Name: fabrication of three-dimensional paper-based microfluidic devices for immunoassays
Uploaded: 2017-03-09T14:00:00
Description: Watch this Scientific Journal Video about Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays at JoVE.com

This work was supported by Tufts University and by a generous gift from Dr. James Kanagy. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. (DGE-1325256) that was awarded to S.C.F. D.J.W. was supported by a U.S. Department of Education GAANN fellowship. We thank Dr. Jeremy Schonhorn (JanaCare), Dr. Jason Rolland (Carbon3D), and Rachel Deraney (Brown University) for helping develop the design of the three-dimensional paper-based microfluidic device and immunoassay.

1. Preparation of Paper-based Microfluidic Device Layers
<ol>
	<li>Prepare patterns for layers of paper, nylon, and adhesive using a graphic design software program.6 Each layer may have a different pattern. 
	NOTE: The pattern may include alignment holes that are not required for a functional paper-based immunoassay, but assist with the reproducible manufacture of three-dimensional devices. Placement of these holes will differ if devices are assembled individually, in strips, or as full sh...

<ol>
	<li>Martinez, A. W., Phillips, S. T., Wiley, B. J., Gupta, M., 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=FLASH:+A+rapid+method+for+prototyping+paper-based+microfluidic+devices.">FLASH: A rapid method for prototyping paper-based microfluidic devices.</a> Lab Chip. 8 (12), 2146-2150 (2008).</li><li>Carrilho, E., Martinez, A. W., 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=Understanding+wax+printing:+a+simple+micropatterning+process+for+paper-based+microfluidic+devices.">Understanding wax printing: a simple micropatterning process for paper-based microfluidic devices.</a> Anal. Chem. 81 (16), 7091-7095 (2009).</li><li>Martinez, A. W., Phillips, S. T., Butte, M. J., Whitesides, G. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+paper-based+analytical+devices+(&#181;PADs)+and+micro+total+analysis+systems+(&#181;TAS):+Development,+applications+and+future+trends.">Microfluidic paper-based analytical devices (&#181;PADs) and micro total analysis systems (&#181;TAS): Development, applications and future trends.</a> Chromatographia. 76, 1201-1214 (2013).</li><li>Pollock, N. R., 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=A+paper-based+multiplexed+transaminase+test+for+low-cost,+point-of-care+liver+function+testing.">A paper-based multiplexed transaminase test for low-cost, point-of-care liver function testing.</a> Sci. Transl. Med. 4 (152), 152ra129 (2012).</li><li>Mentele, M. M., Cunningham, J., Koehler, K., Volckens, J., Henry, C. 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J., 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=Measuring+markers+of+liver+function+using+a+micro-patterned+paper+device+designed+for+blood+from+a+fingerprick.">Measuring markers of liver function using a micro-patterned paper device designed for blood from a fingerprick.</a> Anal Chem. 84 (6), 2883-2891 (2012).</li><li>Nie, Z., Deiss, F., Liu, X., Akbulut, O., 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=Integration+of+paper-based+microfluidic+devices+with+commercial+electrochemical+readers.">Integration of paper-based microfluidic devices with commercial electrochemical readers.</a> Lab Chip. 10 (22), 3163-3169 (2010).</li><li>Martinez, A. W., 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=Programmable+diagnostic+devices+made+from+paper+and+tape.">Programmable diagnostic devices made from paper and tape.</a> Lab Chip. 10 (19), 2499-2504 (2010).</li><li>Connelly, J. T., Rolland, J. P., 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=&#34;Paper+machine&#34;+for+molecular+diagnostics.">&#34;Paper machine&#34; for molecular diagnostics.</a> Anal. Chem. 87 (15), 7595-7601 (2015).</li><li>Schonhorn, J. E., Fernandes, S. C., Rajaratnam, A., Deraney, R. N., Rolland, J. P., Mace, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+device+architecture+for+three-dimensional,+patterned+paper+immunoassays.">A device architecture for three-dimensional, patterned paper immunoassays.</a> Lab Chip. 14 (24), 4653-4658 (2014).</li><li>Fernandes, S. C., Logounov, G. S., Munro, J. B., Mace, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+indirect+immunoassay+formats+on+a+common+paper-based+microfluidic+device+architecture.">Comparison of three indirect immunoassay formats on a common paper-based microfluidic device architecture.</a> Anal. Methods. 8 (26), 5204-5211 (2016).</li><li>Deraney, R. N., Mace, C. R., Rolland, J. P., Multiplexed Schonhorn, J. E. <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+immunoassay+for+detection+of+malaria+and+dengue+fever.">patterned-paper immunoassay for detection of malaria and dengue fever.</a> Anal. Chem. 88 (12), 6161-6165 (2016).</li><li>Abramoff, M., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics Int. 11 (7), 36-42 (2004).</li><li>Derda, R., 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=Multizone+paper+platform+for+3D+cell+cultures.">Multizone paper platform for 3D cell cultures.</a> PLoS ONE. 6 (5), e18940 (2011).</li><li>Mace, C. R., Deraney, R. 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Soc. 133 (44), 17564-17566 (2011).</li><li>Kalish, B., Tsutsui, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Adhesive+patterning+to+construct+3D+paper+microfluidic+devices.">Using Adhesive patterning to construct 3D paper microfluidic devices.</a> J. Vis. Exp. (110), e53805 (2016).</li><li>Scida, K., Cunningham, J. C., Renault, C., Richards, I., Crooks, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+sensitive,+and+quantitative+electrochemical+detection+method+for+paper+analytical+devices.">Simple, sensitive, and quantitative electrochemical detection method for paper analytical devices.</a> Anal. Chem. 86 (13), 6501-6507 (2014).</li><li>Lewis, G. G., DiTucci, M. J., Baker, M. S., Phillips, S. 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Identifying a reproducible manufacturing strategy is an essential component of assay development.22 We use a sequential, layer-by-layer approach to manufacture three-dimensional paper-based microfluidic devices. In contrast to those methods that apply folding or origami techniques to produce multilayer devices from a single sheet of paper23,24 additive manufacturing offers a number of advantages: (i) Multiple materials can be incorporated ...

