High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood

Abstract

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%–90.0%) and excellent linear performance (R2 = 0.8189–0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM− tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Dielectrophoresis assisted loading and unloading of microwells for impedance spectroscopy

“…Morimoto et al. used immunofluorescent labeling on cells and isolated them in microwells, where molecular analyses of cells were performed . Kobayashi et al.…”

Dielectrophoresis for Biomedical Sciences Applications: A Review

“…Conversely, the DEP techniques also provide a great advantages by preserving the correlation of specificity and sensitivity forcing the compromise of high-throughput and highly specific isolation of bio particles label free applications with minimal sample preparations [197]. The high specific and specificity identification of target bio- particles from complex biological fluid such as blood [198] and urine [199] can be used as a solid proof that DEP methods have broad potential as powerful diagnostic tools.…”

Unmasking tumor heterogeneity and clonal evolution by single-cell analysis

“…The HB-chip uses calibrated microfluidic flow patterns to drive cells to come in contact with the antibody-coated walls of the device, thereby reducing cell collisions and improving target cell capture efficiency. A commercial microfluidic circuitry chip DEPArray System (Menarini Silicon Biosystems, Inc.) containing an array of individually controllable electrodes to create a dielectrophoretic (DEP) cage around each cell for single CTC isolation is also available [65] . Besides isolation of CTCs from blood, the microfluidic platform can also be used for single-cell isolation from other tissues [66,67] .…”