Label-free purification and characterization of optogenetically engineered cells using optically-induced dielectrophoresis
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Optogenetically engineered cell population obtained by heterogeneous gene expression plays a vital role in life science, medicine, and biohybrid robotics, and purification and characterization are essential to enhance its application performance. However, the existing cell purification methods suffer from complex sample preparation or inevitable damage and pollution. The efficient and nondestructive label-free purification and characterization of the optogenetically engineered cells, HEK293-ChR2 cells, is provided here using an optically-induced dielectrophoresis (ODEP)-based approach. The distinctive crossover frequencies of the engineered cells and the unmodified cells enable effective separation due to the opposite DEP forces on them. The ODEP-based approach can greatly improve the purity of the separated cell population and especially, the ratio of the engineered cells in the separated cell population can be enhanced by 275% at a low transfection rate. The size and the membrane capacitance of the separated cell population decreases and increases, respectively, as the ratio of the engineered cells grows in the cell population, indicating that successful expression of ChR2 in a single HEK293 cell makes its size and membrane capacitance smaller and larger, respectively. The results of biohybrid imaging with the optogenetically engineered cells demonstrated that cell purification can improve the imaging quality. This work proves that the separation and purification of engineered cells are of great significance for their application in practice.Keywords:
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Abstract Issue no. 18 is a special issue on “Dielectrophoresis – Part II” consisting of 16 contributions distributed over 4 distinct parts. In addition, this issue presents a FAST TRACK paper dealing with electrokinetic DNA transport in 20 nm‐high nanoslits. “The first special issue on Dielectrophoresis (Electrophoresis, 2011, 32, no. 17) presented 16 contributions, covering topics in fundamentals, nanoscale dielectrophoresis and cell dielectrophoresis. This issue presents part II of “Dielectrophoresis 2011” with articles covering (i) Fundamentals, (ii) Cell Dielectrophoresis, (iii) Biomedical and clinical dielectrophoresis and (iv) Applications. The first section on Fundamentals includes a valuable review article that provides an overview of the mathematical modeling and recent applications of dielectrophoresis systems. This section also contains four research articles that study particle focusing with electrodes and insulators, pH gradients, fluid streaming and multipart devices. The second section analyzes Cell Dielectrophoresis, beginning with a review contribution on the application of dielectrophoresis for cell characterization, manipulation, separation and patterning. Three additional research articles complement the second section, covering cell electrofusion, cell pairing and cell immobilization. The third section explores the great potential of dielectrophoresis for biomedical and clinical applications; with contributions on dielectrophoretic exploration and manipulation of red blood cells and cancer cells. The fourth and last section presents two valuable contributions on non‐conventional dielectrophoresis applications on filtering engine oil and microfluidic mixing, demonstrating its great flexibility. We believe these two issues of “Dielectrophoresis 2011”, represent a unique collection of fine articles on this important and growing subject. The contributions compiled here range from solid and noteworthy reviews to novel and exciting research articles.” Featured articles include: FAST TRACK: Electrokinetic DNA transport in 20 nm high nanoslits: Evidence for movement through a wall‐adsorbed polymer nanogel. (( 10.1002/elps.201100278 )) Quantification of pH gradients and implications in insulator‐based dielectrophoresis of biomolecules. (( 10.1002/elps.201100090 )) Real‐time cell electrophysiology using a multi‐channel dielectrophoretic‐dot (DEP‐Dot) microelectrode array. (( 10.1002/elps.201100033 )) Microfluidic mixing using contactless dielectrophoresis (cDEP) (( 10.1002/elps.201100171 ))
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Electric fields interaction with living cells is commonly used in lab-on-chips. Indeed AC electrokinetic techniques (dielectrophoresis, electrorotation and traveling wave dielectrophoresis) are used to handle, trap or separate biological entities (eukaryotic cells, bacteria, yeasts, algae) in microfluidic devices.
Several studies have shown how electric fields can be used to discriminate cell depending on their dielectric properties, which represents a growing interest for many biomedical applications (target cell identification from an heterogeneous biological sample, different stages of cancer disease diagnosis,...).
