Ultrasensitive nuclease activity and inhibition assay using microchip electrophoresis with laser induced fluorescence detection. Issue 8 (8th February 2016)

• Record Type: Journal Article
• Authors: Qin, Yingfeng; Zhao, Jingjin; Huang, Yong; Li, Shuting; Zhao, Shulin
• Is Part Of: Analytical methods. Volume 8:Issue 8(2016)
• Page Start: 1852
• Page End: 1857
• Publication Date: 2016-02-08
• Subjects: Chemistry, Analytic -- Periodicals; Analytical biochemistry -- Periodicals; Chemical laboratories -- Standards -- Periodicals; 543.1905
• DOI: 10.1039/c5ay02831k
• Languages: English
• ISSNs: 1759-9660

Abstract

Abstract : A novel microchip electrophoresis method with laser induced fluorescence detection was developed for ultrasensitive detection of nuclease activity and inhibitors. Abstract : A novel microchip electrophoresis method with laser induced fluorescence detection (MCE-LIF) was developed for ultrasensitive detection of nuclease activity and inhibitors. S1 nuclease was chosen as the model nuclease to demonstrate the proof-of-concept of the proposed approach. In this assay, the fluorescein-labeled ssDNA (FAM-ssDNA) was used as a signal reporter. In the presence of nuclease, FAM-ssDNA was digested to 5-FAM-nucleoside monophosphates and short oligonucleotide fragments. However, the cleavage reaction of FAM-ssDNA with nuclease was prohibited in the presence of inhibitors. Detection of S1 nuclease activity and inhibitors can be achieved by separating FAM-ssDNA and FAM-5-nucleoside monophosphates, and detecting the fluorescence intensity of FAM-5-nucleoside monophosphates in the MCE-LIF platform. The calibration curve showed a linearity in the range of S1 nuclease concentrations from 0.002 to 0.2 U mL −1 . Based on a signal/noise ratio (S/N) of 3, the detection limit was estimated to be 0.001 U mL −1, which was about 1–3 orders of magnitude more sensitive than those of the developed approaches. The proposed method was low-cost and simple in its operation, and the capabilities for target detection from complex fluids and screening of nuclease inhibitors were verified.