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Orientationally immobilized enzyme microreactors (OIMERs), embedded in microfluidic paper-based analytical devices (μPADs) were developed for improved detection of pesticide residues in food. Acetylcholinesterase (AChE) was orientationally immobilized on the reusable Part I of the μPADs, using the specific affinity binding of concanavalin A (Con A) to a glycosyl group on AChE. Using the disposable Part II, facile colorimetric quantification was performed with a smartphone and software, or qualitative detection by a naked-eye visual test. The AChE immobilized in OIMERs not only had improved activity and stability, but also high sensitivity, with a limit of detection as low as (0.007 ± 0.003) μg/mL. The method was used to detect pesticides residues in real vegetable samples; the recovery (88.6-102.7%) showed high reliability for pesticide residues detection in foods. A molecular docking study and an enzyme kinetic analysis were conducted to characterize the mechanism of action of the OIMERs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Smartphone has long been considered as one excellent platform for disease screening and diagnosis, especially when combined with microfluidic paper-based analytical devices (μPADs) that feature low cost, ease of use, and pump-free operations. In this paper, we report a deep learning-assisted smartphone platform for ultra-accurate testing of paper-based microfluidic colorimetric enzyme-linked immunosorbent assay (c-ELISA). Different from existing smartphone-based μPAD platforms, whose sensing reliability is suffered from uncontrolled ambient lighting conditions, our platform is able to eliminate those random lighting influences for enhanced sensing accuracy. We first constructed a dataset that contains c-ELISA results (n = 2048) of rabbit IgG as the model target on μPADs under eight controlled lighting conditions. Those images are then used to train four different mainstream deep learning algorithms. By training with these images, the deep learning algorithms can well eliminate the influences of lighting conditions. Among them, the GoogLeNet algorithm gives the highest accuracy (>97%) in quantitative rabbit IgG concentration classification/prediction, which also provides 4% higher area under curve (AUC) value than that of the traditional curve fitting results analysis method. In addition, we fully automate the whole sensing process and achieve the "image in, answer out" to maximize the convenience of the smartphone. A simple and user-friendly smartphone application has been developed that controls the whole process. This newly developed platform further enhances the sensing performance of μPADs for use by laypersons in low-resource areas and can be facilely adapted to the real disease protein biomarkers detection by c-ELISA on μPADs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nowadays, food safety has become a major concern for the sustainability of global public health. Through the production and distribution steps, food can be contaminated by either chemical hazards or pathogens, and the determination of these plays a critical role in the processes of ensuring food safety. Therefore, the development of analytical tools that can provide rapid screening of these hazards is highly necessary. Microfluidic paper-based analytical devices (µPADs) have advanced significantly in recent years as they are rapid and low-cost analytical screening tools for testing contaminated food products. This review focuses on recent developments of µPADs for various applications in the food safety field. A description of the fabrication of selected papers is briefly discussed, and evaluation of the μPADs' performance with regard to their precision and accuracy as well as their limits of detection is critically assessed. The advantages and disadvantages of these devices are highlighted.

Herein, the applicability of electromembrane extraction (EME), as an efficient and paper-compatible separation technique, was envisaged over customized microfluidic paper-based analytical devices (μPADs). The utility of EME was assessed on 2D planar and 3D origami structures using different types of electrodes including stainless steel and paper-based electrodes. The overall separation procedure was integrated to colorimetric detection demonstrated for copper ions as the model analyte. According to the obtained results, EME based on 3D design of μPADs could effectively be performed under low applied voltage. Using 3D architecture, the analyte could be quantified within the range of 40.0-1500.0 μg L -1 with limit of detection down to 20.0 μg L -1 using smart phone camera as signal read-out. The proposed platform showed remarkable compatibility with direct analysis from untreated real samples of human blood and spring water., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Microfluidic paper-based analytical devices (µPADs) represent one of the promising green analytical strategies for low-cost and simple determination of various analytes. The actual task is the development of such devices for quantitation of antioxidants, e.g., flavonoids. In this paper, possibilities of a novel three-reagent µPAD including silver nitrate, 4-nitrophenyldiazonium tetrafluoroborate, and iron(III) chloride as reagents are assessed with respect to the determination of dihydroquercetin. It is shown that all the three reagents produce different colorimetric responses that can be detected by a mini-spectrophotometer-monitor calibrator or by a smartphone. The method is applicable to direct measuring high contents of dihydroquercetin (the linearity range is 0.026-1 mg mL -1 , and the limit of detection is 7.7 µg mL -1 ), which is favorable for many dietary supplements. The analysis of a food supplement was possible with the relative standard deviations of 9-26%, which is satisfactory for quantitative and semiquantitative determinations. It was found that plotting a calibration graph in 3D space of the three reagents' responses allows us to distinguish dihydroquercetin from its close structural analogue, quercetin.

