Paper-based Diagnostic Devices Research Articles

Solute imbibition in paper strip: Pore-scale insights into the concentration-dependent permeability

Capillary wicking in a thicker gel blot microfluidics paper has been investigated through a combination of an analytical framework, experiments, and numerical simulations. The primary objectives of this work are to investigate the concentration-dependent wicking process inside thicker microfluidic paper and to estimate the concentration-dependent permeability using both theoretical models and experimental data. An additional goal is to estimate the parameters for saturation-dependent flow modeling in thicker microfluidic paper. To comprehend the wicking phenomenon on thicker gel blot paper, a series of experiments employing aqueous food dye solutions at varying concentrations has been conducted. In order to calculate the temporal wicking length analytically, the Brinkman-extended Darcy equation is implemented. By modifying the permeability expression for a simple rectangular unidirectional fiber cell and pure liquid, the expression of effective permeability for the analytical framework has also been introduced. The concentrations of the food dye solutions appear to have a substantial influence on the wicking phenomenon. Effective permeability and wicking length have been found to follow a decreasing pattern at lower concentrations while both increase at higher values. Intriguingly, employing a microfluidics paper with a relatively greater thickness facilitates the visualization of the fluid front. This phenomenon is identified by the formation of an acute angle at intermediate time instants, while the fluid front angle assumes an angle nearly ∼90° during smaller and higher time instants. In order to evaluate the saturation-dependent capillary pressure and permeability, the empirical correlation of concentration-dependent Brooks and Corey parameters is additionally determined experimentally. These parameters are subsequently employed in numerical simulations to illustrate the saturation-dependent flow field using Richards’ equation. Furthermore, numerical simulations based on these estimated model parameters have been conducted, and it turns out that the saturation field has an excellent agreement with the experimental results. The results of the current study can be used to design low-cost paper-based diagnostic devices for usage in healthcare and environmental applications.

Advances in paper based isothermal nucleic acid amplification tests for water-related infectious diseases

Water-associated or water-related infectious disease outbreaks are caused by pathogens such as bacteria, viruses, and protozoa, which can be transmitted through contaminated water sources, poor sanitation practices, or insect vectors. Low- and middle-income countries bear the major burden of these infections due to inadequate hygiene and subpar laboratory facilities, making it challenging to monitor and detect infections in a timely manner. However, even developed countries are not immune to these diseases, as inadequate wastewater management and contaminated drinking water supplies can also contribute to disease outbreaks. Nucleic acid amplification tests have proven to be effective for early disease intervention and surveillance of both new and existing diseases. In recent years, paper-based diagnostic devices have made significant progress and become an essential tool in detecting and managing water-associated infectious diseases. In this review, we have highlighted the importance of paper and its variants as a diagnostic tool and discussed the properties, designs, modifications, and various paper-based device formats developed and used for detecting water-associated pathogens.

Carbon nanotubes modified conductive ink for application to paper-based electrochemical biosensors for pathogenic DNA detection

Multiwalled carbon nanotubes modified conductive ink for paper-based gonorrheal diagnostic device.

Identifying Hypocalcemia in Dairy Cattle by Combining 3D Printing and Paper Diagnostics

This paper describes the design, fabrication, and validation of a paper-based diagnostic device for the rapid diagnosis of hypocalcemia in dairy cattle at the point-of-care (POC). The device incorporates a 3D printed calcium ion-selective membrane (ISM) as the sensing element for free—unbound—calcium in real bovine whole blood samples. With a linear response range of 100 mM to 97.7 μM, the sensor covers the clinically relevant concentrations of Ca2+ associated with both healthy cattle as well as those suffering from hypocalcemia. The components of the Ca2+ ion-selective electrodes were successfully translated to a paper-based device to provide a sensing platform that is simple to use, disposable, and low-cost, and is therefore well-situated for applications at the POC. The paper-based calcium sensor showed a Nernstian response between 10 mM and 100 μM and required only 12 μl of sample to perform a measurement, which can be accomplished in less than two minutes without the need for time-consuming separation steps. The performance of the paper-based Ca2+ sensor was validated using the commercially available epoc® Blood Analysis System, which provided results within 5% of the data obtained with 3D printed Ca2+-ISM integrated paper-based device.

