| 1. |
- Bui, Lan, 1981-
(författare)
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Synthesis of Substituted Alkanethiols Intended for Protein Immobilization : Chelate Associated Photochemistry (CAP)
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The first and main part of this thesis is focused on the design and synthesis of photo-activableand metal chelating alkanethiols. Chelate associated photochemistry (CAP) is a novel conceptof combining two well-known protein (ligand) immobilization strategies to obtain a sensorsurface of covalently bound ligand with defined orientation. This includes nitrilotriacetic acid(NTA) which is used to capture and pre-orientate histidine-tagged proteins to the sensorsurface, followed by UV activation of a neighboring photo-crosslinking agent, benzophenone(BP), to covalently bind the ligand in this favorable orientation. Our results (paper 1) indicatethat up to 55% more activity of the ligand is achieved with the CAP concept compared to theactivity of the randomly oriented ligand (immobilized only by BP). This also yields a surfacethat is more robust compared to if only NTA is used. The photo cross-linking withbenzophenone (BP) adduct is limited to a distance range of 3Å, it is therefore favorable tocapture the ligand before reacting with surface bound BP-adduct. The surface consists of anexcess of ethylene glycols (known for its protein-repellent properties) to prevent non-specificprotein binding, thereby increase the specificity of the sensor surface. With this obtainedsurface chemistry we hope to contribute to the development of large-scale screening systemsand microarrays based on His-tagged labeled biomolecules. This will be used in a number ofapplications such as proteomics-related applications, including drug discovery, the discoveryof lead compounds and characterization of protein-protein interactions.The second part of this thesis describes the effect of the synthetic N-(3-oxododecanoyl)-Lhomoserinelactone (30-C12-HSL) on eukaryotic cells. 30-C12-HSL is a natural occurringsignal substance in the bacterium Pseudomonas aeruginosa, and this signal molecule isinvolved in the regulation of bacterial growth. Pseudomonas aeruginosa has been consideredas a common cause of infections in hospitals, especially in patients with compromisedimmune systems. Since the 30-C12-HSL can diffuse freely cross the cell membranes, it isexpected to have influence on the host cell behaviour. Herein, we study how the eukaryoticcells respond to the bacterial signal molecule, 30-C12-HSL. Our results (paper 2) indicatethat 30-C12-HSL disrupt the adherens junctions in human epithelial cells. The disruption iscaused by a hyperphosphorylation of the adherens junction proteins (protein complexbetween epithelial tissues). This suggests the bacterial signals are sensed by that the host cells.
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| 2. |
- Ericsson, Emma, 1981-, et al.
(författare)
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Controlled Orientation and Covalent Attachment of Proteins on Biosensor Surfaces by Chelation Assisted Photoimmobilization
- 2013
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- This report presents a novel method for uniform orientation and covalent attachment of proteins to sensing surfaces, termed Chelation Assisted Photoimmobilization (CAP). Alkanethiols terminated with either nitrilotriacetic acid (NTA), benzophenone (BP) or oligo(ethylene glycol) were synthesized and mixed self-assembled monolayers (SAMs) were prepared on gold and thoroughly characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry and contact angle goniometry. In the process of CAP, NTA chelates Ni2+ and the complex coordinates a His-tagged ligand in an oriented assembly. The ligand is then photoimmobilized via BP, which forms covalent bonds upon UV light activation. The CAP concept was demonstrated using human IgG-Fc modified with C-terminal hexahistidine tags (His-IgGFc) as the ligand and protein A as the analyte.In the development of affinity biosensors, uniform orientation of ligand molecules where all analyte binding sites are accessible is often preferred to random orientation. In order to monitor the effect of ligand orientation on analyte response, the ligand-analyte interaction was quantified by surface plasmon resonance analysis, both in the case of CAP and when the ligand was attached by conventional amine coupling on surfaces presenting NTA. Responses were adjusted for differences in ligand immobilization level using IRAS. The normalized analyte response with randomly oriented ligand was 2.5 times higher than that with ligand immobilized by CAP, probably due to molecular crowding effects on the surface and the fact that His-IgGFc is bivalent for protein A. This is a reminder that many other factors than orientation alone may play a decisive role in analyte binding on biosensor surfaces.
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| 3. |
- Ericsson, Emma, et al.
(författare)
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Oriented Protein Immobilization by Chelate Associated Photochemistry
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We demonstrate herein the synthesis, characterization and application of a novel chelateassociated photochemistry (CAP) for oriented and robust attachment of biomolecular ligandsto sensing surfaces. The chelation agent is nitrilotriacetic acid (NTA) which is capable ofcoordinating two histidine (His) molecules in the presence of Nickel. Therefore a ligandmodified with a His-sequence can be attached to NTA to form an oriented assembly ofligands on the sensor surface. The ligand is then covalently bound to the surface via a nearbyphotolabile benzophenone (BP) which attacks C-H bonds upon UV light activation. Theligand is then available for analyte interaction. The synthesized compounds used in this studyare based on the well-known organosulphur surface chemistry for proper attachment to goldsurfaces. Besides the two BP and NTA alkane thiols/disulphides we also synthesized a fillermolecule with an oligo (ethylene glycol) (OEG) tail to fine tune the surface composition andto reduce non-specific binding. Results from surface plasmon resonance (SPR) measurementsusing a Biacore 3000 instrument indicate that up to 55% larger analyte response is obtainedwith CAP as compared to the response obtained with the random orientation achieved byphotoimmobilization alone.
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