Over the past decades, paper-based lateral flow immunoassays (LFIAs) have been extensively developed for rapid, facile, and low-cost detection of a wide array of target analytes in a point-of-care manner. Conventional home pregnancy tests are the most significant example of LFAs, which detect elevated concentrations of human chorionic gonadotrophin (hCG) in body fluids to identify early pregnancy. In this work, we have upgraded these platforms to a higher version by developing a customized microfluidic paper-based analytical device (μPAD), as the new generation of paper-based point-of-care platforms, for colorimetric immunosensing. This will offer a cost-efficient and environmentally friendly alternative platform for paper-based immunosensing, eliminating the need for nitrocellulose (NC) membrane as the substrate material. The performance of the developed platform is demonstrated by detection of hCG (as a model case) in urine samples and subsequently indicating positive or negative pregnancy. A dual-functional silane-based composite was used to treat filter paper in order to enhance the colorimetric signal intensity in the detection zones of μPADs. In addition, microfluidic pathways were designed in a manner to provide the desired regulated fluid flow, generating sufficient incubation time (delays) at the designated detection zones, and consequently enhancing the obtained signal intensity. The presented approaches allow to overcome the existing limitations of μPADs in immunosensing and will broaden their applicability to a wider range of assays. Although, the application of the developed hCG μPAD assay is mainly in qualitative (i.e., positive or negative) detection of pregnancy, the semi-quantitative measurement of hCG was also investigated, indicating the viability of this assay for sensitive detection of the target hCG analyte within the related physiological range (i.e., 10-500 ng/mL) with a LOD value down to 10 ng/mL.

Microfluidic paper-based analytical devices (µPADs) have become promising tools offering various analytical applications for chemical and biological assays at the point-of-care (POC). Compared to traditional microfluidic devices, µPADs offer notable advantages; they are cost-effective, easily fabricated, disposable, and portable. Because of our better understanding and advanced engineering of µPADs, multistep assays, high detection sensitivity, and rapid result readout have become possible, and recently developed µPADs have gained extensive interest in parallel analyses to detect biomarkers of interest. In this review, we focus on recent developments in order to achieve µPADs with high-throughput capability. We discuss existing fabrication techniques and designs, and we introduce and discuss current detection methods and their applications to multiplexed detection assays in relation to clinical diagnosis, drug analysis and screening, environmental monitoring, and food and beverage quality control. A summary with future perspectives for µPADs is also presented., Competing Interests: The authors declare no conflicts of interest.

