Microfluidic Paper Research Articles

An efficient protocol to use iron oxide nanoparticles in microfluidic paper device for arsenic detection

This method describes a rapid ecofriendly and affordable method for detecting arsenic in the water sample. The system designed works on the principle that involves generation of arsine due to reduction of arsenic by bare and cysteine capped iron oxide nanoparticles and its further reaction with silver nitrate present on the microfluidic paper analytical device (µPAD). Change in the color of µPAD from colorless to reddish brown is a result of reaction between arsine gas and silver nitrate, and is the detection criteria. The sample solution of arsenic was prepared in lemon juice to provide the required acidic environment for hydride generation. This proposed method has detection limit of 0.01?ppm (10?ppb) and 1?ppm for cysteine capped and bare iron oxide nanoparticles respectively. This is for the first time that iron oxide nanoparticles are being used for detection and reduction arsenic species in environmental sample. The same device can be used for on-site detection in an ecofriendly manner.

Roll-to-roll fabrication of integrated PDMS-paper microfluidics for nucleic acid amplification.

Microfluidic-based integrated molecular diagnostic systems, which are automated, sensitive, specific, user-friendly, robust, rapid, easy-to-use, and portable, can revolutionize future medicine. Current research and development largely relies on polydimethylsiloxane (PDMS) to fabricate microfluidic devices. Since the transition from the proof-of-principle phase to clinical studies requires a vast number of integrated microfluidic devices, there is a need for a high-volume manufacturing method of silicone-based microfluidics. Here we present the first roll-to-roll (R2R) thermal imprinting method to fabricate integrated PDMS-paper microfluidics for molecular diagnostics, which allows production of tens of thousands of replicates in an hour. In order to validate the replicated molecular diagnostic platforms, on-chip amplification of viral ribonucleic acid (RNA) with loop-mediated isothermal amplification (LAMP) was demonstrated. These low-cost, rapid and accurate molecular diagnostic platforms will generate a wide range of applications in preventive personalized medicine, global healthcare, agriculture, food, environment, water monitoring, and global biosecurity.

Short timescale wetting and penetration on porous sheets measured with ultrasound, direct absorption and contact angle.

In this study the short timescale penetration and spreading of liquids on porous sheets is investigated. Three measurement techniques are evaluated: ultrasonic liquid penetration measurement (ULP), contact angle measurement (CA) and scanning absorptiometry (SA). With each of these techniques liquid penetration as well as surface wetting can be measured. A quantitative comparison between the methods is carried out. For our studies we are using model liquids with tuneable surface tension, viscosity and surface energy which are the governing parameters for pore flow according to the Lucas–Washburn equation. Scanning absorptiometry turns out to be an adequate tool for direct measurement for liquid penetration. Ultrasonic liquid penetration showed a stable correlation (R2 = 0.70) to SA and thus also gives a suitable indication on the liquid penetration behaviour. Absorption of individual microliter drops measured in the CA instrument showed different results than the other two measurements. For characterisation of the wetting behaviour the measurement techniques gave substantially different results. We thus conclude that ULP and SA do not capture the wetting behaviour of liquids on paper in the same way as conventional contact angle measurement, it is unclear if their results are meaningful. Finally we are proposing two parameters indicating a combination of liquid penetration and wetting, the slope of the contact angle over time dθ/dt and a contact angle calculated from SA. These two parameters are moderately correlated, supporting the idea that they are indeed capturing a combination of liquid penetration and wetting. While our investigations are restricted to paper, we believe that the methods investigated here are generally applicable to study liquid absorption in thin porous media like microfluidic paper based analytical devices, thin porous storage media, membranes and the like. Our findings are highlighting the importance to have a match in timescale (time for penetration and wetting) and size scale (liquid amount supplied) between the testing method and the actual use case of the material, when analyzing wetting and penetration on porous materials.

