ReviewTechnical aspects and challenges of colorimetric detection with microfluidic paper-based analytical devices (μPADs) - A review
Introduction
According to the World Health Organization (WHO) in the ASSURED Challenge [1], access to equipment cannot constitute a barrier against the performance of diagnostic tests, especially for in-development countries and other resource-limited locations worldwide. Microfluidic paper-based analytical devices (μPADs) [2], [3] can meet most of these requirements, due to their intrinsic characteristics of low-cost, ease-of-use, and portability [4].
As stated by Cate et al. [4], detection is one of the most important steps in paper-based assays to quantify and/or identify the presence of the analyte of interest. Colorimetry is widely regarded as the most suitable detection technique to integrate with μPADs, due to its simplicity and compatibility with relatively low-cost reporting systems, including smartphones [3] and scanners [2], [3]. The pioneer work on digital imaging for analytical purposes goes back to 2000's, when Byrne et al. [5] correctly foresaw the use of digital tools for qualitative and quantitative measurements using colorimetric reactions. Since then, and with the breakthrough research developed by the Whitesides' group at Harvard University [2], [3], [6], the area of qualitative and quantitative analysis using colorimetric reactions on μPADs has experienced great development, with Cate et al. [4] estimating that over 1000 papers were published on the topic from 2012 to 2014.
The versatility of μPADs gives them a myriad of applications, which have recently been reviewed by multiple groups. As Cate et al. [4] asserted in their review, the large amount of research dealing with paper-based devices in the last few years prevents a comprehensive review of all aspects of μPADs systems. Recently Meredith et al. [7] have reviewed the use of paper-based devices for environmental analysis and the related colorimetric readout strategies. Yetisen et al. [8] have reviewed the use of paper-based devices for point-of-care applications, also contemplating colorimetric readouts. An opportunity in the area appears to be the use of μPADs for forensic analysis. The classical guideline from Johns et al. [9] presents a list of colorimetric spot tests relevant to forensic analysis (9 spot tests covering 200 compounds). Making use of chemometric tools, such as those presented by Salles et al. [10], it may be possible to screen a large number of compounds within minutes using a single device.
This review focuses on developments and challenges of colorimetric detection and correlated techniques on μPADs and other assays carried out in a paper-based platform, 10 years after the seminal technology of μPADs was first presented in literature [2]. We initially discuss different approaches to generate color on paper-based assays, addressing methods to improve color generation and homogeneity. Then follows a discussion on how to measure color change using multiple reporting systems and a comparison among these reporting systems. Finally, we critically evaluate data-handling methods and how they effect assay results. We also include thoughts and insights for future development of research on the topic that might finally enable the use of low-cost diagnostic devices by those who need them most.
Section snippets
Assay chemistry
When paper-based analytical assays were introduced around 2007 [2], [3], [6] goals included incorporation of bioassays [2], [3], [6], [11] and to explore colorimetric spot tests developed by Fritz Feigl [11], [12] with a technological approach, using information technology (IT) communication equipment such as scanners and cell phone cameras [13]. In this section we discuss different approaches to generate color on paper-based devices, including indirect color generation through coupled
Color homogeneity
Color homogeneity is important for colorimetric readouts in μPADs because it enables better measurements with improved figures of merit of the analytical method. Some parameters affect the homogeneity of the signal, including device design, assay implementation and the choice and modifications of the substrate. Digitalization and data processing also impact homogeneity and are addressed separately later in the review (section 8.3 Signal homogeneity assessment). Here, we review the impacts of
Reporting systems
After the colorimetric assay has been performed, it is necessary to read off the information displayed in the μPAD. This is a critical step for equipment-dependent quantitative readouts, but can be performed for qualitative and semi-quantitative readouts as well (section 7 Results readout), depending on the purpose. The information obtained can be performed by a standard analytical chemical instrumental technique, such as diffuse reflectance spectroscopy, or by IT communication equipment
Image analysis
After the colorimetric assay has been performed and digitized, it is necessary to analyze the digital image, obtaining the color information contained in each pixel. Not all pixels in the image contain information relevant to the colorimetric test (for example, the black hydrophobic barriers in Fig. 8a or the central channel of Fig. 7b). It is therefore necessary to identify the correct testing regions and obtain the information for that group of pixels. Here, we review automated and manual
Results readout
As stated by Cate et al. [4], the outputs of colorimetric spot tests or lateral flow assays can be analyzed by three distinct approaches:
- i)
Qualitative readout, in which there is a change in the coloration in the reaction zone due to the presence of the analyte of interest, with a YES/NO output [8], [110], [111];
- ii)
Semi-quantitative readout, in which the colorimetric output of the reaction zone is compared with a pre-established calibration curve, giving an estimate of the concentration range of the
Data handling
There are multiple ways to obtain and evaluate colorimetric data from μPADs and determine the figures of merit of the analytical method. Grudpan et al. [13] present and describe equations for the various color spaces, and Capitán-Vallvey et al. [107] present a comprehensive list of work dealing with computer vision-based analytical (CVAC) procedures and how data can be processed in distinct color spaces (RGB, HSV, CIE, CIELAB).
