A Unified Charge Storage Mechanism to Rationalize the Electrochemical Behavior of Quinone-Based Organic Electrodes in Aqueous Rechargeable Batteries.

Due to their eco-sustainability and versatility, organic electrodes are promising candidates for large-scale energy storage in rechargeable aqueous batteries. This is notably the case of aqueous hybrid batteries that pair the low voltage of a zinc anode with the high voltage of a quinone-based (or analogue of quinone-based) organic cathode. However, the mechanisms governing their charge-discharge cycles remain poorly understood and are even a matter of debate and controversy. No consensus exists on the charge carrier in mild aqueous electrolytes, especially when working in an electrolyte containing a multivalent metal cation such as Zn2+. In this study, we comprehensively investigate the electrochemical reactivity of two model quinones, chloranil, and duroquinone, either diluted in solution or incorporated into carbon-based composite electrodes. We demonstrate that a common nine-member square scheme proton-coupled electron transfer mechanism allows us to fully describe and rationalize their electrochemical behavior in relation to the pH and chemical composition of the aqueous electrolyte. Additionally, we highlight the crucial role played by the pKas associated with the reduced states of quinones in determining the nature of the charge carrier that compensates for the negative charges reversibly injected in the active material. Finally, contrary to the widely reported findings for Zn/organic batteries, we unequivocally establish that the predominant solid-state charge carriers in Zn2+-based mild aqueous electrolytes are not multivalent Zn2+ cations but rather protons supplied by the weakly acidic hexaaqua metal ions (i.e., [Zn(H2O)6]2+]).

The Role of Al3+ -Based Aqueous Electrolytes in the Charge Storage Mechanism of MnOx Cathodes.

Rechargeable aqueous aluminium batteries are the subject of growing interest, however, the charge storage mechanisms at manganese oxide-based cathodes remain poorly understood. In essense, every study proposes a different mechanism. Here, an in situ spectroelectrochemical methodology is used to unambiguously demonstrate that reversible proton-coupled MnO2 -to-Mn2+ conversion is the main charge storage mechanism occurring at MnO2 cathodes for a range of slightly acidic Al3+ -based aqueous electrolytes, with the Al3+ hexaaquo complex playing the key role of proton donor. In Zn/MnO2 assemblies, this mechanism is associated with high gravimetric capacities and discharge potentials, up to 560 mAh g-1 and 1.65 V respectively, attractive efficiencies (CE > 99.5% and EE > 82%) and excellent cyclability (almost 100% capacity retention over 1 400 cycles at 2 A g-1 ). Finally, a critical analysis of the data previously published on MnOx cathodes in Al3+ -based aqueous electrolytes is conducted to conclude on a universal charge storage mechanism, i.e., the reversible electrodissolution/electrodeposition of MnO2 .

Specific Versus Non-specific Response in Exponential Molecular Amplification from Cross-Catalysis: Modeling the Influence of Background Amplifications on the Analytical Performances.

Molecule based signal amplifications relying on an autocatalytic process may represent an ideal strategy for the development of ultrasensitive analytical or bioanalytical assays, the main reason being the exponential nature of the amplification. However, to take full advantage of such amplification rates, high stability of the starting co-reactants is required in order to avoid any undesirable background amplification. Here, on the basis of a simple kinetic model of cross-catalysis including a certain degree of intrinsic instability of co-reactants, we highlight the key parameters governing the analytical response of the system and discuss the analytical performances that are expected from a given kinetic set. In particular, we show how the detection limit is directly related to the relative instability of reactants within each catalytic loop. The model is validated with an experimental dataset and is intended to serve as a guide in the design and optimization of autocatalytic molecular-based amplification systems with improved analytical performances.

Exponential Molecular Amplification by H2 O2 -Mediated Autocatalytic Deprotection of Boronic Ester Probes to Redox Cyclers.