Water-based alkyl ketene dimer ink for user-friendly patterning in paper microfluidics

We propose the use of water-based alkyl ketene dimer (AKD) ink for fast and user-friendly patterning of paper microfluidic devices either manually or using an inexpensive XY-plotter. The ink was produced by dissolving hydrophobic AKD in chloroform and emulsifying the solution in water. The emulsification was performed in a warm water bath, which led to an increased rate of the evaporation of chloroform. Subsequent cooling led to the final product, an aqueous suspension of fine AKD particles. The effects of surfactant and AKD concentrations, emulsification procedure, and cooling approach on final ink properties are presented, along with an optimized protocol for its formulation. This hydrophobic agent was applied onto paper using a plotter pen, after which the paper was heated to allow spreading of AKD molecules and chemical bonding with cellulose. A paper surface patterned with the ink (10 g L−1 AKD) yielded a contact angle of 135.6° for water. Unlike organic solvent-based solutions of AKD, this AKD ink does not require a fume hood for its use. Moreover, it is compatible with plastic patterning tools, due to the effective removal of chloroform in the production process to less than 2% of the total volume. Furthermore, this water-based ink is easy to prepare and use. Finally, the AKD ink can also be used for the fabrication of so-called selectively permeable barriers for use in paper microfluidic networks. These are barriers that stop the flow of water through paper, but are permeable to solvents with lower surface energies. We applied the AKD ink to confine and preconcentrate sample on paper, and demonstrated the use of this approach to achieve higher detection sensitivities in paper spray ionization-mass spectrometry (PSI-MS). Our patterning approach can be employed outside of the analytical lab or machine workshop for fast prototyping and small-scale production of paper-based analytical tools, for use in limited-resource labs or in the field.

Wavelengths and Lifetimes of Paper Autofluorescence: A Simple Substrate Screening Process to Enhance the Sensitivity of Fluorescence-Based Assays in Paper.

Porous media made of nitrocellulose and glass fiber are common "paper" substrates for lateral flow assays, microfluidic paper analytical devices and other point-of-care diagnostic assays. Such assays commonly use optical labels such as gold nanoparticles, latex beads, or fluorescent nanoparticles to visualize the presence of analytes. Fluorescent labels are commonly used in bioassays to enhance sensitivity, but autoluminescence of the paper substrate worsens signal-to-noise ratios of fluorescence-based assays. To date, there exists no systematic investigation of autoluminescence wavelengths or lifetimes of porous membranes used in lateral flow assays. In response, we quantified the autoluminescence of commonly used porous materials across the visible spectrum via excitation-emission spectroscopy and time-resolved fluorescence spectroscopy, and demonstrate that autoluminescence is solely due to autofluorescence with lifetimes of about 5 ns in the visible spectrum. Counterintuitively, we found that spectroscopy alone does not provide sufficient information to select candidate paper substrates for fluorophore-labeled assays. Therefore, we developed a simple quantitative framework to select a low-fluorescence substrate that minimizes both the overlap of paper and fluorophore emission spectra and the fluorescence intensity on an imaging system of interest (such as a gel imager). Use of this framework was shown to lower the limit of detection of an influenza A nucleoprotein immunoassay by over 50%. The tools developed in this manuscript enable assay developers to screen appropriate, low-fluorescence porous substrates and enhance the sensitivity of membrane-based fluorescence assays.

Flexible and disposable paper- and plastic-based gel micropads for nematode handling, imaging, and chemical testing.

Today, the area of point-of-care diagnostics is synonymous with paper microfluidics where cheap, disposable, and on-the-spot detection toolkits are being developed for a variety of chemical tests. In this work, we present a novel application of microfluidic paper-based analytical devices (μPADs) to study the behavior of a small model nematode, Caenorhabditis elegans. We describe schemes of μPAD fabrication on paper and plastic substrates where membranes are created in agarose and Pluronic gel. Methods are demonstrated for loading, visualizing, and transferring single and multiple nematodes. Using an anthelmintic drug, levamisole, we show that chemical testing on C. elegans is easily performed because of the open device structure. A custom program is written to automatically recognize individual worms on the μPADs and extract locomotion parameters in real-time. The combination of μPADs and the nematode tracking program provides a relatively low-cost, simple-to-fabricate imaging and screening assay (compared to standard agarose plates or polymeric microfluidic devices) for non-microfluidic, nematode laboratories.

