| Περίληψη: | Τhe dynamics of biomolecules in their native solutions have been attracting increasing attention over the last decade and are studied by various techniques. It has been established that those water molecules in close proximity to the biomolecule are dynamically retarded and have different properties compared to the outer-lying bulk water. In recent years, the terahertz (THz) spectral region from 300 to 3000GHz has seen a flurry of research activity in the areas of biology and biophysics. The resurgence is stimulated in large part by the rapid growth of new technologies needed to access this region and the unique sensitivity of this radiation to probe weak interand intra-molecular interactions of biomolecules that are involved in all the biomolecular processes. A new biosensing approach based on electromagnetic waves at THz frequencies is described in this Master Thesis. This approach overcomes fundamental limitations of THz radiation which are:
Strong water absorption and lack of efficient active devices, which induce low levels of sensitivity
Long wavelength and limited confinement, which lead to use of excessive sample volumes
The novel THz biosensor used is favored by a versatile detection principle of hydration water dielectric response change to biomolecular processes. For this reason, this approach facilitates a label-free, immobilization-free, non-invasive, non-ionising and real-time biosensing method.
This Master Thesis focus on the high potential of this sensor as a bioanalytical tool in pharmaceutical and medical applications. The THz biosensor performance is evaluated with lysozyme, a large protein via monitoring four biomolecular processes in liquid-phase. Protein conformation is highly correlated with its hydration shell,
which can be detected by THz waves. To elucidate the possible direct probing of biomolecule’s dynamics, concentration study, denaturation, crystallization and protein binding are performed.
The first part of this work is devoted to mechanical and microfluidic alterations performed in order to improve and stabilize the THz sensor’s operation. In the second part, a number of different solutions were prepared in advance and samples of 7μL were exposed to THz analysis. Preliminary results in biomolecule’s concentration and conformation changes are presented and discussed in detail. The lowest detection limit achieved by the THz sensor is of the order of 2.5mg/mL for lysozyme. Conclusions for dielectric permittivity changes due to changes of protein hydration shell during the conformational studies (denaturation, crystallization, antigennanobody
complex) are obscured based on these primary measurements. In a nutshell, the present work corresponds to a primary study for the determination of possible applications of this THz biosensor technique. With further improvements and more experimental results proved its potential, THz sensor is envisioned to be applied in analytical instrumentation for life sciences and pharmaceutical industry.
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