Developing a Cost-Effective Bioassay to Detect Alkaline Phosphatase Activity and Generating White Light Emission from a Single Nano-Assembly by Conjugating Vitamin B6 Cofactors with Lysozyme-Stabilized Fluorescent Gold Nanoclusters

In this work, lysozyme-stabilized fluorescent gold nanoclusters (Lyso-AuNCs) and pyridoxal 5′-phosphate (PLP) as a monophosphate ester substrate were used to develop a highly selective and cost-effective bioassay for the detection of alkaline phosphatase (ALP) activity. The vitamin B6 cofactor, PLP, was conjugated with the red-emitting nanoclusters to obtain a probe, PLP_Lyso-AuNCs, via forming a Schiff base linkage between the free amino group of lysozyme and the aldehyde group of PLP. At pH = 10.08, addition of ALP to the yellow-emitting PLP_Lyso-AuNCs solution catalyzed the hydrolysis of PLP and converted it into pyridoxal, which produced a distinct ratiometric fluorescence response and the fluorescent color turned pale white. Using the probe, PLP_Lyso-AuNCs, the ALP activity could be detected down to 0.002 U/L. Further, the changes in the fluorescent color intensity (red, blue, and green) of PLP_Lyso-AuNCs were recorded with the back camera of a smartphone to quantify the ALP activity. Both the fluorimetric and smartphone approaches gave satisfactory recovery percentage, when the practical utility of PLP_Lyso-AuNCs was applied to quantify the ALP activity in environmental and biological samples, such as river and lab tap water, blood plasma, and serum. Finally, a pure white light-emitting nano-assembly was developed by conjugating optimized amounts of both PLP and pyridoxal over the surface of Lyso-AuNCs.