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This paper introduces the concept of "Drawing-PADs" (Drawing paper-based microfluidic analytical devices) allowing to intuitively evaluate the urinary albumin (Alb) index, a clinically important parameter used for the early detection of renal deficiencies related to diabetes, among others. To enable regular monitoring of the Alb index, a simple examination method suitable for self-diagnosis is highly desirable. The Drawing-PADs rely on the simultaneous naked eye detection of Alb and creatinine (Cre) on a single device according to the distance-based microfluidic PAD (μPAD) approach. The Alb index is visualized by simply drawing a straight line connecting the top of two color-changed assay channel sections (Alb and Cre channels), followed by visually confirming the position of the intercept of the drawn straight line. The semiquantitative Alb index evaluation performed with Drawing-PADs does not require any equipment such as a camera, software, or a color reference chart. The obtained results are independent of the sample volume and are not influenced by changes in the absolute Alb and Cre concentrations caused by urine excretion variations, making spot urine assays possible. Classification of Alb index values according to clinically relevant criteria (normoalbuminuria, microalbuminuria, and macroalbuminuria) is readily achieved within 15 min and has been validated for 15 human urine samples including diabetic patients and healthy volunteers.

Paper-based microfluidic devices have advanced significantly in recent years as they are affordable, automated with capillary action, portable, and biodegradable diagnostic platforms for a variety of health, environmental, and food quality applications. In terms of commercialization, however, paper-based microfluidics still have to overcome significant challenges to become an authentic point-of-care testing format with the advanced capabilities of analyte purification, multiplex analysis, quantification, and detection with high sensitivity and selectivity. Moreover, fluid flow manipulation for multistep integration, which involves valving and flow velocity control, is also a critical parameter to achieve high-performance devices. Considering these limitations, the aim of this review is to (i) comprehensively analyze the fabrication techniques of microfluidic paper-based analytical devices, (ii) provide a theoretical background and various methods for fluid flow manipulation, and (iii) highlight the recent detection techniques developed for various applications, including their advantages and disadvantages. [ABSTRACT FROM AUTHOR]

The rapid development of information and communication technology provides an opportunity for scientists to develop a quantitative analytical method that is built on a mobile gadget as a detector. In this paper, we report a low-cost, simple and portable analytical method for mercury ion quantification based on digital image colorimetry coupled with a smartphone application. A small amount of silver nanoparticles (AgNPs) was used as a colorimetric agent that is selective only to mercury ions. The yellowish brown color of AgNPs instantly changed to colorless after the addition of mercury ions due to a redox reaction. To increase the portability, we attached the AgNPs onto a medium to create a paper-based analytical device. The final data processing of the colorimetric analysis was conducted using an android application available on the Google Play Store, called "Mercury Detector". The proposed method has good sensitivity, with a detection limit of 0.86 ppb, which is comparable to those of bulk and more expensive instruments. This allows for the detection of mercury ions as low as 2 ppb (10 nM), which is also the maximum contaminant level permitted in drinking water by the US Environmental Protection Agency. The proposed method was applied to real samples that provide satisfactory results on accuracy (2.4%) and precision (2.5%). [ABSTRACT FROM AUTHOR]

The electrophoresis of ampholytes such as amino acids on a paper device is difficult because of the variation of pH distribution in time. On the basis of this observation, we propose a paper-based analytical device (PAD) with origami structure. By folding a filter paper, a low operation voltage of 5 V was achieved, where the power was supplied by a 5 V 1.5 A portable power bank through the USB type A receptacle. As a demonstration, we carried out the electrophoretic separation of pI markers (pI 5.5 and 8.7). The separation was achieved within 4 min before the pH distribution on the paper varied. Though the separation distance was small, it could be increased by expanding the origami structure. This result indicates that our proposed PAD is useful for electrophoretic separation on a paper device. [ABSTRACT FROM AUTHOR]

