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

Abstract

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.

Regulation of the Enzymatic Activities of Lysozyme by the Surface Ligands of Ultrasmall Gold Nanoclusters: The Role of Hydrophobic Interactions

“…The ADAs, which could be encapsulated in polymeric micelle and released under acidic condition, showed better physiological clearance while maintaining serum pharmacokinetics. Moreover, Sahoo et al synthesized a series of fluorescent gold nanoclusters and copper nanoclusters and applied them for the detection of alkaline phosphatase (ALP), salicylaldehyde, VB 6 cofactor pyridoxal 5′-phosphate (PLP), and nitroaromatics. ,,, The gold/copper nanoclusters exhibited a new fluorescence band and showed selective fluorescence enhancement when interacting with these analytes, exhibiting good applicability as nanoprobes.…”

Cascade Detection of Pyridoxal 5′-Phosphate and Al3+ Ions Based on Dual-Functionalized Red-Emitting Copper Nanoclusters

“…The fluorescent NCs are also investigated to develop multi-color emitting devices . The multi-color emitting ability of β-CD-GSH-CuNCs in the presence of PLP and Al 3+ prompted us to examine the fluorescent color coordinates in the Commission Internationale de l’Éclair-age (CIE) 1931 chromaticity diagram.…”

Decorating Vitamin B6 Cofactor over Beta-Cyclodextrin Stabilized Silver Nanoparticles through Inclusion Complexation for Fluorescent Turn-On Detection of Hydrazine

“…These spectral changes may have occurred due to the inclusion of PLP within the hydrophobic cavity of β-CD functionalized over AgNPs, followed by the possible functionalization of PLP on the surface of AgNPs. − The FT-IR spectra of β-CD-AgNPs and PLP decorated β-CD-AgNPs resembled each other (SI Figure S1c), which clearly delineating the formation of inclusion complex between PLP and β-CD. Because of the vital role of PLP in several biochemical pathways, the assessment of the cellular PLP level was used as a biomarker for the diagnosis of several health problems in clinical practices. , Therefore, the PLP directed distinct and noticeable spectral changes of β-CD-AgNPs prompted us to perform the fluorescence titration experiment. As shown in Figure a, the addition of PLP into the weakly emissive β-CD-AgNPs solution resulted an instantaneous fluorescence enhancement at 532 nm.…”

“…Because of the vital role of PLP in several biochemical pathways, the assessment of the cellular PLP level was used as a biomarker for the diagnosis of several health problems in clinical practices. 49,50 Therefore, the PLP directed distinct and noticeable spectral changes of β-CD-AgNPs prompted us to perform the fluorescence titration experiment. As shown in Figure 2a, the addition of PLP into the weakly emissive β-CD-AgNPs solution resulted an instantaneous fluorescence enhancement at 532 nm.…”