Activationless Electron Transfer of Redox-DNA in Electrochemical Nanogaps.

Our recent discovery of decreased reorganization energy in electrode-tethered redox-DNA systems prompts inquiries into the origin of this phenomenon and suggests its potential use to lower the activation energy of electrochemical reactions. Here, we show that the confinement of the DNA chain in a nanogap amplifies this effect to an extent to which it nearly abolishes the intrinsic activation energy of electron transfer. Employing electrochemical atomic force microscopy (AFM-SECM), we create sub-10 nm nanogaps between a planar electrode surface bearing end-anchored ferrocenylated DNA chains and an incoming microelectrode tip. The redox cycling of the DNA's ferrocenyl (Fc) moiety between the surface and the tip generates a measurable current at the scale of ∼10 molecules. Our experimental findings are rigorously interpreted through theoretical modeling and original molecular dynamics simulations (Q-Biol code). Several intriguing findings emerge from our investigation: (i) The electron transport resulting from DNA dynamics is many times faster than predicted by simple diffusion considerations. (ii) The current in the nanogap is solely governed by the electron transfer rate at the electrodes. (iii) This rate rapidly saturates as overpotentials applied to the nanogap electrodes increase, implying near-complete suppression of the reorganization energy for the oxidation/reduction of the Fc heads within confined DNA. Furthermore, evidence is presented that this may constitute a general, previously unforeseen, behavior of redox polymer chains in electrochemical nanogaps.

Ballistic Brownian Motion of Nanoconfined DNA.

Theoretical treatments of polymer dynamics in liquid generally start with the basic assumption that motion at the smallest scale is heavily overdamped; therefore, inertia can be neglected. We report on the Brownian motion of tethered DNA under nanoconfinement, which was analyzed by molecular dynamics simulation and nanoelectrochemistry-based single-electron shuttle experiments. Our results show a transition into the ballistic Brownian motion regime for short DNA in sub-5 nm gaps, with quality coefficients as high as 2 for double-stranded DNA, an effect mainly attributed to a drastic increase in stiffness. The possibility for DNA to enter the underdamped regime could have profound implications on our understanding of the energetics of biomolecular engines such as the replication machinery, which operates in nanocavities that are a few nanometers wide.

Electrochemical Shot Noise of a Redox Monolayer.

Redox monolayers are the base for a wide variety of devices including high-frequency molecular diodes or biomolecular sensors. We introduce a formalism to describe the electrochemical shot noise of such a monolayer, confirmed experimentally at room temperature in liquid. The proposed method, carried out at equilibrium, avoids parasitic capacitance, increases the sensitivity, and allows us to obtain quantitative information such as the electronic coupling (or standard electron transfer rates), its dispersion, and the number of molecules. Unlike in solid-state physics, the homogeneity in energy levels and transfer rates in the monolayer yields a Lorentzian spectrum. This first step for shot noise studies in molecular electrochemical systems opens perspectives for quantum transport studies in a liquid environment at room temperature as well as highly sensitive measurements for bioelectrochemical sensors.

Electrochemical response of surface-attached redox DNA governed by low activation energy electron transfer kinetics.

The mechanism responsible for electron transport within layers of redox DNA anchored to electrodes has been extensively studied over the last twenty years, but remains controversial. Herein, we thoroughly study the electrochemical behavior of a series of short, model, ferrocene (Fc) end-labeled dT oligonucleotides, terminally attached to gold electrodes, using high scan rate cyclic voltammetry complemented by molecular dynamics simulations. We evidence that the electrochemical response of both single-stranded and duplexed oligonucleotides is controlled by the electron transfer kinetics at the electrode, obeying Marcus theory, but with reorganization energies considerably lowered by the attachment of the ferrocene to the electrode via the DNA chain. This so far unreported effect, that we attribute to a slower relaxation of water around Fc, uniquely shapes the electrochemical response of Fc-DNA strands and, being markedly dissimilar for single-stranded and duplexed DNA, contributes to the signaling mechanism of E-DNA sensors.

