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Micro- and nano-patterned gold structures for selective immobilization of proteins

Arafeh, Manar

Das Ziel dieser Arbeit ist nano- und mikrostrukturierte Gold Nanopartikeln auf Silizium- und Polyethylenglykoloberflächen herzustellen. Diese Oberflächen sind sehr notwendig um biofunktionelle Moleküle zu immobilizieren. Nanostrukturierte Gold Nanopartikeln auf Silizium wurde erreichet durch die “Blockcopolymermizelle” Methode. Mit dieser Methode konnten Nanopartikeln mit definierter Größe, Position und Abstand auf der Siliziumoberfläche immobilisiert werden. Die nanostrukturierten Partikeln wurden danach durch in Wasser gequollenes Polyethylenglykol auf Polyethylenglykoloberfläche transportiert. Gold Nanopartikeln mit verschiedenen Größen und Strukturen wurden synthetisiert und charakterisiert um sie auf amino-funktionalisierter Siliziumoberfläche zu selbstorganisieren. Die Nanopartikeln wurden danach durch Linker Moleküle auf Polyethylenglykoloberfläche transportiert. Mikrostrukturen aus Gold Nanopartikeln auf Siliziumoberflächen wurde erreicht durch Mikrokontaktdrucken und Selbstorganisieren. Weiterhin wurden Mikrostrukruren aus Gold Nanopartikeln im PEG-Polymer, durch Füllen der PEG-Replikatkanäle mit Komposit aus Gold Nanopartikeln und PEG, erreicht. Spezifisch funktionalisierte Moleküle wurden entworfen und synthetisiert, um sie auf der Oberfläche der Gold-Nanopartikel zu adsorbieren.
The current project comprises the synthesis and characterization of different morphologies of metal nanoparticles (e.g. gold nanoparticles). These morphologies range from spherical nanoparticles to more complex one, such as one, two and three dimensional nanostructures, in addition, to hollow urchin-like nanoparticles. The different morphologies of metal nanostructures possess interesting physical properties indicated by the position of their localized surface plasmon resonance (LSPR) peak which appears in the visible to near IR region depending on the size, shape and composition of the nanostructure. Furthermore, metal nanoparticles show high chemical affinity toward specific chemical functionalities namely; thiol and amine. While thiol functionality tends to form strong and stable binding to metal nanoparticles, amine shows weak and reversible binding. The characteristic chemical features of those nanoparticles allow them to self-assemble onto amino functionalized silicon substrate. Further functionalization of those nanoparticles with specific linker molecule through thiol moiety from one side and double bond from the other side to be crosslinked with polyethylene glycol prepolymer under UV-light, allows their facial transference from silicon surface to the surface of PEG polymer. This strategy aims to create specific binding sites composed of different morphologies and compositions of nanostructures to anchor biofunctional molecules on a protein repellent background. The ultimate objective of this work is to generate a number of nano- and micro-patterned gold nanoparticles templates for controlled and selective immobilization of biofunctional molecules. Nano-pattern of gold nanoparticles was fabricated onto two dimensional conductive silicon substrate by “Block copolymer micelle” nanolithography with highly defined size, position and spaces between the nanoparticles. The nano-pattern was then transferred to the surface of PEG polymer using simple benchtop technique and without aid of any linker molecule by employing the swelling property of PEG hydrogel as a driving force for the transference process. Moreover, micro-pattern of gold nanoparticles on silicon was achieved by a novel method that combines µ-contact printing approach with self-assembly to produce micro-stripes of gold nanoparticles. While, the coverage of the nanoparticles is high within the micro-pattern, the nanoparticles were rarely found in the areas in between. Furthermore, micro-pattern of a composite material composed of Au NPs and PEG beside other areas of PEG was fabricated using Fill Molding In Capillaries of a PEG replica mold to obtain a micro-pattern of gold nanoparticles in three dimensional substrate. While gold nanoparticles serve as protein’s binding sites within the composite matrix, PEG hydrogel in the adjacent areas act as protein’s non-adhesive surface. As a model of fluorophore’s containing biofunctional molecules, an organic dye composed of fluorescein moiety were functionalized with an appropriate linker molecule. The linker shows selective binding to the surface of gold nanoparticles through thiol functionality, where the length of the linker was carefully adjusted to decrease fluorescence quenching that would result as a consequence of direct binding between the fluorophore and gold surface. The model aims to investigate our ability to resolve fluorescence light coming out of the adsorbed fluorophore on the nanoparticles. Green fluorescent protein was selected to represent biofunctional molecules to be immobilized onto our nano- and micro-patterned gold nanoparticles surfaces by getting the advantage that its immobilization event could be monitored through fluorescence microscopy. To achieve this goal thiolated nitrilotriacetic acid molecule (NTA) is being synthesized for specific binding to gold nanoparticles through thiol group. NTA-Ni(II) will reside at the other end of the linker for selective binding to his-tag protein in GFP. This concept of GFP immobilization is currently being worked on.