Microfluidic Paper-based Analytical Devices Research Articles

An exonuclease I-assisted quencher-free 2-aminopurine aptasensor based on a multipath paper-based device for ultrasensitive detection of kanamycin

A quencher-free multipath microfluidic paper-based analytical device (µPAD) was constructed for ultrasensitive detection of kanamycin based on exonuclease I (Exo I)-assisted signal enhancement of 2-aminopurine (2-AP). Here, Exo I, a single-stranded DNA-specific nuclease, was introduced to fully liberate 2-AP mononucleotides to greatly enhance biosensing sensitivity. 2-AP, a fluorescent adenine analogue embedded in single-stranded DNA (ssDNA), was employed as the detection signal source. The fluorescence of 2-AP is strong in the mononucleotide state, while it can have low fluorescence and even no fluorescence in ssDNA and dsDNA, respectively. The 2-AP fluorescence probe included 2-AP DNA and kanamycin aptamer. When kanamycin was present, binding occurred between kanamycin and the aptamer, leading to ssDNA, which was further digested by Exo I. In this case, free 2-AP mononucleotides were liberated, indicating strong fluorescence. In addition, the captured kanamycin was released for binding with the new aptamer, which resulted in the formation of a binding-hydrolysis-release cycle with the aid of Exo I. Under optimal conditions, this µPAD exhibited sensitive and multipath detection of kanamycin at concentrations as low as 1.26 × 10−14 M with a wide range of 10−13–10−7 M. Furthermore, satisfactory results were achieved for analysing spiked kanamycin in milk and honey samples. This strategy is a very promising tool for monitoring antibiotics and evaluating the safety of animal-derived foods.

A Microfluidic Paper-Based Lateral Flow Device for Quantitative ELISA

This study presents an innovative lateral flow microfluidic paper-based analytical device (μPAD) designed for conducting quantitative paper-based enzyme-linked immunosorbent assays (p-ELISA), seamlessly executing conventional ELISA steps in a paper-based format. The p-ELISA device utilizes a passive fluidic circuit with functional elements such as a multi-bi-material cantilever (B-MaC) assembly, delay channels, and a buffer zone, all enclosed within housing for autonomous, sequential loading of critical reagents onto the detection zone. This novel approach not only demonstrates a rapid assay completion time of under 30 min, but also boasts reduced reagent requirements, minimal equipment needs, and broad applicability across clinical diagnostics and environmental surveillance. Through detailed descriptions of the design, materials, and fabrication methods for the multi-directional flow assay (MDFA), this manuscript highlights the device’s potential for complex biochemical analyses in a user-friendly and versatile format. Analytical performance evaluation, including a limit of detection (LOD) of 8.4 pM for Rabbit IgG, benchmarks the device’s efficacy compared to existing p-ELISA methodologies. This pioneering work lays the groundwork for future advancements in autonomous diagnostics, aiming to enhance global health outcomes through accessible and reliable testing solutions.

Preconcentrating extractive polymeric network in paper-based sensing of copper

The concept of using an extractive polymeric sensing network on a paper-based substrate for the pretreatment of samples in the colorimetric analysis of heavy metals is demonstrated with the determination of Cu2+ in aqueous samples. The newly developed paper-based device combines the advantages of both liquid–liquid extraction-based separation and the analytical capabilities of microfluidic paper-based analytical devices (μPADs). The preconcentrating extractive polymeric network composed of 53.5 wt% poly(vinyl chloride) (PVC), 45 wt% extractant (di-(2-ethylhexyl)phosphoric acid (D2EHPA)) and 1.5 wt% dye (1-(2′-pyridylazo)-2-naphthol (PAN)), coated on filter paper, shows good water-wicking properties, allowing the transmission of high sample volumes. This results in significant analyte preconcentration. The extractant (D2EHPA) selectively binds Fe3+ and Zn2+ which otherwise will compete with Cu2+ in its colour reaction with PAN at the chosen pH of 3.0. The device’s capacity to process higher sample volumes resulting in higher sensitivity is increased by the integration of several filter paper absorption layers. The optimized device is characterized by a limit of detection (LOD) for tap and environmental waters of 3.4 and 9.6 µg/L, respectively; stability of 51 days if stored in a refrigerator; and total analysis time (from sample insertion to colour detection) of less than 5 min.

