Showing papers by "Tuhin Subhra Santra published in 2013"

Recent Trends on Micro/Nanofluidic Single Cell Electroporation

Abstract: The behaviors of cell to cell or cell to environment with their organelles and their intracellular physical or biochemical effects are still not fully understood. Analyzing millions of cells together cannot provide detailed information, such as cell proliferation, differentiation or different responses to external stimuli and intracellular reaction. Thus, single cell level research is becoming a pioneering research area that unveils the interaction details in high temporal and spatial resolution among cells. To analyze the cellular function, single cell electroporation can be conducted by employing a miniaturized device, whose dimension should be similar to that of a single cell. Micro/nanofluidic devices can fulfill this requirement for single cell electroporation. This device is not only useful for cell lysis, cell to cell fusion or separation, insertion of drug, DNA and antibodies inside single cell, but also it can control biochemical, electrical and mechanical parameters using electroporation technique. This device provides better performance such as high transfection efficiency, high cell viability, lower Joule heating effect, less sample contamination, lower toxicity during electroporation experiment when compared to bulk electroporation process. In addition, single organelles within a cell can be analyzed selectively by reducing the electrode size and gap at nanoscale level. This advanced technique can deliver (in/out) biomolecules precisely through a small membrane area (micro to nanoscale area) of the single cell, known as localized single cell membrane electroporation (LSCMEP). These articles emphasize the recent progress in micro/nanofluidic single cell electroporation, which is potentially beneficial for high-efficient therapeutic and delivery applications or understanding cell to cell interaction.

Tuning nano electric field to affect restrictive membrane area on localized single cell nano-electroporation

Abstract: Interaction of electric field with biological cells is an important phenomenon for field induced drug delivery system. We demonstrate a selective and localized single cell nano-electroporation (LSCNEP) by applying an intense electric field on a submicron region of the single cell membrane, which can effectively allow high efficient molecular delivery but low cell damage. The delivery rate is controlled by adjusting transmembrane potential and manipulating membrane status. Thermal and ionic influences are deteriorated from the cell membrane by dielectric passivation. Either reversible or irreversible by LSCNEP can fully controlled with potential applications in medical diagnostics and biological studies.

Electroporation Based Drug Delivery and Its Applications

Abstract: When a certain strong electrical pulse applied across a cell or tissue, the structures of the cell or tissue would be rearranged to cause the permeabilization of the cell membrane, named in early 1980’s “electroporation”[1]. The theoretical and experimental studies of electric field effects on living cells with their bilayer lipid membrane has been studies in 1960’s to 1970’s century [1-6]. During these years, the researches were primarily dealt with reversible and irreversible membrane breakdown in vitro. Based on these research, the first gene transfer by custom-built electroporation chamber on murine cells was performed by Neumann et al. in 1982 [7]. When electric field (E≈0.2V, Usually 0.5-1V) applied across the cell membrane, a significant amount of electrical conductivity can increase on the cell plasma membrane. As a result, this electric field can create primary membrane “nanopores” with minimum 1 nm radius, which can transport small amount of ions such as Na+ and Clthrough this mem‐ brane “nanopores”. The essential features of electroporation included (a) short electric pulse application (b) lipid bilayer charging (c) structural rearrangements within the cell mem‐ brane (d) water-filled membrane structures, which can perforate the membrane (“aqueous pathways” or pores) and (e) increment of molecular and ionic transportation [8]. In conven‐ tional electroporation (Bulk electroporation) technique, an external high electric field pulses were applied to millions of cells in suspension together in-between two large electrodes. When this electric field was above the critical breakdown potential of the cell, a strong polarization of the cell membrane occur due to the high external electric field. Applying a very high electric field could be resulted in the formation of millions of pores into the cell membrane simultaneously without reversibility [9]. Several methods other than electropora‐ tion can be used for gene transfer like microprecipitates, microinjection, sonoporation,

Nanofocused electric field for localized single cell nanoelectroporation with membrane reversibility

Abstract: Despite the significant research in electroporation, high electric field was applied to the whole cells resulted in permeabilizing the membrane of millions of cells without reversibility [1]. To deliver biomolecules through the specific region of the cell membrane with high cell viability and high transfection rate is important for various biological and therapeutic applications.This report presents a new type localized single cell membrane electroporation (LSCMEP), at specific region of the single cell with the application of 800 μs electric pulse. The ITO nano-electrodes with 100nm thickness and 500 nm gap between two electrodes can generate an intense electric field to track biomolecules inside HeLa cell in our studies. This small gap between two nano-electrodes can neglect thermal effect on cell membrane and permit reversible electroporation with high cell viability (90%) and minimum effected electroporation region (0.48 μm). Our approach successfully delivers biomolecules through a specific region of single cell with high transfection rate (82%) and high cell viability. This process, not only generates well-controlled nano-pores allowing rapid recovery of cell membrane, but also it provides a clear optical path potentially tracking of drugs to deliver inside single cell.

Nano-electrode based chip

Abstract: The present invention disclosed a nano-electrode based transparent chip, which is applicable to perform a micro fluidic substance diffused through a specific region of a single cell membrane by localized electroporation technique with membrane reversibility. The chip comprises a silicon-based layer, different structural layers, an insulating layer and a micro fluidic layer. When the individual single cell was placed on the gap between a plurality of triangular-shaped nano-electrodes with nano-tip, then electric field can intense on a specific region of the individual single cell causes the formation of nano-pores on the membrane, resulting to deliver drugs, DNA molecules from outside of the cell to the inside of the cell with a high transfection rate and a high cell viability. This technique not only generates well-controlled nano-pores to allow rapid recovery of the cell membrane, but also provides clear optical path for potentially monitoring/tracing the drugs into the cell.