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Micromachines
Volume 4
Issue 3
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Micromachines, Volume 4, Issue 3 (September 2013) – 4 articles , Pages 286-356

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4447 KiB  
Review
Recent Trends on Micro/Nanofluidic Single Cell Electroporation
by Tuhin Subhra Santra and Fang Gang Tseng
Micromachines 2013, 4(3), 333-356; https://doi.org/10.3390/mi4030333 - 6 Sep 2013
Cited by 58 | Viewed by 17398
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Micro/Nanofluidic Devices for Single Cell Analysis)
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Figure 1

Figure 1
<p>The bulk electroporation apparatus <span class="html-italic">in vitro</span> experiment with cross sectional view of two metal electrodes. The distance between two large electrodes varies from millimeter to centimeter range. To manufacture of this device is simple but the voltage requirement is very high to permeabilize of millions of cells together due to large distance between two electrodes (a) cells are in suspension within the cuvette, where two metal electrodes are fixed inside the cuvette for electroporation experiment (b) cells are suspension and metal electrodes can introduce from outside to inside of this cuvette. Figure has been redrawn from reference [<a href="#B31-micromachines-04-00333" class="html-bibr">31</a>].</p> Full article ">Figure 2
<p>Electric field distribution for single cell electroporation where induced transmembrane potential is maximum at the cell pole and minimum at the equator.</p> Full article ">Figure 3
<p>Single cell electroporation (SCEP), where an external electric field applied outside of the single cell (<b>a</b>) formation of pores due to electric field application (maximum pores open at poles and minimum pores open at the equators due to different field strength at different positions of the membrane); (<b>b</b>) after withdrawing the pulse, cell membranes reseal again and biomolecules enter successfully inside the single cell.</p> Full article ">Figure 4
<p>Localized single cell membrane electroporation (LSCMEP) where electric field is applied in a specific region (nano-scale region) of the cell membrane. (<b>a</b>) During electroporation, membrane pores open and biomolecules deliver from outside to inside of the single cell; (<b>b</b>) After electroporation cell membranes reseal again and biomolecules entered successfully inside the single cell. Permission to reprint obtained from Springer [<a href="#B27-micromachines-04-00333" class="html-bibr">27</a>].</p> Full article ">Figure 5
<p>Microfluidic SCEP with cell trapping. Figure has been redrawn with reference [<a href="#B26-micromachines-04-00333" class="html-bibr">26</a>,<a href="#B66-micromachines-04-00333" class="html-bibr">66</a>].</p> Full article ">Figure 6
<p>(<b>a</b>) Concept of flow through micro-electroporation chip; (<b>b</b>) Optical image of micro-channel, micro-hole and electrodes. Permission to reprint obtained from Elsevier [<a href="#B78-micromachines-04-00333" class="html-bibr">78</a>].</p> Full article ">Figure 7
<p>The schematic steps for electrophoresis driven cell loading protocol (<b>a</b>) preconcentrate; (<b>b</b>) membrane electroporation; (<b>c</b>) apply electrophoretic driving force. Figure has been redrawn with reference [<a href="#B82-micromachines-04-00333" class="html-bibr">82</a>].</p> Full article ">Figure 8
<p>Layout and performance of a droplet based microfluidic device (<b>a</b>) two microelectrodes, where cells can be positioned for electroporation; (<b>b</b>) after electroporation, a droplet with a cell at the end of the device. Permission to reprint obtained from American Chemical Society (ACS) [<a href="#B87-micromachines-04-00333" class="html-bibr">87</a>].</p> Full article ">Figure 9
