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Rational Selection of Substrates to Improve Color Intensity and Uniformity on Microfluidic Paper-Based Analytical Devices
Abstract
A systematic investigation was conducted to study the effect of paper type on the analytical performance of a series of microfluidic paper-based analytical devices (μPADs) fabricated using a CO2 laser engraver. Samples included three different grades of Whatman chromatography paper, and three grades of Whatman filter paper. According to the data collected and the characterization performed, different papers offer a wide range of flow rate, thickness, and pore size. After optimizing the channel widths on the μPAD, the focus of this study was directed towards the color intensity and color uniformity formed during a colorimetric enzymatic reaction. According to the results herein described, the type of paper and the volume of reagents dispensed in each detection zone can determine the color intensity and uniformity. Therefore, the objective of this communication is to provide rational guidelines for the selection of paper substrates for the fabrication of μPADs.
... A critical issue in the development of a paper-based colorimetric assay is the selection of the appropriate detection substrate. 26 Thus we evaluated the optimal substrate on which our paper microzone BPB assay is to be performed (data not shown). In the rst attempts, we used Whatman G1F paper since a comparative study including 6 grades of Whatman paper demonstrated that it represents an optimal substrate in the fabrication of PADs. ...
... In the rst attempts, we used Whatman G1F paper since a comparative study including 6 grades of Whatman paper demonstrated that it represents an optimal substrate in the fabrication of PADs. 26 Surprisingly, at variance with previous studies, the colour development was barely detectable in Whatman G1F paper. The lower concentration of the dye used in our experiments may be responsible for this difference. ...
... As previously suggested, the reason for this evidence relies on the higher colour intensity and gradient when the substrate in the detection zone moves to a complete saturation. 26 We reached a better performance with loading volumes equal to or higher than 20 mL, when the GF/F circles are completely soaked. Based on these results, at least under the experimental conditions described here, we recommend the use of a total loading volume of 20 mL. ...
This study describes the development of a paper microzone colorimetric assay embedded on a 3D printed support for quantifying total protein content in different biological matrices and foods. The aim was to develop an accurate and reliable method, ensuring at the same time the possibility of customizability, facility of use, wide applicability, and reduced analysis for both time and costs. The device consists of a 3D printed thermoplastic polyurethane support housing the detection substrate (GF/F glass microfiber). The bromophenol blue (BPB) assay was optimized in this substrate to quantify total protein content. The analytical performance, assessed through image analysis, indicated that the hue factor of the HSV colour space represents the best analytical signal (r2>0.98%). The optimized assay ensures a sufficiently low limit of detection (0.05 mg mL-1), and an accuracy between 92% and 95%. The bioanalytical feasibility was demonstrated through total protein concentration measurement in different biological matrices (bee venom and mouse brain tissue), and foods (soya milk, cow’s milk and protein supplements). The obtained values showed a strong agreement with those derived from a standard spectrophotometric analysis. Overall, the paper microzone BPB assay may represent an important contribution to protein quantification technology, and could significantly impact many areas, such as quality control analysis and pre-clinical laboratory analysis.
... The Whatman chromatography paper and filter paper in different grades were studied. The results showed that the thinnest chromatography paper generated the highest color gradient, while increasing the volume of reagents could weaken the color gradient and enhance the mean color intensity (Fig. 3b) [39]. The authors attributed the higher color inhomogeneity to the enzymes having weak affinity with cellulose and being displaced to the edge of the reaction zone via the chromatographic effect of the sample wicking. ...
... This opinion was opposite to the findings of Coltro s Group, as discussed above (Fig. 2d) that the color heterogeneity was verified to be caused by the mobility of small chromogenic molecules (I and I 3 ) instead of enzymes. Besides, the conclusion of Evans et al. that the thicker substrates yielded poor color intensity [39] was contrary to the opinion of Zhang et al. [38] aforementioned that blotter paper exhibited high color intensity results. The possible reason may be various reaction systems (e.g., enzymatic assays, chelation of heavy metal ions) performed differently on different cellulose paper substrates. ...
... (b) Highest color gradients forming on the thinnest paper matrix (ii). Reprinted with permission from ref [39]. Copyright 2014 RSC. ...
Colorimetric paper-based analytical devices (PADs) have been widely fabricated due to the ease of construction of colorimetric reactions on paper and the high compatibility with low-cost and easy-to-use imaging devices for on-site analysis and results recording. However, the further advance of colorimetric PADs is limited by the detection sensitivity and other intrinsic limitations of paper regarding the color quality. This review summarizes the strategies for improving the colorimetric performances (color intensity and uniformity) of PADs. In particular, the principles of controlling liquid-paper interaction for the color intensity enhancement, developed in printing research, are discussed in relation to PAD designs. The influence of the paper assaying environment to the paper assaying selectivity is discussed through a case study for the first time. Image capture techniques and image process algorithms are related to the accuracy and sensitivity of the colored results, which are carefully introduced. Finally, based on the tendency of improvement of colorimetric PADs for practical applications, we summarized the designs that enhance the portability of colorimetric PADs. It is believed that the new links and opinions provided in this review may be useful to researchers to address the challenges in designing future colorimetric PADs with improved sensitivities for field use.
... 19 Filter paper, blotting paper, and chromatography papers are among the most widely used paper substrates for fabricating PADs. 9,20 Paper is a low-cost and ubiquitous material with a wide range of choices. Whatman grade 1 chromatographic paper and Whatman no. 1 filter paper have been widely applied for the development of PADs. ...
... Whatman grade 1 chromatographic paper and Whatman no. 1 filter paper have been widely applied for the development of PADs. 20 These papers are made of cellulose (>98%). Whatman grade 1 chromatography paper has a clean surface, uniform thickness, high hygroscopic properties, wicking properties, flow rate, and cost effectiveness. ...
... The thickness of P5 was similar to that of Whatman grade 1 paper (180 μm), and the remaining four samples were thicker than the Whatman grade 1 paper. 20 However, P1 and P2 had similar thickness to that fo Whatman grade 4, P5 was thinner, and P3 and P4 were even thicker than Whatman grade 4 paper (205 μm). 20 The gram per square meter (GSM) of papers, which is also known as grammage, ranged widely from 49 g/m 2 (P1) to 117.8 g/m 2 (P4). ...
Paper analytical devices (PADs) are a class of low-cost, portable, and easy-to-use platform for several analytical tests in clinical diagnostics, environmental pollution monitoring, and food and drug safety screening. These devices are primarily made from cellulosic paper. Considering the importance of eco-friendly and local or distributed manufacturing of devices realized during the COVID-19 pandemic, we systematically studied the potential of handmade Nepali paper to be used in fabricating PADs in this work. We characterized five different handmade papers made from locally available plant fibers using an eco-friendly method and used them to fabricate PADs for determining the drug quality. The thickness, grammage, and apparent density of the paper samples ranged from 198.6 to 314.8 μm, 49.1 to 117.8 g/m2, and 0.23 to 0.43 g/cm3, respectively. The moisture content, water filtration, and wicking speed ranged from 5.8 to 7.1%, 35.7 to 156.7, and 0.062 to 0.124 mms-1, respectively. Furthermore, the water contact angle and porosity ranged from 76.6 to 112.1° and 79 to 83%, respectively. The best paper sample (P5) was chosen to fabricate PADs for the determination of metformin, an antidiabetic drug. The metformin assay on PADs followed a linear range from 0.0625 to 0.5 mg/mL. The assay had a limit of detection and limit of quantitation of 0.05 and 0.18 mg/mL, respectively. The average amount of metformin concentration in samples collected from local pharmacies (n = 20) was 465.6 ± 15.1 mg/tablet. When compared with the spectrophotometric method, PAD assay correctly predicted the concentration of 90% samples. The PAD assay on handmade paper may provide a low-cost and easy-to-use system for screening the quality of drugs and other point-of-need applications.
... Paper is a low-cost and ubiquitous material with a wide range of choices. Whatman Grade 1 chromatographic paper and Whatman No. 1 filter paper are made of cellulose (>98%) and have been widely applied for the development of PADs 20 . Whatman Grade 1 chromatography paper has clean surface, uniform thickness, high hygroscopic property, wicking properties, flow rate, and cost effectiveness 21 . ...
... Concentration of metformin in samples was estimated by using regression equation of calibration curve. However, P1 and P2 had similar thickness to Whatman grade 4, P5 was thinner and P3 and P4 were even thicker than Whatman grade 4 paper (205 μm) 20 . ...
... The porosity of handmade paper samples was in the range of ~79 % (P2) to 83 % (P1). The porosity of Whatman qualitative filter papers is reported in the range of ~64 % (grade 2) to 68% (grade1) 20 . Papers with high porosity increase the absorbance of ink and help the ink to dry quickly. ...
The COVID-19 pandemic has highlighted the need of eco-friendly and locally or distributed manufacturing of diagnostic and safety products. Here, we characterized five handmade papers for their potential application to make paper analytical device (PADs). The handmade papers were made from locally available plant fiber using eco-friendly method. Thickness, grammage, and apparent density of the paper samples ranged from 198 μm to 314 μm, 49 g/m2 to 117.8 g/m2, and 0.23 to 0.39 g/cm3, respectively. Moisture content, water filtration and wicking speed ranged from 5.2% to 7.1%, 35.7 to 156.7, and 0.062 to 0.124 mms-1, respectively. Further, water contact angle and porosity ranged from 76˚ to 112˚ and 79% to 83%, respectively. The best paper sample one was chosen to fabricate PADs which were used for the determination of metformin. The metformin assay on PADs followed linear range from 0.0625 to 0.5 mg/mL. The assay had limit of detection and limit of quantitation of 0.05 mg/mL and 0.18 mg/mL respectively. The new method was used to test metformin samples (n=20) collected from local pharmacies. The average amount of metformin concentration in samples was 465.6 ± 15.1mg/tablet. Three samples did not meet the regulatory standards. When compared with spectrophotometric method, PADs assay correctly predicted 18 out of 20 samples. The PADs assay on handmade paper may provide a low-cost and easy-to-use system to screening the quality of drugs and other point-of-need applications.
... The intrinsic properties of paper affect the capillary flow, the immobilisation and release of reagents, the development of readout signals, among others. Examples on how the paper properties have been modified to have an effect on some of these aspects include the control of fluid flow through paper by modifying e.g., the paper porosity [78], fibre length [115], and fibre hydrophobicity [69,181], the use of papers with higher surface area for increased loading capacity of reagents [182], and the better colour uniformity and intensity in colorimetric readouts obtained with thinner papers [183]. There is common acknowledgement that a deeper understanding and control of the intrinsic properties of paper is essential for the commercial success of paper-based microfluidic sensors [50,53,54]. ...
... Pulp suspensions for printing were prepared by blending sheets of Whatman 1 filter paper in water. Figure average fibre length [220] and 19 μm fibre width [183] for Whatman 1 filter paper (before blending), it can be noted that while the fibre width is preserved, the blending procedure results in a shortening of the average length of the fibres, likely due to cutting with the blender blades. The method used for pulping has been shown in the literature to have a significant effect on the formed paper properties, including the paper wet strength and its capacity to drive flow by capillarity. ...
