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Coagulation disorders can lead to dangerous bleeding or clot formation. Since centralized laboratories cannot provide rapid results for timely treatment intervention in patients with coagulation disorders, the existence of coagulation sensors for the general public is necessary. The purpose of the present research is to design and fabricate a novel, portable, low-cost, paper-based colorimetric sensor for measuring blood prothrombin time.

Bacterial infections remain a global threat, particularly in low-resource settings, where access to accurate and timely diagnosis is limited. Point-of-care nucleic acid amplification tests have shown great promise in addressing this challenge. However, their dependence on complex traditional sample preparation methods remains a major challenge. To address this limitation, we present a paper-based sample preparation device that integrates bacterial cell lysis, DNA purification, and concentration using an electrokinetic technique called isotachophoresis (ITP). This is the first device that (i) integrates electrochemical bacterial lysis with ITP and (ii) demonstrates the focusing of whole bacterial genomic DNA (gDNA) in paper. Characterization with buffers showed that the paper-based ITP sample preparation module (p-ITPrep) concentrated bacterial gDNA with an average concentration factor of 12×, and DNA could be extracted from a sample containing as few as 10 2 CFU mL -1 Mycobacterium smegmatis ( Msm ). From complex biological matrices - human saliva, human blood serum, and artificial urine - p-ITPrep extracted DNA from samples containing 10 2 CFU Msm per mL saliva or artificial urine and 10 3 CFU Msm per mL serum within 20 min. The extraction procedure involved only 3 user steps, in contrast to conventional solid phase extraction kits that require more than 10 user steps. p-ITPrep may provide a simple, inexpensive, and versatile alternative to conventional multi-step nucleic acid extraction protocols for point-of-care diagnostics.

Alzheimer's disease (AD) is the leading cause of dementia worldwide, and the development of early screening methods can address its significant health and social consequences. In this paper, we present a rotary-valve assisted paper-based immunoassay device (RAPID) for early screening of AD, featuring a highly integrated on-chip rotary micro-valve that enables fully automated and efficient detection of the AD biomarker (amyloid beta 42, Aβ42) in artificial plasma. The microfluidic paper-based analytical device (μPAD) of the RAPID pre-stores the required assay reagents on a μPAD and automatically controls the liquid flow through a single valve. Once the test sample is added, the test reagents are sequentially transferred to the test area in the order set by the enzyme-linked immunosorbent assay (ELISA) protocol. In addition, the RAPID can remotely control the operation of the μPAD valve via a micro-servomotor, quantify the signals generated, display the results, and wirelessly transmit the data to a smartphone. To calibrate the RAPID, we performed a sandwich ELISA for Aβ42 in artificial plasma, and obtained a low limit of detection (LOD) of 9.6 pg mL -1 , a coefficient of determination (COD) of 0.994, and an individual assay time of ∼30 minutes. In addition, we simulated 24 artificial samples to quantify Aβ42 protein concentrations in artificial plasma samples. The results show good consistency between the conventional ELISA and RAPID detection. The experimental results demonstrate that the RAPID is expected to promote further popularization of the screening of early-stage AD.

Developing low-cost self-service portable sensors to detect viruses is an important step in combating the spread of viral outbreaks. Here, we describe the development of an aptamer-free paper-based molecularly imprinted sensor for the instrument-free detection of influenza virus A (H5N1). In this sensor, Whatman paper loaded with Fe 3 O 4 nanoparticles (WP@Fe 3 O 4 ) was prepared as a substrate upon which silicon imprinting occurred in the presence of the template virus H5N1. After removal of the template virus, Fe 3 O 4 which exposed in the imprinted cavities was able to catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide to form blue TMB + ions. Therefore, the concentration of virus can be semi-quantified by the color change of the solution after the catalytic reaction in the absence of any instruments. The color reaction can be clearly observed within 20 min. In addition, the remaining TMB could be quantified fluorometrically, with a limit of detection of 1.16 fM and an imprinting factor of 4.7. As far as we know, this sensor detects the target with the highest sensitivity that has yet been achieved in aptamer-free molecular imprinting sensors. Importantly, the cost of materials used for each sensor was as low as 4 cents (0.23 Yuan) per sensor. This sensitive self-service sensor, which is relatively easy and inexpensive to produce, will provide an effective new avenue for the rapid detection of viruses.

An acute wound is the most common type of skin injury. Developing wound dressings with excellent mechanical properties, wound protection, comfort, angiogenic capacity and therapeutic effects is significant for effective treatments, yet remains challenging. Herein, we have designed a novel HAP-Alg composite dressing comprising a complementary ultralong hydroxyapatite (HAP) nanowire bio-paper and calcium alginate hydrogel. The HAP bio-paper assembled by ultralong HAP nanowires, in contrast to typical brittle HAP bio-ceramics, exhibits a highly flexible and interwoven structure to enhance the mechanical and protective performance of an alginate hydrogel, and the alginate matrix creates a moist environment for skin regeneration. Therefore, the HAP-Alg composite dressing presents good mechanical properties and high resistance to swelling and shrinkage, along with a reliable bacterial shielding ability. In addition, its moisturizing effect can deliver bioactive calcium ions to promote angiogenesis, accelerate re-epithelialization and reduce scar formation. In vitro studies reveal that the HAP-Alg composite dressing has excellent biocompatibility, promotes cell migration and angiogenesis, and enhances calcium ion influx. In vivo wound models further prove the ability of the HAP-Alg composite dressing to accelerate wound closure, enhance collagen deposition, and induce neovascularization. This work demonstrates that the HAP-Alg composite dressing offers a promising wound dressing for acute wound treatment and protection.

Dipicolinic acid (DPA) is a key biomarker of bacterial spores. In this study, we present a novel distance-based paper analytical device (d-PAD) for the fluorescence sensing of DPA. The detection mechanism relies on the complexation of ofloxacin (OFL) with Cu 2+ ions, where Cu 2+ quenches the fluorescence of OFL via static quenching. Upon the introduction of DPA, it interacts with the OFL-Cu 2+ complex, resulting in an enhanced fluorescence signal from OFL. The assay demonstrated a limit of detection (LOD) of 0.08 μM over a range of 0.6-120 μM, as measured using a spectrofluorometer. The d-PAD was designed for efficient reagent transport through capillary action on paper substrates, allowing for rapid on-site DPA analysis without requiring advanced laboratory equipment. The length of the fluorescent bands on the d-PADs was proportional to the concentration of DPA, providing a simple and effective readout method. With a sensitivity of 0.6 μM, the device shows a strong response to varying DPA concentrations. This distance-based platform offers a straightforward and quantitative approach to result interpretation, making it a promising tool for detecting bacterial spores in real samples. The development and optimization of this paper-based microfluidic assay represent a significant step forward in portable diagnostic technologies.

