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

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DOIResolve DOI: http://doi.org/10.1109/JPROC.2003.820551
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TypeArticle
Journal titleProceedings of the IEEE
ISSN0018-9219
Volume92
Issue1
Pages115125; # of pages: 11
Subjectmicrofluidics, galactosidase, lab on a chip, single-cell analysis
AbstractThe 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 ∼100 and ∼40 μm/s were used under protein denaturing [35 mM sodium dodecylsulfate (SDS)] and nondenaturing (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 fluorogenic substrate fluorescein-di-β-D-galactopyranoside (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 1 h or more in order to generate significant signal in a flow cytometer.
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Identifier1007-1799
NPARC number12329152
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Record identifier04ca6679-9724-466f-92df-59747c08dea4
Record created2009-09-10
Record modified2016-05-09
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