Search

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 (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.

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

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.

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.

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.

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.

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.

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.

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.

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%.

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.

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.

Binding protein scaffolds, such as rcSso7d, have been investigated for use in diagnostic tests; however, the functional performance of rcSso7d has not yet been studied in comparison to antibodies. Here, we assessed the analyte-binding capabilities of rcSso7d and antibodies on cellulose with samples in buffer and 100% human serum.

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.

Infections caused by foodborne microorganisms are a great threat to the global environment and public healthcare today. Thus, rapid, portable and sensitive assays that can realize the identification of foodborne bacteria are highly desired. In this study, a smart fluorescent and colorimetric dual-readout sensing system has been established for simple and rapid E. coli determination by utilizing the Cu2+-triggered oxidation of o-phenylenediamine (OPD). Initially, Cu2+ can oxidize OPD to OPDox, resulting in an orange-yellow fluorescence and visible pale-yellow color. However, E. coli can effectively reduce Cu2+ into Cu+, inhibiting the Cu2+-triggered oxidation of OPD to OPDox. Consequently, the introduction of E. coli can turn off both the fluorescence and the UV-vis absorbance signals of the OPD-Cu2+ system, illustrating an original mechanism for fluorescent and colorimetric dual-channel detection of E. coli. Moreover, a filter paper-based visual sensor was built and coupled with OPD-Cu2+ solution under the assistance of a UV lamp. The as-prepared sensor can detect E. coli quantitatively with the help of a typical smartphone color-scanning application (APP). Thus, this study offers a valid dual-mode assay for sensitive and on-site visible detection of E. coli, guaranteeing the reliability of the results and is more attractive for practical use. Graphical Abstract Schematic illustration of the smartphone-integrated sensing system for fluorescent and colorimetric dual-channel detection of E. coli based on the Cu2+-OPD system.

Zinc is an essential transition metal nutrient for bacterial survival and growth but may become toxic when present at elevated levels. The Gram-positive bacterial pathogen Streptococcus pneumoniae is sensitive to zinc poisoning, which results in growth inhibition and lower resistance to oxidative stress. Streptococcus pneumoniae has a relatively high manganese requirement, and zinc toxicity in this pathogen has been attributed to the coordination of Zn(II) at the Mn(II) site of the solute-binding protein (SBP) PsaA, which prevents Mn(II) uptake by the PsaABC transport system. In this work, we investigate the Zn(II)-binding properties of pneumococcal PsaA and staphylococcal MntC, a related SBP expressed by another Gram-positive bacterial pathogen, Staphylococcus aureus, which contributes to Mn(II) uptake. X-ray absorption spectroscopic studies demonstrate that both SBPs harbor Zn(II) sites best described as five-coordinate, and metal-binding studies in solution show that both SBPs bind Zn(II) reversibly with sub-nanomolar affinities. Moreover, both SBPs exhibit a strong thermodynamic preference for Zn(II) ions, which readily displace bound Mn(II) ions from these proteins. We also evaluate the Zn(II) competition between these SBPs and the human S100 protein calprotectin (CP, S100A8/S100A9 oligomer), an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP can sequester Zn(II) from PsaA and MntC, which facilitates Mn(II) binding to the SBPs. These results demonstrate that CP can inhibit Zn(II) poisoning of the SBPs and provide molecular insight into how S100 proteins may inadvertently benefit bacterial pathogens rather than the host.

