Teflon wet-on technology for single-cell isolation on digital microfluidic chips: Advancing genomic heterogeneity...

Single-cell analysis is crucial for uncovering cellular heterogeneity, particularly in cancer research, where traditional bulk methods often miss rare but critical subpopulations. Digital microfluidics (DMF) offers precise droplet manipulation for single-cell isolation, but existing hydrophilic patterning techniques, such as teflon lift-off, face challenges including complex fabrication, delamination risks, and high operational voltages that may compromise cell integrity and omics analysis accuracy. Here, we present a novel teflon wet-on technology for fabricating hydrophilic patterns on DMF chips, eliminating lift-off steps while ensuring robustness and simplicity. By optimizing plasma treatment (80W, 4 min) and SU-8 photolithography, we achieved well-defined hydrophilic patterns that enable efficient droplet actuation at low voltages (70-90V), avoiding potential DNA damage associated with higher voltages (≥100V). Systematic evaluation of parameters: including pattern dimensions, cell concentration, actuation voltage, and cell size-revealed optimal conditions for high-efficiency single-cell isolation. Using this platform, we successfully isolated individual MDA-MB-231 cells and performed genomic analysis, revealing significant intercellular heterogeneity in mutations. The teflon wet-on method outperforms conventional lift-off techniques in simplicity, stability, and biocompatibility, making it a particularly suitable tool for the study of rare and valuable cells, such as circulating tumor cells or primary patient-derived samples, with applications in precision medicine, cancer research, and other single-cell analyses requiring high-resolution manipulation and high recovery rates.