Efficient Chemisorption of Organophosphorous Redox Probes on Indium Tin Oxide Surfaces under Mild Conditions

We report a mild and straightforward one-step chemical surface functionalization of indium tin oxide (ITO) electrodes by redox-active molecules bearing an organophosphoryl anchoring group (i.e., alkyl phosphate or alkyl phosphonate group). The method takes advantage of simple passive adsorption in an aqueous solution at room temperature. We show that organophosphorus compounds can adsorb much more strongly and stably on an ITO surface than analogous redox-active molecules bearing a carboxylate or a boronate moiety. We provide evidence, through quantitative electrochemical characterization (i.e., by cyclic voltammetry) of the adsorbed organophosphoryl redox-active molecules, of the occurrence of three different adsorbate fractions on ITO, exhibiting different stabilities on the surface. Among these three fractions, one is observed to be strongly chemisorbed, exhibiting high stability and resistance to desorption/hydrolysis in a free-redox probe aqueous buffer. We attribute this remarkable stability to the formation of chemical bonds between the organophosphorus anchoring group and the metal oxide surface, likely occurring through a heterocondensation reaction in water. From XPS analysis, we also demonstrate that the surface coverage of the chemisorbed molecules is highly affected by the degree of surface hydroxylation, a parameter that can be tuned by simply preconditioning the freshly cleaned ITO surfaces in water. The lower the relative surface hydroxide density on ITO, the higher was the surface coverage of the chemisorbed species. This behavior is in line with a chemisorption mechanism involving coordination of a deprotonated phosphoryl oxygen atom to the non-hydroxylated acidic metal sites of ITO

Chronoabsorptometry To Investigate Conduction-Band-Mediated Electron Transfer in Mesoporous TiO₂ Thin Films

Chronoabsorptometry response of transparent mesoporous semiconductive metal oxide film loaded with a redox-active dye is established in the framework of fast electron diffusion. It is shown that the effect of uncompensated resistance is to delay the electron accumulation in the semiconductive thin film. Consequently, this effect must be duly taken into account when interfacial charge transfer rates between the adsorbed redox species and the porous semiconductor are extracted from chronoabsorptometry responses. This is illustrated with the quantitative analysis of the chronoabsorptometry responses of a heme-based redox probe strongly adsorbed in highly ordered mesoporous TiO₂ thin films (prepared from evaporation-induced self-assembly, EISA). It is shown that the reduction of the redox probe originates from the conduction band and not from the localized traps in the bandgap. These results are in quantitative agreement with differential cyclic voltabsorptometry recently reported by us, thus showing full consistency of both methods

Switching On/Off the Chemisorption of Thioctic-Based Self-Assembled Monolayers on Gold by Applying a Moderate Cathodic/Anodic Potential

An <i>in situ</i> and real-time electrochemical method has been devised for quantitatively monitoring the self-assembly of a ferrocene-labeled cyclic disulfide derivative (i.e., a thioctic acid derivative) on a polycrystalline gold electrode under electrode polarization. Taking advantage of the high sensitivity, specificity, accuracy, and temporal resolution of this method, we were able to demonstrate an unexpectedly facilitated formation of the redox-active SAM when the electrode was held at a moderate cathodic potential (−0.4 V vs SCE in CH₃CN), affording a saturated monolayer from only micromolar solutions in less than 10 min, and a totally impeded SAM growth when the electrode was polarized at a slightly anodic potential (+0.5 V vs SCE in CH₃CN). This method literally allows for switching on/off the formation of SAMs under “soft” conditions. Moreover the cyclic disulfide-based SAM was completely desorbed at this potential contrary to the facilitated deposition of a ferrocene-labeled alkanethiol. Such a strikingly contrasting behavior could be explained by an energetically favored release of the thioctic-based SAM through homolytic cleavage of the Au–S bond followed by intramolecular cyclization of the generated thiyl diradicals. Moreover, the absence of a discernible transient faradaic current response during the potential-assisted adsorption/desorption of the redox-labeled cyclic disulfide led us to conclude in a potential-dependent reversible surface reaction where no electron is released or consumed. These results provide new insights into the formation of disulfide-based SAMs on gold but also raise some fundamental questions about the intimate mechanism involved in the facilitated adsorption/desorption of SAMs under electrode polarization. Finally, the possibility to easily and selectively address the formation/removal of thioctic-based SAMs on gold by applying a moderate cathodic/anodic potential offers another degree of freedom in tailoring their properties and in controlling their self-assembly, nanostructuration, and/or release

