Saturation Equation: An Analytical Expression for Partial Saturation during Wicking Flow in Paper Microfluidic Channels.

The design and fabrication of paper-based microfluidic devices is critically dependent on modeling fluid flow through porous paper membranes. A commonly observed phenomenon is partial saturation, i.e., regions of the paper membrane not being filled completely due to pores of different sizes. The most comprehensive model to date of partial saturation during wicking flow in paper is the Richards equation. However, the solution to the Richards equation requires numerical solvers like COMSOL, which makes it largely inaccessible to the paper microfluidics and lateral flow assay community. There is therefore a need for a simple and appropriate model of partial saturation in paper membranes, easily usable by the wider research community. In the current work, we present an approach to model paper membranes as a bundle of parallel capillaries whose radii follow a two-parameter log-normal distribution. Application of the Washburn equation to the bundle provides a distribution of fluid fronts, which can be used to calculate saturation. Using this approach, we developed the "saturation equation"─an explicit analytical expression to calculate saturation as a function of space and time in 1D wicking flow. Experimentally obtained data for spatiotemporal saturation for four different paper materials were fit to this analytical model to obtain parameters for each material. Results obtained from this analytical model match well with both experimental data and numerical results obtained from the Richards equation. The availability of an explicit analytical expression for partial saturation will enable incorporation of the critical phenomenon of partial saturation in the design of paper microfluidic devices.

Tuberculosis Diagnosis Using Isothermal Nucleic Acid Amplification in a Paper-and-Plastic Device.

Nucleic acid (DNA/RNA) amplification technologies are indispensable for applications like disease diagnostics, forensics, epidemiology, evolutionary biology, vaccine development, and therapeutics. While polymerase chain reaction (PCR) has deeply penetrated the abovementioned fields and has been commercially successful, two major common disadvantages are exorbitant costs of associated equipment, which create concerning roadblocks in terms of affordability and accessibility. This work describes development of an inexpensive, portable, easy-to-use and deliverable-to-end-users, nucleic acid amplification technology for infectious disease diagnosis. The device uses loop-mediated isothermal amplification (LAMP) and cell phone-based fluorescence imaging to enable nucleic acid amplification and detection. A regular lab incubator and a custom-made low-cost imaging box are the only two additional equipment required for testing. Material cost for a 12-test zone device was $0.88, and cost of reagents per reaction was $0.43. First successful application of the device was demonstrated for tuberculosis diagnosis with clinical sensitivity of 100% and clinical specificity of 68.75% for testing of 30 clinical patient samples.

In situ synthesis of reagents in paper-based analytical devices using paper stacking.

This article demonstrates a technique for the in situ synthesis of an insoluble analytical reagent in paper analytical devices, using paper stacking. Previously, our group has demonstrated that stacking a fast-wicking paper membrane on top of a slow-wicking paper membrane containing dried reagents enables the uniform rehydration of the dried reagents over large areas. This technique is utilized here to fabricate distance-based sweat chloride quantification strips, which requires the synthesis of insoluble silver chromate as an analytical reagent in paper. The in situ generation of silver chromate for sweat chloride detection was previously accomplished by manually dipping a hydrophobically patterned paper channel into multiple precursor solutions with intermittent washing and drying. Compared to the previous technique, the stacking method obviates the need for (i) patterning hydrophobic barriers in paper for creation of flow channels, and (ii) multiple dipping steps that need large reagent volumes. The method is amenable to large scale manufacturing as the insoluble reagent can be synthesized uniformly over large paper areas and can then be cut into multiple sensing strips. The developed sensor has a limit of detection of ∼0.3 mM and a wide linear dynamic range of 0-120 mM for the detection of chloride ions, which enables the diagnosis of cystic fibrosis, characterized by sweat chloride levels greater than 60 mM. This simple technique of in situ synthesis of insoluble analytical reagents in paper could enable expanding the range of analytical chemistries that may be performed in paper-based analytical devices.

A large-volume sputum dry storage and transportation device for molecular and culture-based diagnosis of tuberculosis.

