β-galactosidase assays of single-cell lysates on a microchip: A complementary method for enzymatic analysis of single cells

Gregor Ocvirk, Hossein Salimi-Moosavi, Rod J. Szarka, Edgar A. Arriaga, Per E. Andersson, Richard Smith, Norman J. Dovichi, D. Jed Harrison

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

The use of microfluidic glass chips for continuous single-cell lysis and assay of internal β-Galactosidase (β-Gal) content is described. Cells were transported single file toward a Y-shaped mixing junction at which lytic agents were introduced by suction. Flow velocities of z∼ 100 and ∼40 μm/s were used under protein denaturing [35 mM sodium dodecylsulfate (SDS)] and nondenaluring (0.1% Triton X-100) conditions, respectively. Complete and reproducible lysis of individual cells on-chip occurred within 30 s using Triton X-100 and 2 s when using SDS. Optimal concentrations of lysis and enzyme substrate reagents were determined using microtitre plate and chip-based procedures. Fluorescence peaks. due to the enzymatic product fluorescein mono-β-D-galactopyranoside (FMG), were detected downstream of the mixing and cell lysis point for the reaction of β-Gal with 200 μM of the fluorngenic substrate fluorescein-di-β-D-gatactopyranoside (FDG). FMG fluorescence was observed from cells preincubated with FDG off-chip then subsequently lysed on-chip with SDS. Unincubated cells were mixed on-chip with both FDG and Triton X-100, each individual cell generating FMG fluorescence downstream of the mixing point detected within 2 min of mixing. In contrast, viable cells incubated with FDG required I h or more in order to generate significant signal in a flow cytometer.

Original languageEnglish (US)
Pages (from-to)115-125
Number of pages11
JournalProceedings of the IEEE
Volume92
Issue number1
DOIs
StatePublished - Jan 2004

Keywords

  • Galaclosidase
  • Lab on a chip
  • Microfluidics
  • Single-cell analysis

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    Ocvirk, G., Salimi-Moosavi, H., Szarka, R. J., Arriaga, E. A., Andersson, P. E., Smith, R., Dovichi, N. J., & Harrison, D. J. (2004). β-galactosidase assays of single-cell lysates on a microchip: A complementary method for enzymatic analysis of single cells. Proceedings of the IEEE, 92(1), 115-125. https://doi.org/10.1109/JPROC.2003.820551