Digital microfluidics (DMF) is a platform that enables the automated manipulation of individual droplets of sizes ranging from nanoliter to microliter and can be coupled with numerous techniques, including colorimetry. However, although the DMF electrode architecture is highly versatile, its integration with different analytical methods often requires either changes in sample access, top plate design, or the integration of supplementary equipment into the system. As an alternative to overcome these challenges, this study proposes a simple integration between paper-based analytical devices (PADs) and DMF for automated and eco-friendly sample processing aiming at the colorimetric detection of creatinine (CR, an important biomarker for kidney disease) in artificial urine. An optimized and selective Jaffé reaction was performed on the device, and the reaction products were delivered to the PAD, which was subsequently analyzed with a bench scanner. The optimal operational parameters on the DMF platform were a reaction time of 45 s with circular mixing and image capture after 5 min. Under optimized conditions, a linear behavior was obtained for creatinine concentrations ranging from 2 to 32 mg dL -1 , with limits of detection and quantitation equal to 1.4 mg dL -1 and 2.0 mg dL -1 , respectively. For the concentration range tested, the relative standard deviation varied from 2.5 to 11.0%, considering four measurements per concentration. CR-spiked synthetic urine samples were subjected to analysis via DMF-PAD and the spectrophotometric reference method. The concentrations of CR determined using both analytical techniques were close to the theoretical values, with the resultant standard deviations of 2-9% and 1-4% for DMF-PADs and spectrophotometry, respectively. Furthermore, the recovery values were within the acceptable range, with DMF-PADs yielding 96-108% and spectrophotometry producing 95-102%. Finally, the greenness of the DMF-PAD and spectrophotometry methods was evaluated using the Analytical Greenness (AGREE) metric software, in which 0.71 and 0.51 scores were obtained, respectively. This indicates that the proposed method presents a higher greenness level, mainly due to its miniaturized characteristics using a smaller volume of reagent and sample and the possibility of automation, thus reducing user exposure to potentially toxic substances. Therefore, the DMF-PADs demonstrated great potential for application in the clinical analysis of creatinine, aiding in routine tests by introducing an automated, simple, and environmentally friendly process.

Pesticides are primarily used in agriculture to protect crops and extend their longevity. However, pesticide exposure has been linked to various acute and chronic health effects, raising significant environmental concerns. Current detection methods are often expensive and time-consuming, relying on complex instruments. Although enzyme-inhibition-based microfluidic paper-based analytical device (mPAD) platforms offer an easier alternative, they suffer from slow analyte transport and analyte adsorption issues in microchannels. Consequently, there is a need for a fast, simple, and cost-effective point-of-need platform for pesticide sensing. In this study, we present a rapid microfluidic platform for on-site pesticide residue detection. Unlike traditional mPAD platforms, our system transports pesticide samples through hollow capillary channels within seconds without adsorption of pesticides in the microchannels. While much research has focused on acetylcholinesterase inhibition on paper, this study is the first to introduce a tyrosinase inhibition-based assay on a paper platform for pesticide detection. Ziram, a representative dithiocarbamate pesticide, was detected using a colorimetric enzymatic inhibition assay. A limit of detection (LoD) of 1.5 ppm was obtained. In this study, we optimized the fast-flow device, assessed its stability and susceptibility to various interferences, and conducted real-sample tests using glove extraction to evaluate its capability in real-world settings. Spike recovery analysis revealed an extraction efficiency of 82.5% to 87.5% for leather gloves and 68.9% to 71.9% for nitrile gloves. This platform demonstrates strong selectivity against interferences, with the enzyme retaining 90% activity even after a week under the established storage protocols with room for further investigation. While primarily a proof of concept, this device shows promise as an additional tool for pesticide detection, with potential future integration into multiplexed devices.

In this article, we present the first demonstration of a distance-based paper analytical device (dPAD) for uric acid quantification in human urine samples with instrument-free readout and user-friendliness for the rapid diagnosis and prognosis of various related diseases. By employing gold nanoparticles (AuNPs) as a peroxidase-like nanozyme, our proposed technique eliminates the utilization of horseradish peroxidase (HRP), making the device cost-effective and stable. In our dPAD, uric acid in the sample is oxidized by the uricase enzyme and subsequently catalysed with AuNPs in the sample zone, generating hydroxyl radicals (˙OH). Then, the produced ˙OH reacts with 3,3'-diaminobenzidine (DAB) to form poly DAB (oxDAB), resulting in a coloured distance signal in the detection zone of the dPAD. The variation of the distance of the observed red-brown colour is directly proportional to the uric acid concentration. Our sensor exhibited a linear range from 0.50 to 6.0 mmol L -1 ( R 2 = 0.9922) with a detection limit (LOD) of 0.25 mmol L -1 , covering the clinical range of uric acid in urine. Hence, there is no need for additional sample preparation or dilution. Additionally, this assay is highly selective, with no interferences. We also found that this approach could accurately and precisely determine uric acid in human control samples with the recovery ranging from 99.37 to 100.35 with the highest RSD of 4.05%. Our method is comparable with the use of a commercially available uric acid sensor at a 95% confidence interval. Consequently, the developed dPAD offers numerous advantages such as cost-effectiveness, simplicity, and ease of operation with unskilled individuals. Furthermore, this concept can be applied for extensive biosensing applications in monitoring other biomarkers as an alternative analytical point-of-care (POC) device.

