Ascorbic acid-amplified robust fluorescent assay for versatile detection of acid phosphatase activity

Acid phosphatase (ACP) serves as a crucial clinical biomarker for early detection of various diseases, most notably prostate cancer (PCa), which ranks as the second most common cancer among men worldwide and remains typically asymptomatic in its initial developmental phase. Enzyme-triggered fluorescent reactions under mild conditions offer significant potential for advancing in-situ enzyme activity assays. Despite this promise, the development of acid phosphatase (ACP)-specific detection systems remains challenging due to the scarcity of efficient fluorescent response mechanisms and undesirable fluorescence under acidic conditions.

In this work, we demonstrate a simple but pH-robust polyethyleneimine (PEI)-ascorbic acid (AA) fluorescence generation system exhibiting remarkable adaptability across wide pH value ranges. The developed system exhibits high specificity toward AA coupled with extended linear range (2-200 μM) and high sensitivity (LOD, 0.32 μM). By coupling this prominent fluorogenic reaction with enzymatic hydrolysis of ascorbic acid 2-phosphate, we established two operational modes for ACP quantification. A sequential two-step detection approach demonstrating superior analytical performance (linear range: 0.2-11 mU/mL; LOD: 0.017 mU/mL), surpassing current literature values and an innovative single-step one-pot strategy achieved through synchronizing enzymatic and fluorescent reactions (linear range: 1-60 mU/mL; LOD: 0.050 mU/mL), enabling rapid analysis with high simplicity. The excellent assay performance was successfully applied for the direct ACP determination in serum samples with high accuracy.

A generalizable in-situ fluorescence framework adaptable for quantifying diverse AA-dependent biomolecules with low-background and high-specificity was proposed through a green, facile route. This research provides general guidelines for developing fluorescent turn-on strategy specifically tailored for ACP detection, and is of great significance for facilitating biosensing applications of in-situ fluorescent reactions.