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 ....

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....

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

List of materials used in this article

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