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.

A Prototype Antibody-based Biosensor for Measurement of Salivary MMP-8 in Periodontitis using Surface Acoustic Wave Technology.

Periodontitis is an economically important disease which is highly prevalent worldwide. Current diagnostic approaches are time-consuming and require interpretation of multiple aspects of clinical and radiographic assessment. Chair-side monitoring of inflammatory mediators of periodontitis could provide immediate information about disease activity, which can inform patient management. We aimed to develop a novel prototype biosensor to measure salivary matrix metalloproteinase-8 (MMP-8) using specific antibodies and surface acoustic wave (SAW) technology. The analytical performance of the prototype biosensor was compared to standard enzyme-linked immunosorbent assay (ELISA) using unstimulated saliva samples obtained from patients with periodontitis before and after non-surgical treatment (N = 58), patients with gingivitis (N = 54) and periodontally healthy volunteers (N = 65). Receiver operator characteristic (ROC) analysis for distinguishing periodontitis from health revealed an almost identical performance between the sensor and ELISA assays (area under curve values (AUC): ELISA 0.93; SAW 0.89). Furthermore, both analytical approaches yielded readouts which distinguished between heath, gingivitis and periodontitis, correlated identically with clinical measures of periodontal disease and recorded similar post-treatment decreases in salivary MMP-8 in periodontitis. The assay time for our prototype device is 20 minutes. The prototype SAW biosensor is a novel and rapid method of monitoring periodontitis which delivers similar analytical performance to conventional laboratory assays.

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 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.

Engineered self-assembling monolayers for label free detection of influenza nucleoprotein.

Integrating nanotechnology into useable devices requires a combination of bottom up and top down methodology. Often the techniques to measure and control these different components are entirely different, so methods that can analyse the nanoscale component in situ are of increasing importance. Here we describe a strategy that employs a self-assembling monolayer of engineered protein chimeras to display an array of oriented antibodies (IgG) on a microelectronic device for the label free detection of influenza nucleoprotein. The structural and functional properties of the bio-interface were characterised by a range of physical techniques including surface plasmon resonance, quartz-crystal microbalance and neutron reflectometry. This combination of methods reveals a 13.5 nm thick engineered-monolayer that (i) self-assembles on gold surfaces, (ii) captures IgG with high affinity in a defined orientation and (iii) specifically recognises the influenza A nucleoprotein. Furthermore we also show that this non-covalent self-assembled structure can render the dissociation of bound IgG irreversible by chemical crosslinking in situ without affecting the IgG function. The methods can thus describe in detail the transition from soluble engineered molecules with nanometre dimensions to an array that demonstrates the principles of a working influenza sensor.

Reversible non-stick behaviour of a bacterial protein polymer provides a tuneable molecular mimic for cell and tissue engineering.

Yersina pestis, the bubonic plague bacterium, is coated with a polymeric protein hydrogel for protection from host defences. The protein, which is robust and non-stick, resembles structures found in many eukaryotic extracellular-matrix proteins. Cells grown on the natural polymer cannot adhere and grow poorly; however, when cell-adhesion motifs are inserted into the protein, the cells proliferate.

Self-assembly of protein monolayers engineered for improved monoclonal immunoglobulin G binding.

Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG) to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated.

The structural orientation of antibody layers bound to engineered biosensor surfaces.

This paper describes a membrane protein array that binds immunoglobulin G at its constant regions whilst leaving the variable regions free to bind antigen. The scaffold of the array is the transmembrane domain of outer membrane protein A (tOmpA) from Escherichia coli engineered to assemble as an oriented monolayer on gold surfaces via a single cysteine residue. Other protein domains can be fused to the N and C termini of the scaffold. In this study we use circularly permuted ctOmpA fused to two Z domains of Staphylococcus aureus protein A (ZZctOmpA) to create the immunoglobulin G-binding array. The solution structure of the engineered proteins was assessed by circular dichroism spectroscopy. Assembly of the array, attachment of antibodies and antigen binding were measured using surface plasmon resonance and neutron reflection. Compared to mouse IgG2, polyclonal IgG from rabbit bound very strongly to ZZctOmpA and the dissociation of the immunoglobulin was slow enough to allow neutron reflection studies of the assembled layer with antigen. Using both magnetic and isotopic contrasts a complete layer by layer model was defined which revealed that the 223 Å high layer contains antibodies in an upright orientation.

Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reflection.

Protein arrays are used in a wide range of applications. The array described here binds IgG antibodies, produced in rabbit, to gold surfaces via a scaffold protein. The scaffold protein is a fusion of the monomeric E. coli porin outer membrane protein A (OmpA) and the Z domain of Staphylococcus aureus protein A. The OmpA binds to gold surfaces via a cysteine residue in a periplasmic turn and the Z domain binds immunoglobulins via their constant region. Polarised Neutron Reflection is used to probe the structure perpendicular to the gold surface at each stage of the assembly of the arrays. Polarised neutrons are used as this provides a means of achieving extra contrast in samples having a magnetic metal layer under the gold surface. This contrast is attained without resorting to hydrogen/deuterium exchange in the biological layer. Polarised Neutron Reflection allows for the modelling of many and complex layers with good fits. The total thickness of the biological layer immobilised on the gold surface is found to be 187 A and the layer can thus far be separated into its lipid, protein and solvent parts.

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides.

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.

A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans.

Streptococcus mutans produces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called 'A' repeats. The S. mutans genome sequence was searched for ORFs containing 'A' repeats, and one novel gene, gbpD, which appears to be unique to the mutans group of streptococci, was identified. The GbpD sequence revealed the presence of three 'A' repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with a K(D) of 2-3 nM. Construction of truncated derivatives of GbpD confirmed that the 'A' repeat region was essential for binding. Furthermore, a gbpD knockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose. The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to the alpha/beta hydrolase family (including lipases and carboxylesterases). GbpD contains the two regions typical of these enzymes: a GxSxG active site 'lipase box' and an 'oxyanion hole'. GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity. The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid. The gbpD mutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity of S. mutans and another plaque bacterium, Streptococcus sanguinis. GbpD bound to and released FFA from lipoteichoic acid (LTA) of S. sanguinis, but had no effect on LTA from S. mutans. These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque biofilm.