Technologies for preservation of specimens in the absence of cold chains are essential for optimum utilization of existing laboratory services in the developing world. We present a prototype called specimen transportation tube (SPECTRA-tube) for the collection, exposure-free drying, ambient transportation, and liquid state recovery of large-volume (>1 mL) specimens. Specimens introduced into the SPECTRA-tube are dried in glass fiber membranes, which are critical for efficient liquid-state sample recovery by rehydration and centrifugation. SPECTRA-tube is demonstrated for the dry storage of sputum for tuberculosis detection. Mycobacterium smegmatis (Msm)-spiked mock sputum dried in a native Standard 17 glass fiber was stable for molecular testing after 10 day storage at 45 °C and for culture testing after 10- and 5-day storage at 37 °C and 45 °C, respectively. Compatibility with human sputum storage was demonstrated by dry storing 1.2 mL Mycobacterium bovis-spiked human sputum in a SPECTRA-tube for 5 days at room temperature. We have thus demonstrated the first workflow for dry storage of sputum followed by molecular and culture testing. Compared to existing specimen dry storage technologies, SPECTRA-tube significantly increases the volume of liquid specimens that can be transported in the dry state and enables the recovery of the entire sample in the liquid state, rendering it compatible with conventional downstream analysis methods.

Paper-microfluidic signal-enhanced immunoassays.

Over the past decade, paper-based microfluidic devices have become popular for their simplicity and ability to conduct diagnostic tests at a low cost. An important class of diagnostic assays that paper-based analytical devices have been used for is immunoassays. The lateral flow immunoassay (LFIA), of which the home pregnancy test is the most prominent example, has been one of the most commercially successful membrane-based diagnostic tests. Yet, the analytical sensitivity of LFIAs is lower than the corresponding laboratory technique called ELISA (enzyme-linked immunoassay). As a consequence, traditional LFIAs fail to deliver on the promise of bedside diagnostic testing for many applications. Recognizing this shortcoming, several new developments have been made by researchers to enhance the sensitivity of membrane-based immunoassays. In this chapter, we present the various strategies that have been employed to this end. In the end, we present a brief SWOT analysis to guide future work in this area.

Unreacted Labeled PCR Primers Inhibit the Signal in a Nucleic Acid Lateral Flow Assay as Elucidated by a Transport Reaction Model.

Factors that affect the performance of the nucleic acid lateral flow assay (NALFA) have not been well studied. In this work, we identify two important phenomena that negatively affect signal intensities during the detection of PCR products using NALFA: (i) the presence of unreacted PCR primers, and (ii) the presence of excess PCR amplicons. This is the first report that highlights the negative effect of unreacted PCR primers on NALFA. The negative effect of excess amplicons, while not explicitly reported for NALFAs, emanates from an identical phenomenon in lateral flow immunoassays known as the "hook effect". We show that the above effects may be alleviated by increasing the concentration of capture antibodies at the test line and the concentration of reporter moieties (gold nanoparticles). To demonstrate these, we utilized a PCR assay in which both primers were end-labeled, to generate dually end-labeled (bi-labeled) PCR amplicons of 230 bp length. To provide mechanistic understanding of these phenomena, we present the first transport-reaction model of NALFA, the results of which qualitatively matched all observed phenomena. Based on these results, we provide recommendations for the optimal design of PCR for NALFA detection.

Barrier-Free Microfluidic Paper Analytical Devices for Multiplex Colorimetric Detection of Analytes.

In recent years, microfluidic paper analytical devices (µPADs) have been extensively utilized to conduct multiplex colorimetric assays. Despite their simple and user-friendly operation, the need for patterning paper with wax or other physical barriers to create flow channels makes large-scale manufacturing cumbersome. Moreover, convection of rehydrated reagents in the test zones leads to nonuniform colorimetric signals, which makes quantification difficult. To overcome these challenges, we present a device called a barrier-free µPAD (BF-µPAD) that consists of a stack of two paper membranes having different wicking rates-the top layer acting as a fluid distributing layer and the bottom layer containing reagents for colorimetric detection. Multiple analytes can be detected using this assembly without the need to pattern either layer with wax or other barriers. In one embodiment, a device is capable of delivering the sample fluid to 20 distinct dried reagent spots stored on an 8 cm × 2 cm membrane in as few as 30 s. The multiplexing feature of the BF-µPAD is demonstrated for colorimetric detection of salivary thiocyanate, protein, glucose, and nitrite. Most importantly, the device improves the limit of detection of colorimetric assays performed on conventional µPADs by more than 3.5×. To understand fluid imbibition in the paper assembly, the device geometry is modeled in COMSOL Multiphysics using the Richards equation; the results obtained provide insights into the nonintuitive flow pattern producing perfectly uniform signals in the barrier-free assembly.