The increasing demand in healthcare for accessible and cost-effective analytical tools is driving the development of reliable platforms to the customization of therapy according to individual patient drug serum levels, e.g. of anti-psychotics in schizophrenia. A modifier-free microfluidic paper-based electroanalytical device (μPED) holds promise as a portable, sensitive, and affordable solution. While many studies focus on the working electrode catalysts, improvements by engineering aspects e.g. of the electrode arrangement are less reported. In our study, we demonstrate the enhanced capabilities of the 3D electrode layout of μPED compared to 2D μPED arrangements. We especially show that screen printing can be employed to prepare 3D μPEDs. We conducted a comparison of different 2D and 3D electrode arrangements utilizing cyclic voltammetry in [Fe(CN) 6 ] 3-/4- , along with square-wave voltammetry for clozapine (CLZ) sensing. Our findings reveal that the utilization of the 3D μPED leads to an increase in both the electrochemically active surface area and the electron transfer rate. Consequently, this enhancement contributes to improve sensitivity in the CLZ sensing. The 3D μPED clearly outperforms the 2D μPED arrangement in terms of signal strength. With the 3D μPED under the optimized conditions, a linear dose-response for a concentration range from 7.0 to 100 μM was achieved. The limit of detection and sensitivity was determined to be 1.47 μM and 1.69 μA μM -1 cm -2 , respectively. This evaluation is conducted in the context of detection and determination of CLZ in a human blood serum sample. These findings underscore the potential of the 3D μPED for future applications in pharmacokinetic analyses and clinical tests to personalize the management of schizophrenia.

Conventional affinity-based colorimetric enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for the detection of biomarkers. However, rapid point-of-care (POC) detection of multiple cancer biomarkers by conventional ELISA is limited by long incubation time, large reagent volume, and costly instrumentation along with low sensitivity due to the nature of colorimetric methods. Herein, we have developed a reusable and cost-effective paper-in-polymer-pond (PiPP) hybrid microfluidic microplate for ultrasensitive and high-throughput multiplexed detection of disease biomarkers within an hour without using specialized instruments. A piece of pre-patterned chromatography paper placed in the PMMA polymer pond facilitates rapid protein immobilization to avoid intricate surface modifications of polymer and can be changed with a fresh paper layer to reuse the device. Reagents can be simply delivered from the top PMMA layer to multiple microwells in the middle PMMA layer via flow-through microwells, thereby increasing the efficiency of washing and avoiding repeated manual pipetting or costly robots. Quantitative colorimetric analysis was achieved by calculating the brightness of images scanned by an office scanner or a smartphone camera. Sandwich-type immunoassay was performed in the PiPP hybrid device after the optimization of multiple assay conditions. Limits of detection of 0.32 ng mL -1 for carcinoembryonic antigen (CEA) and 0.20 ng mL -1 for prostate-specific antigen (PSA) were obtained, which were about 10-fold better than those of commercial ELISA kits. We envisage that this simple but versatile hybrid device can have broad applications in various bioassays in resource-limited settings.

Bacillus cereus is a foodborne opportunistic pathogen commonly found in humans and animals. It produces various toxins, causing frequent food safety incidents. Therefore, establishing a fast and accurate method for detecting B. cereus enterotoxin is crucial for disease diagnosis and food safety. In this study, Haemolysin BL comprising Hbl B and L2 was obtained from a prokaryotic expression system and then used to immunize mice for antibody preparation. Paired antibodies 2A10-5C7 against Hbl B and 1E2-10A4 against Hbl L2 were screened using the chessboard method and then used to construct a double-antibody sandwich detection method and a lateral flow immunochromatographic assay (LFIA) to quantify the concentrations of Hbl B and L2 in PBS and milk. The limits of detection for Hbl B and L2 in milk were 0.74 ng mL -1 and 1 ng mL -1 with detection ranges of 1.48-645.5 ng mL -1 and 2.33-391.5 ng mL -1 . The spiked recoveries ranged from 82.2% to 105.67% and there was no cross-reactivity with common microbial toxins. The established LFIA was low in cost and rapid and was comparable with commercially available detection kits for food samples.

Isothermal nucleic acid amplification tests (NAATs) are a vital tool for point-of-care (POC) diagnostics. These assays are well-suited for rapid, low-cost POC diagnostics for infectious diseases compared to traditional PCR tests conducted in central laboratories. There has been significant development of POC NAATs using paper-based diagnostic devices because they provide an affordable, user-friendly, and easy to store format; however, the difficulties in integrating separate liquid components, resuspending dried reagents, and achieving a low limit of detection hinder their use in commercial applications. Several studies report low assay efficiencies, poor detection output, and poorer limits of detection in porous membranes compared to traditional tube-based protocols. Recombinase polymerase amplification is a rapid, isothermal NAAT that is highly suited for POC applications, but requires viscous reaction conditions that has poor performance when amplifying in a porous paper membrane. In this work, we show that we can dramatically improve the performance of membrane-based recombinase polymerase amplification (RPA) of HIV-1 DNA and viral RNA by employing a coin cell-based vibration mixing platform. We achieve a limit of detection of 12 copies of DNA per reaction, nearly 50% reduction in time to threshold (from ∼10 minutes to ∼5 minutes), and an overall fluorescence output increase up to 16-fold when compared to unmixed experiments. This active mixing strategy enables reactions where the target and reaction cofactors are isolated from each other prior to the reaction. We also demonstrate amplification using a low-cost vibration motor for both temperature control and mixing, without the requirement of any additional heating components.

Although CRISPR-based nucleic acid detection has great potential in point-of-care testing due to its simplicity, it has been rarely integrated into paper-based analytical devices (PADs), which are attractive platforms to simplify assays. This work introduces a CRISPR-assisted nucleic acid quantification approach integrated into a PAD with signal readout by a personal glucose meter (PGM). Retention of magnetic beads by filter paper and pre-deposition of all required reagents by freeze-drying stabilized with trehalose enabled the indirect quantification of human papilloma virus (HPV) DNA through a PGM readout without complicated user intervention and complex reagent handling. The calculated limit of detection was 57 pM, which is comparable with other amplification-free CRISPR-based assays detecting nucleic acids. The fully integrated device exhibited good storage stability for up to 4 weeks, suggesting its applicability toward practical point-of-care nucleic acid quantification.

