A novel biotinylated surface designed for QCM-D applications
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Control of protein immobilization at sensor surfaces is of great interest within various scientific fields, since it enables studies of specific biomolecular interactions. To achieve this, one must be able to immobilize proteins with retained native structure, while minimizing non-specific protein binding. The high affinity interaction between streptavidin (SA) and biotin is extensively used as a linker between a surface, where SA is immobilized, and the (biotinylated) molecule of interest. Self- assembled monolayers (SAMs) of poly- and oligo ethylene glycol (PEG and OEG) derivatives have been proven in literature to minimize non-specific protein binding, and biotin-exposing SAMs have been shown efficient for immobilization of SA.
The aim of this master's thesis project was to develop biotinylated gold surfaces for quartz crystal microbalance with dissipation monitoring (QCM-D) applications through the self-assembly of mixed monolayers of thiolated OEG (or PEG) derivatives with or without a terminal biotin head group. For this, different thiol compounds were to be compared and evaluated. For the systems under study, the required biotin density for maximum specific SA immobilization was to be established, while keeping the non-specific serum adsorption at a minimum. Model experiments with biotinylated proteins immobilized to the SA-functionalized surfaces were to be performed to evaluate the possibilities for commercialization.
A protocol for the preparation of a novel biotinylated surface was developed based on the immersion of gold substrates in an ethanolic incubation solution of dithiols with OEG chains (SS-OEG and SS-OEG-biotin, 99:1) and found to give reproducible results with respect to low non-specific protein binding and immobilization of a monolayer of SA. The modified surfaces allowed for subsequent immobilization of biotinylated bovine serum albumin (bBSA) and biotinylated plasminogen (bPLG). PLG was the subject of a challenging case study, using a combination of QCM-D and surface plasmon resonance (SPR), where the immobilized protein was subjected to low molecular weight ligands that were believed to induce conformational changes. The high control of the surface chemistry allowed for the interpretation of the increased dissipation shift upon ligand binding in terms of conformational changes.
An obstacle before commercialization of the described biotinylated surfaces is that they do not seem stable for storage > 7 days. The reasons for this have to be investigated further.
Place, publisher, year, edition, pages
2009. , 57 p.
Quartz crystal microbalance with dissipation monitoring, QCM-D, Self-assembled monolayer, SAM, thiol, ethylene glycol, OEG, PEG, biosensor, surface chemistry, biotin, streptavidin, ellipsometry, contact angle goniometry, surface plasmon resonance, SPR, impedance spectroscopy, IS, sensor surface
Atom and Molecular Physics and Optics
IdentifiersURN: urn:nbn:se:liu:diva-19250ISRN: LITH-IFM-A-EX--09/2199--SEOAI: oai:DiVA.org:liu-19250DiVA: diva2:223812
Subject / course
Master of Science
UppsokPhysics, Chemistry, Mathematics
Svedhem, Sofia, PhD
Enander, Karin, PhD