Enzymatic activity of individual bioelectrocatalytic viral nanoparticles: dependence of catalysis on the viral scaffold and its length.

The enzymatic activity of tobacco mosaic virus (TMV) nanorod particles decorated with an integrated electro-catalytic system, comprising the quinoprotein glucose-dehydrogenase (PQQ-GDH) enzyme and ferrocenylated PEG chains as redox mediators, is probed at the individual virion scale by atomic force microscopy-scanning electrochemical atomic force microscopy (AFM-SECM). A marked dependence of the catalytic activity on the particle length is observed. This finding can be explained by electron propagation along the viral backbone, resulting from electron exchange between ferrocene moieties, coupled with enzymatic catalysis. Thus, the use of a simple 1D diffusion/reaction model allows the determination of the kinetic parameters of the virus-supported enzyme. Comparative analysis of the catalytic behavior of the Fc-PEG/PQQ-GDH system assembled on two differing viral scaffolds, TMV (this work) and bacteriophage-fd (previous work), reveals two distinct kinetic effects of scaffolding: An enhancement of catalysis that does not depend on the virus type and a modulation of substrate inhibition that depends on the virus type. AFM-SECM detection of the enzymatic activity of a few tens of PQQ-GDH molecules, decorating a 40 nm-long viral domain, is also demonstrated, a record in terms of the lowest number of enzyme molecules interrogated by an electrochemical imaging technique.

Converting Any Faradaic Current Generated at an Electrode under Potentiostatic Control into a Remote Fluorescence Signal.

We describe the development of an original faradaic current-to-fluorescence conversion scheme. The proposed instrumental strategy consists of coupling the electrochemical reaction of any species at an electrode under potentiostatic control with the fluorescence emission of a species produced at the counter electrode. In order to experimentally validate this scheme, the fluorogenic species resazurin is chosen as a fluorescent reporter molecule, and its complex reduction mechanism is first studied in unprecedented detail. This kinetic study is carried out by recording simultaneous cyclic voltammograms and voltfluorograms at the same electrode. Numerical simulations are used to account for the experimental current and fluorescence signals, to analyze their degree of correlation, and to decipher their relation to resazurin reduction kinetics. It is then shown that, provided that the reduction of resazurin takes place at a micrometer-sized electrode, the fluorescence emission perfectly tracks the faradaic current. By implementing this ideal configuration at the counter electrode of a potentiostatic setup, it is finally demonstrated that the oxidation reaction of a nonfluorescent species at the working electrode can be quantitatively transduced into simultaneous emission of fluorescence.

Redox-Immunofunctionalized Potyvirus Nanoparticles for High-Resolution Imaging by AFM-SECM Correlative Microscopy.

We present in this chapter a new experimental approach allowing the high resolution imaging of immune complexes on virus particles. Combined atomic force-electrochemical microscopy (AFM-SECM) is used to image the presence of ferrocene functionalized specific antibodies on the surface of potyvirus particles. For this purpose, potyviruses, flexuous filamentous phytoviruses with a high aspect ratio, have been chosen. This technique allows analysis of the distribution of antibody labeling over the virus population. But, more importantly, it opens up the imaging of immune complexes decorating a single viral particle. Finally, its high resolution allows the characterization in situ of the ultrastructure of a single immune complex on the particle.

Electrochemical Imaging of Dense Molecular Nanoarrays.

