Electroporation through a micro-fabricated orifice and its application to the measurement of cell response to external stimuli

Abstract

A device is developed for low-voltage electroporation using field constriction at a micro-orifice, and the application to the real-time measurement of single cell response is demonstrated. The device consists of a pair of electrodes separated by an insulator film having a regularly arranged array of micro-fabricated orifices with a typical diameter of 1–2 µm. Cells are immobilized at the orifices by aspiration, and a pulse voltage is applied. The field lines, being unable to penetrate the insulator, go into the orifices and create a field constriction. This means that most voltage drop occurs in the vicinity of the orifice, and is imposed locally on the membrane in contact with the orifice. Hence, electroporation can be achieved regardless of the cell size, shape or orientation. The experimental verification is made with human monocytes, and uptake of a fluorescence dye is observed with pulses as low as 1 V, and almost 100% yield is achieved at 1.5 V. Then the dynamic response of a myocyte to external stimuli is measured. When the substrates for the metabolic cycle are fed by the method, a clear increase in fluorescence emission from the resultant NADH is observed.

On-chip testing device for electrochemotherapeutic effects on human breast cells

“…Besides two common characteristics of low amplitude voltage source and high heat dissipation rate, each microdevice has its own characteristics of single cell EP (Huang and Rubinsky 2003;Khine et al 2005;Khine et al 2007), electrophoresis assisted EP (Huang et al 2007), focused electric field by geometrical constriction (Kurosawa et al 2006;Wang and Lu 2006;Fei et al 2007;Kim et al 2007b), and parallelized EP process (Kim et al 2007a). However, the applications have been confined in the gene transfection and cell lysis areas.…”

Micro-/nanofluidics based cell electroporation

“…1, those micro-devices usually consist of a large flow channel for cells to freely pass and one or an array of embedded constrictions (e.g., orifices or side microchannels with the key dimensions about half of the cell size or smaller to help trap cells at desired locations). [42][43][44][45][46][47][48][49] Electrodes were generally closely spaced and patterned so that a low potential difference is enough for electroporation and/or measurements of other electrical signal (e.g., electric conductivity or impedance) at the presence of electrolysis disturbance. To firmly hold cells, a gentle vacuum was sometimes used to pull a small portion of cells inside the microscale constrictions.…”

“…To firmly hold cells, a gentle vacuum was sometimes used to pull a small portion of cells inside the microscale constrictions. [46][47][48][49][55][56][57][58] If cell lysis is necessary, more efficient lysis electrode designs such as interdigitated electrodes with a saw-tooth structure could be used. Besides trapping cells with physical contact, optical tweezers technology (a technology that creates radiation pressure with an intensified laser beam to facilitate non-contact optical trapping to colloids) was also introduced to help grab and relocate cells remotely in electroporation micro-devices ( Fig.…”

“…The recent emerging of micro-/nanotechnologies opened new routes to create better electrode designs, improved operation platforms, and more advanced regulations and controls on the electric pulses, cells, and pH and/or temperature variations. [25][26][27] A number of new electroporation systems with micro-/nanoscale features, including microfluidicelectroporation (MEP) and nanofluidic-electroporation (NEP), have been introduced, which offer various advantages over available commercial systems towards improved performance (i.e., high transfection efficiency and high cell viability) from the following aspects: (1) in situ monitoring of intracellular content transport in the electroporation process and dynamics at the single cell level, [28][29][30][31][32][33][34][35][36][37][38][39] (2) very low potential differences (can be as low as 1 V/cm) while sufficient to upset the cell membrane to avoid unwanted electrochemical reactions, pH variations, Joule heating, and gas bubble formation, [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49] (3) better accuracy and flexibility on cell handling and manipulation to achieve dosage control and specific treatment for different sizes of cell population. These new approaches have been applied to cell lysis, cell fusion, and cell electroporation with focuses varying from single cell analysis to large scale process towards clinic use.…”

A pressure drop correlation for low Reynolds number Newtonian flows through a rectangular orifice in a similarly shaped micro-channel

“…Rectangular orifices within such channels are commonly encountered in microfluidics systems in the form of microvalves and micro-pumps (Au et al 2011), micro-coolers (Jin et al 2011), emulsifiers (Skurtys & Aguilera 2008), cell filters (Bhagat et al 2010), flow cytometers (Lee et al 2011), and cell analysers (Kurosawa et al 2006;Li et al 2007;Gel et al 2009). We have also recently suggested use of an orifice as a distributor for micro-fluidized beds (Zivkovic et al 2010), which we are currently developing as a means of increasing mixing, heat and mass transfer in micro-reactor systems and, additionally, sensitivity in analytical applications (Zivkovic et al 2012).…”