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Electrochemically-assisted protein crystallisation: Applications to biosensors

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Protein/enzyme immobilization on electrode surfaces has been limited to soluble biomolecules. However, recent developments in protein crystallisation have created an interest in the study of solid-state electrochemistry of protein single crystals in order to understand the mechanisms of crystal growth coupling these biological crystals to fluid cells for atomic force microscopy (AFM) investigations. Unfortunately, the fixation of these monocrystals to an electrode surface is difficult since the monocrystals break easily under mechanical pressure, and therefore they cannot be immobilised in the same way as inorganic crystals. From this point of view, it is feasible to grow ex situ redox metalloprotein single crystals (such as catalases, ferritins, cytochromes, etc) so that they can be introduced into the fluid cell of the atomic force microscope (AFM) to investigate electron-transfer processes, mechanisms of crystal growth or get technological applications of the biological crystals as biosensors. In order to immobilize these biological crystals or particles, it is shown how the use polypyrrole (ppy) films deposited on highly oriented pyrolytic graphite electrodes (HOPG) or Indium Tin Oxide electrodes (ITO) allow us to perform structural investigations via EC-AFM techniques.

In summary, the basic strategies to control the kinetics and transport phenomena of the crystal growth process are reviewed, as well as novel methods to induce either nucleation or protein crystal growth via electrochemical processes (protein electrochemically-assisted protein crystallisation). Additionally, recent advances, where low direct/alternant current or voltages are applied to the crystal growth cell to obtain high-quality single crystals for X-ray diffraction, are also discussed. This also permitted to develop a first prototype of a solid-state electron-transfer device based on biological crystals. Finally, using the electrochemistry modulus of the AFM , cyclic voltammetry techniques were performed in order to characterize the electron-transfer response on the surface of these crystals attached to the electrode. The potential applications of these methods for the development of protein-based biosensors are discussed in this seminar.

This talk is part of the Experimental and Computational Aspects of Structural Biology and Applications to Drug Discovery series.

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