Probing Cysteine Reactivity in Proteins by Mass Spectrometric EC-Tagging

The on-line electrochemical tagging (EC-tagging) of cysteine residues in proteins during mass spectrometry is studied to probe the cysteine environment. Benzoquinone probes electrogenerated at a microspray electrode react with the thiol functions of the proteins within a microchannel and the products are analyzed by mass spectrometry. The fundamentals of the technique are discussed, with a focus on the kinetic aspects. The EC-tagging efficiency of the cysteine residues in proteins is used to probe their environment. Experiments with unmodified proteins and their chemically reduced forms highlight the strong effect of the cysteine site reactivity on the tagging efficiencies. This study highlights relevant parameters for such on-line electrochemical derivatization/MS detection strategies

CdSe Sensitized Thin Aqueous Films: Probing the Potential Distribution Inside Multilayer Assemblies

Ultrathin polypeptide multilayer films are assembled by the sequential electrostatic adsorption of monolayers of poly-L-lysine and poly-L-glutamic acid onto carboxylic acid terminated alkanethiol-modified gold surfaces. The polypeptide multilayer films are hydrophilic, can incorporate electroactive species such as ferri/ferrocyanide, and are stable when immersed in organic solvents such as 1,2-dichloroethane. Cadmium selenide quantum dots stabilized by negatively charged citrate groups are electrostatically attached to the multilayer film assembly in order to act as photoactive species. Photocurrent responses originating from the CdSe sensitized ultrathin multilayer film are investigated as functions of the applied potential, the thickness of the film and the presence of quenchers in the organic phase. A theoretical model is proposed in order to analyze the kinetics of the photoinduced electron-transfer reactions and to probe the potential distribution within the film

Solvent Effect on Redox Properties of Hexanethiolate Monolayer-Protected Gold Nanoclusters

The capacitance of monolayer-protected gold nanoclusters (MPCs), CMPC, in solution has been theoretically reconsidered from an electrostatic viewpoint, in which an MPC is considered as an isolated charged sphere within two dielectric layers, the intrinsic coating monolayer, and the bulk solvent. The model predicts that the bulk solvent provides an important contribution to CMPC and influences the redox properties of MPCs. This theoretical prediction is then examined experimentally by comparing the redox properties of MPCs in four organic solvents: 1,2-dichloroethane (DCE), dichloromethane (DCM), chlorobenzene (CB), and toluene (TOL), in all of which MPCs have excellent solubility. Furthermore, this set of organic solvents features a dielectric constant in a range from 10.37 (DCE) to 2.38 (TOL), which is wide enough to probe the solvent effect. In these organic solvents, tetrahexylammonium bis(trifluoromethylsulfonyl)imide (THATf2N) is used as the supporting electrolyte. Cyclic and differential pulse voltammetric results provide concrete evidence that, despite the monolayer protection, the solvent plays a significant effect on the properties of MPCs in solution

Antioxidant Sensors Based on DNA-Modified Electrodes

TiO2/ITO modified electrodes were developed to quantitatively photooxidize adsorbed ds-DNA and to study the effect of antioxidants as ds-DNA protecting agents. TiO2 films are used for efficient ds-DNA immobilization, for ds- DNA oxidation through photogenerated hydroxyl radicals, and as electrodes for amperometric sensing. The films, prepared by a sol-gel process, are deposited on ITO glass electrodes. Damages occurring after ds-DNA oxidation by ROS are detected by adding MB as an intercalant probe and by monitoring the electrochemical reduction current of the intercalated redox probe. The MB electrochemical signal is found to be sensitive enough to monitor ds-DNA structure changes, and the electrochemical sensor has been applied to the evaluation of the antioxidant properties of glutathione and gallic acid

Electroacoustic miniaturized DNA-biosensor

A micrometer-sized electroacoustic DNA-biosensor was developed. The device included a thin semi-crystalline polyethylene terephthalate (PET) dielectric layer with two Ag microband electrodes on one side and a DNA thiol-labeled monolayer adsorbed on a gold surface on the other. A resonance wave was observed at 29 MHz with a network analyzer, upon AC voltage application between the two Ag electrodes, corresponding to electromechanical coupling induced by molecular dipoles of the PET polymer chain in the dielectric layer. It was found that the device size and geometry were well adapted to detect DNA hybridization, by measuring the capacity of the resonance response evolution: hybridization induced polarization of the dielectric material that affected the electromechanical coupling established in the dielectric layer. The 0.2 mm2 sensor sensitive area allows detection in small volumes and still has higher detection levels for bioanalytical applications, the non-contact configuration adopted avoids electric faradic reactions that may damage biosensor sensitive layers, and finally, PET is a costless raw material, easy to process and well adapted for large scale production. The well-balanced technological and economic advantages of this kind of device make it a good candidate for biochip integration

Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically-modified animal models, together with biochemical and pharmacological approaches, we provide a high resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington’s disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases

Fingerprint imaging by scanning electrochemical microscopy

An efficient strategy for visualizing human fingerprints on a poly(vinyldene difluoride) membrane (PVDF) by sanning electrochemical microscopy (SECM) has been developed. Compared to a classical ink fingerprint image, here the ink is replaced by an aqueous solution of bovine serum albumin (BSA). After placing the ‘‘inked’’ finger on a PVDF membrane, the latent image is stained by silver nitrate and the fingerprint is imaged electrochemically using potassium hexachloroiridate (III) (K3IrCl6) as a redox mediator. SECM images with an area of 5 mm · 3 mm have been recorded with a high-resolution using a 25-lm-diameter Pt disk-shaped microelectrode. Pores in the skin (40–120 lm in diameter) and relative locations of ridges were clearly observed. The factors relevant to the quality of fingerprint images are discussed