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). As a result, when galactose is sensed in the presence of H₂O₂, the colored response of the AuNS-PVP-GalDH assay transforms from turquoise blue to light pink, and then to wine red in a concentration-dependent manner discernible to the human eye. This competitive AuNS-PVP-GalDH assay could be a viable analytical tool for rapid and convenient galactose quantification in resource-limited areas.