A novel microfluidic paper-based analytical device with dual colorimetric and electrochemical detection (dual μPAD) was developed for the assessment of transferrin saturation (TSAT) in samples from ischemic stroke patients. TSAT was calculated from the ratio between transferrin-bound iron, which was colorimetrically measured, and the total iron-binding capacity, which was electrochemically measured. To this end, a μPAD was smartly designed, which integrated both colorimetric and electrochemical detection reservoirs, communicating via a microchannel acting as a chemical reactor, and with preloading/storing capabilities (reagent-free device). This approach allowed the dual and simultaneous determination of both parameters, providing an improvement in the reliability of the results due to an independent signal principle and processing. The μPADs were validated by analyzing a certified reference material, showing excellent accuracy ( E r ≤ 5%) and precision (RSD ≤ 2%). Then they were applied to the analysis of diagnosed serum samples from ischemic stroke patients. The results were compared to those provided by a free-interference method (urea-PAGE). Impressively, both methods exhibited a good correlation ( r = 0.96, p < 0.05) and no significant differences were found between them (slope 1.0 ± 0.1 and the intercept 1 ± 4, p < 0.05), demonstrating the excellent accuracy of our approach during the analysis of complex samples from ischemic stroke patients, using just 90 μL of clinical samples and taking less than 90 min in comparison with the 18 hours required by the urea-PAGE approach. The developed fully integrated colorimetric-electrochemical μPAD is a promising ready to use reagent-free device for the point-of-care testing of TSAT, which can be used to assist physicians in the fast diagnosis and prognosis of ischemic strokes, where the decision-time is crucial for the patient's survival.

The first step in blood testing necessitates blood separation to obtain an adequate volume of plasma. Traditional centrifugation is bulky, expensive and electricity-powered, which is not suitable for micro-scale blood plasma separation in point-of-care testing (POCT) cases. Microfluidic paper-based plasma separation devices present a promising alternative for plasma separation in such occasions. However, they are limited in terms of plasma yield, which hinders analyte detection. Herein, we proposed a humidity-enhanced paper-based microfluidic plasma separation method to address this issue. Specifically, paper was first treated by blood-typing antibodies, then samples of whole blood were introduced into the prepared paper. After waiting for 5 min for RBC agglutination and plasma wicking under high humidity, micro-scale plasma separation from whole blood was achieved. As a result, an extremely high plasma yield of up to 60.1% could be separated from whole blood through using Xuan-paper. Meanwhile, the purity of plasma could reach 99.99%. Finally, this innovative approach was effortlessly integrated into distance-based glucose concentration detection, enabling rapid determination of blood glucose levels through naked-eye observation. Considering the simplicity and inexpensiveness of this method, we believe that this technology could be integrated to more paper-based microfluidic analytical devices for rapid and accurate detection of plasma analytes in POCT.

Paper-based analytical devices (PADs) are very convenient for determining biomarkers in point-of-care (POC) diagnosis while requiring sample pre-treatment or impurity separation. This study reports a novel hydrogel-coupled, paper-based analytical device (PAD) for separation-free H 2 O 2 colorimetric detection in both aqueous solution and cell lysis with sample-to-answer analysis by directly loading into the sample test zone. By encapsulating an inorganic mimic enzyme and chromogenic substrate into the sodium alginate (SA) hydrogel, amplification of the color signal after catalyzing the substrate could be achieved. Taking advantage of the nanoscale porous structure of the hydrogel and the lateral flow channel of the PAD, large interference fragments or bio-macromolecules are prevented from diffusing into the chromogenic reaction, whereas the small target molecules enter the sensing region to trigger the catalytic reaction. This method demonstrated a rapid and accurate analysis with a limit of detection as low as 0.06 mM and detection selectivity. Our proposed device requires no enzyme and is separation-free, portable, easy-to-fabricate, and low-cost, and may offer a platform for quantitative or qualitative analysis of other analytes in body fluids for POC applications.

Paper-based rapid diagnostic tests (RDTs) are an essential component of modern healthcare, particularly for the management of infectious diseases. Despite their utility, these capillary-driven RDTs are compromised by high failure rates, primarily caused by user error. This limits their utility in complex assays that require multiple user operations. Here, we demonstrate how this issue can be directly addressed through continuous electrochemical monitoring of reagent flow inside an RDT using embedded graphenized electrodes. Our method relies on applying short voltage pulses and measuring variations in capacitive discharge currents to precisely determine the flow times of injected samples and reagents. This information is reported to the user, guiding them through the testing process, highlighting failure cases and ultimately decreasing errors. Significantly, the same electrodes can be used to quantify electrochemical signals from immunoassays, providing an integrated solution for both monitoring assays and reporting results. We demonstrate the applicability of this approach in a serology test for the detection of anti-SARS-CoV-2 IgG in clinical serum samples. This method paves the way towards "smart" RDTs able to continuously monitor the testing process and improve the robustness of point-of-care diagnostics.

Nowadays, kidney dysfunction is a common health issue due to the modernized lifestyle. Even though medications are commercially available to treat kidney diseases, early diagnosis is crucial and challenging. Clinically, measuring urine creatinine and pH has gained significant interest as a way to diagnose kidney diseases early. In the present work, we attempted to develop a low-cost, robust, accurate and naked-eye colorimetric method to determine both creatinine levels and pH variations in artificial urine samples using a simple 3D-printed hybrid microfluidic device. Creatinine was detected by the incorporation of the traditional Jaffe test onto the hybrid paper-PMMA microfluidic device and pH (4-8) was measured by a simple anthocyanin test. Notably, the tests were established without employing any sophisticated or costly instrument clusters. The developed 3D-printed microfluidic probe showed a limit of detection (LOD) of 0.04 mM for creatinine over a concentration range of 1-10 mM, with a regression coefficient ( R 2 ) of 0.995 in laboratory conditions. Interestingly, the experimental data obtained with artificial urine exhibited a wide linear range from 0.1 mM to 5 mM under different pH values ranging from 4 to 8 in the presence of matrices commonly found in urine samples other than proteins, indicating the potential use of this method in pre-clinical analysis. Since the wide linear range of urine creatinine in artificial urine samples falls well below the clinically relevant concentrations in humans (0.07-0.27 mM), the developed lab-on-chip device is further suitable for clinical evaluation with proper ethical clearance. This 3D-printed hybrid microfluidic colorimetry-based creatinine detection and pH indicator platform can be beneficial in the healthcare sector due to the on-site testing capability, cost-effectiveness, ease of use, robustness, and instrument-free approach.

