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Review work on the challenges of paper-based NAATs covering sample-to-answer procedures along with the three main types of clinical samples as well as broader operational, scale up, and regulatory aspects of device development and implementation.

We demonstrate a highly efficient DLD separation device and process that is driven by a paper wick yet allows direct collection of products from reservoirs.

Paper-based microfluidics offers a promising way to in situ sweat detection for non-invasive continuous monitoring. This passage reviews and prospects its development regarding materials, structuring, fabrication, modular design and application.

This work describes a terbium luminescence-based protocol to selectively detect gallate-derived green tea polyphenols on a supramolecular gel immobilised paper platform for the first time. This user-friendly, inexpensive (� 0.0015) approach requires very low sample volumes for the analysis. The developed strategy enables simultaneous detection of gallate polyphenols in multiple tea samples with the potential for practical applications. © 2020 Wiley-VCH GmbH

This work presents a novel cellulose-based aptasensor for the colorimetric detection of a cancer biomarker, osteopontin (OPN), in point-of-care (PoC) analysis. For this purpose, the cellulose paper was chemically modified with (mercaptopropyl)methyldimetoxisilane to attach the thiolated aptamer, which acts as a biological detection layer. The surface modification was checked by Fourier transform infrared spectroscopy and thermogravimetric analysis. Colorimetric detection was performed using a conventional staining solution, Bradford reagent. The colour analysis was performed by evaluating the RGB coordinates provided by the ImageJ program from the photographs taken with a smartphone. Overall, the biosensor shows good sensitivity with a wide linear range (R=0.998) of 51000ng/mL and a detection limit lower than 5ng/mL in buffer and commercial human serum solution after 30 minutes of incubation. In addition, this aptasensor shows good selectivity to some interfering species such as bovine serum albumin and recombinant OPN. Analytical data obtained from spiked serum samples confirm the accuracy of the method. Importantly, it is a broad-spectrum method that tends to meet the criteria of REASSURED (real-time connectivity, ease of sampling, affordability, specificity, ease of use, speed and robustness, device freedom, and deliverability) for global testing., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Ana Cláudia Pereira is a recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Bladder cancer is the fourth most common cancer in men, and it is becoming a prevalent malignancy. Most of the regular clinical examinations are prompt evaluations with cystoscopy, renal function testing, which require high-precision instrument, well-trained operators, and high cost. In this study, a microfluidic paper-based analytical device (μPAD) was fabricated to detect nuclear matrix protein 22 (NMP22) and bladder cancer antigen (BTA) from the urine samples. Urine samples were collected from 11 bladder cancer patients and 10 well-beings as experiment and control groups, respectively, to verify the working efficiency of μPAD. A remarkable checkout efficiency of up to 90.91% was found from the results. Meanwhile, this method is feasible for home-based self-detection from urine samples within 10 min for the total process, which provides a new way for quick, economical, and convenient tumor diagnosis, prognosis evaluation, and drug response.

This work presents an unconventional use of capacitively coupled contactless conductivity detector (C4D) for detection of gas absorption by moist paper with potential application for chemical analysis. To be suitable for measuring conductivity of moist paper absorbent, the C4D sensor was therefore designed in planar configuration. A layer of dry filter paper, only 20 mm × 25 mm in size, was placed on the C4D sensor and the device installed inside a specifically designed vaporization chamber. A vial (16 mm i.d., 8 mm high) containing a 150-μL solution of sodium bicarbonate was placed alongside. The filter paper was loaded with 110 μL of deionized water through an injection hole in the cover lid. A 100-μL aliquot of 2 M hydrochloric acid solution was directly dispensed into the vial through a second hole in the lid to generate CO2 gas from the bicarbonate solution. It was observed that the C4D sensor gave real-time response that corresponded to the absorption of the gas and subsequent production of H+ and HCO3− in the moist paper. The monitored signal reached a constant value at 160 s after the addition of the acid. Chemistry of the absorption process and equivalent circuit for the C4D are proposed. Direct measurement of cement powder was chosen to demonstrate the potential use of this device for quantifying the CaCO3 content of the cement. The calibration curve for 0.5–3 mg CaCO3 was linear for signals recorded at 160 s: Vdc = (0.172 ± 0.005) · (mg CaCO3) + (0.016 ± 0.009), with coefficient of determination of 0.9965. Linear calibrations were also observed when the signals were monitored at various time less than 160 s. The limit of quantitation (3 SD of intercept/slope) was 0.17 mg CaCO3. The method provided acceptable precision with %RSD of 4.6 (2 mg CaCO3, n = 10).

