Microfluidic Paper-based Analytical Devices Research Articles

Development of Microfluidic Paper-Based Analytical Devices (µPADs) for Determination of Cd2+, Pb2+, and Cu2+ Ions in Mineral Water

Microfluidic paper-based analytical devices (µPADs) have been successfully developed using dual detection: electrochemical and colorimetric systems. The µPADs have the potential to be used as Pb(II), Cd(II), and Cu(II) sensors to test the quality of water. The fabrication process uses hot embossing and screen-printing methods. The working electrode in the electrochemical zone was enhanced by the bismuth metal deposition process, while in the colorimetric zone, the gold nanoparticles modified with thioctic acid and dansylhydrazine (TA-Au-DNS) were used as a colorimetric sensor to detect Cu. The basic material of µPADs was characterized using a Fourier-transform infrared (FTIR) and a contact angle meter (CAM). In the electrochemical zone, the signals of square wave anodic stripping voltammetry (SWASV) resulted in good detection of Pb(II) and Cd(II) (from 0 to 100 ppb) with a limit of detection of 1.588 and 1.42 ppb, respectively. In the colorimetric zone, the performance of TA-Au-DNS for detecting Cu metal was obtained from readings through the red-green-blue (RGB) sensor as a miniature of µPADs reader. The LOD, LOQ, and average Vx0 (linearity values) in the detection of Cu(II) (from 58 to 100 ppb) are 8.51 ppb, 28.36 ppb, and 0.41%, respectively.

µPAD-based colorimetric nanogold aptasensor for CRP and IL-6 detection as sepsis biomarkers

Point-of-care testing is defined as medical diagnostic testing at or near the time and place of a patient. The integration of microfluidics with functionalized nanomaterials provides a rapid and sensitive detection method for biomarkers. Due to their optical characteristics, gold nanoparticles (GNPs) and aptamers can be used in a colorimetric technique. In this research, accurate and quick aptasensors were created using aptamers for two sepsis biomarkers, interleukin-6 (IL-6) and C-reactive protein (CRP). Aptamers were combined with GNPs to create aptasensors, which were then used to analyze various biomarker concentrations after aptamer-protein docking on the online HDOCK server to verify aptamer-protein interactions. To improve the results' stability and ease of use during the reaction time, microfluidic paper-based analytical devices (µPAD) was employed. The linear diagnostic ranges in this method were 50–1000 mg L−1 and 1–25 ng/L, and limit of detection values were 1.9 mg L−1 and 0.07 ng/L for CRP and IL-6, respectively; These values decreased when µPADs were used and became more suitable to diagnosis of sepsis. Two aptasensors were highly stable, reproducible, accurate, and recoverable. Our result suggests that colorimetric GNPs can be used to detect a specific analyte through color changes visible to the naked eye for monitoring purposes or as simple optical detectors for quantitative measurements.

Construction of fluorescent sensor array and three-dimensional microfluidic paper based analytical device for specific identification and visual determination of antibiotics in food.

For antibiotics misuse since the global outbreak of COVID 19, a novel strategy for discriminating and detecting antibiotics is proposed based on the graphene quantum dots with multi-doped heteroatoms including F, N and P (M-GQDs), which exhibit blue emission (419.0nm) under the excitation of 336.0nm. Specifically, the fluorescence of M-GQDs is quenched by tetracyclines (TCs) owing to inner filter effect (IFE) and enhanced by alkane-modified fluoroquinolones (AFQs), which is attributed to restricted conformational rotation based on π-π stacking, hydrogen-bonding and electrostatic interactions. Meanwhile, the electron-accepting property of oxazine ring in oxazine-modified fluoroquinolones (OFQs) increases emission peak at 498.0nm and decreases emission peak at 419.0nm as the color changes from blue to cyan. Moreover, a cascade system integrated with 3D microfluidic paper-based analytical device (3D-μPAD) is applied successfully for visually distinguishing three antibiotics, which shows great potential and versatility of M-GQDs for food safety monitoring.

