Sensitive and selective label-free alkaline phosphatase detection based on DNA hairpin probe

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Alkaline phosphatase (ALP) is a crucial enzyme in various biological processes.
• Accurate detection of ALP activity is vital for diagnosing diseases and monitoring biological functions.
• Existing biosensing methods may lack simplicity, sensitivity, or applicability in complex biological matrices.

Purpose of the Study:

• To develop a novel, label-free, and signal-on fluorescent biosensor for alkaline phosphatase.
• To utilize SYBR Green I (SG) for fluorescence amplification in the biosensing system.
• To establish a sensitive and selective method for detecting ALP activity in complex biological fluids.

Main Methods:

• A DNA hairpin probe (HP) was designed to be dephosphorylated by ALP.
• Lambda exonuclease (λ exo) activity was inhibited by the dephosphorylated HP.
• SYBR Green I (SG) was used to detect the formation of double-stranded DNA (dsDNA), leading to a signal-on fluorescence response.

Main Results:

• The developed biosensor architecture is label-free, simple, and exhibits a signal-on fluorescence output.
• The method demonstrated high sensitivity and selectivity for ALP detection.
• A wide linear response range from 0.4 to 200 U/mL was achieved.
• A low detection limit of 0.05 U/mL for ALP was established.
• The biosensor successfully detected ALP activity in complex biological fluids.

Conclusions:

• The SYBR Green I-assisted fluorescence amplification method provides a robust platform for ALP biosensing.
• This novel approach offers a simple, sensitive, and selective assay for quantitative ALP detection.
• The biosensor's applicability in complex biological fluids highlights its potential for real-world diagnostic applications.