Modeling-Guided Design of Paper Microfluidic Networks: A Case Study of Sequential Fluid Delivery.

Paper-based microfluidic devices are popular for their ability to automate multistep assays for chemical or biological sensing at a low cost, but the design of paper microfluidic networks has largely relied on experimental trial and error. A few mathematical models of flow through paper microfluidic devices have been developed and have succeeded in explaining experimental flow behavior. However, the reverse engineering problem of designing complex paper networks guided by appropriate mathematical models is largely unsolved. In this article, we demonstrate that a two-dimensional paper network (2DPN) designed to sequentially deliver three fluids to a test zone on the device can be computationally designed and experimentally implemented without experimental trial and error. This was accomplished by three new developments in modeling flow through paper networks: (i) coupling of the Richards equation of flow through porous media to the species transport equation, (ii) modeling flow through assemblies of multiple paper materials (test membrane and wicking pad), and (iii) incorporating limited-volume fluid sources. We demonstrate the application of this model in the optimal design of a paper-based signal-enhanced immunoassay for a malaria protein, PfHRP2. This work lays the foundation for the development of a computational design toolbox to aid in the design of paper microfluidic networks.

A stoichiometric and pseudo kinetic model of loop mediated isothermal amplification.

Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications, enabling amplification of both DNA and RNA (with the assistance of reverse transcriptase). The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays.

Paper Stacks for Uniform Rehydration of Dried Reagents in Paper Microfluidic Devices.

Spatially uniform reconstitution of dried reagents is critical to the function of paper microfluidic devices. Advancing fluid fronts in paper microfluidic devices drive (convect) and concentrate rehydrated reagents to the edges, causing steep chemical gradients and imperfect mixing. This largely unsolved problem in paper microfluidics is exacerbated by increasing device dimensions. In this article, we demonstrate that mixing of dried reagents with a rehydrating fluid in paper microfluidics may be significantly enhanced by stacking paper layers having different wicking rates. Compared to single-layer paper membranes, stacking reduced the "non-reactive area", i.e. area in which the reconstituted reagents did not interact with the rehydrating fluid, by as much as 97% in large (8 cm × 2 cm) paper membranes. A paper stack was designed to collect ~0.9 ml liquid sample and uniformly mix it with dried reagents. Applications of this technology are demonstrated in two areas: (i) collection and dry storage of sputum samples for tuberculosis testing, and (ii) salivary glucose detection using an enzymatic assay and colorimetric readout. Maximizing the interaction of liquids with dried reagents is central to enhancing the performance of all paper microfluidic devices; this technique is therefore likely to find important applications in paper microfluidics.

A modular paper-and-plastic device for tuberculosis nucleic acid amplification testing in limited-resource settings.

We present a prototype for conducting rapid, inexpensive and point-of-care-compatible nucleic acid amplification tests (NAATs) for tuberculosis (TB). The fluorescent isothermal paper-and-plastic NAAT (FLIPP-NAAT) uses paper-based loop mediated isothermal amplification (LAMP) for DNA detection. The cost of materials required to build a 12-test-zone device is $0.88 and the cost of reagents per reaction is $0.43. An inexpensive imaging platform enables filter-free fluorescence detection of amplified DNA using a cell-phone camera. FLIPP-NAAT can be operated by an untrained user and only requires a regular laboratory incubator as ancillary equipment. All reagents can be dry-stored in the device, facilitating storage and transportation without cold chains. The device design is modular and the assay demonstrated high specificity to Mycobacterium tuberculosis (Mtb), analytical sensitivity of the order of 10 copies of Mtb gDNA, and tolerance to complex samples. The clinical sensitivity and specificity of sputum-based FLIPP NAAT tests were 100% (zero false negatives) and 68.75% (five false positives), respectively (N = 30), using Xpert MTB/RIF assay as the reference standard. FLIPP-NAAT has the potential to provide affordable and accessible molecular diagnostics for TB in low- and middle-income countries, when used in conjunction with an appropriate sample preparation technique. Although demonstrated for the detection of TB, FLIPP-NAAT is a platform technology for amplification of any nucleic acid sequence.