The use of paper as a sensing platform has great potential for point-of-care diagnostics in low-resource settings. However, there is a need for approaches that enable a higher level of control and customisation of the paper properties and reduce the cost of production and immobilisation of bioreagents onto paper. This thesis explores whether introducing digital printing into papermaking in combination with the recombinant protein production of paper-binding bioreagents could provide an accessible, scalable tool for the fabrication of affordable paper-based microfluidic biosensors with customised and varying paper properties. A novel printing method based on the localised vacuum-driven filtration of fibre suspensions (‘vacuum-driven printing’) was explored for the direct formation and patterning of engineered paper. An analytical model based on incompressible cake filtration theory was derived to predict the final thickness of the printed paper as a function of printing parameters and showed good agreement with the experimental data. This model enabled the rational design of complex 2D paper patterns with controlled and varying thickness profile that could be formed in a single printing process. The recombinant fusion of a cellulose-binding domain (CBD) into the protein reagent enabled its one-step immobilisation and purification onto paper fibres directly from the crude lysate, significantly reducing the cost and number of downstream processing steps required for the production of the bioreagent. The specific immobilisation provided by the CBD helped retain the protein’s diagnostic activity during air drying and long-term dry storage, even after four months at 20-37°C, when compared to direct physisorption of the protein reagent onto paper. The protein-bound fibres could be directly patterned with the vacuum-driven printing technique into the detection zone of a paper-based assay, forming bioactive paper. A proof-of-concept paper-based microfluidic biosensor was fabricated in a single vacuum-driven printing process, using unmodified paper fibres to form microfluidic channels of varying thickness, and protein-bound fibres to form bioactive paper at the detection zone of the assay. This research demonstrated a simplified, accessible, and lower-cost pathway to the production of paper-based biosensors with customised properties.
... Most of the reported work on µPADs utilized either Whatman filter paper of various grades or chromatography paper as substrates [27,39,[42][43][44]. Evan et al. reported the importance of paper substrate physical properties to get homogenous colour development in glucose sensor by using cellulose fiber [45]. Their focus was on investigating the effect of pore size, paper thickness, and device dimension for glucose detection. ...
Rapid on-site monitoring of nitrite as a diagnostic biomarker has drawn considerable attention in recent years. However, conventional detection techniques face limitations in terms of portability and ease of usage. In this work microfluidic paper based colorimetric sensor (µPCS) is developed for detection of nitrite from simulated saliva sample using Griess reagent. We discovered that the chemical nature of the filter papers has significant effect on the analytical sensitivity of nitrite detection. Among different paper substrate tested, the glass microfiber filter (GMF) paper exhibits exceptional detection sensitivity. The µPCS was fabricated using GMF as the substrate material through a lamination process. We have fabricated a portable light box using 3D printing and coupled it with µPCS device for image acquisition. This helps to maintain uniform lighting and avoid external interference during image acquisition. Under optimal conditions, a linear change in response with respect to nitrite concentration was observed for 0.5–200 µM with a limit of detection of 3.22 µM and a limit of quantification of 10.72 µM. The proposed method was validated with UV–Vis spectrophotometer. The device developed in this work showed good accuracy (recovery of 103–113%), and precision (RSD < 9.21%). This work demonstrates an end-to-end portable procedure for nitrite detection and its potential as a preliminary screening device in clinical applications.
Graphical Abstract
... ; https://doi.org/10.1101/2024.01.27.577541 doi: bioRxiv preprint conduct any such similar assays is imperative due of its exceptional compatibility for using simplified fabrication techniques (such as laser printing) and hence aiding in the reduction of overall cost per device. Also, it exhibits a rather consistent distribution of pores and shows faster transfer rates that offer better analytical performance which can be utilized to overcome the limitations of µPADs [36]. ...
Increased evaporative losses and flow obstructions can present substantial impediments to current paper analytical devices (µPADs) for efficient on-site testing of biological fluids. Strategic enhancements in wicking rates of paper may thereby counter these limitations and enable on-demand healthcare monitoring. Therefore, herein we have leveraged the features of paper fold-crease regions, for the very first time, and developed a novel fast-flowing platform using laser printing to accelerate fluid flow through paper. A series of extensive experiments have been conducted to optimize the design and maximize wicking rates of µPADs for smaller liquid volumes, making it well-suited for analysing biofluids. The investigation delves into structural alterations within the creased regions, employing both static and dynamic force application strategies. A first-generation Washburn type model in excellent agreement with the experimental findings is developed, providing a comprehensive insight into the fundamental physics involved. Finally, the folded channels are utilized for a distance-based hematocrit sensor employing grade-1 filter paper at very low-cost, simplified fabrication, lesser sample volume and faster analysis. The findings of this work unveil a plethora of potentialities for employing paper and paper folds to develop affordable medical devices with advanced analytical functionalities, specifically tailored for the resource-constrained settings.
Graphical Abstract
... Although all of the proposed strategies showed a more homogeneous color distribution, they required important efforts such as the chemical modification of a surface and the synthesis of nanomaterials. To overcome this issue, Evans et al. [59] reported that a proper selection of the paper support to optimize the flow resistance improves the uniformity of the color distribution. Martinez et al. [60] and Morbioli et al. [61] worked on the optimization reaction area, proposing a diamond shape and an origami geometry, respectively. ...
The need for providing rapid and, possibly, on-the-spot analytical results in the case of intoxication has prompted researchers to develop rapid, sensitive, and cost-effective methods and analytical devices suitable for use in nonspecialized laboratories and at the point of need (PON). In recent years, the technology of paper-based microfluidic analytical devices (μPADs) has undergone rapid development and now provides a feasible, low-cost alternative to traditional rapid tests for detecting harmful compounds. In fact, µPADs have been developed to detect toxic molecules (arsenic, cyanide, ethanol, and nitrite), drugs, and drugs of abuse (benzodiazepines, cathinones, cocaine, fentanyl, ketamine, MDMA, morphine, synthetic cannabinoids, tetrahydrocannabinol, and xylazine), and also psychoactive substances used for drug-facilitated crimes (flunitrazepam, gamma-hydroxybutyric acid (GHB), ketamine, metamizole, midazolam, and scopolamine). The present report critically evaluates the recent developments in paper-based devices, particularly in detection methods, and how these new analytical tools have been tested in forensic and clinical toxicology, also including future perspectives on their application, such as multisensing paper-based devices, microfluidic paper-based separation, and wearable paper-based sensors.
... First, four kinds of paper substrates, such as Whatman filter paper No. 5 (WFP-5), quantitative filter paper (fast flow), WFP-4, and chromatography paper with particle retentions at 2.5, 80-120, 20-25, and 11 μm, were employed to prepare paper pads using rhodamine B as sample solution. Smaller particle retention can offer moderate and uniform flow to obtain homogenous impregnation of solvent on paper substrate (Bhardwaj et al., 2019;Evans et al., 2014). As a result, the test zone of WFP-5 displayed the best color intensity and homogenous impregnation ( Figure S11A). ...
Herbicide residuals have posed severe threat to food safety and public health; thus, the development of smart, portable, efficient, and accurate on‐site detection system is in urgent demand. Here, a multi‐emitting fluorescent system‐assisted lab‐in‐a‐syringe (LIS) device was developed for the on‐site and background‐free determination of 2,4‐dichlorophenoxyacetic acid (2,4‐D). 2,4‐D‐responsive cascade reactions were constructed: 2,4‐D could specifically suppress alkaline phosphatase (ALP) activity and restrain the generation of ascorbic acid (AA), MnO 2 nanosheets would oxidize AA into dehydroascorbic acid that subsequently reacts with o ‐phenylenediamine (OPD) to generate blue‐emission product ( λ em , 435 nm), whereas remained MnO 2 nanosheets and OPD further produced yellow‐emission product ( λ em , 570 nm). Red‐emission carbon dots (RCDs, λ em , 680 nm), synthesized via the solvothermal treatment of cilantro, were introduced to construct a multi‐emitting fluorescent system. Then, four paper pads, respectively, dropped with ALP, sodium l ‐ascorbyl‐2‐phosphate, MnO 2 nanosheets + RCDs, and OPD were held into reusable plastic filters and assembled paper‐based LIS device to trigger cascade reactions (total analysis time, 65 min) and eliminate background interference. As a result, with the support of color recognizer application in smartphone, fluorescent color on detection pads from blue–purple to red to yellow was achieved for the sensitive and visual detection of 2,4‐D with LOD of 5.0 μg L ⁻¹ , recoveries of 94.6%–106.8%, and relative standard deviations of 2.3%–6.8%. Obviously, this strategy provides a robust, visual, and background‐free platform for 2,4‐D detection, which expands application prospects in the field of herbicide analysis.
... All of these quantities-contact angle, pore size, porosity, thickness-will vary depending on the type of paper chosen, and can noticeably affect an assay's performance [75]. In the articles surveyed by the current review, Whatman 1 filter paper and Whatman 1 CHR were by far the most commonly used papers. ...
... In first research about μPADs, hydrophobic lines were fabricated by photolithography method [1]. Currently, fabrication technique of hydrophobic lines has been developed and divided into four main groups [2]: handcrafting [3], cutting/shaping [4][5][6][7], mask usage [1,8,9] and printing [10][11][12][13][14][15][16]. Among these methods, inkjet printing is considered as a promising method because of numerous reasons such as simple process, costeffectiveness, suitability for mass-production, etc [17]. ...
Microfluidic paper-based channels play an important role in microfluidic paper-based analytical devices ( μ PADs). There are some fabrication methods which could be utilised to fabricate microfluidic channels on paper substrate. Among these methods, inkjet printing process is considered as a promising fabrication method with many advantages such as low-cost, material saving, high precision, etc. The aim of this work is to apply inkjet printing technology to fabricate paper channels of μ PADs. A new design of μ PAD was proposed in this paper to demonstrate how to fabricate inkjet-printed hydrophobic lines to make paper-based biosensor. Biological target of our μ PADs is human chOrionic gonadotropin (hCG). Colorimetric signals from μ PADs were captured by digital camera and measured by ImageJ software, which showed that these μ PADs can determine hCG in the range from 1,000 to 10,000 ng ml ⁻¹ . These results showed that piezoelectric inkjet printing technology can fabricate 250 μ m-width hydrophobic lines on paper substrate, helping in fabricating μ PADs in next applications.
... For instance, hydrophobic wax, which leads to a high contact angle, surrounding a porous medium has been reported to impede the imbibition. This is due to the increase of flow resistance from the presence of non-wetting surfaces [34,35,36]. Hong and Kim [37] and Jafry et al. [38] provide more rigorous analyses and discussions on this phenomenon based on the considerations of different capillary forces on the wetting and non-wetting surfaces. ...
