This study is supported by a project entitled &#34;Cooperative Research Program for Agriculture Science &#38; Technology Development (Project No. PJ0011642)&#34; funded by the Rural Development Administration, Republic of Korea. This research was also partly supported by a grant (NRF-2017R1A2B4012253) of the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT &#38; Future Planning, Republic of Korea. This research was also supported by a grant (N0000717) of the Education program for Creative and Industrial Convergence funded by the Ministry of Trade, Industry and Energy, Republic of Korea.

1. Fabrication of a PDMS Microfluidic Platform
<ol>
	<li>Prepare 20 mL of liquid PDMS prepolymer by mixing base polymer and catalyst in a volume ratio of 10:1. Pour 10 mL of the liquid PDMS onto a prepared SU-8 mold on a silicon wafer for the upper part of the microfluidic network. For the bottom flat part, pour the same volume of liquid PDMS on the silicon wafer without a mold structure. 
	Note: The microfluidic network is designed in a CAD program and then converted into a photomask in order to fabricate a master using the typical photolithography process (See Supplemental Figures). This master is comprised of the negative photoresist SU-8 mold on the silicon wafer11.</li>
	<li>Place two silicon wafers coated with liquid PDMS prepolymer on the hot plate and cure at 75 &#176;C for 30 min.
	<ol>
		<li>Manually peel off the cured PDMS layer from the SU-8 mold. Align a 1.5 mm diameter round hole punching tool to the oil port on the replicated microfluidic network for interfacing micron-scale flow channels with the macro fluid samples. Punch out the through-hole manually.</li>
		<li>Repeat this punching process three times for the formation of the two solutions and the outlet port.</li>
	</ol>
	</li>
	<li>Perform hydrophilic surface treatment on both the upper and bottom PDMS layers using a hand-held corona treater12 for several seconds per sample.
	<ol>
		<li>Stack two plasma-treated PDMS layers and heat at 90 &#176;C for 30 minutes using a hot plate for the PDMS-to-PDMS bonding process. Supply the pressurized water using the syringe pump into three inlet ports for the structural bonding and leakage testing of the fabricated device.</li>
	</ol>
	</li>
</ol>
2. Production of Polyacrylamide Oligo-Microspheres
<ol>
	<li>Prepare bead-mixture detailed in Table 1.</li>
	<li>Vortex and briefly centrifuge the standard ssDNA acrydite labeled probe (Ap, 100 &#956;M) and acrylamide:bis (19:1) stock solution.</li>
	<li>Prepare solution I by mixing 25 &#956;L of 40% acrylamide bis solution, 10 &#956;L of ssDNA (acrydite probe), 10 &#956;L of 5x TBE buffer (1.1 M Tris; 900 mM borate; 25 mM EDTA), and 5 &#956;L of water. Prepare solution II with 50 &#956;L of 20% ammonium persulfate.</li>
	<li>Prepare two syringes individually filled with solution I and solution II, and mount them onto the pump to introduce solution flows into the microfluidic platform. Prepare mineral oil mixed with 0.4% TEMED for surface solidification of the microsphere. 
	Note: TEMED is well-known as a free radical stabilizer. Free radicals can accelerate the rate of polymer formation with ammonium persulfate (APS) in order to catalyze acrylamide polymerization.</li>
	<li>Fill a glass bottle with 4 mL of the mineral oil to generate a continuous flow.</li>
	<li>Insert two tubes to two ports in the cap of the glass bottle as a microfluidic reservoir: the pneumatic port for applying the compressed air into the glass bottle from the air compressor and the fluid port for supplying the pressurized oil to the micro channel network from the glass bottle. Connect tubes between the glass bottle and the oil port in the microfluidic device.
	<ol>
		<li>Connect the tubes to the two solution ports in the microfluidic device in order to supply the two solutions from the syringe pumps. Insert tubes to the outlet port in order to transfer the generated microsphere into the beaker.</li>
	</ol>
	</li>
	<li>Set the flow rate of the syringe pump to 0.4 - 0.7 mL/h. Adjust compressed air pressure of the compressor using a regulator (82 - 116 kPa). Set the rotational speed (500 rpm) of the magnetic stirrer bar in the glass beaker on a hot plate. Operate the syringe pump and supply the compressed air generated by an external compressor into the glass bottle using the ON/OFF control of an electromagnetic valve13.</li>
	<li>Observe the formation of microspheres in the flow-focusing geometry and solidification of generated microspheres in the glass beaker with a digital microscope. 
	Note: The size and production speed of the microsphere depend on the flowrate of solutions and the pressure applied for the mineral oil flow (Table 2).</li>
</ol>
3. Performing Polyacrylamide Oligo-Microspheres Counts
<ol>
	<li>For hemocytometer quantification, take a small amount (approximately 100 &#956;L) of aqueous solution of polyacrylamide oligo-microspheres and place on the glass hemocytometer, and gently fill the microsphere suspension up the well of the counting chamber. 
	Note: For further details, see http://www.abcam.com/protocols/counting-cells-using-a-haemocytometer.</li>
