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Modular Protein Engineering Approach to the Functionalization of Gold Nanoparticles for Use in Clinical Diagnostics

Lookup NU author(s): Tim Robson, Dr Alexandra Solovyova, Professor Jeremy LakeyORCiD

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

Functional protein–gold nanoparticle (AuNP) conjugates have a wide variety of applications including biosensing and drug delivery. Correct protein orientation, which is important to maintain functionality on the nanoparticle surface, can be difficult to achieve in practice, and dedicated protein scaffolds have been used on planar gold surfaces to drive the self-assembly of oriented protein arrays. Here we use the transmembrane domain of Escherichia coli outer membrane protein A (OmpATM) to create protein–AuNP conjugates. The addition of a single cysteine residue into a periplasmic loop, to create cysOmpATM, drives oriented assembly and increased equilibrium binding. As the protein surface concentration increases, the sulfur–gold bond in cysOmpATM creates a more densely populated AuNP surface than the poorly organized wtOmpATM layer. The functionalization of AuNP improved both their stability and homogeneity. This was further exploited using multidomain protein chimeras, based on cysOmpATM, which were shown to form ordered protein arrays with their functional domains displayed away from the AuNP surface. A fusion with protein G was shown to specifically bind antibodies via their Fc region. Next, an in vitro selected single chain antibody (scFv)-cysOmpATM fusion protein, bound to AuNP, detected influenza A nucleoprotein, a widely used antigen in diagnostic assays. Finally, using the same scFv-cysOmpATM–AuNP conjugates, a prototype lateral flow assay for influenza demonstrated the utility of fully recombinant self-assembling sensor layers. By simultaneously removing the need for both animal antibodies and a separate immobilization procedure, this technology could greatly simplify the development of a range of in vitro diagnostics.


Publication metadata

Author(s): Robson T, Shah DSH, Solovyova AS, Lakey JH

Publication type: Article

Publication status: Published

Journal: ACS Applied Nano Materials

Year: 2018

Volume: 1

Issue: 7

Pages: 3590-3599

Print publication date: 27/07/2018

Online publication date: 28/06/2018

Acceptance date: 28/06/2018

Date deposited: 13/08/2018

ISSN (electronic): 2574-0970

Publisher: American Chemical Society

URL: https://doi.org/10.1021/acsanm.8b00737

DOI: 10.1021/acsanm.8b00737


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Funding

Funder referenceFunder name
064345/Z/01/Z
Wellcome Trust

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