Microfluidic Paper-based Analytical Devices Research Articles

Detecting Glucose Levels in Blood Plasma and Artificial Tear by Au(I) Complex on the Carbopol Polymer: A Microfluidic Paper-Based Method.

We report on a selective paper-based method and a microfluidic paper-based analytical device (μPAD) for the detection of human plasma glucose and tear glucose using carbopol polymer-encapsulated Au(I) complex (AuC2C6H4OMe)2(Ph2P(C6H4)3PPh2), (B5). To the best of our knowledge, this demonstrates for the first time the glucose sensing based on dual emission, i.e., fluorescence and phosphorescence, of a single type molecule on the carbopol polymer. Upon addition of human blood treated with anticoagulants to μPADs, plasma is separated from the blood and flows into the response region of the μPADs to react with carbopol polymer-encapsulated B5, in which the ratiometric luminescence is analyzed. The plasma glucose concentration can be quantitively detected at 1.0–50.0 mM on paper, and tear glucose can be detected at 0.1–4.0 mM on μPADs. Owing to the structural design, this device has superior ratiometric changes of dual emission over other Au(I) complexes for signal transduction. The encapsulation of carbopol polymer also offers long-term storage stability. In tear measurement, carbopol polymer is not only used to encapsulate enzyme to remain the enzyme’s activity, but also played as a glue (or media) to connect microfluidic channel and response region. This further improves the sensitivity and limit of detection for glucose. Moreover, this sensor provides a faster response time, a wider range for glucose sensing than reported previously, and no statistical difference of the data from a commercial glucometer, allowing for practical diagnosis of diabetes and healthy individuals.

Triple-Indicator-Based Multidimensional Colorimetric Sensing Platform for Heavy Metal Ion Detections.

Heavy metals are highly toxic at trace levels and their pollution has shown great threat to the environment and public health worldwide where current detection methods require expensive instrumentation and laborious operation, which can only be accomplished in centralized laboratories. Herein, we report a low-cost, paper-based microfluidic analytical device (μPAD) for facile, portable, and disposable monitoring of mercury, lead, chromium, nickel, copper, and iron ions. Triple indicators or ligands that contain ions or molecules are preloaded on the μPADs and upon addition of a metal ion, the colorimetric indicators will elicit color changes observed by the naked eyes. The color features were quantitatively analyzed in a three-dimensional space of red, green, and blue or the RGB-space using digital imaging and color calibration techniques. The sensing platform offers higher accuracy for cross references, and is capable of simultaneous detection and discrimination of different metal ions in even real water samples. It demonstrates great potential for semiquantitative and even qualitative analysis with a sensitivity below the safe limit concentrations, and a controlled error range.

Uncovering the Formation of Color Gradients for Glucose Colorimetric Assays on Microfluidic Paper-Based Analytical Devices by Mass Spectrometry Imaging.

This study describes the use of mass spectrometry imaging with matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) to understand the color gradient generation commonly seen in microfluidic paper-based analytical devices (μPADs). The formation of color gradients significantly impacts assay sensitivity and reproducibility with μPADs but the mechanism for formation is poorly understood. The glucose enzymatic assay using potassium iodide (KI) as a chromogenic agent was selected to investigate the color gradient generated across a detection spot. Colorimetric measurements revealed that the relative standard deviation for the recorded pixel intensities ranged between 34 and 40%, compromising the analytical reliability. While a variety of hypotheses have been generated to explain this phenomenon, few studies have attempted to elucidate the mechanisms associated with its formation. Mass spectrometry imaging using MALDI and DESI was applied to understand the nonuniform color distribution on the detection zone. MALDI experiments were first explored to monitor the spatial distribution of the glucose oxidase and horseradish peroxidase mixture, before and after lateral flow assay with and without KI. MALDI(+)-TOF data revealed uniform enzyme distribution on the detection spots. On the other hand, after the complete assay DESI(-) measurements revealed a heterogeneous shape indicating the presence of iodide and triiodide ions at the zone edge. The reaction product (I3-) is transported by lateral flow toward the zone edge, generating the color gradient. Mass spectrometry imaging has been used for the first time to prove that color gradient forms as result of the mobility small molecules and not the enzyme distribution on μPAD surface.