Intracellular copper [Cu(I)] has been hypothesized to play role in the differentiation of the neurons. This necessitates understanding the role of Cu(I) not only in the neurons but also in the glia considering their anatomical proximity, contribution towards ion homeostasis, and neurodegeneration. In this study, we did a systematic investigation of the changes in the cellular copper homeostasis during neuronal and glial differentiation and the pathways triggered by them. Our study demonstrates increased mRNA for the plasma membrane copper transporter CTR1 leading to increased Cu(I) during the neuronal (PC-12) differentiation. ATP7A is retained in the trans-Golgi network (TGN) despite high Cu(I) demonstrating its utilization towards the neuronal differentiation. Intracellular copper triggers pathways essential for neurite generation and ERK1/2 activation during the neuronal differentiation. ERK1/2 activation also accompanies the differentiation of the foetal brain derived neuronal progenitor cells. The study demonstrates that ERK1/2 phosphorylation is essential for the viability of the neurons. In contrast, differentiated C-6 (glia) cells contain low intracellular copper and significant downregulation of the ERK1/2 phosphorylation demonstrating that ERK1/2 activation does not regulate the viability of the glia. But ATP7A shows vesicular localization despite low copper in the glia. In addition to the TGN, ATP7A localizes into RAB11 positive recycling endosomes in the glial neurites. Our study demonstrates the role of copper dependent ERK1/2 phosphorylation in the neuronal viability. Whereas glial differentiation largely involves sequestration of Cu(I) into the endosomes potentially (i) for ready release and (ii) rendering cytosolic copper unavailable for pathways like the ERK1/2 activation.

Nickel (Ni) is an essential yet toxic trace element. Although a cofactor for many metalloenzymes, nickel function and metabolism is not fully explored in eukaryotes. Molecular biology and metallomic methods were utilized to explore the new physiological functions of nickel in Saccharomyces cerevisiae. Here we showed that MTM1 knockout cells displayed much stronger nickel tolerance than wild-type cells and mitochondrial accumulations of Ni and Fe of mtm1Δ cells dramatically decreased compared to wild-type cells when exposed to excess nickel. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely attenuated and restored through Ni supplementation in media or total protein. SOD2 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain but was decreased by Ni supplementation. MTM1 knockout afforded resistance to excess nickel mediated through reactive oxygen species levels. Meanwhile, additional Ni showed no significant effect on the localization of Mtm1p. Our study reveals the MTM1 gene plays an important role in nickel homeostasis and identifies a novel function of nickel in promoting Sod2p activity in yeast cells.

Rising concern about the use of non-permitted colourants, in common food items such as dals and green vegetables sold in Indian markets, have led to a demand for low-cost point-of-use chemical analysis tools. Conventional food-analysis techniques involving tedious sample preparation protocols are not suited for in-field applications. Surface Enhanced Raman Spectroscopy (SERS) is an analytical technique that is well-suited for point-of-use chemical analysis with molecular level detection capability, which can also serve as a quality assurance tool for businesses. Effective and rapid signal collection from a large-area sample within a field-setting using disposable, low-cost SERS substrates is a key challenge in implementing such a solution. Herein, we demonstrate the use of inkjet-printed thin films comprising of robust nanostructured-silver as flexible, paper-based SERS (P-SERS) swabs for the direct detection of Metanil Yellow (MY) from toor dal (yellow split pigeon peas) samples and Malachite Green (MG) from green peas and green chillies. The macroscopic uniformity of these thin-films in combination with a portable Raman spectrometer equipped with orbital raster scanning (ORS™) technology for signal collection results in an unprecedented precision (RSD ∼ 1.6%) upon characterizing samples saturated with Rhodamine-6G (R6G), a standard Raman probe. As several food-cleansing products have appeared in the marketplace, the adulterant removal efficacy of some commercially available ‘washes’ as well as products such as ‘ozoniser’, which was determined by SERS characterization of swabs before and after use, is also reported.

A novel all-in-one paper-based sampling concept for mass spectrometric bottom-up protein analysis is here demonstrated in a chip format integrating instant immunocapture, protein reduction, - alkylation and tryptic digestion all in-device. Conventional laboratory grade filter paper was coated with the polymer 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) with a subsequent covalent immobilization of the monoclonal antibody E27 targeting the biomarker human chorionic gonadotropin (hCG). In-device protein reduction and alkylation was optimized with regards to reagent concentration and reaction pH. The sampling concept showed a high degree of performance between 10 and 1000 ng/mL (R2 > 0.99) by a five-point calibration curve sampled with hCG spiked to human serum samples and freshly collected whole blood samples, respectively. LOD (experimentally obtained at 100 pg/mL (2.64 pM/0.9 IU/L)) was demonstrated to be up to ten times lower with more than six times faster sample preparation than what has previously been reported for on-paper analysis of hCG in human serum samples.

