Name: fabricating cotton analytical devices
Uploaded: 2016-08-30T13:00:00
Description: Watch this Scientific Journal Video about Fabricating Cotton Analytical Devices at JoVE.com

This work was supported in part by grants from Taiwan&#39;s Ministry of Science and Technology (MOST 104-2628-E-007-001-MY3 (CMC)), and Taichung Veterans General Hospital (<span style="color: rgb(34, 34, 34); font-family: arial, sans-serif; font-size: 12.8px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; -webkit-text-stroke-width: 0px; display: inline !important; float: none; background-color: rgb(255, 255, 255);">TCVGH-1056904C (MYH)).

CAUTION: Proper laboratory hygiene practice is required. Gloves and universal precautions are required when using this POC device. Contamination of results or infection may occur if adequate sterilization procedures are not carried out properly.
1. Prepare Test Strip Devices
<ol>
	<li>Determine the hydrophobicity of the exterior layer of the cleansing cotton by contact angle measurement8 (Figure 3).</li>
	<li>Fabricate the cotton-based analytical device by cutting cleansing cotton int...

<ol>
	<li>Yager, P., Domingo, G. J., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+diagnostics+for+global+health.">Point-of-care diagnostics for global health.</a> Annu. Rev. Biomed. Eng. 10, 107-144 (2008).</li><li>Chin, C. D., Linder, V., Sia, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commercialization+of+microfluidic+point-of-care+diagnostic+devices.">Commercialization of microfluidic point-of-care diagnostic devices.</a> Lab Chip. 12, 2118-2134 (2012).</li><li>Lu, Y., Shi, W., Jiang, L., Qin, J., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+of+paper-based+microfluidics+with+wax+for+low-cost,+portable+bioassay.">Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay.</a> Electrophoresis. 30 (9), 1497-1500 (2009).</li><li>Ballerini, D. R., Li, X., Shen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+control+concepts+for+thread-based+microfluidic+devices.">Flow control concepts for thread-based microfluidic devices.</a> Biomicrofluidics. 5 (1), 014105 (2011).</li><li>Lin, S., 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=Cotton-based+Diagnostic+Devices.">Cotton-based Diagnostic Devices.</a> Scientific reports. 4, 6976-6976 (2013).</li><li>Hsu, M. Y., 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=Monitoring+the+VEGF+level+in+aqueous+humor+of+patients+with+ophthalmologically+relevant+diseases+via+ultrahigh+sensitive+paper-based+ELISA.">Monitoring the VEGF level in aqueous humor of patients with ophthalmologically relevant diseases via ultrahigh sensitive paper-based ELISA.</a> Biomaterials. 35 (12), 3729-3735 (2014).</li><li>Hsu, M. Y., 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=Monitoring+VEGF+levels+with+low-volume+sampling+in+major+vision-threatening+diseases:+age-related+macular+degeneration+and+diabetic+retinopathy.">Monitoring VEGF levels with low-volume sampling in major vision-threatening diseases: age-related macular degeneration and diabetic retinopathy.</a> Lab Chip. 15 (11), 2357-2363 (2015).</li><li>Kwok, D., Neumann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+angle+measurement+and+contact+angle+interpretation.">Contact angle measurement and contact angle interpretation.</a> Adv. Colloid Interface Sci. 81 (3), 167-249 (1999).</li><li>Martinez, A. W., Phillips, S. T., Butte, M. J., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+paper+as+a+platform+for+inexpensive,+low-volume,+portable+bioassays.">Patterned paper as a platform for inexpensive, low-volume, portable bioassays.</a> Angew. Chem. Int. Ed. Engl. 46 (8), 1318-1320 (2007).</li><li>Li, X., Tian, J. F., Shen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+biomarker+assay+with+microfluidic+paper-based+analytical+devices.">Quantitative biomarker assay with microfluidic paper-based analytical devices.</a> Anal. Bioanal. Chem. 396 (1), 495-501 (2010).</li><li>Coad, S., Friedman, B., Geoffrion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+Urinalysis.">Understanding Urinalysis.</a> medscape. 7 (3), 269-279 (2012).</li><li>Kupka, T., 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=Accuracy+of+urine+urobilinogen+and+bilirubin+assays+in+predicting+liver+function+test+abnormalities.">Accuracy of urine urobilinogen and bilirubin assays in predicting liver function test abnormalities.</a> Ann. Emerg. Med. 16 (11), 1231-1235 (1987).</li><li>Binder, L., 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=Failure+of+prediction+of+liver+function+test+abnormalities+with+the+urine+urobilinogen+and+urine+bilirubin+assays.">Failure of prediction of liver function test abnormalities with the urine urobilinogen and urine bilirubin assays.</a> Arch. Pathol. Lab. Med. 113 (1), 73-76 (1989).</li><li>Gubala, V., 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=Point+of+care+diagnostics:+status+and+future.">Point of care diagnostics: status and future.</a> Anal. chem. 84 (2), 487-515 (2011).</li><li>Vaidya, V. S., 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=Urinary+biomarkers+for+sensitive+and+specific+detection+of+acute+kidney+injury+in+humans.">Urinary biomarkers for sensitive and specific detection of acute kidney injury in humans.</a> Clin. Transl. Sci. 1 (3), 200-208 (2008).</li></ol>