Caenorhabditis elegans is an in vivo model known for its easy handling and maintenance and lack of associated ethical issues. The release of chitinase can be used to monitor the egg-laying stage in C. elegans. The aim of this study was to develop a simple and cost-effective device to monitor the activity of chitinase in embryos of C. elegans. Colloid chitin azure (CCA), a substrate for chitinase, was preimmobilized on the detection area of paper, forming a purple region, to generate a CCA paper-based analytical device (CCA-PAD). The degradation of CCA by chitinase could be observed as the purple color became faint and the filter paper eventually became colorless. Under the optimum conditions, the proposed device quantified the chitinase enzyme in the range of 15.625-125 mU/mL within 48 h (R2 = 0.993). In this work, 10 young adult-staged wild-type C. elegans (Bristol N2) worms were analyzed on the CCA-PAD, which was supplemented with the laboratory food source E. coli OP50 on a gauze layer. The same strain treated with 5-fluoro-2'-deoxyuridine was used to prevent egg production in C. elegans. A significant difference in the color intensity was observed between these two groups at the end of the experiment (P =

Polygalacturonase (PG) activity in plants can serve as an important index for plant disease. However, the conventional method to detect PG activity is a complex process and requires a skilled technician and expensive analytical equipment. In this study, a paper-based colorimetric sensor was developed based on the principle of the ruthenium red (RR) dye method for easy and simple measurement of PG activity. The proposed paper-based sensor has a three-layer structure for detection of PG activity in samples. The sensor sensitivity was enhanced by optimizing the pH of the sodium acetate buffer used in polygalacturonic acid (PGA)-RR complex formation and the reaction temperature for PG and the PGA-RR complex. Further, for quantitative analysis of PG activity, Delta RGB analysis was conducted to detect color changes in the sensing window of the sensor. Results presented that the linear measurement range of the paper sensor was 0.02-0.1 unit with the limit of detection of 0.02 unit, which showed a similar detection range, but a lower detection limit, compared to the spectrophotometry. Furthermore, PG activity based on culture condition was measured using samples from Sclerotium cepivorum to verify the potential application of the developed paper-based sensor in the field. The measured activity showed no statistically significant difference from the values obtained from the spectrophotometry at 95% confidence level. Therefore, the paper-based colorimetric sensor can be used to predict plant diseases in Allium crops during the stage of pathogen invasion, potentially contributing to the improvement of crop production.

In this paper, nanochitin was used as an alternative natural nanomaterial to combine with cellulose fibers for fabricating high-strength paper. Two typical chitin nanowhiskers having contrasting sign of surface charge were compared to evaluate the enhancement performance on paper in details. The results show that nanochitin with positive charges on the surface has a significant effect on the strength properties of the prepared paper, especially on wet strength. When the dosage of chitin nanowhiskers was 2%, the wet strength index was increased to 2.48 N·m/g, which is important for paper-based analytical devices with the common use in liquid analysis. Typical colorimetric glucose assays were successfully performed, suggesting the improved analytical performance on these prepared paper.

Microfluidic paper-based analytical devices (µPADs) take the paper as a base material and integrate nanoscale microchannel on it for multiple detections. Its unique properties like low cost, portability, simple operation, and easy to save make it better than the traditional microfluidic chips. While designed originally for point-of-care medical diagnostics, µPADs have attracted the attention of many researchers in the fields of environmental monitoring, water quality, and food safety. The novelty of this paper is to present a detailed overview of µPADs for clinical applications. Firstly, a brief introduction to production methods, characteristics, and applications of these methods have been given. Secondly, the basic implementation, working principles, and corresponding performance of detection methods of clinical devices have been discussed, which enable the µPADs to detect biomarkers, human cells, bacteria, and viruses in a short time. Lastly, the factors that limit µPADs commercial applications, and their future research directions have also been briefly summarized.

Microfluidic paper-based analytical devices (μPADs) allow user-friendly and portable chemical determinations, although they provide limited applicability due to insufficient sensitivity. Several approaches have been proposed to address poor sensitivity in μPADs, but they frequently require bulky equipment for power and/or read-outs. Universal serial buses (USB) are an attractive alternative to less portable power sources and are currently available in many common electronic devices. Here, USB-powered μPADs (USB μPADs) are proposed as a fusion of both technologies to improve performance without adding instrumental complexity. Two ITP USB μPADs were developed, both powered by a 5 V potential provided through standard USB ports. The first device was fabricated using the origami approach. Its operation was analyzed experimentally and numerically, yielding a two-order-of-magnitude sample focusing in 15 min. The second ITP USB μPAD is a novel design, which was numerically prototyped with the aim of handling larger sample volumes. The reservoirs were moved away from the ITP channel and capillary action was used to drive the sample and electrolytes to the separation zone, predicting 25-fold sample focusing in 10 min. USB μPADs are expected to be adopted by minimally-trained personnel in sensitive areas like resource-limited settings, the point-of-care and in emergencies. Fil: Schaumburg, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentina Fil: Carrell, Cody S.. State University of Colorado - Fort Collins; Estados Unidos Fil: Berli, Claudio Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Henry, Charles S.. State University of Colorado - Fort Collins; Estados Unidos

