Electrochemical and Electrokinetic Tools for Surface Activity Characterization and Proteomics Analysis

Scanning electrochemical microscopy (SECM) has developed into an excellent and versatile technique to image heterogeneous chemical reactivities on a surface. Therefore SECM applications cover a wide range of different fields such as biology and forensic sciences. One interesting application that has been found in proteomics, and explored herein, is the use of SECM as a read-out tool for protein microseparations. Proteomic research is a long process where two main steps are contained: separation of one protein from a protein mixture and detection of the separated proteins with a high sensitivity and if required selectivity. Shortening the experimental time of each one of these steps while their quality is kept or improved, should be the strategy to win this exciting race. Although microelectrophoresis affords faster protein separation, the smaller amount of sample employed requires a more sensitive protein detection method. In the first part of the present thesis an approach to solve the latter milestone has been found based on the coupling of miniaturized electrophoresis and SECM. As a result, a complete miniaturized (i.e. 1 cm × 0.5 cm) isoelectric focusing (IEF) protein separation was scanned by SECM, providing protein detection with a high sensitivity and high resolution. Additionally, protein detection by SECM was performed by different strategies ranging from general to selective approaches based on the tagging of free cysteines and other nucleophiles in proteins and peptides by benzoquinone. The tagged proteins are detected by the mediated reduction of benzoquinone with a redox species produced electrochemically at the SECM tip. After careful optimization, a sensitivity in the low ng mm-2 range was reached for bovine serum albumin. One of the major advantages of the present technique is that the selectivity of the protein tagging can be tuned by changing the pH of the reaction media. Depending on the requirements, cysteine selective or general detection can therefore be achieved with a high sensitivity. Despite the time reduction achieved with microelectrophoresis and the successful coupling with SECM for sensitive protein detection, conventional SECM setups are limited to scan line by line the whole studied area carrying long experimental times. Additionally, since the response of the sensing microelectrode depends on the probe-substrate distance, discrimination between surface reactivity and topographic artifacts coming from the sample topology is not obvious. The outlined above SECM drawbacks motivate us in the second part of this thesis to address those points. Therefore, some recent developments that show how SECM can be used for reactivity analysis of large, corrugated, tilted and dry surfaces are presented. This extension of SECM is made possible by the use of specialized microelectrode probes fabricated in a soft polymer film integrating for instance, microfluidic systems for delivering microliter volumes of redox-active mediator on a dry sample. These soft structures are then scanned in a contact mode on the substrate, and the originated current from the redox cycling of a mediator is used to construct a reactivity image of the specimen. Due to the use of probes containing individually addressable multiplexed electrodes, it is possible to substantially decrease the recording time while a high quality image resolution is maintained. The electrochemical characterization of the proposed probes was performed by cyclic voltammetry, approach curves and lateral line scans over insulating and conductive substrates of different roughness

Proteins/peptides purification by a three-well OFFGEL electrophoresis with immobilized ultra narrow pH gradient gels

Purification and desalting of protein and peptide samples by a three-well OFFGEL electrophoresis with immobilized ultra narrow pH gradient gels is proposed as a fast preparative strategy for proteomics. The gist of this strategy is to separate the proteins and peptides according to their isoelectric point and to isolate those of a given pI value equal to the mean pH value of the gel. The present approach has been demonstrated both on protein mixtures and a digested Escherichia coli protein extract. UV-Vis spectroscopy, MALDI-MS, SDS-PAGE and LC-MS/MS were employed for the quantitative and qualitative characterization of the separation results. The electrophoretic methodology has been simulated by finite element methods

Soft Microelectrode Linear Array for Scanning Electrochemical Microscopy

A linear array of eight individual addressable microelectrodes has been developed in order to perform highthroughput scanning electrochemical microscopy (SECM) imaging of large sample areas in contact regime. Similar to previous reports, the soft microelectrode array was fabricated by ablating microchannels on a polyethylene terephthalate (PET) film and filling them with carbon ink. Improvements have been achieved by using a 5 μm thick Parylene coating that allows for smaller working distances, as the probe was mounted with the Parylene coating facing the sample surface. Additionally, the application of a SECM holder allows scanning in contact regime with a tilted probe, reducing the topographic effects and assuring the probe bending direction. The main advantage of the soft microelectrode array is the considerable decrease in the experimental time needed for imaging large sample areas. Additionally, soft microelectrode arrays are very stable and can be used several times, since the electrode surface can be regenerated by blade cutting. Cyclic voltammograms and approach curves were recorded in order to assess the electrochemical properties of the device. An SECM image of a gold on glass chip was obtained with high resolution and sensitivity, proving the feasibility of soft microelectrode arrays to detect localized surface activity. Finite element method (FEM) simulations were performed in order to establish the effect of diffusion layer overlapping between neighboring electrodes on the respective approach curves

