A highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity has been developed based on special click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA). Pyrophosphate ion (PPi) can coordinate with Cu2+ to form stable PPi/Cu2+ complex and Cu2+ in the complex cannot be reduced to Cu+. The addition of PPase causes the hydrolysis of PPi into orthophosphate (Pi) and therefore induces the releasing of Cu2+ from the stable PPi/Cu2+ complex, and the free Cu2+ is easily reduced to Cu+ by sodium ascorbate. Then Cu+ catalyzes the cyclization reaction between the specially designed 5'-azide and 3'-alkyne tagged padlock probes through Cu+ catalyzed azide-alkyne cycloaddition (CuAAC), which in turn initiates the hyperbranched rolling circle amplification (HRCA). Given that the CLT-HRCA products contain large amounts of double-stranded DNAs (dsDNAs), the addition of SYBR Green I resulted in the enhanced fluorescence signal. There was a linear relationship between the enhanced fluorescence intensity and the logarithm PPase activity ranging from 0.05 to 25 mU with a detection limit of 0.02 mU. Such proposed biosensor has been successfully applied to screen the potential PPase inhibitors and has accessed the related inhibit ability with high efficiency.
Keywords: Click ligation trigger hyperbranched rolling circle amplification; Inorganic pyrophosphatase; Inorganic pyrophosphatase inhibitors.