Paraquat intoxication is characterized by acute kidney injury and multi-organ failure, causing substantial mortality and morbidity. This study aims to develop a 2-in-1 paper-based analytical device to detect the concentrations of paraquat and creatinine in human serum, which can help clinicians diagnose patients with paraquat poisoning in a more rapid and geographically unrestricted manner. The procedure involves fabrication of a paper-based analytical device, i.e., printing of design on a filter paper, heating of wax-printed micro zone plates so as molten wax diffusing into and completely through the paper to the other side, forming hydrophobic boundaries that could act as detection zones for the paraquat colorimetric assay, and finally analysis using ImageJ software. The paper employed a colorimetric sodium dithionite assay to indicate the paraquat level in a buffer or human serum system in less than 10 min. In this study, colorimetric changes into blue color could be observed by the naked eye. By curve fitting models of sodium dithionite in normal human serum, we evaluated the serum paraquat levels for five paraquat patients. In the sodium dithionate assay, the measured serum paraquat concentrations in patients 1–5 were 22.59, 5.99, 26.52, 35.19 and 25.00 ppm, respectively. On the other hand, by curve fitting models of the creatinine assay in normal human serum, the measured serum creatinine concentrations were 16.10, 12.92, 13.82, 13.58 and 12.20 ppm, respectively. We found that the analytical performance of this device can compete with the standard of Clinical Laboratory of Chang Gung Memorial Hospital, with a less complicated sample preparation process and more rapid results. In conclusion, this 2-in-1 paper-based analytical device has the advantage of being simple and cheap, enabling rapid detection of paraquat intoxication as well as assessment of renal prognosis. [ABSTRACT FROM AUTHOR]

Cronobacter spp. are recognized as a world-wide emerging opportunistic food-borne pathogens that can persist in various food and processing environment for a long time. Therefore, the prevention and detection of them are particularly important. In this study, a micro-spot paper-based analytical device (μPADs) was created by combining PVC pad with filter paper. Detection is achieved by measuring the color change (from colorless to indigo) when a species-specific enzyme associated with the Cronobacter spp. of interest reacts with a chromogenic substrate. When combined with the optimization of specific enrichment process, the method allows for a testing time of 10 h or less and is capable of detecting live bacteria on the inoculated surface of samples in concentrations as low as 10 1  CFU cm -2 . We are surprised to discover that C. dublinensis species and their subspecies had the highest ability to produce α-glucosidase in all genus of Cronobacter spp.. This work demonstrated that the manufacturing method is novel, simple, well reproducible (RSD less than 5%) and low-cost (less than $ 0.15 per micro-spot)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Colorimetric detection with smartphones is ideal for point-of-care measurements because the signal reader is easily available. Densitometric detection schemes enable semiquantitative measurements but require a lightproof box to control photographic conditions and/or extensive data treatment to extract information. Approaches based on pattern recognition are not so sensitive to light artifacts but can only yield a yes/no type of answer when the signal is above or below a certain threshold. Here, we introduce a new method for detecting different concentrations of proteins as well as light artifacts with origami immunosensors and digital logic. The origami design consists of a folded piece of paper with three identical biorecognition sites so that one drop of sample generates three colorimetric signals simultaneously. The three colorimetric signals are then evaluated with an augmented reality app that generates a virtual semaphore that sequentially turns on its green, yellow, and red lights depending on the concentration of analyte. These three Boolean variables pass through "and" and "not" logic gates in a 3-to-8 decoder that enables the semiquantitative detection of proteins and adds a failsafe against erroneous results. The proposed method can detect the model analyte mouse IgG with a limit of detection and sensitivity comparable to densitometry performed under light-controlled conditions. It can also detect the sepsis biomarker procalcitonin at clinically relevant concentrations. With our approach, the detection is performed in real time, and signal processing is not required, which makes it suitable for rapid analyses by nonspecialists at the point of need.

