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The existence of pesticide residues in the hydrosphere, biosphere, and anthroposphere can cause acute or chronic diseases and deteriorate the environment. Therefore, efficient detection of pesticide residues is of great significance to prevent food poisoning, control food pollution, and protect human lives by recognizing their distribution and concentration. Herein, a novel smartphone-coupled three-layered paper-based microfluidic chip is proposed as a facile platform to detect the pesticides. The stereoscopic capillary-driven fluid transport is enabled by the three-layered microfluidic chip configuration. The detection mechanism is based on the enzyme inhibition reaction and the chromatic reaction. The detection results are obtained by a smartphone and figured out by colorimetric quantitative analysis. Taking advantages of the above merits, we demonstrate the utilization of this smartphone-coupled three-layered paper-based microfluidic chip for the effective analysis of typical pesticides (profenofo and methomyl). The linear ranges of profenofo and methomyl are 0.27-2.1 μmol L

The prevalence of diabetes has increased over the past years. Therefore, developing minimally invasive, user-friendly and cost-effective glucose biosensors is necessary especially in low-income and developing countries. Cellulose paper-based analytical devices have attracted the attention of many researchers due to affordability, not requiring trained personnel, and complex equipment. This paper describes a microfluidic paper-based analytical device for the detection of glucose in tear with the naked eye. The paper-based biosensor fabricated by laser CO2, and GOx/HRP enzymatic solution coupled with TMB was utilized as reagents. A sample volume of 10 µl was needed for the biosensor operation and the results were observable within 5 minutes. To evaluate the device performance, color intensity-based and distance-based results were analyzed by ImageJ and Tracker. Distance-based results showed a linear behavior in the range of 0.1–0.6 mM with an R2 = 0.967 and LOD of 0.2 mM. The results could be perceived by the naked eye without any need to further equipment or trained personnel in a relatively short time (3–5 minutes). Moreover, glucose concentration could be obtained non-invasively by tears collected by this µPAD.

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/greenyellow/yellow/yellowred/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

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.

Bio-based additives received significant attention in pulp and paper properties improvement. For this, the most cited biochemical Cellulose Nano Fibrils (CNFs) and Cationic Starch (CS) were experimentally compared with the most declared synthetic chemical, Cationic Polyacrylamide (CPAM). SEM images showed better paper surface filling by the utilization of the chemicals. The three studied polymers, in solely or combination mechanism, improved mainly bagasse pulp and paper properties compared to the blank sample, except for pulp drainage, which decreased by CNFs to lower volumes presumably due to its intrinsic characteristics. Cationic polymers (CP) compared to CP/CNFs approaches increased pulp retention and drainage but decreased paper density and strengths. The best pulp retention and drainage achieved by CS followed by CPAM, while paper air persistency, density, and strength properties evaluated highest by CP/CNFs followed by CNFs. Generally, CS revealed a more significant improvement in pulp and paper properties than CPAM either with or without CNFs.

As a crucial biomarker for some diseases, the determination of cholesterol in human serum is of great significance for the diagnosis and prevention of these diseases. Hence, a portable cholesterol detection method is necessary for clinical and domestic applications. Here, a portable paper sensor was designed for cholesterol detection by modifying screen-printed electrode (SPE) with nanoporous gold (NPG). To achieve the reliable cholesterol detection, a synergistic strategy was proposed based on the oxidation of cholesterol by cholesterol oxidase (ChOx) and the reduction of oxidation product (H2O2) by NPG. Compared to existing electrochemical sensors, the resulting paper sensor exhibited a wider linear response in a range from 50 μM to 6 mM as well as a higher sensitivity of 32.68 μA mM−1 cm−2 with a lower detection limit of 8.36 μM. Moreover, the portable paper sensor presented strong anti-interference capability and stability in the detection of cholesterol in human serum, and the data detected by the portable paper sensor were consistent with that obtained by an automatic biochemical analyzer. These unique performances confirmed that the proposed paper sensor was a sensitive, reliable, and portable cholesterol detection method, making it a good choice for cholesterol detection.

Urinary tract infections (UTIs) make up a significant proportion of the global burden of disease in vulnerable groups and tend to substantially impair the quality of life of those affected, making timely detection of UTIs a priority for public health. However, economic and societal barriers drastically reduce accessibility of traditional lab-based testing methods for critical patient groups in low-resource areas, negatively affecting their overall healthcare outcomes. As a result, cellulose-based materials such as paper and thread have garnered significant interest among researchers as substrates for so-called frugal analytical devices which leverage the material’s portability and adaptability for facile and reproducible diagnoses of UTIs. Although the field may be only in its infancy, strategies aimed at commercial penetration can appreciably increase access to more healthcare options for at-risk people. In this review, we catalogue recent advances in devices that use cellulose-based materials as the primary housing or medium for UTI detection and chart out trends in the field. We also explore different modalities employed for detection, with particular emphasis on their ability to be ported onto discreet casings such as sanitary products. Graphical abstract

Carbon fiber paper (CFP) is commonly used as a proton exchange membrane in fuel cells due to its prominent areal electrosorption capacity, exceptional conductivity and excellent chemical stability. In this paper, we first explored the feasibility of carbon fiber paper as a specific paper substrate in paper spray ionization mass spectrometry (PSI-MS). The results demonstrated that CFPSI-MS combines the merits of PSI and carbon fiber ionization (CFI) and exhibits better performance of various compound analyses than either of these techniques alone. The application of CFP can highly enhance the signal stability and detection sensitivity of a diverse array of analytes, especially in negative ionization mode. The ion intensity of target analytes such as saccharides and flavonoids was improved 2-90-fold. Many nonpolar/low-polarity analytes, such as polycyclic aromatic hydrocarbons, which are difficult to ionize by traditional PSI-MS, were successfully detected by CFPSI-MS with a 2.5 kV high voltage. Moreover, CFPSI-MS presents high sensitivity in semiquantitative analysis, and the limits of detection (LODs) of cyclic adenosine monophosphate (CAMP), naringin and tivantinib in whole blood were improved 2-100-fold compared to those in traditional PSI-MS. In real sample analysis, CFPSI-MS also exhibits excellent capability in human breath analysis and blood metabolomic profiling.