High-throughput deposition of chemical reagents via pen-plotting technique for microfluidic paper-based analytical devices

The deposition of chemical reagent inks on paper is a crucial step in the development and fabrication of microfluidic paper-based analytical devices (μPADs). A pen-plotting approach, delivering chemical ink deposition using technical pens filled with reagents and inserted into a desktop electronic plotter, is shown herein to be a versatile, low-cost, simple, rapid, reproducible, and high-throughput solution. The volume of the deposited ink was quantified gravimetrically, confirming that nanoliter volumes of reagents can be deposited reproducibly (e.g. 7.55 ± 0.37 nL/mm for a plotting speed of 10 cm/s) in detection zones of μPADs, typically spatially defined using wax printing. This approach was further investigated with regard to deposition of reagents in different geometrical forms (circular and linear), so demonstrating its applicability for preparation of μPADs with flexible design and application. By adjusting the plotting speed for linear deposition, lines with a relatively large range of widths (≈628–1192 μm) were created. Circular deposition was also demonstrated via delivery of reagents within wax printed circular fluidic barriers of a range of diameters (inner diameter = 1.5–7 mm). These capabilities were practically demonstrated via the fabrication of μPADs, based upon differing detection principles for determination of aluminum in natural waters using Morin as the fluorescent reagent. Traditional μPADs based on digital image colorimetry (DIC) were produced using circular deposition, whilst distance-based μPADs exploited linear deposition. Both types of μPADs developed using this method showed excellent precision for determination of trace concentrations of aluminium (average RSDs = 3.38% and 6.45%, and LODs = 0.5 ng (0.25 ppm) and 2 ng (0.5 ppm), for traditional and distance-based detection, respectively).

A novel combination of quick response code and microfluidic paper-based analytical devices for rapid and quantitative detection.

Nowadays, there is great interest in the use of microfluidic paper-based analytical devices (μPADs) for low-cost diagnostics. In most cases, the test equipment is needed. Here we report a new type of device-independent detection method based on timer-paper-based analytical devices, which can be tested by smartphone, and its application for cholesterol detection. The Quick Response code was designed as the timer component of this device. Wax printing method was employed to print the pattern on filter paper. The color and enzyme reagents have been immobilized in the hydrophilic channel to complete the colorimetric detection for cholesterol. Under laminar flow conditions of the cellulose network, the liquid volume of detection zone has been quantified by monitoring the fluid residence time on different area of the timer-paper-based devices. The precise monitoring of detection time can promote the accuracy of colorimetric detection. This is very important for the quantitative detection of paper-based analytical devices. One significant outcome of this report is that simple and accurate timer can be used for detection process self-clocking. The factors of total detection time have been investigated. The linear range of the calibration curve was 3.0 ~ 6.0mmolL-1, with correlation coefficient of 0.9956. With the characteristic of easy to use, low cost and accurate monitoring of detection time, this kind of timer-paper-based devices can be applied to cholesterol or other substances rapid detection.

Graphene nanocomposites modified electrochemical aptamer sensor for rapid and highly sensitive detection of prostate specific antigen

Prostate specific antigen (PSA) is a widely used marker for the diagnosis of prostate cancer, and the increasing attention has been attracted on the development of rapid assay using biosensing technology. However, it remains challenging for the sensitive and selective detection of PSA in clinical samples. Here, we report a label-free microfluidic paper-based analytical device for highly sensitive electrochemical detection of PSA. The paper device was fabricated with wax printing to generate hydrophobic and hydrophilic layers for the construction of microfluidic channel, followed by screen-printing of three electrodes including working, counter and reference electrode. Gold nanoparticles (AuNPs)/reduced graphene oxide (rGO)/thionine (THI) nano composites were synthesized and characterized, which were coated onto working electrodes for the immobilization of DNA aptamer probe. THI servers as the electrochemical mediator to transduce the biological recognition between DNA aptamer and PSA, and the excellent conductivity of AuNPs and rGO also play a significant role of electron transfer, leading to a sensitive detection for PSA, able to detect PSA as low as 10 pg mL−1, with a linear range from 0.05 to 200 ng mL−1. We demonstrated that our electrochemical sensor for the detection of clinical serum samples, indicating that our sensor would provide a new platform for low cost, sensitive and point-of-care diagnosis of prostate cancer.

