Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

Abstract

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresisA microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As 2 O 3 ) at low concentration (1-2 mM). Buthionine sulfoximine (BSO), in combination with As 2 O 3 enhanced the decrease of reduced GSH to a great extent. The combined treatment of As 2 O 3 and hydrogen peroxide (H 2 O 2 ) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.

Characterizing doxorubicin‐induced apoptosis in HepG2 cells using an integrated microfluidic device

“…Recently, the advances in soft lithography enabled inexpensive devices to be fabricated by molding the fluidic components of channels, valves, fluidic multiplexers, mixers, and even peristaltic pumps into transparent monolithic devices for performing complex operations at the nanoliter scale. The seamless integration of active control elements into a microfluidic platform offers the ability to perform diverse types of multiplexed bioassays, such as cell sorting [10], chemotaxis [11], gene expression [12], differentiation [13,14], and biochemical separation [15][16][17].…”

The use of naphthalene‐2,3‐dicarboxaldehyde for the analysis of primary amines using high‐performance liquid chromatography and capillary electrophoresis

“…The method was validated to study the relationship between the ROS formation and the GSH level in cell cultures (Parmentier et al, 1999). Recent works using the same fluorogenic dyes in CE-LIF were achieved to measure the intracellular ROS and GSH levels in acute promyelocytic leukaemia (Qin et al, 2005). In this study, the arsenic trioxide (As 2 O 3 ) treatment was tested to observe the applicability and feasibility of the method for monitoring the intracellular GSH and ROS levels in apoptotic leukaemia cells.…”

Recent developments in CE and CEC of peptides

“…The sensitivity of the method was sufficient for determination of GSH and GSSG in human plasma and tobacco leaves. The other CE-LIF techniques were applied to simultaneous and rapid determination of GSH and reactive oxygen species in individual red blod cells [202,203], in apoptotic leukemia cells [204], and in probiotic bacteria [205].…”