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Beyond Wicking: Expanding the Role of Patterned Paper as the Foundation for an Analytical Platform While a number of assays for soluble analytes have been developed using paper-based microfluidic devices, the detection and analysis of blood cells has remained an outstanding challenge. In this Feature, we discuss how the properties of paper determine the performance of paper-based microfluidic devices and permit the design of cellular assays, which can ultimately impact disparities in healthcare that exist in limited-resource settings. Syrena C. Fernandes, Jenna A. Walz, Daniel J. Wilson, Jessica C. Brooks, and Charles R. Mace* Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, Massachusetts 02155, United States S Supporting Information *
address these concerns, The World Health Organization/ Sexually Transmitted Diseases Diagnostics Initiative (WHO/ SDI) developed the ASSURED criteria to determine if diagnostic tests are suitable for deployment in limited-resource settings or at the point-of-care.3 ASSURED stands for Af fordable, Sensitive (i.e., low probability of false negatives), Specif ic (i.e., low probability of false positives), User-f riendly (i.e., easy to use, requiring minimal training), Rapid and robust (i.e., short time to result and stable in a variety of environments), Equipment-f ree, and Deliverable to end-users. It is important to note that the ASSURED criteria are guidelines and not absolute requirements. First and foremost, the assays have to work. They must also demonstrate a positive trajectory for future development and translation. Academic researchers are given some latitude in meeting the conditions of ASSURED. For example, affordability can be a difficult topic to address directly because, in addition to the uncertainties regarding the economies of scale when procuring raw materials in large volumes, the final cost of a commercial assay may also include expenses associated with operating a manufacturing facility, licensing patents, performing pilot studies, and filing for regulatory approval, among others. Additionally, the definition of what constitutes “equipment” is somewhat debatable. One perspective restricts equipment to peripheral devices (e.g., battery-powered readers) that enable a measurement. Such devices potentially add cost and complexity to an assay. However, another point-of-view may consider consumables (e.g., pipets, swabs, or dropper bottles of buffers) as supplemental materials that exclude assays from being considered truly equipment-free. In general, an ideal assay will simplify the user experience, be cost-effective, require minimal supplies, and provide the desired measurement in a manner that informs a diagnosis or treatment decision. An archetypal biomedical device for use at the point-of-care is the dried blood spot (DBS) card. DBS cards operate using a very simple principle: a small volume of blood (e.g., from a fingerstick or heel stick) is applied to a porous filter paper card where it is subsequently allowed to dry, which prepares the sample for transport to a clinical laboratory. Guide marks or
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here are many situations in which measurements must be made outside of a laboratory setting or in the field. Access to technology that enables these measurements without sacrificing analytical performance is therefore necessary. This capability often requires the successful integration of sample preparation, liquid handling, and signal transduction within a single device, which may come at a significant expense to the user. However, a limited number of options exist for scenarios when the cost of a device is just as critical of a criterion as its performance. How can we develop cost-effective devices to perform measurements that have a direct impact on the quality of healthcare? Furthermore, what if the sensitivity and specificity of these assays cannot be compromised?1,2 To © 2017 American Chemical Society
Published: April 13, 2017 5654
DOI: 10.1021/acs.analchem.6b03860 Anal. Chem. 2017, 89, 5654−5664
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Analytical Chemistry
stacked or folded layers of paper)40,41 (Figure 1). Patterning also enables the storage of reagents in defined zones within a
etchings assist a user in applying a standard volume of blood, which is required to generate quantitative (i.e., volumedependent) measurements.4 This one-step method permits the storage of blood and plasma with minimal degradation of desired analytes prior to performing analyses off-site. DBS cards have a century-long history of use in clinical settings5 with a wide range of applications including screening for phenylketonuria in newborns,6 detection of hemoglobin variants for the determination of Sickle cell anemia,7 determination of genetic disorders, 8 diagnosis of viral infections, 9 and surveillance of diseases in the developing world.10 While DBS cards are very useful for acquiring samples of blood, the cards themselves are intended to function only as a storage medium and do not support assays directly. Instead, clinicians must remove blood components from cards (e.g., by elution) and perform preparative steps prior to evaluating a sample using traditional analytical techniques (e.g., HPLC or PCR). Further, assays requiring intact or viable cells are precluded as a result of the blood spot drying. As demonstrated by DBS cards, paper is compatible with blood samples but requires additional considerations for use beyond storage. It would be desirable to develop a platform that combines sample application, preparation, and analysis without sacrificing cost or accessibility. Paper-based microfluidic devices, which are also known as microfluidic paper analytical devices (μPAD), have the capacity to enable these critical features and fulfill many of the outlined requirements of an analytical platform for use in limited-resource settings: they are inexpensive, portable, disposable, and operationally simple. Techniques using one-dimensional (i.e., lateral flow)11−13 and two-dimensional paper networks (2DPN)14−16 offer comparable attributes to paper-based microfluidic devices (e.g., control of flow and operational simplicity) but are dissimilar enough that they will not be included in this Feature. Previous reviews of this growing field have broadly discussed the invention and development of paper-based microfluidic devices17−21 or focused on specific aspects related to their manufacture, assembly, quantitative readout, and translation beyond the academic laboratory.22−26 As a result, these topics will not be covered in depth here. Instead, this Feature will address two questions regarding the advancement of paper-based microfluidic devices for their application in biological analyses: (i) What properties of paper facilitate its use as a platform for inexpensive microfluidic devices? (ii) How can understanding the properties of paper aid in the development of paper-based tests for the detection of blood cells? Capillary Action Enables Simple Microfluidic Devices. Paper was chosen as the basis of an analytical platform in part due to its low cost and ubiquity, but primarily because its porous structure enables the transport of small volumes of fluids by capillary action.27 In point-of-care diagnostics or bioanalysis, the sample fluid is aqueous: human saliva,28 tears,29 urine,30 blister fluid,31 blood,32 plasma,33 serum,34 hemolysate,35 and vaginal swabs36 have all been analyzed using paperbased microfluidic devices. In contrast to traditional openchannel microfluidic approaches, pumps and external equipment are not required to drive these fluids through paper. Therefore, wicking greatly simplifies the operation of paperbased devices and reduces costs associated with their use (i.e., requires no purchase or maintenance of equipment). By patterning the paper with hydrophobic barriers,37−39 the wicking of fluids can be controlled spatially in two dimensions (i.e., a single sheet of paper) or in three dimensions (i.e.,
Figure 1. Wicking in paper-based microfluidic devices. When a drop of aqueous solution is added to a layer of unpatterned paper, it is able to wick in all directions (A). Patterning paper with hydrophobic barriers (e.g., wax) allows wicking to be controlled and directed toward multiple detection zones (B). Wicking can be controlled in three dimensions using devices prepared from multiple layers of patterned paper (C) or a single, folded layer of patterned paper (D).
device, which supports the development of a myriad of analytical tests including clinical chemistry assays,42 immunoassays,35,43,44 electrochemical assays,45−47 and molecular diagnostic assays (Figure 2).48 In addition to analysis, devices fabricated from patterned paper can support sophisticated functionalities found in traditional microfluidic devices such as valves,49 timers,50 electrical actuation,51 and sample preparation steps, such as separation52 and preconcentration.53 Material Properties Influence the Use of Papers in Microfluidic Devices. Although many porous materials, nitrocellulose membranes,54,55 threads,56−58 textiles,59 and bamboo,60 have been used to develop simple microfluidic devices and assays, paper-based microfluidic devices are predominantly fabricated with chromatography papers. These purely cellulosic papers are derived from natural sources (e.g., cotton linters and wood pulp). Raw materials undergo a number of chemical and mechanical processing steps in order to form sheets of paper. Variations in manufacturing techniques ultimately influence the resulting products, which can be categorized into “grades” based on their material properties (e.g., particle retention size and flow rate) and intended applications (e.g., chromatography or filtration). While some cross-referencing between commercial suppliers exists, hundreds of different grades of papers are available from multiple commercial manufacturers (e.g., Whatman, Ahlstrom, and Hahnemühle). To date, the wicking and patterning capabilities of papers have been prioritized as selection criteria for the production of paper-based microfluidic devices. Far less attention has been paid to fundamental properties of paper that drive wicking and support paper as a platform for advanced 5655
DOI: 10.1021/acs.analchem.6b03860 Anal. Chem. 2017, 89, 5654−5664
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Analytical Chemistry
Figure 2. Analytical tests performed using paper-based microfluidic devices can employ a variety of methods to transduce a signal and enable measurements. Colorimetric (A−C), electrochemical (D,E), and fluorescence (F,G) detection schemes can be integrated readily with paper-based devices. Part A was adapted with permission from ref 27. Copyright 2007 John Wiley & Sons. Part B was adapted from Sci. Transl. Med. 2012, 4, 152ra129 (ref 125). Reprinted with permission from AAAS. Copyright 2012 AAAS. Part C was reproduced from ref 43 with permission of The Royal Society of Chemistry. Copyright 2014 The Royal Society of Chemistry. Part D was reprinted with permission from J. Am. Chem. Soc. 2014, 136, 4616−4623 (ref 46). Copyright 2014 American Chemical Society. Part E was reprinted with permission from Anal. Chem. 2016, 88, 6326−6333 (ref 47). Copyright 2016 American Chemical Society. Part F was reproduced from ref 52 with permission of The Royal Society of Chemistry. Copyright 2014 The Royal Society of Chemistry. Part G was adapted with permission from Anal. Chem. 2015, 87, 7595−7601 (ref 48). Copyright 2015 American Chemical Society.