Segmented Field OFFGEL® Electrophoresis

A multielectrode setup for protein OFFGEL electrophoresis that significantly improves protein separation efficiency has been developed. Here, the electric field is applied by segments between seven electrodes connected in series to six independent power supplies. The aim of this strategy is to distribute evenly the electric field along the multiwell system, and as a consequence to enhance electrophoresis in terms of separation time, resolution, and protein collection efficiency, while minimizing the overall potential difference and therefore the Joule heating. The performances were compared to a standard two-electrode setup for OFFGEL fractionation of a protein mixture, using UV-Vis spectroscopy for quantification and MALDI-MS for identification. The electrophoretic separation process was simulated, and optimized by solving the time-dependent Nernst–Planck differential equation

Seeing Big with Scanning Electrochemical Microscopy

Specialized microelectrode probes fabricated in a soft polymer film now make it possible to use scanning electrochemical microscopy to image the reactivity of large, corrugated, tilted, and dry surfaces

Microfluidic Push-Pull Probe for Scanning Electrochemical Microscopy

This paper presents a microfluidic push-pull probe for scanning electrochemical microscopy (SECM) consisting of a working microelectrode, an integrated counter/reference electrode and two microchannels for pushing and pulling an electrolyte solution to and away from a substrate. With such a configuration, a droplet of a permanently renewed redox mediator solution is maintained just at the probe tip to carry out SECM measurements on initially dry substrates or in microenvironments. For SECM imaging purposes, the probe fabricated in a soft polymer material is used in a contact regime. SECM images of various gold-on-glass samples demonstrate the proof-of-concept of a push-pull probe for local surface activity characterization with high spatial resolution even on vertically oriented substrates. Finite element computations were performed to guide the improvement of the probe sensitivity

Voltammetric determination of extreme standard Gibbs ion transfer energy

A voltammetric methodology to determine the standard Gibbs energy of transfer of highly hydrophobic and hydrophilic ions has been developed. The electrochemical cell used includes a water|1,2-dichloroethane micro-interface supported on a microhole in a thin polymer film separating an electrolyte-free aqueous phase and an organic phase with an electrolyte at low concentrations. The limiting current and the half-wave potential of these organic ions were determined by fitting the initial part of the ion transfer wave. The methodology was validated using ions with known thermodynamic data, and applied to very hydrophobic and very hydrophilic ions that usually cannot be observed within the potential window

Inkjet Printed Nanohydrogel Coated Carbon Nanotubes Electrodes For Matrix Independent Sensing

Polyacrylamide (PA) based hydrogels are used in several applications including polyacrylamide gel electrophoresis and sensing devices. Homogeneous and compact PA films can be prepared based on chemical or photopolymerization processes. However, the accurate and reproducible coating of substrates with nanohydrogel patterns is challenging due to the in situ polymerization and deposition requirements. Herein, we report an inkjet printing (IJP) concept with simultaneously performed UV photopolymerization of a specifically prepared acrylamide/N,N′-methylenebis(acrylamide) containing ink. A prepolymerization step of the hydrogel precursor molecules was implemented in the ink formulation protocol to adjust the viscosity of the ink and to enhance the rate of polymerization during printing. After the optimization of the printing parameters, a nanometer thin PA hydrogel coating with well distributed nanopores was achieved on top of a stand-alone carbon nanotubes (CNTs) pattern. Batches of fully inkjet printed PA/CNT modified electrodes were prepared that showed outstanding improvements for the electrochemical detection of antioxidants in complex matrices such as untreated orange juice and red wine samples thanks to the properties of the PA coating

Ion current rectification and rectification inversion in conical nanopores: a perm-selective view

Ionic transport in charged conical nanopores is known to give rise to ion current rectification. The present study shows that the rectification direction can be inverted when using electrolyte solutions at very low ionic strengths. To elucidate these phenomena, electroneutral conical nanopores containing a perm-selective region at the tip have been investigated and shown to behave like classical charged nanopores. An analytical model is proposed to account for these rectification processes

High-throughput scanning electrochemical microscopy brushing of strongly tilted and curved surfaces

The feasibility of high-throughput scanning electrochemical microscopy (SECM) of strongly tilted (tilt angles ≤4°) and curved substrates (diameter of curvature ≥9 cm) is demonstrated by brushing them with a soft linear array of carbon microelectrodes. This probe made of thin polymeric layers operates in contact regime to follow the topography of highly unconventional SECM samples while keeping an almost constant working distance. Strong slope variations of the sample lead to a slight misalignment between the axes of the positioning system and the sliding direction of the microelectrode arrays. The resulting positional offsets can be predicted and corrected to yield a correct representation of the spatial relation on the surface of the sample. Moreover, a custom-made holder system ideally suited for precise control of the soft probe inclination angle and alignment with the substrate plane was also developed to perform high-throughput SECM imaging of a 1.2 cm2 curved metallic pin within less than 2 h