Sensitive assessment of bilirubin using various color space signals derived from captured images of microfluidic paper-based analytical devices

Many neonates’ manifest hyperbilirubinemia and jaundice during the first week following birth because of bilirubin accumulation in the blood. Newborns with severe hyperbilirubinemia develop neurotoxicity. Therefore, sensitive, accurate, cost-effective, and timely determination of bilirubin levels is highly desired. Herein, we present three simple systems of bilirubin assessment based on spectrophotometric, and digital imaging tools. Image-capturing devices pose a formidable challenge to sophisticated spectrophotometers and field-monitoring devices because of their simplicity and sensitivity. The first and second systems of bilirubin assessment, in solution, relied on spectrophotometric and conventional color responses of digital images collected with conventional or android smartphone camera, with LODs of 0.060–0.082 and LOQs of 0.200–0.273 ppm bilirubin. The third system utilized a microfluidic paper-based analytical device (μ-PAD) that is patterned with a wax printer on Whatman grade-1 chromatographic papers, with linear calibration graphs of up to 2.70,s and LODs of 0.035–0.040 and LOQs of 0.117–0.133 μg bilirubin/μ-PAD.

Microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum

BackgroundCalcium and magnesium ions are highly abundant and important cations in human body. At the same time, both dyscalcemia and dysmagnesemia are frequently encountered in the clinical practice. As deficiency or excess of Ca(II) or Mg(II) can cause severe symptoms, determining these ions in serum is of great importance. Concentration of these ions in biological samples is typically assayed in clinical laboratories with the use of expensive and specialized equipment. Since those methods cannot be easily adapted for self-diagnosis purposes, there is a great need to develop a convenient tool for reliable determination of calcium and magnesium in serum at the point-of-care. ResultsThe colorimetric methods employed for calcium and magnesium analysis were o-cresophtalein complexone assay and xylidyl blue assay, respectively. Analytical signal acquisition was accomplished using an ordinary flatbed scanner or smartphone and free software. For increased user-friendliness the device was optimized to perform simultaneous determination of calcium and magnesium ions in only 10 min. In the optimized conditions, the limit of detection for calcium ions was 0.09 mmol L−1, while for magnesium it was 0.04 mmol L−1. Determination of both ions requires only 4 μL of serum sample. The developed paper-based sensors were validated with control human serum samples and the obtained relative errors for majority of samples were below 20 %. SignificanceIn this paper, a microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum is reported for the first time. Additionally, this is also the first report on colorimetric determination in serum of any of these ions in paper-based format. Simultaneous detection of both ions allows for fast and user-friendly screening of disturbance in calcium and magnesium homeostasis.

CRISPR/Cas14 and G-Quadruplex DNAzyme-Driven Biosensor for Paper-Based Colorimetric Detection of African Swine Fever Virus.

The highly contagious nature and 100% fatality rate contribute to the ongoing and expanding impact of the African swine fever virus (ASFV), causing significant economic losses worldwide. Herein, we developed a cascaded colorimetric detection using the combination of a CRISPR/Cas14a system, G-quadruplex DNAzyme, and microfluidic paper-based analytical device. This CRISPR/Cas14a-G4 biosensor could detect ASFV as low as 5 copies/μL and differentiate the wild-type and mutated ASFV DNA with 2-nt difference. Moreover, this approach was employed to detect ASFV in porcine plasma. A broad linear detection range was observed, and the limit of detection in spiked porcine plasma was calculated to be as low as 42-85 copies/μL. Our results indicate that the developed paper platform exhibits the advantages of high sensitivity, excellent specificity, and low cost, making it promising for clinical applications in the field of DNA disease detection and suitable for popularization in low-resourced areas.

High-Throughput μPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.

