Conventional electroporation approaches show limitations in the delivery of macromolecules in vitro and in vivo. These limitations include low efficiency, noticeable cell damage and nonuniform delivery of cells. Here, we present a simple 3D electroporation platform that enables massively parallel single-cell manipulation and the intracellular delivery of macromolecules and small molecules. A pyramid pit micropore array chip was fabricated based on a silicon wet-etching method. A controllable vacuum system was adopted to trap a single cell on each micropore. Using this chip, safe single-cell electroporation was performed at low voltage. Cargoes of various sizes ranging from oligonucleotides (molecular beacons, 22 bp) to plasmid DNA (CRISPR-Cas9 expression vectors, >9 kb) were delivered into targeted cells with a significantly higher transfection efficiency than that of multiple benchmark methods (e.g., commercial electroporation devices and Lipofectamine). The delivered dose of the chemotherapeutic drug could be controlled by adjusting the applied voltage. By using CRISPR-Cas9 transfection with this system, the p62 gene and CXCR7 gene were knocked out in tumor cells, which effectively inhibited their cellular activity. Overall, this vacuum-assisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy.

传统的电穿孔方法在体外和体内递送大分子方面存在局限性。这些限制包括低效率、明显的细胞损伤和细胞的不均匀递送。在这里，我们展示了一个简单的 3D 电穿孔平台，该平台能够实现大规模并行单细胞操作以及大分子和小分子的细胞内递送。基于硅湿法刻蚀方法制作了金字塔凹坑微孔阵列芯片。采用可控真空系统在每个微孔上捕获单个细胞。使用该芯片，可在低电压下进行安全的单细胞电穿孔。从寡核苷酸（分子信标，22 bp）到质粒 DNA（CRISPR-Cas9 表达载体，> 9 kb) 以比多种基准方法（例如商业电穿孔装置和 Lipofectamine）显着更高的转染效率递送到靶细胞中。可以通过调节施加的电压来控制化疗药物的递送剂量。通过在该系统中使用 CRISPR-Cas9 转染，p62基因和CXCR7基因在肿瘤细胞中被敲除，有效抑制了它们的细胞活性。总体而言，这种真空辅助微孔阵列平台提供了一种简单、高效、高通量的细胞内递送方法，可以促进片上细胞操作、细胞内研究和癌症治疗。