Development of a highly sensitive lateral flow strip device for nucleic acid detection using molecular beacons

Extensive research is focused on the development of highly sensitive, rapid on-site diagnostic devices. The lateral flow strip (LFS) is a paper-based point-of-care diagnostic device, which is highly promising because of its ease of use and low cost. Despite these advantages, LFS device is still less popular than other methods such as enzyme-linked immunosorbent assay (ELISA) or real-time polymerase chain reaction (qPCR) due to its low sensitivity. Here, we have developed a fluorescence-based lateral flow strip (f-LFS) device for DNA detection using a molecular beacon (MB), a short hairpin-forming DNA strand tagged with a fluorophore-quencher pair. Each paper and membrane component of f-LFS device was carefully selected based on their physicochemical properties including porosity, surface functionality, and autofluorescence. The limit of detection (LOD) of this device was substantially improved to 2.1 fg/mL by adding MgCl2 to the reaction buffer and narrowing the test membrane dimension. Also, a portable fluorescence detection system for f-LFS was developed using a multi-pixel photon counter (MPPC), a sensitive detector detecting the signal on site. We anticipate that this highly sensitive paper-based diagnostic device can be utilized for on-site diagnosis of various diseases.

Application of the aqueous two-phase system and nanozyme signal enhancement for the improved detection of Plasmodium lactate dehydrogenase in serum.

Malaria is an infectious disease that can cause severe sickness and death if not diagnosed and treated in a timely manner. The current gold standard technique for malaria diagnosis is microscopy, which requires a dedicated laboratory setting and trained personnel and can have a long time to result. These requirements can be alleviated using paper-based diagnostic devices that enable rapid and inexpensive diagnosis at the point of care, which can allow patients to receive treatment before their symptoms progress when used for early detection of diseases. The lateral-flow immunoassay (LFA) is one such device, but currently available LFAs are susceptible to false negative results caused by low parasite density. To improve sensitivity and detection, we utilized the aqueous two-phase system (ATPS) to concentrate and purify the sample, and nanozyme signal enhancement to increase the intensity of the visible signal on the test strip. We were able to achieve a limit of detection (LOD) of 0.01ng/mL for the malaria biomarker Plasmodium lactate dehydrogenase (pLDH) in human serum using a multi-step assay combining the LFA format with the ATPS and nanozyme signal enhancement.

Identifying Hypocalcemia in Dairy Cattle By Combining 3D Printing and Paper Diagnostics

Electrochemistry is a powerful technique for advancing point-of-care (POC) diagnostics due largely to its ease of use and compatibility with electronic readers that can be controlled by ubiquitous technology such as smartphones or tablets. Specifically, potentiometry, (i.e., the use of ion-selective electrodes) is well-suited for incorporation into POC devices for the following reasons: (i) it possesses high selectivity (through the incorporation of ionophores), (ii) it is multiplexable, and (iii) provides a fast response time. This talk will focus on the development of a paper-based diagnostic device for the identification of hypocalcemia in dairy cattle at the point-of-care. Hypocalcemia is a metabolic disorder resulting from a lower-than-normal concentration of calcium in the bloodstream and can lead to deleterious outcomes such as early herd removal or death. Diagnosis and treatment of hypocalcemia lead to increased expenses on dairy operations, where low-cost and rapid testing solutions are currently not commercially available. Our approach utilizes the highly reproducible and mass-production capabilities of 3D printing to fabricate sensors that can be incorporated into paper substrates, resulting in low-cost and reliable diagnostics devices.

Fabrication of Paper-Based Microfluidics by Spray on Printed Paper.