This study introduces the development of a photothermal-based microfluidic paper analytical device (PT-µPAD) integrated with near-field communication (NFC) technology and smartphone readout for enzyme-free glucose quantification in human samples. With the properties of gold nanoparticles (AuNPs) both as a nanozyme and as a photothermal substrate, there is no need for costly reagents like enzymes or a readout instrumentation for the selective and sensitive detection of glucose. In PT-µPADs, AuNPs are etched by hydrogen peroxide (H 2 O 2 ) generated from glucose catalysis. Photothermal detection from the plasmonic heating of these AuNPs when illuminated by a 533nm LED light source is achieved by inserting the PT-µPAD sensor into a portable NFC platform suitable for smartphone readout. Temperature variation is directly proportional to the glucose concentration. After optimization, we acquired a linear range between 5.0 and 20.0 µmol L -1 ( R 2 = 0.9967) and a limit of detection (LOD) of 25.0 nmol L -1 for glucose. Additionally, while our sensor does not utilize any enzyme, it is remarkably selective to glucose with no effects from interferences. Recovery studies in various human control samples indicated a range of 99.73-102.66% with the highest RSD of 3.53%, making it highly accurate and precise. Moreover, our method is more sensitive than other methods relying on conventional µPADs for glucose sensing. By integrating the potential benefits of microfluidics, nanomaterials as nanozymes, and NFC technology for wireless readout, our sensor demonstrates great promise as an accessible, affordable, and shelf-stable device for glucose quantification. Moreover, this concept can be extended to detect other molecules of interest as a point-of-care (POC) diagnostics device.

We reported a colorimetric paper-based device by integrating the modified acid RNA-cleaving DNAzymes (MaRCD-EC1) for highly sensitive (detection limit = 10 2 CFU mL -1 ), and rapid (within 30 min) detection of E. coli without amplification. This device exhibited a clinical sensitivity of 100% and a specificity of 100% in identifying E. coli -associated urinary tract infections (UTIs) using the clinical urine samples.

Safe, accurate, and reliable analysis of urinary biomarkers is clinically important for early detection and monitoring of the progression of chronic kidney disease (CKD), as it has become one of the world's most prevalent non-communicable diseases. However, current technologies for measuring urinary biomarkers are either time-consuming and limited to well-equipped hospitals or lack the necessary sensitivity for quantitative analysis and post a health risk to frontline practitioners. Here we report a robust paper-based dual functional biosensor, which is integrated with the clinical urine sampling vial, for the simultaneous and quantitative analysis of pH and glucose in urine. The pH sensor was fabricated by electrochemically depositing IrOx onto a paper substrate using optimised parameters, which enabled an ultrahigh sensitivity of 71.58 mV pH -1 . Glucose oxidase (GOx) was used in combination with an electrochemically deposited Prussian blue layer for the detection of glucose, and its performance was enhanced by gold nanoparticles (AuNPs), chitosan, and graphite composites, achieving a sensitivity of 1.5 μA mM -1 . This dual function biosensor was validated using clinical urine samples, where a correlation coefficient of 0.96 for pH and 0.98 for glucose detection was achieved with commercial methods as references. More importantly, the urine sampling vial was kept sealed throughout the sample-to-result process, which minimised the health risk to frontline practitioners and simplified the diagnostic procedures. This diagnostic platform, therefore, holds high promise as a rapid, accurate, safe, and user-friendly point-of-care (POC) technology for the analysis of urinary biomarkers in frontline clinical settings.

A new analytical method for chronic kidney disease (CKD) detection utilizing paper spray mass spectrometry (PS-MS) combined with machine learning is presented. The analytical protocol is rapid and simple, based on metabolic profile alterations in urine. Anonymized raw urine samples were deposited (10 μL each) onto pointed PS-MS sample strips. Without waiting for the sample to dry, 75 μL of acetonitrile and high voltage were applied to the strips, using high resolution mass spectrometry measurement (15 s per sample) with polarity switching to detect a wide range of metabolites. Random forest machine learning was used to classify the resulting data. The diagnostic performance for the potential diagnosis of CKD was evaluated for accuracy, sensitivity, and specificity, achieving results >96% for the training data and >91% for validation and test data sets. Metabolites selected by the classification model as up- or down-regulated in healthy or CKD samples were tentatively identified and in agreement with previously reported literature. The potential utilization of this approach to discriminate albuminuria categories (normo, micro, and macroalbuminuria) was also demonstrated. This study indicates that PS-MS combined with machine learning has the potential to be used as a rapid and simple diagnostic tool for CKD.

Paper-based sensors offer an affordable yet powerful platform for field and point-of-care (POC) testing due to their self-pumping ability and utility for many different analytical measurements. When combined with electrochemical detection using small and portable electronics, sensitivity and selectivity of the paper devices can be improved over naked eye detection without sacrificing portability. Herein, we review how the field of electrochemical paper-based analytical devices (ePADs) has grown since it was introduced a decade ago. We start by reviewing fabrication methods relevant to ePADs with more focus given to the electrode fabrication, which is fundamental for electrochemical sensing. Multiple sensing approaches applicable to ePADs are then discussed and evaluated to present applicability, advantages and challenges associated with each approach. Recent applications of ePADs in the fields of clinical diagnostics, environmental testing, and food analysis are also presented. Finally, we discuss how the current ePAD technologies have progressed to meet the analytical and practical specifications required for field and/or POC applications, as well as challenges and outlook. [ABSTRACT FROM AUTHOR]

Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial β-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 μW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics. [ABSTRACT FROM AUTHOR]

A simple, rapid chemical coating and patterning method was developed and optimized for paper-based substrates for use in paper spray mass spectrometry (PS-MS). A variety of chlorosilanes were explored for coating paper substrates, and their effectiveness in forming hydrophobic surfaces was characterized via contact angle goniometry, scanning electron microscopy, and energy dispersive X-ray spectroscopy. Trichloromethylsilane was selected as the primary coating agent because of the short time required to produce a hydrophobic surface (contact angle > 130°), as well as the ease of patterning. Patterning was performed using 3D-printed masks and an oxygen/plasma cleaner. Optimal mask thickness and oxygen/plasma cleaning parameters were determined to produce channels varying from 0.5 to 2.5 mm in width. The effectiveness of the patterned substrates for PS-MS was determined via analysis of four antiretrovirals: emtricitabine, lamivudine, efavirenz, and dolutegravir. Calibration curves were made for each antiretroviral at varying channel widths, and the limits of detection and limits of quantification for each drug were determined. These results show that this patterning method results in an average 7.2-fold improvement in sensitivity and an average 190-fold improvement in limits of detection over uncoated paper substrates in a neat matrix. In a proof-of-concept experiment, calibration curves were generated for each antiretroviral in urine. A patterned paper substrate with a 2-mm channel resulted in an average 7.4-fold improvement in sensitivity and an average 18-fold improvement in limits of detection over uncoated paper substrates.