<p>Droplet microfluidic electroporation technique (<b>a</b>) schematic diagram; (<b>b</b>) electroporation process of algal cell. Figure has been redrawn from reference [<a href="#B76-micromachines-04-00333" class="html-bibr">76</a>].</p> Full article ">Figure 10
<p>Schematic representation of microfluidic cell lysis device where saw-teeth microelectrodes acting as a dielectrophoresis effect on the device for focusing intracellular material after electroporation. Figure has been redrawn from reference [<a href="#B88-micromachines-04-00333" class="html-bibr">88</a>].</p> Full article ">Figure 11
<p>Microfluidic based flow through electroporation device. Cells from sample reservoir moving to the receiving reservoir and electrical lysis were confined with single cell movement through the lysis section (W<sub>2</sub>). Permission to reprint obtained from Elsevier [<a href="#B71-micromachines-04-00333" class="html-bibr">71</a>].</p> Full article ">Figure 12
<p>Schematic diagram for cell lysis using capillary electrophoresis and mass spectrometry. The arrow indicated direction of electroosmotic flow. Cells flow from cell reservoir (A) to the intersection zone where cells were lysed, then they migrate towards electrospray orifice through separation channel. Figure has been redrawn from reference [<a href="#B95-micromachines-04-00333" class="html-bibr">95</a>].</p> Full article ">Figure 13
<p>(<b>a</b>) bright field image of atomic force microscopy (AFM) tip and the cell in the electroporation medium (cell A is electroporated while cell B and C are about 20 µm away from cell A); (<b>b</b>) Fluorescence image of rat fibroblast cell after electroporation; (<b>c</b>) Confocal fluorescence image of an electroporated cell; (<b>d</b>)<b>–</b>(<b>h</b>) Sequence of real time confocal fluorescence images of rat fibroblast cell after electroporation; (<b>i</b>) Calculated spatial distribution of electric field in the vicinity of the cell being electroporated. Permission to reprint obtained from American Institute of Physics (AIP) [<a href="#B29-micromachines-04-00333" class="html-bibr">29</a>].</p> Full article ">Figure 14
<p>Localized single cell membrane electroporation (LSCMEP) device (<b>a</b>) localized electroporation process between two microelectrodes; (<b>b</b>) multiple number of microelectrodes for LSCMEP process; (<b>c</b>) optical microscope image of ITO microelectrodes; (<b>d</b>) scanning electron microscope image of ITO microelectrodes with microchannel. Permission to reprint obtained from Springer [<a href="#B27-micromachines-04-00333" class="html-bibr">27</a>].</p> Full article ">Figure 15
<p>(<b>a</b>) Schematic diagram with electrical connection of the device and PDMS structure; (<b>b</b>) an array of transistors with nanowires and nanoribbons. Figure has been redrawn from reference [<a href="#B77-micromachines-04-00333" class="html-bibr">77</a>].</p> Full article ">
733 KiB  
Article
Lysis of a Single Cyanobacterium for Whole Genome Amplification
by Eric W. Hall, Samuel Kim, Visham Appadoo and Richard N. Zare
Micromachines 2013, 4(3), 321-332; https://doi.org/10.3390/mi4030321 - 21 Aug 2013
Cited by 11 | Viewed by 8648
Abstract
Bacterial species from natural environments, exhibiting a great degree of genetic diversity that has yet to be characterized, pose a specific challenge to whole genome amplification (WGA) from single cells. A major challenge is establishing an effective, compatible, and controlled lysis protocol. We [...] Read more.
