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

The use of microfluidic glass chips for continuous single-cell lysis and assay of internal (beta)-Galactosidase ((beta)-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 (mu)m/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-(beta)-D-galactopyranoside (FMG), were detected downstream of the mixing and cell lysis point for the reaction of (beta)-Gal with 200 (mu)M of the fluorogenic substrate fluorescein-di-(beta)-Dgalactopyranoside (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.