, thus leading to poorly reproducible data. Much more reproducible and reliable results are finally obtained with the Os(bpy)(3)(3+)/7-deaza-dGTP, a result that is attributed to the much lower anodic potential at which the catalytic oxidation of 7-deaza-dGTP by Os(bpy)(3)(3+) is detected. Under these conditions, an exponential decrease of the catalytic signal as a function of the number of PCR cycles is obtained, allowing definition of a cycle threshold value (C(t)) that correlates inversely with the initial amount of target DNA. A semilogarithmic plot of C(t) with the initial copy number of target DNA gives a standard linear curve similar to that obtained with fluorescent-based real-time PCR. Although the detection limit (10(3) molecules of target DNA in 50 microL) and sensitivity of the electrochemical method is not as high as conventional optical-based real-time PCR, the methodology described here offers many of the advantages of real-time PCR, such as a high dynamic range (over 8-log(10)) and speed, high amplification efficiency (close to 2), and the elimination of post-PCR processing. The method also has the advantage of being very simple, just requiring the use of low-cost single-use electrodes and the addition of a minute amount of redox catalyst into the PCR mixture. Moreover, compared to the other recently developed electrochemical real-time PCR based on solid-phase amplification, the present approach does not require electrode functionalization by a DNA probe. Finally, on account of the relative insensitivity of electrochemical methods to downscaling, the detection scheme is quite promising for use in miniaturized devices and in the development of point-of-care diagnosis applications.

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

In 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 H(2)O(2) as substrate and the single electron donor [Os(III)(bpy)(2)pyCl](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 [Os(II)(bpy)(2)pyCl](+), thus allowing one to detect as low as 30 pM of amplified HCMV DNA fragment.

Fabrication of submicrometer-sized gold electrodes of controlled geometry for scanning electrochemical-atomic force microscopy.

A method for fabricating submicrometer-sized gold electrodes of conical or spherical geometry is described. By generating an electric arc between an etched gold microwire and a tungsten counter electrode, the very end of the gold microwire can be melted and given an overall spherical or conical shape a few hundred nanometers in size. The whole wire is subsequently insulated via the cathodic deposition of electrophoretic paint. By applying a high-voltage pulse to the microwire, the film covering its very end can then be selectively removed, thus exposing a submicrometer-sized electrode surface of predefined geometry. The selective exposure of the preformed end of the microwire is demonstrated by cyclic voltammetry, scanning electron microscopy, and metal electrodeposition experiments. The electrophoretic paint coating provides a low-capacitance, robust insulating film allowing exploration of a very wide potential window in aqueous solution. The submicrometer-sized electrodes can easily be turned into probes suitable for combined scanning electrochemical-atomic force microscopy by bending and flattening the gold microwire so that the tip is borne by a flexible enough arm. The good agreement between theoretical and experimental scanning electrochemical microscopy approach curves thus obtained confirms that only the very end of the tip, of predefined geometry, is exposed to the solution.