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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

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A Colorimetric/Fluorescence Dual Detection for Acid Phosphatase Based on Copper Nanoclusters.

Yuanyuan Cheng¹, Xiujuan Huang¹, Yifeng Tu¹

¹The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren'ai Road, Suzhou, 215123, China.

Journal of Fluorescence

|March 24, 2025

Summary

A novel dual-mode detection method for acid phosphatase (ACP) was developed using copper nanoclusters. This method offers sensitive colorimetric and fluorescent detection of ACP activity in human serum samples.

Keywords:

Acid phosphatase Amplification Copper nanocluster Inner filter effect

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Area of Science:

Biochemistry
Nanotechnology
Analytical Chemistry

Background:

Acid phosphatase (ACP) is an important biomarker for various physiological and pathological conditions.
Existing detection methods for ACP may lack sensitivity or require complex procedures.
Copper nanoclusters (CuNCs) offer unique photoluminescent properties and catalytic activity for sensing applications.

Purpose of the Study:

To develop a simple and effective dual-mode (fluorescent/colorimetric) detection method for acid phosphatase (ACP).
To utilize the peroxidase-like activity of copper nanoclusters for ACP detection.
To validate the method's applicability in analyzing human serum samples.

Main Methods:

Synthesis of photoluminescent copper nanoclusters (CuNCs) with blue emission.
Exploitation of CuNCs' peroxidase-like activity to catalyze TMB oxidation.
Inhibition of catalysis by pyrophosphate (PPi) and its recovery by ACP-mediated hydrolysis of PPi.
Dual detection based on colorimetric changes (absorbance) and fluorescence quenching (inner filter effect).

Main Results:

The developed method provides both colorimetric and fluorescent signals for ACP quantification.
Colorimetric detection showed a linear response in the 0.1-10 U/L range with a 0.01 U/L detection limit.
Fluorimetric detection responded within 0.005-10 U/L with a highly sensitive detection limit of 0.001 U/L.
The method demonstrated facile switching between detection modes and was successfully applied to human serum samples.

Conclusions:

A novel and versatile dual-mode sensing platform for ACP detection has been successfully established.
The method leverages copper nanoclusters' catalytic activity and optical properties for sensitive and selective ACP determination.
The facile switching between colorimetric and fluorimetric modes enhances its adaptability for diverse analytical needs and instrumentation.