Marker-specific sorting of rare cells using dielectrophoresis

Abstract

Current techniques in high-speed cell sorting are limited by the inherent coupling among three competing parameters of performance: throughput, purity, and rare cell recovery. Microfluidics provides an alternate strategy to decouple these parameters through the use of arrayed devices that operate in parallel. To efficiently isolate rare cells from complex mixtures, an electrokinetic sorting methodology was developed that exploits dielectrophoresis (DEP) in microfluidic channels. In this approach, the dielectrophoretic amplitude response of rare target cells is modulated by labeling cells with particles that differ in polarization response. Cell mixtures were interrogated in the DEP-activated cell sorter in a continuous-flow manner, wherein the electric fields were engineered to achieve efficient separation between the dielectrophoretically labeled and unlabeled cells. To demonstrate the efficiency of marker-specific cell separation, DEP-activated cell sorting (DACS) was applied for affinity-based enrichment of rare bacteria expressing a specific surface marker from an excess of nontarget bacteria that do not express this marker. Rare target cells were enriched by >200-fold in a single round of sorting at a single-channel throughput of 10,000 cells per second. DACS offers the potential for automated, surface marker-specific cell sorting in a disposable format that is capable of simultaneously achieving high throughput, purity, and rare cell recovery.

Fig. 1.

Operational principle of DACS. (A) The DACS concept: Cells entering in the sample stream are only deflected into the collection stream if they are labeled with a dielectrophoretically responsive label. (B) Schematic view of the electrode region of the microchannels with sample and buffer inlets, as well as waste and collection outlets.

Fig. 2.

Optical micrographs of the stabilized flow at the inlet and outlet channels. (A) The unlabeled cells follow the streamline and enter the waste channels, and the diffusion of the cells into the buffer stream is minimal. (B) Sequentially captured images of polystyrene beads moving under the influence of DEP deflection. Quadrupole electrodes guide the beads to the center of the microchannel. Total volume flow rate is 240 μl/h. Applied voltage is 20 V peak-to-peak at 500 kHz.

Fig. 3.

Flow pattern of fluorescent cells in the DACS device. (A and B) Optical micrographs of streaming E. coli cells at the inlet (A) and outlet (B) channels. (C) An expanded view using optical microscopy, showing DEP particles entering the collection channel after being focused into the center of the stream. The arrows identify two beads carrying bound cells. Total volume flow rate is 300 μl/h, and the applied voltage is 20 V peak-to-peak at 500 kHz.

Fig. 4.

Enrichment of T7·tag mAb-binding clones as measured by flow cytometry. Induced cells were labeled with 20 nM biotin-T7 mAb and 20 nM streptavidin-phycoerythrin. (A) Unselected population. (B) After one round of DACS. (C) After two rounds of DACS.