High per- and polyfluorinated alkyl substance (PFAS) concentrations have been detected in agricultural soils in Southwest Germany. Discharges of PFAS-contaminated paper sludge and compost are suspected to be the cause of the contamination. Perfluorinated carboxylic acids (PFCAs) have been detected also in groundwater, drinking water, and plants in this area. Recently, previously unknown compounds have been identified by high-resolution mass spectrometry (HRMS). Major contaminants were polyfluorinated dialkylated phosphate esters (diPAPs) and N-ethyl perfluorooctane sulfonamide ethanol–based phosphate diester (diSAmPAP). In this study, HRMS screening for PFAS was applied to 14 soil samples from the contaminated area and 14 impregnated paper samples which were from a similar period than the contamination. The paper samples were characterized by diPAPs (from 4:2/6:2 to 12:2/12:2), fluorotelomer mercapto alkyl phosphates (FTMAPs; 6:2/6:2 to 10:2/10:2), and diSAmPAP. In soil samples, diPAPs and their transformation products (TPs) were the major contaminants, but also FTMAPs, diSAmPAP, and their TPs occurred. The distribution patterns of the carbon chain lengths of the precursor PFAS in soil samples were shown to resemble those in paper samples. This supports the hypothesis that paper sludge is a major source of contamination. The presence of major degradation products like PFCAs, FTSAs, or PFOS and their distribution of carbon chain lengths indicate the activity of biotic or abiotic degradation processes and selective leaching processes from the upper soil horizons. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03463-9.

Isothermal amplification strategies capable of rapid, inexpensive, and accurate nucleic acid detection provide new options for large-scale pathogen detection, disease diagnosis, and genotyping. Here we report a highly sensitive multicomponent XNA-based nucleic acid detection platform that combines analyte preamplification with X10-23-mediated catalysis to detect the viral pathogen responsible for COVID-19. The platform, termed RNA-Encoded Viral Nucleic Acid Analyte Reporter (REVEALR), functions with a detection limit of ≤20 aM (∼10 copies/μL) using conventional fluorescence and paper-based lateral flow readout modalities. With a total assay time of 1 h, REVEALR provides a convenient nucleic acid alternative to equivalent CRISPR-based approaches, which have become popular methods for SARS-CoV-2 detection. The assay shows no cross-reactivity for other in vitro transcribed respiratory viral RNAs and functions with perfect accuracy against COVID-19 patient-derived clinical samples.

Funding Information: The financial support of the Austrian Federal Ministry of Digital and Economic Affairs and the National Foundation for Research, Technology and Development , Austria, is gratefully acknowledged. We also thank the industrial partners Mondi, Canon Production Printing, Kelheim Fibres, and SIG Combibloc for their support. Publisher Copyright: © 2021 Copyright: Copyright 2021 Elsevier B.V., All rights reserved. Hydrophobization of cellulosic materials and particularly paper products is a commonly used procedure to render papers more resistant to water and moisture. Here, we explore the hydrophobization of unsized paper sheets via the gas phase. We employed three different compounds, namely palmitoyl chloride (PCl), trifluoroacetic anhydride/acetic anhydride (TFAA/Ac2O)) and hexamethyldisilazane (HMDS) which were vaporized and allowed to react with the paper sheets via the gas phase. All routes yielded hydrophobic papers with static water contact angles far above 90° and indicated the formation of covalent bonds. The PCl and TFAA approach negatively impacted the mechanical and optical properties of the paper leading to a decrease in tensile strength and yellowing of the sheets. The HMDS modified papers did not exhibit any differences regarding relevant paper technological parameters (mechanical properties, optical properties, porosity) compared to the non-modified sheets. XPS studies revealed that the HMDS modified samples have a rather low silicon content, pointing at the formation of submonolayers of trimethylsilyl groups on the fiber surfaces in the paper network. This was further investigated by penetration dynamic analysis using ultrasonication, which revealed that the whole fiber network has been homogeneously modified with the silyl groups and not only the very outer surface as for the PCl and the TFAA modified papers. This procedure yields a possibility to study the influence of hydrophobicity on paper sheets and their network properties without changing structural and mechanical paper parameters.

Several surveillance studies have reported significantly high level of patulin (PAT), mycotoxin in fruit juices suggesting the possible exposure to human. In vitro studies have showed that PAT can alter the permeability, ion transport and modulates tight junction of intestine. In real scenario, human can be exposed with low levels of PAT for longer duration through different fruits and their products. Hence, keeping this possibility in view, we conducted a study where normal intestinal cells were exposed with non-toxic levels of PAT for longer duration and found that PAT exposure causes cancer-like properties in normal intestinal cells. It is a well-known fact that cancer cells rewired their metabolism for cell growth and survival and metabolites closely depict the phenotypic properties of cells. Here, metabolomic study was performed in the PAT transformed and passage matched non-transformed cells using 1H HRMAS NMR. We have identified 12 significantly up-regulated metabolites, which, interestingly, were majorly amino acids, suggesting that PAT-induced pre-cancerous cells are involved in acquirement of nutrients for high protein turn-over. Furthermore, pathway analysis of metabolomics data indicated that aminoacyl tRNA biosynthesis, D-glutamate metabolism, glyoxylate and dicarboxylate metabolism and nitrogen metabolism were majorly hampered in PAT-induced pre-cancerous properties in normal intestinal cells.

