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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Electrochemical atomic force microscopy using a tip-attached redox mediator for topographic and functional imaging of nanosystems.

We describe the development of a new type of high-resolution atomic force electrochemical microscopy (AFM-SECM), labeled Tarm (for tip-attached redox mediator)/AFM-SECM, where the redox mediator, a ferrocene (Fc), is tethered to the AFM-SECM probe via nanometer long, flexible polyethylene glycol (PEG) chains. It is demonstrated that the tip-attached ferrocene-labeled PEG chains effectively shuttle electrons between the tip and substrate, thus acting as molecular sensors probing the local electrochemical reactivity of a planar substrate. Moreover the Fc-PEGylated AFM-SECM probes can be used for tapping mode imaging, allowing simultaneous recording of electrochemical feedback current and of topography, with a vertical and a lateral resolution in the nanometer range.

Electrochemical atomic-force microscopy using a tip-attached redox mediator. Proof-of-concept and perspectives for functional probing of nanosystems.

This paper presents the first steps toward the development of a new type of high-resolution AFM-SECM microscopy which relies on the use of tip-attached redox-labeled polymer chains as mediators to probe the local electrochemical reactivity of a planar substrate at the nanoscale. Submicrometer-sized combined gold AFM-SECM probes were functionalized by linear, nanometer-sized, flexible, PEG3400 chains bearing a ferrocene (Fc) redox label at their free end. Analysis of the force and current approach curves recorded when such Fc-PEGylated probes (tips) were approached to a bare gold substrate allowed the presence of the Fc-PEG chains at the very tip end of the combined probes to be specifically demonstrated.

Electron transport by molecular motion of redox-DNA strands: unexpectedly slow rotational dynamics of 20-mer ds-DNA chains end-grafted onto surfaces via C6 linkers.

The dynamics of electron transport within molecular layers of 3'-ferrocenylated 20-mer oligonucleotide, 5'-thiol end-grafted onto gold electrode surfaces via a six-carbon (C6) linker, is studied by cyclic voltammetry. Single-stranded Fc-DNA layers are observed to behave as diffusionless systems reflecting the rapid dynamics of the ssDNA strand. Following hybridization, the Fc-dsDNA-C6 layers give rise to a characteristic cyclic voltammetry behavior evidencing that the Fc head is animated by a purely diffusional motion, which is ascribed to free rotation of the rigid DNA duplex around its C6 anchoring linker.

Exploring the motional dynamics of end-grafted DNA oligonucleotides by in situ electrochemical atomic force microscopy.

We introduce herein the use of atomic-force electrochemical microscopy (AFM-SECM) to simultaneously probe locally the conformation and motional dynamics of nanometer-sized single-stranded (ss) and double-stranded (ds) DNA oligonucleotides end-tethered to electrode surfaces. The ss-DNA system studied here consists of a low-density monolayer of (dT)20 oligonucleotides, 5'-thiol end-tethered onto a flat gold surface via a C6 alkyl linker and bearing at their free 3'-end a redox ferrocene label. It is shown that, as a result of the flexibility of the relatively long C6 linker, hinge motion, rather than elastic deformation of the DNA chain, is the major component of the dynamics of both the (dT)20 strand and its post-hybridized (dT-dA)20 duplex.

Accessing the dynamics of end-grafted flexible polymer chains by atomic force-electrochemical microscopy. Theoretical modeling of the approach curves by the elastic bounded diffusion model and Monte Carlo simulations. Evidence for compression-induced lateral chain escape.

The dynamics of a molecular layer of linear poly(ethylene glycol) (PEG) chains of molecular weight 3400, bearing at one end a ferrocene (Fc) label and thiol end-grafted at a low surface coverage onto a gold substrate, is probed using combined atomic force-electrochemical microscopy (AFM-SECM), at the scale of approximately 100 molecules. Force and current approach curves are simultaneously recorded as a force-sensing microelectrode (tip) is inserted within the approximately 10 nm thick, redox labeled, PEG chain layer. Whereas the force approach curve gives access to the structure of the compressed PEG layer, the tip-current, resulting from tip-to-substrate redox cycling of the Fc head of the chain, is controlled by chain dynamics.

Dynamics of electron transport by elastic bending of short DNA duplexes. Experimental study and quantitative modeling of the cyclic voltammetric behavior of 3'-ferrocenyl DNA end-grafted on gold.

The dynamics of electron transport within a molecular monolayer of 3'-ferrocenylated-(dT)(20) strands, 5'-thiol end-grafted onto gold electrode surfaces via a short C2-alkyl linker, is analyzed using cyclic voltammetry as the excitation/measurement technique. It is shown that the single-stranded DNA layer behaves as a diffusionless system, due to the high flexibility of the ss-DNA chain. Upon hybridization by the fully complementary (dA)(20) target, the DNA-modified gold electrode displays a highly unusual voltammetric behavior, the faradaic signal even ultimately switching off at a high enough potential scan rate.

Probing the structure and dynamics of end-grafted flexible polymer chain layers by combined atomic force-electrochemical microscopy. Cyclic voltammetry within nanometer-thick macromolecular poly(ethylene glycol) layers.

The combined atomic force-electrochemical microscopy (AFM-SECM) technique was used in aqueous solution to determine both the static and dynamical properties of nanometer-thick monolayers of poly(ethylene glycol) (PEG) chains end-grafted to a gold substrate surface. Approach of a microelectrode tip from a redox end-labeled PEG layer triggered a tip-to-substrate cycling motion of the chains' free ends as a result of the redox heads' oxidation at the tip and re-reduction at the substrate surface. As few as approximately 200 chains at a time could be addressed in such a way.

3'-Ferrocene-labeled oligonucleotide chains end-tethered to gold electrode surfaces: novel model systems for exploring flexibility of short DNA using cyclic voltammetry.

The flexibility of DNA is of central importance in biology, medicine, materials science, and mechanical engineering. In this study, we report an unprecedented electrochemical approach for investigating the flexibility of a short (typically 20-base), surface end-tethered single-stranded synthetic DNA oligonucleotide and of its postformed DNA duplex, taking as an example the homopolymer (dT)20 sequence in the regime of very high ionic strength ( approximately 1 M).