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 into 5.5 cm x 1 cm pieces with a paper cutter (Figure 2A).</li>
	<li>Drill holes (&#248; = 0.5 cm) in a standard sheet of lamination film in rows of three; the distance between two subsequent holes is 1 cm. 
	NOTE: As many as 36 individual test strips with three test areas each may be made using a standard sheet of lamination film (5.5 cm x 2 cm) (Figure 2B).</li>
	<li>Sandwich the cleansing cotton (characterized by exterior hydrophobicity and interior hydrophilicity) between two pieces of lamination film, i.e., the hole-drilled sheet of lamination film on one side, and an undrilled sheet of lamination film (no holes) on the other side. Place the assembly into a laminator to package the device. This enhances the device&#39;s mechanical strength, and provides an external, impervious layer. 
	NOTE: The diameters of holes will shrink from 0.5 cm to ~ 0.47 cm after lamination. Some slight gaps will remain between the cleansing cotton and lamination film around the perimeter of each hole. These slight gaps facilitate the placement of test pad circles made in a subsequent step.</li>
	<li>Use a pair of scissor to cut individual 5.5 cm x 1 cm test strip devices from the assembly, i.e., the drilled lamination sheet/cotton pad/undrilled lamination sheet &#34;sandwich&#34; (Figure 2C). Make 48 devices total to create standard curves as described later (18 devices for the nitrite assay, 15 devices for the BSA assay, and 15 devices for the urobilinogen assay).</li>
	<li>Use a sharp knife or razor to cut a slit (as a small fluidic channel) from the center of each test area (not covered by lamination film), across and through the hydrophobic layer of test area to the hydrophilic layer of the longitudinal device, being cautious to cut halfway through the depth of the sandwiched device (Figure 2D).</li>
	<li>Apply paper test pads (&#248; = 0.5 cm) by sliding them into the gaps at the perimeter of each hole in the lamination (Figure 2E).</li>
</ol>
2. Prepare Standards and Indicator Solutions for Urinalysis
<ol>
	<li>Prepare 10.0 mM nitrite stock solution by dissolving 69.0 mg sodium nitrite in 100 ml deionized (DI) water. Dilute the nitrite stock solution in DI water to create a series of nitrite concentration standard solutions (2,500, 1,250, 625, 312, 156, and 78 &#181;M) (Figure 4A).</li>
	<li>Prepare 60 &#181;M bovine serum albumin (BSA) stock solution by dissolving 415 mg BSA in 100 ml PBS solution (10 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride; pH 7.0). Dilute the BSA stock solution in DI water to create a series of BSA concentration standard solutions (30, 15, 7.5, 3.75, and 1.875 &#181;M) (Figure 4B).</li>
	<li>Prepare 25 g/L urobilinogen stock solution by dissolving 2.5 g urobilinogen in 100 ml DI water. Dilute stock urobilinogen solution in DI water to create a series of urobilinogen concentration standard solutions (7.8, 15.6, 31.2, 62.5, and 125 mg/L) (Figure 4C).</li>
	<li>Prepare nitrite indicator solution containing 50 mM sulfanilamide, 330 mM citric acid, and 10 mM N-(1-naphthyl) ethylenediamine dihydrochloride.</li>
	<li>Prepare BSA indicator solution containing 250 mM citrate buffer solution (pH 1.8) and 3.3 mM solution of tetrabromophenol blue in 95% ethanol.</li>
	<li>Prepare urobilinogen indicator solution containing 0.1 M 4-Dimethylamine benzaldehyde.</li>
</ol>
3. Prepare Indicator Pads
<ol>
	<li>Cut three circular shaped pads or disks (&#248; = 0.5 cm) from chromatography paper and insert each of them (one for each assay on the three-assay strip) onto each test strip device to create a solution standard curve (section 4).</li>
	<li>At each of the three lamination film holes of the assembly, observe a slight gap between the lamination film and the cotton test pad at the very edges of the cut hole. Note, the hole in the lamination film is slightly smaller in diameter than the paper test pad. 
	NOTE: Together, these features allow the paper test pad to be applied against the cotton test pad portion of the device so that the edges of the paper test pad slip into the slim gap mentioned, i.e., under the edges of the lamination film hole. This holds the paper test pads in place.</li>