A rapid, simple and affordable method for arsenic speciation analysis is described in this work. The proposed methodology involves in situ arsine generation, transfer of the volatile to the headspace and its reaction with silver nitrate at the detection zone of a paper-based analytical device (PAD). Thus, silver nitrate acts as a recognition element for arsine in the paper-based sensor. The chemical reaction between the recognition element and the analyte derivative results in the formation of a colored product which can be detected by scanning the detection zone and data treatment with an image processing and analysis program. Detection and injection zones were defined in the paper substrate by formation of hydrophobic barriers, thus enabling the formation of the volatile derivative without affecting the chemical stability of the recognition element present in the PAD. Experimental parameters influencing the analytical performance of the methodology, namely color mode detection, composition of the paper-based sensor and hydride generation and mass transfer conditions, were evaluated. Under optimal conditions, the proposed method showed limits of detection and quantification of 1.1 and 3.6 ng mL -1 , respectively. Remarkably, the limit of detection of the method reported herein was much lower than the maximum contaminant levels set by both the World Health Organization and the US Environmental Protection Agency for arsenic in drinking water, unlike several commercially available arsenic test kits. The repeatability, expressed as relative standard deviation, was found to be 7.1% (n = 8). The method was validated against the European Reference Material ERM ® -CA615 groundwater and successfully applied to the determination of As(III), As(V) and total inorganic As in different water samples. Furthermore, the method can be used for the screening analysis of total arsenic in waters when a cut-off level of 7 ng mL -1 is used., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An easily performed miniaturized, cheap, selective and sensitive procedure for the determination of H 2 S in fuel oil samples based on a headspace liquid-phase microextraction followed by a cyclic voltammetry detection using a paper-based analytical device (PAD) was developed. A modified wax dipping method was applied to fabricate the PAD. The PAD included hydrophobic zones of sample and supporting electrolyte connecting by hydrophilic channel. The zones of sample and supporting electrolyte were connected with nickel working, platinum auxiliary and Ag/AgCl reference electrodes. The analytical procedure included separation of H 2 S from fuel oil sample based on the headspace liquid-phase microextraction in alkaline solution. Then, sulfide ions solution obtained and supporting electrolyte were dropped on the zones followed by analyte detection at + 0.45 V. Under the optimized conditions, H 2 S concentration in the range from 2 to 20 mg kg -1 had a good linear relation with the peak current. The limit of detection (3σ) was 0.6 mg kg -1 . The procedure was successfully applied to the analysis of fuel oil samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This paper describes the fabrication of and data collection from two microfluidic devices: a microfluidic thread/paper based analytical device (μTPAD) and 3D microfluidic paper-based analytical device (μPAD). Flowing solutions of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI), through each device, on contact with glucose, generated a calibration curve for each platform. The resultant yellow-brown color from the reaction indicates oxidation of iodide to iodine. The devices were dried, scanned, and analyzed yielding a correlation between yellow intensity and glucose concentration. A similar procedure, using an unknown concentration of glucose in artificial urine, is conducted and compared to the calibration curve to obtain the unknown value. Studies to quantify glucose in artificial urine showed good correlation between the theoretical and actual concentrations, as percent differences were ≤13.0%. An ANN was trained on the four-channel CMYK color data from 54 μTPAD and 160 μPAD analysis sites and Pearson correlation coefficients of R = 0.96491 and 0.9739, respectively, were obtained. The ANN was able to correctly classify 94.4% (51 of 54 samples) and 91.2% (146 of 160 samples) of the μTPAD and μPAD analysis sites, respectively. The development of this technology combined with ANN should further facilitate the use of these platforms for colorimetric analysis of other analytes., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electrokinetic stacking (ES) is effective for improving sensitivity of paper-based analytical device (PAD) for charged analytes. In this paper, we successfully demonstrated ES of electrically neutral analytes on PAD, and the performance was characterized by smartphone-based colorimetry and fluorescence. Firstly, SDS from cathode reservoir stacked as a micelle band on an open paper fluidic channel by ES, and the target analyte was swept by the micelle. Meanwhile, the probes at the other side were carried by electroosmotic flow (EOF). Eventually, neutral components preloaded on the channel were concentrated as the narrow stacking band. Taking the rhodamine B as a probe, the effects of EOF, background electrolyte concentration and anionic surfactant concentration were investigated. Fluorescence detection of rhodamine B and colorimetric analysis of Sudan III demonstrated the sensitivity enhanced and its potential for the semi-quantitative test. Under the optimized conditions, fluorescence detection limit of 50 nM of rhodamine B was achieved with a linear range of 1.0-10 μM (R 2 = 0.99). The colorimetric detection limit for Sudan III was 5.2 μM and the linear range was 5-40 μM (R 2 = 0.99). Compared with direct analysis without stacking, the signal levels of rhodamine B and Sudan III were increased by 30-fold and 6-fold, respectively. This study showed that with ES, sensitive and rapid PAD detection of electrically neutral analytes could be achieved., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this work, we studied the feasibility of interaction among gold nanoparticles (AuNPs) and a cationic dye in an aptasensor system for the detection of potassium ions. The presence and absence of potassium in the solution was distinguishable by different colors (between orange and green) appeared after reaction. Cationic dye (Y5GL) acts as a new aggregator for AuNP-based sensors which changes the aggregated AuNP solution color from blue-purple to green. In the presence of K + ions, the aptamer dissociated from the surface of the AuNP so that free AuNPs and cationic dye make the solution green. The aptasensor showed that the analytical linear range was from 10 nM to 50 mM and the detection limit was 4.4 nM. Also, we examined the practicality of this method on a simple paper based platform. The linear range of the colorimetric paper sensor covered of K + concentration from 10 μM to 40 mM and the detection limit of 6.2 μM was obtained. The selectivity of AuNP aggregation-based sensor improved by the use of cationic dye. Rapidity, simplicity, high sensitivity and excellent selectivity made this assay suitable for practical determination of K + in real urine samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