The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (μPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

A digital bar pH indicator based on a microfluidic paper-based analytical device

A pH value of an aqueous medium is an essential parameter in the environment and biology. Nowadays, it is possible to measure using a colorimetric pH paper or an electrochemical pH meter. However, since the two methods have drawbacks of the limited precision in pH determination and the careful management of a glass membrane electrode, respectively, it still remains as a challenge to discover a user-friendly, reliable pH-measuring device. Herein, a novel digital bar pH indicator was developed using a microfluidic paper-based analytical device (μPAD) to determine with the naked eye the pH value of an aqueous sample. The fabricated digital indicator consists of seven independent pH sensors displaying a color change from yellow to greenish blue above specific pH values. These sensing elements were vertically aligned to form a bar shape, and their color-changing pH points were regulated to increase upward from 4.5 to 7.5 with an interval of 0.5 units. For fast and simultaneous detection of the colorimetric sensors, sample addition to an inlet of the μPAD was quickly transported vertically along a hollow paper channel, horizontally divided, and arrived at the detection zones at similar times via delay channels. Through independent experiments, control of the color-changing pH point and flow rate were confirmed by addition of a surfactant additive into the pH indicator and inserting empty spaces in a flow channel, respectively. Reliable operation of the fabricated μPAD was verified using standard pH buffer samples. Finally, the feasibility of our digital bar pH indicator was successfully demonstrated for determining the pH value of a urine sample under the resolution of 0.5 units.

An enrichment system for glycoproteins using microfluidic paper-based analytical device with colorimetric detection

We introduce a facile method for glycoprotein enrichment, separation, and detection using microfluidic paper-based analytical devices (μPADs). The μPADs comprise two distinct components: the enrichment region (Chip I) and the detection section (Chip II). The reusable Chip I is fabricated by immobilizing 3-aminophenylboronic acid (APBA) onto a circular paper substrate, demonstrating exceptional glycoprotein affinity. In disposable Chip II, glycoproteins react with periodic acid, generating carbonyl compounds that produce a purple-colored product upon reacting with the Schiff reagent. Alterations in color intensity are captured using an ordinary cell phone and are subsequently analyzed using Photoshop software. Observations note a linear increase in average color intensities from 1.0 × 10−3 to 5.0 × 10−3 mol/L, with an R2 value of 0.9927. The relative standard deviations (RSDs) of inter-day (n = 5) and intra-day (n = 5) assessments stand at 1.5 % and 1.4 %, respectively. Our proposed approach can effectively enrich an ovalbumin (OVA) solution with concentrations as low as 1.0 × 10−11 mol/L, resulting in enrichment rates of 107 to 108 times. The interference deviations of five substances relative to OVA lie within the range of − 2.0 % to + 2.0 %. The methodology successfully enriches glycoproteins from egg white samples diluted by a factor of 1 × 106, demonstrating that the proposed method is remarkably suitable for enriching glycoproteins from authentic samples.

Current diagnostics and biomarkers for arboviral infections (a review on Dengue, Zika, West Nile and Chikungunya viruses)

Arboviral infections, transmitted to humans primarily through arthropod vectors, constitute a significant global health threat. Arboviruses, such as Dengue, Zika, Chikungunya, and West Nile viruses, continue to cause widespread outbreaks, necessitating advanced diagnostic tools. Emerging technologies including Lab On A Chip (LOC), Lab On A Disc (LOAD), Microfluidic Paper-Based Analytical Devices (µPADS), Lateral Flow Devices, CRISPR-CAS 12/13, Quartz crystal microbalance (QCM), and Nano-Technology are evaluated for their potential to enhance arboviral diagnosis, offering rapid, accurate, and point-of-care solutions. Furthermore, the identification of robust biomarkers, including Inflammatory Cytokines, Antibodies, Endothelial Activation Products and Indicators of Tissue or Organ Damage, is crucial for improving the understanding of disease pathogenesis, prognosis, and treatment response. A comprehensive analysis of potential diagnostics and biomarkers for arboviral infections sheds light on the evolving strategies to combat these medically significant diseases, ultimately contributing to more effective surveillance, diagnosis and management worldwide.

