Tu28 - Integrated On-Chip Impedance Cytometry for Inline Optimization of Dielectrophoretic Cell Separation Using Biophysical Metrics

In fifteen words or less, explain the significance of this contribution (Novel Aspect).: Label-free, inline monitoring enables real-time optimization of DEP cell separation by biophysical phenotyping.

Abstract Text:

Microfluidic cell enrichment using dielectrophoresis (DEP), based on biophysical and electrical metrics, is typically optimized through on-chip fluorescence microscopy or off-chip flow cytometry. These methods rely on fluorescent labeling and function as endpoint assays, limiting use of separated cells for downstream studies. Integrating on-chip cytometry enables inline control of separations to enrich cells by metrics such as viability, poration, and size, while minimizing sample loss and cross-contamination with off-chip analysis. Single-cell impedance cytometry, with its compact footprint, can be integrated downstream of DEP for label-free quantification of separated fractions. However, DEP’s requirement for low-conductivity media degrades impedance signal quality and may cause irreversible cell trapping. Additionally, high channel depths—used to improve DEP throughput—can reduce resolution of single-cell impedance signals, posing a challenge for accurate inline analysis.

We present an integrated microfluidic device to couple dielectrophoretic cell separation with downstream multichannel impedance cytometry for single-cell phenotypic detection of the separated fractions in the same media. Specifically, viscoelastic flows are used for cell focusing over the channel depth, so that the dielectrophoretic separation throughput is maintained, while the downstream impedance signal sensitivity in low conductivity media can be enhanced by increasing the impedance acquisition voltage without irreversible cell capture to enable measurement of the separated fractions. In this manner, by normalization of single-cell impedance data against co-flowing polystyrene beads of known size, we optimize the viscoelastic flow conditions (cells in 0.5% w/w of 0.6 MDa poly-ethylene-oxide at 1-10 µL/min) and impedance acquisition frequencies (50 kHz in low s_med of 55-220 µS/cm that permits dielectrophoresis) for setting thresholds in impedance phase (positive for live vs. negative for apoptotic cells) and magnitude (broad cell size distribution for live cells vs. narrow size distribution of small size for apoptotic cells) for single-cell phenotypic distinction. This is applied to optimize the dielectrophoretic enrichment of live floating cells released from adhered pancreatic cancer cell cultures thereby resembling circulating tumor cell phenotypes, so that downstream impedance cytometry can be used to monitor the separated fractions for feedback towards dielectrophoretic selection of live cells within specific size ranges and with minimized transmembrane voltage-induced cell damage.