In this work, the imbibition velocity of molten silver into a porous nickel interlayer on a nonwetting sapphire substrate is investigated. Several imbibition models for porous media that use bulk characteristics and material properties are employed to predict the imbibition front location as a function of time. The model predictions are compared to experimental observations. It is found that pore size distribution is a better predictor of imbibition velocity than correlations based on permeability and porosity. The tortuosity of the microstructure is also found to have a significant effect and should be considered. A new model accounting for the different liquid contact angles on the underlying substrate and the porous interlayer material is proposed and achieves better agreement with the experimental observations.
... Analytica Chimica Acta xxx (xxxx) xxx slower rate, which results in poor color development in μPADs. Using thinner substrates such as grade 1 filter papers and chromatography papers, solution transfer will be faster and analytical performance is increased [53]. ...
Paper-based analytical devices (PADs) have shown great promise for point-of-care testing and on-site detection of analytes with chemical, biochemical, and environmental importance owing to their low cost, convenience, scalability, portability, and biocompatibility. The World Health Organization stated that sensors should meet the ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable). Paper-based optical sensors meet most of these criteria, making them in high demand and applicable in remote areas. Optical PADs outputs are obtained by different means, such as dyes, nanostructures, redox agents, and pH indicators. The outstanding physical and chemical characteristics of nanostructures, their intense signals, and tunable optical properties make them ideal for many sensing platforms, including paper-based ones. This review focuses primarily on paper-based nanosensors using various nanostructures to fabricate and produce optical signals for visualization. We describe the fundamentals and state of the art of PADs and comprehensively explain the following topics: paper types as the substrate of PADs, PAD fabrication approaches, nanostructure stabilization on PADs, signal acquisition, data handling, interpretation of results, sensing mechanisms, and application areas. We also discuss future trends and strategies to enable PADs to reach their full potential and increase their commercialization opportunities.
... Environmental pollution has continuously been a major threat due to fast-growing anthropogenic activities resulting from civilization and industrialization Lau et al., 2020;Podgorski and Berg, 2020;Santos et al., 2021). Associated burden of diseases and death arising from global air and water pollution poses a great challenge on public health, especially in underdeveloped regions and countries (Evans et al., 2014;Mahaqi et al., 2021;Yang et al., 2022). For instance, more than four millions of deaths related with gastrointestinal diseases may be attributed to water contamination in the United States (Colford et al., 2006). ...
Effective environmental monitoring has become a worldwide concern, requiring the development of novel tools to deal with pollution risks and manage natural resources. However, a majority of current assessment methods are still costly and labor-intensive. Thanks to the rapid advancements in microfluidic technology over the past few decades, great efforts have been made to develop miniaturized tools for rapid and efficient environmental monitoring. Compared to traditional large-scale devices, microfluidic approaches provide several advantages such as low sample and energy consumption, shortened analysis time and adaptabilities to onsite applications. More importantly, it provides a low-cost solution for onsite environmental assessment leveraging the ubiquitous materials such as paper and plastics, and cost-effective fabrication methods such as inkjet printing and drawing. At present, devices that are disposable, reproducible, and capable of mass production have been developed and manufactured for a wide spectrum of applications related to environmental monitoring. This review summarizes the recent advances of low-cost microfluidics in the field of environmental monitoring. Initially, common low-cost materials and fabrication technologies are introduced, providing a perspective on the currently available low-cost microfluidic manufacturing techniques. The latest applications towards effective environmental monitoring and assessment in water quality, air quality, soil nutrients, microorganisms, and other applications are then reviewed. Finally, current challenges on materials and fabrication technologies and research opportunities are discussed to inspire future innovations.
... Considering such attribute, the manual filling of the paper zone can be achieved with ease and more rapidly, avoiding significant irreproducibility that may arise during the modification step. Such a feature becomes advantageous when comparing to the direct drop-wise modification of paper zones commonly employed for the fabrication of paper microdevices, since it may lead to increased inhomogeneity in the modified reaction zone due to the coffee ring effect (de Freitas, de Souza, Rodrigues Neto, Vasconcelos, Abdelnur, Vaz, et al., 2018; Evans, Gabriel, Coltro, & Garcia, 2014;Soda, Robinson, Cherubini, & Bakker, 2020). Non-skilled users can fabricate this sensing platform in a more reproducible and simpler manner by drawing instead of manipulating micropipettes. ...
... The paper-based resistors use a unique method of tunable ink deposition on 180 μm-thick filter paper. 18 Sheets were patterned with wax to create paper wells with wax boundaries extending through the depth of the paper substrate. These wells were filled with conductive ink based on a blend of organic conductive polymer poly(3,4ethylenedioxythiophene) and polystyrenesulfonate (PE-DOT:PSS). ...
Humanity's excessive production of material waste poses a critical environmental threat, and the problem is only escalating, especially in the past few decades with the rapid development of powerful electronic tools and persistent consumer desire to upgrade to the newest available technology. The poor disposability of electronics is especially an issue for the newly arising field of single-use devices and sensors, which are often used to evaluate human health and monitor environmental conditions, and for other novel applications. Though impressive in terms of function and convenience, usage of conventional electronic components in these applications would inflict an immense surge in waste and result in higher costs. This work's primary objective is to develop a cost-effective, eco-friendly, all-paper, device for single-use applications that can be easily and safely disposed of through incineration or biodegradation. All electronic components are paper-based and integrated on paper-based printed circuit boards (PCBs), innovatively providing a realistic and practical solution for green electronic platforms. In particular, a methodology is discussed for simultaneously achieving very tunable resistors (20 Ω to 285 kΩ), supercapacitors (∼3.29 mF), and electrolyte-gated field-effect transistors on and within the thickness of a single sheet of paper. Each electronic component is completely integrated into functionalized paper regions and exhibits favorable electrical activity, adjustability, flexibility, and disposability. A simple amplifier circuit is successfully demonstrated within a small area and within the thickness of a single sheet of paper, displaying component versatility and the capability for their fabrication processes to be performed in parallel for efficient and rapid development.
... As μPADs are increasingly finding use in many different applications and more complex chemical and biological tests and assays, a proper choice of the paper type to be used is crucial. It has been experimentally shown that paper-based devices utilizing thicker papers as substrates result in slower fluid flow [49,50]. Several groups have relied on the Lucas-Washburn equation to model fluid flow in paper. ...
The novel paper-based Bi-Material Cantilever (B-MaC) valve allows the autonomous loading and control of multiple fluid reagents which contributes to the accurate operation of paper-based microfluidic devices utilized for biological and chemical sensing applications. In this paper, an extensive parametric study is presented to evaluate the effects of key geometric parameters of the valve, such as paper direction, cantilever width, paper type, tape type, and sample volume, in addition to the effects of relative humidity and temperature on the functionality of the B-MaC and to provide a better understanding of the rate of fluid flow and resulting deflection of the cantilever. Machine direction, cantilever width, paper type, and tape type were found to be important parameters that affect the B-MAC's activation time. It was also observed that the rate of fluid imbibition in the B-MaC is considerably affected by change in humidity for high (55 °C) and low (25 °C) temperatures, while humidity levels have no significant effect during imbibition in the B-MaC at an ambient temperature of 45 °C. It was also found that a minimum distance of 4 mm is required between the B-MaC and the stationary component to prevent accidental activation of the B-MaC prior to sample insertion when relative humidity is higher than 90% and temperature is lower than 35 °C. The rate of fluid imbibition that determines the wetted length of the B-MaC and the final deflection of the cantilever are critical in designing and fabricating point-of-care microfluidic paper-based devices. The B-MaC valve can be utilized in a fluidic circuit to sequentially load several reagents, in addition to the sample to the detection area.
... Table 1 highlights the different types of paper substrates that have been developed for paper-based sensing applications. Analytical separation [61] Electrophoretic separation [62] Soil analysis [63][64][65] Food testing [66] Point of care testing [67] Protein [68] Atmospheric dust [69] Gas detection [70] HIV detection [71] Explosive Sensing [72] Automated DNA extraction and amplification [73] Whatman Compatible with most sensing methods. Super refined cellulose ...
Cancer is one of the major killers across the globe. According to the WHO, more than 10 million people succumbed to cancer in the year 2020 alone. The early detection of cancer is key to reducing the mortality rate. In low- and medium-income countries, the screening facilities are limited due to a scarcity of resources and equipment. Paper-based microfluidics provide a platform for a low-cost, biodegradable micro-total analysis system (µTAS) that can be used for the detection of critical biomarkers for cancer screening. This work aims to review and provide a perspective on various available paper-based methods for cancer screening. The work includes an overview of paper-based sensors, the analytes that can be detected and the detection, and readout methods used.
... First, we optimized the types of paper including Whatman No. 1, No. 114, and lens cleaning tissues No. 105 for immobilization of UOx. As the pore size and thickness differ with the type of paper (Table S1), the result of detection varies due to the differences in solution flow rate on the paper surface and material uniformity 36 . Therefore, appropriate paper selection appears to be critical to improve the current signal of paper-based biosensors. ...
In this study, we introduce a uricase-immobilized paper (UOx-paper) integrated electrochemical sensor for detection of uric acid (UA) in saliva. The UOx was immobilized on the detection zone in the wax-patterned paper substrate. This UOx-paper was integrated with a Prussian blue-modified, screen-printed carbon electrode after electropolymerization of o-phenylenediamine to construct an electrochemical cell for small-volume (20 μL) of samples. First, we optimized the fabrication conditions of UOx-paper. Next, the amperometric response of the UOx-paper-based electrochemical UA sensor was analyzed using a known concentration of UA standard solution in artificial saliva at an applied potential of − 0.1 V (versus Ag pseudo-reference electrode). The UOx-paper based electrochemical UA sensor showed a sensitivity of 4.9 μA·mM−1 in a linear range of 50 to 1000 μM (R2 = 0.998), high selectivity and good reproducibility, as well as a limit of detection of 18.7 μM (0.31 mg/dL) UA. Finally, we quantified the UA levels in undiluted saliva samples of healthy controls (n = 20) and gout patients (n = 8). The levels were correlated with those measured with conventional salivary UA enzymatic assays as well as serum UA levels. The UOx-paper-based electrochemical UA sensor is a user-friendly and convenient tool to assess salivary UA levels.
... For the selection of paper substrates, several physical properties of the paper, which include thickness, porosity, and wicking speed, affect the sample transfer rate within the microfluidic device. Whatman filters are made from pure cellulose and are popular for their uniform thickness and wicking characteristics [114,115]. ...
The illegal use of β-adrenergic agonists during livestock growth poses a threat to public health; the long-term intake of this medication can cause serious physiological side effects and even death. Therefore, rapid detection methods for β-adrenergic agonist residues on-site are required. Traditional detection methods such as liquid chromatography have limitations in terms of expensive instruments and complex operations. In contrast, paper methods are low cost, ubiquitous, and portable, which has led to them becoming the preferred detection method in recent years. Various paper-based fluidic devices have been developed to detect β-adrenergic agonist residues, including lateral flow immunoassays (LFAs) and microfluidic paper-based analytical devices (μPADs). In this review, the application of LFAs for the detection of β-agonists is summarized comprehensively, focusing on the latest advances in novel labeling and detection strategies. The use of μPADs as an analytical platform has attracted interest over the past decade due to their unique advantages and application for detecting β-adrenergic agonists, which are introduced here. Vertical flow immunoassays are also discussed for their shorter assay time and stronger multiplexing capabilities compared with LFAs. Furthermore, the development direction and prospects for the commercialization of paper-based devices are considered, shedding light on the development of point-of-care testing devices for β-adrenergic agonist residue detection.