China, a big paper-making country, produced hundreds of millions of tons of waste paper which contain a lot of fiber every year. Cellulose nanocrystals were extracted from recycled waste paper which can be a high value utilization of secondary fiber. In this paper, cellulose nanocrystals were successfully extracted from waste paper fibers via two different systems, sulfuric acid hydrolysis (SCNCs) and one-step ammonium persulfate (APS) oxidation (OCNCs). This not only broadened the methods of extracting CNCs from waste paper, but also improved the dispersion and reactivity of CNCs. The CNCs products were investigated by FT-IR spectroscopy for functional group structure, X-ray diffraction for crystal structure, TG-DTG for thermal stability and scanning electron microscope, transmission electron microscope for morphology. The results showed that both OCNCs and SCNCs were a rod-like structure. The crystallinity of OCNCs and SCNCs increased to 72.45 and 77.56, but with a low yield of 22.42% and 41.22%, respectively. The result also suggested H2O2 formed by decomposition of APS, selectively oxidized the hydroxyl on the C6 in cellulose to carboxyl, introduced 0.57 mmol/g carboxyl. Successful preparation of CNCs extracted from waste paper can effectively utilize the fiber resources in waste paper, thus transforming into higher economic benefits.

Paper-based microfluidic device was designed with wax-printing to combine potentiometric, fluorimetric and colorimetric detection zones. This newly developed trimodal paper chip has been used for on-site determination of ketamine hydrochloride (KET) as a date rape drug in beverages. The device employed polyaniline nano-dispersion as conducting polymer in ion sensing paper electrodes designed to fit USB plug connector. Carbon dots-gold nanoparticles and cobalt thiocyanate were used in fluorescence and color detection zones, respectively. Cellular phone's camera facilitated the on-site fluorimetric and color detection. The implemented trimodal detection system exhibited specificity for KET detection in the presence of several other beverage interferences i.e., biogenic amines. This innovative sensor brings together analytical figures of merit for effective KET detection in single aliquot of spiked beverages. The proposed paper-based chip also fulfils WHO criteria for point-of-care devices posing the proposed trimodal paper device as an active part for rapid, on-site drug diagnostics and to be applied further for other similar drugs.

Free proline, termed proline, is a biomarker used for diagnosing drought stress in plants. A previously developed proline–ninhydrin reaction-based paper sensor could quickly and easily detect proline, but it was limited by low sensitivity. In this study, we developed an enclosed multilayer paper-based microfluidic sensor with high sensitivity for the quantitative detection of proline in plants. The multilayer paper-based sensor was manufactured using simple wax printing and origami methods, and contained an internal mixing channel to allow good mixing of the proline with ninhydrin, increasing the proline–ninhydrin reactivity and providing accurate and sensitive proline detection. By preloading ninhydrin onto the sample loading area, uniform coloration of the sensing window was achieved, allowing quantitative analysis of various proline concentrations using a constant reaction time. Only the sensing window and sample loading area were exposed to limit sample evaporation and contamination from the external environment. The LOD of the fabricated sensor was 23 μM, which is approximately 29-fold lower than that of the previously proposed paper sensor (657 μM). Samples were extracted from A. thaliana plants subjected to drought stress for proline detection. The proline concentrations measured using the developed paper sensor and a spectrophotometric method were not statistically significant at a confidence level of 95%. Therefore, the developed sensor can be applied to measure proline concentrations precisely in the field with a low detection limit. The developed paper-based sensor can be used to detect the early stages of drought in plants and thus improve crop productivity.

The diagnosis of dengue infection is still a critical factor determining success in the clinical management and treatment of patients. Here, the development of microfluidic paper-based analytical devices (μPADs) utilizing a sandwich immunoassay on wax patterned paper functionalized with anti-dengue NS1 monoclonal antibodies for point-of-care detection of dengue NS1 (DEN-NS1-PAD) is reported. Various assay conditions, including the length of the channel and diluent, were optimized, and the response detected by the naked eye and digitized images within 20–30 min. The DEN-NS1-PAD was successfully tested in the field for detecting dengue NS1 in buffer, cell culture media, and human serum. The limit of detection (LoD) of the DEN-NS1-PAD obtained with the naked eye, scanner, and a smartphone camera was 200, 46.7, and 74.8 ng mL−1, respectively. The repeatability, reproducibility, and stability of the DEN-NS1-PAD were also evaluated. High true specificity and sensitivity in the serum of pediatric patients were observed. These evaluation results confirm that the DEN-NS1-PAD can potentially be used in point-of-care dengue diagnostics, which can significantly impact on the spreading of mosquito-borne diseases, which are likely to become more prevalent with the effects of global warming.

