This work was supported by the Science and Technology Commission of Shanghai Municipality, China (No. 19ZR1477500 and No.18441900400). We gratefully acknowledge financial support from the University of Shanghai for Science and Technology (No.2017KJFZ049).

NOTE: See the Table of Materials for details related to all materials, reagents, and equipment used in this protocol.
1. Fabrication of CF-PCR microfluidic chip
<ol>
	<li>Heat the silicon wafer at 200 &#176;C for 25 min to remove the moisture.</li>
	<li>Dispense 1 mL of SU-8-2075 photoresist per inch of the wafer. Spin it on the silicon wafer using a spin coater at 500 rpm for 5-10 s with an acceleration of 100 rpm/s, and then at 2,000 rpm for 30 s with an acceleration of 500 rpm/s.</li>
	<li>Soft bake the silicon wafer at 65 &#176;C for 3 min, then at 95 &#176;C for 15 min.</li>
	<li>Set 150 - 215 mJ/cm&#178; as the exposure energy for the photolithography machine, and engrave the designed pattern onto the photoresist with a photolithography mask. Place the silicon wafer and the mask ready for exposure.</li>
	<li>Post exposure, bake the silicon wafer at 65 &#176;C for 2 min, and then at 95 &#176;C for 7 min.</li>
	<li>Immerse the silicon wafer in developer solution to remove excess photoresist and take it out when the microchannels can be seen (Figure 1). Use isopropanol to rinse off the residual developer solution.</li>
	<li>Mix the polydimethylsiloxane (PDMS) prepolymer and curing agent at a ratio of 10:1. Pour the mixed PDMS solution into the replica mold and solidify it at 80 &#176;C for 60 min.</li>
	<li>Bond the PDMS microfluidic chip on a slide after activation using a plasma cleaner, and solidify them at 80 &#176;C for 30 min as soon as possible (Figure 2). 
	NOTE: The outer dimensions of the chip are 80.0 mm &#215; 30.0 mm &#215; 0.4 mm. There are 40 serpentine channels (100 &#181;m &#215; 1.46 mm &#215; 100 &#181;m, width &#215; length &#215; depth) in the microfluidic chip.</li>
</ol>
2. Preparation of the PCR solution
<ol>
	<li>Thaw the reagents completely before configuration. Use a vortex mixer and centrifuge to ensure the reagents are well mixed.</li>
	<li>Prepare a centrifuge tube, adding the following components in this order: 33.75 &#181;L of water, 1.0 &#181;L of DNA template, 0.5 &#181;L of primer, 5.0 &#181;L of buffer, 4.0 &#181;L of dNTP mixture, 1.5 &#181;L of Tween 20, 3.5 &#181;L of PVP, and finally 0.25 &#181;L of DNA polymerase. 
	NOTE: Here, the DNA template used is E. coli. The primers for E. coli and the components of the PCR solution are listed in Table 1 and Table 2, respectively.</li>
	<li>Mix the solution by vortexing.</li>
</ol>
3. Construction of the CF-PCR system
<ol>
	<li>Prepare two positive temperature coefficient (PTC) ceramic heaters, two solid-state relays, two PID temperature controllers, two temperature sensors, and a power cord. 
	NOTE: The size of the PTC ceramic heaters depends on the size of the microfluidic chip; the length should be greater than the length of the chip-the length-width-height of the PTC ceramic heaters used here is 10 cm x 2 cm x 0.4 cm.</li>
	<li>Connect the heater to the solid-state relay.</li>
	<li>Connect the solid-state relay to the PID temperature controller.</li>
	<li>Attach the temperature sensor probe to the bottom of the two heaters and connect the terminal to the PID temperature controller.</li>
	<li>Connect two solid-state relays in series and connect the power cord.</li>
	<li>3D print a slot for the two heaters and keep the heaters on the same plane (see Supplemental File 1 for STL files required for 3D printing). 
