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|Autori: ||Rapisarda, Antonino|
|Titolo: ||Localized Surface Plasmon Resonance: Nanoscale Sensing for Processes at Interfaces|
|Abstract: ||This PhD thesis reports the use the emerging surface-sensitive optical technique of localized surface plasmon resonance (LSPR) to characterize the interaction of relevant classes of biomolecules, e.g. peptides, proteins, lipids and DNA strands, at solid-liquid interfaces, with an emphasis on deciphering kinetics and pathways of dynamic adsorption processes.
LSPR-based biosensor exploits the high sensitivity of the plasmon frequency to refractive index changes confined to 5-30 nanometers around the metal nanoparticles deposited on the sensor surface to monitor in situ and in real time the interaction of unlabeled biological molecules skipping the misleading contribution from the bulk of solution affecting conventional optical technique, e.g. SPR and OWLS.
In the present dissertation the advantages of applying this powerful technique are thoroughly demonstrated by investigating four case studies concerning relevant aspects for the biointerfaces science. The case of study 1 will involve the adsorption kinetics of single and binary solution of proteins onto model hydrophilic and hydrophobic surfaces. The analysis of the adsorption kinetics reveals that competitive adsorption occurs, at physiological pH 7.4 and relatively high ionic strength (NaCl 0.1 M), favoring the heavier protein (fibronectin, in our case), which is shown to adsorb faster and in larger amount than the lighter one (human serum albumin, in our case). The case of study 2 will discuss the DNA hybridization process for binary solutions of respectively perfectly matching (PM) and single base mismatching (MM) 93-mer ssDNA from KRAS codon 12, with a surface tethered probe complementary to the PM sequence. Sensitivity down to obtaining down to 10 nM and 13 nM, respectively for PM and MM were obtained, showing that the hybridization process occurs at a lower rate for MM with respect to PM target. The competitive hybridization was accounted for by an inhibition model, where the non-complementary sequences kinetically hinder the hybridization of the perfect matching sequences, owing to their above mentioned affinity constant differences for the same probe. The case of study 3 will cover the kinetics of phospholipid vesicle adsorption on silicon oxide surfaces as function of pH. Two different regimes have been observed for acidic and basic conditions. At low pH, vesicles adsorption showed one-step exponential kinetics. Moreover, no significantly variation of the adsorption rate was observed over the investigated pH range 3-6, suggesting the process is controlled by Van der Waals interactions and steric forces. At high pH, vesicles adsorb showing two-step kinetic. Furthermore, it was observed that the rate of the first step slows down linearly with the increasing of pH, suggesting that the process is primarily driven by vesicle-surface electrostatic repulsion. The case of study 4 will report preliminary results from the study of pH stimuli-responsive smart surfaces, formed by gold nanodisks array of an LSPR sensor chip decorated with Trichogin GA IV and two of its positively-charged analogs, i.e. Lipo-Lys and L20, in which four and eight Lysines positive charged residues have been introduced respectively. The surface-bound peptides exhibit reversible and rapid switching between conformations and can withstand several cycles of swelling and collapsing with no significant loss from the surfaces.
Overall, the results here reported demonstrated the great potential of LSPR technique as a unique tool to monitor specific and non-specific biomolecular interactions at interfaces in application fields ranging from biosensing to materials science.|
|In||Area 02 - Scienze fisiche|
|RPSNNN88M02C351P-Antonino Rapisarda_PhD thesis def_.pdf||Antonino Rapisarda PhD Thesis ||34,29 MB||Adobe PDF||Visualizza/apri
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