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

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 <i>Escherichia coli</i> outer membrane protein A (OmpA_TM) to create protein–AuNP conjugates. The addition of a single cysteine residue into a periplasmic loop, to create cysOmpA_TM, drives oriented assembly and increased equilibrium binding. As the protein surface concentration increases, the sulfur–gold bond in cysOmpA_TM creates a more densely populated AuNP surface than the poorly organized wtOmpA_TM layer. The functionalization of AuNP improved both their stability and homogeneity. This was further exploited using multidomain protein chimeras, based on cysOmpA_TM, 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)-cysOmpA_TM fusion protein, bound to AuNP, detected influenza A nucleoprotein, a widely used antigen in diagnostic assays. Finally, using the same scFv-cysOmpA_TM–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

Fully Aqueous Self-Assembly of a Gold-Nanoparticle-Based Pathogen Sensor

Surface plasmon resonance (SPR) is a very sensitive measure of biomolecular interactions but is generally too expensive for routine analysis of clinical samples. Here we demonstrate the simplified formation of virus-detecting gold nanoparticle (AuNP) assemblies on glass using only aqueous buffers at room temperature. The AuNP assembled on silanized glass and displayed a distinctive absorbance peak due to the localized SPR (LSPR) response of the AuNPs. Next, assembly of a protein engineering scaffold was followed using LSPR and a sensitive neutron reflectometry approach, which measured the formation and structure of the biological layer on the spherical AuNP. Finally, the assembly and function of an artificial flu sensor layer consisting of an in vitro-selected single-chain antibody (scFv)-membrane protein fusion was followed using the LSPR response of AuNPs within glass capillaries. In vitro selection avoids the need for separate animal-derived antibodies and allows for the rapid production of low-cost sensor proteins. This work demonstrates a simple approach to forming oriented arrays of protein sensors on nanostructured surfaces that uses (i) an easily assembled AuNP silane layer, (ii) self-assembly of an oriented protein layer on AuNPs, and (iii) simple highly specific artificial receptor proteins