3D μPAD Research Articles

Evaluation of food μPADs with the new tech perspectives and future prospects

AbstractFood microfluidic paper‐based analytical devices (µPADs) are powerful tools to create total analysis systems and have long been demonstrated to be useful for safety and quality applications. Thanks to new technological innovations food µPADs offer exciting new possibilities. This review introduces how µPADs are obtained from chromatography, filter, or office papers and detect analytes from a food sample. We introduce the most current developments in the use of µPADs with an emphasis on paper types, adapted new technologies, and detection limits. Classifications are also made on food µPADs according to applied innovations and adapted novel technologies. In the first section, simple forms of µPADs for the detection of pathogen, mycotoxin, pesticides, and other food components are discussed with technical details. Then, we introduce multisensing approaches for high throughput analysis and a concise summary will be given. In the case of three‐dimensional (3D) µPADs, the use of 3D is discussed and compared to 2D µPADs in terms of its advantageous with the example of a food colorant test device. In the following section, smartphone adoption to µPADs is introduced in detail with eight different assay examples. Centrifugal platform for nickel assay is demonstrated as an enabling approach with shortened assay times by rotational velocity. The potential of user‐friendly hybrid devices is also summarized in the last part. Finally, we present an outlook to underline the opportunities created by smartphone‐based and intelligent µPADs for food safety and quality with real success perspectives

Hinge-like paper-based dual-channel enzyme-free ratiometric fluorescent microfluidic platform for simultaneous visual detection of carbaryl and glyphosate

On-site multi-pesticide residues detection is particularly urgent and challenging. Here, we fabricated an enzyme-free ratiometric fluorescent detection system in combination with a hinge-like dual-channel 3D microfluidic paper analytical device (3D μPAD) for simultaneous visual detection of carbaryl and glyphosate. Blue-emission 1-naphthol (Em. 470 nm) was hydrolyzed from carbaryl, while yellow-emission 2,3-diaminophenazine (Em. 570 nm) was produced with the aid of Cu2+ for glyphosate sensing. Inner-filter effect between 1-naphthol or 2,3-diaminophenazine and green-emission carbon dots (Em. 510 nm) realized two ratiometric fluorescent detection systems. Remarkable color variation of green–blue for carbaryl (50.00–1100 μΜ) and yellow–green for glyphosate (5.00–600 μΜ) were observed on a dual-channel 3D μPAD without crosstalk. Their detection limits were 1.11 and 0.63 μΜ, respectively. The strategy realized simultaneous visual detection of carbaryl and glyphosate in food/herbal with excellent accuracy (spiked recoveries, 91.00–107.2%), high precision (RSD ≤ 8.43%), and superior selectivity.

A 3D microfluidic paper-based analytical device with smartphone-assisted colorimetric detection for iodine speciation in seaweed samples

The present work reports on the development of a 3D origami microfluidic paper-based analytical device (3D µPAD) for the determination of iodide and iodate in edible seaweeds by smartphone-based colorimetric detection. In addition, a methacrylate holder was designed and fabricated to enhance interlayer contact in 3D µPADs, obtaining excellent sensitivity and precision, also allowing real-time monitoring in a straightforward and expeditious way. The reported assay, based on the formation of a blue colored triiodide-starch complex at the detection areas of the 3D µPAD, represents an affordable, fast and greener alternative for the simultaneous determination of inorganic iodine species. Under optimal conditions, the proposed method showed limits of detection and quantification of 9.8 and 32.7 µM for I- and 0.6 and 1.8 µM for IO3-, respectively. The repeatability, expressed as relative standard deviation, was 1.7% and 3.3% for I- and IO3-, respectively. The proposed 3D µPAD was applied to the determination of iodine species in extracts of edible seaweeds and related food additives, showing satisfactory recoveries (90–109%).

Threaded 3D microfluidic paper analytical device-based ratiometric fluorescent sensor for background-free and visual detection of organophosphorus pesticides

