研究目的
To develop a sensitive and selective fluorometric method for determining the activity of alkaline phosphatase (ALP) and its inhibitors using nitrogen and boron co-doped carbon dots as a fluorescent probe.
研究成果
The developed fluorometric method using boron and nitrogen co-doped carbon dots is effective for sensitive and selective detection of ALP activity and its inhibitors. It offers advantages such as good water solubility, low toxicity, simple synthesis, and cost-effectiveness, making it suitable for bioanalytical applications, including use in human serum samples. Future studies could focus on further optimizing the detection time and expanding the method to other enzyme systems.
研究不足
The method requires a longer incubation time of 2 hours, which may not be ideal for rapid detection applications. Potential areas for optimization include reducing the detection time and improving the stability of the carbon dots in complex biological matrices.
1：Experimental Design and Method Selection:
The method is based on the boronic acid-triggered specific reaction with cis-diols, where boronic acid-modified carbon dots bind to ascorbic acid generated from ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate, leading to aggregation and fluorescence quenching.
2：Sample Selection and Data Sources:
ALP enzyme, ascorbic acid 2-phosphate, and inhibitors like Na3VO4 were used; human serum samples were tested for practical application.
3：List of Experimental Equipment and Materials:
Carbon dots synthesized from 3-aminophenylboronic acid, reagents from Aladdin Reagent Company, Shanghai Macklin Biochemical Co., Ltd., Sangon Biotech Co., Ltd., and Sigma-Aldrich; equipment includes RF-6000 spectrofluorometer, JEM-1400 TEM, ESCALAB 250Xi XPS spectrometer, Nicolet 380 FT-IR spectrophotometer.
4：Experimental Procedures and Operational Workflow:
For ALP detection, mix DEA buffer, MgCl2, 2-AAP, ALP, C-dots, and water; incubate at 37°C for 2 h; measure fluorescence. For inhibitor detection, pre-incubate ALP with Na3VO4, then add 2-AAP and C-dots, incubate, and measure.
5：Data Analysis Methods:
Fluorescence intensity measured at 540 nm with excitation at 490 nm; calculations for (F0-F)/F0, detection limit based on signal-to-noise ratio, IC50 for inhibitors, and statistical analysis for recoveries and RSD.
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