An Integrated Microfluidic Device for Monitoring Changes in Nitric Oxide Production in Single T-Lymphocyte (Jurkat) Cells

Author(s)

Metto, Eve C. ^[1] ; Evans, Karsten ^[1] ; Barney, Patrick ^[1] ; Culbertson, Anne H. ^[1] ; Gunasekara, Dulan B. ^{[2, 3]} ; Caruso, Giuseppe ^{[3, 4]} ; Huvey, Matthew K. ^{[3, 5, 6]} ; Fracassi da Silva, Jose Alberto ^{[7, 3]} ; Lunte, Susan M. ^{[2, 3, 5]} ; Culbertson, Christopher T. ^[1]

Affiliation

^[1] Kansas State Univ, Dept Chem, Manhattan, KS 66506 - USA

^[2] Univ Kansas, Dept Chem, Lawrence, KS 66045 - USA

^[3] Univ Kansas, Ralph N Adams Inst Bioanalyt Chem, Lawrence, KS 66047 - USA

^[4] Univ Catania, Sect Biochem & Mol Biol, Dept Chem Sci, I-95124 Catania - Italy

^[5] Univ Kansas, Dept Pharmaceut Chem, Lawrence, KS 66047 - USA

^[6] Akermin Inc, St Louis, MO 63132 - USA

^[7] Univ Estadual Campinas, Inst Chem, BR-13083970 Sao Paulo - Brazil

• Document type: Journal article
• Source: Analytical Chemistry; v. 85, n. 21, p. 10188-10195, NOV 5 2013.
• Web of Science Citations: 29

Abstract

A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a 2-fold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO. (AU)

• FAPESP's process: 10/01046-6 - Development of microfluidic devices with electrochemical detection for in vitro single cell analysis of nitrogen reactive species
• Grantee: José Alberto Fracassi da Silva
• Support Opportunities: Scholarships abroad - Research