With the increasing concerns of food safety and environmental protection, it is desirable to develop reliable, effective, and portable sensors for detection of organophosphorus pesticides (OPs). Here, a cascade reaction system integrated with threaded 3D microfluidic paper analytical device (3D μPAD) was firstly developed for background-free and visual detection of OPs in agricultural samples. Butyrylcholinesterase (BChE) hydrolyzed acetylcholine into thiocholine (TCh), which reduced MnO2 nanosheets into Mn2+. With addition of OPs, BChE activity was irreversibly inhibited, and the generation of TCh and the reduction of MnO2 nanosheets were prevented. Then the remaining MnO2 nanosheets oxidized o-phenylenediamine into 2,3-diaminophenazine with yellow-emission fluorescence, which quenched the fluorescence intensity of red-emission carbon dots (RCDs) via inner-filter effect. Based on above mechanism, a ratiometric fluorescent system was established for OPs detection. Threaded 3D μPAD consisted of 4 layers, which allowed to load and/or add reagents to trigger the cascade reaction system for OPs detection. The fluorescent images presented distinguishable color variations from red to yellow with dichlorvos concentrations ranging from 2.5 to 120 μg L−1, and the limit of detection was 1.0 μg L−1. In the practical samples testing, threaded 3D μPAD can eliminate background influence on fluorescent signal for OPs detection. Threaded 3D μPAD integrated with ratiometric sensing platform has merits of accuracy response, facile operation, and background-free detection, which supplies a new alternative approach for on-site pesticide detection.

Engineering a monolithic 3D paper-based analytical device (μPAD) by stereolithography 3D printing and sequential digital masks for efficient 3D mixing and dopamine detection

Stereolithography 3D printing and digital masks in sequence are used herein to manufacture 3D µPAD in a monolithic layer of paper within 1 s. Multiple experiments were systematically conducted to further understand the fundamentals and limitation of this manufacturing process, and a novel 3D mixer and a fluorescent chemosensor assay used to determine dopamine concentration at high-alkaline pH conditions were successfully demonstrated. Experiment results showed: (1) the 3D mixer is superior than conventional 2D mixer regarding mixing performance and repeatability, (2) dopamine detection and pH indicator can be realized with fewer operation steps and shorter processing time, (3) a truly 3D µPAD, including multilayer microchannels within a monolithic paper substrate, can be created with fewer reagent loss and higher transportation efficiency, (4) a smallest microchannel, 100 µm wide microchannel, can be successfully created on µPAD. These results clearly proved that this proposed 3D µPAD is not only a novel format of µPAD, but it can also simplify the operation steps for an analytical assay while improving the mixing phenomenon via 3D mixer and using minimum sample volume. • Using SLA 3D printing and multiple digital masks to manufacture monolithic 3D µPAD. • 3D mixer is superior than 2D mixer regarding mixing performance and repeatability. • A truly 3D µPAD can result fewer reagent loss and higher transportation efficiency. • Dopamine detection and pH indicator can be realized with fewer operation steps. • A smallest microchannel, 100 µm wide microchannel, was successfully created.

Microfluidic Paper-Based Preconcentration and Retrieval for Rapid Ribonucleic Acid Biomarker Detection and Visualization.

Microfluidic paper-based analytical devices (μPADs) have attracted significant attention in the field of point-of-care (POC) diagnostics. However, the heterogeneous structure of the paper often impairs the limit of detection (LOD) for low-abundance targets when those targets are directly analyzed. One viable solution to bypass this limitation is to elevate the target concentration above the LOD on-site to reach a valid readout. Here, we developed a 3D μPADs preconcentrator (3D-μP2) to increase sample concentration by electrokinetic trapping and demonstrated its application in increasing the LOD of a downstream colorimetric assay. The three-dimensional (3D) structure of this device was composed of a loading pad, a vertical fluid path formed by stacked absorbent pads, and an ion-selective membrane of PEDOT:PSS. This novel design facilitates fast preconcentration, high capacity in sample processing, and easy target retrieval. The concentration of an exemplary target, a single-stranded DNA sequence, was increased up to 170-fold within 80 s. The LOD of the colorimetric assay to verify the DNA target was increased 3 orders of magnitude with a preconcentrated sample compared to the control. The device and its analysis equipment used in this study were all cheap and portable. Thus, the 3D-μP2 can be a powerful POC tool for sample pretreatment in resource-limited areas.

On paper synthesis of multifunctional CeO2 nanoparticles@Fe-MOF composite as a multi-enzyme cascade platform for multiplex colorimetric detection of glucose, fructose, sucrose, and maltose