An aptamer-based paper microfluidic device for the colorimetric determination of cocaine.

A method utilizing paper microfluidics coupled with gold nanoparticles and two anticocaine aptamers has been developed to detect seized cocaine samples. The ready-to-use format involves the use of a paper strip that produces a color change resulting from the salt-induced aggregation of gold nanoparticles producing a visible color change indicating the presence of the drug. This format is specific for the detection of cocaine. The visual LOD for the method was 2.5μg and the camera based LOD was 2.36μg. The operation of the device is easy and rapid, and does not require extensive training or instrumentation. All of the materials utilized in the device are safe and environmental friendly. This device should prove a useful tool for the screening of forensic samples.

μPAD Fluorescence Scattering Immunoagglutination Assay for Cancer Biomarkers from Blood and Serum.

A microfluidic paper analytical device (μPAD) was created for the sensitive quantification of cancer antigens, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), from human whole blood and serum, toward diagnosis and prognosis of colorectal cancer. Anti-CEA and anti-CA 19-9 antibodies were covalently linked to submicron, fluorescent polystyrene particles, loaded, and then dried in the center of the μPAD channel. CEA- or CA 19-9-spiked blood or serum samples were loaded to the inlet of μPAD, and subsequent immunoagglutination changed the fluorescent scatter signals upon ultraviolet (UV) excitation. The total assay time was about 1 min. Detection limits were 1 pg/mL for CEA and 0.1 U/mL for CA 19-9 from both 10% diluted blood and undiluted serum. The use of UV excitation and subsequent fluorescence scattering enabled much higher double-normalized intensities (up to 1.28-3.51, compared with 1.067 with the elastic Mie scatter detection), successful detection in the presence of blood or serum, and distinct multiplex assays with minimum cross-reaction of antibodies. The results with undiluted serum showed the larger dynamic range and smaller standard errors, which can be attributed to the presence of serum proteins, functioning as a stabilizer or a passivating protein for the particles within paper fibers.

Multi-Normalization and Interpolation Protocol to Improve Norovirus Immunoagglutination Assay from Paper Microfluidics with Smartphone Detection.

Norovirus (NoV) is one of the leading causes of acute gastroenteritis, affecting 685 million people per year around the world. The best preventive measure is to screen water for possible NoV contamination, not from infected humans, preferably using rapid and field-deployable diagnostic methods. While enzyme immunoassays (EIAs) can be used for such detection, the low infectious dose as well as the generally inferior sensitivity and low titer of available NoV antibodies render critical challenges in using EIAs toward NoV detection. In this work, we demonstrated smartphone-based Mie scatter detection of NoV with immunoagglutinated latex particles on paper microfluidic chips. Using only three different concentrations of anti-NoV-conjugated particles, we were able to construct a single standard curve that covered seven orders of magnitude of NoV antigen concentrations. Multiple normalization steps and interpolation procedures were developed to estimate the optimum amount of antibody-conjugated particles that matched to the target NoV concentration. A very low detection limit of 10 pg/mL was achieved without using any concentration or enrichment steps. This method can also be adapted for detection of any other virus pathogens whose antibodies possess low sensitivity and low antibody titer.

Modifier-Free Microfluidic Electrochemical Sensor for Heavy-Metal Detection.