	NOTE: Leave a 12 mm air gap between the two heaters.</li>
	<li>Place the microfluidic chip on the two heaters.</li>
	<li>Prepare a syringe pump and a syringe, fix the syringe on the syringe pump, connect a silicone tubing (0.8 mm inner diameter [ID]) with a syringe, and connect a steel needle (0.7 mm ID) to the top of the silicone tubing (Figure 3).</li>
	<li>Insert the steel needle into the inlet of the microfluidic chip.</li>
	<li>Place a pipette tip at the outlet of the chip to collect the PCR products.</li>
</ol>
4. Construction of the CE system
<ol>
	<li>Use a high-voltage power supply to generate a pulsed-field electric field; note the positive and negative electrodes.</li>
	<li>Use a mercury lamp as the light source and filter the excitation wavelength from the mercury lamp through a filter.</li>
	<li>Place the capillary on the microscope stage.</li>
	<li>Collect the fluorescence emission with the objective, and then detect it using an R928 photomultiplier tube (PMT). Observe the microscope and the capillary. Turn on the light under dark room conditions; excitation light is collected by the objective.</li>
	<li>Use the self-developed LABVIEW software to control the power supply and complete the data acquisition (described in steps 6.3-6.6). See&#160;https://github.com/starliyan/labview/blob/main/CZE20170723.vi. 
	&#8203;NOTE: All experiments were performed in a dark room, and the schematic of the CE system is shown in Figure 4.</li>
</ol>
5. Run the PCR solution
<ol>
	<li>Preset the temperature of the heaters of the CF-PCR system at 65 &#176;C and 95 &#176;C.</li>
	<li>Place the microfluidic chip on the two heating blocks.</li>
	<li>Insert the tip of the silicone tube into a centrifuge tube containing 50 &#181;L of the PCR solution (from step 2.3). Pull the syringe plunger to slowly withdraw the solution. Fix the syringe on a syringe pump. Insert the steel needle into the inlet of the microfluidic chip.</li>
	<li>Set the flow rate of the pump to 10 &#181;L/min and press the start button to push the solution in the microchannel at the inlet of the microchip.</li>
	<li>Collect the PCR products at the outlet of the microfluidic chip. 
	&#8203;NOTE: Rinse the channel with ultrapure water before and after the PCR to remove impurities.</li>
</ol>
6. Detection of the PCR products by this CE system built in-house
<ol>
	<li>Prepare a capillary with an 8 cm total length and a 6 cm effective length.</li>
	<li>Prepare the separation buffer by mixing 100 &#181;L of 1% hydroxyethylcellulose (HEC, w/v), 2 &#181;L of 100x SYBR Green I, and 98 &#181;L of ultrapure water to obtain 0.5% HEC (w/v) containing 1x SYBR Green I.</li>
	<li>Fill the capillary with the prepared separation buffer using a vacuum pump.</li>
	<li>Input the injection voltage (800 V) and the injection time (2.0 s) on the software interface, click the start button, and wait for the PCR products to be electrodynamically introduced into the capillary at 100 V/cm (2.0 s). 
	NOTE: In this step, the PCR products are placed on the negative electrode and the buffer is placed on the positive electrode.</li>
	<li>Input the DC voltage (800 V) on the software interface, click the start button, and run the electrophoresis at 100 V/cm of electric field strength. 
	NOTE: In this step, both the positive and negative electrodes are placed with buffer.</li>
	<li>Click the stop button after all DNA fragments are separated in the capillary.</li>
	<li>Flush the capillary with sterilized water for 1 min after each run.</li>
</ol>

