Redox-Controlled Energy Transfer Quenching of Fluorophore-Labeled DNA SAMs Enables In Situ Study of These Complex Electrochemical Interfaces.

Interfaces modified by a molecular monolayer can be challenging to study, particularly in situ, requiring novel approaches. Coupling electrochemical and optical approaches can be useful when signals are correlated. Here we detail a methodology that uses redox electrochemistry to control surface-based fluorescence intensity for detecting DNA hybridization and studying the uniformity of the surface response. A mixed composition single-strand DNA SAM was prepared using potential-assisted thiol exchange with two alkylthiol-modified ssDNAs that were either labeled with a fluorophore (AlexaFluor488) or a methylene blue (MB) redox tag. A significant change in fluorescence was observed when reducing MB to colorless leuco-MB. In situ fluorescence microscopy on a single-crystal gold bead electrode showed that fluorescence intensity depended on (1) the potential controlling the oxidation state of MB, (2) the surface density of DNA, (3) the MB:AlexFluor488 ratio in the DNA SAM, and (4) the local environment around the DNA SAM. MB efficiently quenched AlexaFluor488 fluorescence. Reduction of MB showed a significant increase in fluorescence resulting from a decrease in quenching or energy transfer efficiency. Hybridization of DNA SAMs with its unlabeled complement showed a large increase in fluorescence due to MB reduction for surfaces with sufficient DNA coverage. Comparing electrochemical-fluorescence measurements to electrochemical (SWV) measurements showed an improvement in detection of a small fraction of hybridized DNA SAM for surfaces with optimal DNA SAM composition and coverage. Additionally, this coupled electrochemical redox-fluorescence microscopy method can measure the spatial heterogeneity of electron-transfer kinetics and the influence of the local interfacial environment.

A snapshot of the electrochemical reaction layer by using 3 dimensionally resolved fluorescence mapping.

The coupling between electrochemistry and fluorescence confocal laser scanning microscopy (FCLSM) allows deciphering the electrochemical and/or redox reactivity of electroactive fluorophores. This is demonstrated with phenoxazine electrofluorogenic species frequently used in bioassays by mapping the variation of fluorescence intensity with respect to the distance from the electrode. The electrochemical conversion of resorufin dye (RF) to non-fluorescent dihydroresorufin (DH) leads to a sharp decrease of the fluorescence signal in the vicinity of the electrode. In contrast, the direct reduction of resazurin (RZ) to DH leads to an unexpected maximum fluorescence intensity localized further away from the surface. This observation indicates that the initial electron transfer (heterogeneous) is followed by a chemical comproportionation step (homogeneous), leading to the formation of RF within the diffusion layer with a characteristic concentration profile. Therefore, in situ FCLSM affords a direct way to monitor such chemical reactivity in space and to decipher a new redox pathway that cannot be resolved solely by electrochemical means.

Grafting of Oligo(ethylene glycol)-Functionalized Calix[4]arene-Tetradiazonium Salts for Antifouling Germanium and Gold Surfaces.

Biosensors that can determine protein concentration and structure are highly desired for biomedical applications. For the development of such biosensors, the use of Fourier transform infrared (FTIR) spectroscopy with the attenuated internal total reflection (ATR) configuration is particularly attractive, but it requires appropriate surface functionalization of the ATR optical element. Indeed, the surface has to specifically interact with a target protein in close contact with the optical element and must display antifouling properties to prevent nonspecific adsorption of other proteins. Here, we report robust monolayers of calix[4]arenes bearing oligo(ethylene glycol) (oEG) chains, which were grafted on germanium and gold surfaces via their tetradiazonium salts. The formation of monolayers of oEGylated calix[4]arenes was confirmed by AFM, IR, and contact angle measurements. The antifouling properties of these modified surfaces were studied by ATR-FTIR spectroscopy and fluorescence microscopy, and the nonspecific absorption of bovine serum albumin was found to be reduced by 85% compared to that of unmodified germanium. In other words, the organic coating by oEGylated calix[4]arenes provides remarkable antifouling properties, opening the way for the design of germanium- or gold-based biosensors.

Electrochemical Monitoring of the Reversible Folding of Surface-Immobilized DNA i-Motifs.

Two cytosine (C) rich DNA sequences folding in i-motif upon protonation of C at low pH have been immobilized at gold electrodes to study the impact of the electrode|electrolyte interface on the stability of the noncanonical DNA secondary structure. The effects of the molecular composition and environment on the melting and folding of the structures immobilized at the gold surface have been compared to the properties of the DNA strands in solution. The DNA folding into i-motif upon protonation, both at the surface and in solution, results in a significant variation of the charge density which is monitored electrochemically through the electrostatic interactions between the DNA strand and the electroactive hexaammineruthenium(III). This method is shown to be sufficiently sensitive to distinguish hemiprotonated folded state and single strand unfolded state of i-motif. The pH of melting has been determined for both sequences in the bulk and at the gold|electrolyte interface. The results evidence a stabilizing effect of the interface on i-motif structure, whereby the pH of melting is higher for the sequences immobilized at the surface. The reversibility and precision of the electrochemical model described here allows a clear and simple characterization of DNA structures and does not require any labeling of the sequence.