This study reports an economical and portable point-of-care (POC) monitoring device based on artificial multi-enzyme cascade systems for multiple detection purposes. The device was made up of a disposable three dimensional microfluidic paper-based analytical device (3D μPAD) with multiple detection zones and a smartphone readout. On-paper synthesis of a multifunctional mimetic composite, based on the CeO2 nanoparticles embedded in the amino-functionalized Fe metal-organic frameworks (CeO2@NH2-MIL-88B(Fe)), for cascade reactions was the main achievement of this work. The 3D μPAD was applied for simultaneous quantification of glucose, fructose, sucrose and maltose, and the detection process consisted of the enzymatic reaction of each sugar by anchored enzymes on the metal-organic frameworks (MOF) and successive oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). Utilizing the new artificial mimicking system improved the color development uniformity and resulted in a reliable detection tool, with excellent detection limits in the range of 20–280 μM. It was directly applied to analyze the sugars levels of human total blood, urine, semen, honey and juice samples with the relative errors of less than 7.7% compared with the HPLC method. The cost-effective and easy-to-use μPAD has a great potential to be used in either medical diagnostics or the food industry. Also, it can be considered as a competitive POC method for patients in disadvantaged communities or emergencies.

Photo‐Adjustable TiO2‐Paper as a Smart Substrate for Paper‐Based Analytical Devices

AbstractMicrofluidic paper‐based analytical devices (μPADs) are commonly used in point‐of‐care testing (POCT) due to their advantages of low cost and easy operation. However, the fabrication and potential environmental pollution of μPADs are the universal and pendent concerns. In this respect, a simple and novel method is developed for preparing photo‐adjustable μPADs using the photocatalytic properties of TiO2. A superhydrophobic TiO2‐paper substrate with unique photoactivity is fabricated. The flow channels on the superhydrophobic TiO2‐paper substrate can be quickly generated and on‐demand edited by a simple UV irradiation process, to fabricate various 2D and 3D μPADs for specific applications. After usage, the residual organics and biological specimens on μPADs can be easily bleached via TiO2‐assited degradation under UV or sunlight, avoiding the risk of potential environment pollution and disease transmission. The successful application of such novel substrate in the fabrication of smart and safe μPADs for sweat analysis and motion monitoring is shown.

3D origami paper-based ratiometric fluorescent microfluidic device for visual point-of-care detection of alkaline phosphatase and butyrylcholinesterase

On-site multiplex enzyme detection is crucial for diagnosis, therapeutics and prognostic. To date, it is still a daunting challenge to develop portable, low-cost, and efficient multi-enzyme detection methods. Herein, a novel sample-in-result-out platform integrating ratiometric fluorescent assays with 3D origami microfluidic paper-based device (μPAD) was developed for simultaneous visual point-of-care testing (POCT) of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Cascade catalytic reaction with the same two fluorescent signal indicators was rationally designed to ratiometric fluorescent detection of ALP and BChE: substrate of ALP (pyrophosphate) and product of BChE (thiocholine) can strongly complex with Cu2+, Cu2+ oxidizes o-phenylenediamine to fluorescent 2,3-diaminophenazine (oxOPD) (emission, 565 nm), oxOPD quenches the fluorescence of carbon dots (CDs, emission at 445 nm) via inner filter effect, thus oxOPD/CDs values are relevant to ALP and BChE activities. Then 3D origami μPAD composing of four layers and two parallel channels was fabricated and simply prepared by one-step plotting with black oil-based marker and specific metal molds. After simple folding and unfolding neighboring layers to sequentially initiate reactions of pre-loaded reagents, fluorescent images on the detection zone can be captured by smartphone and analyzed by red-green-blue software for quantitative analysis. Under optimal conditions, the proposed platform was successfully performed to detect ALP and BChE with activity difference at 3 orders of magnitude in human serum samples without any pretreatment procedures. Excellent selectivity, good precision, favorable linear range, and high accuracy were exhibited. Importantly, the platform opens a promising horizon for high-throughput POCT of multiplex biomarkers.

Local photo-polymer deposition-assisted fabrication of multilayer paper-based devices

In this paper, we report on the use of a local acrylate-based negative photo-polymer deposition technique for the fabrication of 3D microfluidic paper-based analytical devices (3D-μPADs) where the sample flows in both the lateral and vertical directions through multiple stacked layers of porous materials. A simple and inexpensive manufacturing method was used, which is based on the local deposition of a photo-polymer (deposition speed 30 mm/s) on a porous cellulose paper substrate followed by the subsequent exposure (scanning speed 30 mm/s) to a laser source (fibre coupled continuous wave at 405 nm with maximum power of 60 mW), to stack four layers of cellulose paper and make 3D multilayer μPADs. With this technique, we provide a pathway to eliminate the limitations that other reported methods have during the fabrication of μPADs such as the need for multiple sophisticated alignments between adjoining layers and the use of additional tools to ensure adequate contact between the layers. In this study, we demonstrate the usefulness of our four-layer 3D-μPAD for simultaneous detection of three analytes, namely BSA, glucose, nitrite spiked in artificial urine and also the pH of the tested sample, through single step colorimetric assays with the limit of detection found at 0.4 mg/mL for BSA, 14.5 μg/mL for glucose and 2.5 μg/mL for nitrite. Our 3D-μPAD fabrication methodology can also be adapted in more complex analytical assays where multiple steps are needed for applications in point-of-care diagnostics.

