Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip.

A microfluidic system was developed for the analysis of single biological cells, with functional integration of cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser induced fluorescence (LIF) detection in microfabricated channels of a single glass chip. Channels were 12 microm deep and 48 microm wide, with a simple crossed-channel design. The effective separation channel length was 35 mm. During sampling with a cell suspension (cell population 1.2 x 10(5) cells per mL in physiological salt solution), differential hydrostatic pressure (created by adjusting liquid levels in the four reservoirs) was used to control cell flow exclusively through the channel crossing. Single cell loading into the separation channel was achieved by electrophoretic means by applying a set of potentials at the four reservoirs, counteracting the hydrostatic flow. A special docking (adhering) procedure for the loaded cell was applied before lysis by repeatedly connecting and disconnecting a set of low potentials, allowing precise positioning of the cell within the separation channel. Cell lysis was then effected within 40 ms under an applied CE separation voltage of 1.4 kV (280 V cm(-1)) within the working electrolyte (pH 9.2 borate buffer) without additional lysates. The docked lysing approach reduced dispersion of released intracellular constituents, and significantly improved the reproducibility of CE separations. Glutathione (GSH) was used as a model intracellular component in single human erythrocyte cells. NDA derivatized GSH was detected using LIF. A throughput of 15 samples h(-1), a retention time precision of 2.4% RSD was obtained for 14 consecutively injected cells. The average cellular concentration of GSH in human erythrocytes was found to be 7.2 [times] 10(-4)+/- 3.3 x 10(-4) M (63 +/- 29 amol per cell). The average separation efficiency for GSH in lysed cells was 2.13 x 10(6)+/- 0.4 x 10(6) plates per m, and was about a factor of 5 higher than those obtained with GSH standards using pinched injection.

[1]  G. Gradl,et al.  A 3-D microelectrode system for handling and caging single cells and particles , 1999 .

[2]  J. Jorgenson,et al.  Quantitative analysis of individual neurons by open tubular liquid chromatography with voltammetric detection. , 1989, Analytical chemistry.

[3]  J. Michael Ramsey,et al.  Effects of injection schemes and column geometry on the performance of microchip electrophoresis devices , 1994 .

[4]  Paul Yager,et al.  Cell lysis and protein extraction in a microfluidic device with detection by a fluorogenic enzyme assay. , 2002, Analytical chemistry.

[5]  A. Meister.  Glutathione metabolism and its selective modification. , 1988, The Journal of biological chemistry.

[6]  E. Yeung,et al.  Simultaneous monitoring of glutathione and major proteins in single erythrocytes , 1995 .

[7]  Paul C. H. Li,et al.  Transport, manipulation, and reaction of biological cells on-chip using electrokinetic effects. , 1997, Analytical chemistry.

[8]  Mengsu Yang,et al.  Cell docking and on-chip monitoring of cellular reactions with a controlled concentration gradient on a microfluidic device. , 2002, Analytical chemistry.

[9]  S J Lillard,et al.  Continuous cell introduction for the analysis of individual cells by capillary electrophoresis. , 2001, Analytical chemistry.

[10]  R N Zare,et al.  Use of 2,3-naphthalenedicarboxaldehyde derivatization for single-cell analysis of glutathione by capillary electrophoresis and histochemical localization by fluorescence microscopy. , 1995, Analytical chemistry.

[11]  Alan P. Morrison,et al.  Development of a microfluidic device for fluorescence activated cell sorting , 2002 .

[12]  E. Yeung.  Study of single cells by using capillary electrophoresis and native fluorescence detection. , 1999, Journal of chromatography. A.

[13]  J. Jorgenson,et al.  Microcolumn separations and the analysis of single cells. , 1989, Science.

[14]  Fumihito Arai,et al.  High‐speed separation system of randomly suspended single living cells by laser trap and dielectrophoresis , 2001, Electrophoresis.

[15]  R. Ferrari,et al.  Improved procedure for determining glutathione in plasma as an index of myocardial oxidative stress. , 1987, Clinical Chemistry.

[16]  Sheri J. Lillard,et al.  New approaches to single-cell analysis by capillary electrophoresis , 2001 .

[17]  G. Matioli,et al.  Electrophoresis of Hemoglobin in Single Erythrocytes , 1965, Science.

[18]  N. Dovichi,et al.  Methyl-beta-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence for separation and detection of phospholipids. , 2000, Journal of chromatography. A.

[19]  W. Jin,et al.  Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection , 2000, Electrophoresis.

[20]  Aaron R Wheeler,et al.  Microfluidic device for single-cell analysis. , 2003, Analytical chemistry.

[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]  D. Graves,et al.  Powerful tools for genetic analysis come of age. , 1999, Trends in biotechnology.

[23]  N. Dovichi,et al.  Instrumentation for chemical cytometry. , 2000, Analytical chemistry.