Colorimetric determination of the activity of alkaline phosphatase based on the use of Cu(II)-modulated G-quadruplex-based DNAzymes

A colorimetric detection scheme is introduced for the determination of alkaline phosphatase (ALP) activity based on Cu(II)-modulated G-quadruplex-based DNAzymes. It is exploiting the strong affinity of Cu(II) for pyrophosphate (PPi) upon which the cofactor PPi is trapped by Cu(II). Hence, the activity of the DNAzyme is inhibited. ALP catalyzes the hydrolysis of PPi, causing the release of Cu(II). DNAzyme, in turn, is activated and catalyzes the cleavage of the DNA probe substrate. The released G-rich sequence folds into the G-quadruplex, which can bind hemin and catalyze the oxidation of 2,2'-azinobis (3-ethylbenzothiozoline)-6-sulfonate (ABTS), and this leads to an increase in absorbance at 420 nm. Absorbance increases linearly with increasing ALP activity in 0.07 to 300 U.L-1 range, with a 70 mU.L-1 detection limit. The method was applied in ALP inhibition tests and to the determination of ALP activity in spiked serum samples where it gave satisfactory results.