Pseudoalteromonas (BB2-AT2) is a ubiquitous marine heterotroph, often associated with labile organic carbon sources in the ocean (e.g. phytoplankton blooms and sinking particles). Heterotrophs hydrolyze exported photosynthetic materials, components of the biological carbon pump, with the use of diverse metalloenzymes containing zinc (Zn), manganese (Mn), cobalt (Co), and nickel (Ni). Studies on the metal requirements and cytosolic utilization of metals for marine heterotrophs are scarce, despite their relevance to global carbon cycling. Here, we characterized the Zn, Mn, Co, and Ni metallome of BB2-AT2. We found that the Zn metallome is complex and cytosolic Zn is associated with numerous proteins for transcription (47.2% of the metallome, obtained from singular value decomposition of the metalloproteomic data), translation (33.5%), proteolysis (12.8%), and alkaline phosphatase activity (6.4%). Numerous proteolytic enzymes also appear to be putatively associated with Mn, and to a lesser extent, Co. Putative identification of the Ni-associated proteins, phosphoglucomutase and a protein in the cupin superfamily, provides new insights for Ni utilization in marine heterotrophs. BB2-AT2 relies on numerous transition metals for proteolytic and phosphatase activities, inferring an adaptative potential to metal limitation. Our field observations of increased alkaline phosphatase activity upon addition of Zn in field incubations suggest that such metal limitation operates in sinking particulate material collected from sediment traps. Taken together, this study improves our understanding of the Zn, Mn, Co, and Ni metallome of marine heterotrophic bacteria and provides novel and mechanistic frameworks for understanding the influence of nutrient limitation on biogeochemical cycling.

In this report, we investigate the toxicity of the ionophore thiomaltol (Htma) and Cu salts to melanoma. Divalent metal complexes of thiomaltol display toxicity against A375 melanoma cell culture resulting in a distinct apoptotic response at submicromolar concentrations, with toxicity of Cu(tma)2 > Zn(tma)2 >> Ni(tma)2. In metal-chelated media, Htma treatment shows little toxicity, but the combination with supplemental CuCl2, termed Cu/Htma treatment, results in toxicity that increases with suprastoichiometric concentrations of CuCl2 and correlates with the accumulation of intracellular copper. Electron microscopy and confocal laser scanning microscopy of Cu/Htma treated cells shows a rapid accumulation of copper within lysosomes over the course of hours, concurrent with the onset of apoptosis. A buildup of ubiquitinated proteins due to proteasome inhibition is seen on the same timescale and correlates with increases of copper without additional Htma.

To ensure safe drinking water, it is necessary to have a simple method by which the probable pollutants are detected at the point of distribution. Nitrite contamination in water near agricultural locations could be an environmental concern due to its deleterious effects on the human population. The development of a frugal paper-based microfluidic sensor could be desirable to achieve the societal objective of providing safe drinking water. This work describes the development of a facile and cost-effective microfluidic paper-based sensor for quantitative estimation of nitrite in aquatic environments. A simple punching machine was used for fabrication and rapid prototyping of paper-based sensors without the need of any specialized equipment or patterning techniques. A reusable 3D printed platform served as the support for simultaneous testing of multiple samples. The nitrite estimation was carried out with smartphone-assisted digital image acquisition and colorimetric analysis. Under optimized experimental conditions, the variation in average grayscale intensity with concentration of nitrite was linear in the range from 0.1 to 10 ppm. The limits of detection and quantitation were 0.12 ppm and 0.35 ppm respectively. The reproducibility, expressed as relative standard deviation was 1.31%. The selectivity of nitrite detection method was determined by performing interference studies with commonly existing co-ions in water, such as bicarbonates, chloride and sulphate. The paper-based sensor was successfully applied for estimation of nitrite in actual water samples and showed high recoveries in the range of 83.5-109%. The results were in good agreement with those obtained using spectrophotometry. The developed paper-based sensor method, by virtue of its simplicity, ease of fabrication and use, could be readily extended for detection of multiple analytes in resource-limited settings.