Experimental Measurement of Parameters Governing Flow Rates and Partial Saturation in Paper-Based Microfluidic Devices.

Paper-based microfluidic devices are rapidly becoming popular as a platform for developing point-of-care medical diagnostic tests. However, the design of these devices largely relies on trial and error, owing to a lack of proper understanding of fluid flow through porous membranes. Any porous material having pores of multiple sizes contains partially saturated regions, i.e., regions where less than 100% of the pores are filled with fluid. The capillary pressure and permeability of the material change as a function of the extent of saturation. Although methods to measure these relationships have been developed in other fields of study, these methods have not yet been adapted for paper for use by the larger community of analytical chemists. In the current work, we present a set of experimental methods that can be used to measure the relationships between capillary pressure, permeability, and saturation for any commercially available paper membrane. These experiments can be performed using commonly available lab instruments. We further demonstrate the use of the Richards equation in modeling imbibition into two-dimensional paper networks, thus adding new capability to the field. Predictions of spatiotemporal saturation from the model were in strong agreement with experimental measurements. To make these methods readily accessible to a wide community of chemists, biologists, and clinicians, we present the first report of a simple protocol to measure the flow rates considering the effect of partial saturation. Use of this protocol could drastically reduce the trial and error involved in designing paper-based microfluidic devices.

Paper-based nucleic acid amplification tests for point-of-care diagnostics.

There has been a recent resurgence in the use of paper as a substrate for developing point-of-care medical diagnostic tests, possibly triggered by expiring patents published in the 1990s. A hallmark of this resurgence has been the development of advanced shapes and structures made from paper to conduct multi-step fluidic operations using the wicking action of porous materials. Such devices indicate a distinct improvement over lateral flow immunoassays, which are restricted to conducting one-step operations. New developments in paper-based diagnostic devices have triggered interest in the development of paper-based point-of-care nucleic acid amplification tests (NAATs). NAATs can identify extremely low levels of specific nucleic acid sequences from clinical samples and are the most sensitive of all available tests for infectious disease diagnosis. Because traditional PCR-based NAATs require expensive instruments, the development of portable paper-based NAAT's has become an exciting field of research. This article aims to review and analyse the current state of development of paper-based NAATs. We project paper-based NAATs as miniaturized chemical processes and shed light on various schemes of operation used for converting the multiple steps of the chemical processes into paper microfluidic devices. We conclude by elaborating on the challenges that must be overcome in the near future so that progress can be made towards the development of fully functional and commercial paper-based NAATs.

Investigations into the cancer stem cell niche using in-vitro 3-D tumor models and microfluidics.

The concept of Cancer Stem Cells (CSCs) and the CSC Niche/Tumor Microenvironment (TME) as the central driving force behind tumor progression and maintenance has garnered much attention in recent years. Concomitantly, the widespread adoption of 3D tissue models, organotypic co-cultures, and the revolutionary microfluidic technology has resulted in a plethora of ground-breaking fundamental discoveries and has enabled investigations which were previously unfeasible. A large number of existing review papers concern themselves with either a broad look at the TME and CSC Niche, or on the studies undertaken on a particular niche component alone. In this article, we attempt to bring out a harmonic, expansive look at the concept of CSCs, the TME, and the various advancements in answering key biological queries enabled by these emerging new technologies. Our primary goal is to present a fundamental understanding of CSCs, as well as the CSC niche, and elucidate note-worthy examples of investigations being carried out with regard to each of the major TME components, along with our insights into the potential for further research. We hope that this serves as an impetus to new, as well as existing researchers in this area, to gain fresh perspectives on the CSC niche, as well as provide them with a glimpse at the kind of progress being made using 3D tumor models and microfluidic devices.