Smartphone-Based Colorimetric Microfluidic Paper-Based Analytical Device for On-Site Detection of Calcium Ions in Milk Samples

Milk is a major dietary source of calcium; thus, accurate and precise quantification of calcium in milk and other dairy products is necessary for quality control. Conventional instrumental methods are sensitive and accurate but require expensive instrumentation, sample pretreatment, skilled personnel, and a long detection time. Herein, a microfluidic paper-based analytical device (µPAD) was developed to quantify calcium ions (Ca2+) in milk using a smartphone as a detector. The method utilized a complexometric reaction between Ca2+ and ethylene diamine tetraacetic acid with murexide as an indicator. The response was the average color intensity at the detection zones measured using the Color Picker application. The proposed µPAD exhibited a good linear detection range (1 to 10 mM), a low detection limit of 0.42 mM, and a short analysis time of 2 min. The determination of Ca2+ in milk samples using the proposed µPAD was consistent with a potentiometric method. The proposed µPAD demonstrates many advantages, including rapid detection, acceptable accuracy, low cost, low sample volume, and suitability for on-site quantification of Ca2+ in real samples.

Adsorption enrichment integrated with paper-based devices for detection of trace levels of hexavalent chromium in water samples.

In the present study, a sensitive microfluidic paper-based analytical device (μ-PADs) integrated with adsorption enrichment procedure was developed to analyze Cr(VI) in water samples. The affecting factors, including pH and amounts of reagents were optimized. The limit of detection of 0.0015mg L-1 and linear range of 0.005-2mg L-1 were achieved with good intra- and inter-day precision of 5.1 and 7.6% RSD, respectively. The results obtained by the proposed method were validated by inductively coupled plasma-optical emission spectrometry (ICP-OES). The recoveries of the present method and ICP-OES were ranged from 96.3 to 109.0‬% and 106.0 to 109.7%, respectively. The two sets of (μ-PADs and ICP-OES) results were in a good agreement as paired t-test indicated no significant differences. The proposed method could be utilized for analyzing trace levels of Cr(VI) in water samples in the absence of conventional analytical instruments.

Microfluidic Paper-Based Device Incorporated with Silica Nanoparticles for Iodide Quantification in Marine Source Dietary Supplements.

Iodine is an essential micronutrient for humans due to its fundamental role in the biosynthesis of thyroid hormones. As a key parameter to assess health conditions, iodine intake needs to be monitored to ascertain and prevent iodine deficiency. Iodine is available from various food sources (such as seaweed, fish, and seafood, among others) and dietary supplements (multivitamins or mineral supplements). In this work, a microfluidic paper-based analytical device (μPAD) to quantify iodide in seaweed and dietary supplements is described. The developed μPAD is a small microfluidic device that emerges as quite relevant in terms of its analytical capacity. The quantification of iodide is based on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide in the presence of iodine, which acts as the catalyst to produce the blue form of TMB. Additionally, powder silica was used to intensify and uniformize the colour of the obtained product. Following optimization, the developed μPAD enabled iodide quantification within the range of 10-100 µM, with a detection limit of 3 µM, and was successfully applied to seaweeds and dietary supplements. The device represents a valuable tool for point-of-care analysis, can be used by untrained personnel at home, and is easily disposable, low-cost, and user-friendly.

Fluid Flow Dynamics in Partially Saturated Paper.