The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that nonspecific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high-affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase, and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione (GSH). In agreement, inclusion of GSH accelerated the reaction in a concentration-dependent manner. The implications of abundant high-affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with GSH in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.

Copper (Cu) is a key transition metal that is involved in many important biological processes in a cell. Cu is also utilized by the immune system to hamper pathogen growth during infection. However, genome-level knowledge on the mechanisms involved in adaptation to Cu stress is limited. Here, we report the results of a genome-wide reverse genetic screen for Cu-responsive phenotypes in Escherichia coli. Our screen has identified novel genes involved in adaptation to Cu stress in E. coli. We detected multiple genes involved in the biosynthesis and uptake of enterobactin, a siderophore utilized for high-affinity TonB-dependent acquisition of iron (Fe), as critical players in survival under Cu intoxication. We demonstrated the specificity of Cu-dependent killing by chelation of Cu and by genetic complementation of tonB. Notably, TonB is involved in protection from Cu in both laboratory and uropathogenic strains of E. coli. Cu stress leads to increased expression of the genes involved in Fe uptake, indicating that Fur regulon is derepressed during exposure to excess Cu. Trace element analyses revealed that Fe homeostasis is dysregulated during Cu stress. Taken together, our data supports a model in which lack of enterobactin-dependent Fe uptake leads to exacerbation of Cu toxicity, and elucidates the intricate connection between the homeostasis of Cu and Fe in a bacterial cell., Graphical Abstract Graphical AbstractCopper Stress and Iron Homeostasis in E. coli.

Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158 ± 57.7 femtograms (fg) to 65.7 ± 29.7 fg; calcium decreased from 216 ± 67.4 to 154.3 ± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4 ± 12.2 to 47.2 ± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

Bacillus cereus is one of the most common foodborne pathogens found in various kinds of staple foods such as rice and wheat. A rapid and accurate detection method for this pathogen is highly desirable for the sustainable production of relevant food products. While several classical and molecular-based detection methods are available for the identification of B. cereus, they suffered one or more limitations such as the requirement for a tedious and time-consuming process, less than ideal specificity, and the lack of portability. Herein, we developed the first paper-based sensing device that exhibits high species specificity with sufficiently low limit of detection for the visual detection of specific DNA sequences of B. cereus. The success is attributed to the strategic planning of fabrication in various dimensions including thorough bioinformatics search for highly specific genes, the use of the pyrrolidinyl peptide nucleic acid (PNA) probe whose selectivity advantage is well documented, and an effective PNA immobilization and DNA-binding visualization method with an internal cross-checking system for validating the results. Testing in rice matrices indicates that the sensor is capable of detecting and distinguishing B. cereus from other bacterial species. Hence, this paper-based sensor has potential to be adopted as a practical means to detect B. cereus in food industries.

Sprague Dawley rats were exposed to beryllium sulfate (BeSO4), and proteomic and bioinformatic techniques were applied to screen for differentially expressed proteins in their lung tissue and serum. A total of 12 coexpression modules were constructed for 18 samples with 2333 proteins. Four modules were found to have significant differences in the regulation of protein coexpression modules in the serum following exposure to BeSO4. A further three modules had significant differences in the regulation of protein coexpression modules in the lung tissues. Five modules with good correlation were obtained by calculating the gene significance and module membership values, whereas these module Hub proteins included: Hspbp1, Rps15a, Srsf2, Hadhb, Elmo3, Armt1, Rpl18, Afap1L1, Eif3d, Eif3c, and Rps3. The five proteins correlating highest with the Hub proteins in the lung tissue and serum samples were obtained using string analysis. KEGG and GO enrichment analyses showed that these proteins are mainly involved in ribosome formation, apoptosis, cell cycle regulation, and tumor necrosis factor regulation. By analyzing the biological functions of these proteins, proteins that can be used as biomarkers, such as Akt1, Prpf19, Cct2, and Rpl18, are finally obtained.