The aim of the present work is to explore the combination of atomic force electrochemical microscopy, operated in molecule touching mode (Mt/AFM-SECM), and of dense nanodot arrays, for designing an electrochemically addressable molecular nanoarray platform. A high density nanoarray of single grained gold nanodots (∼15 nm-diameter nanoparticles, 100 nm pitch) is decorated by a model molecular system, consisting of ferrocene (Fc) labeled polyethylene glycol (PEG) disulfide chains. We show that the high resolution of Mt/AFM-SECM enables the electrochemical interrogation of several hundreds of individual nanodots in a single image acquisition. As a result, the statistical dispersion of the nanodot molecular occupancy by Fc-PEG chains can be reliably quantified, evidencing that as little as a few tens of copies of redox-labeled macromolecules immobilized on individual nanodots can be detected. The electrochemical reactivity of individual nanodots can also be reliably sampled over a large population of nanodots. We evidence that the heterogeneous rate constant characterizing the electron transfer between the nanodots and the Fc heads displays some quantifiable variability but that the electron transfer remains in any case in the quasi-reversible regime. Overall, we demonstrate that Mt/AFM-SECM enables high throughput reading of dense nanoarrays, with a sensitivity and a read-out speed considerably higher than previously reported for scanning electrochemical microscopy (SECM) imaging of molecular microarrays.

The Potyvirus Particle Recruits the Plant Translation Initiation Factor eIF4E by Means of the VPg covalently Linked to the Viral RNA.

The viral protein genome-linked (VPg) of potyviruses is a protein covalently linked to the 5' end of viral RNA. It interacts with eIF4E, a component of the cellular translation initiation complex. It has been suggested that the 5' RNA-linked VPg could mimic the cellular mRNA cap, promoting synthesis of viral proteins. Here, we report evidence for recruitment of the plant eIF4E by Lettuce mosaic virus (LMV, potyvirus) particles via the 5' RNA-linked VPg. Analysis of the viral population was performed by enzyme-linked immunosorbent assay-based tests, either with crude extracts of LMV-infected tissues or purified viral particles. In both cases, LMV-VPg and LMV-eIF4E subpopulations could be detected. After reaching a maximum within the first 2 weeks postinoculation, these populations decreased and very few labeled particles were found later than 3 weeks postinoculation. The central domain of VPg (CD-VPg) was found to be exposed at the surface of the particles. Using a purified recombinant lettuce eIF4E and CD-VPg-specific antibodies, we demonstrate that the plant factor binds to the VPg via its central domain. Moreover, the plant eIF4E factor could be imaged at one end of the particles purified from LMV plant extracts, by immunoredox atomic force microscopy coupled to scanning electrochemical microscopy. We discuss the biological significance of these results.

Scaffolding of Enzymes on Virus Nanoarrays: Effects of Confinement and Virus Organization on Biocatalysis.

Organizing active enzyme molecules on nanometer-sized scaffolds is a promising strategy for designing highly efficient supported catalytic systems for biosynthetic and sensing applications. This is achieved by designing model nanoscale enzymatic platforms followed by thorough analysis of the catalytic activity. Herein, the virus fd bacteriophage is considered as an enzyme nanocarrier to study the scaffolding effects on enzymatic activity. Nanoarrays of randomly oriented, or directionally patterned, fd bacteriophage virus are functionalized with the enzyme glucose oxidase (GOx), using an immunological assembly strategy, directly on a gold electrode support. The scaffolding process on the virus capsid is monitored in situ by AFM (atomic force microscopy) imaging, while cyclic voltammetry is used to interrogate the catalytic activity of the resulting functional GOx-fd nanoarrays. Kinetic analysis reveals the ability to modulate the activity of GOx via nanocarrier patterning. The results evidence, for the first time, enhancement of the enzymatic activity due to scaffolding on a filamentous viral particle.

Electrochemical atomic force microscopy imaging of redox-immunomarked proteins on native potyviruses: from subparticle to single-protein resolution.

We show herein that electrochemical atomic force microscopy (AFM-SECM), operated in molecule touching (Mt) mode and combined with redox immunomarking, enables the in situ mapping of the distribution of proteins on individual virus particles and makes localization of individual viral proteins possible. Acquisition of a topography image allows isolated virus particles to be identified and structurally characterized, while simultaneous acquisition of a current image allows the sought after protein, marked by redox antibodies, to be selectively located. We concomitantly show that Mt/AFM-SECM, due to its single-particle resolution, can also uniquely reveal the way redox functionalization endowed to viral particles is distributed both statistically among the viruses and spatially over individual virus particles. This possibility makes Mt/AFM-SECM a unique tool for viral nanotechnology.

