Target sequence of single cells captured by a polymeric microfluidic device

Detecting rare circulating tumor cells (CTCs), malignant cells of primary site origin, in the bloodstream is difficult. We previously constructed a polymeric microfluidic device with high capture efficiency for lung cancer cell lines using antibodies against the epithelial cell adhesion molecule (EpCAM). In this study, we investigated a method for extracting DNA from single cells captured in a microfluidic device and performed targeted sequencing using the Cancer Hotspot Panel v2. Additionally, we employed a fixation method, which enabled more efficient sequencing of the EpCAM-chip-captured cells than existing fixation methods. We used blood samples obtained from three patients with lung cancer to assess the clinical applicability of our method. Directly sequenced samples revealed better coverage uniformity than samples subjected to whole-genome sequencing. Direct sequencing of cells fixed with preserver fluid and 100% ethanol was performed accurately with high coverage uniformity. Our method demonstrated a sensitivity of 99.4%, specificity of 99.5%, and area under the curve of 1 (allele frequency cut-off, 18%). Our approach suggests that single cells captured in a microfluidic device are sufficient for genetic mutation analysis.