This paper presents a microfluidic device devoted to the determination of a single cell electro-physiological properties combining dielectrophoresis force for the cell trapping, with electrorotation experiments to extract the dielectric properties of the cell. A microfluidic device has been designed for this purpose and evaluated with two different cell lines.
Combining dielectrophoresis and electrorotation experiments allows reproducible measurement by avoiding possible perturbations or interactions with other cells duting the analysis within the biodevice. External sollicitations applied to biological cell might be monitored in ‘real-time’ in such microfluidic platform.
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AC-dielelectrophoresis is utilized inside a lab-on-a-chip device to separate particles and cells. Dielectrophoresis is the movement of particles in a non-uniform electric field due to the interaction of the particle's dipole and spatial gradient of the electric field. Dielectrophoresis is a subtle solution to manipulate particles and cells at microscale due to its favorable scaling for reduced size of the system. Dielectrophoresis is applicable with both DC and AC fields. DC-dielectrophoresis only depends on the electrical conductivities of the particle and the medium. AC-dielectrohoresis depends on the permittivities of the particle and the medium, and the field frequency. AC-dielectrophoresis is richer in the sense that both positive and negative dielectrophoretic force can be generated for biological particles by tuning the field frequency. The dielectrophoretic force depends on the size and the electrical properties of the particles and the suspending medium which makes the separation of particles and cells based on their size and based on their electrical properties possible. In this dissertation, the continuous separation of particles and cells based on their size and based on their electrical properties is achieved inside a lab-on-a-chip device. PDMS (polydimethylsiloxane) microchannels are fabricated using soft lithography technique. The flow is induced by pressure gradient. Simple, 3D electrodes which are fabricated by a simple and inexpensive technique extended from soft-lithographic fabrication are used to achieve a localized, non-uniform electric field. Dielectrophoretic force is generated in the transverse direction to the flow by inserting 3D electrodes along the channel side walls. The localized electric field is important to reduce the Joule heating and any adverse effects on biological particles due to the interaction of particles with the electric field. Latex particles of different size and mixture of white blood cells (which have a typical size of 8-12 micron) and yeast cells (which have a typical size of 3-5 micron) is separated based on their size difference. The separation based on electrical properties is demonstrated by means of the separation of 10 micron latex particles and white blood cells. A numerical simulation based on Lagrangian tracking method is used to simulate the particle trajectories. The present designs have the feature of using simple electrodes like DC-dielectrohoretic devices and of using low electrical potential like AC-dielectrophoretic devices; they are unique in a sense that the effect of the electric field is confined in a small area which means a very short time for the interaction of the particles with the electric field.
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The expanding field of AC Electrokinetics offers a number of opportunities for the separation and characterisation of cells. Here the authors consider two methods, Dielectrophoresis and Travelling Wave Dielectrophoresis. Dielectrophoresis is a non-invasive technique that has been used to probe the internal dielectric properties of single cells by inducing a motive force on that cell. By controlling the conditions so that the force exerted on particles of different types is significantly different, a mixture of different cell types can be sorted into groups. Travelling Wave Dielectrophoresis is better able to discriminate small changes in cell properties than conventional Dielectrophoresis. It is also used as a means of transporting cells around electrode arrays. Here the authors illustrate how these technologies and others can be integrated to form an on-chip cell sorter capable of performing Dielectrophoresis Activated Cell Sorting (DACS).