A rapid, instrument-free, sample-to-result nucleic acid amplification test.

The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).

Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis.

We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest existing isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in <30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.

A versatile valving toolkit for automating fluidic operations in paper microfluidic devices.

Failure to utilize valving and automation techniques has restricted the complexity of fluidic operations that can be performed in paper microfluidic devices. We developed a toolkit of paper microfluidic valves and methods for automatic valve actuation using movable paper strips and fluid-triggered expanding elements. To the best of our knowledge, this is the first functional demonstration of this valving strategy in paper microfluidics. After introduction of fluids on devices, valves can actuate automatically after a) a certain period of time, or b) the passage of a certain volume of fluid. Timing of valve actuation can be tuned with greater than 8.5% accuracy by changing lengths of timing wicks, and we present timed on-valves, off-valves, and diversion (channel-switching) valves. The actuators require ~30 µl fluid to actuate and the time required to switch from one state to another ranges from ~5 s for short to ~50 s for longer wicks. For volume-metered actuation, the size of a metering pad can be adjusted to tune actuation volume, and we present two methods - both methods can achieve greater than 9% accuracy. Finally, we demonstrate the use of these valves in a device that conducts a multi-step assay for the detection of the malaria protein PfHRP2. Although slightly more complex than devices that do not have moving parts, this valving and automation toolkit considerably expands the capabilities of paper microfluidic devices. Components of this toolkit can be used to conduct arbitrarily complex, multi-step fluidic operations on paper-based devices, as demonstrated in the malaria assay device.

Swab sample transfer for point-of-care diagnostics: characterization of swab types and manual agitation methods.

BACKGROUND: The global need for disease detection and control has increased effort to engineer point-of-care (POC) tests that are simple, robust, affordable, and non-instrumented. In many POC tests, sample collection involves swabbing the site (e.g., nose, skin), agitating the swab in a fluid to release the sample, and transferring the fluid to a device for analysis. Poor performance in sample transfer can reduce sensitivity and reproducibility. METHODS: In this study, we compared bacterial release efficiency of seven swab types using manual-agitation methods typical of POC devices. Transfer efficiency was measured using quantitative PCR (qPCR) for Staphylococcus aureus under conditions representing a range of sampling scenarios: 1) spiking low-volume samples onto the swab, 2) submerging the swab in excess-volume samples, and 3) swabbing dried sample from a surface. RESULTS: Excess-volume samples gave the expected recovery for most swabs (based on tip fluid capacity); a polyurethane swab showed enhanced recovery, suggesting an ability to accumulate organisms during sampling. Dry samples led to recovery of ∼20-30% for all swabs tested, suggesting that swab structure and volume is less important when organisms are applied to the outer swab surface. Low-volume samples led to the widest range of transfer efficiencies between swab types. Rayon swabs (63 µL capacity) performed well for excess-volume samples, but showed poor recovery for low-volume samples. Nylon (100 µL) and polyester swabs (27 µL) showed intermediate recovery for low-volume and excess-volume samples. Polyurethane swabs (16 µL) showed excellent recovery for all sample types. This work demonstrates that swab transfer efficiency can be affected by swab material, structure, and fluid capacity and details of the sample. Results and quantitative analysis methods from this study will assist POC assay developers in selecting appropriate swab types and transfer methods.

Tunable-delay shunts for paper microfluidic devices.

We demonstrate a novel method for controlling fluid flow in paper-based devices. The method delays fluid progress through a porous channel by diverting fluid into an absorbent pad-based shunt placed into contact with the channel. Parameters to control the delay include the length and the thickness of the shunt. Using this method, reproducible delays ranging from 3 to 20 min were achieved. A simple electrical circuit model was presented and used to predict the delays in a system. Results from the model showed good agreement with experimental observations. Finally, the shunts were used for the sequential delivery of fluids to a detection zone in a point-of-care compatible folding card device using biochemical reagents for the amplified detection of the malaria protein PfHRP2.