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 sheets. The software program used to design patterns may vary based on the choice of patterning technique (e.g., photolithography, wax printing, or cutting).6</li>
	<li>Spray the work area with a solution of 70% (v/v) ethanol and water. Wipe the work area with a clean paper towel.</li>
</ol>
2. Preparation of Paper Layers: Sample Addition, Conjugate Storage, Incubation, and Wash Layers
<ol>
	<li>Prepare layers of qualitative filter paper using a large tabletop paper cutter. Cut a stock sheet of paper into a standard paper size to facilitate patterning using a solid ink (wax) printer. For example, a single 460 x 570 mm2 sheet can make 4 sheets of US Letter paper (8.5 x 11 inches2). Handle paper with clean gloves at all times to minimize contamination.</li>
	<li>Load a cut sheet of chromatography paper into the printer tray. Print previously designed layers (see Figure 1). 
	NOTE: A pattern can be printed directly onto this sheet using the automatic feed. Only one sheet of paper should be printed at a time to avoid paper jams. For all layers, use the &#34;Enhanced&#34; print settings.</li>
</ol>
3. Preparation of Nylon Membrane Layer: Capture Layer
<ol>
	<li>Cut the stock roll of nylon membrane into sheets (7.5 x 10 inches2) using a tabletop paper cutter. Take 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. 
	NOTE: Cut sheets are narrower than US Letter paper. Because nylon membranes are thin and fragile, they cannot be processed by the printer directly and require support. Details are discussed below.</li>
	<li>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 the positioning of the nylon membrane. The light box aids the alignment of multiple layers.</li>
	<li>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.</li>
	<li>Place a cut sheet of nylon membrane onto the clean piece of copy paper. Make sure that 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. 
	NOTE: Make sure that the nylon membrane is flat and smooth so that there are no problems with printing (e.g., paper jams or uneven printing of wax). Wax may be printed on the tape where the nylon membrane is attached to the copy paper. If this occurs, areas where nylon is incompletely patterned due to tape coverage should be discarded. For future preparations, larger pieces of nylon membrane can be used to avoid this printing error.</li>
	<li>Load a sheet of nylon membrane (supported by the copy paper affixed to it) into the manual feed printer tray. Print only one sheet of nylon membrane at a time. 
	NOTE: There are no preparation steps required for the blot layer, as it is not patterned.</li>
</ol>
4. Creating Hydrophobic Barriers in the Printed Layer
<ol>
	<li>Tape the printed layers onto an acrylic frame for even heating above and below the layer when placed in a gravity convection oven. Keep the nylon membrane taped to the support sheet of copy paper until after the wax is melted and hydrophobic barriers are formed. 
	NOTE: The acrylic frame is a custom-made, laser cut piece of 1/2&#34; thick acrylic plastic. Two frame sizes depending on the number of devices being fabricated were used. The outer border of the smaller frame measures 11 5/8&#34; x 2 3/4&#34;, and the inner hole of the frame measures 10 3/8&#34; x 1 3/4&#34;. The outer border of the larger frame measures 11 5/8&#34; x 8 7/8&#34;, and the inner hole of the frame measures 10 1/4&#34; x 7 7/8&#34;. The open, inner space allows for even melting of wax through the entire thickness of the paper.</li>
	<li>Place the layers in the oven at 150 &#176;C for 30 sec until the wax melts into the thickness of the paper. Confirm that the wax has permeated the thickness of the paper by turning it over and checking for imperfections in the design. 
	NOTE: Forced air ovens or hot plates may also be used to melt the solid wax ink. Melting times or temperatures may vary depending on the heating method.</li>
	<li>Remove the paper and nylon membrane from the acrylic frame. Also, remove nylon membrane from support sheet of copy paper.</li>
</ol>
5. Preparation of Adhesive Layers
<ol>
	<li>Pattern double-sided sheets of adhesive films using a robotic knife plotter, using design files previously prepared (step 1.1). Protect any exposed adhesive surface using a sheet of wax liner. 
	NOTE: The double-sided adhesive should be patterned with holes that allow the sample to flow through layers as a continuous fluidic pathway. The wax liner is easily removed from the adhesive, and serves to protect it from contamination and tearing during cutting. A laser cutter or die press may also be used to pattern layers of adhesive films.</li>
</ol>
6. Backing of Device Layers with Adhesive
<ol>
	<li>Spray the light box with a solution of 70% (v/v) ethanol and water. Wipe with a clean paper towel.</li>
	<li>Tape a patterned layer of paper or nylon membrane that needs to be backed with adhesive onto the light box with the printed side down.</li>
	<li>Peel one side of protective liner from the patterned sheet of adhesive and affix it to the layer of paper or nylon membrane. Use the light box to ensure proper alignment of patterns. Press together. Place the partially assembled device into a protective slip. 
	NOTE: The protective slip is a folded piece of lamination film backing that protects the devices from contamination or damage by ensuring that they do not contact the laminator rollers.</li>
	<li>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. 
	NOTE: Air pockets between the layers of the device can interfere with device integrity and wicking reproducibility by causing leaks.</li>
</ol>
7. Treatment of Conjugate Layer with Reagents for Immunoassays Prior to Device Assembly
<ol>
	<li>Tape conjugate layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.</li>
	<li>Add 2.5 &#181;l of 100 mg/ml bovine serum albumin (BSA) in 1x phosphate buffered saline (PBS) to the hydrophilic zone on the conjugate layer. Allow it to dry at room temperature for 2 min and then at 65 &#176;C for 5 min. 
	NOTE: This volume is just enough to wet the zone of the paper. The BSA solution helps to prevent aggregation of the colloidal nanoparticles during the drying process, which will facilitate the release of the nanoparticles when the paper and reagents are rehydrated by the sample.</li>
	<li>Add 5 &#181;l of 5 O.D. colloidal gold nanoparticle conjugated to anti-&#946;-hCG antibody, and repeat the drying process. 
	NOTE: The units of concentration of colloidal gold nanoparticles are often expressed as optical density (O.D.) as measured by absorbance at &#955; = 540 nm. No treatment is required for the wick pad before device assembly in Section 10.</li>
</ol>
8. Treatment of Lateral Channel with Reagent for Immunoassays Prior to Device Assembly
<ol>
	<li>Tape lateral channel layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.</li>
	<li>Add 10 &#181;l of blocking agent (5 mg/ml non-fat milk and 0.1% (v/v) Tween 20 in 1x PBS) to treat the lateral channel. Repeat the same drying process (2 min at room temperature and then at 65 &#176;C for 5 min) as the conjugate layer.</li>
</ol>
9. Treatment of Capture Layer with Reagents for Immunoassays Prior to Device Assembly
<ol>
	<li>Tape capture layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.</li>
	<li>Treat the capture layer with 5 &#181;L of 1 mg/ml anti-&#945;-hCG antibody and then allow the sample to dry at room temperature for 2 min followed by 8 min at 65 &#176;C.</li>
	<li>Add 2 &#181;L of blocking agent (5 mg/ml non-fat milk and 0.1% (v/v) Tween 20 in 1x PBS). Repeat the drying process for the capture layer. 
	NOTE: This amount is appropriate to coat the papers without occluding the pores of the nylon membrane, which can happen when too much blocking agent is used.</li>
</ol>
10. Assembly of Three-dimensional Paper-based Microfluidic Devices
<ol>
	<li>Tape the wash layer to the light box (printed side facing upwards). If alignment holes are used, remove them from subsequent layers using a handheld hole-punch tool.</li>
	<li>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. Press the two layers together. Avoid touching hydrophilic zones to minimize contamination or damage to the device. Tweezers may be used to assist assembly.</li>
	<li>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.</li>
	<li>Place the partially assembled device into a protective slip and firmly affix the layers together using a laminator.</li>
	<li>Remove the protective film on the back of the wash layer and affix the blot layer to the bottom of the device. Repeat lamination step 10.4 to complete the assembly of the three-dimensional paper-based microfluidic device. Cut desired number of devices from strips or sheets of fully-assembled devices using scissors. 
	NOTE: Full sheets of devices, strips of devices, or single devices may be prepared using a similar approach.</li>
</ol>
11. Performing a Paper-based Immunoassay
<ol>
	<li>Add 20 &#181;l of a sample to the hydrophilic zone on top of the device (i.e., the sample layer).</li>
	<li>Wait for the sample to wick completely into the device, then add 15 &#181;l of wash buffer (0.05% v/v Tween 20 in 1x phosphate buffered saline). After the first aliquot of wash buffer has wicked completely into the device, add a second 15 &#181;l aliquot of wash buffer. 
	NOTE: The wash buffer has completely wicked into the device when the liquid droplet has disappeared, showing no meniscus on the surface of the paper. The assay is complete when the second aliquot of wash buffer has completely entered the device.</li>
	<li>To reveal the results of the assay, peel away the three top layers of the device using tweezers to expose the capture layer.
	<ol>
		<li>Interpret the results of the assay qualitatively by observing the presence or absence of color. Alternatively, image the readout layer using a desktop scanner and use image processing software or algorithms to quantify results and characterize the distributions of intensity within a detection zone.20</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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