Paper is widely available in a range of formulations or grades, can be functionalized to tune its properties, and can transport fluids autonomously by capillary action or wicking. If paper is patterned with a hydrophobic substance (e.g., photoresist1 or wax2), the wicking of fluids can be controlled spatially within a layer of paper. For example, an applied aqueous sample can be directed into a number of different zones to react with chemical and biochemical reagents stored within the paper. These paper-based microfluidic devices have been demonstrated to be a useful platform for the development of portable and inexpensive analytical assays3,4,5,6,7. Applications of paper-based microfluidic devices include point-of-care diagnostics8, monitoring of environmental contaminants9, detection of counterfeit pharmaceuticals10, and delocalized healthcare (or &#34;telemedicine&#34;) in limited-resource settings11.
Multiple layers of patterned paper can be assembled into an integrated device where hydrophilic zones from neighboring layers (i.e., above or below) connect to form continuous fluidic networks whose inlets and outlets may be coupled or left independent.12 Each layer can comprise a unique pattern, which enables the spatial separation of reagents and multiple assays to be performed on a single device. The resulting three-dimensional microfluidic device is not only capable of wicking fluids to enable analytical assays (e.g., liver function tests13 and electrochemical detection of small molecules14), but it can also support a number of sophisticated functions (e.g., valves15 and simple machines16) common to traditional microfluidic approaches. Importantly, because paper wicks fluids by capillary action, these devices can be operated with minimal effort from the user.
Since reagents can be stored within the three-dimensional architecture of a paper-based device, complex protocols can be reduced to a single addition of aqueous sample to a device. Recently, we introduced a general three-dimensional device architecture that can be used for the development of paper-based immunoassays using the wax-printing technique to create patterned layers.17,18 These studies focused on how aspects related to the design of the device-number of stacked layers used, composition of the layers, and the pattern of the three-dimensional microfluidic network-controlled the overall performance of the immunoassay. Ultimately, we were able to use these design rules to facilitate the rapid development of a multiplexed immunoassay19. In this manuscript, a previously developed immunoassay for human chorionic gonadotropin (hCG; pregnancy hormone)17 is used as an example to illustrate the strategies that we have developed for the assembly and manufacture of three-dimensional paper-based immunoassays. Accordingly, we focus on the assembly and operation of a device rather than the development of an assay.
In a sandwich immunoassay, which is the format used to detect hCG, a capture antibody specific to one subunit of the hormone is coated onto a solid substrate, which is then blocked to limit the non-specific adsorption of a sample or any subsequent reagent. This substrate is most often a polystyrene microwell plate (e.g., for an enzyme-linked immunosorbent assay or ELISA). The sample is then added to a well and allowed to incubate for a period of time. After rigorous washing, an antibody specific to the other subunit of hCG is added and allowed to incubate. This detection antibody may be conjugated to a colloidal particle, enzyme, or fluorophore in order to produce a measurable signal. The well is again washed prior to interpreting the results of an assay (e.g., using a plate reader). While commercial kits rely on this time-consuming multistep process, all of these steps can be performed rapidly in paper-based microfluidic devices with minimal intervention to the user.
The device used for the hCG immunoassay comprises six active layers, which are, from top to bottom, used for sample addition, conjugate storage, incubation, capture, wash, and blot (Figure 1). The sample addition layer is made from qualitative filter paper. It facilitates the introduction of a liquid sample and protects the reagents in the conjugate layer from contamination from the environment or accidental contact by the user. The conjugate layer (qualitative filter paper) holds the color-producing reagent (e.g., colloidal gold-labeled antibody) for the immunoassay. The incubation layer (qualitative filter paper) allows the sample to travel laterally within the plane of the paper to promote binding of the analyte with reagents before reaching the next layer, the capture layer. The capture layer (nylon membrane) contains ligands specific for the analyte adsorbed to the material. After the assay is completed, this layer is revealed to enable visualization of the completed immunocomplex. The wash layer (qualitative filter paper) draws excess fluids including free conjugate reagents away from the face of the capture layer into the blot layer (thick chromatography paper). The six-layer device is held together by five layers of patterned, double-sided adhesive: four layers of permanent adhesive maintain the integrity of the assembled device and one layer of removable adhesive facilitates peeling of the device to inspect the results of the immunoassay on the capture layer.
For the purpose of this manuscript, we use only negative and positive control samples of hCG (0 mIU/mL and 81 mIU/mL, respectively) to provide representative results of a paper-based immunoassay, which permits a dedicated discussion of the relationship between fabrication methods and the performance of a device. In addition to demonstrating how to manufacture devices successfully, we highlight several manufacturing errors that could lead to the failure of a device or irreproducible assay results. The protocol and discussion detailed in this manuscript will provide researchers with valuable insight into how paper-based immunoassays are designed and fabricated. While we focus our demonstration on immunoassays, we anticipate that the guidelines presented herein will be broadly useful for the manufacture of three-dimensional paper-based microfluidic devices.