... Cellulose-LFA were assembled using 27.8 mm length strips of Whatman N. 1 paper, a cellulose based (>98%) porous material defined by 15 µm fibers that has a basis weight of 87 g/m 2 ( Figure S2 (Supplementary Materials)). This paper features pores with a size distribution centered around 5 µm and spanning the 1-19 µm range [31][32][33][34]. Aqueous solutions wick through Whatman N. 1 cellulose strips with flow times of the order of~484 ± 69 s/4 cm, which are larger than the flow times that were obtained with typical NC membranes (e.g., 75-240 s/4 cm, [14]). ...
... The choice of the paper substrate while designing a sensor is determined by the proposed application (Sajid et al., 2015). The wicking ability of paper is governed by its porosity, pore size, fiber orientation, density, and thickness (Liana et al., 2012;Böhm et al., 2014;Evans et al., 2014;Walji and MacDonald, 2016;Tenda et al., 2016). Filter paper is the most commonly used paperfluidic substrate (Liana et al., 2012;Tenda et al., 2016;Mentele et al., 2012;Liu et al., 2015), while charged organic dyes are the most popular model analytes to study flow characteristics on paper (Osborn et al., 2010;Carrilho et al., 2009;Ota et al., 2018). ...
Understanding the fluid flow on paperfluidic substrates is crucial due to an increasing interest in affordable sensors for diagnosis, environmental testing, food testing, etc. Charged dyes are the most commonly used model analytes for characterizing the fluid flow in paperfluidic devices. Here, we propose a framework to quantify the concentration-dependent loss during transport of four charged dyes (e.g., rhodamine B, methylene blue, tartrazine and amaranth). At concentrations lower than a critical value (Cc), electrostatic interaction of the dye molecules with the filter paper retards the flow of cationic dyes rhodamine B and methylene blue, resulting in 75%–99% sample loss. We determined Cc to be 20 μM for rhodamine B and 5 mM for methylene blue. The large difference in their Cc values is explained by the molecular structures of these dyes. While anionic dyes (e.g., tartrazine and amaranth) are not retarded during flow on filter paper due to negligible electrostatic interaction with the substrate, they form strong coffee ring patterns in the detection zone when the dye concentration is below Cc. We introduced a new parameter called the ‘coffee ring index’ (CRI) to quantify the non-uniform sample distribution in the detection zone, and from this, determined Cc to be 250 μM for amaranth and 1.25 mM for tartrazine. Finally, we demonstrated that an increase in the dye viscosity by adding 30% glycerol can lower Cc of tartrazine from 1.25 mM to 100 μM. These results provide useful guidelines for the paperfluidics community to design more effective sensors.
A fluorescent pyrazinium-based 1-benzyl-3,5-diphenylpyrazin-1-ium bromide (BPPyz) chemosensor was synthesized and well-characterized. A significant reduction in blue emission of BPPyz was observed in the presence of TNP as compared to other nitroaromatic compounds, indicating high selectivity towards TNP. In the presence of sulfite ions, BPPyz showed fluorescence quenching and rapid naked-eye detection with a significant color change. The sensing mechanism was investigated through UV–visible studies, time-resolved fluorescence results, and density functional theory (DFT) calculations. The quenching constants (KSV) are 4.12 × 10⁵ M⁻¹ for TNP and 3.8 × 10⁵ M⁻¹ for sulfite with the detection limits of 9.5 nM and 46.17 nM for TNP and sulfite, respectively. The selectivity of BPPyz towards TNP was ascribed to the ground state charge transfer complex (GSC) formation and resonance energy transfer. Sulfite ion detection involved the formation of a GSC through hydrogen bonding with the pyrazinium proton.
This work discusses surface modification of cellulose paper specimens for compatibility with nitrogen and sulfur co‐doped carbon dots (NSCDs) for lead ion sensing. The interaction of carbon dots (CDs) and cellulose fibers was investigated using silane or chitosan‐modified cellulose papers. It was found that modified papers could reduce undesirable redistribution of CDs, during paper drying. Also, only chitosan‐modified filter paper was suitable for the successful immobilization of NSCDs. The effect of paper type, chitosan amount, pH, and NSCDs concentration was also studied, and a Whatman No. 42 filter paper modified with chitosan (1% w/v), pH 8.0, and an NSCD concentration of 2.5 g L ⁻¹ being selected for further studies. The sensor exhibited high selectivity for lead(II) compared with other metal ions because lead(II) resulted in the most significant changes in the emitted light intensity. Variations in NSCDs fluorescence were measured using a fluorescence imaging system. The NSCDs‐paper sensor showed a linear relationship between mean fluorescence intensity and lead(II) in the concentration range of 5.00–1.25 × 10 ² μmol L ⁻¹ with a correlation coefficient ( R ² ) of 0.9988 and a detection limit of 4.50 μmol L ⁻¹ . The suggested method showed satisfying results for lead(II) determination in different samples as a fast and low‐cost approach with on‐site application.
The colorimetric paper-based device using lipase inhibition assay was developed for rapid and visual detection of orlistat in weight loss supplements. The paper device with five circular detection zones was simply fabricated from filter paper No 1 using a low-cost paper craft puncher with a design of a flower-like shape. The enzymatic reaction on the detection zone employed a small volume of a substrate, a-naphthyl acetate, a sample, and a lipase enzyme. After incubation, the Fast Blue B solution was used as a chromogenic reagent. The decrease in purple color can be observed by the naked eye, in the presence of orlistat. Under optimized conditions, the paper device showed satisfactory sensitivity and selectivity. The device was applied for rapid screening of orlistat in weight loss supplements and the results agreed with those obtained from TLC analysis.
Among the analytical tools, paper-based analytical devices (PADs) have become a leading alternative for point-of care testing (POCT). In this study, PADs were fabricated using an office laser printer. Then, the paper zone was modified with graphene oxide (GO) and pyrene derivatives, which provide a sufficient amount of carboxylic groups for conjugating antibodies. At an optimal pH, antibodies were covalently bound onto carboxylated cellulose surface in an oriented manner through a two-step strategy: electrostatic adsorption was followed by EDC/NHS coupling. α-fetoprotein (AFP) as a detection model, we compared with cellulose powder modified and unmodified paper zone. The results showed the color intensity and color uniformity on GO modified paper was improved. The activity of immobilized antibodies on GO/1-pyrenebutyric acid (GO/PBA) modified was three times higher than that of GO modified and about 1.8-fold higher than that of GO/1-pyrenecarboxylic acid (GO/PCA) modified. The GO/PBA modified paper-based immunoassay has enhanced sensitivity and low detection limit. A linear correlation between color intensity and concentration of AFP in the range of 0.01～16.5 ng mL-1 with a detection limit of 9.0 pg mL-1 were achieved, respectively. The obtained results point towards rapid, sensitive, and specific early diagnosis of liver cancer at the point of care and other low-resource settings.
Paper-based analytical devices (PADs) are powerful platforms for point-of-need testing since they are inexpensive devices fabricated in different shapes and miniaturized sizes, ensuring better portability. Additionally, the readout and detection systems can be accomplished with portable devices, allying with the features of both systems. These devices have been introduced as promising analytical platforms to meet critical demands involving rapid, reliable, and simple testing. They have been applied to monitor species related to environmental, health, and food issues. Herein, an outline of chronological events involving PADs is first reported. This work also introduces insights into fundamental parameters to engineer new analytical platforms, including the paper type and device operation. The discussions involve the main analytical techniques used as detection systems, such as colorimetry, fluorescence, and electrochemistry. It also showed recent advances involving PADs, especially combining optical and electrochemical detection into a single device. Dual/combined detection systems can overcome individual barriers of the analytical techniques, making possible simultaneous determinations, or enhancing the devices’ sensitivity and/or selectivity. In addition, this review reports on distance-based detection, which is also considered a trend in analytical chemistry. Distance-based detection offers instrument-free analyses and avoids user interpretation errors, which are outstanding features for analyses at the point of need, especially for resource-limited regions. Finally, this review provides a critical overview of the practical specifications of the recent analytical platforms involving PADs, demonstrating their challenges. Therefore, this work can be a highly useful reference for new research and innovation.
Graphical Abstract
A digital colorimetric sensor was developed for the on-site measurement of iodate in food-grade salt. It involved a paper-based analytical device (PAD) containing an immobilized iodide/starch reagent and a smartphone as the detection system. Iodate ions produce a blue-black coloration on the PAD, the smartphone captures a digital image of the PAD, and an installed mobile app performs an analysis to generate color values related to the intensity of the color in the PAD. Parameters in the image acquisition and PAD preparation were optimized, and a matrix-matched calibration plot was employed for quantification. The plot exhibited a linear response in the concentration range of 10 to 100 mg iodate kg-1 of salt, and a detection limit of 2.20 mg kg-1 was determined. The present method was applied to analyze real samples, and the results agreed well with those obtained using the reference method at the 95% confidence level.
Using microfluidic paper-based analytical devices has attracted considerable attention in recent years. This is mainly due to their low cost, availability, portability, simple design, high selectivity, and sensitivity. Owing to their specific substrates and catalytic functions, enzymes are the most commonly used bioactive agents in μPADs. Enzymatic μPADs are various in design, fabrication, and detection methods. This paper provides a comprehensive review of the development of enzymatic μPADs by considering the methods of detection and fabrication. Particularly, techniques for mass production of these enzymatic μPADs for use in different fields such as medicine, environment, agriculture, and food industries are critically discussed. This paper aims to provide a critical review of μPADs and discuss different fabrication methods as the central parts of the μPADs production categorized into printable and non-printable methods. In addition, state-of-the-art technologies such as fully printed enzymatic μPADs for rapid, low-cost, and mass production and improvement have been considered.
Purpose
The purpose of this paper is to predict a suitable paper substrate which has high capillary pressure with the tendency of subsequent fluid wrenching in onward direction for the fabrication of microfluidics device application.
Design/methodology/approach
The experiment has been done on the Whatman TM grade 1, Whatman TM chromatography and nitrocellulose paper samples which are made by GE Healthcare Life Sciences. The structural characterization of paper samples for surface properties has been done by scanning electron microscope and ImageJ software. Identification of functional groups on the surface of samples has been done by Fourier transform infrared analysis. A finite elemental analysis has also been performed by using the “Multiphase Flow in Porous Media” module of the COMSOL Multiphysics tool which combines Darcy’s law and Phase Transport in Porous Media interface.
Findings
Experimentally, it has been concluded that the paper substrate for flexible microfluidic device application must have large number of internal (intra- and interfiber) pores with fewer void spaces (external pores) that have high capillary pressure to propel the fluid in onward direction with narrow paper fiber channel.
Originality/value
Surface structure has a dynamic impact in paper substrate utilization in multiple applications such as paper manufacturing, printing process and microfluidics applications.