Kinetic Rotating Droplet Electrochemistry: A Simple and Versatile Method for Reaction Progress Kinetic Analysis in Microliter Volumes

Here, we demonstrate a new generic, affordable, simple, versatile, sensitive, and easy-to-implement electrochemical kinetic method for monitoring, in real time, the progress of a chemical or biological reaction in a microdrop of a few tens of microliters, with a kinetic time resolution of ca. 1 s. The methodology is based on a fast injection and mixing of a reactant solution (1–10 μL) in a reaction droplet (15–50 μL) rapidly rotated over the surface of a nonmoving working electrode and on the recording of the ensuing transient faradaic current associated with the transformation of one of the components. Rapid rotation of the droplet was ensured mechanically by a rotating rod brought in contact atop the droplet. This simple setup makes it possible to mix reactants efficiently and rotate the droplet at a high spin rate, hence generating a well-defined hydrodynamic steady-state convection layer at the underlying stationary electrode. The features afforded by this new kinetic method were investigated for three different reaction schemes: (i) the chemical oxidative deprotection of a boronic ester by H₂O₂, (ii) a biomolecular binding recognition between a small target and an aptamer, and (iii) the inhibition of the redox-mediated catalytic cycle of horseradish peroxidase (HRP) by its substrate H₂O₂. For the small target/aptamer binding reaction, the kinetic and thermodynamic parameters were recovered from rational analysis of the kinetic plots, whereas for the HRP catalytic/inhibition reaction, the experimental amperometric kinetic plots were reproduced from numerical simulations. From the best fits of simulations to the experimental data, the kinetics rate constants primarily associated with the inactivation/reactivation pathways of the enzyme were retrieved. The ability to perform kinetics in microliter-size samples makes this methodology particularly attractive for reactions involving low-abundance or expensive reagents

Ultimate Single-Copy DNA Detection Using Real-Time Electrochemical LAMP

Herein, we report the first successful detection of single DNA copies using real-time electrochemical loop-mediated isothermal amplification (LAMP). Toward this end, real-time electrochemical LAMP amplifications of bacteriophage M13mp18 DNA and of a genomic DNA sequence from the pathogen Flavobacterium columnare were systematically run each with four different redox reporters (i.e., three intercalating reporters with different ds-DNA binding strengths and one nonintercalating probe able to sense the pyrophosphate ion concentration produced during LAMP). In order to provide a reliable, rapid assessment of the LAMP performances, the experiments were carried out using a prototype high-throughput house-built automatized electrochemical read-out device, optimized for parallel monitoring of up to 48 samples. The electrochemical results were then compared to those achieved with conventional real-time fluorescence LAMP and PCR methods (using commercially available fluorescent dyes and standard benchtop equipment). The best electrochemical performances were obtained with the two strongest intercalating redox reporters, affording analytical sensitivity and detection limits comparable to real-time fluorescence LAMP. These electrochemical reporters enabled us to assemble highly accurate, log–linear calibration curves in only 30 min, over a six-decade dynamic concentration range with detection limits as low as two DNA copies of genomic target in 50 μL. These are to our knowledge the best results so far reported for electrochemical-based real-time LAMP, rivaling those achieved with fluorescence-based real-time LAMPs. The major advantages of our electrochemical LAMP approach over the fluorescence-based LAMP method include miniaturization, portability, robustness, low cost, and the ability to work with nontransparent reaction mixtures

Unraveling the Mechanism of Catalytic Reduction of O₂ by Microperoxidase-11 Adsorbed within a Transparent 3D-Nanoporous ITO Film

