The through-hole micropore array was visualized by phase contrast
The through-hole micropore array was visualized by phase contrast. demonstrate key aspects of the platform for gene transfer, screening and detection of targeted intracellular markers in living cells. and applications due to its simplicity and potential to transfect large numbers of cells.[10,11] A number of electroporation systems have been developed and commercialized.[12-15] For example, bulk electroporation (BEP) is a techniques in which millions of cells are simultaneously shocked with a high voltage between two electrodes. A serious drawback of this approach, however, is that a large fraction of the cells are damaged due to the nonuniform and hazardous electric-fields that affect individual cells. Therefore, three critical aspects – transfection efficiency, alpha-Hederin gene delivery to targeted cells and cell viability – are not guaranteed [16-17] with the BEP approach. Microchannel electroporation (MEP) provides a means to overcome these drawbacks by offering a gentler environment where each cell is porated under more controlled conditions.[17-19] By confining individual cells at a microscale pore, the electric field strength across the pore increases by several orders over those achieved by BEP.[20-22] Thus not only are low voltages (< 10 V) sufficient for cell poration [17,18,23-25], but delivery into the cell is confined to regions determined by the size of the pore. Furthermore, MEP offers the potential for versatile lab-on-chip systems that integrate cell-manipulation and real-time detection followed by cell transfer, thereby paving the road for comprehensive analysis of cellular behaviors in response to environment, signal pathways, cell-cell interactions and cellular dynamics in the post-transfection stage.  Currently most MEP designs, however, only facilitate single-cell electroporation,[18,19,23,24] which is inadequate for clinical applications that require high throughput. Amongst recent approaches [17,18,24,27-29], microfluidic electroporation devices often operate in a sequential manner, and thus could be less conducive to scale-up for clinical applications. On the other hand, 3D microchannel electroporation (3D MEP) could achieve high throughput by handling thousands of cells on a planar (X,Y) membrane while the applied electric field and transfection are in the CED vertical (Z) direction. [24,31-33] However, a critical requirement that is currently lacking for 3D MEP is an efficient approach to manipulate and safely align a large number of individual cells with an array of micropores for high throughput transfection at a low voltage. In this work we report on the application of a versatile 3D MEP – magnetic alpha-Hederin tweezers (MT) based system capable of realizing the three important aspects of (a) individual-cell based electroporation, (b) high throughput transfection, and (c) retention of cell viability. To efficiently place a cell at a single micropore, an array of thin Permalloy (NiFe) magnetic disks fabricated on a silicon wafer are utilized as an effective alpha-Hederin multiplexed magnetic tweezers. Magnetically labeled cells are remotely controlled by weak external magnetic fields which operate over the entire array enabling simultaneous manipulation of tens of thousands of cells. Additionally, the weak magnetic fields (< 150 G) do not generate heat nor adversely damage the cells, concerns that arise with manipulation associated with other techniques, including vacuum force [24, 31-33], which is difficult to optimize without serious cell membrane damage, and optical tweezers, which is burdened by low throughput[35-37] and laser-induced Joule heating. The present magnetic tweezers-based approach illustrates parallel manipulation, localization, electroporation, and subsequent transport of the transfected cells. The versatility of the approach with its potential for pre-clinical studies and gene therapy is demonstrated with several distinct cell types and transfection reagents. A highlight is the delivery of the GATA2 molecular beacon alpha-Hederin (MB) for detection of GATA2 mRNA expression. The GATA2 family.