<table border="0" cellpadding="0" cellspacing="0" width="1059">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Illustrator CC</td>
			<td style="width:135px;">Adobe</td>
			<td style="width:175px;"></td>
			<td style="width:304px;">to design patterns for layers of paper and adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Xerox ColorQube 8580 printer</td>
			<td style="width:135px;">Amazon</td>
			<td style="width:175px;">B00R92C9DI</td>
			<td>to print wax patterns onto layers of paper and Nylon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Isotemp General Purpose Heating and Drying Oven</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:175px;">15-103-0509</td>
			<td>to melt wax into paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artograph LightTracer</td>
			<td style="width:135px;">Amazon</td>
			<td style="width:175px;">B000KNHRH6</td>
			<td>to assist with alignment of layers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Apache AL13P laminator</td>
			<td style="width:135px;">Amazon</td>
			<td style="width:175px;">B00AXHSZU2</td>
			<td>to laminate layers together</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graphtec CE6000 Cutting Plotter</td>
			<td style="width:135px;">Graphtec America</td>
			<td style="width:175px;">CE6000-40</td>
			<td>to pattern adhesive films</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swingline paper cutter</td>
			<td style="width:135px;">Amazon</td>
			<td style="width:175px;">B0006VNY4C</td>
			<td>to cut paper or devices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epson Perfection V500 photo scanner</td>
			<td style="width:135px;">Amazon</td>
			<td style="width:175px;">B000VG4AY0</td>
			<td>to scan images of readout layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">economy plier-action hole punch</td>
			<td style="width:135px;">McMaster-Carr</td>
			<td>3488A9</td>
			<td>to remove alignment holes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Whatman chromatogrpahy paper, Grade 4</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>WHA1004917</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Fisherbrand chromatography paper (thick)&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>05-714-4</td>
			<td>to function as blot layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Immunodyne ABC (0.45 &#181;m pore size )</td>
			<td style="width:135px;">Pall Corporation</td>
			<td>NBCHI3R</td>
			<td>to function as material for capture layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">removable/permanent adhesive-double faced liner</td>
			<td style="width:135px;">FLEXcon</td>
			<td style="width:175px;">DF021621</td>
			<td>to facilitate peeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">permanent adhesive-double faced liner</td>
			<td style="width:135px;">FLEXcon</td>
			<td style="width:175px;">DF051521</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:447px;">wax liner</td>
			<td style="width:135px;">FLEXcon</td>
			<td style="width:175px;">FLEXMARK 80 D/F PFW LINER</td>
			<td>to assist with patterning adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">acrylic sheet</td>
			<td style="width:135px;">McMaster-Carr</td>
			<td style="width:175px;">8560K266&#160;</td>
			<td>to fabricate frame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">self-adhesive sheets</td>
			<td style="width:135px;">Fellowes</td>
			<td style="width:175px;">CRC52215</td>
			<td>to use as protective slip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">absolute ethanol</td>
			<td style="width:135px;">VWR</td>
			<td style="width:175px;">89125-172</td>
			<td>to sanitize work area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">bovine serum albumin</td>
			<td style="width:135px;">AMRESCO</td>
			<td style="width:175px;">0332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sekisui Diagnostics OSOM hCG Urine Controls</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>22-071-066</td>
			<td>to use as positive and negative samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-&#946;-hCG monoclonal antibody colloidal gold conjugate (clone 1)</td>
			<td style="width:135px;">Arista Biologicals&#160;</td>
			<td>CGBCG-0701</td>
			<td>to treat conjugate layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-&#945;-hCG antibody</td>
			<td style="width:135px;">Arista Biologicals&#160;</td>
			<td>ABACG-0500</td>
			<td>to treat capture layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">10X phosphate buffered saline</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:175px;">BP3991</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Oxoid skim milk powder</td>
			<td style="width:135px;">Thermo Scientific</td>
			<td style="width:175px;">OXLP0031B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Tween 20</td>
			<td style="width:135px;">AMRESCO</td>
			<td style="width:175px;">M147</td>
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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.