Herein, a new molecular autocatalytic reaction scheme based on a H2 O2 -mediated deprotection of a boronate ester probe into a redox cycling compound is described, generating an exponential signal gain in the presence of O2 and a reducing agent or enzyme. For such a purpose, new chemosensing probes built around a naphthoquinone/naphthohydroquinone redox-active core, masked by a self-immolative boronic ester protecting group, were designed. With these probes, typical autocatalytic kinetic traces with characteristic lags and exponential phases were obtained by using either UV/Visible or fluorescence optical detection, or by using electrochemical monitoring. Detection of concentrations as low as 0.5 µm H2 O2 and 0.5 nm of a naphthoquinone derivative were achieved in a relatively short time (<1 h). From kinetic analysis of the two cross-activated catalytic loops associated with the autocatalysis, the key parameters governing the autocatalytic reaction network were determined, indirectly showing that the analytical performances are currently limited by the slow nonspecific self-deprotection of boronate probes. Collectively, the present results demonstrate the potential of this new exponential molecular amplification strategy, which, owing to its generic nature and modularity, is quite promising for coupling to a wide range of bioassays involving H2 O2 or redox cycling compounds, or for use as a new building block in the development of more complex chemical reaction networks.

Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3'- versus 5'-End Sequence Modification.

Nucleic acid aptamers are involved in a broad field of applications ranging from therapeutics to analytics. Deciphering the binding mechanisms between aptamers and small ligands is therefore crucial to improve and optimize existing applications and to develop new ones. Particularly interesting is the enantiospecific binding mechanism involving small molecules with nonprestructured aptamers. One archetypal example is the chiral binding between l-tyrosinamide and its 49-mer aptamer for which neither structural nor mechanistic information is available. In the present work, we have taken advantage of a multiple analytical characterization strategy (i.e., using electroanalytical techniques such as kinetic rotating droplet electrochemistry, fluorescence polarization, isothermal titration calorimetry, and quartz crystal microbalance) for interpreting the nature of binding process. Screening of the binding thermodynamics and kinetics with a wide range of aptamer sequences revealed the lack of symmetry between the two ends of the 23-mer minimal binding sequence, showing an unprecedented influence of the 5' aptamer modification on the bimolecular binding rate constant kon and no significant effect on the dissociation rate constant koff. The results we have obtained lead us to conclude that the enantiospecific binding reaction occurs through an induced-fit mechanism, wherein the ligand promotes a primary nucleation binding step near the 5'-end of the aptamer followed by a directional folding of the aptamer around its target from 5'-end to 3'-end. Functionalization of the 5'-end position by a chemical label, a polydA tail, a protein, or a surface influences the kinetic/thermodynamic constants up to 2 orders of magnitude in the extreme case of a surface immobilized aptamer, while significantly weaker effect is observed for a 3'-end modification. The reason is that steric hindrance must be overcome to nucleate the binding complex in the presence of a modification near the nucleation site.

Real-time electrochemical LAMP: a rational comparative study of different DNA intercalating and non-intercalating redox probes.

We present a comparative study of ten redox-active probes for use in real-time electrochemical loop-mediated isothermal amplification (LAMP). Our main objectives were to establish the criteria that need to be fulfilled for minimizing some of the current limitations of the technique and to provide future guidelines in the search for ideal redox reporters. To ensure a reliable comparative study, each redox probe was tested under similar conditions using the same LAMP reaction and the same entirely automatized custom-made real-time electrochemical device (designed for electrochemically monitoring in real-time and in parallel up to 48 LAMP samples). Electrochemical melt curve analyses were recorded immediately at the end of each LAMP reaction. Our results show that there are a number of intercalating and non-intercalating redox compounds suitable for real-time electrochemical LAMP and that the best candidates are those able to intercalate strongly into ds-DNA but not too much to avoid inhibition of the LAMP reaction. The strongest intercalating redox probes were finally shown to provide higher LAMP sensitivity, speed, greater signal amplitude, and cleaner-cut DNA melting curves than the non-intercalating molecules.

