Synthetic heme-proteins in biosensors development

Abstract

A biosensor is an analytical device that relies on a biological recognition element, such as an enzyme, an antibody, a DNA fragment, which communicates with a signal transducer, thus providing a measurable response related to the concentration of an analyte. Currently, electrochemical biosensors, which exploit an electron transfer (ET) event at a solid electrode for the signal transduction, are the larger and most promising biosensor category in terms of ease of construction, costs, versatility, tunability and miniaturization (up to the nanoscale) for in vivo applications. A real breakthrough toward the development of electrochemical systems with superior selectivity, stability and durability is represented by the third generation biosensors. These devices exploit the unmediated direct electron transfer occurring at interfaces made up of an electrode immobilized redox-active specie, with a current transduction of the biological interaction, resulting in an amperometric detection of the analyte. An attractive feature of systems based on direct electron transfer is the possibility of modulating the properties of the analytical device using protein modification with genetic or chemical engineering techniques. Efficient direct electron transfer (DET) reactions have been reported for a restricted number of redox enzymes. The majority of these redox enzymes contain metal sites and particularly heme. Nevertheless, several studies based on heme-proteins adsorbed or immobilized on various electrodes showed that high-molecular weight enzymes are not amenable to direct electrical communication with the electrode. Our strategy to overcome the above limitations is the exploitation of artificial low-molecular weight proteins, designed on rational bases, to possess the required activity. In our laboratory, a new class of heme-peptide conjugate, named Mimochromes, have been developed, with the aim of understanding the effects of the peptide chain composition and conformation in modulating the heme redox potential. The main features of these molecules are the covalent structure and the well defined helical conformation of the peptide chains linked to the deuteroporphyrin ring. The presented work exploits the use of two different class of Mimochromes, the penta-coordinated Fe(III)-Mimochrome VI and the esa-coordinated Fe(III)-Mimochrome IV Lys3, in biosensor technology. These two artificial molecules were used as bio-recognition elements/signal transducers for biosensing. The aim of this thesis was to develop and optimise catalytic and affinity-based biosensors for the rapid screening of target analytes in complex matrices. The electrochemical properties of the two Mimochromes were studied by voltammetric techniques, then they were suitably immobilized on a gold electrode surface to achieve the biosensors construction and finally the analyte recognition was performed.