A simple and low-cost paper-based device for simultaneous determination of hematocrit and hemoglobin levels in point-of-care settings

Hematocrit and hemoglobin levels are common blood tests used to assess the overall health status of patients as well as the oxygen-carrying capacity of the blood. The gold-standard technologies to determine the hematocrit and hemoglobin levels are precise and fast, but they are not pragmatic in extreme point-of-care settings due to the prohibitive cost, process complexity, and sophisticated facilities. Therefore, here, we report the development of a microfluidic paper-based analytical device to determine hematocrit and hemoglobin levels simultaneously. This device exploits the area of the stain formed by a spreading drop of 20 μl of whole blood on Whatman filter paper immobilized with sodium chloride and ethylenediaminetetraacetic acid for hematocrit level, whereas for hemoglobin concentration, it utilizes the gray color intensity of a 20 μl droplet of a mixture of blood and de-ionized water. The performance of the device is verified by comparing with gold-standard results of the automated hematology analyzer, showing a high degree of correlation (R2) of 0.9651 and 0.9701 for hematocrit and hemoglobin levels, respectively. The bias and standard deviation of the differences between the two measurements for hematocrit determination are 0.002 and 1.005, respectively, while the bias and standard deviation of differences between the two methods for hemoglobin determination are 0.065 and 0.398, respectively. This device is likely to provide a simple, fast, disposable, and inexpensive tool to determine the hematocrit and hemoglobin levels in resource-constraint settings.

Smartphone-integrated colorimetric and microfluidic paper-based analytical devices for the trace-level detection of permethrin.

Permethrin is a pyrethroid pesticide and insect repellent that prevents mosquito-borne infections like dengue and malaria in tropical areas. This work describes a new colorimetric sensor based on metronidazole-stabilized silver nanoparticles (MTZ-AgNPs) for the first rapid, sensitive, and selective permethrin detection. The MTZ-AgNPs-based colorimetric sensor has a limit of detection (LOD) of 0.0104µM and a limit of quantification (LOQ) of 0.0348µM, respectively. The sensor is further integrated with smartphone and microfluidic fabrication of paper-based analytical devices (µPADs) for real-time and on-site detection of permethrin. Under optimal settings, no potential environmental contaminants interfere with permethrin detection, confirming its high selectivity. Finally, the practical applicability of sensors is confirmed in real tomato and apple extract samples. The US environmental protection agency's recommended UPLC method validated the detection efficiency of the proposed colorimetric sensor. The % recoveries from UPLC and MTZ-AgNPs suggest that the present sensor can quantitatively analyze permethrin in real samples.

Dual-spot ratiometric microfluidic paper-based analytical device for accuracy and precision improvement

Instrumental and environmental fluctuations are common sources of error in smartphone-based optical detection, significantly affecting the accuracy of analytical measurements. In this regard, spotting the sample and reference simultaneously and in close proximity compensates for the fluctuations. This “dual-spot” design is similar to the double-beam technique used in spectrophotometry, which reduces fluctuations in the results. The underlying hypothesis is that any instrumental and/or environmental factors influencing the color intensity in the detection zones will similarly impact the color intensity in the control zone under the same conditions. To test our design, a ratiometric microfluidic paper-based analytical device (μPAD), functionalized with a mixture of green-emissive carbon dots (CDs) and red-emissive ethidium bromide, was developed for the selective detection of ascorbic acid (AA). The green emission of the CDs is quenched by both AA and Fe3+; NaF was thus loaded onto the 3D connector as a masking agent to remove the interference effect of the Fe3+ ions. The color variations were monitored under a UV lamp, using a smartphone to capture the images, and the RGB intensities were processed using the Color Grab application. The proposed double-spot method greatly enhanced the analytical precision and accuracy of the device. A linear working range from 0 to 125 μM was obtained, and the limit of detection was 2.71 μM. The μPAD was successfully used for the detection of AA in human serum, with recoveries from 87.27 to 98.52 %.