Probing the conformation and 2D-distribution of pyrene-terminated redox-labeled poly(ethylene glycol) chains end-adsorbed on HOPG using cyclic voltammetry and atomic force electrochemical microscopy.

The present paper aims at illustrating how end-attachment of water-soluble flexible chains bearing a terminal functional group onto graphene-like surfaces has to be carefully tuned to ensure the proper positioning of the functional moiety with respect to the anchoring surface. The model experimental system considered here consists of a layer of poly(ethylene glycol) (PEG) chains, bearing an adsorbing pyrene foot and a ferrocene (Fc) redox functional head, self-assembled onto highly oriented pyrolytic graphite (HOPG). Cyclic voltammetry is used to accurately measure the chain coverage and gain insights into the microenvironment experienced by the Fc heads. Molecule-touching atomic force electrochemical microscopy (Mt/AFM-SECM) is used to simultaneously probe the chain conformation and the position of the Fc heads within the layer, and also to map the 2D-distribution of the chains over the surface. This multiscale electrochemical approach allows us to show that whereas Fc-PEG-pyrene readily self-assembles to form extremely homogeneous layers, the strongly hydrophobic nature of graphite planes results in a complex coverage-dependent structure of the PEG layer due to the interaction of the ferrocene label with the HOPG surface. It is shown that, even though pyrene is known to adsorb particularly strongly onto HOPG, the more weakly adsorbing terminal ferrocene can also act as the chain anchoring moiety especially at low coverage. However we show that beyond a critical coverage value the Fc-PEG-pyrene chains adopt an ideal "foot-on" end-attached conformation allowing the Fc head to explore a volume away from the surface solely limited by the PEG chain elasticity.

Probing individual redox PEGylated gold nanoparticles by electrochemical--atomic force microscopy.

Electrochemical-atomic force microscopy (AFM-SECM) was used to simultaneously probe the physical and electrochemical properties of individual ~20 nm sized gold nanoparticles functionalized by redox-labeled PEG chains. The redox PEGylated nanoparticles were assembled onto a gold electrode surface, forming a random nanoarray, and interrogated in situ by a combined AFM-SECM nanoelectrode probe. We show that, in this so-called mediator-tethered (Mt) mode, AFM-SECM affords the nanometer resolution required for resolving the position of individual nanoparticles and measuring their size, while simultaneously electrochemically directly contacting the redox-PEG chains they bear. The dual measurement of the size and current response of single nanoparticles uniquely allows the statistical distribution in grafting density of PEG on the nanoparticles to be determined and correlated to the nanoparticle diameter. Moreover, because of its high spatial resolution, Mt/AFM-SECM allows "visualizing" simultaneously but independently the PEG corona and the gold core of individual nanoparticles. Beyond demonstrating the achievement of single-nanoparticle resolution using an electrochemical microscopy technique, the results reported here also pave the way toward using Mt/AFM-SECM for imaging nano-objects bearing any kind of suitably redox-labeled (bio)macromolecules.

Nano-Electrochemistry and Nano-Electrografting with an Original Combined AFM-SECM.

This study demonstrates the advantages of the combination between atomic force microscopy and scanning electrochemical microscopy. The combined technique can perform nano-electrochemical measurements onto agarose surface and nano-electrografting of non-conducting polymers onto conducting surfaces. This work was achieved by manufacturing an original Atomic Force Microscopy-Scanning ElectroChemical Microscopy (AFM-SECM) electrode. The capabilities of the AFM-SECM-electrode were tested with the nano-electrografting of vinylic monomers initiated by aryl diazonium salts. Nano-electrochemical and technical processes were thoroughly described, so as to allow experiments reproducing. A plausible explanation of chemical and electrochemical mechanisms, leading to the nano-grafting process, was reported. This combined technique represents the first step towards improved nano-processes for the nano-electrografting.