<ol>
	<li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-in-one+microfluidic+device+for+on-site+diagnosis+of+pathogens+based+on+an+integrated+continuous+flow+PCR+and+electrophoresis+biochip.">All-in-one microfluidic device for on-site diagnosis of pathogens based on an integrated continuous flow PCR and electrophoresis biochip.</a> Lab on a Chip. 19 (16), 2663-2668 (2019).</li><li>Crews, N., Wittwer, C., Gale, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous-flow+thermal+gradient+PCR.">Continuous-flow thermal gradient PCR.</a> Biomedical Microdevices. 10 (2), 187-195 (2008).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+portable+continuous+flow+PCR+microfluidic+chip+for+DNA+replication.">Design and fabrication of portable continuous flow PCR microfluidic chip for DNA replication.</a> Biomedical Microdevices. 22 (1), 5 (2019).</li><li>Kim, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+characterization+of+a+PDMS-glass+hybrid+continuous-flow+PCR+chip.">Fabrication and characterization of a PDMS-glass hybrid continuous-flow PCR chip.</a> Biochemical Engineering Journal. 29 (1-2), 91-97 (2006).</li><li>Shen, K., Chen, X., Guo, M., Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microchip-based+PCR+device+using+flexible+printed+circuit+technology.">A microchip-based PCR device using flexible printed circuit technology.</a> Sensors and Actuators B: Chemical. 105 (2), 251-258 (2005).</li><li>Harstad, R. K., Johnson, A. C., Weisenberger, M. M., Bowser, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis.">Capillary Electrophoresis.</a> Analytical Chemistry. 88 (1), 299-319 (2016).</li><li>Redman, E. A., Mellors, J. S., Starkey, J. A., Ramsey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intact+antibody+drug+conjugate+variants+using+microfluidic+capillary+electrophoresis-mass+spectrometry.">Characterization of intact antibody drug conjugate variants using microfluidic capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 88 (4), 2220-2226 (2016).</li><li>Britz-Mckibbin, P., Kranack, A. R., Paprica, A., Chen, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assay+for+epinephrine+in+dental+anesthetic+solutions+by+capillary+electrophoresis.">Quantitative assay for epinephrine in dental anesthetic solutions by capillary electrophoresis.</a> Analyst. 123 (7), 1461-1463 (1998).</li><li>Maeda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+real-time+PCR+using+TaqMan+and+SYBR+Green+for+Actinobacillus+actinomycetemcomitans,+Porphyromonas+gingivalis,+Prevotella+intermedia,+tetQgene+and+total+bacteria.">Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQgene and total bacteria.</a> FEMS Immunology and Medical Microbiology. 39 (1), 81-86 (2003).</li><li>Hajba, L., Guttman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+column+coatings+for+capillary+electrophoresis+of+proteins.">Recent advances in column coatings for capillary electrophoresis of proteins.</a> TrAC Trends in Analytical Chemistry. 90, 38-44 (2017).</li><li>Kleparnik, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+combination+of+capillary+electrophoresis+with+mass+spectrometry:+methodology+and+theory.">Recent advances in combination of capillary electrophoresis with mass spectrometry: methodology and theory.</a> Electrophoresis. 36 (1), 159-178 (2015).</li></ol>