Chemiluminescence Immunoassays for Simultaneous Detection of Three Heart Disease Biomarkers Using Magnetic Carbon Composites and Three-Dimensional Microfluidic Paper-Based Device

Chemiluminescence (CL) immunoassays for simultaneous detection of early acute myocardial infarction (AMI) biomarkers, including copeptin, heart-type fatty acid binding protein (h-FABP), and cardiac troponin I (cTnI), were developed by using Co2+/N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized magnetic carbon composite (Co2+-ABEI-Fe3O4@void@C) as an interface and a three-dimensional microfluidic paper-based device (3D μPAD) as a detection system. For CL immunoassays, Co2+-ABEI-Fe3O4@void@C was assembled with chitosan (CS) and gold nanoparticle-conjugated antibody (Au-Ab) sequentially to form the sensing platform (Co2+-ABEI-Fe3O4@void@C-CS/Au-Ab). In the presence of antigen (Ag), Ag was captured by the sensing interface to form an immunocomplex, leading to an increase in CL intensity due to the catalysis of -COO- existing in Ag. A 3D μPAD with three detection zones for simultaneous detection of copeptin, h-FABP, and cTnI was designed and fabricated to obtain time-resolved CL signals. Three kinds of immunocomplexes formed with copeptin, h-FABP, and cTnI were added to three detection zones of 3D μPAD, respectively. After injecting H2O2, three time-resolved CL signals were generated in one CL detection run by virtue of time-delayed transport of H2O2 to different detection zones. The three time-resolved CL signals were used for the simultaneous determination of copeptin, h-FABP, and cTnI. The detection limit of copeptin, h-FABP, and cTnI was 0.40 pg/mL, 0.32 pg/mL, and 0.50 pg/mL, respectively, which is at least 1 order of magnitude lower than most of the reported immunoassays. The immunoassays could be directly used for the detection of copeptin, h-FABP, and cTnI in human serum samples. The proposed immunoassays are simple, fast, sensitive, and selective, and are of great application potential in early diagnosis and treatment of AMI.

Multiplexed chemiluminescence determination of three acute myocardial infarction biomarkers based on microfluidic paper-based immunodevice dual amplified by multifunctionalized gold nanoparticles

Acute myocardial infarction (AMI) causes significant mortality and morbidity. The determination of multiple AMI biomarkers is very important for the timely diagnosis of AMI. In this work, simultaneous determination of three AMI biomarkers were achieved by virtue of a three-dimensional (3D) microfluidic paper-analytical device (μPAD) with temporally resolved chemiluminescence (CL) emissions for the first time. A dual-signal amplification strategy was introduced including by employing primary antibody functionalized gold nanoparticles (Ab1-GNPs) immobilized on the detection zone as amplified capture probes, and Co(II) catalyst, secondary antibody, luminol multifunctionalized gold nanoparticles (Co(II)-Ab2-luminol-GNPs) with excellent CL activity as amplified signal probes. CL immunoreactions were performed at three detection zone of the fabricated 3D μPAD by assembling Ab1-GNPs, antigen, and Co(II)-Ab2-luminol-GNPs to form sandwich-type immunocomplexes. Auto separated CL signals with temporal resolution were obtained by time delayed transport of H2O2 to different detection zones for multiplexed analysis. The CL signal obtained by using Co(II)-Ab2-luminol-GNPs as signal probe (10576 a.u.) were about 20-fold higher than that by using conventional horseradish peroxidase labeled antibody modified luminol-GNPs as signal probe (531 a.u.). Finally, three AMI biomarkers including heart-type fatty acid-binding protein (H-FABP), cardiac troponin I (cTnI) and copeptin were quantitatively analyzed in one CL detection run by reading the CL intensity of the obtained three CL emission peaks. The detection range were ultra-wide ranged from 0.1 pg/mL to 1 μg/mL, 0.5 pg/mL to 1 μg/mL and 1 pg/mL to 1 mg/mL with the detection limits down to 0.06 pg/mL, 0.3 pg/mL and 0.4 pg/mL for H-FABP, cTnI and copeptin detection, respectively. The developed μPAD based immunoassay performing multiplexed analysis ability, high sensitivity, ultra-wide dynamic range, favorable selectivity, accessible accuracy and reproducibility, have great application potential for the early diagnosis of AMI.