Since the monumental work conducted by Whitesides et al. in 2007, research and development of paper-based microfluidics has been widely carried out, with its applications ranging from chemical and biological detection and analysis, to environmental monitoring and food-safety inspection. Paper-based microfluidics possesses several competitive advantages over other substrate materials, such as being simple, inexpensive, power-free for fluid transport, lightweight, biodegradable, biocompatible, good for colorimetric tests, flammable for easy disposal of used paper-based diagnostic devices by incineration, and being chemically modifiable. Myriad methods have been demonstrated to fabricate paper-based microfluidics, such as solid wax printing, cutting, photolithography, microembossing, etc. In this study, fabrication of paper-based microfluidics was demonstrated by spray on the printed paper. Different from the normally used filter papers, printing paper, which is much more accessible and cheaper, was utilized as the substrate material. The toner was intended to serve as the mask and the patterned hydrophobic barrier was formed after spray and heating. The processing parameters such as toner coverage on the printing paper, properties of the hydrophobic spray, surface properties of the paper, and curing temperature and time were systematically investigated. It was found that, after repetitive printing four times, the toner was able to prevent the hydrophobic spray (the mixture of PDMS and ethyl acetate) from wicking through the printing paper. The overall processing time for fabrication of paper-based microfluidic chips was less than 10 min and the technique is potentially scalable. Glucose detection was conducted using the microfluidic paper-based analytical devices (µPADs) as fabricated and a linear relationship was obtained between 1 and 10 mM.

Development of a Tetrazolium-Derived Paper-Based Diagnostic Device as an Early, Alternative Bacteria Screening Tool.

(1) Background: The complexity, amount of time, and the large amount of resource required to perform gold-standard bacteria culture procedures makes it difficult to perform timely pathogenic analyses, especially in areas where such resources are not readily available. A paper-based biochemical analytical tool can potentially tackle problems economically in terms of time and convenience, potentially finding utility in applications where simple and timely detection of bacteria is necessary; (2) Methods: The utility of paper-based MTT-PMS strips was tested using a simple colorimetric analytical methodology; (3) Results: Sufficient evidence was obtained to suggest that the strips can potentially be used as a rapid and convenient early, alternative bacteria screening tool for a variety of applications; (4) Conclusions: The potential of strips for the rapid detection of bacteria compared to standard bacteria culture is a key advantage in certain clinical, agricultural, and environmental applications.

Paper-based diagnostic platforms and devices

Paper-based analytical devices have become lately “must have” components in equipment and instrumental designed for point-of-care applications, especially when they are used in tandem with microfluidic platforms. Nowadays, paper-based electrochemical devices (PEDs) represent the first choice in the development of lab-on-a-chip biosensors because of their benefits in biomedical diagnosis in terms of simplicity, affordability, portability, and disposability. Moreover, cellulose is a biodegradable and biocompatible substrate, ideal for building disposable devices for use in remote locations or low-resource settings. Despite their low costs and simplicity, PEDs must face a tough challenge—meeting the affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users criteria. The latest achievements in microfluidic PEDs for clinical diagnosis will be critically discussed, putting emphasis on innovative assay formats and methods for surface modification. • Paper-based electrochemical devices for biomedical applications. • Printed electrodes integrated on 2D and 3D paper configurations. • Conductive nanocomposites used for enhanced performance. • Innovative formats with paper biosensors adapted for point-of-care-testing.

Paper-Based Microfluidic Devices: Low-Cost Platforms for Rapid Biochemical Detection.