Heavy-metal pollution poses severe threat to ecological systems and presents a great challenge for global sustainability. Portable point-of-care sensing platform for detection/monitoring of heavy-metal pollution in the environment is urgently demanded. Herein, a highly sensitive, robust, and low-cost microfluidic electrochemical carbon-based sensor (μCS) for detection of trace heavy metals is presented. The miniaturized μCS devices are based on a microfluidic paper channel combined with a novel three-dimensional layout with working and counter electrodes facing each other and analyte flowing along the microfluidic channel between these two electrodes. Pristine graphite foil free of any surface modifier is not only used as the electronically conductive pad but also directly employed as the working electrode for fabricating the μCS. The resulting simple and portable device was applied in Cd2+ and Pb2+ detection using square-wave anodic stripping voltammetry. Detection limits down to 1.2 μg/L for Cd2+ and 1.8 μg/L for Pb2+ can be achieved over the μCS. The μCS devices are also found to be highly robust, and 10 repetitive measurements with a single μCS device resulted to be highly reproducible.

Matrix-Assisted Three-Dimensional Printing of Cellulose Nanofibers for Paper Microfluidics.

A cellulose nanofiber (CNF), one of the most attractive green bioresources, was adopted for construction of microfluidic devices using matrix-assisted three-dimensional (3D) printing. CNF hydrogels can support structures printed using CAD design in a 3D hydrogel environment with the appropriate combination of rheological properties between the CNF hydrogel and ink materials. Amazingly, the structure printed freely in the bulky CNF hydrogels was able to retain its highly resolved 3D features in an ultrathin two-dimensional (2D) paper using a simple drying process. The dimensional change in the CNF hydrogels from 3D to 2D resulted from simple dehydration of the CNFs and provided transparent, stackable paper-based 3D channel devices. As a proof of principle, the rheological properties of the CNF hydrogels, the 3D structure of the ink, the formation of channels by evacuation of the ink, and the highly localized selectivity of the devices are described.

Recent developments in microfluidic paper-, cloth-, and thread-based electrochemical devices for analytical chemistry

AbstractThe attractive structural and mechanical properties of cellulose substrates (paper, cloth, and thread), including passive fluid transport, biocompatibility, durability, and flexibility, have attracted researchers in the past few decades to explore them as alternative microfluidic platforms. The incorporation of electrochemical (EC) sensing broadened their use for applications such as clinical diagnosis, pharmaceutical chemical analyses, food quality, and environmental monitoring. This article provides a review on the microfluidic devices constructed on paper, cloth, and thread substrates. It begins with an overview on paper-based microfluidic devices, followed by an in-depth review on the various applications of EC detection incorporated on paper-based microfluidic devices reported to date. The review on paper-based microfluidic devices attempts to convey a few perspective directions that cloth- and thread-based microfluidic devices may take in its development. Finally, the research efforts on the development and evaluation, as well as current limitations of cloth- and thread-based microfluidic devices are discussed. Microfluidic devices constructed on paper, cloth, and thread substrates are still at an early development stage (prototype) requiring several improvements in terms of fabrication, analytical techniques, and performance to become mature platforms that can be adapted and commercialized as real world products. However, they hold a promising potential as wearable devices.

Lab on paper chip integrated with Si@GNRs for electroanalysis of diazepam

We describe herein the fabrication of an electrochemical microfluidic paper based device (EμPAD) for the detection of diazepam, a sedative, anxiety-relieving and muscle-relaxing drug. To achieve it, silica coated gold nanorods (Si@GNRs) were synthesized and drop casted on an electrochemical microfluidic paper based device (EμPAD) for the detection of diazepam. The synthesized composites were characterized by recording its images in scanning electron microscope (SEM) and transmission electron microscope (TEM). The experimental results confirmed that Si@GNRs had good electrocatalytic activity towards diazepam. The modified paper based electrode showed a stable electrochemical response for diazepam in the concentration range of 3.5 nM to 3.5 mM. EμPAD offers many advantageous features such as facile approach, economical and have potential for commercialization. Si@GNRs modified EμPAD was also employed for determination of diazepam in spiked human urine samples. Reported facile lab paper approach integrated with Si@GNRs could be well applied for the determination of serum metabolites.

Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics

The most performing techniques enabling early diagnosis of infectious diseases rely on nucleic acid detection. Today, because of their high technicality and cost, nucleic acid amplification tests (NAAT) are of benefit only to a small fraction of developing countries population. By reducing costs, simplifying procedures and enabling multiplexing, paper microfluidics has the potential to considerably facilitate their accessibility. However, most of the studies performed in this area have not quit the lab. This letter brings NAAT on paper closer to the field, by using clinical samples and operating in a resource-limited setting. We first performed isothermal reverse transcription and Recombinase Polymerase Amplification (RT-RPA) of synthetic Ribonucleic Acid (RNA) of Ebola virus using paper microfluidics devices. We further applied this method in Guinea to detect the presence of Ebola virus in human sample RNA extracts, with minimal facilities (carry-on detection device and freeze-dried reagents on paper). RT-RPA results were available in few minutes and demonstrate a sensitivity of 90.0% compared to the gold-standard RT-PCR on a set of 43 patient samples. Furthermore, the realization of a nine-spot multilayered device achieving the parallel detection of three distinct RNA sequences opens a route toward the detection of multiple viral strains or pathogens.

Paper-based microfluidic devices: A complex low-cost material in high-tech applications

Abstract

A novel direct and cost effective method for fabricating paper-based microfluidic device by commercial eye pencil and its application for determining simultaneous calcium and magnesium

This article presents a novel, simple and low-cost method for the fabrication of microfluidic paper using sticker templates with specific patterns and a highly accessible waterproof eye pencil. The entire fabrication process could be finished in 10min by heating the patterned paper at 150° without using complicated instruments. Important parameters in the fabrication of microfluidic paper such as type of paper, heating time, melting temperature, reagent and sample volume were optimized. To verify the applicability of the fabricated microfluidic paper, colorimetric assays were performed for simultaneous analysis of calcium and magnesium by single and multiple indicators and water hardness was determined. The limit of detection is 8.3mgL−1 and 1.0mgL−1, and the relative standard deviation is 8.3% and 5.9% for calcium and magnesium, respectively. A linear range from 10 to 100mgL−1 for calcium and two linear ranges of 4–20mgL−1 and 20–100mgL−1 for magnesium were obtained in the paper based microfluidic device. The concentration of calcium and magnesium were successfully determined in tap, river, mineral and household purifier water samples.

Time-Dependent Model for Fluid Flow in Porous Materials with Multiple Pore Sizes.

An understanding of fluid transport through porous materials is critical for the development of lateral flow assays and analytical devices based on paper microfluidics. Models of fluid transport within porous materials often assume a single capillary pressure and permeability value for the material, implying that the material comprises a single pore size and that the porous material is fully saturated behind the visible wetted front. As a result, current models can lead to inaccuracies when modeling transport over long distances and/or times. A new transport model is presented that incorporates a range of pore sizes to more accurately predict the capillary transport of fluid in porous materials. The model effectively predicts the time-dependent saturation of rectangular strips of Whatman filter no. 1 paper using the manufacturer's data, published pore-size distribution measurements, and the fluid's properties.

March 2017: This Month in JoVE: Human Embryo Vitrification, Paper Microfluidic Devices, Virtual Hand/Face Illusions, and Quantification of Amyloid-Beta for Alzheimer's Diagnosis