The correct interpretation of the result from a point-of-care device is crucial for an accurate and rapid diagnosis to guide subsequent treatment. Lateral flow tests (LFTs) use a well-established format that was designed to simplify the user experience. However, the LFT device architecture is inherently limited to detecting analytes that can be captured by molecular recognition. Microfluidic paper-based analytical devices (μPADs), like LFTs, have the potential to be used in diagnostic applications at the point of care. However, μPADs have not gained significant traction outside of academic laboratories, in part, because they have often demonstrated a lack of homogeneous shape or color in signal outputs, which consequently can lead to inaccurate interpretation of results by users. Here, we demonstrate a new class of μPADs that form colorimetric signals at the interfaces of converging liquid fronts ( i.e. , lines) to control where colorimetric signals are formed without relying on capture techniques. We demonstrate our approach by developing assays for three classes of analytes-an ion, an enzyme, and a small molecule-to measure using iron(III), acetylcholinesterase, and lactate, respectively. Additionally, we show these devices have the potential to support multiplexed assays by generating multiple lines in a common readout zone. These results highlight the ability of this new paper-based device architecture to aid the interpretation of assays that create soluble products by using flow to constrain those colorimetric products in a familiar, line-format output.

Lab-on-a-paper-based devices are promising alternatives to the existing arduous techniques for point-of-need monitoring. The present work reports an instant and facile method to produce a microfluidic paper-based analytical device (μPAD). The fabricated μPAD has been used to detect hypochlorite (OCl - ) by incorporating newly synthesized chromo-fluorogenic ratiometric probes 1 and 2 into the sample reception zone. The probes showed high selectivity and fast response (<10 s) toward OCl - with an excellent linear relationship in the concentration range of 0-100 μM. The concentration-dependent fluorometric change driven by the reaction of 1@μPAD with OCl - has been monitored using gel-doc imaging systems, which is unprecedented. Digitizing the intensity of the colour solution with different mathematical models of colour has developed a straightforward method for monitoring OCl - without any interference from its competitors. 1@μPAD can detect OCl - at ∼10 times lower than the WHO recommended limit. The detection limit of 1@μPAD via a digital camera-based fluorescence technique was found to be better over digital camera-based cuvette assays. Therefore, 1@μPAD has been successfully utilized to monitor OCl - in actual environmental water samples with portability, ease of use, and sensitivity. The analytical RSD was found to be ≤3% based on fluorimetric detection using 1@μPAD. The chemodosimetric reaction between OCl - and the probe was evidenced by UV-vis and fluorescence spectroscopy, 1 H NMR, and ESI-MS. The rapid response time, biocompatibility, low cytotoxicity, 100% aqueous solubility, ratiometric feature, and exclusive OCl - selectivity over other competitive ROS/RNS successfully lead to the application of the probes for bioimaging of exogenous as well as endogenous OCl - in normal cells (HEK293) and cancerous cells (HeLa).

The lack of sensitivity is a major problem among microfluidic paper-based analytical devices (μPADs) for early disease detection and diagnosis. Accordingly, the present study presents a method for improving the enrichment factor of low-concentration biomarkers by using shallow paper-based channels realized through a double-sided wax-printing process. In addition, the enrichment factor is further enhanced by exploiting the ion concentration polarization (ICP) effect on the cathodic side of the nanoporous membrane, in which a stationary sample plug is obtained. The occurrence of ICP on the shallow-channel μPAD is confirmed by measuring the current–voltage response as the external voltage is increased from 0 to 210 V (or the field strength from 0 to 1.05 × 104 V m−1) over 600 s. In addition, to the best of our knowledge, the electroosmotic flow (EOF) speed on the μPAD fabricated with a wax-channel is measured for the first time using a current monitoring method. The experimental results show that for a fluorescein sample, the concentration factor is increased from 130-fold in a conventional full-thickness paper channel to 944-fold in the proposed shallow channel. Furthermore, for a fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) sample, the proposed shallow-channel μPAD achieves an 835-fold improvement in the concentration factor. The concentration technique presented here provides a novel strategy for enhancing the detection sensitivity of μPAD applications. [ABSTRACT FROM AUTHOR]

This communication describes the development of polyvinyl chloride electrochemical system in which a paper layer loaded with reagents is inserted into the device, demonstrating a new concept of a paper card-like pad for a reagent-free and easy measurement of the target analyte in solution. This device detects glucose in artificial tears in the range of 0.2-2 mM with a detection limit of 50 μM by simply adding the artificial tears to the paper card-like pad. The novel configuration goes beyond the state of the art, widening the application range of paper in the design of smart analytical devices.

A scalable, and cost-effective method was employed to prepare self-supported CuSn bimetallic catalyst on carbon paper. The obtained CuSn catalyst demonstrates high faradaic efficiency of CO around or above 90% at a broad potential range from -0.7 to -1.8 V vs. reversible hydrogen electrode, greatly surpassing Cu or Sn counterparts.

Microfluidic paper-based analytical devices (microPADs) are emerging as simple-to-use, low-cost point-of-care testing platforms. Such devices are mostly fabricated at present by creating hydrophobic barriers using wax or photoresist patterning on porous paper sheets. Even though devices fabricated using these methods are used and tested with a wide variety of analytes, still they pose many serious practical limitations for low-cost automated mass fabrication for their widespread applicability. We present an affordable and simple two-step process - cut and heat (CH-microPADs) - for the selective fabrication of hydrophilic channels and reservoirs on a wide variety of porous media such as tissue/printing/filter paper and cloth types, such as cotton and polyester, by a lamination process. The technique presents many advantages as compared to existing commonly used methods. The devices possess excellent mechanical strength against bending, folding and twisting, making them virtually unbreakable. They are structurally flexible and show good chemical resistance to various solvents, acids and bases, presenting widespread applicability in areas such as clinical diagnostics, biological sensing applications, food processing, and the chemical industry. Fabricated paper media 96 well-plate CH-microPAD configurations were tested for cell culture applications using mice embryonic fibroblasts and detection of proteins and enzymes using ELISA. With a simple two-step process and minimal human intervention, the technique presents a promising step towards mass fabrication of inexpensive disposable diagnostic devices for both resource-limited and developed regions.