Figure 3. Material and chemical properties of papers influence the design and performance of paper-based microfluidic devices and their application as analytical sensors. Key properties include pore size (A,B), surface area (C), wet strength (D,E), stiffness (F), and chemical reactivity (G,H). Papers considered here are predominantly manufactured from cellulosic materials (center inset). Parts A, B, and G were adapted with permission from ref 64. Copyright 2014 John Wiley & Sons. Part C was adapted with permission from ref 74. Copyright 2016 John Wiley & Sons. Part D was reprinted with permission from Anal. Chem. 2016, 88, 6161−6165 (ref 35). Copyright 2016 American Chemical Society. Part E was adapted from ref 80 with permission from Royal Society of Chemistry. Copyright 2010 Royal Society of Chemistry. Part F was adapted with permission from ref 51. Copyright 2016 John Wiley & Sons. Part H was adapted with permission from ref 91. Copyright 2014 John Wiley & Sons.
bioanalysis. Dedicated characterizations of these properties (e.g., surface area and pore size distribution) do not commonly
accompany published reports of developed assays, which makes designing paper-based analytical devices largely an empirical 5656
DOI: 10.1021/acs.analchem.6b03860 Anal. Chem. 2017, 89, 5654−5664
Feature
Analytical Chemistry
be used as a means to evaluate surface area. However, grammage is expressed as an area density, which ultimately neglects the contributions of paper thickness and porosity, and assumes that the materials used to prepare the paper have identical densities (kg/m3). Surface area plays a prominent role in paper-based assays that rely on the irreversible adsorption of capture ligands (e.g., immunoassays). In these applications, the total surface area of cellulose fibers within a test zone functions as the solid phase for binding tagged analyte/reporter complexes and transducing a measurable signal. The concept of adsorption is not limited to biomolecules. Cellulose papers are hygroscopic and the ability to detect the reversible adsorption of water through changes in the conductivity of paper has recently been demonstrated as an effective sensor for respiration (Figure 3C).74 In this example, the performance of the paper-based device was, in part, attributed to the grammage of the papers used: sensors fabricated with Whatman grade 3MM (185 g/m2) had a superior sensitivity than those made using Whatman grade 1 (87 g/m2). (iv) Color. In the production of cellulose-based papers, lignin and other impurities are removed to ensure that the final product is colorless. For example, the presence of lignin can cause discoloration and eventual yellowing due to reactions with light.75 Papers with minimal coloration are particularly desirable for the development of paper-based microfluidic devices that rely on colorimetric outputs. Since many applications of paper-based sensors anticipate that a user will interpret the results of an assay by eye using a change in color as an output, a white test zone provides the ideal background for differentiating negative samples from those containing a small amount of analyte. For those analyses aided by acquiring an image, any background color produced by the paper will limit the available dynamic range of a quantitative measurement. Moreover, image analysis requires careful consideration of the color or temperature (in Kelvin) of the light used to illuminate the device (e.g., LED), the sensitivity of the detector (e.g., smartphone CCD), and the color space (e.g., RGB or HSV) required to perform a measurement.76,77 (v) Wet Strength. Cellulosic papers that start from a relatively pure state can be treated with additives that alter their mechanical properties. Certain applications in filtration may require paper with an improved wet strength in order to resist bursting when positive pressure (e.g., from a rubber diaphragm) or negative pressure (i.e., from a vacuum) is applied to the paper. For example, burst strengths for Whatman brand filter papers, which differ if a paper is wet or dry, can span a range from approximately 0.3−44 psi.78 Some paperbased microfluidic devices can be subjected to considerable wear from user handling and thus require sufficient wet strength to minimize damage caused by a user and ensure that the performance of an assay is not compromised. For example, devices that require delamination (i.e., by peeling)79 or disassembly (i.e., by unfolding)41 to interpret the results of assays must be constructed using materials that can tolerate physical manipulation (Figure 3D). In addition, some applications require the user to apply pressure to defined zones of a device in order to activate desired fluidic conduits (e.g., programmable “on buttons” triggered with a pen) (Figure 3E).80 Such devices may become damaged, which would invalidate a measurement, if they were fabricated from materials with a relatively poor wet strength. (vi) Stiffness. Stiffness (or the related elastic modulus) is an additional property related to the ability of paper to be
process and overly reliant on literature precedent. An improved understanding of how the material properties of paper influence methods for device fabrication and, ultimately, assay performance will have a substantial impact on the development of future paper-based analytical sensors. Below, we discuss seven properties of paper that may be important to consider during design and fabrication of paper-based microfluidic devices (Figure 3): (i) Pore Size. The pores of cellulose-based papers are highly heterogeneous. The pore structure of paper can be characterized by mercury intrusion porosimetry (MIP),61 gas adsorption,62 microcomputed tomography (μCT),63 and scanning electron microscopy (SEM) (Figure 3A,B).64 MIP and gas adsorption use pressure measurements to calculate pore sizes in the micrometer (or larger) and nanometer ranges, respectively, while μCT (volume) and SEM (surface) are imaging techniques that can be used alongside computational algorithms to calculate pore sizes in the micrometer range. Because many chromatography papers are used in qualitative and quantitative filtration applications, pore size data provided by the manufacturer are typically expressed as “particle retention sizes” that are determined experimentally. A particle retention size suggests applications in filtration, which is a traditional use of paper in gravimetric analysis or particle recovery. Generally, particle retention sizes in chromatography papers range from