On-site rapid monitoring of nitrite as an assessment indicator of the environment, food, and physiological systems has drawn extensive attention. Here, electrokinetic stacking (ES) was combined with colorimetric reaction on a paper-based device (PAD) to achieve colorless nitrite detection with smartphone. In this paper, nitrite was stacked on the paper fluidic channel as a narrow band by electrokinetic stacking. Then, Griess reagent was introduced to visualize the stacking band. Under optimal conditions, the sensitivity of nitrite was 160-fold increased within 5 min. A linear response in the range of 0.075 to 1.0 μg mL -1 (R 2  = 0.99) and a limit of detection (LOD) of 73 ng mL -1 (0.86 μM) were obtained. The LOD was 10 times lower than the reported PAD, and close to that achieved by a desktop spectrophotometer. The applicability was demonstrated by nitrite detection from saliva and water with good selectivity, adding 100 times more concentrated co-ions. High recovery (91.0~108.7%) and reasonable intra-day and inter-day reproducibility (RSD < 9%) were obtained. This work shows that the sensitivity of colorless analyte detection-based colorimetric reaction can be effectively enhanced by integration of ES on a PAD. Graphical abstract Schematic of the experimental setups (left) and the corresponding images (right) of the actual portable device.

Soil-transmitted helminth (STH) infections are a global health issue affecting nearly one-third of the world's population. As most endemic areas of STH are impoverished countries or regions with limited healthcare resources, the accurate diagnosis of STH requires analytical tools that are not only quantitative, but also portable, inexpensive, and with no or minimal demand for external instrument. Herein, we introduce a novel paper-based diagnostic device, termed quantitative paper-based DNA reader (qPDR), capable of quantifying STH at the molecular level by measuring distance as readout, thus eliminating the need for external readers. On the basis of the unique interfacial interaction of a DNA intercalating dye, SYBR Green I, with native cellulose on a chromatographic paper, qPDR allows the distance-based quantification of minute amounts of double-stranded DNA as short as 6 min. By integrating qPDR with polymerase chain reactions that were performed using a smartphone-controlled portable thermal cycler, we were able to quantify minute amount of genetic markers from adult worms of an STH (Trichuris trichiura) that were expelled post-treatment by infected children living in the rural areas of Honduras.

In this work, we report a highly sensitive colorimetric sensing strategy for cancer biomarker diagnosis using gold nanoparticles (AuNPs) labeled with biotinylated poly(adenine) ssDNA sequences and streptavidin-horseradish peroxidase for enzymatic signal enhancement. By adopting this DNA-AuNP nanoconjugate sensing strategy, we were able to eliminate the complicated and costly thiol-binding process typically used to modify AuNP surfaces with ssDNA. In addition, different antibodies can be introduced to the AuNP surfaced via electrostatic interactions to provide highly specific recognition sites for biomolecular sensing. Moreover, multiple, simultaneous tests can be rapidly performed with low sample consumption by incorporating these surface-modified AuNPs into a paper-based analytical device that can be read using just a smartphone. As a result of these innovations, we were able to achieve a detection limit of 10 pg/mL for a prostate specific antigen in a test that could be completed in as little as 15 min. These results suggest that the proposed paper platform possesses the capability for sensitive, high-throughput, and on-site prognosis in resource-limited settings.

