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Electroimmobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purification
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Technology, Department of Engineering Sciences. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Technology, Department of Engineering Sciences, Electronics. Fasta tillståndets elektronik.
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2005 (English)In: Anal Biochem, Vol. 341, no 1, 89-93 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2005. Vol. 341, no 1, 89-93 p.
Identifiers
URN: urn:nbn:se:uu:diva-26506OAI: oai:DiVA.org:uu-26506DiVA: diva2:54280
Available from: 2007-02-19 Created: 2007-02-19 Last updated: 2011-01-12
In thesis
1. Development of Electroacoustic Sensors for Biomolecular Interaction Analysis
Open this publication in new window or tab >>Development of Electroacoustic Sensors for Biomolecular Interaction Analysis
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biomolecular interaction analysis to determine the kinetics and affinity between interacting partners is important for the fundamental understanding of biology, as well as for the development of new pharmaceutical substances. A quartz crystal microbalance instrument suitable for kinetics and affinity analyses of interaction events was developed. The functionality of the sensor system was demonstrated by development of an assay for relative affinity determination of lectin-carbohydrate interactions.

Sensor surfaces allowing for effective immobilization of one interacting partner is a key functionality of a biosensor. Here, three different surfaces and immobilization methods were studied. First, optimized preparation conditions for sensor surfaces based on carboxyl-terminated self assembled monolayers were developed and were demonstrated to provide highly functional biosensor surfaces with low non-specific binding. Second, a method allowing for immobilization of very acidic biomolecules based on the use of an electric field was developed and evaluated. The electric field made it possible to immobilize the highly acidic C-peptide on a carboxylated surface. Third, a method for antibody immobilization on a carboxyl surface was optimized and the influence of immobilization pH on the immobilization level and antigen binding capacity was thoroughly assessed. The method showed high reproducibility for a set of antibodies and allowed for antibody immobilization also at low pH.

Three broadly different strategies to increase the sensitivity of electroacoustic sensors were explored. A QCM sensor with small resonator electrodes and reduced flow cell dimensions was demonstrated to improve the mass transport rate to the sensor surface. The use of polymers on QCM sensor surfaces to enhance the sensor response was shown to increase the response of an antibody-antigen model system more than ten-fold. Moreover, the application of high frequency thin film bulk acoustic resonators for biosensing was evaluated with respect to sensing range from the surface. The linear detection range of the thin film resonator was determined to be more than sufficient for biosensor applications involving, for instance, antibody-antigen interactions. Finally, a setup for combined frequency and resistance measurements was developed and was found to provide time resolved data suitable for kinetics determination.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 68 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 658
Keyword
biosensor, protein interactions, kinetics, affinity, QCM, quartz crystal microbalance, piezoelectric resonators, dissipation, motional resistance
National Category
Other Industrial Biotechnology Analytical Chemistry
Research subject
Engineering Science with specialization in Microsystems Technology
Identifiers
urn:nbn:se:uu:diva-107211 (URN)978-91-554-7572-7 (ISBN)
Public defence
2009-09-11, Å80101, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:30 (English)
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wisenet
Available from: 2009-08-20 Created: 2009-07-29 Last updated: 2014-11-25Bibliographically approved

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Andersson, Henrik

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