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.
Keywords: Scanning Electrochemical Microscopy ; Protein Detection ; Microfabrication by laser ablation ; microscopie électrochimique à Balayage ; détection de protéines ; microfabrication par ablation laserThèse École polytechnique fédérale de Lausanne EPFL, n° 4846 (2010)
Programme doctoral Chimie et Génie chimique
Faculté des sciences de base
Institut des sciences et ingénierie chimiques
Laboratoire d'électrochimie physique et analytique
Record created on 2010-08-26, modified on 2016-08-08