	<li>Use a microscope and hand tally counter to count microspheres in one set of 16 squares. Then, move the hemocytometer to the next set of the chamber and carry on counting until all four sets of 16 corners are counted.</li>
	<li>Determine the average microsphere count and calculate the number of microspheres in the original bead suspension.</li>
	<li>Transfer 100 &#956;L of microspheres to a 1.5 mL microcentrifuge tube. Remove the supernatant through gentle centrifuging (400 x g) and pipetting.</li>
</ol>
4. Performing DNA Hybridization on the Surface of Polyacrylamide Oligomicrosphere
Note: An identical DNA probe with a 5&#8217;-NH2-group instead of 5&#8217;-acrytide modification is added into solution I and tested for Ap-containing microspheres in parallel. DNA hybridization results are shown in Figure 2. The Cy3-labeled complementary oligonucleotide probes (cAp) solution should be placed in a dark room.
<ol>
	<li>Resuspend Cy3-cAp using 100 &#956;L of 1xTE buffer (TE buffer: 10 mM Tris and 1&#956;M EDTA, pH 8.0) in order to achieve a final concentration of 100 &#956;M. For example, resuspend 1 pmol of cAp in 100 mL of TE buffer and transfer to a microcentrifuge tube covered with aluminum foil. 
	Note: For strand sequences, see Table 3.</li>
	<li>Add 100 &#956;M of Cy3-cAp to a sterile 1.5 mL microcentrifuge tube containing Ap-copolymerized microspheres.</li>
	<li>Tap the tube a few times to mix and incubate at room temperature in the dark for 1 h.</li>
	<li>Discard the supernatant and remove residual buffer through pipetting.</li>
	<li>Rinse three times with 500 &#956;L of TE buffer.</li>
	<li>Resuspend microspheres by gently tapping the microcentrifuge tube. Then, repeat step 4.4.</li>
	<li>Place microspheres on the glass slide (75 mm x 50 mm) and cover with aluminum foil prior to imaging.</li>
</ol>
5. Asymmetric PCR for Amplifying ssDNA
<ol>
	<li>Prepare an asymmetric PCR reagent mix for amplifying ssDNA to be analyzed. Thaw the reagents in Table 4 on ice. Do not keep the Taq polymerase enzyme (50 U/&#181;L) on ice, but rather store it at -20 &#176;C until needed.</li>
	<li>Gently vortex all reagents and then briefly centrifuge tubes at 10,000 x g for 10 s.</li>
	<li>Combine all reagents as described in Table 4.</li>
	<li>Place samples into a thermocycler and start the asymmetric PCR under the following conditions: 25 cycles (95 &#176;C for 30 s, 52 &#176;C for 30 s, and 72 &#176;C for 30 s), 85 &#176;C for 5 min, hold at 4 &#176;C.</li>
</ol>
6. Microsphere-PCR for Amplifying ssDNA
Note: This section describes the protocol for amplifying ssDNA in a PCR reaction tube. Microsphere-PCR reactions were performed in 50 &#956;L of reaction volume. The detailed sequences used to amplify ssDNA are listed in Table 5. In this case, Ap on the surface of microspheres can anneal to random DNA templates in a complementary manner. This is a very important step for producing complementary DNA strands (antisense DNA strand, Figure 3). The DNA extended is used as a template for microsphere-PCR amplification.
<ol>
	<li>Prepare microsphere-PCR reagent mix. Thaw the reagents in Table 6 on ice; however, do not keep the Taq polymerase enzyme on ice. Store it at -20 &#176;C until needed.</li>
	<li>Gently vortex all reagents and then briefly centrifuge tubes at 10,000 x g for 10 s.</li>
	<li>Obtain approximately ~25 microspheres through microscopic counting. 
	Note: The number of microspheres in one reaction tube are calculated using a light microscope with 40X magnification. About ~25 microspheres are used for microsphere-PCR amplification. More detailed information is in Step 3.</li>
	<li>Combine all reagents as described in Table 6.</li>
	<li>Place samples into a thermocycler and start the asymmetric PCR under the following conditions: 25 cycles (95 &#176;C for 30 s, 52 &#176;C for 30 s, and 72 &#176;C for 30 s), 85 &#176;C for 5 min, hold at 4 &#176;C.</li>
	<li>Following amplification, add 8 &#956;L of 6x loading buffer and load 15 &#956;L of each sample into 2% agarose gel. Then, perform electrophoresis at 100 V for 35 min in 1x TAE (Tris-acetate-EDTA, 40 mM Tris acetate, 1 mM EDTA, pH 8.2) buffer.</li>
</ol>
7. Confocal Microscopy Acquisition
Note: The results of microsphere-DNA probe hybridization are imaged under a confocal microscope. Image analysis is performed using ImageJ.
<ol>
	<li>Fix hybridized microspheres to the stage of the microscope in a holder.</li>
	<li>Select the laser (Helium/Neon laser, 543 nm line) and turn it on in laser control.</li>
	<li>Select the objective lens in microscope control.</li>
	<li>Select the desired filter for Cy3 and channel in configuration control.</li>
	<li>Start the experiment and observe the sample. Settings for the confocal microscope are summarized in Table 7.</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Xenopus+laevis+mitochondrial+single-stranded+DNA-binding+protein+on+primer-template+binding+and+3'--&gt;5'+exonuclease+activity+of+DNA+polymerase+gamma.">Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'--&gt;5' exonuclease activity of DNA polymerase gamma.</a> Journal of Biological Chemistry. 271 (31), 18939-18946 (1996).</li><li>Yu, X., Cheng, G., Zhou, M. D., Zheng, S. 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The authors have no conflicts of interest to disclose.