Lysine is one of the essential amino acids and plays a vital role in the growth, development and health of pigs. Blood lysine concentration is a direct indication of lysine status; however, current methods can not satisfy the demands for rapid and on-site lysine concentration measurement of swine serum. Here, we developed blue-emissive nitrogen-doped carbon dots as a fluorescence probe for the determination of lysine with high fluorescence quantum yield, stability, sensitivity and specificity. The carbon dots were entrapped within hydrogel microstructures to fabricate microfluidic chips for rapid assay for lysine quantification. We further developed an imaging attachment to integrate the microfluidic chip and a smartphone into a portable point-of-care testing platform. This platform requires only 3 μL sample and has a linear detection range of 25 to 300 μmol/L with a limit of detection less than 16 μmol/L, which covers the normal range of lysine concentration in swine serum. We tested lysine concentration in swine serum using this platform with high accuracy, low sample consumption, and within 3 min. Together, these results may provide a rapid and portable platform for dynamic monitoring of swine lysine status and contribute to precise feed formula modulation with low-protein diet strategy.
A µPAD to determine the total polyphenol content (TPC) by Folin-Ciocalteau (FC) method, and antioxidant capacity by ABTS method, coupling an in-situ pretreatment zone with Carrez reagents is proposed. Conditions of FC, K2S2O8, ABTS reagents concentrations, pH, and incubation were established using 2k factorial designs. The proposed µPAD showed linearities upper than 0.99 in ranges 0.150-2.000 mg L⁻¹ and 0.100-1.000 mg L⁻¹ for FC and ABTS methods, respectively, detection limits were lower than 0.135 mg L⁻¹, precision lower than 13.47 %, and recoveries between 82.27 % and 114.60 %. Ten different food samples were analyzed to demonstrate the applicability of the µPAD, and the results were compared with those obtained by the batch methods, showing no significant differences (p > 0.05). The µPAD in-situ sample treatment allowed removing interferences before the TPC and ABTS detection avoiding overestimation of the results.
Conventional detectors are mostly made up of complicated structures that are hard to use. A paper-based microfluidic chip, however, combines the advantages of being small, efficient, easy to process, and environmentally friendly. The paper-based microfluidic chips for biomedical applications focus on efficiency, accuracy, integration, and innovation. Therefore, continuous progress is observed in the transition from single-channel detection to multi-channel detection and in the shift from qualitative detection to quantitative detection. These developments improved the efficiency and accuracy of single-cell substance detection. Paper-based microfluidic chips can provide insight into a variety of fields, including biomedicine and other related fields. This review looks at how paper-based microfluidic chips are prepared, analyzed, and used to help with both biomedical development and functional integration, ideally at the same time.
In this review, we provide an overview of the nanopaper biosensors used for point of care (POC) diagnostics in various fields such as biomedical, environmental, food safety, and agriculture. The lateral flow assays (LFAs) comprising nanoparticles have drawn the interest of researchers due to their high sensitivity, low cost, lower time consumption, lack of equipment, and easy handling characteristics. These assays have become an integral part of the health sector all over the world over a short time period and are extensively being engaged in diagnosis of viral infections in regions low on resources. A large number of innovative approaches have been introduced in making these test-strips or nanopaper biosensors as quickly as possible, because of their ease of operation, cheaper cost, and quick results, even with the simplest setups or in pathological laboratories, etc. Keeping all this in mind, we have reviewed the nanoparticles based lateral flow test strips (LFTS), their composition, working methods, reaction mechanisms, detection methods, and applications in different fields. We also provide an overview of paper-based microfluidic analytical devices (μPADs) and our perspective about the future trends which may facilitate understanding their applications.
Soft, skin-mounted microfluidic devices can collect microliter volumes of eccrine sweat and are capable of in situ real-time analysis of different biomarkers to assess physiological state and health. Chrono-analysis of sweat can be implemented to monitor temporal variations of biomarker concentrations over a certain period of interest. Conventional methods used to capture sweat or some of the newly developed microfluidic platforms for sweat collection and analysis are based on absorbent pads. They suffer from evaporation, leading to considerable deviations in the concentration of the biomarkers. Here, a paper-integrated microfluidic device is presented for sequential analysis of sweat that is easy to fabricate and does not include air exits for each reservoir, which reduces undesirable effects of sweat evaporation. Furthermore, the high capillary force of filter paper is leveraged to route the liquid into the chambers in a sequential fashion and allow further chemical analysis. The employed design of the paper-embedded microfluidic device successfully samples and analyzes artificial sweat sequentially for flow rates up to 5 μL min⁻¹ without showing any leakage. We demonstrated the performance of the device, employing colorimetric assays for chrono-analysis of glucose standard solutions at concentrations in the range of 10–100 mM and pH of sweat during exercise. The results reveal the presented approach's functionality and potential to analyze the concentration of biomarkers over a certain period sequentially.
Microfluidic paper-based analytical devices (μ-PADs) have the potential for simple and rapid disease diagnostic testing through the implementation of colorimetric assays using an external camera and image analysis; this is particularly significant in countries with limited access to medical facilities and in point-of-care diagnostics. The attractiveness of these devices arises from their relative ease of fabrication, low cost, portability, and the absence of external power source requirements. Typical device structures have hydrophilic channels through which liquids can flow and react with reagents located in reaction zones; channels are bounded by either hydrophobic porous material that prevents wetting, or by non-porous walls. μ-PADs have shown their potential in fulfilling the ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid, and robust, Equipment-free and Deliverable to those who need it) for diagnostic methods stipulated by World Health Organization (WHO). A variety of studies have demonstrated that μ-PADs can be used to detect analytes with relevance for disease diagnostics, to evaluate food quality and monitor environmental conditions, and for blood screening and testing.
In spite of the promising initial results, μ-PADs are yet not fully commercialized because of the multiple technical challenges such as—1) improper μ-PAD packaging which leads to sample contamination and sample lose due to evaporation 2) Lack of flow control in flow channels, which restricts μ-PAD applications for multistep protocols and hinders the automation of μ-PAD functions 3) Need for sample preparation steps prior to μ-PAD applications, which increases device reliance on external equipment and is not ideal for usage in low-resource environments 4) Poor signal quality in detection zones of μ-PADs,
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which leads to unreliable results 5) Effect of sample matrix and external lighting on colorimetric signals which further decreases the reliability of assay results generated using μ-PADs.
In this thesis, we have tackled the challenges explained above by developing a novel μ-PAD fabrication method using plasma processes. We developed a two-step process to create both semi- and fully enclosed flow channels in a single layer of paper. We demonstrated that μ-PADs made using fully enclosed channels can be easily packaged using low-cost adhesive tape to prevent sample contamination and evaporation. We validated the proposed fabrication method by designing a low-cost colorimetric glucose sensor that can measure glucose concentration in clinically relevant range.
We also developed a μ-PAD with flow control functionality to enable automated multistep fluid handling protocols that are required for complex assays such as ELISA. As, μ-PADs with flow control functionality were also fully enclosed, safe and effective packaging of the device could be achieved using simple plastic adhesive tape. To validate the incorporation of flow control functionality, we designed a μ-PAD that can sequentially deliver three different reagents, each with different pH, to a detection zone spotted with a pH indicator; change in indicator color was observed over time as different reagents sequentially arrived to the detection zone.
Finally, we developed a method to fabricate semi-enclosed μ-PADs directly on commercially available blood separation membrane to incorporate plasma separation functionality on the device itself. This reduces the need to rely on external equipment like centrifuges, which are often used to separate blood plasma from whole blood. Additionally,
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the detection zones of the semi-enclosed μ-PADs on the blood separation membrane were modified with cellulose nanocrystals to enhance color signal quality by increasing the homogeneity of the colorimetric signals across the detection zone. The complete μ-PAD was then used for colorimetric detection of glucose and albumin simultaneously in a whole blood sample. One disadvantage of the blood separation membrane is its brittleness, but we showed that the semi-enclosed μ-PADs can be easily supported by plastic adhesive tape layer to make them mechanically more robust as stand-alone devices, a critical factor for on-field device application. Finally, we tested the compatibility of semi-enclosed μ-PADs with a cell-free expression system to semi-quantitatively measure zinc concentration in a whole blood sample without using any external equipment. We were able to demonstrate the compatibility of cell-free expression system with fiber-based porous substrates. We were also able to show that μ-PADs fabricated on chromatography substrate can detect the presence/absence of analyte in the sample. However, dose-dependent distinct colorimetric signals were not obtained. Further, it was shown that patterned LF1 membranes are not compatible with cell-free expression system in its current form and further membrane treatment is required to generate any colorimetric signals on this substrate. Because we incorporated parallel calibration reactions in the device, we envisioned that the final device design will be able to inhibit external lighting effects, thus producing more reliable colorimetric signals.
With the goal of creating a multipurpose platform for electrogenerated luminescence, a single electrode electrochemical system was designed, developed, and validated. Glow sticks were used as the source of the luminophore, which was used as the optical reporter for the biosensor. A smartphone was used as the detector to quantify the electrochemiluminescence emissions. A disposable paper-based device was designed and used as a two-compartment electrochemical reaction cell, affording the possibility to individually optimize the sensing and detection reactions. This sensor assembly was tested under different conditions, showing acceptable performance both in the determination of hydrogen peroxide concentrations, to evaluate rancidity markers in edible oil samples, and to quantify the glucose concentration in soft drinks. The analytical performance of the single electrode, electrochemiluminescent device showed a limit of detection for hydrogen peroxide of 1.02 μM, with a working range between 0.4 μM and 150 mM. The proposed approach represents the first example of a system that combines paper-based devices, single electrode electrochemistry, electrochemiluminescence, and smartphone image sensing. As such, it not only provides a convenient platform for the development of a variety of analytical applications but also broaden the versatility of ePADs.
Herein, a fully paper-based biobattery composed of four microbial fuel cell (MFC) units is evaluated, one that can be prepared in advance, stored, and quickly activated with virtually any available fluid. The biobattery uniquely utilizes Bacillus subtilis endospores as the storable anodic biocatalyst; the dormant, robust nature of B. subtilis endospores should allow for device preinoculation with spores followed by prolonged storage of the fully fabricated paper battery until needed. A germinant paper layer strategically fabricated above the spore-loaded anode layer contains all of the necessary chemical germinants and nutrient components required for the endospores to begin germination, exit dormancy, and return to fully metabolic vegetative bacterial cells that can generate electrical energy. This mechanism allows for the battery to be simply initiated via a wide range of available liquids. Bioelectricity generation of the battery is successfully demonstrated after introduction of a variety of artificial bodily fluids, including saliva, sweat, and urine, along with tap water. Since the biobattery has the capability of serially linking all 4 of its MFCs through simple dynamic folding, the device’s total power output can be greatly enhanced; a single biobattery is able to achieve 0.56 V and 2.4 μW, which is beyond the ratings required for their intended application in single-use, disposable sensors. Therefore, this concept of integrating 4 spore-based MFCs into a single biobattery device with a built-in germinant layer offers a potential solution for stable, long-term storable power sources, displaying feasibility for integration with low power, disposable sensor applications.