Kinetics of enzyme action on surface-attached substrates: a practical guide to progress curve analysis in any kinetic situation.

In the present work, exact kinetic equations describing the action of an enzyme in solution on a substrate attached to a surface have been derived in the framework of the Michaelis-Menten mechanism but without resorting to the often-used steady-state approximation. The here-derived kinetic equations are cast in a workable format, allowing us to introduce a simple and universal procedure for the quantitative analysis of enzyme surface kinetics that is valid for any kinetic situation. The results presented here should allow experimentalists studying the kinetics of enzyme action on immobilized substrates to analyze their data in a perfectly rigorous way.

Optimizing electrode-attached redox-peptide systems for kinetic characterization of protease action on immobilized substrates. Observation of dissimilar behavior of trypsin and thrombin enzymes.

In this work, we experimentally address the issue of optimizing gold electrode attached ferrocene (Fc)-peptide systems for kinetic measurements of protease action. Considering human α-thrombin and bovine trypsin as proteases of interest, we show that the recurring problem of incomplete cleavage of the peptide layer by these enzymes can be solved by using ultraflat template-stripped gold, instead of polished polycrystalline gold, as the Fc-peptide bearing electrode material. We describe how these fragile surfaces can be mounted in a rotating disk configuration so that enzyme mass transfer no longer limits the overall measured cleavage kinetics. Finally, we demonstrate that, once the system has been optimized, in situ real-time cyclic voltammetry monitoring of the protease action can yield high-quality kinetic data, showing no sign of interfering effects. The cleavage progress curves then closely match the Langmuirian variation expected for a kinetically controlled surface process. Global fit of the progress curves yield accurate values of the peptide cleavage rate for both trypsin and thrombin. It is shown that, whereas trypsin action on the surface-attached peptide closely follows Michaelis-Menten kinetics, thrombin displays a specific and unexpected behavior characterized by a nearly enzyme-concentration-independent cleavage rate in the subnanomolar enzyme concentration range. The reason for this behavior has still to be clarified, but its occurrence may limit the sensitivity of thrombin sensors based on Fc-peptide layers.

High-resolution mapping of redox-immunomarked proteins using electrochemical-atomic force microscopy in molecule touching mode.

We explore the possibility of using molecule touching atomic force electrochemical microcopy (Mt/AFM-SECM) for high-resolution mapping of proteins on conducting surfaces. The proposed imaging strategy relies on making surface-immobilized proteins electrochemically "visible" via redox-immunomarking by specific antibodies conjugated to poly(ethylene glycol) (PEG) chains terminated by redox ferrocene (Fc) heads. The flexibility and length of the PEG chains are such that, upon approaching a combined AFM-SECM microelectrode tip toward the surface, the Fc moieties can efficiently shuttle electrons from the surface to the tip. The so-generated SECM positive feedback tip current allows the specific localized detection of the sought protein molecules on the surface. This new electrochemical imaging scheme is validated experimentally on the basis of a model system consisting of mouse IgGs adsorbed onto electrode surfaces and recognized by Fc-PEG-labeled antimouse antibodies. In order to estimate the resolution of Mt/AFM-SECM for protein imaging, regular arrays of submicrometer-sized spots of mouse IgGs are fabricated onto gold electrode surfaces using particle lithography. The Fc-PEG-immunomarked mouse IgG spots are imaged by Mt/AFM-SECM operated in tapping mode. Both an electrochemical image, reflecting the surface distribution of the redox-labeled IgGs, and a topography image are then simultaneously and independently acquired, with a demonstrated resolution in the ~100 nm range. The strength of Mt/AFM-SECM imaging is to combine the nanometric resolution of AFM with the selectivity of the electrochemical detection, potentially allowing individual target proteins to be identified amidst similarly sized "nano objects" present on a conducting surface.

Optimized hand fabricated AFM probes for simultaneous topographical and electrochemical tapping mode imaging.

