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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 ∼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.