Hun Tingting, Zhang Yi, Xu Qingmei, Huang Dong, Wang Qi, Li Zhihong, Wang Wei
School of Integrated Circuits, Peking University, Beijing 100871, China.
National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Beijing 100871, China.
Micromachines (Basel). 2022 Apr 26;13(5):672. doi: 10.3390/mi13050672.
Labeling-assisted visualization is a powerful strategy to track circulating tumor cells (CTCs) for mechanism study (e.g., tumor metastasis). Due to the rarity of CTCs in the whole blood, efficient simultaneous enrichment and labeling of CTCs are needed. Hereby, novel in situ electroporation on a previously-developed micropore-arrayed filter (PERFECT filter) is proposed. Benefiting from the ultra-small-thickness and high-porosity of the filter plus high precision pore diameter, target rare tumor cells were enriched with less damage and uniform size distribution, contributing to enhanced molecular delivery efficiency and cell viability in the downstream electroporation. Various biomolecules (e.g., small molecule dyes, plasmids, and functional proteins) were used to verify this in situ electroporation system. High labeling efficiency (74.08 ± 2.94%) and high viability (81.15 ± 3.04%, verified via live/dead staining) were achieved by optimizing the parameters of electric field strength and pulse number, ensuring the labeled tumor cells can be used for further culture and down-stream analysis. In addition, high specificity (99.03 ± 1.67%) probing of tumor cells was further achieved by introducing fluorescent dye-conjugated antibodies into target cells. The whole procedure, including cell separation and electroporation, can be finished quickly (<10 min). The proposed in situ electroporation on the PERFECT filter system has great potential to track CTCs for tumor metastasis studies.
标记辅助可视化是一种用于追踪循环肿瘤细胞（CTC）以进行机制研究（如肿瘤转移）的强大策略。由于全血中CTC数量稀少，因此需要对CTC进行高效的同步富集和标记。在此，我们提出了一种在先前开发的微孔阵列滤膜（PERFECT滤膜）上进行的新型原位电穿孔技术。得益于滤膜的超薄厚度、高孔隙率以及高精度的孔径，目标稀有肿瘤细胞得以在较少损伤且尺寸分布均匀的情况下被富集，这有助于提高下游电穿孔过程中的分子递送效率和细胞活力。使用了各种生物分子（如小分子染料、质粒和功能蛋白）来验证这种原位电穿孔系统。通过优化电场强度和脉冲数参数，实现了高标记效率（74.08 ± 2.94%）和高活力（通过活/死染色验证为81.15 ± 3.04%），确保标记后的肿瘤细胞可用于进一步培养和下游分析。此外，通过将荧光染料偶联抗体引入靶细胞，进一步实现了对肿瘤细胞的高特异性探测（99.03 ± 1.67%）。整个过程，包括细胞分离和电穿孔，可在短时间内（<10分钟）完成。所提出的PERFECT滤膜原位电穿孔系统在追踪CTC以进行肿瘤转移研究方面具有巨大潜力。
