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Controlled Orientation and Covalent Attachment of Proteins on Biosensor Surfaces by Chelation Assisted Photoimmobilization
Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, 52074 Aachen, Germany.
Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
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2013 (English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
2013.
Keyword [en]
Biosensor, Surface chemistry, Protein immobilization, Orientation
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:liu:diva-87929OAI: oai:DiVA.org:liu-87929DiVA: diva2:601398
Available from: 2013-01-29 Created: 2013-01-28 Last updated: 2013-01-31
In thesis
1. Biosensor surface chemistry for oriented protein immobilization and biochip patterning
Open this publication in new window or tab >>Biosensor surface chemistry for oriented protein immobilization and biochip patterning
2013 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

This licentiate thesis is focused on two methods for protein immobilization to biosensor surfaces for future applications in protein microarray formats. The common denominator is a surface chemistry based on a gold substrate with a self-assembled monolayer (SAM) of functionalized alkanethiolates. Both methods involve photochemistry, in the first case for direct immobilization of proteins to the surface, in the other for grafting a hydrogel, which is then used for protein immobilization.

Paper I describes the development and characterization of Chelation Assisted Photoimmobilization (CAP), a three-component surface chemistry that allows for covalent attachment and controlled orientation of the immobilized recognition molecule (ligand) and thereby provides a robust sensor surface for detection of analyte in solution. The concept was demonstrated using His-tagged IgG-Fc as the ligand and protein A as the analyte. Surprisingly, as concluded from IR spectroscopy and surface plasmon resonance (SPR) analysis, the binding ability of this bivalent ligand was found to be more than two times higher with random orientation obtained by amine coupling than with homogeneous orientation obtained by CAP. It is suggested that a multivalent ligand is less sensitive to orientation effects than a monovalent ligand and that island formation of the alkanethiolates used for CAP results in a locally high ligand density and steric hindrance.

Paper II describes the development of nanoscale hydrogel structures. These were photografted on a SAM pattern obtained by dip-pen nanolithography (DPN) and subsequent backfilling. The hydrogel grew fast on the hydrophilic patterns and slower on the hydrophobic background, which contained a buried oligo(ethylene glycol) (OEG) chain. Using IR spectroscopy, it was found that the OEG part was degraded during UV light irradiation and acted as a sacrificial layer. In this process other OEG residues were exposed and acted as new starting points for the self-initiated photografting and photopolymerization (SIPGP). A biotin derivative was immobilized to the hydrogel density pattern and interaction with streptavidin was demonstrated by epifluorescence microscopy.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. 50 p.
Series
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1573
Keyword
Biochip, biosensor, hydrogel, ligand, orientation, patterning, photochemistry, protein immobilization, surface chemistry, self-assembled monolayer, SAM
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-88102 (URN)978-91-7519-698-5 (ISBN)
Presentation
2013-02-28, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 13:15 (English)
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Available from: 2013-01-31 Created: 2013-01-30 Last updated: 2013-01-31Bibliographically approved

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Ericsson, EmmaEnander, KarinBui, LanLundström, IngemarKonradsson, PeterLiedberg, Bo

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