Hydrogen sulfide (H2S) is known to participate in bacteria-induced inflammatory response in periodontal diseases. Therefore, it is necessary to quantify H2S produced by oral bacteria for diagnosis and treatment of oral diseases including halitosis and periodontal disease. In this study, we introduce a paper-based colorimetric assay for detecting bacterial H2S utilizing silver/Nafion/polyvinylpyrrolidone membrane and a 96-well microplate. This H2S-sensing paper showed a good sensitivity (8.27 blue channel intensity/μM H2S, R2 = 0.9996), which was higher than that of lead acetate paper (6.05 blue channel intensity/μM H2S, R2 = 0.9959). We analyzed the difference in H2S concentration released from four kinds of oral bacteria (Eikenella corrodens, Streptococcus sobrinus, Streptococcus mutans, and Lactobacillus casei). Finally, the H2S level in Eikenella corrodens while varying the concentration of cysteine and treatment time was quantified. This paper-based colorimetric assay can be utilized as a simple and effective tool for in vitro screening of H2S-producing ability of many bacteria as well as salivary H2S analysis.

In the current study, we have utilized semi-enclosed, leak-proof, microfluidic paper-based analytical devices (μPAD's) modified with isatin conjugated chitosan as specific colorimetric reagent for the detection of proline. Proline is one of the globally accepted stress biomarker in plants and also one of the prominent amino acid present in wine and some processed food. Quantification of proline is regularly required in agriculture field, food and wine industries. Specific interaction of isatin with proline, uniform film forming ability of chitosan which results in uniform coloration and the presence of leak-proof layer which prevent the diffusion of colorimetric reagent deeper resulted in enhancement of color signal intensity at the reaction zone were utilized. Further, the images of the μPAD's were captured using smartphone with 3D printed imaging box which houses smartphone and μPAD's. This platform utilizes smartphone flash for uniform illumination and ensures constant positioning of μPAD's to capture images. This greatly enhances the sensitivity and accuracy of our platform. Compared to previously published highly sensitive multi-layer, paper-based platform for detection of proline, current method has enhanced detection range (∼7 fold) and has comparable limit of detection of 23.75 μM. Moreover, the developed μPAD's platform has reduced optimum reaction temperature and time compared to previous work. The developed paper based platform was utilized for evaluation of proline content in young Arabidopsis plants which are subjected to water stress for 5 days. The devised paper-based methods have the potential to be applicable for the on-site evaluation of various stresses in plants.

Abiogenic metals Pb and Hg are highly toxic since chronic and/or acute exposure often leads to severe neuropathologies. Mn2+ is an essential metal ion but in excess can impair neuronal function. In this study, we address in vitro the interactions between neuronal calcium sensor 1 (NCS1) and divalent cations. Results showed that non-physiological ions (Pb2+ and Mn2+) bind to EF-hands in NCS1 with nanomolar affinity and lower equilibrium dissociation constant than the physiological Ca2+ ion. (Kd, Pb2+ = 7.0 ± 1.0 nM; Kd, Mn2+ = 34.0 ± 6.0 nM; K). Native ultra-high resolution mass spectrometry (FT-ICR MS) and trapped ion mobility spectrometry—mass spectrometry (nESI-TIMS-MS) studies provided the NCS1-metal complex compositions—up to four Ca2+ or Mn2+ ions and three Pb2+ ions (M⋅Pb1-3Ca1-3, M⋅Mn1-4Ca1-2, and M⋅Ca1-4) were observed in complex—and similarity across the mobility profiles suggests that the overall native structure is preserved regardless of the number and type of cations. However, the non-physiological metal ions (Pb2+, Mn2+, and Hg2+) binding to NCS1 leads to more efficient quenching of Trp emission and a decrease in W30 and W103 solvent exposure compared to the apo and Ca2+ bound form, although the secondary structural rearrangement and exposure of hydrophobic sites are analogous to those for Ca2+ bound protein. Only Pb2+ and Hg2+ binding to EF-hands leads to the NCS1 dimerization whereas Mn2+ bound NCS1 remains in the monomeric form, suggesting that other factors in addition to metal ion coordination, are required for protein dimerization.

Graphical abstract Enzyme-linked immunosorbent assay (ELISA) is widely used for the detection of disease biomarkers. However, it utilizes time-consuming procedures and expensive instruments, making it infeasible for point-of-care (POC) analysis especially in resource-limited settings. In this work, a multicolorimetric ELISA biosensor integrated on a paper/polymer hybrid microfluidic device was developed for rapid visual detection of disease biomarkers at point of care, without using costly equipment. This multicolormetric ELISA platform was built on multiple distinct color variants resulted from the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) and the etching of gold nanorods (AuNRs). The vivid color changes could be easily distinguished by the naked eye, and their red mean values allowed quantitative biomarker detection, without using any sophisticated instruments. When this multicolorimetric ELISA was integrated on a paper/polymer hybrid analytical device, it not only provided integrated processing and high portability but also enabled fast assays in about 50 min due to the unique advantages of paper/polymer hybrid devices. The limit of detection of 9.1 ng/μL of the hepatitis C virus core antigen, a biomarker for hepatitis C, was achieved using this multicolorimetric ELISA platform. This multicolor ELISA analytical device provides a new versatile, user-friendly, affordable, and portable immunosensing platform with high potential for on-site detections of various viruses, proteins, and biomarkers for low-resource settings such as at home, public venues, rural areas, and developing nations. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03359-8.