This study presents an integrated approach to understanding fluid dynamics in Microfluidic Paper-Based Analytical Devices (µPADs), combining empirical investigations with advanced numerical modeling. Paper-based devices are recognized for their low cost, portability, and simplicity and are increasingly applied in health, environmental monitoring, and food quality analysis. However, challenges such as lack of flow control and the need for advanced detection methods have limited their widespread adoption. To address these challenges, our study introduces a novel numerical model that incorporates factors such as pore size, fiber orientation, and porosity, thus providing a comprehensive understanding of fluid dynamics across various saturation levels of paper. Empirical results focused on observing the wetted length in saturated paper substrates. The numerical model, integrating the Highly Simplified Marker and Cell (HSMAC) method and the High Order accuracy scheme Reducing Numerical Error Terms (HORNET) scheme, successfully predicts fluid flow in scenarios challenging for empirical observation, especially at high saturation levels. The model effectively mimicked the Lucas-Washburn relation for dry paper and demonstrated the increasing time requirement for fluid movement with rising saturation levels. It also accurately predicted faster fluid flow in Whatman Grade 4 filter paper compared with Grade 41 due to its larger pore size and forecasted an increased flow rate in the machine direction fiber orientation of Whatman Grade 4. These findings have significant implications for the design and application of µPADs, emphasizing the need for precise control of fluid flow and the consideration of substrate microstructural properties. The study's combination of empirical data and advanced numerical modeling marks a considerable advancement in paper-based microfluidics, offering robust frameworks for future development and optimization of paper-based assays.

Use of a rhodamine-based chelator in a microfluidic paper-based analytical device for the in-situ copper quantification in natural waters

This work describes the development of a microfluidic paper-based analytical device (μPAD) for the determination of copper in fresh and marine waters. A functionalized rhodamine-based chelator was synthesized and used as a chromogenic reagent, forming a highly intense pink complex with the analyte. The aim was to create a paper device that offers optimal performance and provides in-situ, rapid and cost-effective analysis in line with World Health Organization guidelines. The influence on the determination of several physical and chemical parameters was evaluated aiming to achieve the best performance. Under optimised conditions, a linear correlation was established in the range of 0.05–0.50 mg L−1 of copper, with a limit of detection of 10 μg L−1. The accuracy of the proposed method was assessed by comparing the results obtained with the developed μPAD and the results obtained with Inductively Coupled Plasma measurements (RE < 10 %). Recovery studies were also performed using different types of water samples with no need for any prior sample pre-treatment: tap, well, river and seawater. The average recovery percentage of 101 % (RSD = 4.3 %) was obtained, a clear indication of no multiplicative matrix interferences.

Microfluidic paper-based analytical device for the speciation of inorganic nitrogen species

A microfluidic paper-based analytical device (μPAD) utilizing gas-diffusion separation and solid-phase reduction was developed for the first time for the determination of both ammonium and nitrate, which are the dominant inorganic nitrogen species in environmental waters. The device consists of 3 filter paper layers accommodating the sample, reagent and detection zones. The reagent zone is separated from the detection zone by a semipermeable hydrophobic membrane and acts as a solid-phase reactor where nitrate is reduced to ammonia by Devarda's alloy microparticles, integrated into a μPAD for the first time. The detection zone incorporates the acid-base indicators bromothymol blue (BTB) or nitrazine yellow (NY) and changes colour in two steps. Initially the colour change is caused by ammonia generated by the reaction of ammonium and sodium hydroxide in the sample zone. This colour change is followed by a subsequent colour change as a result of the ammonia produced by the reduction of nitrate by the Devarda's alloy microparticles. The corresponding reflectance value changes are used for the quantification of the two inorganic nitrogen species in the ranges 6.5–100.0 or 2.1–15.0 mg N L−1 for ammonium and 18.2–100.0 or 4.2–15.0 mg N L−1 for nitrate when BTB or NY are used, respectively. Under optimal conditions the limits of quantification of ammonium and nitrate in the case of BTB were determined as 6.5 and 18.2 mg N L−1, respectively, while the corresponding values in the case of NY were found to be 2.1 and 4.2 mg N L−1. The newly developed μPAD was stable for 62 days when stored in a freezer and 1 day at ambient temperature. It was validated with a certified reference material and successfully applied to the determination of ammonium and nitrate in spiked environmental water samples and soil extracts.