The authors have no conflicts of interest to declare.

Both PCR and CE are two popular biotechnologies in the analysis of nucleic acids. This paper describes the amplification of E. coli and the detection of the PCR products using the CF-PCR and CE systems, both built in-house. The target gene of E. coli was successfully amplified within 10 min because of the high heat transfer rates. The DNA fragments smaller than 1,500 bp were separated within 8 min (Figure 5). The great advantage of these two techniques is that it can greatl...

Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, thereby amplifying trace amounts of DNA hundreds of millions of times. It has been widely used in clinical diagnosis, medical research, food safety, forensic identification, and other fields. The PCR process mainly consists of three steps: denaturation at 90-95 &#176;C, annealing at 50-60 &#176;C, and extension at 72-77 &#176;C. Thermal cycling is an important part of the PCR process; however, the traditional PCR thermal cycler is not only bulky but also inefficient, requiring approximately 40 min to complete 25 cycles. To overcome these limitations, a continuous-flow PCR (CF-PCR) system was built in-house, based on a microfluidic chip. CF-PCR can greatly save time by driving the PCR solution into microchannels placed on heaters at different temperatures1,2,3,4,5.
As capillary electrophoresis (CE) has many advantages, such as high resolution, high speed, and excellent reproducibility6,7,8,9,10,11, it has become a popular tool in the lab for the analysis of nucleic acids and proteins. However, most labs, especially labs in the developing world, cannot afford this technology because of the high price of the CE instrument. Herein, we have outlined protocols for how to fabricate the CF-PCR microfluidic chip and how to build a versatile CE system in the lab. We also demonstrate the process of amplification of E. coli by this CF-PCR system and the detection of the PCR products by the CE system. By following the procedures described in this protocol, users should be able to fabricate microfluidic chips, prepare PCR solutions, build a CF-PCR system for nucleic acid amplification, and set up a simple CE system, even with limited resources, to separate DNA fragments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 bp DNA ladder</td>
			<td>Takara Bio Inc.</td>
			<td>3422A</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Fast Buffer I</td>
			<td>Takara Bio Inc.</td>
			<td>RR070A</td>
			<td></td>
		</tr>
		<tr>
			<td>10x TBE</td>
			<td>Beijing Solarbio Science &#38; Technology Co., Ltd.</td>
			<td>T1051</td>
			<td></td>
		</tr>
		<tr>
			<td>developer solution</td>
			<td>Alfa Aesar, USA</td>
			<td>L15459</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mixture (2.5 &#956;M)</td>
			<td>Takara Bio Inc.</td>
			<td>RR070A</td>
			<td></td>
		</tr>
		<tr>
			<td>EC-F</td>
			<td>Sangon Biotech, Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EC-R</td>
			<td>Sangon Biotech, Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEC,1300K</td>
			<td>Sigma-Aldrich, USA</td>
			<td>9004-62-0</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropanol</td>
			<td>Aladdin, Shanghai, China</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Olympus, Japan</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>photolithography&#160;</td>
			<td>SUSS MicroTec, Germany</td>
			<td>MJB4</td>
			<td></td>
		</tr>
		<tr>
			<td>photomultiplier tube&#160;</td>
			<td>Hamamatsu Photonics, Japan</td>
			<td>R928</td>
			<td></td>
		</tr>
		<tr>
			<td>photoresist</td>
			<td>MicroChem, USA</td>
			<td>SU-8 2075</td>
			<td></td>
		</tr>
		<tr>
			<td>PID temperature controllers&#160;</td>
			<td>Shanghai, China</td>
			<td>XH-W2023</td>
			<td></td>
		</tr>
		<tr>
			<td>plasma cleaner&#160;</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G-2</td>
			<td></td>
		</tr>
		<tr>
			<td>polyvinyl pyrrolidone (PVP)</td>
			<td>Aladdin, Shanghai, China</td>
			<td>P110608</td>
			<td></td>
		</tr>
		<tr>
			<td>pump</td>
			<td>Harvard Apparatus</td>
			<td>PHD2000</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone tubing&#160;</td>
			<td>BIO-RAD,USA</td>
			<td>7318210</td>
			<td></td>
		</tr>
		<tr>
			<td>solid-state relays</td>
			<td>KZLTD, China</td>
			<td>KS1-25LA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedSTAR HS DNA Polymerase&#160;</td>
			<td>Takara Bio Inc.</td>
			<td>RR070A</td>
			<td></td>
		</tr>
		<tr>
			<td>steel needle</td>
			<td>zhongxinqiheng,Suzhou,China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR GREEN <img alt="Equation 1" src="/files/ftp_upload/63523/63523eq01.jpg" style="margin: auto;" /></td>
			<td>Solarbio, Beijing, China</td>
			<td>SY1020</td>
			<td></td>
		</tr>
		<tr>
			<td>temperature sensors</td>
			<td>EasyShining Technology, Chengdu, China</td>
			<td>TCM-M207</td>
			<td></td>
		</tr>
		<tr>
			<td>Template (E. coli)</td>
			<td>Takara Bio Inc.</td>
			<td>AK601</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Aladdin, Shanghai, China</td>
			<td>T104863</td>
			<td></td>
		</tr>
		<tr>
			<td>voltage power supply&#160;</td>
			<td>Medina, NY, USA</td>
			<td>TREK MODEL 610E</td>
			<td></td>
		</tr>
	</tbody>
</table>

amplification of escherichia coli in a continuous-flow-pcr microfluidic chip and its detection with a capillary electrophoresis system

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Figure 5&#160;represents the electropherogram of the PCR products and the DNA markers. Trace (Figure 5A) is the CE result of the CF-PCR amplified product, trace (Figure 5B) is the CE result of the product amplified by thermal cycling, and trace (Figure 5C) is the CE result of the 100 bp DNA ladder. We first amplified the target gene of E. coli in the CF-PCR system; the PCR solution took ~10 min...

Watch this Scientific Journal Video about Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System at JoVE.com

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular ...

amplification-escherichia-coli-continuous-flow-pcr-microfluidic-chip

Research

JoVE Journal

Bioengineering

1.9K Views.  University of Shanghai for Science and Technology. This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab. 

Video: Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

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 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 ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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 ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...