Bacterial species from natural environments, exhibiting a great degree of genetic diversity that has yet to be characterized, pose a specific challenge to whole genome amplification (WGA) from single cells. A major challenge is establishing an effective, compatible, and controlled lysis protocol. We present a novel lysis protocol that can be used to extract genomic information from a single cyanobacterium of Synechocystis sp. PCC 6803 known to have multilayer cell wall structures that resist conventional lysis methods. Simple but effective strategies for releasing genomic DNA from captured cells while retaining cellular identities for single-cell analysis are presented. Successful sequencing of genetic elements from single-cell amplicons prepared by multiple displacement amplification (MDA) is demonstrated for selected genes (15 loci nearly equally spaced throughout the main chromosome). Full article
(This article belongs to the Special Issue Micro/Nanofluidic Devices for Single Cell Analysis)
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Figure 1

Figure 1
<p>Schematic of the lysis protocol. Cellular layers and chemical reagents used to remove them are shown. The letter D stands for DNA molecules; D in black represents genomic materials originated from captured single cyanobacteria cells whereas D in grey indicates DNA from other cells, either cyanobacteria of interest or different species; this type of DNA is termed “contaminant DNA” in the text for describing single-cell genome amplification experiments.</p> Full article ">Figure 2
<p>Inhibition of 50-µL Multiple displacement amplification (MDA) reactions by lysis reagents. Amplification factor was calculated by dividing the amount of amplified DNA, as quantified via PicoGreen assay, with that of the starting template (50 pg). The MDA reaction with 0.1% SDS yielded an amount of product (&lt;10 pg/µL) that could not be detected within the limits of the PicoGreen assay, meaning that its amplification factor was below 10. Sark and ProK refer to sarkosyl and proteinase K, respectively. Error bars are not shown on this figure but are approximately 10% of each value.</p> Full article ">Figure 3
<p>Removal of extracellular DNA via multiple washings of cell pellets before injecting into the microfluidic device. dsDNA concentration of the supernatant solutions were determined via PicoGreen fluorescence assay. ProK refers to proteinase K.</p> Full article ">Figure 4
<p>dMDA confirmation of extracellular DNA removal via multiple washings of cell pellets before injection into the microfluidic device. The amounts of DNA fragments in the supernatants from the third and fourth successive TES Buffer washes were quantified with dMDA (as explained in Materials &amp; Methods). Fluorescence images of the dMDA chips containing (<b>a</b>) the supernatant from the third wash and (<b>b</b>) its 200-fold dilution with Injection Buffer; (<b>c</b>) and (<b>d</b>) show the same set from the fourth wash, and (<b>e</b>) is the no-template-DNA control result.</p> Full article ">Figure 5
<p>Loci coverage across scMDA samples. The bar chart presents the occurrence of <span class="html-italic">Synechocystis</span>-specific sequences across scMDA samples, broken down by sample and primer sets.</p> Full article ">Figure 6
<p>Nucleic acid fragment content of final sample set washes. The supernatant of the final wash for each sample set was saved and diluted by a factor of 200. Nucleic acid fragments of the supernatants were quantified to obtain an idea of the level of sample exogenous contamination present in each set injection. Panels are lettered by sample ID (<b>a</b>) through (<b>c</b>) while the fourth (<b>d</b>) is a no-template control. Target counts are quantified per microliter of wash analyte (<b>e</b>), with error bars representing upper and lower 95% confidence interval estimates.</p> Full article ">
434 KiB  
Article
Formation of Tunable, Emulsion Micro-Droplets Utilizing Flow-Focusing Channels and a Normally-Closed Micro-Valve
by Jung-Hao Wang and Gwo-Bin Lee
Micromachines 2013, 4(3), 306-320; https://doi.org/10.3390/mi4030306 - 17 Jul 2013
Cited by 11 | Viewed by 10359
Abstract
A mono-dispersed emulsion is of great significance in many chemical, biomedical and industrial applications. The current study reports a new microfluidic chip capable of forming tunable micro-droplets in liquids for emulsification applications. It can precisely generate size-tunable, uniform droplets using flow-focusing channels and [...] Read more.