Due to their unique properties, paper-based biosensors have attracted attention as inexpensive devices for on-site analysis. To achieve fast and sensitive detection of analytes, immobilization of enzymes with high apparent activities on paper is highly desirable; however, this is challenging. Herein, we report an improved approach to attach a malathion degrading enzyme, PoOPHM9, on paper via an interlocking network of Pluronic F127 (PF127)–poly(acrylic acid)–enzyme conjugates. The addition of PF127 improved retention of enzymatic activity as the apparent kinetic constant Vmax of the immobilized enzyme increased two-fold compared with the paper prepared without PF127. The PF127–poly(acrylic acid)–PoOPHM9 papers provided rapid colorimetric detection of malathion at 0.1–50 mM. The detection was completed within 5 min using a smartphone and image analysis software. As a proof-of-concept, malathion-contaminated water, plant, and apple samples were analyzed with the papers successfully. This material is promising for on-site rapid analysis of malathion-contaminated samples.

Benzene metabolites (HQ and BQ) are toxic compounds and their presence in human cause alteration in cellular respiration and kidney damage. In the current study, Saccharomyces cerevisiae has been used as a model organism and acute exposure of hydroquinone (HQ) decreased cell growth and increased reactive oxygen species (ROS). The expression of apoptosis regulatory genes (YCA1, NUC1, YSP1 and AIF1) were increased with HQ exposure in the wild-type cells. HQ exposure in the wild-type cells altered both the phospholipid and neutral lipid levels. Phosphatidylcholine is a vital membrane lipid that has a vital role in membrane biogenesis and was increased significantly with HQ. The neutral lipid results were supported with lipid droplets data and mRNA expression study. The phospholipid knockouts (Kennedy pathway) accumulated neutral lipids via the CDP-DAG (cytidine-diphosphate-diacylglycerol) pathway genes both in the presence and absence of HQ.

Hypochlorous acid (HOCl) is generated at a high concentration by activated neutrophils at sites of inflammation in a myeloperoxidase catalyzed reaction. The increased and sustained production of HOCl at inflammatory sites may lead to tissue injury and this process is believed to play an important role in the progression of several diseases like chronic inflammation, atherosclerosis and some types of cancers. We have examined the effect of HOCl on human red blood cells (RBCs) under in vitro conditions. Treatment of RBC with different concentrations of HOCl (0.05–2.5 mM) at 37°C resulted in decreased activities of major antioxidant enzymes while the antioxidant power of RBC was weakened, as shown by lowered metal-reducing and free radical quenching ability of HOCl treated cells. RBC plasma membrane redox system was also inhibited suggesting membrane damage. The enzymes of glucose metabolism were inhibited indicating deranged energy metabolism. Electron microscopic images showed gross morphological changes in HOCl treated RBC. These results show that HOCl causes major alterations in the cellular antioxidant defense system and inhibition of glycolytic pathways, which increase the susceptibility of RBC to oxidative damage.

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. This journal is

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.

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.

There is growing demand for simple to operate, sensitive, on-site quantitative assays to investigate concentrations of drug molecules in pharmaceutical preparations for quality assurance. Here, we report on the development of two colorimetric analysis methods for the study the antibiotic doxycycline hyclate (DOX) and the nasal decongestant oxymetazoline hydrochloride (OXY), in solution as well as in their respective formulations. We compare a UV/vis spectrophotometry method with a color change recorded on a microfluidic paper-based analytical device (μPAD). Detection is based on the pharmaceutical compounds coupling with diazotized 4-aminoacetophenone (DAAP) under alkaline conditions to produce colored azo-dye products. These azo-compounds were monitored by absorbance at 425 nm for DOX and 521 nm for OXY, with linear calibration graphs in the concentration range of 0.5–35 mg L−1 (DOX) and 1.0–40 mg L−1 (OXY) and limits of detection of 0.24 mg L−1 (DOX) and 0.32 mg L−1 (OXY). For the μPAD method, color intensity was measured from photographs and a linear increase was observed at concentrations from above approximately 15 mg L−1 for both compounds and up to 35 mg L−1 for DOX and 40 mg L−1 for OXY. The developed methods were also applied to the formulated pharmaceuticals and no interference was found from the excipient. Thus, the paper-based device provides an inexpensive, simple alternative approach for use outside centralized laboratories with semi-quantitative capability.

There is a pressing need around the world to develop novel functional biodegradable materials to separate oil/water mixtures and emulsions completely. Recently, superhydrophilicity and underwater superoleophobicity materials have been attracted attention due to their high efficiency in oil/water separation. However, it is still a challenge to prepare materials that combine oil/water separation and water purification in an environment-friendly way. In this work, biodegradable starch-based nanospheres (SNPs) coated filter paper was prepared in a low-cost, simple, and environmentally friendly manner. The SNPs coating could not only help to change the wettability of the substrate material but also build the hierarchical micro and nano structures which are conducive to separation and purification process. After modification by coating SNPs, the filter paper exhibited excellent performance in a wide range of oil/water mixtures or emulsions separation and the wettability of the filter paper could be regulated by adjusting the pH value. The modified filter paper presented good recyclability after several separation process. Furthermore, the as-prepared filter paper could also remove water-soluble contaminants during the oil/water separation process, thus realizing to combine separation and purification process in one single step. This biodegradable starch-based separating material with good separation performance, stability and recyclability has significant application potential in practical separation and purification process.