A new method for making superhydrophobic (SH) paper based on spraying a nanocomposite film is developed. Furthermore, manipulating the wetting characteristics of SH paper has been demonstrated through a new method, i.e.printing solid grey patterns of different intensities with simple printing technology (home or office grade ink-jet and laser printers). It has been found that for a range of ink intensities (0–85%), water drop mobility can be changed at a different rate (almost independently) from repellency. The repellency of water decreases minimally up to 85% ink intensity with a sharp decrease up to 100% ink intensity. Drop mobility remains constant up to 30% ink intensity with a steady decrease up to 100% ink intensity. It was observed that using ink-jet or laser printing would yield different results for the change of mobility or repellency with higher amounts of ink/toner used. Being able to achieve almost independent control of water drop mobility over water drop repellency on SH paper would allow inexpensive lab-on-paper devices to be used for sampling, mixing and transport of liquids. [ABSTRACT FROM AUTHOR]

We report a method for fabricating inexpensive microfluidic platforms on paper using laser treatment. Any paper with a hydrophobic surface coating (e.g., parchment paper, wax paper, palette paper) can be used for this purpose. We were able to selectively modify the surface structure and property (hydrophobic to hydrophilic) of several such papers using a CO2laser. We created patterns down to a minimum feature size of 62 ± 1 µm. The modified surface exhibited a highly porous structure which helped to trap/localize chemical and biological aqueous reagents for analysis. The treated surfaces were stable over time and were used to self-assemble arrays of aqueous droplets. Furthermore, we selectively deposited silica microparticles on patterned areas to allow lateral diffusion from one end of a channel to the other. Finally, we demonstrated the applicability of this platform to perform chemical reactions using luminol-based hemoglobin detection. [ABSTRACT FROM AUTHOR]

This paper describes the fabrication and the performance of microfluidic paper-based electrochemical sensing devices (we call the microfluidic paper-based electrochemical devices, µPEDs). The µPEDs comprise paper-based microfluidic channels patterned by photolithography or wax printing, and electrodes screen-printed from conducting inks (e.g., carbon or Ag/AgCl). We demonstrated that the µPEDs are capable of quantifying the concentrations of various analytes (e.g., heavy-metal ions and glucose) in aqueous solutions. This low-cost analytical device should be useful for applications in public health, environmental monitoring, and the developing world. [ABSTRACT FROM AUTHOR]

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells viaSAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics. [ABSTRACT FROM AUTHOR]

This article describes FLASH (Fast Lithographic Activation of Sheets), a rapid method for laboratory prototyping of microfluidic devices in paper. Paper-based microfluidic devices are emerging as a new technology for applications in diagnostics for the developing world, where low cost and simplicity are essential. FLASH is based on photolithography, but requires only a UV lamp and a hotplate; no clean-room or special facilities are required (FLASH patterning can even be performed in sunlight if a UV lamp and hotplate are unavailable). The method provides channels in paper with dimensions as small as 200 µm in width and 70 µm in height; the height is defined by the thickness of the paper. Photomasks for patterning paper-based microfluidic devices can be printed using an ink-jet printer or photocopier, or drawn by hand using a waterproof black pen. FLASH provides a straightforward method for prototyping paper-based microfluidic devices in regions where the technological support for conventional photolithography is not available. [ABSTRACT FROM AUTHOR]

Microfluidic paper-based analytical devices (μPADs) have attracted great attention as potential candidates for point-of-care testing (POCT). Nevertheless, only a limited number of μPADs expected to satisfy the standard of Clinical Laboratory Improvement Amendments (CLIA) waived tests as issued by the US Food and Drug Administration (FDA) have been reported. This work introduces a "traffic light type μPAD", enabling highly intuitive semi-quantitative equipment-free naked-eye readout with no need for calibration, subjective interpretation or calculation. Assay results are displayed as traffic light colours reporting 5 analyte concentration levels (green/green & yellow/yellow/yellow & red/red). The device has been designed to never display all three colours simultaneously, eliminating any risk for misinterpretation. The mechanism relies on the modulation of sample flow through a network of paperfluidic channels modified with a hydrophobic to hydrophilic phase-switching substance responsive to H 2 O 2 . User operation is limited to sample application, followed by observing a clear and time-independent traffic light signal after approximately 10-30 min. Multiple factors influencing the H 2 O 2 concentration-dependent appearance of a specific traffic light signal were studied. Making use of the possibilities for customising the concentration threshold levels for traffic light colour appearance, quantification of glucose at 5 levels in a clinically relevant concentration range was demonstrated in artificial urine as a model proof-of-concept. This platform is expected to offer the possibility for the future detection of other important metabolites.

In the present study, iodide-mediated 3,3',5,5'-tetramethylbenzidine (TMB)-H 2 O 2 reaction system was applied to a microfluidic paper-based analytical device (μPAD) for non-enzymatic colorimetric determination of H 2 O 2 . The proposed system is portable and incorporates a μPAD with a machine learning-based smartphone app. A smartphone app called " Hi-perox Sens " capable of image capture, cropping and processing was developed to make the system simple and user-friendly. Briefly, circular μPADs were designed and tested with varying concentrations of H 2 O 2 . Following the color change, the images of the μPADs were taken with four different smartphones under seven different illumination conditions. In order to make the system more robust and adaptive against illumination variation and camera optics, the images were first processed for feature extraction and then used to train machine learning classifiers. According to the results, TMB + KI showed the highest classification accuracy (97.8%) with inter-phone repeatability at t = 30 s under versatile illumination and maintained its accuracy for 10 minutes. In addition, the performance of the system was also comparable to two different commercially available H 2 O 2 kits in real samples.

Detection and characterization of DNA damage plays a critical role in genotoxicity testing, drug screening, and environmental health. We developed a fully integrated origami paper-based analytical device (oPAD) for measuring DNA damage. This simple device allows on-paper cell lysis, DNA extraction, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction and signal readout with simple operation steps, enabling rapid (within 30 min) and high throughput assessment of multiple DNA damages induced by exogenous chemical agents.

