Fluorescence biosensor for inorganic pyrophosphatase activity.
Anal Bioanal Chem
; 409(4): 999-1005, 2017 Feb.
Article
en En
| MEDLINE
| ID: mdl-27858125
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Técnicas Biosensibles
/
Pirofosfatasa Inorgánica
/
Colorantes Fluorescentes
Idioma:
En
Revista:
Anal Bioanal Chem
Año:
2017
Tipo del documento:
Article
País de afiliación:
China