1. Single cell nano-electroporation
Single cell analysis is potentially beneficial to understand cell to cell heterogeneity from population of cells together, which is a key feature for disease analysis such as cancer metastasis drug response of tumor cells and etc. We demonstrate an efficient and fast method for multi-nanolocalized single cell nanoelectroporation, where electroporation take place on a multiple positions together with tens of nanometer confined region on single cell membrane using ITO nano-electrodes array. The gap between two nano-electrodes are 60 nm with triangle tip diameter is 40 nm, which can intense an electric field in a narrow region of single cell membrane to deliver biomolecules with high transfection rate and high cell viability. In this study, we successfully deliver dyes, QDs, mRNA, EGFP and different plasmids into single cell with different field strengths and pulse durations. This new approach can allow us to analyze different dyes/biomolecules interaction in single living cell with high ability of spatial, temporal, and qualitative dosage control which potentially applicable for medical diagnostics and biological cell studies.
Figure: Fabrication process of a nano-electrode-based transparent chip: (a) fabrication process step; (b) SEM image after wet chemical etch of ITO lines; (c) FIB etched ITO nano-electrode (electrode width = 2 Ám, electrode gap = 500 nm and electrode thickness = 90 nm, the depth of the gap from electrode surface = 3 Ám to make as microfluidic channel); (d) final packaging of fabricated ITO nano-electrode based transparent chip (left figure). Schematic representation of the localized single cell electroporation device (a) without SiO2 passivation layer (b) with SiO2 passivation layer. (c) fabrication process step of the device without SiO2 layer (d) microscopy image of the ITO electrode (e) Scanning Electron Microscope (SEM) image of the ITO lines (f) Focused Ion Beam (FIB) etched ITO electrode (g) an ITO electrode with microfluidic channel formation by using FIB (h) Final packaging of the localized single cell electroporation chip.
2. Photporation based intracellular/extracellular drug delivery
This research present nanosecond pulse laser induced plasmonic photoporation for a high efficient intracellular delivery with high cell viability using nano-corrugated mushroom shape gold nanoparticles (nm-AuNPs). In our study, poly ethylene glycol (PEG) modified nm-AuNPs bind with different cancer cells as well as embroynic stem cells easily create transient membrane pores due to high surface plasmon generated nanobubbles upon pulsed laser illumination and thus cargos such as dyes, Quantum Dots (QDs) and plasmids can be gently delivered into cells. We found uptake efficiency and cell viability can depends upon laser fluence and concentration of nm-AuNPs. The higher laser energy produce higher intracellular uptake for AGS cells with close to 100% cell viability at 480 nm wavelength.
Fig: Nanosecond pulse laser mediated photoporation for high efficient intracellular delivery with high cell viability using nanocorrugated mushroom shape gold nanoparticles (nm-AuNPs).
3. Single cell Mechanoporation
In this work, we are developing single cell mechanoporation for intracellular delivery, which is a compact, easy to use, massively parallel, single cell mechanoporation platform that not only overcomes the throughput limitation but also it can provide very high delivery efficiency with high cell viability.
4. Diamond-like nanocomposite thin films and its biomedical applications
Diamond-like nanocomposite (DLN) thin films are deposited on glass (pyrex/silicon) substrate by plasma enhanced chemical vapor deposition (PECVD) technique using different combinations of hexamethyldisiloxane (HMDSO) and hexamethyldisilazane (HMDSN) gas precursors. The surface morphology of the DLN films has been investigated by atomic force microscopy (AFM). Nanoparticles on DLN films were analyzed by high resolution transmission electron microscopy (HRTEM). Fourier transform infrared spectroscopy FTIR, Raman spectroscopy, and x-ray photoelectron spectroscopy XPS were used to determine the structural change within the DLN films. The hardness and friction coefficient of the films were measured by nanoindentation and scratch test techniques, respectively. The biocompatibility of the films was verified using different cancer cell adhesion on micro pattern DLN films (using microfabrication process) and western blot experiment. This research emphasizes on the possible biomedical applications of DLN films such as biosensors for diagnostics and therapies, surgical instruments, prosthetic replacements etc.
Fig: (a) optical image of CL1(0) cell attachment on micropattern DLN surface (b) cell nucleus stain with hochest (c) deep red plasma membrane (d) merge image of cell nucleus and plasma membrane.