Microfluidic paper-based analytical devices (μPADs) have been widely explored for point-of-care testing due to their simplicity, low cost, and portability. μPADs with multiple-step reactions usually require precise flow control, especially flow-delay. This paper reports the numerical, mathematical, and experimental studies of flow delay through wax valves surrounded by PDMS walls on paper microfluidics. The predried surfactant in the sample zone diffuses into the liquid sample which can therefore flow through the wax valves. The delay time is automatically regulated by the diffusion of the surfactant after sample loading. The numerical study suggested that both the elevated contact angle and the reduced porosity and pore size in the wax printed region could effectively prevent water but allow liquids with lower contact angles ( e.g. , surfactant solutions) to flow through. The PDMS walls fabricated using a low-cost liquid dispenser effectively prevented the leakage of surfactant solutions. By controlling the quantity, diffusion distance, and type of the surfactant predried on the chip, the system successfully achieved a delay time ranging from 1.6 to 20 minutes. A mathematical model involving the above parameters was developed based on Fick's second law to predict the delay time. Finally, the flow-delay systems were applied in sequential mixing and distance-based detection of either glucose or alcohol. Linear ranges of 1-100 mg dL -1 and 1-40 mg dL -1 were achieved for glucose and alcohol, respectively. The lower limit detection (LOD) of glucose and alcohol was 1 mg dL -1 . The LOD of glucose was only 1/11 of that detected using μPADs without flow control, indicating the advantage of controlling fluid flow. The systematic findings in this study provide critical guidelines for the development and applications of wax valves in automatic flow delay for point-of-care testing.

The article focuses on controlled transport of reagents within paper devices using two dimensional (2D) paper networks. The geometry of the paper network and dissolvable barriers makes them able to transport multiple analytes and reagents. It states these paper-based lateral flow immunoassay strips analyse only a single analyte per strip but their utility can be increased using highly sensitive particle labels like quantum dots. It also offers information on an experiment and its result.

A novel miniaturized and sustainable platform exploiting two merged paper-based substrates has been applied for the programmable pre-concentration of analytes of interest and electrochemical detection of mercury traces in drinking water using printable sensor strips. This strategy represents a novel versatile possibility in merging humble materials maximizing their impacts on analytical and remediation challenges.

Leukocyte esterase (LE) is a useful marker that can be used in establishing a diagnosis of urinary tract infections (UTIs). The development of a UTI diagnostic method with quantitative determinations of biomarkers across all age groups is becoming more important. In this report, microfluidic resistance sensors based on silver ink (Ag ink) and silver ink mixed with ZnO nanoparticles (Ag-ZnO ink) were synthesized and coated on cellulose paper, namely LE-Ag-μPADs and LE-Ag-ZnO-μPADs, respectively, for the sensitive detection of LE. The microfluidic design increases the precision of data and further allows for quantitative determination and early detection of LE in human urine. The quantification of LE relies on the change in the resistance readout coating with Ag ink as well as Ag-ZnO ink in the detection zone. A mixture of 3-(N-tosyl-l-alaninyloxy)-5-phenylpyrrole (PE) and 1-diazo-2-naphthol-4-sulfonic acid (DAS) was deposited in the sample zone to selectively recognize LE, and the resulting nonconductive products, i.e., azo compounds, further reacted with the Ag ink and Ag-ZnO ink to increase resistance. The quantitative detectable LE concentrations between 2 to 32 (×5.2 U mL-1), i.e. ≈12 to 108 μg L-1, cover the commercial dipstick range of trace, +1 and +2. The minimum detectable concentration of LE in urine was 1 (×5.2 U mL-1). The lower concentrations of LE detectable by LE-Ag-μPADs (1-8 × 5.2 U mL-1) are below the value achieved with the ELISA LE kit. Urine samples from inpatients with indwelling urinary catheters were used, and the LE levels measured by the present device were highly correlated with those determined by a commercial urine analyser.

Negative ion mode paper spray mass spectrometry (PS-MS) suffers from intense background noise and unstable MS signal. For the first time, we reported fluorinated boron nitride nanosheet (h-FBN) assisted negative ion PS-MS for the detection of a series of molecules. We demonstrated that the introduction of h-FBN can greatly improve the detection sensitivity and signal stability in the negative ion mode.

Infectious diseases cause millions of deaths annually in the developing world. Recently, microfluidic paper-based analytical devices (μPADs) have been developed to diagnose such diseases, as these tests are low cost, biocompatible, and simple to fabricate. However, current μPADs are difficult to use in resource-limited areas due to their reliance on external instrumentation to measure and analyze the test results. In this work, we propose an electricity and external instrumentation-free μPAD sensor based on the colorimetric enzyme-linked immunosorbent assay (ELISA) for the diagnosis of infectious disease (3D-tPADs). Designed based on the principle of origami, the proposed μPAD enables the sequential steps of the colorimetric ELISA test to be completed in just ∼10 min. In addition, in order to obtain an accurate ELISA result without using any instrument, we have integrated an electricity-free "timer" within the μPAD that can be controlled by the buffer viscosity and fluid path volume to indicate the appropriate times for washing and color development steps, which can avoid false positive or false negative results caused by an extended or shortened amount of washing and development times. Due to the low background noise and high positive signal intensity of the μPAD, positive and negative detection results can be distinguished by just the naked eye. Furthermore, the ELISA result can be semi-quantified by comparing the results shown on the μPAD with a color chart diagram with a detection limit of HIV type 1(HIV-1) p24 antigen as low as 0.03 ng mL-1. These results demonstrate the proposed sensor can perform infectious disease diagnosis without external instrumentation or electricity, extending the application of the μPAD test for on-site detection and use in resource-limited settings.

Lab-on-paper, or microfluidic paper-based analytical devices (μPADs), use paper as a substrate material, and are patterned with a system of microchannels, reaction zones and sensing elements to perform analysis and detection. The sample transfer in such devices is performed by capillary action. As a result, external driving forces are not required, and hence the size and cost of the device are significantly reduced. Lab-on-paper devices have thus attracted significant attention for point-of-care medical diagnostic purposes in recent years, particularly in less-developed regions of the world lacking medical resources and infrastructures. This review discusses the major advances in lab-on-paper technology for blood analysis and diagnosis in the past five years. The review focuses particularly on the many clinical applications of lab-on-paper devices, including diabetes diagnosis, acute myocardial infarction (AMI) detection, kidney function diagnosis, liver function diagnosis, cholesterol and triglyceride (TG) analysis, sickle-cell disease (SCD) and phenylketonuria (PKU) analysis, virus analysis, C-reactive protein (CRP) analysis, blood ion analysis, cancer factor analysis, and drug analysis. The review commences by introducing the basic transmission principles, fabrication methods, structural characteristics, detection techniques, and sample pretreatment process of modern lab-on-paper devices. A comprehensive review of the most recent applications of lab-on-paper devices to the diagnosis of common human diseases using blood samples is then presented. The review concludes with a brief summary of the main challenges and opportunities facing the lab-on-paper technology field in the coming years.