Contaminants of dsDNA are a major issue in ssDNA amplification. It remains difficult to minimize dsDNA amplification in conventional asymmetric PCR amplification15. In addition, although technical improvements for generating ssDNA have enabled us to increase the efficiency of sample throughput, ssDNA isolation is still problematic due to its high costs and incomplete purification yields.
Asymmetric PCR is one of the most challenging methods used when working with ssDNA....

Single-stranded DNA (ssDNA) has been extensively considered as a molecular recognition element (MRE) due to its intrinsic properties for DNA-DNA hybridization1,2. The development of ssDNA synthetic systems can lead to biological applications such as DNA microarrays3, oligotherapeutics, diagnostics, and integrated molecular sensing based on complementary interactions4,5.
To date, micrometer-scale polymer particles have been successfully demonstrated using microfluidic devices. Several microfluidic techniques have been proven to be powerful for producing highly homogenous microspheres on continuous flow in the microchannel environment6,7.
In the study of Lee et al.8, a droplet-based microfluidic platform for the microfluidic synthesis of copolymerizable oligo-microsphere and ssDNA amplification was reported. The microfluidic platform consists of two PDMS (polydimethylsiloxane) layers: an upper part with a microfluidic channel network for generating microsphere and a bottom flat part. These consist of three kinds of PDMS fluidic channels: 1) a flow focusing channel for droplet generation, 2) a serpentine channel for mixing two solutions, and 3) a sequential polymerization channel for microsphere solidification. Once two immiscible flows are introduced into a single PDMS fluidic channel, the flows can be forced through the narrow orifice structure. The flow behaviors such as channel geometry, flow-rate, and viscosity affect the size and morphology of the microsphere. Therefore, the main liquid stream can be divided into microscale monospheres9,10.
Here, a detailed microsphere-PCR protocol is provided for the amplification of ssDNA. First, a droplet-based microfluidic device design process is described. Then, the way in which polyacrylamide microspheres can be functionalized with random DNA template in a complementary manner is explained. Finally, a microsphere-PCR protocol for amplifying ssDNA is shown.

