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Identifier

000433309

Title

Acoustic and optical characterization of soft biological matter and biopolymers

Alternative Title

Ακουστικός και οπτικός χαρακτηρισμός μαλακής βιολογικής ύλης και βιοπολυμερών.

Author

Χρονάκη, Δήμητρα Ι

Thesis advisor

Γκιζελή, Ηλέκτρα

Reviewer

Τσόρτος, Αχιλλέας
Χαλεπάκης, Γεώργιος
Αθανασάκη, Ειρήνη

Abstract

In this thesis the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) is used in combination with other biophysical techniques in order to monitor the adsorption of various biological entities at the solid/liquid interface. The samples of interest are soft biological matter (cells) and biopolymers (polymers and proteins); effort is placed on the study and characterization of the interactions between the sample and the underlying substrate. The QCM-D was used in order to discriminate between two thyroid cell phenotypes, a normal and tumor one. Three surfaces were exploited as a substrate for adhesion studies: bare titanium, bare gold and fibrinogen-coated gold. Their adhesion to the three surfaces was monitored in real-time by QCM-D and the acoustic signals obtained were the frequency change, energy dissipation change, acoustic ratio ΔD/ΔF and rate of cell binding on the surface. In parallel, scanning electron microscopy (SEM) images were used to observe the morphology of the adhered cells on the three surfaces. The combination of both techniques provided valuable insight regarding cell adhesion. Our results suggest that the application of two surfaces for cell adhesion experiments can discriminate with accuracy between the two particular cell types and potentially form a platform for discrimination between normal and cancer thyroid cell types. The QCM-D combined with a spectroscopic ellipsometer was employed for in-situ monitoring of the adsorption of the copolymer PLL-g-PEG on gold surface and the subsequent binding of DNA molecules. Acoustic and optical data modeling gave an insight into the architecture of the copolymer and the bound DNA molecules; the “combined” thickness, “wet” and “dry” mass and the hydration of the formed film. By combining this new information with the acoustic ratio values obtained for the films we concluded that DNA is lying rather flat on the PLL-g-PEG copolymer. Overall, property of the PLL-g-PEG copolymer to attract DNA molecules (due to the positively charged poly-lysine backbone) and at the same time to repel proteins through the grafted PEG units was confirmed. We highlight that the exceptional PLL-g-PEG copolymer architecture enabled the specific binding of DNA from whole Salmonela Thyphimurium cells in complex samples such as milk. The combined QCM-D/Ellipsometry technique was used to monitor in real-time the adsorption of avidin, streptavidin and neutravidin on gold surface. Acoustic and optical data modeling provided information about film properties such as refractive index, “dry” mass, “wet” mass and water content. Also, FTIR-ATR spectroscopy measurements were used to elucidate the protein secondary structure in the bulk and upon adsorption to gold surface; Raman spectroscopy measurements were only used for validation of the bulk secondary structure percentages obtained by FTIR-ATR. The acoustic data suggest that the adsorption of neutravidin on gold surface is quite different than avidin and streptavidin. Neutravidin always exhibited higher dissipation and acoustic ratio values than avidin and streptavidin. The low acoustic ratio values of avidin and streptavidin imply a tight interaction with gold surface which probably leads to higher decrease of β-sheet content than in the case of neutravidin. Acoustic and optical data modeling suggest that neutravidin films contain more water than avidin and streptavidin films. Moreover, the neutravidin films possibly contain more types and/or amount of water molecules than avidin and streptavidin films as deduced by the acoustic and spectroscopic data.

Language

English

Subject

Acoustic biosensor (QCM-D)

Biophysics

Cells

Infrared spectroscopy (FTIR-ATR)

Polymers

Proteins

Spectroscopic ellipsometry

Ακουστικός βιοαισθητήρας (QCM-D)

Βιοφυσική