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.

Unraveling the charge transfer/electron transport in mesoporous semiconductive TiO2 films by voltabsorptometry.

In this work, we demonstrate that chronoabsorptometry and more specifically cyclic voltabsorptometry are particularly well suited techniques for acquiring a comprehensive understanding of the dynamics of electron transfer/charge transport within a transparent mesoporous semiconductive metal oxide film loaded with a redox-active dye. This is illustrated with the quantitative analysis of the spectroelectrochemical responses of two distinct heme-based redox probes adsorbed in highly-ordered mesoporous TiO2 thin films (prepared from evaporation-induced self-assembly, EISA). On the basis of a finite linear diffusion-reaction model as well as the establishment of the analytical expressions governing the limiting cases, it was possible to quantitatively analyse, predict and interpret the unusual voltabsorptometric responses of the adsorbed redox species as a function of the potential applied to the semiconductive film (i.e., as a function of the transition from an insulating to a conductive state or vice versa). In particular, we were able to accurately determine the interfacial charge transfer rates between the adsorbed redox species and the porous semiconductor. Another important and unexpected finding, inferred from the voltabsorptograms, is an interfacial electron transfer process predominantly governed by the extended conduction band states of the EISA TiO2 film and not by the localized traps in the bandgap. This is a significant result that contrasts those previously observed for dye-sensitized solar cells formed of randomly sintered TiO2 nanoparticles, a behaviour that was ascribed to a particularly low density of localized surface states in EISA TiO2. The present methodology also provides a unique and straightforward access to an activation-driving force relationship according to the Marcus theory, thus opening new opportunities not only to investigate the driving-force effects on electron recombination dynamics in dye-sensitized solar cells but also to study the electron transfer/transport mechanisms in heterogeneous photoelectrocatalytic systems combining nanostructured semiconductor electrodes and heterogeneous redox-active catalysts.

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 (Kb = 2.4 × 10(10) M(-1)) and association rate (kon = 2 × 10(6) 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.

Rational design of a redox-labeled chiral target for an enantioselective aptamer-based electrochemical binding assay.

A series of redox-labeled L-tyrosinamide (L-Tym) derivatives was prepared and the nature of the functional group and the chain length of the spacer were systematically varied in a step-by-step affinity optimization process of the tracer for the L-Tym aptamer. The choice of the labeling position on L-Tym proved to be crucial for the molecular recognition event, which could be monitored by cyclic voltammetry and is based on the different diffusion rates of free and bound targets in solution. From this screening approach an efficient electroactive tracer emerged. Comparable dissociation constants Kd were obtained for the unlabeled and labeled targets in direct or competitive binding assays. The enantiomeric tracer was prepared and its enantioselective recognition by the corresponding anti-D-Tym aptamer was demonstrated. The access to both enantiomeric tracer molecules opens the door for the development of one-pot determination of the enantiomeric excess when using different labels with well-separated redox potentials for each enantiomer.

Multivalent-Ion versus Proton Insertion into Nanostructured Electrochromic WO3 from Mild Aqueous Electrolytes.

Mild aqueous electrolytes containing multivalent metal salts are currently scrutinized for the development of ecosustainable energy-related devices. However, the role of soluble multivalent metal ions in the electrochemical reactivity of transition metal oxides is a matter of debate, especially when they are performed in protic aqueous electrolytes. Here, we have compared, by means of (spectro)electrochemistry, the reversible electrochromic reduction of transparent nanostructured γ-WO3 thin films in mild aqueous electrolytes of various chemical composition and pH. This study reveals that reversible proton insertion is the only charge storage mechanism over a large pH range and that it is effective for aqueous electrolytes prepared from either organic (such as acetic acid) or inorganic (such as solvated multivalent cations) Bro̷nsted acids. By refuting charge storage mechanisms relying on the reversible insertion of multivalent metal ions, notably in aqueous electrolytes based on Al3+ ions or a mixture of Al3+ and Zn2+ ions, these fundamental results pave the way for the rational development of electrolytes and active materials for a range of aqueous-based devices, such as the emerging concept of an energy-saving smart window, which we also address in this study.