This report provides perspectives concerning dual roles of serum ferritin as a measure of both iron status and of inflammation. We suggest benefits of a lower range of serum ferritin as has occurred for total serum cholesterol and fasting blood glucose levels. Observations during a prospective randomized study using phlebotomy in patients with peripheral arterial disease (PAD) offered unique insights into dual roles of serum ferritin both as an iron status marker and acute phase reactant. Robust positive associations between serum ferritin, interleukin 6 [IL-6], tissue necrosis factor-alpha (TNF-a), and high sensitivity C-reactive protein (hsCRP) were discovered. Elevated serum ferritin and IL-6 levels associated with increased mortality and with reduced mortality at ferritin levels < 100 ng/mL. Epidemiologic studies demonstrate similar outcomes. Extremely elevated ferritin and IL-6 levels also occur in individuals with high mortality due to SARS-CoV-2 infection. Disordered iron metabolism reflected by a high range of serum ferritin level signals disease severity and outcomes. Based upon experimental and epidemiologic data, we suggest testing the hypotheses that optimal ferritin levels for cardiovascular mortality reduction range from 20—100 ng/mL with % transferrin levels from 20–50%, to ensure adequate iron status and that ferritin levels above 194 ng/mL associate with all-cause mortality in population cohorts., Graphical Abstract Graphical Abstract

Rapid diagnosis by using small, simple, and portable devices could represent one of the best strategies to limit the damage and contain the spread of viral, bacterial or protozoa diseases, principally when they can be transmitted by air and are highly contagious, as some respiratory viruses are. The presence of antibodies in blood or serum samples is not the best option for deciding when a person must be quarantined to stop transmission of disease, given that cured patients have antibodies, so the best diagnosis methods rely on the use of nucleic acid amplification procedures. Here we present a very simple device and detection principle, based on paper discs coupled to contactless conductivity (C4D) sensors, can provide fast and easy diagnostics that are needed when an epidemic outbreak develops. The paper device presented here solves one of the main drawbacks that nucleic acid amplification tests have when they are performed outside of central laboratories. As the device is sealed before amplification and integrally disposed in this way, amplimers release cannot occur, allowing repetitive testing in the physician’s practice, ambulances, or other places that are not prepared to avoid cross-contamination of new samples. The use of very low volume samples allows efficient reagent use and the development of low cost, simple, and disposable point-of-care diagnostic systems., Author summary In 2005, the World Health Organization (WHO) recognized Chagas Disease as a neglected tropical disease. Meanwhile the serological tests, recommended by WHO, can be performed for chronic disease diagnosis, the nucleic acid amplification tests must be performed for the detection of the acute phase of the disease. Although the existing laboratory diagnosis tests for Chagas Disease are sensitive and highly reproducible, they cannot be performed in rural, low infrastructure environments, where this disease prevails. In this sense, the use of simple and portable analytical devices may be able to offer an affordable solution to this problem, allowing fast sampling, diagnosis and treatment prescription in one simple and fast intervention, as the performed by short term medical missions. In this study we show for the first time a diagnosis test comprising low cost materials and employing a contactless and label-free conductivity detection system that is used to read the result of a nucleic acid amplification reaction. The test showed high sensitivity for Chagas Disease diagnosis showing the potential to be used in rural and low income places.

This paper describes the development of two microfluidic paper-based analytical devices (μPADs), one well-based and the other based on a lateral flow assay (LFA) configuration, to detect glucose via a colorimetric assay using the solid metal-organic framework (MOF) Zr-PCN-222(Fe), to encapsulate glucose oxidase (GOx). The well-based platform consisted of laminate sheets and multiple layers of wax-printed chromatography paper. Solutions of KI and glucose placed into the well flowed through the device and reacted with the GOx@MOF species sandwiched between the paper layers realizing a yellow-brown color. The LFA platform consisted of chromatography paper between parafilm and polyvinyl acetate (PVA) layers. GOx@MOFs spotted on the paper subjected to solutions of KI and glucose yielded a brown color. The devices were then dried, scanned, and analyzed yielding a correlation between average inverse yellow intensity and glucose concentrations. The development of these devices employing MOFs as biomimetic catalysts should further expand the applications of microfluidic technologies for sensors a variety of analytes.

