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A particulate platform for bioluminescent immunosensing
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry. (Biologisk struktur och funktion)
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2007 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 22, 8601-8607 p.Article in journal (Refereed) Published
Abstract [en]

The present study examines pyruvate kinase-conjugated antibodies for potential use in EUSA applications. The conjugates had an acceptable stability, and the coupling inflicted only minor impairment on the kinase activity. To mimic the setup of an immunoassay under development, a test antigen (BSA) was attached to polystyrene nanoparticles. This arrangement was found to be suitable as solid support for presentation of antigens in sensitive bioluminescence assays. The nanoparticles were well characterized in terms of protein surface load and were used to establish the number of conjugate complexes needed to generate a detectable signal. Under the biochemical conditions employed here, the detection limit of the pyruvate kinase conjugate lies in the femtomole range.

Place, publisher, year, edition, pages
2007. Vol. 79, no 22, 8601-8607 p.
Keyword [en]
Enzyme, Transferases, Biosensor, Chemical sensor, Pyruvate kinase, Detection limit, Protein, Chemical analysis, Bioluminescence, Nanoparticle, Immunological method, Stability, Antibody, Immunosensor
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-96290DOI: 10.1021/ac0715118ISI: 000250937500023PubMedID: 17883280OAI: oai:DiVA.org:uu-96290DiVA: diva2:170812
Available from: 2007-10-17 Created: 2007-10-17 Last updated: 2011-01-18Bibliographically approved
In thesis
1. Nanoscale Reaction Systems
Open this publication in new window or tab >>Nanoscale Reaction Systems
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system.

The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 66 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 350
Life sciences and physical sciences, nanoparticles, Sedimentation Field-Flow Fractionation, bioaffinity, multilayered functionalization, protein attachment, diagnostics, bioluminescence, NATURVETENSKAP
urn:nbn:se:uu:diva-8249 (URN)978-91-554-6983-2 (ISBN)
Public defence
2007-11-08, B42, BMC, Uppsala, 13:15
Available from: 2007-10-17 Created: 2007-10-17Bibliographically approved

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