In this work, we prepared novel cellulosic paper by incorporating tannin into the kraft pulp for potential application in active food packaging. The kraft pulp fibers were firstly periodate oxidized to obtain the dialdehyde cellulosic fibers, and then reacted with varied dosages of tannin to incorporate them into the fibers by covalent bondings between aldehyde groups on cellulose and active hydrogen on tannin. Handsheets were prepared using the tannin incorporated fibers through papermaking process and the properties were characterized. The percentage of tannin in the paper increased with the increase of the tannin dosage. FT-IR spectra confirmed the successful incorporation of tannin into the cellulosic fibers. It was found that paper after incorporation of tannin turned to be surface hydrophobic with contact angles higher than 90°, which may probably due to the covalent bonds between tannin and cellulose. The handsheets show high antioxidative and UV-shielding properties, which both increased with the increase of the tannin percentage in the paper. Water vapor transmission rate (WVTR) decreased after the incorporation of tannin, and this could facilitate its application in food packaging. The breaking length of tannin incorporated paper decreased insignificantly, less than 10% with the tannin percentage as high as 45%.

Starch and derivatives thereof have proven their usefulness in paper coating processes. Among these derivatives, cationic starch has been widely used in the paper industry as a flocculation, dispersion and ink fixing agent. In another context, nanoscale cellulosic materials have been shown to improve the strength, retention of fillers, the barrier properties of packaging paper products, and printing qualities. This review summarizes the recent studies on the general components used in paper coating, describes the conventional and alternative synthetic processes of cationic starches and nanocellulose, and deals with their current and potential applications in papermaking, focusing primarily on surface treatments. Moreover, environmental applications have been considered to expand the understanding and usefulness of these materials. Further research on modified polysaccharides is encouraged to replace, in a feasible way, petro-based components of coating formulations, and to provide paper surfaces with new properties.

Cancer is a major cause of mortality and morbidity worldwide. Detection and quantification of cancer biomarkers plays a critical role in cancer early diagnosis, screening, and treatment. Clinicians, particularly in developing countries, deal with high costs and limited resources for diagnostic systems. Using low-cost substrates to develop sensor devices could be very helpful. The interest in paper-based sensors with colorimetric detection increased exponentially in the last decade as they meet the criteria for point-of-care (PoC) devices. Cellulose and different nanomaterials have been used as substrate and colorimetric probes, respectively, for these types of devices in their different designs as spot tests, lateral-flow assays, dipsticks, and microfluidic paper-based devices (μPADs), offering low-cost and disposable devices. However, the main challenge with these devices is their low sensitivity and lack of efficiency in performing quantitative measurements. This review includes an overview of the use of paper for the development of sensing devices focusing on colorimetric detection and their application to cancer biomarkers. We highlight recent works reporting the use of paper in the development of colorimetric sensors for cancer biomarkers, such as proteins, nucleic acids, and others. Finally, we discuss the main advantages of these types of devices and highlight their major pitfalls., This research was funded by Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of UIDB/04469/2020 unit and through Mariana Carneiro PhD grant reference SFRH/BD/131959/2017.

[EN] Low cost and user-friendly paper microfluidic devices, combined with DNA-based biosensors with binding capacities for specific molecules, have been proposed for the developing of novel platforms that ease and speed-up the process of cell secretion monitoring. In this work, we present the first cellulose microfluidic paper-based analytical device for the single-step detection of cell secreted Vascular Endothelial Growth Factor through a self-reporting Structure Switching Signaling Aptamer. A three-part Structure Switching Signaling Aptamer was designed with an aptameric sequence specific for VEGF, which provides a quantifiable fluorescent signal through the displacement of a quencher upon VEGF recognition. The VEGF biosensor was integrated in cellulose paper, enabling the homogenous distribution of the sensor in the paper substrate and the detection of as low as 0.34ng of VEGF in 30min through fluorescence intensity analysis. As a proof-of-concept, the biosensor was incorporated in a microfluidic paper-based analytical device format containing a VEGF detection zone and a control zone, which was applied for the detection of cell secreted VEGF in the supernatant of mesenchymal stem cells culture plates, demonstrating its potential use in cell biology research. Authors acknowledge funding support from Basque Government, under Grupos Consolidados with Grant No. IT1271-19, from “Ministerio de Ciencia y Educación de España” under grant PID2020-120313GB-I00 /AIE /10.13039/501100011033, from the University of the Basque Country and the Spanish Government under the program “Margarita Salas” funded by “Unión Europea-Next Generation EU” . LBD and FBL personally acknowledge funds from the DNASURF (H2020-MSCA-RISE-778001) project. The authors thank for technical and human support provided by PhD Maite Alvarez from DNA Bank Service (SGIker) of the University of the Basque Country (UPV/EHU) and European funding (ERDF and ESF). Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN). Authors thank Dr. Alberto Gorrochategui from “Clínica Dermatológica Ercilla (Bilbao)” for providing the hair follicles from where hHF-MSCs were extracted. Finally, authors also wish to thank the technical assistance from the Nanopharmagene research group at the University of the Basque Country (UPV/EHU). FB-L and LBD acknowledge the “Red de Microfluídica Española” RED2018-102829-T.