We developed low-cost, portable paper-based diagnostic devices for detection of human immunoglobulin M (IgM) and immunoglobulin G (IgG) in serum without any sample preparation. These devices can be used to help identify presence of diseases, used to provide rapid results (<5 minutes), readily used by untrained personnel, employed in austere environments, configured to obtain multiplexed assays, and easily disposed of. We successfully accomplished colorimetric detection of human IgG and human IgM using a sandwich-style assay within the microfluidic paper device via vertical flow immunoassay configuration. The reaction zone in the wax printed paper layer is a small circular pattern. Gold nanoparticles conjugated with anti-human IgG and IgM antibodies have been used for colorimetric detection of IgG or IgM by naked eye. Colorimetric signal can be precisely quantified through implementation of image analysis software which can be developed as an app for a smartphone. The size of the device is 2 cm × 2 cm × 1 mm. Colorimetric detection of human IgG was accomplished at 100 fg/mL concentration using a gold nanoparticle-conjugated anti-human IgG antibody. The developed platform has a dynamic range of IgM and IgG concentrations between 0.1 pg/mL and 100 μg/mL. These devices provided a color readout in <5 minutes using 20 µL of serum. We also demonstrated that the devices show a significant degree of ruggedness and temperature stability as they were able to provide satisfactory results (detection of 0.1 pg/mL IgG) after 14 days of long stability and shelf-life experiment at an elevated temperature of at least 50 ˚C-the shelf life can be as long as 180 days under ambient conditions for detection of 100 µg/mL IgG. Because of the inherent simplicity of the device operation and their ease of use, there is no variation between samples and users of the device. This low-cost approach enables multiplexing with >1 measurement performed in parallel at the same time. We anticipate that because of the sensitivity, specificity, ease of use, and overall reliability, this approach will become a standard for diagnosis of diseases and health conditions.

One-Step Hot Microembossing for Fabrication of Paper-Based Microfluidic Chips in 10 Seconds.

In recent years, microfluidic paper-based analytical devices (µPADs) have been developed because they are simple, inexpensive and power-free for low-cost chemical, biological and environmental detection. Moreover, paper is lightweight; easy to stack, store and transport; biodegradable; biocompatible; good for colorimetric tests; flammable for easy disposal of used paper-based diagnostic devices by incineration; and can be chemically modified. Different methods have been demonstrated to fabricate µPADs such as solid wax printing, craft cutting, photolithography, etc. In this study, one-step hot microembossing was proposed and demonstrated to fabricate µPADs. The processing parameters like embossing temperature, pressure and time were systematically investigated. It was found that, at 55 °C embossing temperature, the embossing pressure ranging from 10 to 14 MPa could be applied and the embossing time was only 5 s. This led to the overall processing time for fabrication of µPADs within 10 s. Glucose detection was conducted using the µPADs as fabricated, and a linear relationship was obtained between 5 and 50 mM.

Thermal analysis of loop mediated isothermal DNA amplification (LAMP) based point-of-care diagnostic device

Loop-Mediated Isothermal Amplification reaction or LAMP has been identified as a feasible DNA amplification process. Paper-based LAMP diagnostic devices offer several advantages for point-of-care (POC) applications. LAMP occurs at a constant temperature of higher limits, which remains a major challenge on-site. This calls for an efficient thermal design which is beneficial in maintaining a constant high temperature at POC locations. The present study reports thermal design and energy consumption analysis of a paper-based LAMP POC device for the first time using CFD analysis. The POC design consists of a rectangular porous membrane embedded on a rectangular solid material, heated from the side walls. Isothermal and discrete heating with different horizontal and vertical clearance configurations are used as a design parameter. CFD analysis of different thermal configurations is carried out in COMSOL Multiphysics platform to solve the energy and convection–diffusion-reaction equation in porous media. The thermal analysis demonstrated that two discrete heating configurations with high horizontal clearance are effective in reducing energy consumption. It was inferred that two portable AA batteries are sufficient for the POC device operation. Overall, the current work successfully demonstrates the energy optimization and speed of operation of the POC medical device by detailed thermal analysis.

Point-of-care semen analysis of patients with infertility via smartphone and colorimetric paper-based diagnostic device.