This work presents a novel development that exploits the concept of in-situ gas-separation together with a specific enzymatic colorimetric detection to produce a portable biosensor called “Blood Alcohol Micro-pad” for direct quantitation of ethanol in whole blood. The thin square device (25 mm × 25 mm × 1.8 mm) comprises two layers of patterned filter paper held together with a double-sided mounting tape with an 8-mm circular hole (the headspace). In operation, the reagent is deposited on one layer and covered with sticky tape. Then 8 μL of a blood sample is dispensed onto the opposite layer and covered with sticky tape. Diffusion of ethanol across the 1.6 mm narrow headspace permits selective detection of ethanol by the enzymatic reagents deposited on the opposite layer. This reagent zone contains alcohol oxidase, horseradish peroxidase and 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, as the chromogenic reagent. The color intensity, measured from the recorded digital image, resulting from the enzymatic assay of ethanol, correlates with the concentration of blood alcohol. The results obtained with spiked mice and sheep blood samples, using an external calibration in the range of 1–120 mg dL−1ethanol, gave recoveries of 93.2–104.4% (n = 12). The “Blood Alcohol Micro-pad” gave good precision with %RSD

Cytochrome P450 (CYP450), and in particular CYP3A4, is the most abundantly expressed CYP450 isozyme implicated in many drug-drug and medicinal plant-drug interactions. Therefore, incorporation of CYP3A4 enzyme screening at an early stage of drug discovery is preferable in order to avoid enzymatic interactions. Here we present for the first time a paper-based CYP3A4 immobilized sol-gel-derived a platform using resorufin benzyl ether as a fluorogenic enzyme substrate used to investigate enzyme activity. The fluorescence intensity of the product can be simply quantified by using a handheld digital microscope and an image analysis software. The limit of quantitation was 0.35 μM with good precision (RSDs 0.05), while offering simplicity and lower cost. Kinetic parameters of the immobilized CYP3A4 in sol-gel coated paper were calculated from the Lineweaver-Burk plot, including Michaelis constant (Km) and maximum velocity (Vmax), which were 2.71 ± 0.35 μM and 0.43 ± 0.05 μM/min, respectively. Moreover, a functional test of these devices was conducted by assessments of known CYP3A4 inhibitors (i.e. ketoconazole, itraconazole) and inducers (i.e. phenytoin, carbamazepine). To further demonstrate the broad range of uses, the devices were utilized to assay plant extracts i.e. Areca catechu seeds, Camellia sinensis leaves, Eclipta prostrata aerial part, providing results in good agreement with previous studies. Furthermore, the sol-gel immobilized enzyme stored at 4 °C can increase storage stability, offering the activity of 86.3 ± 0.4% after 3-weeks storage, equivalent to the activity of the free enzyme solution after 1-week storage. The developed paper-based devices offer versatility, portability and low-cost.

In this study, we developed a point-of-care assay platform with simultaneous detection and self-calibration capabilities for multiple targets based on a microfluidic paper-based analytical device (μPAD). This system is easily manufactured using a wax printing method on chromatographic paper. The design pattern consists of a zone of detection and a calibrant zone for controlled loading using wax barriers with different thicknesses. We showed the utility and applicability of this approach by a proof-of-concept study for two clinically important markers: glucose and lactate. With the naked eye, the results could be fully distinguished and recorded to evaluate the analytical performance with a flatbed scanner. The detection limits of glucose and lactate were 0.3125 mM and 0.2975 mM, respectively, and simultaneous detection was possible from a small sample (0.4 μL) with high sensitivity. Furthermore, this device has a self-calibration function, which minimizes the influence of environmental conditions (i.e., ambient light intensity, temperature, humidity, and pressure). Therefore, the developed multiplex paper-based device is promising for clinical multianalyte point-of-care testing since it is easy to manufacture, cost-effective, user-friendly, and highly sensitive.

A novel 3D paper-based microfluidic screen-printed electrode (SPE) composed of two layers was constructed by photolithography and screen-printing technology. Aldehyde functionalized hydrophilic zone of the counter and reference electrodes layer was prepared for glucose oxidase immobilization. Highly conductive Prussian blue deposited reduced graphene oxide-tetraethylene pentamine (rGO-TEPA/PB) modified paper working electrode layer can be used as an electrochemical sensitive membrane for quantitative detection of hydrogen peroxide (H2O2), which was the enzyme-catalyzed reaction product. Therefore, this 3D paper-based microfluidic electrochemical biosensor can be used for quantitative detection of glucose. Under optimum conditions, the proposed biosensor can be used for quantitative determination of glucose over a wide linear range of 0.1 mM∼25 mM with detection limit of 25 μM. Finally, the 3D paper-based microfluidic electrochemical biosensor was applied to determine glucose in human sweat and blood, and the obtained results were in good consistency with values measured by Roche's blood glucose meter. In addition, the proposed 3D paper-based electrochemical device showed good repeatability, stability, and anti-interference, which would be of great potential to monitor glucose in complex biological fluids.

