1. Nano-localized single-cell nano-electroporation
The ability to deliver foreign cargo into single living cells is of great interest in cell biology and therapeutic research. Here, we have reported single or multiple positions based nano-localized single-cell nano-electroporation platform. The device consists of an array of triangular shape ITO nano-electrodes with a 70 nm gap between two nano-electrodes and each of having 40 nm tip diameter. The voltage is applied between nano-electrodes to generate an intense electric field, which electroporates multiple nano-localized regions of the targeted single-cell membrane, and biomolecules gently deliver into cells by pressurizing pumping flow, without affecting cell viability. The platform successfully delivers dyes, QDs, and plasmids into different cell types with the variation of field strength, pulse duration, and the number of pulses. This new approach allows us to analyze different biomolecular delivery into single living cells with high transfection efficiency (> 96%, for CL1-0 cell), and high cell viability (~98%), which are potentially beneficial for cellular therapy and diagnostics purpose.
Figure 1. (a). Schematic of nano-localized single-cell nano-electroporation (NL-SCNEP) chip. (b) Optical microscopic image of 100 nm thin suspended Si3N4 membrane (800 µm × 800 µm) with patterned ITO lines. (c) Scanning electron microscopy (SEM) image of ITO lines. (d) Focused Ion Beam (FIB) cut ITO lines to form ITO nano-electrodes. (e) An SEM image of ITO Nano-electrode with 70-nm gaps. (f) Single-cell attachment on top of the ITO nano-electrodes before the experiment. (g) The final packaged nano-electroporation device with a Printed Circuit Board (PCB) (h-i) PI dye delivery using ms and µs pulse (j) plasmid delivery using ms pulse.
2. 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 �m, electrode gap = 500 nm and electrode thickness = 90 nm, the depth of the gap from electrode surface = 3 �m 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.
3. Photporation based drug delivery/ Single-cell photoporation
In intracellular delivery, the introduction of foreign biomolecules into cells with high transfection efficiency and high cell viability is a challenging task for therapeutics, diagnostics, diseases analysis and many biological and biomedical research. We reported infrared (IR) pulse laser-activated high efficient massively parallel intracellular delivery using an array of titanium micro-dish (TMD) device. The IR laser was espoused on an array of TMD device, which induces heat generation, resulting in the formation of cavitation bubbles at the cell membrane surface and create transient membrane pores to deliver biomolecules into cells by a simple diffusion process. We successfully delivered dyes and the different size of dextran in different cell types. Our platform has the ability to transfect more than 1000000 cells in parallel fashion or even more within a minute. The best results were achieved using propidium iodide dye and dextran 3000 MW delivery in SiHa cells with 96% and 98% delivery efficiency and 98% and nearly 100% cell viability. The device is compact, easy to use, and potentially applicable for cellular therapy and diagnostics purposes.
Figure: Schematic of photoporation based massively parallel intracellular delivery (Left Figure), microfabrication (Middle Figure) and parallel intracellular delivery in different cancer cells (Right Figure)
4. Laser based cell therapy
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 embryonic 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 CL1-0 cells with close to 100% cell viability near infrared wavelength.
Figure: Schematic of laser based cell therapy (First Figure), COMSOL Multiphysics simulation of core shell nm-AuNPs (Second Figure), nm-AuNPs fabrication using micro/nano fabrication and cell attachment (Third Figure), Intracellular delivery in different cancer cells and stem cell (Forth Figure).
5. 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.
6. Micro-contact cell printing technologies
In order to pattern substrates, Polydimethylsiloxane (PDMS) micro pillar structure is fabricated using SU8 mold using micro/nanofabrication technique. These PDMS structures are used as stamp to print fluorescence ink/ cell specific proteins on the substrate. Then cell was cultured on substrate and align cells in desired pattern with high cell viability. This cell printing technique is very useful for different cellular study such as cellular heterogeneity, cell-cell interactions, cell-environment interactions, cell signaling, diseases analysis and different omics analysis.
Figure: (a) Schematic of PDMS microstamp and proteins/fluorescence stamping on substrate (b) successful experimental results of different fluorescence stamping on substrate.
7. Tissue engineering and regenerative medicine
Cells show bidirectional interaction with Extracellular matrix (ECM) through specialized membrane receptors like integrin to sense mechanical and chemical stimulus. The ECM structure and cell alignment is varying from organ to organ based on its specific functional requirements. Under specific mechanical or chemical stimulus, cells can secrete proteins and enzymes to build or remodel ECM structure. Currently, we are developing 2D cell patterning for alignment of cells in a specific pattern by providing different chemical stimulus to cells. To achieve cell alignment micro contact printing technique for substrate patterning is employed. Cells are seeded on pre patterned substrates. Based on the cell affinity to particular chemical stimulus, they get aligned on the substrate. After array of cell (single or multiple) alignment, this platform are using for tissue engineering and regenerative medicine purpose.
Figure: Formation of array of cells.
8. Microfluidic neuronal platform for neuron regenerative studies
Understanding the basic mechanisms of neural regeneration after injury is a pre-requisite for developing appropriate treatments. This research aims the fabrication of compartmentalized microfluidic chip to isolate and grow axons ultimately delivering a platform for analysing neuronal response to axonal directionality. It also helps in monitoring the axon-neural scaffold interfacing for neuro-regenerative perspective. These neuro-scaffolds comprise of electro spun polymeric nanofibers. The microfluidics chamber allows the flexibility of focussing particularly on axons for exposing them to external stimuli on glass surface or on scaffolds.
Figure: Culture of neurons extending axons on compartmentalized microfluidic device
9. Development of 3D scaffold for periodontal regeneration
Owing to unique morphology and properties Graphene foam is gaining interest in various tissue engineering applications. While mimicking the layered native tissue structures, biggest drawback is separation or loose integration of various layers, therefore leading to failure of the scaffold and functionality. But generation of layered functionally gradient structure having core of graphene foam solves this issue along with providing better connection and organization of cells and cellular signals. This scaffold also provides the higher degree of freedom to optimize the polymer reinforcement and therefore tuning biological and physical properties. Particularly, for periodontitis, the prevalence of the disease is very high nationally and globally. Every clinic will have minimum of one or two patients every day and every department of each college will have a minimum of 20-25% of the patients attending every day with periodontitis. Therefore the close mimicry of natural tissues, degenerated in the diseased condition can be regenerated with the application of proposed functionally gradient scaffold. So society and market wise we feel it will be of great benefit.
Figure: SEM of 3D scaffold layers (First row, Figure a-c) and florescence images showing MG-63 cells growing on 3D scaffold layer. (Figure a, second row) cross section of the scaffold. (Figure b and c, second row) Cells are stained with Calcein (green) dye to show cell viability. Arrows indicate cells growing on scaffold.
10. Nanostructure materials/Implantable devices for biomedical applications
We are developing different nanostructures such as nanotubes, nanocones, nanoflowers, nanoporus, nanofibers material on commercially pure titanium, titanium alloys, magnesium alloys for biomedical applications. Currently, the Nanotubes are using for controlled or localized drug delivery applications using different physical methods such as electroporation, photoporation, machoanoporation, microinjection and etc. The fabrication of nanofibers using electrospinning are using for wound healing applications.
Figure: SEM image of fabricated electrospun PCL fibers (Figure a-d, First column). Fabrication of TiO2 monolayer (Figure a-b, second column) and TiO2 double layer (Figure a-b, second column).
11. Micro-patterened 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, surgical instruments, prosthetic replacements etc.
Figure: Contact angle and surface roughness measurement of DLN films (First column), Micro-patterned DLN films for cells adhesion study (second column).