Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400–800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.

毫秒级脉冲标准电穿孔已被用作将药物或遗传探针递送至细胞的有效工具，而纳秒级脉冲的不可逆电穿孔被研究以改变细胞内活性，以诱导脉冲诱导的细胞凋亡。在这项工作中采用了长纳秒脉冲和标准毫秒脉冲的组合处理，以帮助促进DNA质粒快速穿过细胞质膜和核膜，从而促进贴壁细胞和悬浮细胞的转基因表达水平和动力学。发现持续时间为400-800 ns的纳秒脉冲可有效破坏核膜，促进质粒DNA的核传递。额外的微流体操作进一步有助于抑制负面影响，例如焦耳加热和普通纳秒脉冲处理产生的气泡，这些不良影响会导致高毒性和/或无效的转染。在具有不同脉冲持续时间的两种类型的处理之间具有适当的顺序并且几乎没有延迟对于保证有效性至关重要：GFP质粒的转染效率提高2倍或更高且转基因表达动力学迅速而细胞活力不受影响。这种新的电穿孔方法的实施可能有益于许多需要有效递送外源探针的生物学研究和临床实践。在具有不同脉冲持续时间的两种类型的处理之间具有适当的顺序并且几乎没有延迟对于保证有效性至关重要：GFP质粒的转染效率提高2倍或更高且转基因表达动力学迅速而细胞活力不受影响。这种新的电穿孔方法的实施可能有益于许多需要有效递送外源探针的生物学研究和临床实践。在具有不同脉冲持续时间的两种类型的处理之间具有适当的顺序并且几乎没有延迟对于保证有效性至关重要：GFP质粒的转染效率提高2倍或更高且转基因表达动力学迅速而细胞活力不受影响。这种新的电穿孔方法的实施可能有益于许多需要有效递送外源探针的生物学研究和临床实践。