Photothermal biosensing integrated with microfluidic paper-based analytical device for sensitive quantification of sarcosine

This article presents the development of a photothermal biosensing integrated with microfluidic paper-based analytical device (PT-μPAD) as a quantitative biosensor method for monitoring sarcosine in human control urine, plasma, and serum samples. The device utilizes gold nanoparticles (AuNPs) as both a peroxidase-like nanozyme and a photothermal substrate to enable sarcosine detection. In our PT-μPAD, hydrogen peroxide (H2O2) is generated through the oxidation of sarcosine by a sarcosine oxidase (SOx) enzyme. Subsequently, the H2O2 flows through the paper microchannels to the detection zone, where it etches the pre-deposited AuNPs, inducing a temperature change upon exposure by a 532 nm laser. The temperature variation is then measured using a portable and inexpensive infrared thermometer. Under optimized conditions, we obtained a linear range between 10.0 and 40.0 nmol L−1 (R2 = 0.9954) and a detection limit (LOD) of 32.0 pmol L−1. These values fall within the clinical range for sarcosine monitoring in prostate cancer diagnostics in humans. Moreover, our approach exhibits high selectivity without interfering effects. Recovery studies in various human control samples demonstrated a range of 99.05–102.11 % with the highest RSD of 2.25 %. The PT-μPAD was further validated for sarcosine determination in human control urine and compared with a commercial ELISA assay, revealing no significant difference between these two methods at a 95 % confidence level. Overall, our proposed sarcosine biosensor is well-suited for prostate cancer monitoring, given its affordability, sensitivity, and user-friendliness, even for unskilled individuals. Moreover, this strategy has promising prospects for broader applications, potentially detecting various biomarkers as a point-of-care (POC) diagnostic tool.

Microfluidic paper-based analytical device with a preconcentration system for the measurement of anionic surfactants using an optode detector.

The paper discusses the development of a low-cost, simple, and sensitive on-site measurement system for anionic surfactants (AS), specifically sodium alkylbenzene sulfonate (LAS), using a microfluidic paper-based analytical device (μPAD) with an optode as a detector. The need arises due to the discharge of AS-containing wastewater into natural environments, posing risks to aquatic organisms. Traditional methods for AS measurement have drawbacks like the use of harmful solvents, time-consuming procedures, and the need for expensive equipment, prompting the exploration of alternative approaches. The μPAD incorporates a sample solution preconcentration system using filter paper modified with chitosan. The optode, a chemical sensor detecting analytes optically, is employed for AS detection. When a large volume of AS is added to a positively charged modified filter paper with chitosan, the AS is adsorbed and concentrated on the filter paper. The concentrated AS is eluted with a small volume of alkaline solution, and the eluted AS is detected by the optode in the μPAD. The μPAD with preconcentration provides improved sensitivity and a broader range of detection compared to the method without preconcentration. In the present μPAD method, linear alkylbenzenesulfonate (LAS) in the concentration range of 0.1 to 10 μmol dm-3 was measured. The developed μPAD offers advantages such as portability, cost-effectiveness, and negligible interference from coexisting substances in environmental water samples. The μPAD method was applied for the determination of LAS in tap water and river water.

A dual colorimetric-electrochemical microfluidic paper-based analytical device for point-of-care testing of ischemic strokes.

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 (Er ≤ 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.

A paper-based point-of-care device for the detection of cysteine using gold nanoparticles from whole blood.

We present a colorimetric probe based on polyvinylpyrrolidone-capped gold nanoparticles (PVP-AuNPs) that is sensitive and selective for cysteine (Cys). A microfluidic paper-based analytical device (μ-PAD) with embedded dried PVP-AuNPs at the polyethersulfone (PES) paper surface is used for Cys detection. When thiol molecules attach to PVP-AuNPs in the presence of Cys, they clump together, and this causes the solution's color to shift from red to blue within 5 minutes. The device is capable of detecting Cys levels between 1.0 μM and 50.0 μM with a limit of detection (LOD) of 0.2 μM under optimized conditions. The stability of the μ-PAD was tested for 100 days, demonstrating re-dispersibility to detect Cys levels in blood. Dried PVP-AuNP-μPADs were integrated with blood plasma separation modules for point-of-care (POC) Cys detection. Consequently, the device shows potential as a self-sustaining, quantification platform with a recovery percentage ranging from 98.44 to 111.9 in clinical samples.