<table>
	<tbody>
		<tr>
			<td>liquid polydimethylsiloxane, PDMS</td>
			<td>Dow Corning Inc.</td>
			<td>Sylgard 184</td>
			<td>Components of chip</td>
		</tr>
		<tr>
			<td>40% Acrylamide:bis solution (19:1)</td>
			<td>Bio-rad</td>
			<td>1610140</td>
			<td>Components of Copolymerizable oligo-microsphere</td>
		</tr>
		<tr>
			<td>Ammonium persulfate, APS</td>
			<td>Sigma Aldrich</td>
			<td>A3678</td>
			<td>Hardener of acrylamide:bis solution</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine, TEMED</td>
			<td>Sigma Aldrich</td>
			<td>T9281</td>
			<td>Catalyst of ammonium persulfate</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma Aldrich</td>
			<td>M5904</td>
			<td>Table 1. Solution III. Component of microsphere reagents</td>
		</tr>
		<tr>
			<td>Cy3 labeled complementary oligonucleotide probes</td>
			<td>Bioneer</td>
			<td>synthesized</td>
			<td>Table 3. Sequence information&#160;</td>
		</tr>
		<tr>
			<td>ssDNA acrydite labeled probe</td>
			<td>Bioneer</td>
			<td>synthesized</td>
			<td>Table 1. Solution I. Component of microsphere reagents</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Biosesang</td>
			<td>&#160;T1016</td>
			<td>Components of TE buffer, pH buffer solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>EDS</td>
			<td>Components of TE buffer, removal of ion (Ca2+)</td>
		</tr>
		<tr>
			<td>Ex taq</td>
			<td>Takara</td>
			<td>RR001A</td>
			<td>ssDNA amplification</td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td>LSM 510</td>
			<td>Identifying oligonucleotides expossure of microsphere surface</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Nikon Instruments Inc.</td>
			<td>eclipse 80i</td>
			<td>Caculating number of microspheres</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-rad</td>
			<td>1861096</td>
			<td>ssDNA amplification</td>
		</tr>
		<tr>
			<td>Hand-held Corona Treater</td>
			<td>Electro-Technic</td>
			<td>BD-20AC Laboratory Corona Treater</td>
			<td>Hydrophilic surface treatment</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>As one</td>
			<td>HI-1000</td>
			<td>heating plate for curing of liquid PDMS</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>kd Scientific</td>
			<td>78-1100</td>
			<td>Uniform flow of Solution I and Solution II</td>
		</tr>
		<tr>
			<td>Compressor</td>
			<td>Kohands</td>
			<td>KC-250A</td>
			<td>Flow control of Solution III</td>
		</tr>
		<tr>
			<td>Bright-Line Hemacytometer</td>
			<td>Sigma Aldrich</td>
			<td>Z359629</td>
			<td>Caculating number of microspheres</td>
		</tr>
	</tbody>
</table>

a droplet-based microfluidic approach and microsphere-pcr amplification for single-stranded dna amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

The fabricated polymeric droplet-based microfluidic platform consists of two PDMS layers (Figure 1a). Three kinds of microfluidic channel networks are used for generating microspheres: 1) Flow-focusing geometry as shown in Figure 1b, 2) a serpentine channel for mixing solution I and solution II, and 3) a polymerization channel for microsphere solidification. The height of all channels was 60 &#956;m. The channel length for mixing...

Watch this Scientific Journal Video about A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons at JoVE.com

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.5K Views.  Chungbuk National University. This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Video: A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...