Microfluidic paper-based devices (μPADs) and wearable devices have been highly studied to be used as diagnostic tools due to their advantages such as simplicity and ability to provide instrument-free fast results. Diseases such as periodontitis and diabetes mellitus can potentially be detected through these devices by the detection of important biomarkers. This study describes the development of μPADs through craft cutter printing for glucose and nitrite salivary diagnostics. In addition, the use of μPADs integrated into a mouthguard as a wearable sensor for glucose monitoring is also presented. μPADs were designed to contain two detection zones for glucose and nitrite assays and a sampling zone interconnected by microfluidic channels. Initially, the analytical performance of the proposed μPADs was investigated and it provided linear behavior (r2 ≥ 0.994) in the concentration ranges between 0 to 2.0 mmol L−1 and 0 to 400 μmol L−1 for glucose and nitrite, respectively. Under the optimized conditions, the limits of detection achieved for glucose and nitrite were 27 μmol L−1 and 7 μmol L−1, respectively. Human saliva samples were collected from healthy individuals and patients previously diagnosed with periodontitis or diabetes and then analyzed on the proposed μPADs. The results found using μPADs revealed higher glucose concentration values in saliva collected from patients diagnosed with diabetes mellitus and greater nitrite concentrations in saliva collected from patients diagnosed with periodontitis, as expected. The results obtained on μPADs did not differ statistically from those measured by spectrophotometry. With the aim of developing paper-based wearable sensors, μPADs were integrated, for the first time, into a silicone mouthguard using a 3D-printed holder. The proof of concept was successfully demonstrated through the monitoring of the glucose concentration in saliva after the ingestion of chocolate. According to the results reported herein, paper-based microfluidic devices offer great potential for salivary diagnostics, making their integration into a silicone mouthguard possible, generating simple, low-cost, instrument-free, and powerful wearable sensors.

Analytical strategies to ensure the quality and safety of food products without the need for skilled personnel are highly required. The simplicity of glucose strips for diabetes monitoring should be translated to the agri-food sector, ensuring easy evaluation of certain molecules and/or the freshness of a beverage/foodstuff. In addition to the well-known advantages that are characteristic of electroanalytical methods over other methods, paper-based materials are being used to further reduce the gap between complex laboratory testing and simple point-of-need testing. This article highlights some of the most recent advances in the development of paper-based electrochemical approaches for food surveillance, specifically focusing on the use of novel paper-based materials. Two examples are discussed: the development of a miniaturized biosensor realized on copy paper for the quantification of ethanol in commercial beers, and the measurement of ascorbic acid in food supplements within printed electronics supports. Paper-based materials have the potential to lower economic costs, simplify the operative tasks, and most importantly reduce waste generation. The continued combination of manufacturing methods and functional (smart) materials will facilitate the implementation of food analysis at the point of need.

Isocitrate dehydrogenase (IDH) I and II mutations in gliomas cause an abnormal accumulation of 2-hydroxyglutarate (2-HG) in these tumor cells. These mutations have potential prognostic value in that knowledge of the mutation status can lead to improved surgical resection. Information on mutation status obtained by immunohistochemistry or genomic analysis is not available during surgery. We report a rapid extraction nanoelectrospray ionization (nESI) method of determining 2-HG. This should allow the determination of IDH mutation status to be performed intraoperatively, within minutes, using a miniature mass spectrometer. This study demonstrates that the combination of tandem mass spectrometry with low-resolution mass spectrometry allows this analysis to be performed with confidence. Graphical Abstract.

Significance Populations living in remote rural communities would benefit from rapid, highly sensitive molecular, DNA-based diagnostics to inform the correct and timely treatment of infectious diseases. Such information is also becoming increasingly relevant in global efforts for disease elimination, where the testing of asymptomatic patients is now seen as being important for the identification of disease reservoirs. However, healthcare workers face practical and logistical problems in the implementation of such tests, which often involve complex instrumentation and centralized laboratories. Here we describe innovations in paper microfluidics that enable low-cost, multiplexed DNA-based diagnostics for malaria, delivered, in a first-in-human study, in schools in rural Uganda., Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in