Self-cleaning paper-based microfluidic biosensor employing DNAzyme and semiconducting single-walled carbon nanotube for copper ion detection

Microfluidic paper-based analytical device (μPAD) offers a simple and efficient platform for point-of-care monitoring, which can be beneficial for copper determination in livestock feed and manure. However, common cellulose paper has excellent hydrophilicity, causing μPAD is accompanied by poor mechanical properties, short service life, and low sensitivity. Here, a self-cleaning paper-based microfluidic biosensor for Cu2+ determination was proposed to overcome the mentioned shortages in the application. Polymeric octadecyl trichlorosilane was synthesized and decorated on cellulose paper to form hydrophobic paper, which can improve the hydrophobicity, self-cleaning, and pollution ability. In addition, hydrophobic paper, semiconducting single-walled carbon nanotube, and DNAzyme through the chemical bond were employed to fabricate a self-cleaning paper-based microfluidic biosensor. The properties were investigated using scanning electron microscopy, Raman, and electrochemical methods. The detecting parameters were also optimized. It could measure the Cu2+ concentration from 1 nM to 100 μM, and the detection limit was 0.65 nM. The self-cleaning paper-based microfluidic biosensor was applied to detect Cu2+ concentration in livestock feed and manure that can meet the requirements for fast screening and detection.

A Microfluidic Paper-based Analytical Device for Simultaneous Measurement of Albumin, Creatinine and Uric Acid in Urine based on Standard Addition Method

This work describes a simple method for the simultaneous measurement of urinary albumin, creatinine and uric acid using a standard addition approach on a microfluidic paper-based analytical device (µPAD). Hydrophobic barrier of the µPAD was created by stamping indelible ink onto a filter paper. The µPAD was designed with a flower-liked configuration. After aliquoting urine to a central sample zone, the sample flowed to ten surrounding channels, namely “the inner channels”, where the blank (water) or standard solutions had been added. The inner channels were connected to circular reagent zones where reagents had been immobilized. Tetrabromophenolphthalein ethyl ester, alkaline picrate, and a mixed solution of ferric chloride and ferric cyanide were used as the chromogenic reagents for the colorimetric detections of albumin, creatinine, and uric acid, respectively. Each reagent zone was linked with a circular detection zone through a second channel, namely “the outer channels”. The blue-, orange- and greenish-blue colored products were observed in the detection zones for the measurement of albumin, creatinine, and uric acid, correspondingly. A digital image of the µPAD was captured with a mobile phone. The color intensities were evaluated by ImageJTM and were employed for the quantitative analyses. The standard addition calibrations were found to be linear (r2 > 0.99) for the spiked analyte concentrations up to 100 mg L-1 albumin, 1000 mg L-1 creatinine, and 50 mg dL-1 uric acid. The paper platform provided high precision (RSD < 5 %) and good analytical recovery (91.8-109.7 %). Under paired t-test, the results obtained by the developed µPAD and the validating methods were not significantly different at 95 % confidence (albumin: tstat = -0.130, tcri = 3.182, creatinine: tstat = -0.133, tcri = 3.182, and uric acid: tstat = 1.119, tcri = 3.182).

Colorimetric detection of glucose with smartphone-coupled µPADs: harnessing machine learning algorithms in variable lighting environments

Recent advances in microfluidic paper-based analytical devices (µPADs) have shown immense potential for point-of-care testing (POCT) in resource-constrained settings, especially for monitoring glucose levels in patients with diabetes. However, the practical adoption of smartphone-coupled µPADs has been limited due to the variability in ambient lighting conditions, which affects the colorimetric detection. In this study, we have developed a machine learning-based algorithm together with a flash/no-flash technic, adaptable to various µPADs and capable of accurately monitoring plasma glucose levels across a wide range (25 µM to 30 mM), addressing issues related to environmental illumination variations, and shooting angles.The algorithm is implemented in an Android application, Gluco estimator. It utilizes images captured by smartphones and incorporates intelligent feature engineering. The XYZ color space is adopted for device-independent analysis. KI and TMB color indicators are used to enhance resolution for low and high glucose concentrations, respectively. The EBC classifier, combined with a handcrafted feature set, exhibits outstanding performance, achieving an accuracy of 95% and 91% for TMB and KI, respectively. Additionally, multiple linear regression (MLR) yields high reliability among regression models, with R2 values of 0.95 at 0.25 µM to 3 mM and 0.97 at 3–30 mM. This work represents a significant stride toward realizing a portable, high-resolution, and reliable smartphone-coupled µPAD in uncontrolled environments.