Male infertility affects millions of males worldwide and is rising in prevalence due to social and environmental conditions. However, men often feel too embarrassed to receive a semen analysis in the hospital due to social stigmas. To overcome this problem, we developed a 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide test strip to distinguish semen samples with low total motile sperm concentration from those with normal motile sperm concentration. This is a point‐of‐care colorimetric semen analytical method with a one‐step, inexpensive, equipment‐free evaluation process, and adequate accuracy validated in a 42‐sample clinical trial. In this study, results were evaluated visually and with a smartphone application. Using visual observation methods, the area under the curve (AUC) was 0.71 (95% of confidence interval = 0.55–0.86; p = 0.021), sensitivity was 41%, specificity was 95%, positive predictive value was 90%, negative predictive value (NPV) was 59.4%, and accuracy was 67%. Using a smartphone recording and analytical system, AUC was 0.766 (95% of confidence interval = 0.612–0.92; p = 0.003), sensitivity was 96%, specificity was 65%, PPV was 75%, NPV was 92.9%, and accuracy was 80.9%. This work demonstrated a screening tool that could elevate semen analysis to the level of routine healthcare and provide for private, in‐home self‐assessment.

Recent Advances of Fluid Manipulation Technologies in Microfluidic Paper-Based Analytical Devices (μPADs) toward Multi-Step Assays.

Microfluidic paper-based analytical devices (μPADs) have been suggested as alternatives for developing countries with suboptimal medical conditions because of their low diagnostic cost, high portability, and disposable characteristics. Recently, paper-based diagnostic devices enabling multi-step assays have been drawing attention, as they allow complicated tests, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which were previously only conducted in the laboratory, to be performed on-site. In addition, user convenience and price of paper-based diagnostic devices are other competitive points over other point-of-care testing (POCT) devices, which are more critical in developing countries. Fluid manipulation technologies in paper play a key role in realizing multi-step assays via μPADs, and the expansion of biochemical applications will provide developing countries with more medical benefits. Therefore, we herein aimed to investigate recent fluid manipulation technologies utilized in paper-based devices and to introduce various approaches adopting several principles to control fluids on papers. Fluid manipulation technologies are classified into passive and active methods. While passive valves are structurally simple and easy to fabricate, they are difficult to control in terms of flow at a specific spatiotemporal condition. On the contrary, active valves are more complicated and mostly require external systems, but they provide much freedom of fluid manipulation and programmable operation. Both technologies have been revolutionized in the way to compensate for their limitations, and their advances will lead to improved performance of μPADs, increasing the level of healthcare around the world.

Anomalous diffusion in an electrolyte saturated paper matrix.

Diffusion of colored dye on water saturated paper substrates has been traditionally exploited with great skill by renowned water color artists. The same physics finds more recent practical applications in paper-based diagnostic devices deploying chemicals that react with a bodily fluid yielding colorimetric signals for disease detection. During spontaneous imbibition through the tortuous pathways of a porous electrolyte saturated paper matrix, a dye molecule undergoes diffusion in a complex network of pores. The advancing front forms a strongly correlated interface that propagates diffusively but with an enhanced effective diffusivity. We measure this effective diffusivity and show that it is several orders of magnitude greater than the free solution diffusivity and has a significant dependence on the solution pH and salt concentration in the background electrolyte. We attribute this to electrically mediated interfacial interactions between the ionic species in the liquid dye and spontaneous surface charges developed at porous interfaces, and introduce a simple theory to explain this phenomenon.

Paper-based human neutrophil elastase detection device for clinical wound monitoring.

Paper-based diagnostic devices have been widely applied to assess the presence and status of a variety of clinical diseases by analyzing samples such as urine or blood. Due to their low cost, user-friendliness, and convenience, they have been used as point-of-care (POC) devices in countries lacking resources or energy. Despite wide-ranging research and implementation, paper-based devices have not previously been developed for wound analysis. Here, we discuss the successful development of such a tool to facilitate simple and rapid wound status assessment. The purpose of this study was to develop a paper-based elastase detection device (PEDD) for clinical wound assessment that specifically examines human neutrophil elastase (HNE), one of the most abundant serine proteases found in chronic wounds. The first step in this study was an examination of different paper substrate types (i.e., chromatography paper and filter paper) to determine which provided the best protease immobilization and colorimetric response. We then used a wax printing approach to create hydrophobic and hydrophilic regions and designated test zones created on both chromatography and filter papers. This allowed us to physically immobilize both substrate and protease within the desired test zone regions. This PEDD which demonstrated good sensitivity (0.631 μg mL-1, in a wound fluid system) can be used to monitor protease activity expressed in wounds. After developing this device, we examined samples from 9 patients presenting a total of 7 acute and 4 chronic wounds to determine wound HNE concentration. We believe that this study may be widely applicable in both academic and commercial sciences, including the development of practical POC detection devices.

