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Generic Phosphatase Activity Detection using Zinc Mediated Aggregation Modulation of Polypeptide-Modified Gold Nanoparticles
Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-7001-9415
2014 (English)In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 6, no 23, 14204-14212 p.Article in journal (Refereed) Published
Abstract [en]

A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme. Phosphatase activity generates inorganic phosphate that forms an insoluble complex with Zn2+. In a sample containing a preset concentration of Zn2+, phosphatase activity will markedly reduce the concentration of dissolved Zn2+ from the original value, which in turn affects the aggregation of gold nanoparticles functionalized with a designed Zn2+ responsive polypeptide. The change in nanoparticle stability thus provides a rapid and sensitive readout of the phosphatase activity. The assay is not limited to a particular enzyme or enzyme substrate, which is demonstrated using three completely different phosphatases and five different substrates, and thus constitutes a highly interesting system for drug screening and diagnostics.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2014. Vol. 6, no 23, 14204-14212 p.
Keyword [en]
Phosphatase, gold nanoparticle, assay, chelation, polypeptide, zinc
National Category
Chemical Sciences
URN: urn:nbn:se:liu:diva-106718DOI: 10.1039/c4nr02791dISI: 000344997600018OAI: diva2:718089
Available from: 2014-05-19 Created: 2014-05-19 Last updated: 2015-02-25Bibliographically approved
In thesis
1. Polypeptide functionalized gold nanoparticles for bioanalytical applications
Open this publication in new window or tab >>Polypeptide functionalized gold nanoparticles for bioanalytical applications
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Detection strategies that allow for simple, rapid, cost efficient and sensitive monitoring of proteins and their interactions with biomolecules are of great importance in drug development and diagnostics. This thesis describes the development of bioanalytical applications based on the tunable self-assembly of gold nanoparticles functionalized with a de novo designed polypeptide. Strategies for protein affinity sensing and for detection of several fundamentally important biological processes have been investigated, including Zn2+-mediated coordination between polypeptides and low molecular weight chelants and protease and phosphatase activity.

A Zn2+ responsive synthetic polypeptide designed to fold into a helix-loop-helix motif and dimerize into a four-helix bundle has been used to control the stability and self-assembly of gold nanoparticles. This polypeptide has a high negative net charge at neutral pH as a consequence of its many glutamic acid residues, efficiently preventing folding and dimerization due to charge repulsion. Zn2+ coordination provides a means to trigger folding and dimerization at neutral pH. The polypeptide can be readily attached to gold nanoparticles via a cysteine residue in the loop region, retaining its folding properties and responsiveness to Zn2+. The polypeptide functionalized gold nanoparticles display excellent colloidal stability but aggregate reversibly after addition of millimolar concentrations of Zn2+. Aggregates are dense with a defined interparticle distance corresponding to the size of the four-helix bundle, resulting in a distinct red shift of the localized surface plasmon resonance band.

Three completely different strategies for colorimetric biosensing have been developed, all being based on the same responsive hybrid nanomaterial. In the first strategy a synthetic receptor was co-immobilized on the gold nanoparticles together with the Zn2+ responsive polypeptide. Protein analyte binding to the receptor could be detected as this interaction sterically prevented aggregation induced by Zn2+. In the second strategy the reduction in colloidal stability caused by specific proteolytic cleavage of the immobilized polypeptide was exploited to monitor the enzymatic activity. The third strategy utilized the sensitivity of the system to small variations in Zn2+ concentration. The presence of low molecular weight chelants was found to influence the mode of aggregation, both by sequestering Zn2+ and through the formation of ternary complexes involving the polypeptides, which prevented dimerization and thus aggregation. This approach was further developed into a generic concept for phosphatase detection exploiting the different affinity of enzyme substrates and reaction products for Zn2+.

The flexibility of the different detection schemes enables detection of a large number of analytes by exploiting the tunable stability of the nanoparticles and the possibilities to effectively decouple the recognition event and the nanoparticle stability modulation.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. 62 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1598
National Category
Natural Sciences
urn:nbn:se:liu:diva-106719 (URN)10.3384/diss.diva-106719 (DOI)978-91-7519-321-2 (print) (ISBN)
Public defence
2014-06-12, Plank, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (English)
Available from: 2014-05-19 Created: 2014-05-19 Last updated: 2014-10-08Bibliographically approved

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