Microfluidic Devices for the High-Throughput Chemical Analysis of Cells

Abstract

A microfluidic device is reported that integrated cell handling, rapid cell lysis, and electrophoretic separation and detection of fluorescent cytosolic dyes. The device function was demonstrated using Jurkat cells that were loaded with the fluorogenic dyes - carboxyfluorescein diacetate, Oregon green carboxylic acid diacetate, or Calcein AM. The loaded cells were hydrodynamically transported from the cell-containing reservoir to a region on the microfluidic device where they were focused and then rapidly lysed using an electric field. Complete lysis was accomplished in <33 ms. The hydrolyzed, fluorescent dyes in the cell lysate were automatically injected into a separation channel on the device and detected 3 mm downstream of the injection point. The total separation time was approximately 2.2 s with absolute migration time reproducibilities of <1% and efficiencies ranging from 2300 to 4000 theoretical plates. Results from 139 cells are reported. A small fraction of these cells, approximately 9%, were found to enzymatically hydrolyze the loaded dyes in a manner significantly different from the majority of the cells. Cell analysis rates of 7-12 cells/min were demonstrated and are >100 times faster than those reported using standard bench-scale capillary electrophoresis.

Molded polyethylene glycol microstructures for capturing cells within microfluidic channels

“…2,3 As the cells flow through the channel, they are potentially lysed, and the intracellular components of the cells are analyzed. 4,5 This approach is, however, potentially limited to non-adherent cells and transient analysis since unanchored cells exit the system and elude follow-up characterization. An alternative approach to analyze adherent cells is to immobilize cells within channels.…”

Alkaline hemolysis fragility is dependent on cell shape: Results from a morphology tracker

“…The distance between electrodes was 600 mm, resulting in a field strength of 43 V/cm, an order of magnitude below minimum fields needed for electroporation-based lysis, with reported ranges of 0.3 kV/cm) (14) to 0.9 kV/ cm (15). The chamber width and length were 1.5 mm 3 5 mm.…”

Chemical Analysis of Single Mammalian Cells with Microfluidics

“…Once the cell is positioned in the entrance to the separation channel, electric field strengths >600 V/cm will completely lyse it within 40 ms (25). A second device that uses electrical fields to induce cell lysis uses an ac po- tential on top of a dc potential applied to the separation channel (26). The constant 675 V/cm dc potential is supplemented with a ±225 V/cm ac potential that allows for the rapid lysis of Jurkat T cells during the field-strength peak (~900 V/cm) while minimizing the amount of Joule heating that frequently occurs at such high field strengths.…”

“…These disruptions of electroosmotic flow lead to irreproducible migration times and could possibly cause a reduction in the signal. To prevent these effects, emulsification agents (to solubilize lipid bilayers) and polyethylene glycol (to reduce protein adsorption) can be used to improve microchip durability and performance (26).…”

“…Another method of controlling reproducibility is to simultaneously detect the cell or lysate via fluorescence as it enters the separation channel (26). The RSDs for Oregon Green hydrolyzed within Jurkat T cells analyzed in this manner are <1% with separation efficiencies of 500,000-1.3 million plates/m.…”