We report a smartphone–paper-based sensor impregnated with cetyltrimethylammonium bromide modified silver nanoparticles (AgNPs/CTAB) for determination of Fe3+ in water and blood plasma samples. The methodology for determination of Fe3+ is based on the change in signal intensity of AgNPs/CTAB fabricated on a paper substrate after the deposition of analyte, using a smartphone followed by processing with ImageJ software. The mechanism of sensing for detection and determination of Fe3+ is based on the discoloration of AgNPs which impregnated the paper substrate. The discoloration is attributed to the electron transfer reaction taking place on the surface of NPs in the presence of CTAB. Fe3+ was determined when the paper was impregnated with 1 mM AgNPs for 5 min of reaction time and the substrate was kept under acidic conditions. The linear range for determination of total iron in terms of Fe3+ was 50–900 μg L−1 with a limit of determination (LOD) of 20 μg L−1 and coefficient of variation (CV) of 3.2%. The good relative recovery of 91.3–95.0% and interference studies showed the selectivity of the method for determination of total iron in water and blood plasma samples. Smartphone–paper-based sensors have advantages of simplicity, rapidity, user-friendliness, low cost, and miniaturization of the method for on-site determination of total iron compared to methods that require sophisticated analytical instruments.

Regulating the fluid flow in microfluidic devices enables a wide range of assay protocols for analytical applications. A programmable, photo-paper-based microfluidic device fabricated by using a method of cutting and laminating, followed by printing, is reported. The flow distance of fluid in the photo-paper-based channel was linearly proportional to time. By printing silver nanoparticle (AgNP) and poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene] (PTFE) patterns on the surface of a photo-paper-based channel, we were able to either increase or decrease the fluid flow in the fabricated microfluidic devices, while maintaining the linearity in the flow distance-time relation. In comparison to the speed of fluid flow in a pristine channel, by using hydrophilic AgNP patterns, we were able to increase the speed in the channel by up to 15 times while we were able to slow the speed by a factor of 3 when using hydrophobic PTFE dots. We then further demonstrated a single-step protocol for detecting glucose and a multi-step protocol for detecting methyl paraoxon (MPO) with our methods in photo-paper-based microfluidic devices. This approach can lead to improved fluid handling techniques to achieve a wide range of complex, but programmable, assays without the need for any additional auxiliary devices for automated operation.

Cobalt(ii) phthalocyanine (CoPc) was suspended in aqueous medium and the colloidal system was used as catalyst for the electrochemical determination of hydrogen peroxide on paper-based electrodes modified with carbon nanomaterials. H2O2 was oxidised at 0.275 V vs. Ag pseudoreference electrode. This system was adapted to develop a glucose sensor with glucose oxidase immobilized on the cellulose electrode. CoPc suspended nanoparticles acted as nanoenzyme mimicking peroxidase activity and were combined with different carbon nanomaterials to form hybrids with optimised catalytic performance. GO-CoPc paper-based electrodes yielded the best results with a linear range of ∼12 μM to 49 mM for H2O2 and 0.1 mM to 1 mM for glucose. Glucose was determined in physiological serum and juice samples with recoveries of 93.3 and 94.2% respectively. CoPc could replace HRP for the catalytic sensing of H2O2, without the need to be dissolved. This material can be used in situ in a simple protocol with other nanomaterials for electrode modification. The sensor described has the advantage of easy preparation, using the catalyst in colloidal form, long term stability, and versatility to be adapted to other low cost and disposable enzymatic systems.

Paper-based diagnostic devices have been widely applied to assess the presence and status of a variety of clinical diseases by analyzing samples such as urine or blood. Due to their low cost, user-friendliness, and convenience, they have been used as point-of-care (POC) devices in countries lacking resources or energy. Despite wide-ranging research and implementation, paper-based devices have not previously been developed for wound analysis. Here, we discuss the successful development of such a tool to facilitate simple and rapid wound status assessment. The purpose of this study was to develop a paper-based elastase detection device (PEDD) for clinical wound assessment that specifically examines human neutrophil elastase (HNE), one of the most abundant serine proteases found in chronic wounds. The first step in this study was an examination of different paper substrate types (i.e., chromatography paper and filter paper) to determine which provided the best protease immobilization and colorimetric response. We then used a wax printing approach to create hydrophobic and hydrophilic regions and designated test zones created on both chromatography and filter papers. This allowed us to physically immobilize both substrate and protease within the desired test zone regions. This PEDD which demonstrated good sensitivity (0.631 μg mL-1, in a wound fluid system) can be used to monitor protease activity expressed in wounds. After developing this device, we examined samples from 9 patients presenting a total of 7 acute and 4 chronic wounds to determine wound HNE concentration. We believe that this study may be widely applicable in both academic and commercial sciences, including the development of practical POC detection devices.

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng μL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.

In the present article we report a new hybrid microfluidic device (hyFlow) comprising a disposable paper electrode and a three-dimensional (3D) printed plastic chip for the electrochemical detection of a magnetic bead-silver nanoparticle (MB-AgNP) bioconjugate. This hybrid device evolved due to the difficulty of incorporating micron-scale MBs into paper-only fluidic devices. Specifically, paper fluidic devices can entrap MB-containing conjugates within their cellulose or nitrocellulose fiber matrix. The hyFlow system was designed to minimize such issues and transport MB conjugates more efficiently to the electrochemical detection zone of the device. The hyFlow system retains the benefit of fluid transport by pressure-driven flow, however, no pump is required for its operation. The hyFlow device is capable of detecting either pre-formed MB-AgNP conjugates or conjugates formed in-situ formation. The detection limit of AgNPs using this device is 12 pM, which represents just 22 AgNPs per MB.