Nanoporous films of indium tin oxide (ITO), with thicknesses ranging from 250 nm to 2 μm, were prepared by Glancing Angle Deposition (GLAD) and used as highly sensitive transparent 3D-electrodes for quantitatively interrogating, by time-resolved spectroelectrochemistry, the reactivity of microperoxidase-11 (MP-11) adsorbed within such films. The capacitive current densities of these 3D-electrodes as well as the amount of adsorbed MP-11 were shown to be linearly correlated to the GLAD ITO film thickness, indicating a homogeneous distribution of MP-11 across the film as well as homogeneous film porosity. Under saturating adsorption conditions, MP-11 film concentration as high as 60 mM was reached. This is equivalent to a stack of 110 monolayers of MP-11 per micrometer film thickness. This high MP-11 film loading combined with the excellent ITO film conductivity has allowed the simultaneous characterization of the heterogeneous one-electron transfer dynamics of the MP-11 Fe^III/Fe^II redox couple by cyclic voltammetry and cyclic voltabsorptometry, up to a scan rate of few volts per second with a satisfactory single-scan signal-to-noise ratio. The potency of the method to unravel complex redox coupled chemical reactions was also demonstrated with the catalytic reduction of oxygen by MP-11. In the presence of O₂, cross-correlation of electrochemical and spectroscopic data has allowed us to determine the key kinetics and thermodynamics parameters of the redox catalysis that otherwise could not be easily extracted using conventional protein film voltammetry. On the basis of numerical simulations of cyclic voltammograms and voltabsorptograms and within the framework of different plausible catalytic reaction schemes including appropriate approximations, it was shown possible to discriminate between different possible catalytic pathways and to identify the relevant catalytic cycle. In addition, from the best fits of simulations to the experimental voltammograms and voltabsorptograms, the partition coefficient of O₂ for the ITO film as well as the values of two kinetic rate constants could be extracted. It was finally concluded that the catalytic reduction of O₂ by MP-11 adsorbed within nanoporous ITO films occurs via a 2-electron mechanism with the formation of an intermediate Fe^III–OOH adduct characterized by a decay rate of 11 s^–1. The spectroelectroanalytical strategy presented here opens new opportunities for characterizing complex redox-coupled chemical reactions not only with redox proteins, but also with redox biomimetic systems and catalysts. It might also be of great interest for the development and optimization of new spectroelectrochemical sensors and biosensors, or eventually new photoelectrocatalytic systems or biofuel cells

Heterogeneous Reconstitution of the PQQ-Dependent Glucose Dehydrogenase Immobilized on an Electrode: A Sensitive Strategy for PQQ Detection Down to Picomolar Levels

A highly sensitive electroanalytical method for determination of PQQ in solution down to subpicomolar concentrations is proposed. It is based on the heterogeneous reconstitution of the PQQ-dependent glucose dehydrogenase (PQQ-GDH) through the specific binding of its pyrroloquinoline quinone (PQQ) cofactor to the apoenzyme anchored on an electrode surface. It is shown from kinetics analysis of both the enzyme catalytic responses and enzyme surface-reconstitution process (achieved by cyclic voltammetry under redox-mediated catalysis) that the selected immobilization strategy (i.e., through an avidin/biotin linkage) is well-suited to immobilize a nearly saturated apoenzyme monolayer on the electrode surface with an almost fully preserved PQQ binding properties and catalytic activity. From measurement of the overall rate constants controlling the steady-state catalytic current responses of the surface-reconstituted PQQ-GDH and determination of the PQQ equilibrium binding (<i>K</i>_b = 2.4 × 10¹⁰ M^–1) and association rate (<i>k</i>_on = 2 × 10⁶ M^–1 s^–1) constants with the immobilized apoenzyme, the analytical performances of the method could be rationally evaluated, and the signal amplification for PQQ detection down to the picomolar levels is well-predicted. These performances outperform by several orders of magnitude the direct electrochemical detection of PQQ in solution and by 1 to 2 orders the detection limits previously achieved by UV–vis spectroscopic detection of the homogeneous PQQ-GDH reconstitution