Screening of angiotensin converting enzyme inhibitors from natural products via origami microfluidic paper-based analytical devices with colorimetric detection

We report the screening of angiotensin converting enzyme (ACE) inhibitors on an origami microfluidic paper-based analytical device (μPAD) using colorimetric detection. The hydrolysis product reacts with ninhydrin, resulting in a purple color at the detection zones. Images of the μPADs are captured using a common cell phone and analyzed with Photoshop software. This platform allows six independent colorimetric reactions to take place simultaneously, and the IC50 values can be obtained in a single run within 22 min. The relative standard deviations of inhibition efficiencies are generally lower than 4.0 % (n = 5). The IC50 values of captopril and five products from natural plants were obtained and corresponded well with UV methods. The relative deviations between the two methods are within the range of −5 % to +5 %. This work is a proof-of-concept successfully demonstrating the use of μPADs technology to screen enzyme inhibitors from natural products.

Accelerated naked-eye identification of cations using non-plasmonic amplification by functionalized GQDs: A novel photochemical microfluidic sensor for environmental/mineral analysis using Lab-on-paper technology

This study describes the synthesis of various types of thiolated-graphene quantum dots (GQDs) for the simultaneous multi-sensing of heavy metal ions using one-droplet microfluidic paper-based analytical devices (OD-µPADs). For the first time, functionalized GQDs were synthesized and stabilized on the surface of hydrophobic fiber-glass with simple manufacturing and assembly strategy towards colorimetric chemosensing of ions including iron, copper, cobalt, mercury, chrome, manganese, and nickel. For this purpose, GQDs are functionalized by various organic species including cysteamine, decanethiol, and mercaptopropionic acid (MPA) towards specific electrostatic interaction with metal ions of Fe (II), Cu (II), Co(II), Hg (II), Mn (II), and Ni (II). Therefore, an innovative GQDs-based miniaturized chemo-system was proposed for the environmental analysis. Additionally, the results were confirmed using fluorescence spectrometry as an optical detection method. Finally, this simple and efficient colorimetric platform was used for facile metal ions detection in human urine models, and environmental fluids, which can provide a commercial device for sensing contaminants by non-professional users in biomedical/environmental mixture. As an economical technique, this platform presents a “mix-and-sense” system without dye (as a side substance) or chemical treatments. The low instrumental requirements, the color uniformity, and the disposability of OD-μPADs approve the commercial application of the engineered ion-chemosensor.

Dual Chromatic Laser-Printed Microfluidic Paper-Based Analytical Device (μPAD) for the Detection of Atrazine in Water

Dual Chromatic Laser-Printed Microfluidic Paper-Based Analytical Device (μPAD) for the Detection of Atrazine in Water

Atomically Dispersed Manganese on Graphene Nanosheets as Biocompatible Nanozyme for Glutathione Detection in Liver Tissue Lysate Using Microfluidic Paper-based Analytical Devices.

Recently, single atom catalysts (SACs) featuring M-Nx (M = metal) active sites on carbon support have drawn considerable attention due to their promising enzyme-like catalytic properties. However, typical synthesis methods of SACs often involve energy-intensive carbonization processes. Herein, we report a facile one-pot, low-temperature, wet impregnation method to fully utilize M-N4 sites of manganese phthalocyanine (MnPc) by decorating molecular MnPc over the sheets of graphene nanoplatelets (GNP). The synthesized MnPc@GNP exhibits remarkable peroxidase-mimic catalytic activity toward the oxidation of chromogenic 3,3',5,5'-tetramethylbenzidine (TMB) substrate owing to the efficient utilization of atomically dispersed Mn and the high surface-to-volume ratio of the porous catalyst. A nanozyme-based colorimetric sensing probe is developed to detect important biomarker glutathione (GSH) within only 5 min in solution phase based on the ability of GSH to effectively inhibit the TMB oxidation. The high sensitivity and selectivity of the developed colorimetric assay enable us to quantitatively determine GSH concentration in different biological fluids. This work, for the first time, reports a rapid MnPc@GNP nanozyme-based colorimetric assay in the solid substrate by fabricating microfluidic paper-based analytical devices (μPADs). GSH is successfully detected on the fabricated μPADs coated with only 6.0 μg of nanozyme containing 1.6 nmol of Mn in the linear range of 0.5-10 μM with a limit of detection of 1.23 μM. This work also demonstrates the quantitative detection of GSH in mice liver tissue lysate using μPADs, which paves the way to develop μPADs for point-of-care testing.