Critical steps in this protocol included determining the appropriate combination of cotton material (with varying hydrophobicity/hydrophilicity) and filter paper (chromatography filter paper or quantitative filter paper). A well-planned and executed device design renders the best performance attributes for colorimetric assays. From our colorimetric assay results, the cotton-based analytical device presented herein demonstrates great potential as a platform for multiple disease detection.
Most ...

The development of point-of-care (POC) diagnostic devices that are affordable, robust, and easily used is imperative for improving global health1,2. In particular, devices composed of cellulose substrates (e.g., paper, thread, and cotton) provide promising analytical platforms for low-cost analysis because of their ubiquity, affordability, ease of use, robustness, and capacity to provide rapid results3-7.
Here, we unveil the development of a cotton-based analytical device that uses a lateral flow-based format for urinalysis. This cotton-based analytical device provides an alternative detection approach with several key advantages: i) fabrication with minimal human effort; ii) low cost; iii) the capacity to be used to conduct multiple, different assays without cross-contamination concerns; iv) device independence, i.e., the ability to be run without additional equipment and/or electricity; and, v) speed (colorimetric assays can be completed within 10 min).
The structure of this cotton-based analytical device can be divided into four parts: i) cotton that is naturally hydrophobic on its external hydrophobic layer; ii) cotton that is internally hydrophilic and serves as a transportation channel for liquid wicking; iii) lamination film that binds and compresses the cotton being used but contains drilled out holes for the placement of reaction/test pads; and, iv) chromatography paper test pads, which are coated/embedded with reactive reagents, placed on the exterior surface of the cotton (specifically, in the space drilled out of the lamination film) as reaction areas for colorimetric assays (i.e., nitrite, total protein, pH, and urobilinogen assays) and results display.
The underlying mechanism of the test is as follows. The cotton-based analytical device is scored with lines that penetrate half way through the depth of the cotton material to create a flow channel that allows sample fluid to reach the reactive pads being used. The absorptive edge of the analytical device is immersed into the target sample, whereupon the solution is wicked along the fluidic channel from the absorption end to the test pads (Figure 1). Because the absorptive strength of the test pad is greater than that of cotton, solutions absorbed by the test pads are firmly contained inside the test pad paper so that there is no reflow back into the fluidic channel, and the colorimetric results become subsequently fixed on the test pad material. At the end of the reaction, colorimetric results are recorded via a desktop scanner, and analyzed via image analyzing software.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma-Aldrich, US</td>
			<td>No. 9048468</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>nitrite&#160;</td>
			<td>Sigma-Aldrich, US</td>
			<td>No. 7632000</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>urobilinogen&#160;</td>
			<td>Santa Cruz Bio, US</td>
			<td>No. SC-296690</td>
			<td></td>
		</tr>
		<tr>
			<td>citrate</td>
			<td>Sigma-Aldrich U.S</td>
			<td>No. 6132043</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>tetrabromophenol blue</td>
			<td>Sigma-Aldrich U.S</td>
			<td>No. 4430255</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>sulfanilamide</td>
			<td>Sigma-Aldrich U.S</td>
			<td>No. 63741</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>citric acid&#160;</td>
			<td>Sigma-Aldrich U.S</td>
			<td>No. 77929</td>
			<td>&#8805; 99.5%</td>
		</tr>
		<tr>
			<td>&#160;N-(1-naphthyl) ethylenediamine dihydrochloride</td>
			<td>Sigma-Aldrich U.S</td>
			<td>No. 1465254</td>
			<td>&#8805; 98%</td>
		</tr>
		<tr>
			<td>4-(Dimethylamine)benzaldehyde</td>
			<td>AlfaAesar, U.S</td>
			<td>No. A11712</td>
			<td>&#8805; 98%</td>
		</tr>
		<tr>
			<td>Methyl Red sodium salt</td>
			<td>sigma, U.S</td>
			<td>No. 114502</td>
			<td>&#8805;95%</td>
		</tr>
		<tr>
			<td>Bromothyle blue</td>
			<td>sigma, U.S</td>
			<td>No. 114413</td>
			<td>&#8805;95%</td>
		</tr>
		<tr>
			<td>Shiseido Cleansing Cotton</td>
			<td>Shiseido, Japan</td>
			<td>No. 79014</td>
			<td></td>
		</tr>
		<tr>
			<td>chromatography paper</td>
			<td>GE Healthcare Whatman, Springfield Mill, UK</td>
			<td>No. 30306132</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic substrate</td>
			<td>lamination film, MAS</td>
			<td>A4</td>
			<td>216 mm &#215; 303 mm</td>
		</tr>
		<tr>
			<td>scanner</td>
			<td>microtek scanmaker</td>
			<td>&#160;i2400</td>
			<td></td>
		</tr>
		<tr>
			<td>paper cutter</td>
			<td>Life paper cutter</td>
			<td>No.306</td>
			<td></td>
		</tr>
		<tr>
			<td>laminator</td>
			<td>AURORA&#160;</td>
			<td>LM4231H</td>
			<td></td>
		</tr>
		<tr>
			<td>laminator film</td>
			<td>UNI LAMI&#160;</td>
			<td>4A</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabricating cotton analytical devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