A mono-dispersed emulsion is of great significance in many chemical, biomedical and industrial applications. The current study reports a new microfluidic chip capable of forming tunable micro-droplets in liquids for emulsification applications. It can precisely generate size-tunable, uniform droplets using flow-focusing channels and a normally-closed valve, which is opened by a pneumatic suction force. Experimental data showed that micro-droplets with a diameter ranging from several to tens of micrometers could be precisely generated with a high uniformity. The droplet size is experimentally found to be dependent on the velocity of the dispersed-phase liquid, which is controlled by the deflection of the suction membrane. Emulsions with droplet sizes ranging from 5.5 to 55 μm are successfully observed. The variation in droplet sizes is from 3.8% to 2.5%. The micro-droplets have a uniform size and droplets smaller than those reported in previous studies are possible with this approach. This new microfluidic device can be promising for emulsification and other related applications. Full article
(This article belongs to the Collection Lab-on-a-Chip)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>(<b>a</b>) Schematic illustration of the microfluidic chip. (<b>b</b>) Close-up view of the microchannels and the NC micro-valve. (<b>c</b>) Cross-sectional view of the microfluidic chip<b>.</b> (<b>d</b>) Schematic illustrations of the operation of the NC micro-valve to control the size of the emulsion micro-droplets.</p> Full article ">Figure 2
<p>(<b>a</b>) Exploded view of the microfluidic chip. (<b>b</b>) Simplified fabrication process of the chip.</p> Full article ">Figure 3
<p>Photographs of (<b>a</b>) the microfluidic chip and (<b>b</b>) the NC valve after assembly.</p> Full article ">Figure 4
<p>(<b>a</b>) The maximum deflection of membrane were measured as (1) 11 μm (for 4.5 psi) and (2) 0.8 μm (for 14.7 psi), respectively. (<b>b</b>) The membrane deflection of the NC micro-valve at different applied absolute pressures. Note that the initial membrane defection is 0.8 μm when the syringe pumps are activated. (<b>c</b>) The upstream and downstream flow velocities were measured in the (1) upstream and (2) downstream regions which were distant form the NC valve with 200 μm, respectively. (<b>d</b>) The downstream velocity of the dispersed-phase liquid (<span class="html-italic">V</span><sub>1</sub>’) after flowing through the NC micro-valve when operated at different suction pressures. The velocity of the continuous-phase liquid (<span class="html-italic">V</span><sub>2</sub>) is set at 50 μm/s. Two experiments with two different upstream (initial) velocities of the dispersed-phase liquid (<span class="html-italic">V</span><sub>1</sub>) of 25 and 30 μm/s, respectively, are performed.</p> Full article ">Figure 5
<p>Formation of micro-droplets using the NC micro-valve with different applied absolute pressures. The size of the emulsion droplets can be precisely controlled using the micro-valve operated at different applied absolute pressures. Average droplet diameters are measured to be (<b>a</b>) 58 μm (for 4.5 psi), (<b>b</b>) 34 μm (for 10.4 psi), (<b>c</b>) 10 μm (for 14.2 psi), and (<b>d</b>) 5.5 μm (for 14.7 psi), respectively. The histograms of size distributions with a total number of 100 micro-droplets for applied absolute pressures of (<b>e</b>) 4.5 psi, (<b>f</b>) 10.4 psi, (<b>g</b>) 14.2 psi, and (<b>h</b>) 14.7 psi, respectively. The CVs in droplet sizes were measured to be 3.8%, 3.3%, 2.9%, and 2.5%, respectively. The <span class="html-italic">V</span><sub>1</sub> and <span class="html-italic">V</span><sub>2</sub> are set at 30 and 50 μm/s, respectively. The droplet diameter without applying any suction force is 5.5 μm.</p> Full article ">Figure 6
<p>The relationship between the average diameters of the droplets and the applied absolute pressures. The <span class="html-italic">V</span><sub>2</sub> is kept constant (50 μm/s). The smallest droplet diameters without opening the micro-valve are 6.5 and 5.5 μm when the <span class="html-italic">V</span><sub>1</sub> are 25 and 30 μm/s, respectively.</p> Full article ">Figure 7
<p>The relationship between the droplet formation frequency and the applied absolute pressures on the suction membrane. The <span class="html-italic">V</span><sub>2</sub> is constant at 50 μm/s.</p> Full article ">Figure 8
<p>The relationship between the average diameter of the droplets and the driving frequency of the EMV at an applied absolute pressure of 10.4 psi. The <span class="html-italic">V</span><sub>2</sub> is 50 μm/s, and the <span class="html-italic">V</span><sub>1</sub> are 30 μm/s and 25 μm/s, respectively.</p> Full article ">
1205 KiB  
Article
Approaches for a Fair Comparison and Benchmarking of Electromagnetic Vibration Energy Harvesters
by Clemens Cepnik and Ulrike Wallrabe
Micromachines 2013, 4(3), 286-305; https://doi.org/10.3390/mi4030286 - 5 Jul 2013
Cited by 6 | Viewed by 5113
Abstract
The performance of more than 60 different electromagnetic energy harvesters described in more than 100 publications is benchmarked. The benchmarking is based on earlier published parameters from literature as well as on two novel parameters introduced in this paper. The former allow to [...] Read more.
