title: Continuous analysis of dye-loaded, single cells on a microfluidic chip.
abstract: Continuous analysis of two dyes loaded into single mammalian cells using laser-based lysis combined with electrophoretic separation was developed and characterized on microfluidic chips. The devices employed hydrodynamic flow to transport cells to a junction where they were mechanically lysed by a laser-generated cavitation bubble. An electric field then attracted the analyte into a separation channel while the membranous remnants passed through the intersection towards a waste reservoir. Phosphatidylcholine (PC)-supported bilayer membrane coatings (SBMs) provided a weakly negatively charged surface and prevented cell fouling from interfering with device performance. Cell lysis using a picosecond-pulsed laser on-chip did not interfere with concurrent electrophoretic separations. The effect of device parameters on performance was evaluated. A ratio of 2 : 1 was found to be optimal for the focusing-channel : flow-channel width and 3 : 1 for the flow-channel : separation-channel width. Migration times decreased with increased electric field strengths up to 333 V cm(-1), at which point the field strength was sufficient to move unlysed cells and cellular debris into the electrophoretic channel. The migration time and full width half-maximum (FWHM) of the peaks were independent of cell velocity for velocities between 0.03 and 0.3 mm s(-1). Separation performance was independent of the exact lysis location when lysis was performed near the outlet of the focusing channel. The migration time for cell-derived fluorescein and fluorescein carboxylate was reproducible with <10% RSD. Automated cell detection and lysis were required to reduce peak FWHM variability to 30% RSD. A maximum throughput of 30 cells min(-1) was achieved. Device stability was demonstrated by analyzing 600 single cells over a 2 h time span.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: K Scott
authors.initials: KS
authors.lastname: Phillips
authors.forename: Hsuan Hong
authors.initials: HH
authors.lastname: Lai
authors.forename: Emily
authors.initials: E
authors.lastname: Johnson
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D000818:Animals; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002469:Cell Separation; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D004560:Electricity; D004586:Electrophoresis; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000378:metabolism*; D005898:Glass / Q000737:chemistry; D057446:Hydrodynamics; D007834:Lasers; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D009842:Oligopeptides / Q000737:chemistry / Q000302:isolation & purification; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D002457:Cell Extracts; D004129:Dimethylpolysiloxanes; D005456:Fluorescent Dyes; D009842:Oligopeptides; C013830:baysilon