Salivary calcium determination with a specially developed microfluidic paper-based device for point-of-care analysis

The calcium monitoring in the body not only anticipates several potential diseases (osteoporosis, kidney stones or high blood pressure) but also helps to improve target therapies and follow-up the patient's health status. Calcium monitoring is essential for the diagnosis of one of the most common endocrine disorders worldwide, namely hyperparathyroidism. So, in this work, a new Point-of-care test (POC-test) using a microfluidic paper-based analytical device (μPAD) for calcium quantification in saliva samples is described. The developed μPAD was based on the colorimetric reaction between calcium and cresolphthalein complexone (CPC) which forms an intense purple colour product. The developed device enabled calcium quantification in the range of 0.27–4.50 mmol/L (11.0–180 mg/L) with a detection limit of 80 µmol/L (3.2 mg/L). The accuracy of the developed μPAD was confirmed by analysing saliva samples (#10) and comparing the results obtained with the atomic absorption spectrometry reference procedure; the relative deviation between the two sets of results was below 10 %. A correlation between salivary calcium content and calcium content in blood was established and it was possible to conclude that salivary calcium concentrations above 1.55 mmol/L is an indicator of hypercalcemia. The developed device was stable for 2 weeks when stored at room temperature in vacuum conditions.

A Facile Method to Fabricate an Enclosed Paper-Based Analytical Device via Double-Sided Patterning for Ionic Contaminant Detection.

Microfluidic paper-based analytical devices (μPADs) have been developed for use in a variety of diagnosis and analysis fields. However, conventional μPADs with an open-channel system have limitations for application as analytical platforms mainly because of the evaporation and contamination of the sample solution. This study demonstrates the design and fabrication of an enclosed three-dimensional(3D)-μPAD and its application as a primary early analysis platform for ionic contaminants. To generate the hydrophobic PDMS barrier, double-sided patterning is carried out using a PDMS blade-coated stamp mold that is fabricated using 3D printing. The selective PDMS patterning can be achieved with controlled PDMS permeation of the cellulose substrate using 3D-designed stamp molds. We find the optimal conditions enabling the formation of enclosed channels, including round shape pattern and inter-pattern distance of 10 mm of stamp design, contact time of 0.5 min, and spacer height of 300 µm of double-sided patterning procedure. As a proof of concept, this enclosed 3D-μPAD is used for the simultaneous colorimetric detection of heavy metal ions in a concentration range of 0.1-2000 ppm, including nickel (Ni2+), copper (Cu2+), mercury (Hg2+), and radioactive isotope cesium-137 ions (Cs+). We confirm that qualitative analysis and image-based quantitative analysis with high reliability are possible through rapid color changes within 3 min. The limits of detection (LOD) for 0.55 ppm of Ni2+, 5.05 ppm of Cu2+, 0.188 ppm of Hg2+, and 0.016 ppm of Cs+ are observed, respectively. In addition, we confirm that the analysis is highly reliable in a wide range of ion concentrations with CV values below 3% for Ni2+ (0.56%), Cu2+ (0.45%), Hg2+ (1.35%), and Cs+ (2.18%). This method could be a promising technique to develop a 3D-μPAD with various applications as a primary early analysis device in the environmental and biological industries.

Electroanalytical paper-based device for reliable detection and quantification of sugars in milk

Knowing the level of sugars in food and beverages is essential because they are related to diseases such as diabetes, and obesity, among others. In this regard, an approach has been designed, characterized, and applied to develop the first all-in-one paper-based microfluidic analytical device for the quantification of sugars in milk samples. In particular, the powerful combination of the paper-based platform, the facile manufacturing, and the on-demand customization, allowed to obtain a stand-alone platform that was capable to store all the reagents necessary for the assay, to treating the sample by removing major interferences due to lipids and proteins, and to visualize the results in minutes. A wax-patterned chromatographic paper was finely designed to provide a reagent-free platform for the use of non-specialists applied towards the analysis of sugars' content (lactose as the main sugar). Sugars were electrochemically quantified down to 0.16 g L−1 with a repeatability of 5%. To strengthen the efficacy of the novel platform, the electrochemical paper-based analytical device has been applied to commercial milk, including skimmed, semi-skimmed, whole, and lactose-free, confirming satisfactory performance with analytical reliability. This paper-based architecture opens novel avenues for portable (bio)sensing of other relevant species, not only in the agri-food field.

