Design of a Microchannel- Nanochannel- Microchannel Array Based Nanoelectroporation System for Precise Gene Transfection
Design of a Microchannel- Nanochannel- Microchannel Array Based Nanoelectroporation System for Precise Gene Transfection
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DOI：
10.1002/smll.201300116
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发表时间：
2014-03-01
期刊：
影响因子：
13.3
通讯作者：
Lee, L. James
中科院分区：
文献类型：
--
作者：
Gao, Keliang;Li, Lei;Lee, L. James
描述了纳米通道电穿孔（NEP）阵列的微/纳米制造工艺及其在非病毒基因转染中质粒精确递送的应用。一个浸梳装置进行了优化，以产生DNA纳米线跨越微脊阵列图案的聚二甲基硅氧烷（PDMS）表面上的产率高达95%。采用基于低粘度树脂1，4-丁二醇二丙烯酸酯（1，4-BDDA）的分子印迹技术将微脊-纳米线-微脊阵列转化为微通道-纳米通道-微通道（MNM）阵列。飞秒激光烧蚀的二次加工应用于缩短微通道的一侧从3000到50 m，以方便细胞加载和卸载。然后将生物芯片密封在具有储液器和微流体通道的包装盒中，以使细胞和质粒加载，并保护生物芯片免受泄漏和污染。NEP后可打开包装箱进行细胞卸载，以便进行后续细胞培养和分析。这些NEP案件可以放在一个纺纱盘和多达十个光盘可以堆叠在一起纺纱。由此产生的离心力可以在3分钟内同时操纵数百或数千个细胞进入NEP阵列的微通道。为了证明其应用，将诱导多能干细胞（iPSC）的13 kbp OSKM质粒注射到小鼠胚胎成纤维细胞（MEFC）中。NEP生物芯片内转染细胞的荧光检测表明，与通过常规的本体电穿孔（BEP）方法进行的类似基因转染相比，递送的剂量高且均匀得多。
A micro/nano-fabrication process of a nanochannel electroporation (NEP) array and its application for precise delivery of plasmid for non-viral gene transfection is described. A dip-combing device is optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4-butanediol diacrylate (1,4-BDDA), adopted to convert the microridge-nanowire-microridge array into a microchannel-nanochannel-microchannel (MNM) array. Secondary machining by femtosecond laser ablation is applied to shorten one side of microchannels from 3000 to 50 m to facilitate cell loading and unloading. The biochip is then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case can be opened for cell unloading after NEP to allow for the follow-up cell culture and analysis. These NEP cases can be placed in a spinning disc and up to ten discs can be piled together for spinning. The resulting centrifugal force can simultaneously manipulate hundreds or thousands of cells into microchannels of NEP arrays within 3 minutes. To demonstrate its application, a 13 kbp OSKM plasmid of induced pluripotent stem cell (iPSC) is injected into mouse embryonic fibroblasts cells (MEFCs). Fluorescence detection of transfected cells within the NEP biochips shows that the delivered dosage is high and much more uniform compared with similar gene transfection carried out by the conventional bulk electroporation (BEP) method.