title: Pyrophosphate-regulated Zn(2+)-dependent DNAzyme activity: an amplified fluorescence sensing strategy for alkaline phosphatase.
abstract: In this work, based on the fact that pyrophosphate (PPi) could regulate the activity of Zn(2+)-dependent DNAzyme, we for the first time report a fluorescence turn-on sensing system for alkaline phosphatase (ALP) with improved sensitivity via nonprotein-enzymatic signal amplification. A catalytic and molecular beacon (CAMB) design was employed to further improve its sensitivity. Taking advantage of the strong interactions between PPi and the Zn(2+), the cofactor Zn(2+) was caged, and the DNAzyme activity was effectively inhibited. The introduction of ALP, however, could catalyze the hydrolysis of PPi and release free Zn(2+), resulting in the activation of DNAzyme to catalyze the cleavage of the molecular beacon substrate with a remarkable increase of fluorescent signal. These optimized designs together allow a high sensitivity for ALP, with a detection limit of 20 pM observed, much lower than previously reported methods. It has also been used for detection of ALP in human serum with satisfactory results, demonstrating its potential applications in clinical diagnosis.
authors.affiliation: The Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165, PR China.
authors.forename: Rong-Mei
authors.initials: RM
authors.lastname: Kong
authors.forename: Ting
authors.initials: T
authors.lastname: Fu
authors.forename: Ni-Na
authors.initials: NN
authors.lastname: Sun
authors.forename: Feng-Li
authors.initials: FL
authors.lastname: Qu
authors.forename: Shu-Fang
authors.initials: SF
authors.lastname: Zhang
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Zhang
mesh_terms: D000469:Alkaline Phosphatase / Q000097:blood* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D021881:DNA, Catalytic / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D057230:Limit of Detection; D013050:Spectrometry, Fluorescence / Q000379:methods; D015032:Zinc / Q000378:metabolism*
chemical_list: D021881:DNA, Catalytic; D011756:Diphosphates; C107241:diphosphoric acid; D000469:Alkaline Phosphatase; D015032:Zinc
keywords: Alkaline phosphatase; DNAzyme; Fluorescence; Pyrophosphate