Obtaining reproducible assay performances in three-dimensional paper-based microfluidic devices relies on a fabrication method that ensures consistency among devices. Towards this goal, we have identified a number of manufacturing processes and material considerations, and discuss them here in the context of demonstrating a paper-based immunoassay. We use a wax printing method to form hydrophobic barriers within paper-based microfluidic devices (Figure 2A).<sup class="xre...

Watch this Scientific Journal Video about Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays at JoVE.com

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

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 ...

The overall goal of this protocol is to demonstrate our process for fabricating three-dimensional paper-based devices, which we use as a platform to develop point of care immunoassays. This method provides a paper-based microfluidics platform with a reliable manufacturing process, such that time and effort can be directed to developing assays instead of designing devices. The main advantage of our technique is that it allows many devices to be prepared in parallel and at a quantity that is desirable for academic research projects.Generally, new users will struggle with this method because there are many variables to consider when fabricating devices, like proper alignment of layers. Mistakes can results in devices that malfunction. A visual demonstration of this method is critical because assembly can be difficult to envision using only the details provided in manuscripts.First, prepare layers of qualitative filter paper by cutting a stock sheet of paper into a standard paper size to facilitate patterning using a wax printer. Load a cut sheet of paper into the printer tray. Then print previously designed layers.Next, cut a stock row of nylon membrane into sheets using a tabletop paper cutter, taking great care in handling the nylon membrane to maintain its integrity and protect against ripping. Store any unused material in a desiccator cabinet, as nylon membranes are moisture sensitive. Using a wax printer, print a capture layer pattern onto a piece of copy paper, and tape it to a light box to serve as a guide for positioning of the nylon membrane.Following this, place a clean sheet of copy paper onto the previously printed sheet of copy paper. Tape the clean sheet of paper to the light box but do not tape the two sheets together. Now, place a cut sheet of nylon membrane onto the clean sheet of copy paper, making sure that the membrane covers the printed area of the bottom layer of copy paper.Tape all four sides of the nylon membrane to the clean sheet of copy paper. Load a sheet of nylon membrane supported by the copy paper affixed to it into the manual feed printer tray. Then print one sheet of nylon membrane at a time.Tape the printed layers onto an acrylic frame for even heating above and below the layer when placed in a gravity convection oven. Now, place the layers in the oven at 150 degrees Celsius for 30 seconds until the wax melts into the thickness of the paper. After removing the paper from the oven, confirm that the wax has permeated the thickness of the paper by turning it over and checking for imperfections in the design.Remove the paper and nylon membrane from the acrylic frame. Then remove the nylon membrane from the support sheet of copy paper using a paper cutter. Pattern double-sided sheets of adhesive films using a robotic knife plotter with previously prepared design files.Following this, tape a pattern layer of paper that needs to be backed with adhesive onto a light box with the printed side down. Peel one side of protective liner from the pattern sheet of adhesive. Press the pattern sheet of adhesive and layer of paper together.Then place the partially assembled device into a protective slip. Next, pass the resulting two layer assembly through an automated laminator to completely press the adhesive and paper together, removing any pockets of air from the adjoined layers. Tape a conjugate layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.Add 2.5 microliters of BSA and one X PBS to the hydrophilic zone on the conjugate layer. After allowing the sample to dry at room temperature for two minutes, dry it at 65 degrees Celsius for five