Beyond Simple Cartoons: Challenges in Characterizing Electrochemical Biosensor Interfaces.

Design and development of surface-based biosensors is challenging given the multidisciplinary nature of this enterprise, which is certainly the case for electrochemical biosensors. Self-assembly approaches are used to modify the surface with capture probes along with electrochemical methods for detection. Complex surface structures are created to improve the probe-target interaction. These multicomponent surface structures are usually idealized in schematic representations. Many rely on the analytical performance of the sensor surface as an indication of the quality of the surface modification strategy. While directly linked to the eventual device, arguments for pursuing a more extensive characterization of the molecular environments at the surface are presented as a path to understanding how to make electrochemical sensors that are more robust, reliable with improved sensitivity. This is a complex task that is most often accomplished using methods that only report the average characteristics of the surface. Less often applied are methods that are sensitive to the probe (or adsorbate) present in nonideal configurations (e.g., aggregates, clusters, nonspecifically adsorbed). Though these structures may compose a small fraction of the overall modified surface, they have an uncertain impact on sensor performance and reliability. Addressing this issue requires application of imaging methods over a variety of length scales (e.g., optical microscopy and/or scanning probe microscopy) that provide valuable insight into the diversity of surface structures and molecular environments present at the sensing interface. Furthermore, using in situ analytical methods, while complex, can be more relevant to the sensing environment. Reliable measurements of the nature and extent of these features are required to assess the impact of these nonideal configurations on the sensing process. The development and use of methods that can characterize complex surface based biosensors is arguably required, highlighting the need for a multidisciplinary approach toward the preparation and analysis of the biosensor surface. In many ways, representing the surface without reliance on overly simplified cartoons will highlight these important considerations for improving sensor characteristics.

NMR Study of the Reductive Decomposition of [BMIm][NTf2 ] at Gold Electrodes and Indirect Electrochemical Conversion of CO2.

Potential controlled electrolyses of [BMIm][NTf2 ] ionic liquid were performed at a gold cathode under nitrogen atmosphere. The structures of the major conversion products of the BMIm+ cation were elucidated on the basis of 1D and 2D nuclear magnetic resonance (NMR) analyses and gas chromatography (GC) analysis of the volatile compounds. Recombination of the imidazol-2-yl radicals, generated at the electrode by single electron transfer, leads to neutral diastereomeric dimers in equal proportions, with a faradaic efficiency of 80 %, while disproportionation of these radicals and/or reaction with hydrogen atoms adsorbed at the electrode generates a neutral monomer with 20 % faradaic efficiency. Both pathways also yield the N-heterocyclic carbene imidazolin-2-ylidene, which is involved in fast proton exchange with the parent BMIm+ cation. The reductive decomposition products of the BMIm+ cation are no longer detected if the pre-electrolysed sample is reacted with CO2 , which undergoes an indirect reduction and generates the carboxylate adduct.

Measuring and Remediating Nonspecific Modifications of Gold Surfaces Using a Coupled in Situ Electrochemical Fluorescence Microscopic Methodology.

In surface-based biosensors, the nonspecific or undesired adsorption of the probe is an important characteristic that is typically difficult to measure and therefore to control or eliminate. A methodology for measuring and then minimizing or eliminating this problem on gold surfaces, readily applicable to many common surface modifications is presented. Combining electrochemical perturbation and fluorescence microscopy, we show that the potential at which the adsorbed species is removed can be used as an estimate of the strength of the adsorbate-surface interaction. This desorption potential can be easily measured through an increase in fluorescence intensity as the potential is manipulated. Furthermore, this method can be used to evaluate strategies for preventing or removing nonspecific adsorption. This is demonstrated for a wide variety of surface modifications, from strongly chemisorbed monolayers such as thiol self-assembled monolayers (SAMs) to physisorbed monolayers as well as for complex surface structures like peptide and DNA mixed-component SAMs. The use of a coadsorption strategy or small magnitude potential-step cycles was shown to significantly decrease the amount of nonspecifically or noncovalently bound probe, creating better defined surfaces.

Direct oxidative pathway from amplex red to resorufin revealed by in situ confocal imaging.

Amplex Red (AR) is a very useful chemical probe that is employed in biochemical assays. In these assays, the non-fluorescent AR is converted to resorufin (RS), which strongly absorbs in the visible region (λabs = 572 nm) and yields strong fluorescence (λfluo = 583 nm). Even if AR is commonly used to report on enzymatic oxidase activities, an increasing number of possible interferences have been reported, thus lowering the accuracy of the so-called AR assay. As a redox-based reaction, we propose here to directly promote the conversion of AR to RS by means of electrochemistry. The process was first assessed by classic electrochemical and spectroelectrochemical investigations. In addition, we imaged the electrochemical conversion of AR to RS at the electrode surface by in situ confocal microscopy. The coupling of methodologies allowed to demonstrate that RS is directly formed from AR by an oxidation step, unlike what was previously reported. This gives a new insight in the deciphering of AR assays' mechanism and about their observed discrepancy.