A paper-based photothermal array using Parafilm to analyze hyperthermia response of tumour cells under local gradient temperature.

Temperature is a critical extrinsic physical parameter that determines cell fate. Hyperthermia therapy has become an efficient treatment for tumor ablation. To understand the response of tumor cells under thermal shocks, we present a paper-based photothermal array that can be conveniently coupled with commercial 96-well cell culture plates. This paper chip device was fabricated in one step using Parafilm® and Kimwipers® based on a heat lamination strategy. Liquid was completely adsorbed and confined within the cellulose fibres of hydrophilic regions. Then, Prussian blue nanoparticles (PB NPs) as the photothermal initiator were introduced into the loading wells, and thermal energy was generated via near infrared (NIR) laser irradiation. After assembling the paper device with a 96-well plate, the temperature of each well could be individually controlled by varying the loading amount of PB NPs and laser irradiation time. As a proof-of-concept study, the effects of local thermal shocks on HeLa cells were investigated using MTT cell viability assay and Live/Dead cell staining. The variation of cell viability could be monitored in situ with controllable temperature elevation. The proposed paper photothermal array loaded with thermal initiators represents an enabling tool for investigating the hyperthermia responses of biological cells. Moreover, the facile fabrication technique for paper patterning is advantageous for customizing high-throughput microfluidic paper-based analytical devices (μPADs) with extremely low cost.

Simple Way To Fabricate Novel Paper-Based Valves Using Plastic Comb Binding Spines

A novel strategy for fabricating the paper-based valves on microfluidic paper-based analytical devices (μPADs) was described to control fluid in a user-friendly way. Initial prototypes of 3D μPADs manipulate the spatial distribution of fluid within the device. The movable paper channel in a different layer could be achieved using the channel's connection or disconnection to realize the valve function using plastic comb binding spines (PCBS). The entire valve manipulation process was similar to a desk calendar that can be flipped over and turned back. It is notable that this kind of PCBS valve can control a fluid in a simple and easy way without the timing setting or any trigger, and this advantage makes it user-friendly for untrained users to carry out the complex and high throughput operations. The reusable plastic comb binding spines greatly reduce the cost of fabricating paper-based valves. To evaluate the performance, the actual samples of Fe (II) and nitrite were successfully analyzed. We hope this method will introduce a new approach to fabrication of paper-based valves on μPADs in the future.

Flow of liquids through paper

The flow of liquids through paper is challenging to model due to the complexity and disordered layout of the fibre matrix. The expanding use and capability of microfluidic paper-based analytical devices ($\unicode[STIX]{x1D707}$PADs) and their requirement for precision has increased the need to accurately predict the flow of liquids through paper. Many studies have developed models and revealed some of the physical mechanisms responsible for the flow behaviour, but we still lack a complete understanding, particularly in relation to how the fluid fills the various voids with a wide range of shapes and sizes in the fibre matrix of paper. In the featured article, Chang et al. (J. Fluid Mech., vol. 845, 2018, 36–50) used a combined experimental and theoretical approach to uncover the importance of the liquid filling the intra-fibre pores, showed that this results in deviation from the flow behaviour predicted by the Lucas–Washburn equation and developed a model which accounts for this effect.

Cellulose Nanofibers as a Module for Paper-Based Microfluidic Analytical Devices: Labile Substance Storage, Processability, and Reaction Field Provision and Control.

While basic concepts of paper-based microfluidic analytical devices (μPADs) have appeared considerably, elemental technology development is worth working from different specialized points of view. Herein, we show that 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized cellulose nanofiber (TOCN) can be reasonably used as a module of μPADs; we adopted a biochemical reaction of indigo formation by oxidation of indoxyl acetate (IDA) via its hydrolysis with acetylcholinesterase (AChE) as an example. First, we demonstrate that TOCN stably stores unstable AChE and IDA when dried and acts as a biochemical reaction field when wet. The thixotropic nature of TOCN aqueous dispersion gave inkjet printability, which will facilitate the μPADs production. The exchange of substances in the wet TOCN network took more time than in solution, but this can be read as control of reaction kinetics. Finally, we constructed semiquantitative μPADs for an organic phosphorus pesticide. We illustrate that by mounting TOCN, various functions can be incorporated on one sheet of paper to lead to the extension of design flexibility and universal use for μPADs.

