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

Summary

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.

Abstract

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.

Introduction

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 assays^3,4,5,6,7. Applications of paper-based microfluidic devices include point-of-care diagnostics⁸, monitoring of environmental contaminants⁹, detection of counterfeit pharmaceuticals¹⁰, and delocalized healthcare (or "telemedicine") 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.

Protocol

1. Preparation of Paper-based Microfluidic Device Layers

1. 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
2. Spray the work area with a solution of 70% (v/v) ethanol and water. Wipe the work area with a clean paper towel.

2. Preparation of Paper Layers: Sample Addition, Conjugate Storage, Incubation, and Wash Layers

1. 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.
2. 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 "Enhanced" print settings.

3. Preparation of Nylon Membrane Layer: Capture Layer

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.

4. Creating Hydrophobic Barriers in the Printed Layer

1. 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" 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" x 2 3/4", and the inner hole of the frame measures 10 3/8" x 1 3/4". The outer border of the larger frame measures 11 5/8" x 8 7/8", and the inner hole of the frame measures 10 1/4" x 7 7/8". The open, inner space allows for even melting of wax through the entire thickness of the paper.
2. Place the layers in the oven at 150 °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.
3. Remove the paper and nylon membrane from the acrylic frame. Also, remove nylon membrane from support sheet of copy paper.

5. Preparation of Adhesive Layers

1. 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.

6. Backing of Device Layers with Adhesive

1. Spray the light box with a solution of 70% (v/v) ethanol and water. Wipe with a clean paper towel.
2. 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.
3. 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.
4. 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.

7. Treatment of Conjugate Layer with Reagents for Immunoassays Prior to Device Assembly

1. Tape conjugate layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.
2. Add 2.5 µl 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 °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.
3. Add 5 µl of 5 O.D. colloidal gold nanoparticle conjugated to anti-β-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 λ = 540 nm. No treatment is required for the wick pad before device assembly in Section 10.

8. Treatment of Lateral Channel with Reagent for Immunoassays Prior to Device Assembly

1. 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.
2. Add 10 µl 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 °C for 5 min) as the conjugate layer.

9. Treatment of Capture Layer with Reagents for Immunoassays Prior to Device Assembly

1. Tape capture layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.
2. Treat the capture layer with 5 µL of 1 mg/ml anti-α-hCG antibody and then allow the sample to dry at room temperature for 2 min followed by 8 min at 65 °C.
3. Add 2 µL 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.

10. Assembly of Three-dimensional Paper-based Microfluidic Devices

1. 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.
2. 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.
3. 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.
4. Place the partially assembled device into a protective slip and firmly affix the layers together using a laminator.
5. 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.

11. Performing a Paper-based Immunoassay

1. Add 20 µl of a sample to the hydrophilic zone on top of the device (i.e., the sample layer).
2. Wait for the sample to wick completely into the device, then add 15 µl 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 µl 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.
3. To reveal the results of the assay, peel away the three top layers of the device using tweezers to expose the capture layer.
   1. 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

Results

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).

Discussion

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 paper^23,24 additive manufacturing offers a number of advantages: (i) Multiple materials can be incorporated ...

Materials

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