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><tbody><tr><td>bovine serum albumin</td><td>Sigma-Aldrich, US</td><td>No. 9048468</td><td>&amp;ge; 99%</td></tr><tr><td>nitrite&amp;nbsp;</td><td>Sigma-Aldrich, US</td><td>No. 7632000</td><td>&amp;ge; 99%</td></tr><tr><td>urobilinogen&amp;nbsp;</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>&amp;ge; 99%</td></tr><tr><td>tetrabromophenol blue</td><td>Sigma-Aldrich U.S</td><td>No. 4430255</td><td>&amp;ge; 99%</td></tr><tr><td>sulfanilamide</td><td>Sigma-Aldrich U.S</td><td>No. 63741</td><td>&amp;ge; 99%</td></tr><tr><td>citric acid&amp;nbsp;</td><td>Sigma-Aldrich U.S</td><td>No. 77929</td><td>&amp;ge; 99.5%</td></tr><tr><td>&amp;nbsp;N-(1-naphthyl) ethylenediamine dihydrochloride</td><td>Sigma-Aldrich U.S</td><td>No. 1465254</td><td>&amp;ge; 98%</td></tr><tr><td>4-(Dimethylamine)benzaldehyde</td><td>AlfaAesar, U.S</td><td>No. A11712</td><td>&amp;ge; 98%</td></tr><tr><td>Methyl Red sodium salt</td><td>sigma, U.S</td><td>No. 114502</td><td>&amp;ge;95%</td></tr><tr><td>Bromothyle blue</td><td>sigma, U.S</td><td>No. 114413</td><td>&amp;ge;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 x 303 mm</td></tr><tr><td>scanner</td><td>microtek scanmaker</td><td>&amp;nbsp;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&amp;nbsp;</td><td>LM4231H</td><td></td></tr><tr><td>laminator film</td><td>UNI LAMI&amp;nbsp;</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

6.7K 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

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Fabrication of Large-area Free-standing Ultrathin Polymer Films

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films. Typically, ultrathin films are prepared using either sacrificial layers, which may damage the film or affect its mechanical properties, or they are made on freshly cleaved mica, a substrate that is difficult to scale. Further, the size of ultrathin film is typically limited to a few square millimeters. In this method, we modify a surface with a polyelectrolyte that alters the strength of adhesion between polymer and deposition substrate. The polyelectrolyte can be shown to remain on the wafer using spectroscopy, and a treated wafer can be used to produce multiple films, indicating that at best minimal amounts of the polyelectrolyte are added to the film. The process has thus far been shown to be limited in scalability only by the size of the coating equipment, and is expected to be readily scalable to industrial processes. In this study, the protocol for making the solutions, preparing the deposition surface, and producing the films is described.

We describe a method for the fabrication of large-area (up to 13 cm diameter) and ultrathin (as thin as 8 nm) polymer films. Instead of using a sacrificial interlayer to delaminate the film from its substrate, we use a self-limiting surface treatment suitable for arbitrarily large areas.

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films.  Typically, ultrathin films are prepared ...

Chemistry

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Fabrication of an Optical Cell Dryer for the Spectroscopic Analysis Cells

Optical cells, which are experimental instruments, are small, square tubes sealed on one side. A sample is placed in this tube, and a measurement is performed with a spectroscope. The materials used for optical cells generally include quartz glass or plastic, but expensive quartz glass is reused by removing substances, other than liquids, to be analyzed that adhere to the interior of the container. In such a case, the optical cells are washed with water or ethanol and dried. Then, the next sample is added and measured. Optical cells are dried naturally or with a manual hairdryer. However, drying takes time, which makes it one of the factors that increase the experiment time. In this study, the objective is to drastically reduce the drying time with a dedicated automatic dryer that can dry multiple optical cells at once. To realize this, a circuit was designed for a microcomputer, and the hardware using it was independently designed and manufactured.

A protocol for fabricating a device for simultaneously drying multiple optical cells is presented.

Optical cells, which are experimental instruments, are small, square tubes sealed on one side. A sample is placed in this tube, and a measurement is ...

Engineering

Hybrid Printing for the Fabrication of Smart Sensors

A method to combine additively manufactured substrates or foils and multilayer inkjet printing for the fabrication of sensor devices is presented. First, three substrates (acrylate, ceramics, and copper) are prepared. To determine the resulting material properties of these substrates, profilometer, contact angle, scanning electron microscope (SEM), and focused ion beam (FIB) measurements are done. The achievable printing resolution and suitable drop volume for each substrate are, then, found through the drop size tests. Then, layers of insulating and conductive ink are inkjet printed alternately to fabricate the target sensor structures. After each printing step, the respective layers are individually treated by photonic curing. The parameters used for the curing of each layer are adapted depending on the printed ink, as well as on the surface properties of the respective substrate. To confirm the resulting conductivity and to determine the quality of the printed surface, four-point probe and profilometer measurements are done. Finally, a measurement set-up and results achieved by such an all-printed sensor system are shown to demonstrate the achievable quality.

Here we present a protocol for the fabrication of inkjet-printed multilayer sensor structures on additively manufactured substrates and foil.

A method to combine additively manufactured substrates or foils and multilayer inkjet printing for the fabrication of sensor devices is presented. First, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Micro-Embossed Fabrication of Nanocellulose Paper-Based Analytical Devices  

Microembossing: A Convenient Process for Fabricating Microchannels on Nanocellulose Paper-Based Microfluidics

Nanopaper, derived from nanofibrillated cellulose, has generated considerable interest as a promising material for microfluidic applications. Its appeal lies in a range of excellent&#160;qualities, including an exceptionally smooth surface, outstanding optical transparency, a uniform nanofiber matrix with nanoscale porosity, and customizable chemical properties. Despite the rapid growth of nanopaper-based microfluidics, the current techniques used to create microchannels on nanopaper, such as 3D printing, spray coating, or manual cutting and assembly, which are crucial for practical applications, still possess certain limitations, notably susceptibility to contamination. Furthermore, these methods are restricted to the production of millimeter-sized channels. This study introduces a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanopaper, achieving a minimum width of 200 &#181;m. The developed microchannel outperforms existing approaches, achieving a fourfold improvement, and can be fabricated within 45 min. Furthermore, fabrication parameters have been optimized, and a convenient quick-reference table is provided for application developers. The proof-of-concept for a laminar mixer, droplet generator, and functional nanopaper-based analytical devices (NanoPADs) designed for Rhodamine B sensing using surface-enhanced Raman spectroscopy was demonstrated. Notably, the NanoPADs exhibited exceptional performance with improved limits of detection. These outstanding results can be attributed to the superior optical properties of nanopaper and the recently developed accurate microembossing method, enabling the integration and fine-tuning of the NanoPADs.

Author Spotlight: Revolutionizing Microfluidics Through Microchannel Fabrication on Nanopaper 

This protocol describes a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanofibrillated cellulose paper, achieving a minimum width of 200 &#181;m.

Nanopaper, derived from nanofibrillated cellulose, has generated considerable interest as a promising material for microfluidic applications. Its appeal ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fabricating Cotton Analytical Devices

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