Insights into the Fabrication and Electrochemical Aspects of Paper Microfluidics-Based Biosensor Module.

Over the past ten years, microfluidic paper-based analytical devices (micro-PADs) have attracted a lot of attention as a viable analytical platform. It is expanding as a result of advances in manufacturing processes and device integration. Conventional microfluidics approaches have some drawbacks, including high costs, lengthy evaluation times, complicated fabrication, and the necessity of experienced employees. Hence, it is extremely important to construct a detection system that is quick, affordable, portable, and efficient. Nowadays, micro-PADs are frequently employed, particularly in electrochemical analyses, to replicate the classic standard laboratory experiments on a miniature paper chip. It has benefits like rapid assessment, small sample consumption, quick reaction, accuracy, and multiplex function. The goal of this review is to examine modern paper microfluidics-based electrochemical sensing devices for the detection of macromolecules, small molecules, and cells in a variety of real samples. The design and fabrication of micro-PADs using conventional and the latest techniques have also been discussed in detail. Lastly, the limitations and potential of these analytical platforms are examined in order to shed light on future research.

Maximizing flow rate in single paper layer, rapid flow microfluidic paper-based analytical devices

Small, single-layer microfluidic paper-based analytical devices (µPADs) offer potential for a range of point-of-care applications; however, they have been limited to low flow rates. Here, we investigate the role of laser cutting paper channels in maximizing flow rate in small profile devices with limited fluid volumes. We demonstrate that branching, laser-cut grooves can provide a 59.23–73.98% improvement in flow rate over a single cut, and a 435% increase over paper alone. These design considerations can be applied to more complex microfluidic devices with the aim of increasing the flow rate, and could be used in stand-alone channels for self-pumping.

μPADs on Centrifugal Microfluidic Discs for Rapid Sample-to-Answer Salivary Diagnostics.

A fully integrated device for salivary detection with a sample-in-answer-out fashion is critical for noninvasive point-of-care testing (POCT), especially for the screening of contagious disease infection. Microfluidic paper-based analytical devices (μPADs) have demonstrated their huge potential in POCT due to their low cost and easy adaptation with other components. This study developed a generic POCT platform by integrating a centrifugal microfluidic disc with μPADs to realize sample-to-answer salivary diagnostics. Specifically, a custom centrifugal microfluidic disc integrated with μPADs is fabricated, which demonstrated a high efficiency in saliva treatment. To demonstrate the capability of the integrated device for salivary analysis, the SARS-CoV-2 Nucleocapsid (N) protein, a reliable biomarker for SARS-CoV-2 acute infection, is used as the model analyte. By the chemical treatment of the μPAD surface, and by optimizing the protein immobilization conditions, the on-disc μPADs were able to detect the SARS-CoV-2 N protein down to 10 pg mL-1 with a dynamic range of 10-1000 pg mL-1 and an assay time of 8 min. The integrated device was successfully used for the quantification of the N protein of pseudovirus in saliva with high specificity and demonstrated a comparable performance to the commercial paper lateral flow assay test strips.

Pronounced effect of lamination on plasma separation from whole blood by microfluidic paper-based analytical devices

Many diseases are detected through blood tests. Currently, most blood tests are done on plasma instead of whole blood because of the interference of blood cells on detection results. Here, we developed a laminated microfluidic paper-based analytical device (L-μPAD) for the separation of plasma from whole blood without using plasma separation membrane (PSM). A lateral flow design consisting of a circular sampling zone and rectangular detection zone was patterned on the paper substrate using laser printing technology. The μPAD was then laminated after impregnation with KCl solution. Lamination and electrolyte addition represented synergistic effects on the separation by controlling the pore size of the paper. In addition, by preventing evaporation on one hand and squeezing paper pores on the other hand, lamination caused longer movement of the separated plasma, the longest plasma path reported so far. The separation process was monitored using colorimetric reagent bromocresol green and scanning electron microscopy. The process of separation was completed in less than 90s without significant hemolysis and the separated plasma was far from the interfering effect of red blood cells. We used the device for the determination of serum albumin. However, it represents the potential for point-of-care testing in multi-assay experiments too.