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Oriented Protein Immobilization by Chelate Associated Photochemistry
Linköpings universitet, Institutionen för fysik, kemi och biologi, Sensorvetenskap och Molekylfysik. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Sensorvetenskap och Molekylfysik. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
Vise andre og tillknytning
(engelsk)Manuskript (Annet vitenskapelig)
Abstract [en]

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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Identifikatorer
URN: urn:nbn:se:liu:diva-20647OAI: oai:DiVA.org:liu-20647DiVA, id: diva2:235434
Tilgjengelig fra: 2009-09-16 Laget: 2009-09-16 Sist oppdatert: 2013-01-30
Inngår i avhandling
1. Synthesis of Substituted Alkanethiols Intended for Protein Immobilization: Chelate Associated Photochemistry (CAP)
Åpne denne publikasjonen i ny fane eller vindu >>Synthesis of Substituted Alkanethiols Intended for Protein Immobilization: Chelate Associated Photochemistry (CAP)
2009 (engelsk)Licentiatavhandling, med artikler (Annet vitenskapelig)
Abstract [en]

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.

sted, utgiver, år, opplag, sider
Linköping: Linköping University Electronic Press, 2009. s. 26
Serie
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1411
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-20648 (URN)LIU-TEK-LIC-2009:19 (Lokal ID)978-91-7393-540-1 (ISBN)LIU-TEK-LIC-2009:19 (Arkivnummer)LIU-TEK-LIC-2009:19 (OAI)
Presentation
2009-10-02, Jordan-Fermi, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2009-09-16 Laget: 2009-09-16 Sist oppdatert: 2020-03-10bibliografisk kontrollert

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