Coliforms are one of the most common families of bacteria responsible for water contamination. Certain coliform strains can be extremely toxic, and even fatal if consumed. Current technologies for coliform detection are expensive, require multiple complicated steps, and can take up to 24 hours to produce accurate results. Recently, open-channel, paper-based microfluidic devices have become popular for rapid, inexpensive, and accurate bioassays. In this work, we have created an integrated microfluidic coliform lysis and detection device by fabricating customizable omniphilic regions via direct printing of omniphilic channels on an omniphobic, fluorinated paper. This paper-based device is the first of its kind to demonstrate successful cell lysing on-chip, as it can allow for the flow and control of both high and low surface tension liquids, including different cell lysing agents. The fabricated microfluidic device was able to successfully detect E. coli, via the presence of the coliform-specific enzyme, β-galactosidase, at a concentration as low as ~10(4) CFU/mL. Further, E. coli at an initial concentration of 1 CFU mL(−1) could be detected after only 6 hours of incubation. We believe that these devices can be readily utilized for real world E. coli contamination detection in multiple applications, including food and water safety.

As enzymes in the outer membrane of the mitochondrion, monoamine oxidases (MAOs) can catalyze the oxidative deamination of monoamines in the human body. According to different substrates, MAOs can be divided into MAO-A and MAO-B. The imbalance of the MAO-A is associated with neurological degeneration, while excess MAO-B activity is closely connected with Parkinson's disease (PD) and Alzheimer's disease (AD); therefore, detection of MAOs is of great significance for the diagnosis and treatment of these diseases. This work reports the multiplexed detection of MAO-A and MAO-B using paper-based devices based on chemiluminescence (CL). The detection limits were 5.01 pg/mL for MAO-A and 8.50 pg/mL for MAO-B in human serum. In addition, we used paper-based devices to detect MAOs in human cells and tissue samples and found that the results of paper-based detection and Western blotting (WB) showed the same trend. While only one antibody can be incubated on the same membrane by WB, multiple antibodies incubated on the same paper enabled simultaneous detection of MAO-A and MAO-B by paper-based devices. The paper-based assay could be used for preliminary early screening of clinical samples for MAOs and can be extended as an alternative to WB for multiplexed detection of various proteins in disease cell or tissue samples.

This study aimed to design and fabricate planar constricted–expanded structures that are integrated into paper-based channels in order to enhance their chaotic advection and improve their mixing performance. Chromatography papers were used to print paper-based microfluidics using a solid-wax printer. Three different constricted–expanded structures—i.e., zigzag, crossed, and curved channels—were designed in order to evaluate their mixing performance in comparison with that of straight channels. A numerical simulation was performed in order to investigate the mixing mechanism, and to examine the ways in which the planar constricted–expanded structures affected the flow patterns. The experimental and numerical results indicated that the proposed structures can successfully mix confluents. The experimental results revealed that the mixing indices (σ) rose from the initial 20.1% (unmixed) to 34.5%, 84.3%, 87.3%, and 92.4% for the straight, zigzag, curved, and cross-shaped channels, respectively. In addition, the numerical calculations showed a reasonable agreement with the experimental results, with a variation in the range of 1.0–11.0%. In future, we hope that the proposed passive paper-based mixers will be a crucial component in the application of paper-based microfluidic devices.

[EN] Nowadays, miniaturization and portability are crucial characteristics that need to be considered for the development of water monitoring systems. In particular, the use of handheld technology, including microfluidics, is exponentially expanding due to its versatility, reduction of reagents and minimization of waste, fast analysis times and portability. Here, a hybrid handheld miniaturized polymer platform with a paper-based microfluidic device was developed for the simultaneous detection of nitrite and nitrate in real samples from both, fresh and seawaters. The platform contains an ionogel-based colorimetric sensor for nitrite detection and a paper-based microfluidic device for the in situ conversion of nitrate to nitrite. The platform was fully characterized in terms of its viability as a portable, cheap and quick pollutant detector at the point of need. The calibration was carried out by multivariate analysis of the color of the sensing areas obtained from a taken picture of the device. The limits of detection and quantification, for nitrite were 0.47 and 0.68mgL-1, while for nitrate were 2.3 and 3.4mgL-1, found to be within the limits allowed by the environmental authorities, for these two pollutants. Finally, the platform was validated with real water samples, demonstrating its potential to monitor nitrite and nitrate concentrations on-site as a first surveillance step before performing extensive analysis. This project has received funding from the European Union Seventh Framework Programme (FP7) for Research, Technological Development and Demonstration under grant agreement no. 604241. The funding support from Gobierno de España, Ministerio de Ciencia y Educación de España” under grant PID2020-120313 GB-I00/AIE/10.13039/501100011033, and Gobierno Vasco Dpto. Educación for the consolidation of the research groups (IT1271-19) are also acknowledged. RC-C acknowledges funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 778001. Special thanks to (SGIker) of the University of the Basque Country (UPV/EHU). FB-L and LB-D acknowledge the “Red de Microfluídica Española” RED2018-102829-T.