Innovative Energy Storage Smart Windows Relying on Mild Aqueous Zn/MnO2 Battery Chemistry.

Rechargeable mild aqueous Zn/MnO2 batteries are currently attracting great interest thanks to their appealing performance/cost ratio. Their operating principle relies on two complementary reversible electrodeposition reactions at the anode and cathode. Transposing this operating principle to transparent conductive windows remains an unexplored facet of this battery chemistry, which is proposed here to address with the development of an innovative bifunctional smart window, combining electrochromic and charge storage properties. The proof-of-concept of such bifunctional Zn/MnO2 smart window is provided using a mild buffered aqueous electrolyte and different architectures. To maximize the device's performance, transparent nanostructured ITO cathodes are used to reversibly electrodeposit a high load of MnO2 (up to 555 mA h m-2 with a CE of 99.5% over 200 cycles, allowing to retrieve an energy density as high as 860 mA h m-2 when coupled with a zinc metal frame), while flat transparent FTO anodes are used to reversibly electrodeposit an homogenous coating of zinc metal (up to ≈280 mA h m-2 with a CE > 95% over 50 cycles). The implementation of these two reversible electrodeposition processes in a single smart window has been successfully achieved, leading for the first time to a dual-tinting energy storage smart window with an optimized face-to-face architecture.

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.

Switching on/off the chemisorption of thioctic-based self-assembled monolayers on gold by applying a moderate cathodic/anodic potential.

An in situ 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.

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 O(2), 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 O(2) 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 O(2) 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.

Simple and highly enantioselective electrochemical aptamer-based binding assay for trace detection of chiral compounds.

A new electrochemical methodology is reported for monitoring in homogeneous solution the enantiospecific binding of a small chiral analyte to an aptamer. The principle relies on the difference of diffusion rates between the targeted molecule and the aptamer/target complex, and thus on the ability to more easily electrochemically detect the former over the latter in a homogeneous solution. This electrochemical detection strategy is significant because, in contrast to the common laborious and time-consuming heterogeneous binding approaches, it is based on a simple and fast homogeneous binding assay which does not call for an aptamer conformational change upon ligand binding. The methodology is here exemplified with the specific chiral recognition of trace amounts of l- or d-tyrosinamide by a 49-mer d- or l-deoxyribooligonucleotide receptor. Detection as low as 0.1% of the minor enantiomer in a nonracemic mixture can be achieved in a very short analysis time (<1 min). The assay finally combines numerous attractive features including simplicity, rapidity, low cost, flexibility, low volume samples (few microliters), and homogeneous format.

Spectroelectrochemical characterization of small hemoproteins adsorbed within nanostructured mesoporous ITO electrodes.

3D nanostructured transparent indium tin oxide (ITO) electrodes prepared by glancing angle deposition (GLAD) were used for the spectroelectrochemical characterization of cytochrome c (Cyt c) and neuroglobin (Nb). These small hemoproteins, involved as electron-transfer partners in the prevention of apoptosis, are oppositely charged at physiological pH and can each be adsorbed within the ITO network under different pH conditions. The resulting modified electrodes were investigated by UV-visible absorption spectroscopy coupled with cyclic voltammetry. By using nondenaturating adsorption conditions, we demonstrate that both proteins are capable of direct electron transfer to the conductive ITO surface, sharing apparent standard potentials similar to those reported in solution. Preservation of the 3D protein structure upon adsorption was confirmed by resonance Raman (rR) spectroscopy. Analysis of the derivative cyclic voltabsorptograms (DCVA) monitored either in the Soret or the Q bands at scan rates up to 1 V s(-1) allowed us to investigate direct interfacial electron transfer kinetics. From the DCVA shape and scan rate dependences, we conclude that the interaction of Cyt c with the ITO surface is more specific than Nb, suggesting an oriented adsorption of Cyt c and a random adsorption of Nb on the ITO surface. At the same time, Cyt c appears more sensitive to the experimental adsorption conditions, and complete denaturation of Cyt c may occur as evidenced from cross-correlation of rR spectroscopy and spectroelectrochemistry.