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 ...

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Bioengineering

15.6K Views.  Tufts University. 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.

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

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

In this manuscript, periodate oxidation and glutaraldehyde cross-linking methods for covalent immobilization of antibodies on paper discs are presented. The binding activity of immobilized antibodies was evaluated. Based on these results, a glutaraldehyde cross-linking method was used to develop a novel paper-based immunoassay for immunoglobulin G (IgG) detection.

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) ...

Biochemistry

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

Engineering

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 ...

Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices

This project aims to develop an easy-to-use and cost-effective platform for the fabrication of precise, multilayer microfluidic devices, which typically can only be achieved using costly equipment in a clean room setting. The key part of the platform is a three dimensionally (3D) printed microscope mask alignment adapter (MMAA) compatible with regular optical microscopes and ultraviolet (UV) light exposure systems. The overall process of creating the device has been vastly simplified because of the work done to optimize the device design. The process entails finding the proper dimensions for the equipment available in the laboratory and 3D-printing the MMAA with the optimized specifications. Experimental results show that the optimized MMAA designed and manufactured by 3D printing performs well with a common microscope and light exposure system. Using a master mold prepared by the 3D-printed MMAA, the resulting microfluidic devices with multilayered structures contain alignment errors of &lt;10 &#181;m, which is sufficient for common microchips. Although human error through transportation of the device to the UV light exposure system can cause larger fabrication errors, the minimal errors achieved in this study are attainable with practice and care. Furthermore, the MMAA can be customized to fit any microscope and UV exposure system by making changes to the modeling file in the 3D printing system. This project provides smaller laboratories with a useful research tool as it only requires the use of equipment that is typically already available to laboratories that produce and use microfluidic devices. The following detailed protocol outlines the design and 3D printing process for the MMAA. In addition, the steps for procuring a multilayer master mold using the MMAA and producing poly(dimethylsiloxane) (PDMS) microfluidic chips is also described herein.

This project allows small laboratories to develop an easy-to-use platform for the fabrication of precise multilayer microfluidic devices. The platform consists of a three-dimensionally printed microscope mask alignment adapter using which multilayer microfluidic devices with alignment errors of &lt;10 &#181;m were achieved.

This project aims to develop an easy-to-use and cost-effective platform for the fabrication of precise, multilayer microfluidic devices, which typically ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

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 ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.