In this paper, the paper cups were used as the research objects, and the machine vision detection technology was combined with different image processing techniques to investigate a non-contact optical automatic detection system to identify the defects of the manufactured paper cups. The combined ring light was used as the light source, an infrared (IR) LED matrix panel was used to provide the IR light to constantly highlight the outer edges of the detected objects, and a multi-grid pixel array was used as the image sensor. The image processing techniques, including the Gaussian filter, Sobel operator, Binarization process, and connected component, were used to enhance the inspection and recognition of the defects existing in the produced paper cups. There were three different detection processes for paper cups, which were divided into internal, external, and bottom image acquisition processes. The present study demonstrated that all the detection processes could clearly detect the surface defect features of the manufactured paper cups, such as dirt, burrs, holes, and uneven thickness. Our study also revealed that the average time for the investigated Automatic Optical Detection to detect the defects on the paper cups was only 0.3 s.

Nowadays, in the clinical, pharmaceutical, and environmental sectors, the development of facile and sensitive analytical methods and/or innovative devices for the follow-up and detection of antibiotics and pharmaceutical formulations, in general, are urgently needed and still challenging. This work declared three vital applications for broad-spectrum nitrofurantoin (macrofuran) antibiotic detection and quantification: A colorimetric method, a coated paper strip-based nano-lanthanum complex prototype and fabrication of smart electronic color sensor device-based coated paper strips. The colorimetric method showed a significant response upon increasing the concentration of the nitrofurantoin in a range between (1.0–100.0 ng/mL) via a visual color change from orange-yellow to red colors degree with detection and quantification limits of 0.175 and 0.53 ng/mL, respectively, whereas the nano-lanthanum complex coated paper strip prototype showed qualitative on-site sensing for nitrofurantoin via naked eye color changes which can be detected anywhere. Moreover, a smart prototype for detecting macrofuran in the means of paper color change in the RGB color component extraction algorithm and the grayscale projection value processing algorithm was fabricated. The change in RGB color on the coated paper strip was detected using an electronic color sensor device. The developed colorimetric method, coated paper strip, and the electronic color sensor device prototype exhibited fast, simple, costless, and selective towards macrofuran over the competing analyzed. As well as, showed good applicability in the different real samples spiked with different concentrations of macrofuran. Graphical abstract

Preservation of paper-based historical artifacts against deterioration due to the presence of bacteria and fungi colonies has been one of the major issues for the importance of protecting the cultural heritage of humankind. Advances in nanotechnology have enabled the implementation of nanomaterials for this purpose. In this work, calcium/chitosan nanoparticles (Ca/CS NPs) were prepared and well-characterized to investigate their potential as a novel approach for preserving paper-based documents. Following the fundamental characterizations, it was found that Ca/CS NPs are spherical nanoparticles with ~65 nm average size and homogenous dispersion (PdI: 0.2). Besides, minimum inhibition concentration results revealed that Ca/CS NPs show a superior antimicrobial effect against specific bacteria and fungi strains commonly found on paper documents compared to the effect of bare chitosan nanoparticles (CS NPs). After the deposition of Ca/CS NPs onto the paper the pH level was increased and stabilized, and only a limited amount of microbial colony formation was observed for up to 20 days. Moreover, molecular docking analysis provided a better insight into the antibacterial and antifungal activities of these nanoparticles. The antimicrobial activity of CS NPs and Ca/CS NPs was investigated through their interactions with E. coli DNA gyrase B and C. albicans dihydrofolate reductase. The binding modes and all possible interactions of active sites were confirmed by in silico molecular docking method. Collectively, our findings revealed that the formulated Ca/CS NPs are promising candidates for preserving paper documents.

Field-amplified stacking (FAS) is a commonly used method for enhancing the sensitivity of charged species from low conductive media in capillary electrophoresis. FAS also showed significant sensitivity enhancement effect on a uniform paper fluidic channel by proper design of the electrolyte. In this paper, a novel method of introducing electric field gradient is proposed by geometry design of a 2D paper fluidic channel, and field amplification effect was successfully demonstrated with reduced requirement on the sample's conductivity. Sensitive colorimetric detection of microalbuminuria (MAU) from urine samples was demonstrated by mobile phone camera. Experimental results showed that, with active electric field motivation, up to 93.5% of the loaded protein probe could be effectively transferred and stacked into a narrow band on the newly designed paper fluidic channel. A limit of detection (LOD) of 6.5 mg‧L−1 HSA was achieved with a dynamic range of 10–300 mg‧L−1 (linear in the range of 10–100 mg‧L−1, R2 = 0.991). Combined with selective staining of albumin with bromophenol blue (BPB), the established method was applied to the detection of MAU from clinical urine samples, and consistent results with that of the clinical method were obtained. With this paper-based analytical device (PAD), MAU from highly conductive urine samples can be directly loaded and detected without any pretreatment. This method provides a way to develop highly sensitive point-of-care test (POCT) for rapid screening of some diseases.