Exponential amplification by redox cross-catalysis and unmasking of doubly protected molecular probes.

The strength of autocatalytic reactions lies in their ability to provide a powerful means of molecular amplification, which can be very useful for improving the analytical performances of a multitude of analytical and bioanalytical methods. However, one of the major difficulties in designing an efficient autocatalytic amplification system is the requirement for reactants that are both highly reactive and chemically stable in order to avoid limitations imposed by undesirable background amplifications. In the present work, we devised a reaction network based on a redox cross-catalysis principle, in which two catalytic loops activate each other. The first loop, catalyzed by H2O2, involves the oxidative deprotection of a naphthylboronate ester probe into a redox-active naphthohydroquinone, which in turn catalyzes the production of H2O2 by redox cycling in the presence of a reducing enzyme/substrate couple. We present here a set of new molecular probes with improved reactivity and stability, resulting in particularly steep sigmoidal kinetic traces and enhanced discrimination between specific and nonspecific responses. This translates into the sensitive detection of H2O2 down to a few nM in less than 10 minutes or a redox cycling compound such as the 2-amino-3-chloro-1,4-naphthoquinone down to 50 pM in less than 30 minutes. The critical reason leading to these remarkably good performances is the extended stability stemming from the double masking of the naphthohydroquinone core by two boronate groups, a counterintuitive strategy if we consider the need for two equivalents of H2O2 for full deprotection. An in-depth study of the mechanism and dynamics of this complex reaction network is conducted in order to better understand, predict and optimize its functioning. From this investigation, the time response as well as detection limit are found to be highly dependent on pH, nature of the buffer, and concentration of the reducing enzyme.

Effect of substrate inhibition and cooperativity on the electrochemical responses of glucose dehydrogenase. Kinetic characterization of wild and mutant types.

Thanks to its insensitivity to dioxygen and to its good catalytic reactivity, and in spite of its poor substrate selectivity, quinoprotein glucose dehydrogenase (PQQ-GDH) plays a prominent role among the redox enzymes that can be used for analytical purposes, such as glucose detection, enzyme-based bioaffinity assays, and the design of biofuel cells. A detailed kinetic analysis of the electrochemical catalytic responses, leading to an unambiguous characterization of each individual steps, seems a priori intractable in view of the interference, on top of the usual ping-pong mechanism, of substrate inhibition and of cooperativity effects between the two identical subunits of the enzyme. Based on simplifications suggested by extended knowledge previously acquired by standard homogeneous kinetics, it is shown that analysis of the catalytic responses obtained by means of electrochemical nondestructive techniques, such as cyclic voltammetry, with ferrocene methanol as a mediator, does allow a full characterization of all individual steps of the catalytic reaction, including substrate inhibition and cooperativity and, thus, allows to decipher the reason that makes the enzyme more efficient when the neighboring subunit is filled with a glucose molecule. As a first practical illustration of this electrochemical approach, comparison of the native enzyme responses with those of a mutant (in which the asparagine amino acid in position 428 has been replaced by a cysteine residue) allowed identification of the elementary steps that makes the mutant type more efficient than the wild type when cooperativity between the two subunits takes place, which is observed at large mediator and substrate concentrations. A route is thus opened to structure-reactivity relationships and therefore to mutagenesis strategies aiming at better performances in terms of catalytic responses and/or substrate selectivity.

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.