title: A label-free near-infrared fluorescent assay for the determination of deoxyribonuclease I activity based on malachite green/G-quadruplexes.
abstract: Owing to the biological and clinical significance of deoxyribonuclease I (DNase I), it is highly desirable to develop near-infrared (NIR) fluorescent assays for the determination of DNase I activity. Here we report a label-free NIR fluorescent assay for selective determination of DNase I activity based on malachite green (MG)/G-quadruplexes. In the presence of Na(+) or K(+), single stranded DNA (ssDNA) is able to form a G-quadruplex structure, thus to increase the rigidity of MG structure and result in a remarkable NIR fluorescence. As DNase I is capable of cleaving all types of DNA indiscriminately to release nucleotide products, the G-quadruplexes are cleaved into oligonucleotides in the presence of DNase I. As a result, the rigidity of MG structure is reduced, and the NIR fluorescence of the solution decreases with increase of DNase I activity, providing a useful platform for low-cost, label-free and convenient detection of DNase I activity. Under the optimum conditions, the proposed label-free NIR fluorescent assay gave a detection limit of 1 u mL(-1), and a relative standard deviation of 3.2% for eleven replicate detections of 50 u mL(-1) DNase I. The proposed assay was applied to the determination of DNase I activity in spiked human urine samples with recoveries from 99.1 to 109.0%.
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, China.
authors.forename: Shao-Kai
authors.initials: SK
authors.lastname: Sun
authors.forename: Bei-Bei
authors.initials: BB
authors.lastname: Wang
authors.forename: Xiu-Ping
authors.initials: XP
authors.lastname: Yan
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism* / Q000652:urine*; D057075:Enzyme Assays / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis*; D054856:G-Quadruplexes; D006801:Humans; D057230:Limit of Detection; D012394:Rosaniline Dyes / Q000032:analysis*; D019265:Spectroscopy, Near-Infrared / Q000379:methods*
chemical_list: D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D012394:Rosaniline Dyes; C005095:malachite green; D003850:Deoxyribonuclease I