	<li>Prepare the Nitrite Assay Portion of the Device.
	<ol>
		<li>Coat the nitrite assay pad with 4 &#181;l of nitrite indicator solution (prepared in step 2.4) and allow it to dry completely at ambient temperature.</li>
	</ol>
	</li>
	<li>Prepare the Total Protein Assay Portion of the Detection Device.
	<ol>
		<li>Coat the total protein assay pad with 4 &#181;l of BSA indicator solution (prepared in step 2.5) and allow it to dry completely at ambient temperature.</li>
	</ol>
	</li>
	<li>Prepare the Uroblinogen Assay Portion of the Detection Device.
	<ol>
		<li>Coat the uroblinogen assay pad with 4 &#181;l of uroblinogen indicator (prepared in step 2.6) and allow it to dry completely at ambient temperature.</li>
	</ol>
	</li>
</ol>
4. Create Standard Curves for the Cotton-based Analytical Device
<ol>
	<li>For each standard prepared, dip the absorptive end of a prepared device (preloaded with assay indicators (see section 3.4)) into the standard solution for 10 sec (the absorption volume ~ 200 - 300 &#181;l) (Figure 2F).</li>
	<li>Place the cotton-based device in an ambient environment for 10 min to complete colorimetric reactions (Figure 2F). Repeat each experiment in triplicate.</li>
	<li>Scan the device to analyze the colorimetric change occurring at each test pad (Figure 2G). Place resulting test pads on the glass of a desktop scanner and scan test pads in RGB mode with 600 dpi of image resolution. Save each image in jpeg format.</li>
	<li>Analyze the scanned image on a computer using image analysis software to determine respective color intensities (Figure 2H).
	<ol>
		<li>Download ImageJ software at http://imagej.nih.gov/ij/. Open the scanned image files. Select the Image in the menu commands, and select &#8220;Type&#8221; to convert RGB images to 8-bit grayscale.</li>
		<li>Use the &#34;Oval&#34; tool in the toolbar to select the test pad analytical area. Select &#34;Analyze&#34; in the menu commands, and select &#34;Measure&#34; to analyze color intensity. Record mean intensity results.</li>
	</ol>
	</li>
	<li>Establish the standard curves of nitrite, total protein, and urobilinogen assays (Figure 2I).
	<ol>
		<li>Indicate different concentrations of standard nitrite, total protein, and urobilinogen solutions and correlated mean intensity results on a two-dimensional coordinate system using stastical software.</li>
		<li>Fit the standard curve for a nitrite assay by a quadratic model and the standard curves for total protein and urobilinogen assays by linear regression models.</li>
	</ol>
	</li>
</ol>
5. Determine Unknown Sample Concentrations Using Established Standard Curve Values
<ol>
	<li>To determine nitrite, total protein, and urobilinogen concentrations in unknown solutions, use the device as described for measuring standard solution concentrations and substitute measured intensity results for nitrite, total protein, and urobilinogen assays into the equations of the models built in step 2.</li>
</ol>

<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>

The authors declare that they have no competing financial interests. S.C.L., C.M.K., F.G.T., and C.M.C. are inventors of a patent (patent No.: US 8691162 B1) owned by National Tsing Hua University on the basis of the work reported in this manuscript. S.C.L., C.M.K., F.G.T., and C.M.C.have patents pending in Taiwan and China based on the work reported in this manuscript. M.Y.H. declares no potential conflict of interest.

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 ...

Research

JoVE Journal

Bioengineering

6.6K Views.  National Tsing Hua University. 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.

Video: Fabricating Cotton Analytical Devices

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

We describe the design of the &#x201C;quick-linker&#x201D; device for easier orthotopic rat liver transplantation.

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

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 ...