A sensitive fluorescence biosensor for alkaline phosphatase activity based on the Cu(II)-dependent DNAzyme

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Alkaline phosphatase (ALP) is crucial for phosphate metabolism.
• Altered ALP levels are indicative of various diseases.
• Sensitive and simple monitoring methods for ALP are necessary.

Purpose of the Study:

• To develop a highly selective and sensitive fluorescent biosensor for ALP activity determination.
• To establish a reliable method for monitoring ALP levels in biological samples.

Main Methods:

• Design of a Cu(II)-dependent DNAzyme (Cu-Enzyme) system composed of two parts labeled with alkyne and azido groups.
• A Cu-substrate (Cu-Sub) strand labeled with a FAM fluorophore and a quencher (BHQ1).
• ALP-induced hydrolysis of AA-P generates AA, reducing Cu(II) to Cu(I), catalyzing DNAzyme assembly and subsequent cleavage of Cu-Sub, releasing FAM for fluorescence detection.

Main Results:

• The biosensor exhibited a low background fluorescence due to FAM quenching.
• Upon ALP addition, a strong fluorescence signal was generated, linearly correlated with ALP concentration (0.36-54.55 U L⁻¹).
• A low detection limit of 0.14 U L⁻¹ (S/N = 3) was achieved.

Conclusions:

• The proposed fluorescent biosensor offers a simple, selective, and sensitive approach for ALP activity determination.
• The method was successfully validated for ALP detection in human serum samples.
• This biosensor holds potential for clinical diagnostics and disease monitoring.