Hemorrhagic transformation of ischemic stroke has devastating consequences, with high mortality and poor functional outcomes. Animal models of ischemic stroke also demonstrate the potential for hemorrhagic transformation, which complicates biochemical characterization, treatment studies, and hinders poststroke functional outcomes in affected subjects. The incidence of hemorrhagic transformation of ischemic stroke in animal model research is not commonly reported. The postmortem brain of such cases presents a complex milieu of biomarkers due to the presence of healthy cells, regions of varying degrees of ischemia, dead and dying cells, dysregulated metabolites, and blood components (especially reactive Fe species released from lysed erythrocytes). To improve the characterization of hemorrhage biomarkers on an ischemic stroke background, we have employed a combination of histology, X-ray fluorescence imaging (XFI), and Fourier transform infrared (FTIR) spectroscopic imaging to assess 122 photothrombotic (ischemic) stroke brains. Rapid freezing preserves brain biomarkers in situ and minimizes metabolic artifacts due to postmortem ischemia. Analysis revealed that 25% of the photothrombotic models had clear signs of hemorrhagic transformation. The XFI and FTIR metabolites provided a quantitative method to differentiate key metabolic regions in these models. Across all hemorrhage cases, it was possible to consistently differentiate otherwise healthy tissue from other metabolically distinct regions, including the ischemic infarct, the ischemic penumbra, blood vessels, sites of hemorrhage, and a region surrounding the hemorrhage core that contained elevated lipid oxidation. Chemical speciation of deposited Fe demonstrates the presence of heme-Fe and accumulation of ferritin.

LarC catalyzes the CTP-dependent insertion of nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), the final biosynthetic step for generating the nickel-pincer nucleotide (NPN) enzyme cofactor. In this study, we characterized a LarC homolog from Moorella thermoacetica (LarCMt) and characterized selected properties of the protein. We ruled out the hypothesis that enzyme inhibition by its product pyrophosphate accounts for its apparent single-turnover activity. Most notably, we identified a cytidinylylated-substrate intermediate that is formed during the reaction of LarCMt. Selected LarCMt variants with substitutions at the predicted CTP-binding site retained substantial amounts of activity, but exhibited greatly reduced levels of the CMP-P2TMN intermediate. In contrast, enhanced amounts of the CMP-P2TMN intermediate were generated when using LarCMt from cells grown on medium without supplemental nickel. On the basis of these results, we propose a functional role for CTP in the unprecedented nickel-insertase reaction during NPN biosynthesis.

Microfluidic paper-based analytical devices (μPADs) have been widely used in point-of-care testing owing to their simple operation, low volume of the sample required, and the lack of the need for an external force. To obtain accurate semi-quantitative or quantitative results, μPADs need to respond to the challenges posed by differences in reaction conditions. In this paper, multi-layer μPADs are fabricated by the imprinting method for the colorimetric detection of C-reactive protein (CRP). Different lighting conditions and shooting angles of scenes are simulated in image acquisition, and the detection-related performance of μPADs is improved by using a machine learning algorithm. The You Only Look Once (YOLO) model is used to identify the areas of reaction in μPADs. This model can observe an image only once to predict the objects present in it and their locations. The YOLO model trained in this study was able to identify all the reaction areas quickly without incurring any error. These reaction areas were categorized by classification algorithms to determine the risk level of CRP concentration. Multi-layer perceptron, convolutional neural network, and residual network algorithms were used for the classification tasks, where the latter yielded the highest accuracy of 96%. It has a promising application prospect in fast recognition and analysis of μPADs.

Propofol is a widely used intravenous anesthetic agent in sedation and general anesthesia. To improve the safety and maintain the depth of anesthesia, it is important to develop a rapid, sensitive, and reliable method to monitor the concentration of propofol in blood during anesthesia continuously. Here, we present a novel strategy based on paper spray ionization-mass spectrometry (PSI-MS) to detect propofol. Samples (in 10 μL) were mixed with methanol as protein precipitation solvent and 2,6-dimethylphenol as internal standard. Protein micro-precipitation was achieved with methanol by vortexing and centrifuging for 5 s each, and propofol was extracted to the supernatant. PSI-MS was performed in negative ionization mode, and MS signal lasted for 1 min. The analysis of a single sample was completed within 2 min. The area ratios of propofol to internal standard were calculated for quantification. Limit of detection of 5.5 ng mL−1 and limit of quantification of 18.2 ng mL−1 were achieved for propofol in whole blood. Calibration curve was linear in the range of 0.02–10 μg mL−1. The developed method was used successfully in monitoring the propofol concentration in 3 patients’ whole blood during anesthesia, showing its further application in controlling and feeding-back target concentration infusion.

Thiocarbamates are a major class of herbicides that were used extensively in the agricultural industry. Toxicological evaluation showed molinate caused reproductive impairment in male rats, whilst others produced behavioural effects at high doses. Rats dosed with molinate either as a single large oral dose of 100 mg/kg or as multiple doses of 50 mg/kg for 7 days produced inhibition of brain acetylcholinesterase (AChE). Molinate and other thiocarbamate herbicides undergo metabolism to form sulphoxides that can carbamoylate thiol’s such as glutathione and proteins. We have chemically synthesised the sulphoxide and sulphone metabolites of six thiocarbamate herbicides and examined their ability to inhibit rat brain and human red cell AChE in vitro. Parent thiocarbamates were inactive, whilst the sulphoxides produced inhibition with IC50’s in the 1–10 mM range, the sulphone metabolites were the most active with IC50’s for molinate, pebulate, EPTC and vernolate in the μM range. Inhibition was both time- and dose-dependent with biomolecular rate constants for the inhibition of the human red cell enzyme of 0.3 × 102 and 2.0 × 102 M−1 min−1 for molinate sulphoxide and sulphone, respectively. No recovery of enzyme activity, with either enzyme, was seen following dilution of the inhibitor to a concentration that does not inhibit the enzyme for up to 24 h at 25°C at pH 7.4. The metabolites of these thiocarbamate herbicides are rather poor inhibitors of AChE when compared to the organophosphorus ester, paraoxon or the monomethylcarbamate, eserine. Unlike eserine the inhibition produced by the thiocarbamates is irreversible.