The incompatibility of most organic solvents with acetylcholinesterase (AChE) inhibition assay normally limits pesticide extraction efficiency in sample pretreatment, which might cause false negatives in real world sample assessment. Herein, a novel method has been developed for an improved AChE inhibition assay via organic solvent extraction combined spontaneous in situ solvent evaporation on microfluidic paper-based analytical devices. Enzyme pre-immobilization procedure was spared and AChE was added to the system after sampling step until a complete in-situ solvent evaporation process was performed on chip. IC 50 levels of the six investigated organophosphate and carbamate pesticides indicated a completely eliminated influence of solvents on AChE behavior with the new method. Most importantly, analytical performances were significantly improved in food sample measurements. Reduction in matrix effect was observed when acetonitrile was adopted for lettuce sample pretreatment instead of water. Studies on different pesticides suggested a remarkably decreased discrimination effect on recoveries from sample pretreatment with the new developed method. The recovery level for phoxim spiked head lettuce samples reached (107.5 ± 14.2) %, in comparison with that of (18.6 ± 1.4) % from water-based extraction. Spiked water and apple juice samples with carbaryl concentration of as low as 0.02 mg L -1 were also successfully recognized with the present method by visual detection. This is the first report on direct sampling of organic extracts for AChE inhibition assay on-chip and it might provide a new perspective for real world sample assessments involving bio-reagents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lactoferrin is an abundant glycoprotein in human body fluids and is known as a biomarker for various diseases. Therefore, point-of-care testing (POCT) for lactoferrin is of interest. Microfluidic paper-based analytical devices (µPADs) have gained a lot of attention as next-generation POCT device candidates, due to their inexpensiveness, operational simplicity, and being safely disposable. This work presents a colorimetric sensing approach for quantitative lactoferrin analysis. The detection mechanism takes advantage of the high affinity of lactoferrin to ferric ions (Fe 3+ ). Lactoferrin is able to displace an indicator from a colorimetric 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP)-Fe 3+ complex, resulting in a color change. A 5-Br-PADAP-Fe 3+ complex was encapsulated into water-dispersible poly(styrene- block -vinylpyrrolidone) particles, whose physical entrapment in the cellulosic fiber network results in the immobilization of the complex to the paper matrix. The complex-encapsulating particles showed a color change response in accordance with lactoferrin concentration. Both color intensity-based paper well plates and distance readout-based µPADs are demonstrated. Color intensity-based devices allowed quantitative analysis of lactoferrin concentrations with a limit of detection of 110 µg/mL, using a smartphone and a color readout app. On the other hand, distance readout-based µPADs showed changes of the length of colored sections in accordance with lactoferrin concentration. In summary, we successfully developed both colorimetric intensity-based paper wells and distance-based µPADs for lactoferrin detection. This work demonstrates a user-friendly colorimetric analysis platform for lactoferrin without requiring lab equipment and expensive antibodies.

Microfluidic paper-based analytical devices (μPADs) for detection of hydrogen peroxide and glucose have been developed. The analytical performance of colorimetric detection using the conventional starch-iodine color reaction has been significantly improved by using gelatin as the surface modifier which retains the enzyme activity in the dry filter paper strip, improves antioxidability, as well as decreases the strong background signal. Under optimal conditions, the color intensities show a good linear relationship with glucose concentration ranging from 0.5 to 5 mM and hydrogen peroxide concentration from 0.5 to 6 mM, with the detection limit of 0.05 mM and 0.1 mM, respectively. In addition, the accuracy of colorimetric sensor has been successfully assessed in detecting glucose from real human serum samples and recovery value ranges from 95.7% to 97%, which are approaching to the glucose oxidase endpoint. The new colorimetric assay exhibits high sensitivity, good selectivity, acceptable stability and reproducibility. The present approach is promising for monitoring glucose for point of care diagnostic applications, especially in regions with resource-limited settings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Microfluidic paper-based devices (μPADs) and wearable devices have been highly studied to be used as diagnostic tools due to their advantages such as simplicity and ability to provide instrument-free fast results. Diseases such as periodontitis and diabetes mellitus can potentially be detected through these devices by the detection of important biomarkers. This study describes the development of μPADs through craft cutter printing for glucose and nitrite salivary diagnostics. In addition, the use of μPADs integrated into a mouthguard as a wearable sensor for glucose monitoring is also presented. μPADs were designed to contain two detection zones for glucose and nitrite assays and a sampling zone interconnected by microfluidic channels. Initially, the analytical performance of the proposed μPADs was investigated and it provided linear behavior (r 2  ≥ 0.994) in the concentration ranges between 0 to 2.0 mmol L -1 and 0 to 400 μmol L -1 for glucose and nitrite, respectively. Under the optimized conditions, the limits of detection achieved for glucose and nitrite were 27 μmol L -1 and 7 μmol L -1 , respectively. Human saliva samples were collected from healthy individuals and patients previously diagnosed with periodontitis or diabetes and then analyzed on the proposed μPADs. The results found using μPADs revealed higher glucose concentration values in saliva collected from patients diagnosed with diabetes mellitus and greater nitrite concentrations in saliva collected from patients diagnosed with periodontitis, as expected. The results obtained on μPADs did not differ statistically from those measured by spectrophotometry. With the aim of developing paper-based wearable sensors, μPADs were integrated, for the first time, into a silicone mouthguard using a 3D-printed holder. The proof of concept was successfully demonstrated through the monitoring of the glucose concentration in saliva after the ingestion of chocolate. According to the results reported herein, paper-based microfluidic devices offer great potential for salivary diagnostics, making their integration into a silicone mouthguard possible, generating simple, low-cost, instrument-free, and powerful wearable sensors.