A colorimetric paper-based analytical device coupled with hollow fiber membrane liquid phase microextraction (HF-LPME) for highly sensitive detection of hexavalent chromium in water samples

A simple and highly sensitive procedure based on the combination of hollow fiber membrane liquid phase microextraction and a microfluidic paper-based analytical device (µPAD) was developed for pre-concentration and determination of hexavalent chromium in water samples. The hexavalent chromium was pre-concentrated using the HF-LPME technique via ion exchange or a coupled transport process through a supported ionic liquid (Aliquat 336) prior to colorimetric detection with diphenylcarbazide on the µPAD. The violet colour could be seen by the naked eye. Images from the µPADs were scanned using a commercial desktop scanner at 600 dpi resolution. ImageJ software was used for quantitative analysis by measuring the intensity values at green colour channel since it gave the best sensitivity among the RGB colour. Under optimal conditions, the calibration curve was linear in the range 10–90 µg L-1, with a limit of detection of 3 µg L-1. The developed method was successfully applied to determine the level of hexavalent chromium spiked into natural water samples at the parts-per-billion (ppb) level, and the results were in good agreement with those obtained using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The developed method was able to improve the detection limit of the conventional µPAD, and was expected to be used for the effective analysis of hexavalent chromium in natural water.

Paper-Based Membraneless Co-Laminar Microfluidic Glucose Biofuel Cell With MWCNT-Fed Bucky Paper Bioelectrodes.

This paper establishes a membraneless, co-laminar flow-based approach to develop a cost-effective microfluidic paper-based analytical device for enzymatic biofuel cell ( PAD-EBFC). The developed PAD-EBFC supporting the self-capillary fluid transport action consists of Y-shaped paper microchannel with the fuel (glucose) and oxidant (O2) streaming in parallel over carbon nanotube-based bucky paper electrodes modified with biocompatible electrocatalytic enzymes, such as glucose oxidase and laccase without any additional redox cofactor. The electrochemical performance for the modified bioelectrodes, i.e., electrocatalytic oxidation and reduction reaction, was carried out using linear sweep voltammetry, cyclic voltammetry, and open circuit potential. The overall performance of PAD-EBFC was evaluated using the polarization studies. Subsequently, the catalytic activity of enzymes on the electrode surface was validated by the scanning electron microscope. This simple and portable PAD-EBFC can generate the maximum power density to the order of /cm2 ( /cm2) at 0.505 V over prolonged durations of around 50 h. Hence, the presented PAD-EBFC shows good power density and stability, leading to its strong potential to power miniaturized microelectronics devices and sensors.

Origami-enabled signal amplification for paper-based colorimetric biosensors

Signal amplification in colorimetric biosensors usually requires labile reagents and/or additional steps, which are not ideal for in-field measurements. Here we show that the limit of detection of a gold nanoparticle-labeled colorimetric immunosensor can be decreased 10-fold by performing the assay on a folded piece of paper. In this approach, the intrinsic porosity of the substrate enables generating multiple signals with a single drop of reagent. This yields combined signals with higher optical density thanks to the semitransparent nature of the wet material. It will be shown that this method can decrease the limit of detection 10 times in a model immunosensor for the detection of immunoglobulins. The procedure is so straightforward that it could be easily implemented to decrease the limit of detection of microfluidic paper-based analytical devices with colorimetric readouts.