Three-dimensional microfluidic paper-based device for multiplexed colorimetric detection of six metal ions combined with use of a smartphone.

A simple double-layered three-dimensional (3D) microfluidic paper-based analytical device (μPAD) was designed for the simultaneous determination of six metal ions-Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II)-for the first time. The 3D μPAD was composed of two paper layers: a top pretreatment layer and a bottom colorimetric detection layer. The sample solution added to the central sample reservoir of the 3D μPAD could be automatically divided into eight flow pathways and be automatically pretreated while flowing through the pretreatment zones located in the microfluidic channels, and automatically carried out the chromogenic reactions after reaching the detection zones. Random diffusion of the chromogenic reagents was effectively prevented by transport of the pretreated sample solution to the detection zones through 3D microfluidic channels with an L-type circuitous flow route design, resulting in highly increased color uniformity and reproducibility. Combined with use of a flat LED lamp as an upward lighting source and a smartphone as a convenient detector, improved color perception, highly enhanced sensitivity, and an extended detection range were obtained. Finally, the double-layered 3D μPAD was applied to the multiplexed determination of the six metal ions in mixtures and environmental samples with satisfactory results. Detection limits as low as 0.2, 0.3, 0.1, 0.03, 0.08, and 0.04 mg/L for Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II) detection, respectively, were achieved, which are about one order of magnitude lower than obtained with previously reported μPADs for the detection of metal ions. The present 3D μPAD is simple, fast, selective, sensitive, and user-friendly, and holds great application potential for multiplexed on-site analysis.

High-resolution temporally resolved chemiluminescence based on double-layered 3D microfluidic paper-based device for multiplexed analysis

In this work, a double-layered three-dimensional (3D) microfluidic paper-based analytical device (μPAD) with high resolution temporally resolved chemiluminescence (CL) emissions were designed for multiplexed CL analysis. The temporally resolved CL emissions were obtained by virtue of the 3D branched microfluidic channel design, which create time delays for luminol transported from one detection zone to another. The peak intensity and peak shape of the temporally resolved CL emissions were quite stable and base-line separated with resolution as high as 21.2–24.4. Then, the fabricated μPAD was applied to multiplexed determination of glucose, lactate, cholesterol, and choline as model analytes. The sample was added to four detection zone modified with CL catalyst cobalt ion and different oxidase by virtue of chitosan. When luminol flowed to μPAD, four temporally resolved CL peaks were successively generated from the cobalt ion catalyzed CL reactions between luminol and generated H2O2 from the specific enzymatic reactions between the oxidase and the analytes. The generated four CL emission peaks in the CL kinetic curve increased in proportion to the concentrations of glucose, lactate, cholesterol, and choline, respectively. Finally, four linear calibration curves were obtained for the detection of glucose (0.01–1.0 mmol/L), lactate (0.02–5.0 mmol/L), cholesterol (0.01–0.4 mmol/L), and choline (0.001–1.0 mmol/L). The detection limits were as low as 8 μmol/L, 15 μmol/L, 6 μmol/L, and 0.07 μmol/L for glucose, lactate, choline, and cholesterol detection, respectively. The present work provides a new strategy for the fabrication of simple and sensitive 3D μPAD with high resolution temporally resolved CL emissions for multiplexed CL analysis, which holds great application potential for point-of-care diagnosis.

Flow Control-based 3D μPADs for Organophosphate Pesticide Detection.

Flow control-based paper devices have recently shown great potential for point-of-need analysis, since they allow for the easy operation of multi-step assays by minimizing user operation. In this work, a wax printing method was evaluated as a means to control liquid flow in 3D microfluidic paper-based analytical devices (μPADs). The resulting flow control-based 3D μPADs were applied to determine paraoxon-ethyl as a typical organophosphate pesticide model system. The analytical procedure is as simple as applying a 200-μL sample solution, resulting in reproducible (relative standard deviation of colorimetric signals from 6 independently fabricated devices, 2.63%) colorimetric signals within 1 h of the assay time with the limit of detection (LOD) reaching 25.0 μg/L. Finally, results obtained for pesticide-spiked water samples analyzed by flow control-based 3D μPADs showed good agreement with those from a conventional HPLC analysis with UV detection.

Simple Way To Fabricate Novel Paper-Based Valves Using Plastic Comb Binding Spines

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.