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After rigorous development over the years, dielectrophoresis has been established as an effective method to manipulate micron and sub-micron sized particles.In particular, it is a promising technology for lab-on-a-chip or micro total analysis system (µTAS) to separate cells for biomedical applications.This technology is based on the knowledge that a particle suspended in a fluid medium experiences a net electrical force, due to a polarization effect, when non-uniform electrical fields are applied across the fluid.By varying the applied electric field frequencies, the magnitude and the direction of the dielectrophoretic forces on the particle can be varied and controlled.When the applied electric field only varies in magnitude over time, the dielectrophoretic force is 1-dimensional.This is commonly referred as conventional dielectrophoresis.When the applied electric field has a varying magnitude and phase, the dielectrophoretic force is 2-dimensional.This is commonly referred as traveling wave dielectrophoresis.While particle separations have been demonstrated with devices based on these two techniques, the separated particles were confined in space.To overcome this issue, fluid flow is generally used to carry the particles.In this investigation, moving dielectrophoresis (mDEP) is introduced for the manipulation and transportation of particles.The moving dielectrophoresis is generated by a series of electrodes which can be individually energized to induce an electric field that moves from one electrode to another.Beside the electric field frequency, the switching speed of the electrode is a second time parameter introduced in moving dielectrophoresis.A major difference of this technique from the traveling wave dielectrophoresis is that the moving speed of the energized electrodes is independent of the electric field frequency.By sequentially energizing the electrodes, a particle can be controlled to move in the same direction.By controlling the electric field frequencies and the energizing of the electrodes, other manipulation techniques like separation, isolation, fractionation and trapping can be achieved.A mathematical model is also presented to provide a theoretical basis for the use of the moving dielectrophoresis.
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The electrodeless dielectrophoretic trapping and concentration of viruses was demonstrated. Dielectrophoresis is the motion of matter caused by polarization effects in a nonuniform electric field. Experiments were performed on a glass chip with insulating posts in order to study the dielectrophoretic behavior of the viruses and extend the application of insulative (electrodeless) Dielectrophoresis (iDEP) to polymeric microdevices. The ultimate goal of his research project is to create a highthroughput EDEP system using polymers as substrate. Only two electrodes were present in the system. In the presence of an applied DC electric field the viruses exhibited streaming and trapping dielectrophoresis.
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Бұл зерттеужұмысындaКaно моделітурaлы жәнеоғaн қaтыстытолықмәліметберілгенжәнеуниверситетстуденттерінебaғыттaлғaн қолдaнбaлы (кейстік)зерттеужүргізілген.АхметЯссaуи университетініңстуденттеріүшін Кaно моделіқолдaнылғaн, олaрдың жоғaры білімберусaпaсынa қоятынмaңыздытaлaптaры, яғнисaпaлық қaжеттіліктері,олaрдың мaңыздылығытурaлы жәнесaпaлық қaжеттіліктерінеқaтыстыөз университетінқaлaй бaғaлaйтындығытурaлы сұрaқтaр қойылғaн. Осы зерттеудіңмaқсaты АхметЯсaуи университетіндетуризмменеджментіжәнеқaржы бaкaлaвриaт бaғдaрлaмaлaрыныңсaпaсынa қaтыстыстуденттердіңқaжеттіліктерінaнықтaу, студенттердіңқaнaғaттaну, қaнaғaттaнбaу дәрежелерінбелгілеу,білімберусaпaсын aнықтaу мен жетілдіружолдaрын тaлдaу болыптaбылaды. Осы мaқсaтқaжетуүшін, ең aлдыменКaно сaуaлнaмaсы түзіліп,116 студенткеқолдaнылдыжәнебілімберугежәнеоның сaпaсынa қaтыстыстуденттердіңтaлaптaры мен қaжеттіліктерітоптықжұмыстaрaрқылыaнықтaлды. Екіншіден,бұл aнықтaлғaн тaлaптaр мен қaжеттіліктерКaно бaғaлaу кестесіменжіктелді.Осылaйшa, сaпa тaлaптaры төрт сaнaтқa бөлінді:болуытиіс, бір өлшемді,тaртымдыжәнебейтaрaп.Соңындa,қaнaғaттaну мен қaнaғaттaнбaудың мәндеріесептелдіжәнестуденттердіңқaнaғaттaну мен қaнaғaттaнбaу деңгейлерінжоғaрылaту мен төмендетудеосытaлaптaр мен қaжеттіліктердіңрөліaйқын aнықтaлды.Түйінсөздер:сaпa, сaпaлық қaжеттіліктер,білімберусaпaсы, Кaно моделі.
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