Electrophoretic separations on Parafilm-paper-based analytical devices

Microfluidic paper-based analytical devices (mPADs) have gained significant attention in recent years for applications ranging from clinical diagnostics to environmental testing. However, separation on mPADs remains challenging to implement, particularly in complex samples. This has revived interest in revisiting paper chromatography and paper electrophoresis in mPADs to address these needs. Here, laminated Parafilm-paper (l-paper) is applied to fabricate electrophoretic devices. This approach yields a free-standing channel, leading to improved peak resolution relative to previous electrophoretic separations in traditional wax-printed mPADs. Major factors influencing the separation, including Joule heating, electroosmotic flow, and electrophoretic mobility, were investigated. As a result of paper’s high ratio of surface area (78%) to pore volume (22%) resulting in slow heat dissipation, a usable applied field strength range of 0–200 V cm−1 was employed to avoid Joule heating. The electroosmotic flow of the system was found to be 2.5 × 10−5 ± 7.7 × 10−7 cm2 V−1 s−1 and the electrophoretic mobility of chlorophenol red was 1.2 × 10−4 ± 7.7 × 10−7 cm2 V−1 s−1. Basic separation protocols were optimized using colorimetric detection of chlorophenol red and indigo carmine dyes as representative molecules. Paper type, channel width, and applied potential were then used to optimize the separations. Addition of an injection port to the device improved resolution and reduced peak broadening. Finally, the separation of fluorescein isothiocyanate (FITC) and l-glutamic acid (Glu) labeled with FITC, was successfully carried out using the l-paper electrophoretic device. Imaging with a microscope was found to achieve reduced peak broadening and increased resolution relative to imaging with a mobile camera, due to elimination of background signal, achieving a 72 ± 4% conjugation of Glu and FITC.

Paper-Based Enzyme Competition Assay for Detecting Falsified β-Lactam Antibiotics.

Falsified and substandard antibiotics are a growing worldwide problem that leads to increased patient mortality and decreased trust in healthcare, and contributes to antimicrobial resistance. Monitoring falsified antibiotics is difficult because most falsified pharmaceuticals are most commonly found in developing countries, where detecting the active ingredient is difficult due to lack of access to complex instrumentation. Herein, we describe the development and optimization of a microfluidic paper-based analytical device (μPAD) to detect the active ingredient in the most falsified class of antibiotics, β-lactams. The assay is based on enzyme competition, making it the first demonstrated competitive enzyme assay reported in paper-based devices. The assay uses nitrocefin, a chromogenic substrate, to compete with β-lactam antibiotics in a reaction with β-lactamase. A yellow color indicates legitimate drugs, while a color change from yellow to red indicates falsified drugs. In addition to testing for the active ingredient, another section of the device was added to test the sample pH to further verify results and identify common falsified ingredients like aspirin or baking soda. Calibration curves for four different antibiotics, including cefazolin, have been generated making it the first paper-based device capable of detecting both cephalosporin and penicillin antibiotics. The μPAD has also been tested with common falsified ingredients and four antibiotics in tablet or injectable form, demonstrating its potential for in-field falsified antibiotic testing.

A chemically functionalized paper-based microfluidic platform for multiplex heavy metal detection

This study presents a simple, fast, and sensitive method for multiplexing heavy metal detection. Using a chemically patterned microfluidic paper-based analytical device (C-μPAD), silane compounds terminating in amine (NH2), carboxyl (COOH), and thiol (SH) were immobilized on a patterned chromatography paper via condensation reactions. Three chromogenic reagents that react distinctly with Ni(II), Cr(VI), and Hg(II) were then covalently coupled to these functional groups. These functionalized C-μPADs were then evaluated for heavy metals ions detection with spiked water samples. Using a single-plex platform, we achieved detection limits as low as 0.24 ppm for Ni(II), 0.18 ppm for Cr(VI) and 0.19 ppm for Hg(II). The detection limits for this single-plex platform were twice as good as those obtained in previous studies using colorimetric analysis due to the formation of homogeneous metal complexes from immobilized chromogenic reagents. Furthermore, by minimizing cross-reaction with chelating agents and maximizing reaction uniformity, multiplex heavy metal ion detection was successfully demonstrated for lake-water samples spiked with various concentrations of heavy metal ions with high precision. This functionalized C-μPAD provides a highly portable and rapid on-site monitoring platform for multiple heavy metal ions detection with extremely high repeatability, which is useful for resource-limited areas and developing countries.

Development of an Electrochemical Paper-Based Analytical Device for Trace Detection of Virus Particles.