The performance of more than 60 different electromagnetic energy harvesters described in more than 100 publications is benchmarked. The benchmarking is based on earlier published parameters from literature as well as on two novel parameters introduced in this paper. The former allow to compare different harvester conversion principles as well as harvesters of different electrodynamic design principles. The latter consider the impact of ambient and boundary conditions for the most important sub-group, namely the resonant electrodynamic harvesters. The special consideration of how the mechanical damping and the energy conversion effectiveness depend on these conditions enables a fairer benchmarking of this common harvester type. High performing prototypes are identified, and the key parameters are provided for explanation. Finally, beneficial design approaches and the main challenges to maximize the output power are pointed out. Full article
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Figure 1

Figure 1
<p>(<b>a</b>) conversion effectiveness <span class="html-italic">ν</span> with respect to scaling factor <span class="html-italic">s</span> according to Equation (<a href="#FD6-micromachines-04-00286" class="html-disp-formula">6</a>) and (<b>b</b>) average power <math display="inline"> <msub> <mi>P</mi> <mrow> <mi>a</mi> <mi>v</mi> <mi>g</mi> </mrow> </msub> </math> at the eigenfrequency according to Equations (<a href="#FD5-micromachines-04-00286" class="html-disp-formula">5</a>) and (<a href="#FD10-micromachines-04-00286" class="html-disp-formula">10</a>) for different <math display="inline"> <mrow> <mi>ν</mi> <mspace width="-0.166667em"/> <mo>(</mo> <mi>V</mi> <mo>=</mo> <mn>1</mn> <mspace width="0.166667em"/> <mi mathvariant="normal">c</mi> <msup> <mi mathvariant="normal">m</mi> <mn>3</mn> </msup> <mo>)</mo> </mrow> </math>. Dashed lines denote slope of limits.</p> Full article ">Figure 2
<p>(<b>a</b>) conversion effectiveness <span class="html-italic">ν</span> with respect to scaling factor <span class="html-italic">s</span> according to Equation (<a href="#FD12-micromachines-04-00286" class="html-disp-formula">12</a>) and (<b>b</b>) average power <math display="inline"> <msub> <mi>P</mi> <mrow> <mi>a</mi> <mi>v</mi> <mi>g</mi> </mrow> </msub> </math> at the eigenfrequency according to Equation (<a href="#FD13-micromachines-04-00286" class="html-disp-formula">13</a>) for different <math display="inline"> <msub> <mi>ν</mi> <mrow> <mi>r</mi> <mi>e</mi> <mi>f</mi> </mrow> </msub> </math>.</p> Full article ">Figure 3
<p>Benchmark plots for electrodynamic harvesters. Plots show the maximum performance of each harvester according to the respective BP as well as available measurement data and with respect to the corresponding acceleration and frequency. Values are proportional to the area of circle and according to <a href="#micromachines-04-00286-t001" class="html-table">Table 1</a>.</p> Full article ">
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