Laminated and infused Parafilm® - paper for paper-based analytical devices.

Numerous fabrication methods have been reported for microfluidic paper-based analytical devices (μPADs) using barrier materials ranging from photoresist to wax. While these methods have been used with wide success, consistently producing small, high-resolution features using materials and methods that are compatible with solvents and surfactants remains a challenge. Two new methods are presented here for generating μPADs with well-defined, high-resolution structures compatible with solvents and surfactant-containing solutions by partially or fully fusing paper with Parafilm® followed by cutting with a CO2 laser cutter. Partial fusion leads to laminated paper (l-paper) while the complete fusion results in infused paper (i-paper). Patterned structures in l-paper were fabricated by selective removal of the paper but not the underlying Parafilm® using a benchtop CO2 laser. Under optimized conditions, a gap as small as 137 ± 22 μm could be generated. Using this approach, a miniaturized paper 384-zone plate, consisting of circular detection elements with a diameter of 1.86 mm, was fabricated in 64 × 43 mm2 area. Furthermore, these ablation-patterned substrates were confirmed to be compatible with surfactant solutions and common organic solvents (methanol, acetonitrile and dimethylformamide), which has been achieved by very few μPAD patterning techniques. Patterns in i-paper were created by completely cutting out zones of the i-paper and then fixing pre-cut paper into these openings similar to the strategy of fitting a jigsaw piece into a puzzle. Upon heating, unmodified paper was readily sealed into these openings due to partial reflow of the paraffin into the paper. This unique and simple bonding method was illustrated by two types of 3D μPADs, a push-on valve and a time-gated flow distributor, without adding adhesive layers. The free-standing jigsaw-patterned sheets showed good structural stability and solution compatibility, which provided a facile alternative method for fabricating complicated μPADs.

A simple method to produce 2D and 3D microfluidic paper-based analytical devices for clinical analysis

This paper describes the fabrication of 2D and 3D microfluidic paper-based analytical devices (μPADs) for monitoring glucose, total protein, and nitrite in blood serum and artificial urine. A new method of cutting and sealing filter paper to construct μPADs was demonstrated. Using an inexpensive home cutter printer soft cellulose-based filter paper was easily and precisely cut to produce pattern hydrophilic microchannels. 2D and 3D μPADs were designed with three detection zones each for the colorimetric detection of the analytes. A small volume of samples was added to the μPADs, which was photographed after 15 min using a digital camera. Both μPADs presented an excellent analytical performance for all analytes. The 2D device was applied in artificial urine samples and reached limits of detection (LODs) of 0.54 mM, 5.19 μM, and 2.34 μM for glucose, protein, and nitrite, respectively. The corresponding LODs of the 3D device applied for detecting the same analytes in artificial blood serum were 0.44 mM, 1.26 μM, and 4.35 μM.

A 3D µPAD based on a multi-enzyme organic-inorganic hybrid nanoflower reactor.

This work reports on the development of a 3D microfluidic paper-based device (3D µPAD) for glucose detection using organic-inorganic hybrid nanoflower technology to immobilize the bi-enzymatic system (glucose oxidase and horseradish peroxidase). The system is based on nanoflowerssupported on cellulose paper (the microreactor zone) coupled to 3,3',5,5'-tetramethylbenzidine (TMB) as the colorimetric probe in the detection zone. We used a digital camera for the quantitative analysis of glucose with the S coordinate of the HSV color space as the analytical parameter. Under optimal operational conditions, linearity was observed for glucose concentrations up to 300 μM, with a detection limit of 15.6 µM. The biosensor is reusable and remains stable for 75 days in conventional storage conditions.

Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices

In this work, we first employ a drying method combining with the bienzyme colorimetric detection of glucose and uric acid on microfluidic paper-based analysis devices (μPADs). The channels of 3D μPADs are also designed by us to get better results. The color results are recorded by both Gel Documentation systems and a common camera. By using Gel Documentation systems, the limits of detection (LOD) of glucose and uric acid are 3.81×10−5M and 4.31×10−5M, respectively one order of magnitude lower than that of the reported methods on μPADs. By using a common camera, the limits of detection (LOD) of glucose and uric acid are 2.13×10−4M and 2.87×10−4M, respectively. Furthermore, the effects of detection conditions have been investigated and discussed comprehensively. Human serum samples are detected with satisfactory results, which are comparable with the clinical testing results. A low-cost, simple and rapid colorimetric method for the simultaneous detection of glucose and uric acid on the μPADs has been developed with enhanced sensitivity.