Lab-on-paper technologies, also known as paper-based analytical devices (PADs), have received increasing attention in the last years, and nowadays, their use has spread to virtually every application area, i.e., medical diagnostic, food safety, environmental monitoring, etc. Advantages inherent to on-field detection, which include avoiding sampling, sample preparation and conventional instrumentation in central labs, are undoubtedly driving many developments in this area. Heavy metals represent an important group of environmental pollutants that require strict controls due to the threat they pose to ecosystems and human health. In this overview, the development of PADs for Hg monitoring, which is considered the most toxic metal in the environment, is addressed. The main emphasis is placed on recognition elements (i.e., organic chromophores/fluorophores, plasmonic nanoparticles, inorganic quantum dots, carbon quantum dots, metal nanoclusters, etc.) employed to provide suitable selectivity and sensitivity. The performance of both microfluidic paper-based analytical devices and paper-based sensors using signal readout by colorimetry and luminescence will be discussed. MInisterio de Ciencia, Innovación y Universidades | Ref. RTI2018-093697-B-I00 Xunta de Galicia | Ref. ED481D-2021-021 Xunta de Galicia | Ref. ED431I 2020/04 European Commission | Ref. P.P0000421S 140.08

Paper-fluidic devices are a popular platform for point-of-care diagnostics due to their low cost, ease of use, and equipment-free detection of target molecules. They are limited, however, by their lack of sensitivity and inability to incorporate more complex processes, such as nucleic acid amplification or enzymatic signal enhancement. To address these limitations, various valves have previously been implemented in paper-fluidic devices to control fluid obstruction and release. However, incorporation of valves into new devices is a highly iterative, time-intensive process due to limited experimental data describing the microscale flow that drives the biophysical reactions in the assay. In this paper, we tested and modeled different geometries of thermally actuated valves to investigate how they can be more easily implemented in an LFIA with precise control of actuation time, flow rate, and flow pattern. We demonstrate that bulk flow measurements alone cannot estimate the highly variable microscale properties and effects on LFIA signal development. To further quantify the microfluidic properties of paper-fluidic devices, micro-particle image velocimetry was used to quantify fluorescent nanoparticle flow through the membranes and demonstrated divergent properties from bulk flow that may explain additional variability in LFIA signal generation. Altogether, we demonstrate that a more robust characterization of paper-fluidic devices can permit fine-tuning of parameters for precise automation of multi-step assays and inform analytical models for more efficient design.

With characters of low cost, portability, easy disposal, and high accuracy, as well as bulky reduced laboratory equipment, paper-based sensors are getting increasing attention for reliable indoor/outdoor onsite detection with nonexpert operation. They have become powerful analysis tools in trace detection with ultra-low detection limits and extremely high accuracy, resulting in their great popularity in medical detection, environmental inspection, and other applications. Herein, we summarize and generalize the recently reported paper-based sensors based on their application for mechanics, biomolecules, food safety, and environmental inspection. Based on the biological, physical, and chemical analytes-sensitive electrical or optical signals, extensive detections of a large number of factors such as humidity, pressure, nucleic acid, protein, sugar, biomarkers, metal ions, and organic/inorganic chemical substances have been reported via paper-based sensors. Challenges faced by the current paper-based sensors from the fundamental problems and practical applications are subsequently analyzed; thus, the future directions of paper-based sensors are specified for their rapid handheld testing.

A lignin-based PU coating was prepared for paper-based green packaging. Two representative diisocyanate were used to prepare the coatings. Due to the rigid aromatic, the physical properties of the TDI system reached the maximum below the lignin content of 40%. The HDI that contains flexible aliphatic chains alleviated the brittleness of coating, and it showed physical advantages when the lignin content was more than 50%. Owing to the high lignin content, the coating presented enhanced thermal stability. After coated with the lignin-based PU coatings, the dry tensile strength of coated paper was improved by 126%. Amazingly, the wet strength was increased from 0.31 to 12.6 MPa with an improvement nearly 40 times. Based on the coordination of lignin, Ag+ was introduced into the PU matrix, which imparted the coating with excellent antibacterial ability. The colony forming units of E. coli and S. aureus were both less than 1. However, no inhibition halo was observed, which indicated that the Ag was firmly anchored on the coating and the antibacterial ability is only available when the bacterial contact the coating surface. The lignin-based PU coating with favorable sustainability and properties shows great potential in paper-based green packaging fields.

An immunochromatographic test strip method for detecting A29 was established. The strips utilizing mAb-7C5 and 5D8 showed the best sensitivity with the lowest LOD, 50 pg mL−1 for A29.

Cellular viability measurements quantify decreased proliferation or increased cytotoxicity caused by drug candidates or potential environmental toxins.