Quantification is a fundamental aspect of performance of an analytical system. Paper-based analytical device (PAD) as an on-site detection platform has drawn wide attention mainly due to its portability and cost effectiveness. In this work, a portable and low-cost PAD for online preconcentration and sensitive determination of fluorescent whitening agent (FWA) was demonstrated, which was consisted of ultra violet light-emitting diode (UV LED), macro-focusing lens, smartphone and miniaturized DC voltage source. Taking a widely used FWA component VBL as the analyte, the performance of the PAD enhanced with electrokinetic stacking (ES) and fluorescence imaging detection was systematically investigated. With ES, the sensitivity of the PAD system was 160-fold enhanced, and a limit of detection (LOD) of 0.06 μg mL -1 was achieved. The dynamic range was 0.1-10.0 μg mL -1 (linear in 0.1-0.7 μg mL -1 , R 2  = 0.99). With manual operation, the relative standard deviation (RSD) of intra-day and inter-day were all below 15%. Eventually, VBL from different napkin samples and toilet paper was determined with average recovery rates in the range of 90%-95% (RSD = 8.0%-12.0%). This work shows that with ES, the sensitivity of PAD can be greatly improved, and a LOD close to a desktop fluorescent spectrophotometer can be achieved as demonstrated by the detection of FWA component., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We have developed a colorimetric sensing strategy employing gold nanoparticles and a paper-based analytical platform for the diagnosis of tuberculosis (TB). By utilizing the surface plasmon resonance effect, we were able to monitor changes in the color of a gold nanoparticle colloid based on the effects of single-stranded DNA probe molecules hybridizing with targeted double-stranded TB DNA. The hybridization event changes the surface charge density of the nanoparticles, causing them to aggregate to various degrees, which modifies the color of the solution in a manner that can be readily measured to determine the concentration of the targeted DNA analyte. In order to adapt this TB diagnosis method to resource-limited settings, we extended this label-free oligonucleotide and unmodified gold nanoparticle solution-based technique to a paper-based system that can be measured using a smartphone to obtain rapid parallel colorimetric results with low reagent consumption and without the need for sophisticated analytical equipment. In this study, we investigated various assay conditions, including the denaturing temperature and time, different oligonucleotide probe sequences, as well as the ratio of single stranded probe and double stranded target DNA. After optimizing these variables, we were able to achieve a detection limit of 1.95 × 10 -2 ng/mL for TB DNA. Furthermore, multiple tests could be performed simultaneously with a 60 min turnaround time.

This paper describes a paper-based analytical device allowing the direct semiquantitative interpretation of the result of a chemical assay in the form of "text". The combined use of a classical colorimetric indicator system and an additional inert colorant enables a versatile text-displaying detection mechanism on a paper device. For proof-of-concept, urinary protein has been selected as a model analytical target. The whole text-displaying paper device has been developed based on printing techniques including wax printing, inkjet printing, and 3D printing. The results of user tests performed with protein (human serum albumin) samples in aqueous standard solutions and human urine demonstrated that the accuracy was comparable for the elaborated paper device (74.7% for standard samples and 66.7% for urine) and a conventional colorimetric urine dipstick (67.2% for standard samples and 65.3% for urine). Storage stability as long as at least 117 days has been confirmed based on software-assisted quantitative color analysis. The developed text-displaying approach is proposed as an alternative simple detection motif for paper-based analytical devices.

In this research, we found that the peroxidase-like activities of noncovalent DNA-Pt hybrid nanoparticles could be obviously blocked, when Pt nanoparticles (PtNPs) were synthesized in situ using DNA as a template. Moreover, this self-assembled synthetic process was very convenient and rapid (within few mintues), and the inhibition mediated by DNA was also very effective. First, by the paper-based analytical device (PAD) we found the catalytic activities of DNA-Pt hybrid nanoparticles exhibited a linear response to the concentration of DNA in the range from 0.0075 to 0.25µM. Then, with the magnetic bead isolated system and target DNA-induced hybridization chain reaction (HCR), we realized the specific target DNA analysis with a low detection of 0.228nM, and demonstrated its effectivity in distinguishing the target DNA from other interferences. To our knowledge, this is the first report that used the nanoassembly between DNA and PtNPs for colorimetric detection of nucleic acids, which was based on DNA-mediated inhibition of catalytic activities of platinum nanoparticles. The results may be useful for understanding the interactions between DNA and metal nanoparticles, and for development of other convenient and effective analytical strategies., (Copyright © 2017. Published by Elsevier B.V.)