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

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

A visual colorimetric microfluidic paper-based analytical device (μPAD) was constructed following the direct synthesis of enzyme-inorganic hybrid nanomaterials on the paper matrix. An inorganic solution of MnSO 4 and KH 2 PO 4 containing a diluted enzyme (glucose oxidase, GOx) was subsequently pipetted onto cellulose paper for the in situ growth of GOx@Mn 3 (PO 4 ) 2 hybrid functional materials. The characterization of the morphology and chemical composition validated the presence of hybrid materials roots in the paper fiber, while the Mn 3 (PO 4 ) 2 of the hybrid provided both a surface for enzyme anchoring and a higher peroxidase-like catalytic activity as compared to the Mn 3 (PO 4 ) 2 crystal that was synthesized without enzyme modulation. This new approach for the in situ growth of an enzyme-inorganic hybrid on a paper matrix eliminates centrifugation and the dry process by casting the solution on paper. The sensing material loading was highly reproducible because of the accuracy and stability of pipetting, which eventually contributed to the reliability of the μPAD. The self-assembled natural and artificial enzyme hybrid on the μPADs specifically detected glucose from a group of interferences, which shows great specificity using this method. Moreover, the colorimetric signal exhibited detection limitation for glucose is 0.01mM, which lies in the physiological range of glucose in biological samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow μPADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model μPAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 μg mL -1 . Graphical Abstract A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported.

Abstract This review reports on recent advances in state-of-the-art detection methods for microfluidic paper-based analytical devices (μPADs). The review commences by describing the materials, fabrication methods and driving mechanisms employed in μPADs. The review then explores the main detection methods and applications of μPADs proposed over the past five years. The discussions focus on seven detection technologies, namely (1) colorimetric detection, (2) fluorescent detection, (3) electrochemical detection and photoelectrochemical detection, (4) chemiluminescent detection, (5) electrochemiluminescent detection, (6) nanoparticle-based detection, and (7) spectrometry detection. Overall, the review provides a useful insight into recent advances in the μPAD detection field and serves as a useful source of reference for further research and innovation in the field. Highlights • Detection methods of microfluidic paper-based analytical devices (μPADs) have been discussed. • The review focuses on proposals for main detection methods and applications of μPADs. • A comprehensive review of the main applications of μPADs over the past five years. [ABSTRACT FROM AUTHOR]

A colorimetric sensing strategy employing gold nanoparticles and a paper assay platform has been developed for tuberculosis diagnosis. Unmodified gold nanoparticles and single-stranded detection oligonucleotides are used to achieve rapid diagnosis without complicated and time-consuming thiolated or other surface-modified probe preparation processes. To eliminate the use of sophisticated equipment for data analysis, the color variance for multiple detection results was simultaneously collected and concentrated on cellulose paper with the data readout transmitted for cloud computing via a smartphone. The results show that the 2.6 nM tuberculosis mycobacterium target sequences extracted from patients can easily be detected, and the turnaround time after the human DNA is extracted from clinical samples was approximately 1 h. [ABSTRACT FROM AUTHOR]