Fluorescence biosensor for inorganic pyrophosphatase activity.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Inorganic pyrophosphatase (PPase) plays a crucial role in various biological processes.
• Developing sensitive and selective methods for PPase activity detection is essential for biochemical research and drug discovery.
• Existing methods may lack the required sensitivity or specificity for certain applications.

Purpose of the Study:

• To develop a highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity.
• To utilize click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA) for signal amplification.
• To apply the developed biosensor for screening potential PPase inhibitors.

Main Methods:

• A fluorescence biosensor was designed based on CLT-HRCA.
• Pyrophosphate ion (PPi) coordination with Cu2+ was used to modulate Cu+ availability.
• Cu+ catalyzed azide-alkyne cycloaddition (CuAAC) initiated HRCA, leading to amplified fluorescence signals with SYBR Green I.

Main Results:

• The biosensor demonstrated a linear response to PPase activity from 0.05 to 25 mU.
• A low detection limit of 0.02 mU was achieved.
• The biosensor was successfully applied to screen potential PPase inhibitors with high efficiency.

Conclusions:

• A novel and efficient fluorescence biosensor for PPase activity detection was successfully developed.
• The CLT-HRCA strategy provides a sensitive and selective platform for enzyme activity assays.
• This biosensor shows promise for high-throughput screening of enzyme inhibitors in drug discovery.