The suitability of NAD(P)/NAD(P)H as redox agents for gold nanostar shape alteration in colourimetric clinical enzymology

Abstract

Biosensors have proven to be a strong instrument for detecting varied targets such as proteins, DNA, small molecules (disease biomarkers), toxins and metabolites with great sensitivity and specificity. The fundamental goal of the research presented in this thesis was to evaluate the applicability of NAD(P)/NAD(P)H as redox agents for gold nanostar shape alteration in colourimetric clinical enzymology. Furthermore, our research aimed to develop simple, low-cost, highly sensitive and specific gold nanostar-based colourimetric biosensor platforms for analyte detection as well as to understand how NAD(P)/NAD(P)H affects colourimetric, redox chemistry and morphological changes in gold nanostars. A seedle 4(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and silver-mediated method was used to synthesise the gold nanostars. However, the procedure was simple, robust, and repeatable, yielding heteromorphic, monodispersed, multi-branched nanostars measuring 37 nm ± 2nm in diameter. The nanostars were found to have varying stabilities in salt, ionic strength and cell culture medium environments after being functionalised with polyethylene glycol (PEG) 8 000, polyethylene oxide (PEO)100 000 and polyvinylpyrrolidone (PVP) 10 000. Our findings show that capping agents influenced gold nanostar stability, functionalisation and colourimetric biosensing capabilities, with PVP capping proving to be the most appropriate option for HEPES mediated gold nanostar synthesis. Gold nanostars' anisotropic structure has shown considerable promise in plasmonic colourimetric sensing. When the colourimetric ability of the new nanostars was evaluated, it was discovered that the colourimetric assays for gold nanostars could be tailored for a specific application by using capping agents, enzymes and the detection solution as colourimetric catalysts. The high energy facets at the terminals of the gold nanostar spikes triggered remodelling to start from there, resulting in the transition from star morphology to quasi-spherical nanoparticles and colourimetric signals. The colourimetric signals obtained were vivid and easily discernible with the naked eye. However, it has been revealed that differences in the morphological properties or the surface nature of gold nanostars strongly affect their plasmonic colourimetric sensing and optical properties. The protein conjugation and immobilisation approach were also found to have an effect on the analyte detection rate. For optimal concentration-dependent detection findings, apoenzymes were co-immobilised with their coenzymes during biosensor fabrication. The biosensors reacted to the neo-formed NAD(P)/NAD(P)H after the addition of NAD(P)/NAD(P)H-dependent enzymes galactose dehydrogenase and aldose reductase, resulting in a sufficient colourimetric gradient based on substrate concentration visible to the naked eye and detectable spectrophotometrically. When the same colourimetric assay parameters were used in an ELISA-type assay, the colourimetric variation between different concentrations of glucose oxidase and galactose oxidase enzymes resulted in a sufficient analyte concentration-dependent vivid colourimetric gradient, as expected. This thesis summarises the most recent studies about the optical and spectroscopic features as well as the effect of NAD(P)/NAD(P)H redox chemistry on gold nanostars. Furthermore, it was found that NAD(P)/NAD(P)H-dependent enzymes could be used in gold nanoparticle-based plasmonic colourimetric assays when hydrogen peroxide-producing enzymes are unavailable. The versatility of technology was demonstrated across a broad spectrum of analytes. We applied it to detect metabolites (glucose and galactose) in a variety of matrices and conditions using plasmonic gold nanostars as both a scaffold and transducer element. The ramifications and scope of these findings have yet to be determined, as the full potential of anisotropic gold nanoparticles is only now being studied. This is critical for the successful development of robust, simple, and low-cost diagnostic equipment for resource-constrained settings. In conclusion, the NAD(P)/NAD(P)H-dependent enzymes immobilised on gold nanostars have the potential to replace costly equipment, reagents and lengthy experiments needed to assess various analytes and related clinical disorders such as diabetes and galactosemia diagnosis.