Detection of cardiovascular disease associated miR-29a using paper-based microfluidics and surface enhanced Raman scattering.

The development of viable point-of-care diagnostic formats is integral to achieving better patient care and improved outcomes. The need for robust and low-cost tests is especially important in under-resourced and rural settings. Perhaps the greatest challenge is ensuring that an untrained individual is capable of operating and interpreting the test, out with a care facility. Here we present a paper-based diagnostic device capable of sensing miR-29a using both colorimetric and surface enhanced Raman scattering (SERS) analysis. Rather, than carry out the two types of analyses in tandem, we envisage that the colorimetric output is easy enough to be interpreted by the untrained-individual administering the test to provide them with qualitative feedback. If deemed positive, the test can be further validated at a centralized care facility using a handheld-Raman spectrometer to provide a semi-quantitative result. Detection of miR-29a, a microRNA associated with myocardial infarction, was achieved at a level of pg μL-1 through the combination of three-dimensional paper-based microfluidics, colorimetric detection, and surface enhanced Raman scattering (SERS) analysis. RGB analysis of the colorimetric output generated from samples containing miR-29a at different concentrations (18-360 pg μL-1) showed differentiation from the control sample, however significant repeat variability indicated that it could not be used for quantifying miR-29a levels. However, the SERS analysis exhibited greater reproducibility at varying concentrations, achieving an LoD of 47 pg μL-1. The union of the paper-based device and the two analysis methods resulted in the production of a sensitive, reproducible and facile, point of care test (POCT), which paves the way for future implementation in the diagnosis of a range of diseases.

A novel naphthofluorescein-based probe for ultrasensitive point-of-care testing of zinc(II) ions and its bioimaging in living cells and zebrafishes.

The combination of fluorescence method with paper-based diagnostic device is quite suitable in point-of-care testing (POCT). Herein, we designed a novel hybrid fluorescein-based probe ZN-2 and investigated its fluorescent properties thoroughly in the detection of Zn2+. In comparison with the fluorescein-based probe ZN-1, ZN-2 displayed better sensitivity, long-wavelength and faster response to Zn2+ within 20min. Interestingly, we could achieve ultrasensitive, high-throughput and visual detection in the POCT analysis of Zn2+ by anchoring this probe ZN-2 on the paper-based device. This device with satisfied performance for Zn2+ detection was achieved in real samples including cytochylema, serum and lake water. Finally, the probe ZN-2 was further applied to visualize and monitor the level changes of Zn2+ in the living cells and zebrafishes.

Paper-Based Diagnostic System Facilitating Escherichia coli Assessments by Duplex Coloration.

Laboratory support for low-resource regions is a rising global issue. As microbiological contamination is closely associated with other issues like food safety, water supply sustainability, and public health, bacterial assessments in this setting need to be improved. Herein, we demonstrate a paper-based diagnostic device for point-of-need testing, in which fecal-indicating Escherichia coli and highly pathogenic E. coli are detected by duplex coloration. This device was functionalized by mixing different chromogenic substrates that reflect each bacterial enzymatic phenotype. In the final part of the paper, we describe this microbiological diagnostic system tested with bacteria-contaminated food samples. The device sensitivity was shown to have greatly reduced the total analysis time (below to 4 h) when combined with an enrichment amplification procedure. Notably, this paper device successfully detected 10 cfu/mL of target bacteria in a contaminated milk sample. Our diagnostic system shows acceptable accuracy, short analysis time, and a user-friendly interface, thereby eliminating demands for high-end equipment and a highly trained staff. We expect that this diagnostic system will be a sustainable solution in supporting microbiological or clinical laboratories in low-income countries.