Ultrasensitive nuclease activity and inhibition assay using microchip electrophoresis with laser induced fluorescence detection

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.