Here's a look at what's coming up in the March 2017 issue of JoVE: The Journal of Visualized Experiments. As the start of spring brings new life, we begin the month investigating exciting new developments in embryo storage for in-vitro fertility treatment. In JoVE Developmental Biology, our authors present a modified technique for vitrification and preservation of human embryos. This aseptic technique uses all FDA-compliant materials including ionomeric resin embryo straws and sterile flexipettes, and allows fast, safe, and repeatable embryo storage independent of operator variation. In addition, with a greater than 95% embryo survival-rate the method contributes to excellent pregnancy outcomes. Paper cards...printers, it's all so 1990's, right? Well, our next video highlight might make you reconsider. In JoVE Bioengineering this month, our authors reveal 3-dimensional paper microfluidic devices for immunoassays. By patterning paper with hydrophobic barriers, the transfer of fluids can be controlled and directed. Stacking multiple layers of patterned paper creates sophisticated 3-D microfluidic networks that can be utilized in cheap and simple bioanalytical assays, such as the detection of Human Chorionic Gonadotriphin, or hCG - the pregnancy hormone. So don't throw out those old Valentine's Day cards yet - upcycle! In JoVE Medicine this month, our authors reveal an accurate procedure for quantifying the plaque-forming amyloid-beta protein in cerebral spinal fluid to provide diagnoses in suspected Alzheimer's cases. Current diagnosis is mostly based on mental state and cognitive testing, with some studies beginning to examine amyloid-beta levels, but this is hampered by variability and reproducibility issues. Here, our authors use a combination of solid-phase extraction and liquid chromatography tandem mass spectrometry to quantify amyloid-beta levels to a high degree of accuracy, quantifying the deposition of the protein in plaques in the brain and enhancing the power for accurate diagnoses. Our final video highlight this month comes from JoVE Behavior, and showcases a new technique to create human body illusions. Here, our authors demonstrate a computer-based virtual hand and face that move in synchrony with the operator's action, creating an illusion that the model is an extension of the participant's own body. This is a useful tool in the study of self-representation, and is an advance over traditional studies requiring demonstrators to mimic actions on participants in time with actions performed on a physical model body part. Ah, just one small step until I realize my dream of becoming a super-fast blue hedgehog. You've just had a sneak peek of the March 2017 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Quantum Dot-Based Molecularly Imprinted Polymers on Three-Dimensional Origami Paper Microfluidic Chip for Fluorescence Detection of Phycocyanin.

In this work, we developed a novel strategy using fluorescent quantum dots (QDs) combined with molecularly imprinted polymers (MIPs) on three-dimensional (3D) origami paper-based microfluidic devices for specific recognition and sensitive detection of phycocyanin. This method can realize the liquid phase of QDs@MIPs being transferred to the solid-phase paper base and achieve easy portability for the analysis. Under optimal conditions, we successfully demonstrated the proposed paper@QDs@MIPs 3D microfluidic chip for the sensitive and selective detection of phycocyanin protein target in a simple and robust manner. Our results revealed that the method exhibited a dynamic response to phycocyanin in the range of 10-50 mg/L with a limit of detection of 2 mg/L. Importantly, this device could provide quantitative information very conveniently and show great potential to be further extended to the detection of other proteins or biomarkers for environmental and food safety research.

Dual-Functional Carbon Dots Pattern on Paper Chips for Fe3+ and Ferritin Analysis in Whole Blood.

Though microfluidic paper analytical devices (μPADs) have attracted paramounting attentions in recent years as promising devices for low cost point-of-care tests, their real applications for blood analysis are still challenged by integrating sample preparation with different detection modes on a same μPAD. Herein, we developed a novel μPAD, which well coupled automatic serum extraction with reliable dual mode iron health tests: fluorescent analysis for Fe3+ and colorimetric ELISA for ferritin. All these functions are made available by in situ carbon dots (CDs) and AuNPs sequential patterning techniques. For CDs immobilization, hydrothermal reaction was taken on paper, to which a patterned through-hole polydimethylsiloxane (PDMS) mask was applied. None fluorescence CDs (nF-CDs) were generated on exposed regions, while the fluorescent CDs (F-CDs) were generated simultaneously on covered regions. Sensitive serum iron quantification was realized on the F-CDs modified regions, where Fe3+ ion can selectively quench the fluorescence of F-CDs. For AuNPs immobilization, electroless plating was taken on nF-CDs modified regions. The resulting AuNPs on nF-CDs layer on one hand triggered the coagulation of blood cells and thus led to the longest ever wicking distance for serum separation, on the other hand facilitated colorimetric enzyme linked immunosorbent assay (ELISA) for detection of serum ferritin. Combining the two readings, the μPAD can provide reliable measurement for serum iron and serum ferritin in whole blood. Furthermore, as CDs and AuNPs modified μPAD has the features of easy handling, low-cost, lightweight, and disposability, it is accounting for a promising prototype for whole blood point-of-care analysis.