minutes. Following this, add five microliters of five OD colloidal gold nanoparticle conjugated to anti-beta hCG antibody, then repeat the drying process.Tape a lateral channel layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame. Add 10 microliters of blocking agent to treat the lateral channel, then repeat the same drying process used for the conjugate layer. Next, tape a capture layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.Treat the capture layer with five microliters of anti-alpha hCG antibody. After allowing the sample to dry at room temperature for two minutes, dry it at 65 degrees Celsius for eight minutes. Add two microliters of blocking agent, then repeat the same drying process used for the capture layer.Tape the wash layer to the light box with the printed side facing upwards. If alignment holes are used, remove them from subsequent layers using a handheld hole punch tool. Remove the protective film on the back of the capture layer to expose the adhesive.Align the capture layer above the wash layer using the alignment holes as a guide, then press the two layers together and avoid touching the hydrophilic zones to minimize contamination or damage to the device. Following this, remove the protective film on the back of the incubation layer to expose the adhesive. Align the incubation layer above the capture layer and press them together.Continue adding layers in this manner until all active layers are assembled. Now, place the partially assembled device into a protective slip and firmly affix the layers together using a laminator. Remove the protective film on the back of the wash layer and affix the blot layer to the bottom of the device.After laminating to complete the assembly of the three-dimensional paper-based microfluidic device, cut the desired number of devices from sheets of fully assembled devices using scissors. Add 20 microliters of a positive control sample of hCG buffer to the hydrophilic zone on top of the device. Once the sample has wicked completely into the device, add 15 microliters of wash buffer.After the first aliquot of wash buffer has wicked completely into the device, add a second 15 microliter aliquot of wash buffer. To reveal the results of the assay, peel away the three top layers of the device using tweezers to expose the capture layer. The wax printing method can be used to form hydrophobic barriers within paper-based microfluidic devices, and produces fluidic pathways with reproducible dimensions, which is critical for assays with repeatable performances and duration times.The performance of the hCG paper-based immunoassay was demonstrated by performing 35 positive and 35 negative assays in parallel. The coefficient of variation for each dataset was determined to be 1%for assays performed using negative samples and 3%for assays performed using positive samples. A misalignment between layers comprising the incubation channel and capture zone can cause the development of an irregular pattern in the positive signal, which may result in a misinterpretation of the qualitative signal.If the wax is not printed in a sufficient amount or not allowed to melt completely through the thickness of the paper, then the integrity of the resulting hydrophobic barriers may be compromised and lead to leaks within the device. Assays that take longer than expected to complete may indicate a malfunction in the fabrication of a device. Paper-based microfluidic devices provide researchers with a versatile platform to develop low cost, point of care analytical tests.While a number of applications exist, the approach we demonstrate here results in a general device architecture to perform immunoassays, which are critical in healthcare. While attempting this procedure, it's important to remember to check for imperfections during the fabrication process and before treating the layers with expensive biochemical reagents. Once mastered, this method, from printing layers to assembling treated layers, can be used to prepare a sheet of functional immunoassays in two hours.After watching this video, you should have a good understanding of how to fabricate three-dimensional paper-based devices, and be comfortable enough to tailor this platform to other assay types of interest.

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