Characterization of molecules of biological interest and their application in biosensors

Abstract

Biomolecules and molecules of biological interest, thanks to their diversified chemical physical properties and their unlimited list of functions in chemical reactions and biological processes, are more and more used in several fields, from fundamental science to technological applications in medicine, agrifood monitoring and detection of environmental pollutant. These perspectives call for new strategies and techniques to investigate the intrinsic properties (electronic structure, conformation, mechanical properties, etc.) of these macromolecules, to handle and to embed them in devices as well as to study their functioning in different environments, from their natural biological settings to the ones relevant for the diverse applications. The study of the intrinsic chemical physical properties can be performed with a high level of accuracy, by a combination of sophisticated experimental and theoretical approaches, on model systems. However, the size of the model systems is often reduced to small building blocks of the large biomolecules and the employed methodologies cannot be directly scaled up to more complex and realistic systems. The motivation of this thesis is to introduce methodologies to study biomolecules and their applications which contribute to fill the gap between small and complex biomolecular systems. The electronic structure and photofragmentation mechanisms of a group of molecules belonging to the oxygen mimetic class of radiosensitisers, which are used in radiotherapy to increase the efficacy and selectivity of the medical treatment, are investigated by several spectroscopic techniques (valence band photoemission, photoelectron-photoion coincidence and appearance energy measurements) and advanced quantum mechanical calculations. Molecules of increasing complexity are characterized in a bottom up approach from model systems (imidazole and nitroimidazole isomers) to real drugs (misonidazole and metronidazole) in order to unravel the correlation between intrinsic properties and their functions in medical applications. To move the investigation towards more complex and realistic systems that can be handled experimentally, the electrospray ionization, ESI, a novel technique for production of beams of multicharged complex molecules has been adopted. A vacuum apparatus equipped with a non- commercial ESI source and electronic optical devices has been designed and characterized comparing computer simulations, performed by SIMION software, and experimental measurements to achieve a realistic and detailed understanding and control of the ion beam. The main purpose of this characterization is to optimise the performances of the instrument, reaching a sufficiently intense ion flux either to perform spectroscopic measurements or to deposit the molecules for making biosensors. The ESI technique at ambient pressure condition has been also used for enzyme immobilisation and biosensors application. Laccase, a well known enzyme, has been used to validate the technique and promising results have been obtained. Indeed it has been demonstrated that at least 75% of enzymatic activity is preserved after ESI deposition, with the additional benefit of well controlled deposition parameters. The activity has reached the stage to produce a working biosensor on commercial screen printed electrodes, whose response is comparable with laccase based biosensor currently available. The results of this thesis have opened up many possibilities for both fundamental and applied future work.