Coupling Electrochemistry with Fluorescence Confocal Microscopy To Investigate Electrochemical Reactivity: A Case Study with the Resazurin-Resorufin Fluorogenic Couple.

The redox couple resazurin-resorufin exhibits electrofluorochromic properties which are investigated herein by absorption and fluorescence spectroelectrochemistry and by electrochemically coupled-fluorescence confocal laser scanning microscopy (EC-CLSM). At pH 10, the highly fluorescent resorufin dye is generated at the electrode surface by the electrochemical reduction of the poorly fluorescent resazurin. Performing EC-CLSM at electrode surfaces allows to monitor spatially resolved electrochemical processes in situ and in real time. Using a small (315 µm diameter) cylindrical electrode, a steady-state diffusion layer builds up under potentiostatic conditions at -0.45 V vs Ag|AgCl. Mapping the fluorescence intensity in 3D by CLSM enables us to reconstruct the relative concentration profile of resorufin around the electrode. The comparison of the experimental diffusion-profile with theoretical predictions demonstrates that spontaneous convection has a direct influence on the actual thickness of the diffusion layer, which is smaller than the value predicted for a purely diffusional transport. This study shows that combining fluorescence CLSM with electrochemistry is a powerful tool to study electrochemical reactivity at a spatially resolved level.

Electrochemical discrimination between G-quadruplex and duplex DNA.

Analytical tools enabling the discrimination between duplex DNA and G-quadruplex DNA are necessary to unravel the biological function(s) of G-quadruplexes. A methodology relying on the electrochemical response of the electroactive hexaammineruthenium(III) cation at DNA-modified surfaces is presented. A characteristic voltammetric peak is evidenced for all the investigated G-quadruplex sequences, encompassing various types of folding and numbers of quartets. In contrast, no such peak is detected for dsDNA sequences. The occurrence of the voltammetric peak is the consequence of a strong association between the hexaammineruthenium ligand and the surface-immobilized G-quadruplexes. The peak potential points to a significant contribution of nonelectrostatic interactions between the electroactive ligand and G-quadruplexes. The very good efficiency of the discrimination methodology is demonstrated by comparing a G-quadruplex and its corresponding duplex.

Imaging redox activity at bipolar electrodes by indirect fluorescence modulation.

Bipolar electrochemistry (BPE) is nowadays well-known but relatively underexploited and still considered as unconventional. It has been used, among others, in the frame of materials science and most importantly has also found very promising applications in analytical chemistry. Here, we extend this emerging field of analytical applications to the development of a new sensing concept based on indirect BPE. This approach is based on the generation of local pH gradients which will allow detecting indirectly redox-active molecules due to a modulation of the fluorescence intensity in the vicinity of a bipolar electrode.

On the interaction between [Ru(NH3)6]3+ and the G-quadruplex forming thrombin binding aptamer sequence.

The interaction between the thrombin binding aptamer (TBA), a G-quadruplex forming DNA sequence, and the electroactive hexaammineruthenium(III) cation has been studied by electrochemical methods and circular dichroism spectroscopy. When TBA is immobilised on a gold surface in a typical aptasensor configuration, the [Ru(NH3)6](3+) cation can be bound to the electrode surface through its interaction with the TBA sequence. This interaction is strong enough to enable the ruthenium complex to remain at the surface when the electrode is immersed in an electrolyte free of [Ru(NH3)6](3+), meaning that the complex does not diffuse back into the solution. A stoichiometry of 2 [Ru(NH3)6](3+) per TBA strand has been determined, indicating that the interaction differs from the conventional, non-specific electrostatic charge compensation, for which a 5 to 1 ratio would be expected between the triply charged cation and the 15 bases sequence. It is shown that this interaction takes place not only at the surface, but also when both TBA and hexaammineruthenium(III) are dissolved in solution. Under such conditions, a similar stoichiometry of 2 [Ru(NH3)6](3+) per TBA strand has been evidenced by two independent methods, namely circular dichroism spectroscopy and differential pulse voltammetry.

Interaction of cytidine 5'-monophosphate with Au(111): an in situ infrared spectroscopic study.

The interaction of cytidine 5'-monophosphate (CMP) with gold surfaces is studied by means of in situ infrared spectroscopy and cyclic voltammetry at the Au(111)|aqueous solution interface. Similar to other nucleic acid components, cytidine 5'-monophosphate is chemisorbed on the surface at positive potentials, and the amount of adsorbed CMP increases with the potential. Subtractively normalized interfacial Fourier-transform infrared spectroscopy (SNIFTIRS) is used to identify the adsorbed and desorbed species. Upon electrochemical desorption, the molecules released in solution are unprotonated on the N3 atom. Striking similarities are found between the spectrum of adsorbed CMP and the solution spectrum of protonated CMP. The origin of such similarities is discussed. The results strongly suggest that chemisorption occurs through the N3 atom of the pyrimidine ring. A comparison is drawn with cytidine, whose electrochemical and spectroscopic behaviors are also investigated.