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<p>Alkaline phosphatase (ALP) is an important clinical biomarker, and rapid activity quantification is needed in routine diagnostics. Here, we report a CoOOH nanosheet–based colorimetric assay for ALP that uses a H₂O₂-free TMB chromogenic module. CoOOH nanosheets catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to produce the blue oxidized product, while ALP hydrolyzes ascorbic acid 2-phosphate (AAP) to generate ascorbic acid (AA), which reduces/decomposes CoOOH and suppresses the TMB signal, resulting in a distinct blue-to-colorless readout. Under optimized conditions, the absorbance response is linear with ALP activity from 0.01 to 25&#xa0;mU&#xa0;mL<sup>−1</sup> with a detection limit of 0.01&#xa0;mU&#xa0;mL<sup>−1</sup>. The assay shows good robustness against common matrix components (e.g., urea, glucose, proteins, and major inorganic ions), whereas thiol-containing reductants can attenuate the signal at elevated levels. Serum recovery tests gave recoveries of 94.30–97.50% with RSDs of 1.9–4.9%, confirming reliability in complex matrices. This method provides a simple, cost-effective, and sensitive approach for ALP activity measurement. In addition, the CoOOH–TMB pair serves as a general H<sub>2</sub>O<sub>2</sub>-free TMB signal module, suggesting potential adaptability to microplate-based ALP-linked immunoassay/ELISA formats.</p> Graphical Abstract <p></p>

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A CoOOH nanosheet–based colorimetric method for sensitive detection of alkaline phosphatase in biological samples

  • 期刊论文

  • 发表日期：

    2026年3月17日

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  • Yijiang Li,
  • Shaoxing Chen,
  • Leijuan Huang,
  • Biying Lin,
  • Zhixiong Cai,
  • Yuanfu Xie

摘要

Alkaline phosphatase (ALP) is an important clinical biomarker, and rapid activity quantification is needed in routine diagnostics. Here, we report a CoOOH nanosheet–based colorimetric assay for ALP that uses a H₂O₂-free TMB chromogenic module. CoOOH nanosheets catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to produce the blue oxidized product, while ALP hydrolyzes ascorbic acid 2-phosphate (AAP) to generate ascorbic acid (AA), which reduces/decomposes CoOOH and suppresses the TMB signal, resulting in a distinct blue-to-colorless readout. Under optimized conditions, the absorbance response is linear with ALP activity from 0.01 to 25 mU mL−1 with a detection limit of 0.01 mU mL−1. The assay shows good robustness against common matrix components (e.g., urea, glucose, proteins, and major inorganic ions), whereas thiol-containing reductants can attenuate the signal at elevated levels. Serum recovery tests gave recoveries of 94.30–97.50% with RSDs of 1.9–4.9%, confirming reliability in complex matrices. This method provides a simple, cost-effective, and sensitive approach for ALP activity measurement. In addition, the CoOOH–TMB pair serves as a general H2O2-free TMB signal module, suggesting potential adaptability to microplate-based ALP-linked immunoassay/ELISA formats.

Graphical Abstract