Xenoestrogens exert antiandrogenic effects on the human androgen receptor. In the analytical field, such antagonists block the detection of testosterone and falsify results obtained by sum parameter assays. Currently, such agonistic versus antagonistic effects are not differentiated in complex mixtures. Oppositely acting hormonal effects present in products of everyday use can only be differentiated after tedious fractionation and isolation of the individual compounds along with subjection of each fraction/compound to the status quo bioassay testing. However, such long-lasting procedures are not suited for routine. Hence, we developed a fast bioanalytical tool that figures out agonists versus antagonists directly in complex mixtures. Exemplarily, 8 cosmetics and 15 thermal papers were analyzed. The determined antagonistic potentials of active compounds found were comparable to the ones of known antagonists (in reference shown for bisphenol A, 4-n-nonylphenol and four parabens). Relevant biological/chromatographic parameters such as cell viability, culture conditions, dose response curves, limits of biological detection/quantification and working range (shown for testosterone, dihydrotestosterone, nandrolone and trenbolone) were investigated to obtain the best sensitivity of the biological detection. The developed and validated method was newly termed reversed phase high-performance thin-layer chromatography planar yeast ant-/agonistic androgen screen (RP-HPTLC-pYAAS bioassay). Results were also compared with the RP-HPTLC-Aliivibrio fischeri bioassay (applied on RP plates for the first time). As proof-of-concept, the transfer to another bioassay (RP-HPTLC-pYES) was successfully demonstrated, analogously termed RP-HPTLC-pYAES bioassay detecting anti-/estrogens (exemplarily shown for evaluation of 4 pharmaceuticals used in breast cancer treatment). The new imaging concept provides (1) detection and differentiation of individual agonistic versus antagonistic effects in the bioprofiles, (2) bioanalytical quantification of their activity potential by scanning densitometry and (3) characterization of unknown bioactive compound zones by hyphenation to high-resolution mass spectrometry. Depending on the hormonal bioassay, 15 samples were analyzed in parallel within 5 h or 6 h (calculated as 20 or 24 min per sample). For the first time, piezoelectric spraying of the yeast cells was successfully demonstrated for the planar yeast–based bioassays.

To improve assessment of risks associated with pharmaceutical contamination of the environment, it is crucial to understand effects and mode of action of drugs in non-target species. The evidence is accumulating that species with well-conserved drug targets are prone to be at risk when exposed to pharmaceuticals. An interesting group of pharmaceuticals released into the environment is imidazoles, antifungal agents with inhibition of ergosterol synthesis as a primary mode of action in fungi. However, imidazoles have also been identified as competitive antagonists of calmodulin (CaM), a calcium-binding protein with phylogenetically conserved structure and function. Therefore, imidazoles would act as CaM inhibitors in various organisms, including those with limited capacity to synthesize sterols, such as arthropods. We hypothesized that effects observed in crustaceans exposed to imidazoles are related to the CaM inhibition and CaM-dependent nitric oxide (NO) synthesis. To test this hypothesis, we measured (i) CaM levels and its gene expression, (ii) NO accumulation and (iii) gene expression of NO synthase (NOS1 and NOS2), in the cladoceran Daphnia magna exposed to miconazole, a model imidazole drug. Whereas significantly increased CaM gene expression and its cellular allocation were observed, supporting the hypothesized mode of action, no changes occurred in either NO synthase expression or NO levels in the exposed animals. These findings suggest that CaM inhibition by miconazole leads to protein overexpression that compensates for the loss in the protein activity, with no measurable downstream effects on NO pathways. The inhibition of CaM in D. magna may have implications for effect assessment of exposure to mixtures of imidazoles in aquatic non-target species.

A laminated paper-based analytical device (LPAD) for histidine detection was fabricated from a chromatography filtration paper and laminate films. Histidine recognition was effected by histidyl-tRNA synthetase (HisRS), and its detection was signaled colorimetrically based on the molybdenum blue reaction. The analytical conditions and detectable concentration range of histidine were examined. The method provided selective quantification from 1 to 100 μM histidine. LPAD fabrication is considerably simple, involving only the craft-cutting of the chromatography filtration paper and laminate film, and is cost-effective.

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.

A sonication of graphite in polysaccharide (pullulan, chitosan and alginate) is one of the viable methods for the preparation of few-layer graphene. However, the effect of these adsorbed polysaccharides on the electrical performance of the produced graphene so far is not yet clear. In order to investigate the present effect of pullulan, chitosan and alginate on the electrical characteristic of resulted graphene, we have produced few-layer graphene using bath sonication of graphite in pullulan, chitosan and alginate medium for the application as electrical conductive ink in strain-sensitive. Data from the TEM reveals the appearance of folded few-layer graphene flakes after sonication for 150 min while the XPS data shows that the chitosan-based graphene possesses the highest carbon-oxygen ratio of 7.2 as compared to that of the pullulan and alginate-based graphene. By subjecting the produced graphene as the ink for paper-based strain sensor, we have discovered that the chitosan-graphene has the best resistivity value (1.66 × 10-3 Ω⋅cm) and demonstrate the highest sensitivity towards strain (GF: 18.6). This result interestingly implies the potential of the reported chitosan-based conductive ink as a strain-sensitive material for future food packaging.

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.

