| Transfection of nucleotides and delivery of other molecules into cells have wide applications in different areas such as cellular imaging, molecular biology analysis, protein production, gene therapy, cell reprogramming and cancer treatment. A perfect transfection method should provide high delivery efficiency and minimal cellular damage, but established protocols often fail to achieve both and compromise between the two due to their stochastic nature. To provide precise cell transfection while limiting the damage, nanochannel electroporation (NEP) was developed based on a micro/nanofluidics device. The nanochannel device could be fabricated by either DNA Combing and Imprinting, or a combined photo/soft-lithography. On the device, the cells and nucleotides were separated into two compartments linked by nanochannels. Individual cells were placed against nanochannels by optical tweezers and electric pulses were applied across the channels to create electrophoretic flow. The nanochannel confined electric field, enhancing the electrophoretic flow and limiting the cell surface exposed to the intense field. Material diffusion through the nanochannel without electric field was negligible, and the dosage of transfection could be controlled by the electric conditions. Apart from nucleotides, other charged materials could also be delivered as long as their dimensions fit in the nanochannels.;Unlike most established methods, the delivery by NEP occurred within 100 milliseconds and was proved to be independent of endocytosis. This characteristic of NEP was utilized to study the biological interactions of non-endocytic multi-walled carbon nanotubes (MWCNTs) and Quantum dots (Q-dots) with BEAS-2B cells. The results showed that Q-dots and MWCNTs induced more oxidation stress when their uptake bypassed endocytosis. The cytotoxicity of MWCNTs mainly rises from the non-endocytic cellular entrance path, through which the toxicity from certain chemical modifications was also more apparent.;When combined with molecular beacons (MBs), NEP could quantitatively analyze nucleotides inside individual cells without cell lysis. The multi-target RNA detection was demonstrated by the simultaneous NEP analysis of DNMT3A/B messenger RNAs in single cells and their downregulations by miR-29b with MBs carrying dyes of different fluorescence. With the NEP-MB platform, the effects of clinically observed CEBPA gene mutations on the expression of miR-181a in leukemia cells were also studied by delivering the mutants via NEP and detecting miR-181a with MB. The results confirmed the upregulation of miR-181a by truncated C/EBPalpha protein. The upregulation effects of plasmid carrying CEBPA mutant and miR-181a gene were also compared and the former showed higher efficiency.;To allow easier cell positioning and the scale-up of NEP, dielectrophoresis based cell loading was developed on the current 2D NEP device as well as a 3D NEP prototype. With a customized amplifier, positive DEP could trap cells at the micro/nanochannel interface with a holding force larger than 10 pN, and negative DEP could be used to collect the cells after NEP. Procedures for parallel and efficient cell loading were developed on both 2D and 3D devices. DEP-NEP was also performed on both designs to show their compatibility. The simplicity, low cost and high-throughput ability of DEP-NEP made it an excellent candidate for transfection of large cell population. |