Viral pathogens are a serious health threat around the world, particularly in resource limited settings, where current sensing approaches are often insufficient and slow, compounding the spread and burden of these pathogens. Here, we describe a label-free, point-of-care approach toward detection of virus particles, based on a microfluidic paper-based analytical device with integrated microwire Au electrodes. The device is initially characterized through capturing of streptavidin modified nanoparticles by biotin-modified microwires. An order of magnitude improvement in detection limits is achieved through use of a microfluidic device over a classical static paper-based device, due to enhanced mass transport and capturing of particles on the modified electrodes. Electrochemical impedance spectroscopy detection of West Nile virus particles was carried out using antibody functionalized Au microwires, achieving a detection limit of 10.2 particles in 50 μL of cell culture media. No increase in signal is found on addition of an excess of a nonspecific target (Sindbis). This detection motif is significantly cheaper (∼$1 per test) and faster (∼30 min) than current methods, while achieving the desired selectivity and sensitivity. This sensing motif represents a general platform for trace detection of a wide range of biological pathogens.

Design keys for paper-based concentration gradient generators

The generation of concentration gradients is an essential operation for several analytical processes implemented on microfluidic paper-based analytical devices. The dynamic gradient formation is based on the transverse dispersion of chemical species across co-flowing streams. In paper channels, this transverse flux of molecules is dominated by mechanical dispersion, which is substantially different than molecular diffusion, which is the mechanism acting in conventional microchannels. Therefore, the design of gradient generators on paper requires strategies different from those used in traditional microfluidics. This work considers the foundations of transverse dispersion in porous substrates to investigate the optimal design of microfluidic paper-based concentration gradient generators (μPGGs) by computer simulations. A set of novel and versatile μPGGs were designed in the format of numerical prototypes, and virtual experiments were run to explore the ranges of operation and the overall performance of such devices. Then physical prototypes were fabricated and experimentally tested in our lab. Finally, some basic rules for the design of optimized μPGGs are proposed. Apart from improving the efficiency of mixers, diluters and μPGGs, the results of this investigation are relevant to attain highly controlled concentration fields on paper-based devices.

Thread/paper- and paper-based microfluidic devices for glucose assays employing artificial neural networks.

This paper describes the fabrication of and data collection from two microfluidic devices: a microfluidic thread/paper based analytical device (μTPAD) and 3D microfluidic paper-based analytical device (μPAD). Flowing solutions of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI), through each device, on contact with glucose, generated a calibration curve for each platform. The resultant yellow-brown color from the reaction indicates oxidation of iodide to iodine. The devices were dried, scanned, and analyzed yielding a correlation between yellow intensity and glucose concentration. A similar procedure, using an unknown concentration of glucose in artificial urine, is conducted and compared to the calibration curve to obtain the unknown value. Studies to quantify glucose in artificial urine showed good correlation between the theoretical and actual concentrations, as percent differences were ≤13.0%. An ANN was trained on the four-channel CMYK color data from 54 μTPAD and 160 μPAD analysis sites and Pearson correlation coefficients of R = 0.96491 and 0.9739, respectively, were obtained. The ANN was able to correctly classify 94.4% (51 of 54 samples) and 91.2% (146 of 160 samples) of the μTPAD and μPAD analysis sites, respectively. The development of this technology combined with ANN should further facilitate the use of these platforms for colorimetric analysis of other analytes.

Features in Microfluidic Paper-Based Devices Made by Laser Cutting: How Small Can They Be?

In this paper, we determine the smallest feature size that enables fluid flow in microfluidic paper-based analytical devices (µPADs) fabricated by laser cutting. The smallest feature sizes fabricated from five commercially available paper types: Whatman filter paper grade 50 (FP-50), Whatman 3MM Chr chromatography paper (3MM Chr), Whatman 1 Chr chromatography paper (1 Chr), Whatman regenerated cellulose membrane 55 (RC-55) and Amershan Protran 0.45 nitrocellulose membrane (NC), were 139 ± 8 µm, 130 ± 11 µm, 103 ± 12 µm, 45 ± 6 µm, and 24 ± 3 µm, respectively, as determined experimentally by successful fluid flow. We found that the fiber width of the paper correlates with the smallest feature size that has the capacity for fluid flow. We also investigated the flow speed of Allura red dye solution through small-scale channels fabricated from different paper types. We found that the flow speed is significantly slower through microscale features and confirmed the similar trends that were reported previously for millimeter-scale channels, namely that wider channels enable quicker flow speed.