Nanolayer and nanolayer by nanolayer deposition of nanofilms of Ag and C using cold plasma in sequences (Ag, Ag-C, Ag-Ag-C), on porous paper, were used to design three disposable stochastic sensors for the assay of amyloid polypeptide from whole blood. The nanofilms were modified with α-cyclodextrin. The test developed using the nanofilm-based disposable stochastic sensors is used for early detection of diabetes. The wider linear concentration range (1.00 × 10−6–1.00 ng mL−1) and the lower limit of quantification (1.00 × 10−6ng mL−1) were obtained using the disposable stochastic sensors based on Ag-C and Ag-Ag-C, while the highest sensitivity (3.19 × 104 s−1/μg mL−1) was recorded using the disposable stochastic sensor based on Ag-Ag-C. The screening methods were fully validated using whole blood samples from confirmed patients, when the recovery of the islet amyloid polypeptide was higher than 98.00%.

A complete mathematical model for electromigration in paper-based analytical devices is derived, based on differential equations describing the motion of fluids by pressure sources and EOF, the transport of charged chemical species, and the electric potential distribution. The porous medium created by the cellulose fibers is considered like a network of tortuous capillaries and represented by macroscopic parameters following an effective medium approach. The equations are obtained starting from their open-channel counterparts, applying scaling laws and, where necessary, including additional terms. With this approach, effective parameters are derived, describing diffusion, mobility, and conductivity for porous media. While the foundations of these phenomena can be found in previous reports, here, all the contributions are analyzed systematically and provided in a comprehensive way. Moreover, a novel electrophoretically driven dispersive transport mechanism in porous materials is proposed. Results of the numerical implementation of the mathematical model are compared with experimental data, showing good agreement and supporting the validity of the proposed model. Finally, the model succeeds in simulating a challenging case of free-flow electrophoresis in paper, involving capillary flow and electrophoretic transport developed in a 2D geometry. 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: 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

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.

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.

Pathogen detection is crucial for human, animal, and environmental health; crop protection; and biosafety. Current culture-based methods have long turnaround times and lack sensitivity. Nucleic acid amplification tests offer high specificity and sensitivity. However, their cost and complexity remain a significant hurdle to their applications in resource-limited settings. Thus, point-of-need molecular diagnostic platforms that can be used by minimally trained personnel are needed. The nuclease protection assay (NPA) is a nucleic acid hybridization-based technique that does not rely on amplification, can be paired with other methods to improve specificity, and has the potential to be developed into a point-of-need device. In traditional NPAs, hybridization of an anti-sense probe to the target sequence is followed by single-strand nuclease digestion. The double-stranded target-probe hybrids are protected from nuclease digestion, precipitated, and visualized using autoradiography or other methods. We have developed a paper-based nuclease protection assay (PB-NPA) that can be implemented in field settings as the detection approach requires limited equipment and technical expertise. The PB-NPA uses a lateral flow format to capture the labeled target-probe hybrids onto a nitrocellulose membrane modified with an anti-label antibody. A colorimetric enzyme-substrate pair is used for signal visualization, producing a test line. The nuclease digestion of non-target and mismatched DNA provides high specificity while signal amplification with the reporter enzyme-substrate provides high sensitivity. We have also developed an on-chip sample pretreatment step utilizing chitosan-modified paper to eliminate possible interferents from the reaction and preconcentrate nucleic acids, thereby significantly reducing the need for auxiliary equipment. Graphical abstract.

The incompatibility of most organic solvents with acetylcholinesterase (AChE) inhibition assay normally limits pesticide extraction efficiency in sample pretreatment, which might cause false negatives in real world sample assessment. Herein, a novel method has been developed for an improved AChE inhibition assay via organic solvent extraction combined spontaneous in situ solvent evaporation on microfluidic paper-based analytical devices. Enzyme pre-immobilization procedure was spared and AChE was added to the system after sampling step until a complete in-situ solvent evaporation process was performed on chip. IC50 levels of the six investigated organophosphate and carbamate pesticides indicated a completely eliminated influence of solvents on AChE behavior with the new method. Most importantly, analytical performances were significantly improved in food sample measurements. Reduction in matrix effect was observed when acetonitrile was adopted for lettuce sample pretreatment instead of water. Studies on different pesticides suggested a remarkably decreased discrimination effect on recoveries from sample pretreatment with the new developed method. The recovery level for phoxim spiked head lettuce samples reached (107.5 ± 14.2) %, in comparison with that of (18.6 ± 1.4) % from water-based extraction. Spiked water and apple juice samples with carbaryl concentration of as low as 0.02 mg L−1 were also successfully recognized with the present method by visual detection. This is the first report on direct sampling of organic extracts for AChE inhibition assay on-chip and it might provide a new perspective for real world sample assessments involving bio-reagents.

Problems of stability were found for biomarkers of alcohol consumption: ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths), and fatty acid ethyl esters (FAEEs) in whole blood. The purpose of this study was to establish a method for the determination of these four kinds of ethanol's non-oxidative metabolites in dried blood spots (DBS) by liquid chromatography tandem mass spectrometry (LC-MS/MS), and to evaluate their stability. In this method, 50 μL of human blood was spotted onto a filter paper for DBS analysis. Samples were extracted by methanol, reconstituted by 2-propanol, and injected into the LC-MS/MS system. Limits of detection were among 0.5–50 ng/mL, and deviations in accuracy and precision were all lower than 15% at three quality control levels. The stability of the four kinds of ethanol non-oxidative metabolites in DBS was investigated during a 90-day range under three temperatures, −20 °C, 4 °C, and 25 °C. EtG and EtS showed a high level of stability in DBS in the 90-day range, regardless of the temperature. FAEEs were unstable after three days. PEths showed stability within 15 days in postmortem DBS and 60 days in antemortem DBS, respectively, at all temperatures.