Microfluidic paper-based analytical devices (μPADs) are promising biosensors that may be used in a variety of bioanalytical applications. A μPAD for automating the competitive enzyme-linked immunosorbent assay (ELISA) of small-sized target detection at the femtogram level using submicroliter samples is reported in this study. The proposed μPAD was integrated with a sucrose valve to automate the sequential delivery of reagents, providing simple control of reagent delivery time and simple operation. The use of a sample solution dropping location at the zones on the device that had been prepared with an antibody-conjugated enzyme before immersion in a running buffer allowed minimization of sample volume to 0.6 μL, while eliminating the possible loss of a target molecule by adsorption on the membrane, thus improving detection sensitivity. Furthermore, the proposed device was successfully applied to the automation of competitive ELISA for the detection of aflatoxin B1 (AFB1), a potent carcinogen that causes substantial health risks to humans worldwide, with a detection limit of 60 femtograms or 0.1 ng/mL. The method developed in this study provides high sensitivity, small sample volume, on-site and equipment-free measurements, low-cost operation, and user-friendliness. This approach could be used to analyze small-sized molecules in the fields of food safety and quality control, environmental monitoring, and clinical diagnostics.
The miniaturization of analysis systems brings about advantages like point-of-care usage, reduce sample volume consumption, and eliminate the need of bulky machines and skilled technicians for operation. Miniaturized analysis systems had also found relevance in application of various fields ranging from chemical analysis, biodefense to wearable electronics. This chapter describes fabrication methods for miniaturized analysis system made of different materials like paper, polymer, glass, and silicon. Different material has its strength and weaknesses and considerations discussed on material choices help push the fabricated analysis systems beyond its limitations. Production of miniaturized systems involves fabrication and assembly of fluidics, electronics, valves, and electronic components at a micro- or nanoscale while taking into considerations conditions unique to the micro- and submicro world. Nowadays, analysis systems also need to integrate components for hyperconnectivity via Internet of Things. Finally, fabrication challenges and potential solutions to improve the performance of these miniaturized analysis systems are also pointed out. The future looks toward fabricating robust multiplexing, calibration free analysis systems that can scale further down the submicron scale while being well suited for mass production and well connected to the internet for better data acquisition, data analysis, and eventually translate to useful interpretation of information for sensor applications in all the different fields.
This chapter presents and discusses the most usual methodologies for colorimetric detection on paper-based analytical devices (PADs). State of the art associated with pioneering, popularity, physical aspect, surface modifications, instrumental and instrumental-free colorimetric measurements are carefully presented. Moreover, a side-by-side comparison among the most usual colorimetric detection modes on PADs was performed using a well-known complexometric reaction model. The pros and cons were discussed to show their advantages for chemical analysis.
Electrochemical paper-based analytical devices (ePADs) are receiving increasing attention in recent years, overcoming the publication number of colorimetric paper-based analytical devices in the literature. This chapter covers recent advances in the manufacturing of ePADs for chemical analysis. Initially, an introduction about its beginning in the literature and mainly practical theoretical principles are discussed. The following sections present how the devices are being fabricated and used in the literature, exploring their advantages and limitations and describing electrochemical sensing detection and future outlooks.
The main focus of this chapter is to discuss the primary materials and processes relevant to the development of paper-based analytical devices, including the steps required for the separation of cellulose from wood and non-wood sources and how some of the additives added to the material could impact the analytical properties of the resulting analytical devices. The chapter also discusses the most important routes for the chemical modification of cellulose, including thermal treatment.
A new technique for the detection of explosives has been developed based on fluorescence quenching of pyrene on paper-based analytical devices (μPADs). Wax barriers were generated (150 °C, 5 min) using ten different colours. Magenta was found as the most suitable wax colour for the generation of the hydrophobic barriers with a nominal width of 120 μm resulting in fully functioning hydrophobic barriers. One microliter of 0.5 mg mL(-1) pyrene dissolved in an 80 : 20 methanol-water solution was deposited on the hydrophobic circle (5 mm diameter) to produce the active microchip device. Under ultra-violet (UV) illumination, ten different organic explosives were detected using the μPAD, with limits of detection ranging from 100-600 ppm. A prototype of a portable battery operated instrument using a 3 W power UV light-emitting-diode (LED) (365 nm) and a photodiode sensor was also built and evaluated for the successful automatic detection of explosives and potential application for field-based screening.
Dipstick and lateral-flow formats have dominated rapid diagnostics over the last three decades. These formats gained popularity in the consumer markets due to their compactness, portability and facile interpretation without external instrumentation. However, lack of quantitation in measurements has challenged the demand of existing assay formats in consumer markets. Recently, paper-based microfluidics has emerged as a multiplexable point-of-care platform which might transcend the capabilities of existing assays in resource-limited settings. However, paper-based microfluidics can enable fluid handling and quantitative analysis for potential applications in healthcare, veterinary medicine, environmental monitoring and food safety. Currently, in its early development stages, paper-based microfluidics is considered a low-cost, lightweight, and disposable technology. The aim of this review is to discuss: (1) fabrication of paper-based microfluidic devices, (2) functionalisation of microfluidic components to increase the capabilities and the performance, (3) introduction of existing detection techniques to the paper platform and (4) exploration of extracting quantitative readouts via handheld devices and camera phones. Additionally, this review includes challenges to scaling up, commercialisation and regulatory issues. The factors which limit paper-based microfluidic devices to become real world products and future directions are also identified.
In this paper, we report a simple, low-cost method for rapid, highly reproductive fabrication of paper-based microfluidics by using a commercially available, minitype CO(2) laser cutting/engraving machine. This method involves only one operation of cutting a piece of paper by laser according to a predesigned pattern. The hollow microstructures formed in the paper are used as the 'hydrophobic barriers' to define the hydrophilic flowing paths. A typical paper device on a 4 cm × 4 cm piece of paper can be fabricated within ∼7-20 s; it is ready for use once the cutting process is finished. The main fabrication parameters such as the applied current and cutting rate of the laser were optimized. The fabrication resolution and multiplexed analytical capability of the hollow microstructure-patterned paper were also characterized.
In developed nations, monitoring for drug-induced liver injury through serial measurements of serum transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] in at-risk individuals is the standard of care. Despite the need, monitoring for drug-related hepatotoxicity in resource-limited settings is often limited by expense and logistics, even for patients at highest risk. This article describes the development and clinical testing of a paper-based, multiplexed microfluidic assay designed for rapid, semiquantitative measurement of AST and ALT in a fingerstick specimen. Using 223 clinical specimens obtained by venipuncture and 10 fingerstick specimens from healthy volunteers, we have shown that our assay can, in 15 min, provide visual measurements of AST and ALT in whole blood or serum, which allow the user to place those values into one of three readout "bins" [<3× upper limit of normal (ULN), 3 to 5× ULN, and >5× ULN, corresponding to tuberculosis/HIV treatment guidelines] with >90% accuracy. These data suggest that the ultimate point-of-care fingerstick device will have high impact on patient care in low-resource settings.
In this paper, we report the progress in using paper sizing chemistry to fabricate patterned paper for chemical and biological
sensing applications. Patterned paper sizing uses paper sizing agents to selectively hydrophobize certain area of a sheet.
The hydrophilic-hydrophobic contrast of the pattern so created has an excellent ability to control capillary penetration of
aqueous liquids in channels of the pattern. Incorporating this idea with digital ink jet printing technique, a new fabrication
method of paper-based microfluidic devices is established. Ink jet printing can deliver biomolecules and chemicals with precision
into the microfluidic patterns to form biological/chemical sensing sites within the patterns, forming the complete sensing
devices. This study shows the potential of combining paper sizing chemistry and ink jet printing to produce paper-based sensors
at low cost and at commercial volume.
KeywordsPaper microfluidics-Ink jet printing-Paper sizing-Low-cost sensors
In this work, we first employ a drying method combining with the bienzyme colorimetric detection of glucose and uric acid on microfluidic paper-based analysis devices (μPADs). The channels of 3D μPADs are also designed by us to get better results. The color results are recorded by both Gel Documentation systems and a common camera. By using Gel Documentation systems, the limits of detection (LOD) of glucose and uric acid are 3.81 × 10(-5)M and 4.31 × 10(-5)M, respectively one order of magnitude lower than that of the reported methods on μPADs. By using a common camera, the limits of detection (LOD) of glucose and uric acid are 2.13 × 10(-4)M and 2.87 × 10(-4)M, respectively. Furthermore, the effects of detection conditions have been investigated and discussed comprehensively. Human serum samples are detected with satisfactory results, which are comparable with the clinical testing results. A low-cost, simple and rapid colorimetric method for the simultaneous detection of glucose and uric acid on the μPADs has been developed with enhanced sensitivity.
This paper describes three-dimensional microfluidic paper-based analytical devices (3-D microPADs) that can be programmed (postfabrication) by the user to generate multiple patterns of flow through them. These devices are programmed by pressing single-use 'on' buttons, using a stylus or a ballpoint pen. Pressing a button closes a small space (gap) between two vertically aligned microfluidic channels, and allows fluids to wick from one channel to the other. These devices are simple to fabricate, and are made entirely out of paper and double-sided adhesive tape. Programmable devices expand the capabilities of microPADs and provide a simple method for controlling the movement of fluids in paper-based channels. They are the conceptual equivalent of field-programmable gate arrays (FPGAs) widely used in electronics.
This article describes the use of microfluidic paper-based analytical devices (μPADs) to perform quantitative chemical assays with internal standards. MicroPADs are well-suited for colorimetric biochemical assays; however, errors can be introduced from the background color of the paper due to batch difference and age, and from color measurement devices. To reduce errors from these sources, a series of standard analyte solutions and the sample solution are assayed on a single device with multiple detection zones simultaneously; an analyte concentration calibration curve can thus be established from the standards. Since the μPAD design allows the colorimetric measurements of the standards and the sample to be conducted simultaneously and under the same condition, errors from the above sources can be minimized. The analytical approach reported in this work shows that μPADs can perform quantitative chemical analysis at very low cost.
Figure
The scanned images of microfluidic paper-based analytical devices for colorimetric multi-analyte detection (left) and quantitative biomarker assay (right)
Electrophoresis 2014, 35, 2325–2332. DOI: 10.1002/elps.elps201300511
In order to demonstrate the versatility of the proposed methodology, a PMMA substrate was engraved following the conditions described in our article (CO 2 laser engraving, raster mode, 1200 dpi) and then imaged using a 3D microscope. We believe these two instruments are very good complements of the traditional instrumentation linked to the fabrication and characterization of microfluidic devices and could provide not only a simple way to produce devices but also a new way to see the surface of the features produced. image
Point-of-care platforms can provide fast responses, decrease the overall cost of the treatment, allow for in-home determinations with or without a trained specialist, and improve the success of the treatment. This is especially true for microfluidic paper-based analytical devices (μPAD), which can enable the development of highly efficient and versatile analytical tools with applications in a variety of biomedical fields. The objective of this work was the development of μPADs to identify and quantify levels of nitrite in saliva, which has been proposed as a potential marker of periodontitis. The devices were fabricated by wax printing and allowed the detection of nitrite by a colorimetric reaction based on a modified version of the Griess reaction. The presented modifications, along with the implementation of a paper-based platform, address many of the common drawbacks (color development, stability, etc.) associated with the Griess reaction and are supported by results related to the design, characterization, and application of the proposed devices. Under the optimized conditions, the proposed devices enable the determination of nitrite in the 10-1000μmolL(-1) range with a limit of detection of 10μmolL(-1) and a sensitivity of 47.5AU [log (μmolL(-1))](-1). In order to demonstrate the potential impact of this technology in the healthcare industry, the devices were applied to the analysis of a series of real samples, covering the relevant clinical range.
