Abstract:
Aptamers are single-stranded DNA or RNA molecules isolated in vitro by a selection
and amplification method. Aptamers bind with high specificity and affinity to a wide
range of target molecules, with dissociation constant comparable to antibodies.
In this work aptamers were employed as a new kind of bio-recognition element in
affinity biosensors for the detection of clinically relevant proteins in heterogeneous
assay, using Piezoelectric Quartz Crystal Microbalance and Surface Plasmon Resonance
as transducers. The work was focused on two case studies, i.e. the Thrombin-binding
aptamer and the aptamer against C-Reactive Protein. From an analytical point of view,
the work was devoted to the optimisation of the analytical performance of a
piezoelectric and an optical aptasensor for Thrombin and C-Reactive Protein detection,
respectively. Efforts towards the application of these aptasensors in complex matrices,
such as human plasma and serum, were also undertaken, in order to demonstrate the
wide applicability of aptamers, as an alternative to antibodies.
In this work, the possibility of introducing a computationally-assisted method to study
aptamer-protein interaction and aptamer selection was also evaluated. For this purpose,
the Thrombin-binding aptamer was chosen as a model and a retrospective docking study
was performed by comparing the affinity of mutated sequences for thrombin with that
of the Thrombin-binding aptamer, on the basis of a computationally-derived binding
score. Finally, the reliability of computational results was tested by experimental
measurements. For this purpose, the Thrombin-binding aptamer and other mutated
sequences, selected on the basis of their binding score, were employed for the
development of optical biosensors and the resulting analytical performances were
compared. Even if further studies should be carried out in order to validate the proposed
computational approach to aptamer selection, this work can have a significant impact on
future aptamers selection for sensors and diagnostics.
