Quantitative Galactose Colorimetric Competitive Assay Based on Galactose Dehydrogenase and Plasmonic Gold Nanostars.

We describe a competitive colorimetric assay that enables rapid and sensitive detection of galactose and reduced nicotinamide adenine dinucleotide (NADH) via colorimetric readouts and demonstrate its usefulness for monitoring NAD+-driven enzymatic reactions. We present a sensitive plasmonic sensing approach for assessing galactose concentration and the presence of NADH using galactose dehydrogenase-immobilized gold nanostars (AuNS-PVP-GalDH). The AuNS-PVP-GalDH assay remains turquoise blue in the absence of galactose and NADH; however, as galactose and NADH concentrations grow, the reaction well color changes to a characteristic red color in the presence of an alkaline environment and a metal ion catalyst (detection solution).

Feasibility of NAD(P)/NAD(P)H as redox agents in enzymatic plasmonic gold nanostar assays for galactose quantification.

Plasmonic colorimetric sensors have emerged as powerful analytical tools in biochemistry due to their localized surface plasmon resonance extinction in the visible range. Here, we describe the feasibility of NAD(P)/NAD(P)H as redox agents in enzymatic plasmonic gold nanostar (AuNS) assays for galactose quantification using three model enzymes, GalDH, AR and GalOx, immobilized separately on polyvinylpyrrolidone-capped AuNS scaffolds. These highly specific, sensitive and selective bioassays induce the transformation of AuNS into quasi-spherical nanoparticles during the biorecognition of galactose in water and synthetic blood matrices.

The Effect of Capping Agents on Gold Nanostar Stability, Functionalization, and Colorimetric Biosensing Capability.

Capping agents (organic ligands, polymers, and surfactants) are pivotal for stabilizing nanoparticles; however, they may influence the surface chemistry, as well as the physico-chemical and biological characteristics, of gold nanostar (AuNS)-based biosensors. In this study, we proved that various capping agents affected capped and bioconjugated AuNS stability, functionality, biocatalysis, and colorimetric readouts. Capped and bioconjugated AuNSs were applied as localized surface plasmon resonance (LSPR)-based HO sensors using glucose oxidase (GOx) as a model enzyme.