Photolithography was used to pattern hydrophobic design on common quantitative filter paper, and then the paper-based micro-zone plates were successfully fabricated. In order to improve its protein-adsorbing capacity for further signal magnification, modified silicon dioxide (SiO2) micro-beads were deposited onto the surface of paper. Indirect-ELISA for goat anti-rabbit IgG was realized on the 36-zone plates. Taking enzyme-linked goat anti-rabbit IgG as an example, loading SiO2 beads could remarkably increase the quantity of the adsorbed protein by 20%–700%. When this method was used for goat anti-rabbit IgG detection, signals were magnified by 20%–150%, and the linear range of detection was from 3 × 10−12 mmol to 3 × 10−8 mmol per zone. In this manner, bio-reagent consumption was reduced to 3–5 μL per zone, test time dropped to 25 min per plate and the cost of fabrication was reduced to $1.1 per plate. This work suggests a new way for development of new diagnosis devices.
Paper-based microfluidic devices have recently garnered an increasing interest in the literature. The majority of these devices were produced by patterning hydrophobic zones in hydrophilic paper via photoresist or wax. Others were created by cutting paper using a laser. Here, we present a fabrication method for producing devices by simple craft-cutting and lamination, in a way similar to making an identification (ID) card. The method employs a digital craft cutter and roll laminator to produce laminated paper-based analytical devices (LPAD). Lamination with a plastic backing provides the mechanical strength for a paper device. The approach of using a craft cutter and laminator makes it possible to rapid-prototype LPAD with no more difficulty than producing a typical ID card, at very low cost. Devices constructed using this method have been exploited for simultaneous detection of bovine serum albumin (BSA) and glucose in synthetic urine with colorimetric assays. Both BSA and glucose are detectable at clinically relevant concentrations, with the detection limit at 2.5 μM for BSA and 0.5 mM for glucose.
Rigid substrate e.g. glassy carbon is typically used in biofuel cells (BFCs). Compared with the substrates, paper-based materials are low cost, lightweight, flexible design and easy to manufacture. In this work, flexible BFCs have been constructed on filter papers coated with a composite material of cellulose and carbon nanotube as binder. For mediator-less electrode reactions, biliribin oxidase and fructose dehydrogenese were employed as cathodic and anodic biocatalysts respectively. The power of a BFC unit reached 4.3 μW while the cathode was the main limitation based on discharge experiments. The BFCs can be designed and constructed to various shapes on paper substrates in series and parallel configuration, a maximum power output of 7.9 μW was produced. The voltage reversal behavior of individual cell in stack has also been investigated for the first time, which was caused by the unit with lowest ability in power generation in stack. This unique BFC obtained here is promising to be used as a kind of flexible power resources for wearable or rolling-up devices in practice.
Lateral flow tests (LFTs) are an ingenious format for rapid and easy-to-use diagnostics, but they are fundamentally limited to assay chemistries that can be reduced to a single chemical step. In contrast, most laboratory diagnostic assays rely on multiple timed steps carried out by a human or a machine. Here, we use dissolvable sugar applied to paper to create programmable flow delays and present a paper network topology that uses these time delays to program automated multi-step fluidic protocols. Solutions of sucrose at different concentrations (10-70% of saturation) were added to paper strips and dried to create fluidic time delays spanning minutes to nearly an hour. A simple folding card format employing sugar delays was shown to automate a four-step fluidic process initiated by a single user activation step (folding the card); this device was used to perform a signal-amplified sandwich immunoassay for a diagnostic biomarker for malaria. The cards are capable of automating multi-step assay protocols normally used in laboratories, but in a rapid, low-cost, and easy-to-use format.
A simplified method for measuring the fluidic resistance (R(fluidic)) of microfluidic channels is presented, in which the electrical resistance (R(elec)) of a channel filled with a conductivity standard solution can be measured and directly correlated to R(fluidic) using a simple equation. Although a slight correction factor could be applied in this system to improve accuracy, results showed that a standard voltage meter could be used without calibration to determine R(fluidic) to within 12% error. Results accurate to within 2% were obtained when a geometric correction factor was applied using these particular channels. When compared to standard flow rate measurements, such as meniscus tracking in outlet tubing, this approach provided a more straightforward alternative and resulted in lower measurement error. The method was validated using 9 different fluidic resistance values (from ∼40 to 600kPasmm(-3)) and over 30 separately fabricated microfluidic devices. Furthermore, since the method is analogous to resistance measurements with a voltage meter in electrical circuits, dynamic R(fluidic) measurements were possible in more complex microfluidic designs. Microchannel R(elec) was shown to dynamically mimic pressure waveforms applied to a membrane in a variable microfluidic resistor. The variable resistor was then used to dynamically control aqueous-in-oil droplet sizes and spacing, providing a unique and convenient control system for droplet-generating devices. This conductivity-based method for fluidic resistance measurement is thus a useful tool for static or real-time characterization of microfluidic systems.
In this technical note, we describe a facile method for one-step fabrication of paper-based microfluidic devices, by simply using commercially available permanent markers and metal templates with specific patterns. The fabrication process involves only a single step of plotting pattern in paper; it can be typically finished within 1 min. The ink marks formed in the patterned paper will act as the hydrophobic barriers to define the hydrophilic flow paths or separate test zones. Various paper devices can be created by using different templates with corresponding patterns. Transparent adhesive tape-sandwiched devices could protect their assay surfaces from potential contamination. In the proof-of-concept experiments, circular paper test zones (~3 mm diameter) were fabricated for colorimetric and quantification detection of prostate-specific antigen (PSA) as a model target, based on dot-immunogold staining assays coupled with gold enhancement amplification. Several serum specimens were additionally evaluated with this new approach and the results were compared with the commercial chemiluminescence immunoassay, validating its feasibility of practical applications. Such a one-step plotting method for paper patterning does not require any specialized equipments and skills, is quite inexpensive and rapid, and thus holds great potential to find wide applications especially in remote regions and resource-limited environments such as small laboratories and private clinics.
Bioactive paper includes a range of potential paper-based materials that can perform analytical functions normally reserved for multi-well plates in the laboratory or for portable electronic devices. Pathogen detection is the most compelling application. Simple paper-based detection, not requiring hardware, has the potential to have impacts in society, ranging from the kitchen to disasters in the developing world. Bioactive-paper research is an emerging field with significant efforts in Canada, USA (Harvard), Finland and Australia.Following a brief introduction to the material and surface properties of paper, I review the literature. Some of the early work exploits the porosity of paper to generate paper-based microfluidics (“paperfluidics”) devices. I exclude from this review printed electronic devices and plastics-supported devices.
This communication describes the first paper-based microfluidic device that is capable of generating its own power when a sample is added to the device. The microfluidic device contains galvanic cells (that we term "fluidic batteries") integrated directly into the microfluidic channels, which provides a direct link between a power source and an analytical function within the device. This capability is demonstrated using an example device that simultaneously powers a surface-mount UV LED and conducts an on-chip fluorescence assay.
Foodborne pathogens are a major public health threat and financial burden for the food industry, individuals, and society, with an estimated 76 million cases of food-related illness occurring in the United States alone each year. Three of the most important causative bacterial agents of foodborne diseases are pathogenic strains of Escherichia coli , Salmonella spp., and Listeria monocytogenes , due to the severity and frequency of illness and disproportionally high number of fatalities. Their continued persistence in food has dictated the ongoing need for faster, simpler, and less expensive analytical systems capable of live pathogen detection in complex samples. Culture techniques for detection and identification of foodborne pathogens require 5-7 days to complete. Major improvements to molecular detection techniques have been introduced recently, including polymerase chain reaction (PCR). These methods can be tedious; require complex, expensive instrumentation; necessitate highly trained personnel; and are not easily amenable to routine screening. Here, a paper-based analytical device (μPAD) has been developed for the detection of E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes in food samples as a screening system. In this work, a paper-based microspot assay was created by use of wax printing on filter paper. Detection is achieved by measuring the color change when an enzyme associated with the pathogen of interest reacts with a chromogenic substrate. When combined with enrichment procedures, the method allows for an enrichment time of 12 h or less and is capable of detecting bacteria in concentrations in inoculated ready-to-eat (RTE) meat as low as 10(1) colony-forming units/cm(2).
This article introduces fully enclosed microfluidic paper-based analytical devices (microPADs) fabricated by printing toner on the top and bottom of the devices using a laser printer. Enclosing paper-based microfluidic channels protects the channels from contamination, contains and protects reagents stored on the device, contains fluids within the channels so that microPADs can be handled and operated more easily, and reduces evaporation of solutions from the channels. These benefits extend the capabilities of microPADs for applications as low-cost point-of-care diagnostic devices.
In this study, a novel microfluidic paper-based chemiluminescence analytical device (μPCAD) with a simultaneous, rapid, sensitive and quantitative response for glucose and uric acid was designed. This novel lab-on-paper biosensor is based on oxidase enzyme reactions (glucose oxidase and urate oxidase, respectively) and the chemiluminescence reaction between a rhodanine derivative and generated hydrogen peroxide in an acid medium. The possible chemiluminescence assay principle of this μPCAD is explained. We found that the simultaneous determination of glucose and uric acid could be achieved by differing the distances that the glucose and uric acid samples traveled. This lab-on-paper biosensor could provide reproducible results upon storage at 4 °C for at least 10 weeks. The application test of our μPCAD was then successfully performed with the simultaneous determination of glucose and uric acid in artificial urine. This study shows the successful integration of the μPCAD and the chemiluminescence method will be an easy-to-use, inexpensive, and portable alternative for point-of-care monitoring.
This Technical Note demonstrates a simple method based on flexographic printing of polystyrene to form liquid guiding boundaries and layers on paper substrates. The method allows formation of hydrophobic barrier structures that partially or completely penetrate through the substrate. This unique property enables one to form very thin fluidic channels on paper, leading to reduced sample volumes required in point-of-care diagnostic devices. The described method is compatible with roll-to-roll flexography units found in many printing houses, making it an ideal method for large-scale production of paper-based fluidic structures.
