title: Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells.
abstract: A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells.
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Shandong Normal University, Jinan, 250014.
authors.forename: Xinyuan
authors.initials: X
authors.lastname: Zhang
authors.forename: Qingling
authors.initials: Q
authors.lastname: Li
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Chen
authors.forename: Hongmin
authors.initials: H
authors.lastname: Li
authors.forename: Kehua
authors.initials: K
authors.lastname: Xu
authors.forename: Lisheng
authors.initials: L
authors.lastname: Zhang
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
mesh_terms: D016521:Electrophoresis, Gel, Pulsed-Field; D005452:Fluoresceins / Q000737:chemistry; D056945:Hep G2 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis*; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*
chemical_list: D005452:Fluoresceins; C543452:bis(p-methylbenzenesulfonyl)dichlorofluorescein; D006861:Hydrogen Peroxide