A new analytical platform based on the use of thin-layer chromatography (TLC) coupled with paper-based analytical device (PAD) was developed for the determination of total capsaicinoids in chilli samples. This newly developed TLC-PAD is simple and low-cost without any requirement of special instrument or skillful person. The analysis consisted of two steps, i.e., extraction of capsaicinoids from chilli samples by using ethanol as solvent and separation of capsaicinoids by thin-layer chromatography (TLC) and elution of capsaicinoids from the TLC plate with in situ colorimetric detection of capsaicinoids on the PAD. For colorimetric detection, Folin-Ciocalteu reagent was used to detect phenolic functional group of capsaicinoids yielding the blue color. The blue color on the PAD was imaged by a scanner followed by evaluation of its grayscale intensity value by ImageJ program. This newly developed TLC-PAD method provided a linear range from 50 to 1000mgL -1 capsaicinoids with the limit of detection as low as 50mgL -1 capsaicinoids. The proposed method was applied to determine capsaicinoids in dried chilli and seasoning powder samples and the results were in good agreement with those obtained by HPLC method., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Here, we report on carbon nanotube paper-based electroanalytical devices. A highly aligned-carbon nanotube (HA-CNT) array, grown using chemical vapor deposition (CVD), was processed to form bi-layered paper with an integrated cellulose-based Origami-chip as the electroanalytical device. We used an inverse-ordered fabrication method from a thick carbon nanotube (CNT) sheet to a thin CNT sheet. A 200-layered HA-CNT sheet and a 100-layered HA-CNT sheet are explored as a working electrode. The device was fabricated using the following methods: (1) cellulose-based paper was patterned using a wax printer, (2) electrical connection was made using a silver ink-based circuit printer, and (3) three electrodes were stacked on a 2D Origami cell. Electrochemical behavior was evaluated using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). We believe that this platform could attract a great deal of interest for use in various chemical and biomedical applications.

"Distance-based" detection motifs on microfluidic paper-based analytical devices (μPADs) allow quantitative analysis without using signal readout instruments in a similar manner to classical analogue thermometers. To realize a cost-effective and calibration-free distance-based assay of lactoferrin in human tear fluid on a μPAD not relying on antibodies or enzymes, we investigated the fluidic mobilities of the target protein and Tb(3+) cations used as the fluorescent detection reagent on surface-modified cellulosic filter papers. Chromatographic elution experiments in a tear-like sample matrix containing electrolytes and proteins revealed a collapse of attractive electrostatic interactions between lactoferrin or Tb(3+) and the cellulosic substrate, which was overcome by the modification of the paper surface with the sulfated polysaccharide ι-carrageenan. The resulting μPAD based on the fluorescence emission distance successfully analyzed 0-4 mg mL(-1) of lactoferrin in complex human tear matrix with a lower limit of detection of 0.1 mg mL(-1) by simple visual inspection. Assay results of 18 human tear samples including ocular disease patients and healthy volunteers showed good correlation to the reference ELISA method with a slope of 0.997 and a regression coefficient of 0.948. The distance-based quantitative signal and the good batch-to-batch fabrication reproducibility relying on printing methods enable quantitative analysis by simply reading out "concentration scale marks" printed on the μPAD without performing any calibration and using any signal readout instrument.

Paper-based analytical devices are fluidic chips fabricated with extremely inexpensive materials, namely paper, thereby allowing their use as a zero-cost analytical device in third-world countries that lack access to expensive diagnostic infrastructures. The aim of this review is to discuss: (1) microfluidic paper-based analytical devices (µPADs) for quantitative analysis, (2) fabrication of two- or three-dimensional µPADs, (3) analytical methods of µPADs, and (4) our opinions regarding the future applications of µPADs for quantitative urinalysis.