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>

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 ...

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

6.6K visualizações.  National Tsing Hua University. 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. B...