We report here the use of multiple indicators for a single analyte for paper-based microfluidic devices (microPAD) in an effort to improve the ability to visually discriminate between analyte concentrations. In existing microPADs, a single dye system is used for the measurement of a single analyte. In our approach, devices are designed to simultaneously quantify analytes using multiple indicators for each analyte improving the accuracy of the assay. The use of multiple indicators for a single analyte allows for different indicator colors to be generated at different analyte concentration ranges as well as increasing the ability to better visually discriminate colors. The principle of our devices is based on the oxidation of indicators by hydrogen peroxide produced by oxidase enzymes specific for each analyte. Each indicator reacts at different peroxide concentrations and therefore analyte concentrations, giving an extended range of operation. To demonstrate the utility of our approach, the mixture of 4-aminoantipyrine and 3,5-dichloro-2-hydroxy-benzenesulfonic acid, o-dianisidine dihydrochloride, potassium iodide, acid black, and acid yellow were chosen as the indicators for simultaneous semi-quantitative measurement of glucose, lactate, and uric acid on a microPAD. Our approach was successfully applied to quantify glucose (0.5-20 mM), lactate (1-25 mM), and uric acid (0.1-7 mM) in clinically relevant ranges. The determination of glucose, lactate, and uric acid in control serum and urine samples was also performed to demonstrate the applicability of this device for biological sample analysis. Finally results for the multi-indicator and single indicator system were compared using untrained readers to demonstrate the improvements in accuracy achieved with the new system.
The interest in low-cost microfluidic platforms as well as emerging microfabrication techniques has increased considerably over the last years. Toner- and paper-based techniques have appeared as two of the most promising platforms for the production of disposable devices for on-chip applications. This review focuses on recent advances in the fabrication techniques and in the analytical/bioanalytical applications of toner and paper-based devices. The discussion is divided in two parts dealing with (i) toner and (ii) paper devices. Examples of miniaturized devices fabricated by using direct-printing or toner transfer masking in polyester-toner, glass, PDMS as well as conductive platforms as recordable compact disks and printed circuit board are presented. The construction and the use of paper-based devices for off-site diagnosis and bioassays are also described to cover this emerging platform for low-cost diagnostics.
This paper described a convenient semiquantitative method for colorimetric detection of protein with self-calibration integrated on the test strip. Hydrophilic paper was employed as microfluidic device for running colorimetric assay, tree-shaped design was developed to ensure uniform microfluidic flow for multiple branches. The approach was validated with bovine serum albumin (BSA) colorimetric detection, and colorimetric results observed by naked eyes were consistent with that from apparatus. The device could be coupled with digital transmission of images for remote monitoring system for diagnosis, food control, and environmental analysis.
We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 μm wide with barriers as narrow as 250 μm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318–1320, 1; Martinez et al., Anal Chem 10:3699–3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606–19611, 3; Lu et al., Electrophoresis 9:1497–1500, 4; Abe et al., Anal Chem 18:6928–6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 μM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 μM) were generated. The time of device fabrication to the time of test results was about 25 min.
Paper-based microfluidic chip illustrating the colorimetric detection of salivary nitrite. Color intensities were quantified using a flatbed scanner and image manipulation software and plotted against concentration to produce calibration curves for the assay
This article describes an exceedingly simple and low-cost method for metering the capillary-driven flow rate of fluids within three-dimensional (3D) microfluidic, paper-based analytical devices (microPADs). Initial prototypes of 3D microPADs control the spatial distribution of fluids within a device, but they provide little control over how quickly (or slowly) fluids move within the device. The methods described in this article provide control over when and how quickly a fluid is distributed into detection zones. These methods are inexpensive (the metering regions are composed of paraffin wax), the devices are easy to fabricate, and they are capable of controlling the flow of fluids to detection zones with precise time delays (e.g., +/-6% of the total wicking time). We anticipate that this type of precise control over fluid distribution rates will be useful particularly for point-of-care assays that require multiple steps (where each step requires that the reagents interact for a defined period of time) or for simultaneously displaying the results of multiple different assays on a single device.
We have fabricated paper- and nitrocellulose-based lateral-flow devices that are shaped in two dimensions by a computer-controlled knife. The resulting star, candelabra, and other structures are spotted with multiple bioassay reagents to produce multiplex lateral-flow assays. We have also fabricated laminar composites in which porous nitrocellulose media are sandwiched between vinyl and polyester plastic films. This minimizes evaporation, protects assay surfaces from contamination and dehydration, and eliminates the need for the conventional hard plastic cassette holders that are typically used to package commercial lateral-flow diagnostic strips. The reported fabrication method is novel, low-cost, and well-suited to (i) fabrication and adoption in resource-poor areas, (ii) prototype development, (iii) high-volume manufacturing, and (iii) improving rates of operator error.
Microfluidic paper-based analytical devices (microPADs) are a new class of point-of-care diagnostic devices that are inexpensive, easy to use, and designed specifically for use in developing countries. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html.).
This article describes a point-of-care (POC) system--comprising a microfluidic, paper-based analytical device (micro-PAD) and a hand-held optical colorimeter--for quantifying the concentration of analytes in biological fluids. The micro-PAD runs colorimetric assays, and consists of paper that has been (i) patterned to expose isolated regions of hydrophilic zones and (ii) wet with an index-matching fluid (e.g., vegetable oil) that is applied using a disposable, plastic sleeve encasement. Measuring transmittance through paper represents a new method of quantitative detection that expands the potential functionality of micro-PADs. This prototype transmittance colorimeter is inexpensive, rugged, and fully self-contained, and thus potentially attractive for use in resource-limited environments and developing countries.
This paper describes 96- and 384-microzone plates fabricated in paper as alternatives to conventional multiwell plates fabricated in molded polymers. Paper-based plates are functionally related to plastic well plates, but they offer new capabilities. For example, paper-microzone plates are thin (approximately 180 microm), require small volumes of sample (5 microL per zone), and can be manufactured from inexpensive materials ($0.05 per plate). The paper-based plates are fabricated by patterning sheets of paper, using photolithography, into hydrophilic zones surrounded by hydrophobic polymeric barriers. This photolithography used an inexpensive formulation photoresist that allows rapid (approximately 15 min) prototyping of paper-based plates. These plates are compatible with conventional microplate readers for quantitative absorbance and fluorescence measurements. The limit of detection per zone loaded for fluorescence was 125 fmol for fluorescein isothiocyanate-labeled bovine serum albumin, and this level corresponds to 0.02 the quantity of analyte per well used to achieve comparable signal-to-noise in a 96-well plastic plate (using a solution of 25 nM labeled protein). The limits of detection for absorbance on paper was approximately 50 pmol per zone for both Coomassie Brilliant Blue and Amaranth dyes; these values were 0.4 that required for the plastic plate. Demonstration of quantitative colorimetric correlations using a scanner or camera to image the zones and to measure the intensity of color, makes it possible to conduct assays without a microplate reader.
Paper-based microfluidic patterns have been demonstrated in recent literature to have a significant potential in developing low-cost analytical devices for telemedicine and general health monitoring. This study reports a new method for making microfluidic patterns on a paper surface using plasma treatment. Paper was first hydrophobized and then treated using plasma in conjunction with a mask. This formed well defined hydrophilic channels on the paper. Paper-based microfluidic systems produced in this way retained the flexibility of paper and a variety of patterns could be formed. A major advantage of this system is that simple functional elements such as switches and filters can be built into the patterns. Examples of these elements are given in this study.
We report the first demonstration of electrochemical detection for paper-based microfluidic devices. Photolithography was used to make microfluidic channels on filter paper, and screen-printing technology was used to fabricate electrodes on the paper-based microfluidic devices. Screen-printed electrodes on paper were characterized using cyclic voltammetry to demonstrate the basic electrochemical performance of the system. The utility of our devices was then demonstrated with the determination of glucose, lactate, and uric acid in biological samples using oxidase enzyme (glucose oxidase, lactate oxidase, and uricase, respectively) reactions. Oxidase enzyme reactions produce H2O2 while decomposing their respective substrates, and therefore a single electrode type is needed for detection of multiple species. Selectivity of the working electrode for H2O2 was improved using Prussian Blue as a redox mediator. The determination of glucose, lactate, and uric acid in control serum samples was performed using chronoamperometry at the optimal detection potential for H2O2 (0 V versus the on-chip Ag/AgCl reference electrode). Levels of glucose and lactate in control serum samples measured using the paper devices were 4.9 +/- 0.6 and 1.2 +/- 0.2 mM (level I control sample), and 16.3 +/- 0.7 and 3.2 +/- 0.3 mM (level II control sample), respectively, and were within error of the values measured using traditional tests. This study shows the successful integration of paper-based microfluidics and electrochemical detection as an easy-to-use, inexpensive, and portable alternative for point of care monitoring.
This paper presents an inkjet printing method for the fabrication of entire microfluidic multianalyte chemical sensing devices made from paper suitable for quantitative analysis, requiring only a single printing apparatus. An inkjet printing device is used for the fabrication of three-dimensional hydrophilic microfluidic patterns (550-mum-wide flow channels) and sensing areas (1.5 mm x 1.5 mm squares) on filter paper, by inkjet etching, and thereby locally dissolving a hydrophobic poly(styrene) layer obtained by soaking of the filter paper in a 1 wt % solution of poly(styrene) in toluene. In a second step, the same inkjet printing device is used to print "chemical sensing inks", comprising the necessary reagents for colorimetric analytical assays, into well-defined areas of the patterned microfluidic paper devices. The arrangement of the patterns, printed inks, and sensing areas was optimized to obtain homogeneous color responses. The results are "all-inkjet-printed" chemical sensing devices for the simultaneous determination of pH, total protein, and glucose in clinically relevant concentration ranges for urine analysis (0.46-46 muM for human serum albumin, 2.8-28.0 mM for glucose, and pH 5-9). Quantitative data are obtained by digital color analysis in the L*a*b* color space by means of a color scanner and a simple computer program.
(Chemical Equation Presented) By the book: A method for patterning paper with photoresist to create well-defined, millimeter-sized channels comprising hydrophilic paper bounded by hydrophobic polymer is described. This type of patterned paper is a prototype of a class of low-cost, portable, and technically simple platforms for running multiplexed bioassays with microliter volumes of a single biological sample.
This article describes a prototype system for quantifying bioassays and for exchanging the results of the assays digitally with physicians located off-site. The system uses paper-based microfluidic devices for running multiple assays simultaneously, camera phones or portable scanners for digitizing the intensity of color associated with each colorimetric assay, and established communications infrastructure for transferring the digital information from the assay site to an off-site laboratory for analysis by a trained medical professional; the diagnosis then can be returned directly to the healthcare provider in the field. The microfluidic devices were fabricated in paper using photolithography and were functionalized with reagents for colorimetric assays. The results of the assays were quantified by comparing the intensities of the color developed in each assay with those of calibration curves. An example of this system quantified clinically relevant concentrations of glucose and protein in artificial urine. The combination of patterned paper, a portable method for obtaining digital images, and a method for exchanging results of the assays with off-site diagnosticians offers new opportunities for inexpensive monitoring of health, especially in situations that require physicians to travel to patients (e.g., in the developing world, in emergency management, and during field operations by the military) to obtain diagnostic information that might be obtained more effectively by less valuable personnel.
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