In this work hybrid AFM-electrochemical (SECM) probes to be used in dynamic atomic force microscopy are presented. These nanosensors are hand fabricated from gold microwires using a simple benchtop method. They display proportions close to commercially available silicon and silicon nitride cantilevers giving comparable performance in terms of resolution and imaging stability. The remarkable characteristic of these hybrid nanosensors is that they allow the coupling of 3D imaging ability and versatility of atomic force microscopy with the power of electrochemical methods. Local measurement of electrochemical-activity of a test sample consisting of gold bands functionalized by redox-labeled nanometer-sized polyethylene glycol chains has been achieved with simultaneous imaging of the 3D surface topography at high resolution. These hybrid AFM-SECM tips are capable of sensing local electrochemical currents down to ∼ 10 fA emphasizing the sensitivity and resolution of this technique.

Touching surface-attached molecules with a microelectrode: mapping the distribution of redox-labeled macromolecules by electrochemical-atomic force microscopy.

We report on the development of a mediator-free electrochemical-atomic force microscopy (AFM-SECM) technique designed for high-resolution imaging of molecular layers of nanometer-sized redox-labeled (macro)molecules immobilized onto electrode surfaces. This new AFM-SECM imaging technique, we call molecule touching atomic force electrochemical microscopy (Mt/AFM-SECM), is based on the direct contact between surface-anchored molecules and an incoming microelectrode (tip). To validate the working-principle of this microscopy, we consider a model system consisting of a monolayer of nanometer long, flexible, polyethylene glycol (PEG) chains covalently attached by one extremity to a gold surface and bearing at their free end a ferrocene (Fc) redox tag. Using Mt/AFM-SECM in tapping mode, i.e., by oscillating the tip so that it comes in intermittent contact with the grafted chains, we show that the substrate topography and the distribution of the redox-tagged PEG chains immobilized on the gold surface can be simultaneously and independently imaged at the sub-100 nm scale. This novel type of SECM imaging may be found useful for characterizing the surface of advanced biosensors which use electrode-grafted, redox-tagged, linear biochains, such as peptides or DNA chains, as sensing elements. In principle, Mt/AFM-SECM should also permit in situ imaging of the distribution of any kind of macromolecules immobilized on electrode surfaces or simply conducting surfaces, provided they are labeled by a suitable redox tag.

Cleavage-sensing redox peptide monolayers for the rapid measurement of the proteolytic activity of trypsin and alpha-thrombin enzymes.

Ferrocene (Fc)-labeled peptides are end-grafted onto gold electrodes via a flexible polyethylene glycol (PEG) linker, and their ability to act as substrates for proteolytic enzymes trypsin and alpha-thrombin is investigated by cyclic voltammetry. It is shown that whereas a short Fc-tetrapeptide substrate is rapidly cleaved by trypsin, a longer Fc-heptapeptide substrate is required for alpha-thrombin detection. However, in both cases it is observed that not all of the Fc-peptide chains present on the electrode surface are cleavable by the proteases and that the cleavage yield is actually controlled by the surface coverage in the Fc-peptide. Surface dilution of the Fc-peptide using a backfilling molecule such as MCH (6-mercapto-1-hexanol) was required to obtain a cleavage yield larger than 80%. The kinetics of Fc-peptide cleavage by trypsin or alpha-thrombin is then shown to be adequately described by Michaelis Menten kinetics, allowing enzymatic constants k(cat) and K(M) to be determined. The obtained rate constant values showed that the affinity of the enzymes for their respective Fc-peptide substrates is very high (i.e., low K(M) values) whereas that for the cleavage step itself is relatively low (low k(cat) values). Partial compensation of these parameters yields a fast response of the Fc-peptide electrodes to the proteases in solution in the 1-1000 nM range. The type of molecule used to backfill the Fc-peptide layers, either MCH or PEG(6) chains, is shown to modulate the activity of the proteases versus the Fc-peptide layers: in particular, the PEG(6) diluent is specifically shown to decrease the ability of alpha-thrombin to cleave its Fc-peptide substrate whereas trypsin activity is unaffected by the presence of PEG chains.