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 vacuumassisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy.

The genetic heterogeneities in cancer cells pose challenges to achieving precise drug treatment in a widely applicable manner. Most single-cell gene analysis methods rely on cell lysis for gene extraction and identification, showing limited capacity to provide the correlation of genetic properties and realtime cellular behaviors. Here, we report a single living cell analysis nanoplatform that enables interrogating gene properties and drug resistance in millions of single cells. We designed a Domino-probe to identify intracellular target RNAs while releasing 10-fold amplified fluorescence signals. An on-chip addressable microwellnanopore array was developed for enhanced electro-delivery of the Domino-probe and in situ observation of cell behaviors. The proofof-concept of the system was validated in primary lung cancer cell samples, revealing the positive-correlation of the ratio of EGFR mutant cells with their drug susceptibilities. This platform provides a high-throughput yet precise tool for exploring the relationship between intracellular genes and cell behaviors at the single-cell level.

used fluorophores, such as organic dyes and fluorescent proteins. This severely limits STED nanoscopy for long-term observation of cellular events. [2] Moreover, the lateral spatial resolution of STED nanoscopy is theoretically depend on STED beam intensity, with a higher intensity resulting in a better resolution. [3] Therefore, highly photostable fluorophores are urgently desired for the wide application of STED imaging.Besides the efforts to develop more photostable dye molecules, in recent years, the application of fluorescent nanoparticles (NPs), such as quantum dots (QDs), aggregation induced emission (AIE) NPs, upconversion NPs (UCNPs) and dye hybrid plasmonic nanoparticles has been the attractive approach for STED imaging. [4] With their distinctive advantages, nanoparticles' limitations are also found for further improvements. For example, QDs and AIE NPs are bright and have enhanced antiphotobleaching properties, but the donutshaped STED beam often cause a dim "halo" by two-photon excited fluorescence, thus reducing signal to noise ratio. [4b,c] UCNPs only require a low-intensity STED beam, nonetheless, they have relatively low quantum yield and rather long lifetimes which reduced the imaging speed of STED nanoscopy. [4d] Dye hybrid plasmonic nanoparticles efficiently reduced depletion power and improved resolution, whereas heating of metal nanoparticles may lead to thermally induced NP motion and destruction of dyes and NPs. [4e,f ] Herein, we reported for the first time, the development of semiconducting polymer dots (Pdots) as a new class of ultrastable and bright imaging probe in STED nanoscopy. With the closely packed chains of hydrophobic semiconducting polymers, Pdots are emerging fluorescent NPs featuring with high brightness, good biocompatibility and fine labeling capability for a broad range of applications in chemical and biomedical sensing. [5] A wide variety of semiconducting polymers have been developed with different molecular structures, emission wavelengths and NP sizes. [6] Recently, small-sized (<13 nm) Pdots (PFO/poly(9,9-dioctylfluorenyl-2,7-diyl), PFBT/poly[(9,-9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-(2,1′,3)-triadiazole)], CN-PPV/poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)], PFTBT5/poly[(9,9-dioctylfluorene)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)]) have been reported for multicolor super-resolution optical fluctuation imaging Stimulated emission depletion (STED) nanoscopy is an emerging superresolution imaging technique, which enables high spatiotemporal characterization of cellular structures and dynamics. However, its current application is limited by the lack of photostable fluorophores, which can endure strong STED illumination. Herein, a type of photobleaching-resistant semiconducting polymer dots (Pdots) for STED imaging is reported. The Pdots are synthesized by nanoprecipitation of the mixture of hydrophobic fluorescent polymer poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorene}-alt-co-{2,5bis(N,N′-diphenylamino)-1,4-phenylene}] (...