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 journalArticlepeer-review

34 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

Bibliographical note

Funding Information:
Manuscript received February 20, 2003; revised June 4, 2003. This work was supported in part by the Natural Sciences and Engineering Research Council of Canada. G. Ocvirk is with Roche Diagnostics, D-68305 Mannheim, Germany. H. Salimi-Moosavi is with ACLARA Biosciences Inc., Mountain View, CA 94043-1432 USA. R. J. Szarka is with Cytovax Biotechnologies Inc., Edmonton, AB, T6E 6S4, Canada, and also with the Alberta Research Council, Edmonton, AB, T6N 1E4, Canada. E. A. Arriaga is with the Department of Chemistry, University of Minnesota, Minneapolis, MN 55455-0431 USA. P. E. Andersson is with Gyros AB, Uppsala Science Park, SE-751 83, Uppsala, Sweden. R. Smith is with the Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2G2, Canada. N. J. Dovichi is with the Department of Chemistry, University of Washington, Seattle, WA 98195-1700 USA. D. J. Harrison is with the Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada. Digital Object Identifier 10.1109/JPROC.2003.820551

Keywords

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

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