Here, we examine the impact of the choice of the color space on data
General strategies for signal-to-noise improvement
Paper-based microfluidic devices enable relative freedom in designs [81] that permits the addition of multiple functionalities. The modification of devices, including the incorporation of pre-concentrators and the addition of reagents in the reactional zone to improve colorimetric readouts [122]. Here we review strategies to improve colorimetric signal.
Concluding remarks
In the almost 10 years since μPADs were first proposed [2] much development has occurred in paper-based microfluidics expanding the capabilities and the applicability of these devices. However, there is still room for improvement and we have indicated potential pathways for such improvement in our discussion of colorimetric readout. Meeting the requirements of WHO in the ASSURED Challenge [1] requires a large dose of creativity and even a larger dose of hard work, but μPADs can meet them. To
Acknowledgements
The authors would like to thank the funding agencies FAPESP (Grant No. 2011/13997-8), CNPq (Grant No. 131306/2013-8 and 205453/2014-7) for the scholarships and the financial support to the Instituto Nacional de Ciência e Tecnologia de Bioanalítica – INCTBio (FAPESP Grant Nr. 2008/57805-2/CNPq Grant Nr. 573672/2008-3), the Georgia Institute of Technology (Georgia Tech) and the State of Georgia, USA. We gratefully acknowledge the use of the laboratory facilities of Dr. Ubirajara Pereira Rodrigues
Giorgio Gianini Morbioli is a Ph.D. student in the School of Chemistry and Biochemistry at the Georgia Institute of Technology. He obtained his BSc (2013) and his MSc (2015) in Chemistry in the Institute of Chemistry of São Carlos at University of São Paulo, Brazil. His main research interests involve instrumentation design and development, microchip capillary electrophoresis and paper-based microfluidic devices.
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Giorgio Gianini Morbioli is a Ph.D. student in the School of Chemistry and Biochemistry at the Georgia Institute of Technology. He obtained his BSc (2013) and his MSc (2015) in Chemistry in the Institute of Chemistry of São Carlos at University of São Paulo, Brazil. His main research interests involve instrumentation design and development, microchip capillary electrophoresis and paper-based microfluidic devices.
Thiago Mazzu-Nascimento received his PhD in Chemistry (2016) from the Institute of Chemistry of São Carlos (IQSC) at University of São Paulo (USP), Brazil. He obtained his BSc (2011) in Biomedicine from Central Paulista University Center (UNICEP), Brazil. His main research interests involve: Laboratory diagnosis, metabolic disorders, epidemiology, biochemical assays, immunoassays and paper-based microfluidic devices.
Amanda Stockton is an Assistant Professor in Chemistry and Biochemistry at Georgia Tech. Prior to this appointment, she worked at the Jet Propulsion Laboratory, California Institute of Technology. Her Ph.D. work was with Richard Mathies at UC Berkeley after she earned a Master's degree in Chemistry from Brown University and a Bachelor's degree in Aerospace Engineering and Chemistry from the Massachusetts Institute of Technology. Dr. Stockton has extensive experience in the use of microcapillary electrophoresis, laser-induced fluorescence (μCE-LIF) to detect exceptionally low levels (sub-pptr) of organic molecules in astrobiologically relevant samples, including those from the Murchison meteorite, Atacama Desert, Saline Valley, Rio Tinto, etc. Her work also includes a significant field-work component, including the FELDSPAR project involving repeated expeditions to volcanic regions of Iceland as a Martian analog study.
Dr. Emanuel Carrilho is Professor of Chemistry at the University of São Paulo (USP), São Carlos, Brazil. He obtained his B.Sc. in Chemistry (1987) and M.Sc. in Analytical Chemistry (1990) from the University of São Paulo (USP) at São Carlos, Brazil. He obtained his Ph.D. at the Northeastern University under the mentoring of Professor Barry L. Karger, from The Barnett Institute, in Boston, MA, in 1997. Dr. Carrilho joined the faculty of the analytical chemistry program of the Institute of Chemistry of São Carlos, USP in 1998, and during 2007–2009 he was a visiting scholar at Harvard University in Professor George M. Whitesides' group. Dr. Carrilho's group has been working on the development of new bioanalytical methods covering the broad aspects of genomics, proteomics, metabolomics for human health and applied microbiology in the search for cancer biomarkers and neglected tropical diseases. The primary goal is to translate the targeted biomarkers research to microfluidic platforms with biosensors and microchip electrophoresis for point-of-care applications. Recently, is developing microfluidic applications for low-cost diagnostics for developing countries using paper-based analytical devices (μPADs), and developing new ultrasensitive contactless conductivity detection.