Selective detection of endogenous thiols using microchip-based flow analysis and mercury/gold amalgam microelectrodes.

This paper describes the fabrication and characterization of thin-layer mercury/gold amalgam microelectrodes and their integration with microchip-based flow injection analysis. This microchip platform allows on-chip injection and lysis of erythrocytes followed by selective detection of intracellular glutathione (GSH) at low potentials. The thin-layer gold microelectrodes were amalgamated by electrodeposition of mercury. The electrodes produced a linear response for both GSH and cysteine in flow injection analysis studies utilizing both off-chip and on-chip injection. Comparative experiments using diamide and on-chip injection were performed to demonstrate the ability of the microchip device to detect changes in GSH concentration. Finally, rabbit erythrocyte samples (2% hematocrit) were injected and lysed on-chip and the amount of GSH detected corresponded to 312 amol/cell, which is in agreement with previously reported values. The selectivity, short time between injection and detection (approximately 5 s), and the continuous introduction of sample to the on-chip injector should enable the study of dynamically changing systems such as the glutathione redox system found in erythrocytes.

[1]  Michelle W. Li,et al.  Microchip-based integration of cell immobilization, electrophoresis, post-column derivatization, and fluorescence detection for monitoring the release of dopamine from PC 12 cells. , 2008, The Analyst.

[2]  D. Spence,et al.  Measuring the simultaneous effects of hypoxia and deformation on ATP release from erythrocytes. , 2008, The Analyst.

[3]  Michael J. Moehlenbrock,et al.  Development of an on-chip injector for microchip-based flow analyses using laminar flow. , 2007, Lab on a chip.

[4]  Dana M Spence,et al.  Addressing a vascular endothelium array with blood components using underlying microfluidic channels. , 2007, Lab on a chip.

[5]  C. Culbertson,et al.  Chemical analysis of single mammalian cells with microfluidics. Strategies for culturing, sorting, trapping, and lysing cells and separating their contents on chips. , 2007, Analytical chemistry.

[6]  Nancy L Allbritton,et al.  CRITICAL REVIEW www.rsc.org/loc | Lab on a Chip Analysis of single mammalian cells on-chip , 2006 .

[7]  D. Spence,et al.  Dynamic monitoring of glutathione in erythrocytes, without a separation step, in the presence of an oxidant insult. , 2006, Analytical chemistry.

[8]  Dana M Spence,et al.  Microfluidic technologies as platforms for performing quantitative cellular analyses in an in vitro environment. , 2006, The Analyst.

[9]  Michael J Moehlenbrock,et al.  Use of microchip-based hydrodynamic focusing to measure the deformation-induced release of ATP from erythrocytes. , 2006, The Analyst.

[10]  D. Spence,et al.  An altered oxidant defense system in red blood cells affects their ability to release nitric oxide-stimulating ATP. , 2006, Molecular bioSystems.

[11]  Dana M. Spence,et al.  A Microchip‐Based System for Immobilizing PC 12 Cells and Amperometrically Detecting Catecholamines Released After Stimulation with Calcium , 2005 .

[12]  Dana M Spence,et al.  Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel. , 2004, The Analyst.

[13]  R. Zare,et al.  Chemical cytometry on a picoliter-scale integrated microfluidic chip. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[14]  P. O'Brien,et al.  Metabolism, not autoxidation, plays a role in α-oxoaldehyde- and reducing sugar-induced erythrocyte GSH depletion: Relevance for diabetes mellitus , 2003, Molecular and Cellular Biochemistry.

[15]  D. Spence,et al.  Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo. , 2003, The Analyst.

[16]  S. Shahrokhian,et al.  Lead phthalocyanine as a selective carrier for preparation of a cysteine-selective electrode. , 2001, Analytical chemistry.

[17]  R. Sprague,et al.  Impaired Release of ATP from Red Blood Cells of Humans with Primary Pulmonary Hypertension , 2001, Experimental biology and medicine.

[18]  C. Henry,et al.  Dual-electrode electrochemical detection for poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips. , 2000, Analytical chemistry.

[19]  R. Zare,et al.  Dual electrochemical detection of cysteine and cystine in capillary zone electrophoresis , 1994 .

[20]  S. Lunte,et al.  Selective detection of free thiols by capillary electrophoresis-electrochemistry using a gold/mercury amalgam microelectrode , 1993 .

[21]  E. Yeung,et al.  Determination of intracellular species at the level of a single erythrocyte via capillary electrophoresis with direct and indirect fluorescence detection. , 1992, Analytical chemistry.

[22]  R. Adams,et al.  Determination of reduced glutathione in guinea pig and rat tissue by HPLC with electrochemical detection. , 1978, Life sciences.

[23]  P. Jocelyn.  The reduction of oxidized glutathione in erythrocyte haemolysates in pernicious anaemia. , 1960, The Biochemical journal.

[24]  Stephen L. R. Ellison,et al.  Standard additions: myth and reality , 2008, The Analyst.

[25]  J. Aaseth,et al.  Glutathione in overweight patients with poorly controlled type 2 diabetes , 2000 .

[26]  S. Lunte,et al.  Tubular-wire dual electrode for detection of thiols and disulfides by capillary electrophoresis/electrochemistry. , 1999, Analytical chemistry.

[27]  C. Costagliola.  Oxidative state of glutathione in red blood cells and plasma of diabetic patients: in vivo and in vitro study. , 1990, Clinical physiology and biochemistry.

[28]  R. Shoup,et al.  Dual electrode liquid chromatography detector for thiols and disulfides , 1983 .