We successfully demonstrated the development of cotton-based analytical devices by using commercially available cleansing cotton characterized by hydrophilic (inner portion) and hydrophobic (exterior portion) properties (Figure 1A). Figure 3 shows the results of contact angle measurement. The hydrophobic interface of exterior cotton was 127.35&#176; &#177;&#160; 4.73&#176;. From a user-friendly perspective, colorimetric assays employed here could be direc...

Watch this Scientific Journal Video about Fabricating Cotton Analytical Devices at JoVE.com

Fabricating Cotton Analytical Devices

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

The overall goal of this protocol is to fabricate an analytic device from an easily-sourced material, cotton, to use for your analysis. The main advantage of this technique is that the device is easy to fabricate. It's very low-cost, can be used for multiple assays, and is quick to assemble.The analytical method with the device is sensitive to detecting sample and reaction contamination. Demonstrating the procedure will be Shang-Chi Lin, who was instrumental in innovating the method. Begin with preparing the test strip devices.First, determine the hydrophobicity of the cleansing cotton for the test strips using a standard contact angle measurement with droplets of deionized water. Then, start fabricating the cotton-based analytical device by cutting cleansing cotton into 5.5 centimeter by one centimeter pieces. Use a paper cutter.The next step is to drill sets of three 0.5-centimeter diameter holes in a sheet of lamination film. Drill the holes in rows with one centimeter between the holes. Each set of holes makes a test strip, and up to 36 strips can be fit on a standard sheet of lamination film.Next, sandwich the cleansing cotton between the hole-drilled sheet of lamination film and an un-drilled sheet of lamination film. Then, use a standard laminator to package the device. Now, cut individual 5.5 by one centimeter test strip devices from the assembly.To make standard curves of three test solutions, 48 strips are needed. On each test area, use a sharp blade to make a small channel from the center of each test area to the hydrophilic layer of the longitudinal device. Be careful to only cut halfway through the device.Now, insert half-centimeter diameter chromatography paper discs into each of the three holes. The lamination film should have a small lip under which the pad edges can be tucked. For a urinalysis strip, coat the nitrite assay pads with nitrite indicator solution.Coat the protein assay pad with BSA indicator solution. And coat the urobilinogen assay pad with urobilinogen indicator. Allow the strips to dry completely at ambient temperatures before proceeding.For each standard prepared, dip the absorptive end of a prepared device into the standard solution for ten seconds. Each strip should soak up 200 to 300 microliters of solution. Prepare three test strips for each standard concentration being tested.Wait ten minutes for the colorimetric reaction to complete at room temperature. Now, scan the devices to analyze each test pad's color. Use a desktop scanner in RGB mode and at 600 dpi.Analyze the scanned image using standard software such as ImageJ. After opening the scanned image files, select the image in the menu commands and select Type to convert RGB images to 8-bit grayscale. Then, use the oval tool in the toolbar to select the test pad analytical area.Next, select Analyze in the menu commands, and select Measure to analyze color intensity. From the mean intensity results, make the standard curves for the assays. In this case, fit the standard curve for the nitrite assay using a quadratic model, and fit the standard curves for total protein and urobilinogen using linear regression models.On the hydrophobic interface of the fabricated device, the contact angle at the exterior cotton was measured 127 degrees, give or take 5 degrees. Using the devices, standard curves were made to establish the limits of detections for each assay. For nitrites, the limit of detection was 0.147 millimolar.For bovine serum albumin, the protein test solution, the limit of detection was 3.7 micromolar. For urobilinogen, the limit of detection was 4.9 milligrams per liter. After watching this video, you should have a good understanding of how to fabricate cotton diagnostic devices with the hydrophobicity of the external layer that helps avoid contamination.Once mastered, this technique can be done in one or two hours, if it is performed properly. While attempting this procedure, it's important to determine the hydrophobicity of the external layer of the cleansing cotton by contact angle measurement.

Research

JoVE Journal

Bioengineering

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

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 ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...