Development of enzymatic assays using a universal detection system based on nanotechnology

Abstract

Nanotechnology offers the technological potential that can be harnessed to deal with the diagnostic challenges in resource-constrained settings. Optical biosensors based on nanotechnology have being explored for biomedical analysis as they are cheaper and use readily available instruments. Signal transduction has been based on metallic nanoparticles in biosensors for optical detection which are simple, rapid and cost-effective. Gold nanostars (AuNSs) were used as a scaffold to design a universal detection system based on enzymeguided changes in nanosensors. The detection was based on H2O2-mediated growth/shapealteration of gold nanostars resulting in colorimetric and spectrophometric changes. This detection strategy enabled the fabrication of two oxidase-based biosensors for glucose and cholesterol which were simple in design, sensitive and rapid in detection, and overall highthroughput. Both were colorimetric and utilised a basic entry-level laboratory spectrophotometer plate reader for analysis. Although a number of synthetic approaches for AuNSs have been reported, the choice of synthesis method depends on a number of experimental parameters and downstream application. Thus, there are still gaps for methods that are appropriate, simple and produce AuNSs suited to their intended purposes. I therefore developed a seedless synthesis strategy for AuNSs that has the advantages of the seeded methods. The method used ascorbic acid as a reducing agent and silver nitrate as an anisotropic growth control assisting agent. AuNSs with multiple branches and diameter of 59 nm were produced. They showed good stability when capped with PVP and modified with an enzyme in relatively strong ionic conditions. I investigated their application in plasmonic sensing by modifying them with glucose oxidase and detection of glucose. The AuNSs were found to be a good scaffold for the enzyme, proved to stable and sensitive as transducers. Thus, the AuNSs showed good promise for further applications in plasmonic biosensing for in vivo biomedical diagnosis. Gold nanoparticles provide a user-friendly and efficient surface for immobilisation of enzymes and proteins. However, one of the major limitations for the implementation of nanobiosensors in clinical use are related to biofunctionalisation of biorecognition elements such as enzymes and antibodies. I designed a novel approach for enzyme bioconjugation to AuNSs where the nanostars were modified with L-cysteine and covalently bound to N-hydroxysulfosuccinimide (sulfo-NHS) activated intermediate glucose oxidase (GOx) to create a stable and sensitive AuNSs-Cys-GOx bioconjugate complex. This strategy demonstrated potential for increased attachment affinity without protein adsorption onto the AuNSs surface. Good dispersity in buffer suspension was observed, as well as stability in high ionic environments. Greater sensitivity in the determination of low concentrations of glucose based on plasmonic and colorimetric detection was observed using the AuNSs-Cys-GOx bioconjugates. Such a novel approach for enzyme immobilisation could lead to the production of nanoparticle-enzyme conjugates could be used in nanobiosensors with real clinical samples for biomedical analyses. Despite the progress made on the design of novel plasmonic colorimetric biosensors there are still significant challenges in their practical application in clinical samples. I optimised and developed a glucose biosensor based on biocatalytic shape-altering of gold nanostars via silver deposition in serum. Improved sensitivity was observed due to nanostars clustering after being functionalised with glucose oxidase (GOx). The biosensor quantified glucose in serum samples with a 1:1000 dilution factor, and colorimetrically distinguished between concentrations. The assay demonstrated good specificity and sensitivity. A rapid assay was developed that could be used for high throughput analyses using either naked eye detection or a basic entry level laboratory spectrophotometry microplate reader. This observation shows the potential for further development of such nanobiosensors that can be validated for practical clinical applications. Future perspectives are on the application of these optimised strategies to other enzyme-based and immunoassays using nanotechnology.