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Polypeptide functionalized gold nanoparticles for bioanalytical applications
Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, The Institute of Technology.
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.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1598
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-106719DOI: 10.3384/diss.diva-106719ISBN: 978-91-7519-321-2 (print)OAI: oai:DiVA.org:liu-106719DiVA: diva2:718097
Public defence
2014-06-12, Plank, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (English)
Opponent
Supervisors
Available from: 2014-05-19 Created: 2014-05-19 Last updated: 2014-10-08Bibliographically approved
List of papers
1. Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors
Open this publication in new window or tab >>Colorimetric Protein Sensing by Controlled Assembly of Gold Nanoparticles Functionalized with Synthetic Receptors
Show others...
2009 (English)In: Small, ISSN 1613-6810, Vol. 5, no 21, 2445-2452 p.Article in journal (Refereed) Published
Abstract [en]

A strategy for colorimetric sensing of proteins, based on the induced assembly of polypeptide-functionalized gold nanoparticles, is described. Recognition was accomplished using a polypeptide sensor scaffold designed to specifically bind the model analyte, human carbonic anhydrase II (HCAII). The extent of particle aggregation, induced by the Zn2+-triggered dimerization and folding of a second polypeptide also present on the surface of the gold nanoparticle, gave a readily detectable colorimetric shift that was dependent on the concentration of the target protein. In the absence of HCAII, particle aggregation resulted in a major redshift of the plasmon peak whereas analyte binding prevented formation of dense aggregates, significantly reducing the magnitude of the redshift. The limit of detection of HCAII was estimated to be around 15 nM. The versatility of the technique was demonstrated using a second model system based on the recognition of a peptide sequence from the tobacco mosaic virus coat protein (TMVP by a recombinant antibody fragment. This strategy is proposed as a generic platform for robust and specific protein analysis that can be further developed for monitoring a wide range of target proteins.

Keyword
Not available.
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-15122 (URN)10.1002/smll.200900530 (DOI)
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2015-05-29Bibliographically approved
2. Peptide functionalized gold nanoparticles for colorimetric detection of matrilysin (MMP-7) activity
Open this publication in new window or tab >>Peptide functionalized gold nanoparticles for colorimetric detection of matrilysin (MMP-7) activity
2013 (English)In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 5, no 19, 8973-8976 p.Article in journal (Refereed) Published
Abstract [en]

A peptide with two cleavage sites for MMP-7 has been synthesized and immobilized on gold nanoparticles (AuNPs) through a cysteine residue. Digestion of the peptide by MMP-7 decreases its size and net charge, which leads to the aggregation of the AuNPs. The color shift caused by aggregation enables a direct and quantitative measurement of the concentration and activity of MMP-7 with an estimated limit of detection of 5 nM (0.1 μg mL−1).

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2013
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-99413 (URN)10.1039/c3nr03006g (DOI)000324500900027 ()
Note

Funding Agencies|School of Materials Science and Engineering, Nanyang Technological University, Singapore||Provost Office, Nanyang Technological University, Singapore||Swedish Foundation for Strategic Research (SSF)||Knut and Alice Wallenberg Foundation (KAW), CeNano||Swedish Research Council (VR)||

Available from: 2013-10-17 Created: 2013-10-17 Last updated: 2017-12-06
3. Probing Zinc-Protein-Chelant Interactions using Gold Nanoparticles Functionalized with Zinc-Responsive Polypeptides
Open this publication in new window or tab >>Probing Zinc-Protein-Chelant Interactions using Gold Nanoparticles Functionalized with Zinc-Responsive Polypeptides
2014 (English)In: Particle & particle systems characterization, ISSN 0934-0866, E-ISSN 1521-4117, Vol. 31, no 11, 1127-1133 p.Article in journal (Refereed) Published
Abstract [en]

The coordination of zinc by proteins and various other organic molecules is essential for numerous biological processes, such as in enzymatic catalysis, metabolism and signal transduction. Presence of small molecular chelants can have a profound effect on the bioavailability of zinc and affect critical Zn2+-protein interactions. Zn2+ chelators are also emerging therapeutics for Alzheimer’s diseases because of their preventive effect on zinc promoted amyloid formation. Despite the importance of zinc-protein-chelant interactions in biology and medicine, probing such interactions is  challenging. Here, we introduce an innovative approach for real-time characterization of zinc-protein-chelant interactions using gold nanoparticles (AuNPs) functionalized with a zinc-responsive protein mimetic polypeptide. The peptide functionalized AuNPs aggregate extensively in the presence of Zn2+, triggered by specific Zn2+-mediated polypeptide dimerization and folding, causing a massive red shift of the plasmon band. Chelants affects the Zn2+- polypeptide interaction and thus the aggregation differently depending on their concentrations, zincbinding affinities and coordination numbers, which affect the position of the plasmon band. This system is a simple and powerful tool that provides extensive information about the interactions of chelants in the formation of Zn2+ coordination complexes and is an interesting platform for development of bioanalytical techniques and characterization of chelation-based therapeutics.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2014
Keyword
Gold nanoparticles, zinc, peptide, chelation
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-106717 (URN)10.1002/ppsc.201400082 (DOI)000344681700003 ()
Available from: 2014-05-19 Created: 2014-05-19 Last updated: 2017-12-05Bibliographically approved
4. Generic Phosphatase Activity Detection using Zinc Mediated Aggregation Modulation of Polypeptide-Modified Gold Nanoparticles
Open this publication in new window or tab >>Generic Phosphatase Activity Detection using Zinc Mediated Aggregation Modulation of Polypeptide-Modified Gold Nanoparticles
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
Keyword
Phosphatase, gold nanoparticle, assay, chelation, polypeptide, zinc
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-106718 (URN)10.1039/c4nr02791d (DOI)000344997600018 ()
Available from: 2014-05-19 Created: 2014-05-19 Last updated: 2017-12-05Bibliographically approved

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