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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.

Aβ42 is one of the most extensively studied blood and Cerebrospinal fluid (CSF) biomarkers for the diagnosis of symptomatic and prodromal Alzheimer’s disease (AD). Because of the heterogeneity and transient nature of Aβ42 oligomers (Aβ42Os), the development of technologies for dynamically detecting changes in the blood or CSF levels of Aβ42 monomers (Aβ42Ms) and Aβ42Os is essential for the accurate diagnosis of AD. The currently commonly used Aβ42 ELISA test kits usually mis-detected the elevated Aβ42Os, leading to incomplete analysis and underestimation of soluble Aβ42, resulting in a comprised performance in AD diagnosis. Herein, we developed a dual-target lateral flow immunoassay (dLFI) using anti-Aβ42 monoclonal antibodies 1F12 and 2C6 for the rapid and point-of-care detection of Aβ42Ms and Aβ42Os in blood samples within 30 min for AD diagnosis. By naked eye observation, the visual detection limit of Aβ42Ms or/and Aβ42Os in dLFI was 154 pg/mL. The test results for dLFI were similar to those observed in the enzyme-linked immunosorbent assay (ELISA). Therefore, this paper-based dLFI provides a practical and rapid method for the on-site detection of two biomarkers in blood or CSF samples without the need for additional expertise or equipment. Graphical Abstract

This work describes the development of a system that combines a derivatization protocol based on the Katritzky reaction with paper spray ionization mass spectrometry (PSI-MS) for the analysis of amino acid neurotransmitters in mouse brain tissues. The system is relatively simple, consisting of spraying the derivatization solution onto a mouse brain section mounted on a glass slide, applying a small volume of solvent to moisten the sample, pressing a triangular paper onto the sample surface to transfer the sample constituents to the paper surface, and using the paper as a substrate for PSI-MS analysis. The Katritzky reaction facilitated the ionization of the amino acids by reacting a pyrylium salt with the amino group of the analytes, forming very stable pyridinium cations, which greatly increased the sensitivity of the PSI-MS analysis. Most of the intensities of the amino acids modified by the Katritzky reaction were more than 10 times greater than the nonderivatized ones. The system was applied for the analysis of brain sections obtained from mice with Parkinson's disease, and the amino acids gamma-aminobutyric acid (GABA) and glycine (Gly), two compounds very well-known in studies of Parkinson's disease, were readily detected. The results suggest that the Katritzky reaction combined with PSI-MS might offer a significant advance in the knowledge on protocols that improve the sensitivity of detection of crucial biological compounds.

Recent studies have evaluated alternatives to the use of live animals in colony health monitoring. Currently, an alternative method that is suitable for all rack types and that has been verified to detect the infectious agents most commonly excluded from mouse colonies is unavailable. We compared the use of filter paper placed on the inside floor of mouse cages to the traditional use of sentinel mice in the detection of several prevalent murine pathogens including mouse hepatitis virus (MHV), murine norovirus (MNV), minute virus of mice (MVM), mouse parvovirus (MPV), Theiler murine encephalomyelitis virus (TMEV), Helicobacter spp., Syphacia obvelata, and Aspiculuris tetraptera. Experimental groups comprised 7 cages containing either 2 pieces of filter paper on the cage floor or 2 ICR sentinel mice. Soiled bedding from pet-store mice was transferred to the experimental cages weekly for 8 wk. At 1 and 2 mo after bedding transfer, the filter papers were evaluated by PCR and sentinel mice were tested by serology and fecal PCR. Filter papers detected all pathogens as effectively (MHV, MNV, MPV, MVM, TMEV S. obvelata, and A. tetraptera) or more effectively (Helicobacter spp.) than sentinel mice at both time points. Filter papers more readily detected pathogens with a high copy number per RT-PCR analysis than a low copy number. Helicobacter spp. were not detected by sentinel mice at either time point. These results indicate that the use of filter paper placed on the interior floor of empty mouse cages and exposed to soiled bedding is efficient in detecting bacteria, endoparasites, and most of the common mouse viruses included in an animal health monitoring program.

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

The present study delineates the preparation, characterization, and application of (3-Aminopropyl)triethoxysilane (APTES)/Caprine liver-derived extracellular matrix (CLECM) coated paper matrix for cell delivery. Here, we exploited positively charged surface of the paper matrix (as imparted by APTES derivatization) to improve the biological responses of the cells. Our results demonstrated that the functionalized paper matrixes favored the adhesion, growth, and proliferation of multiple cell types including normal, transformed, cancerous, and stem cells as compared to the pristine paper matrix. Upon implantation into the mice model, the developed paper matrix supported infiltration of the host cells and vasculature without showing any evidence of significant systemic toxicity. Moreover, the cells cultured on the paper matrix, when delivered to the CAM and mouse models, showed an enhanced vascular network around the substrate, thereby confirming its potential to deliver the cells in vivo. Together, the study confirms that the reported paper-based platform is easy to fabricate, cheap, portable and could efficiently be applied to cell delivery applications for either tissue repair or the development of humanized animal model.

Recently, paper has gained traction in the biotechnology research field due to its ability to be a substrate for 3D cell culture. In this work, we demonstrate the application of paper-based 3D cell culture for rapid and easy screening of the effect of natural compounds on melanin production. Whatman No. 1 filter paper was used as the substrate for B16F10 melanoma cell culture. The use of paper is beneficial for supporting the 3D structure of cells, which makes the result more reliable due to the similarity to in vivo conditions. Furthermore, paper is beneficial for melanin observation due to melanin's black color, which is easily in situ visualized after it is cultured on white paper. Matrigel was used to encapsulate cells before being pipetted onto the paper to prevent the passing of cells through paper pores. The intensity of melanin can then be observed with the naked eye and analyzed by scanning the paper. The analysis process took only 20 minutes, which is faster than that of the conventional absorbance spectroscopy, owing to the elimination of centrifugation, melanin solubilization, and the absorbance measurement step. The color intensity on the paper showed a direct proportion with increased α-MSH concentrations, confirming that the color on the paper was melanin. The 3D structure of cells was confirmed by using a scanning electron microscope. To demonstrate the application of the paper-based scaffold, paper-based 3D cell culture was used for screening the effects of Kojic acid and Arbutin on melanin production, which showed increased anti-melanogenesis effects with increased concentrations of natural compounds. High cell viability was observed over 120 hours. In conclusion, the developed paper-based scaffold can be used for screening the effect of natural compounds on melanin production, as a rapid and simple method with low cost.

(1) Background: The medical practice of acupuncture involves the insertion of a specialized stainless needle into a specific body point, often called an acupoint, to initiate a perceived phenomenon of de-qi sensation. Therefore, the term “de-qi” describes bodily sensations experienced by the recipient during acupuncture, which may include feelings of soreness, heaviness, fullness, numbness, and migration. However, while acupuncture treatments reportedly result in acupoint activation and an increased release of neurotransmitters or cytokines, detecting these substances released into the acupoint microenvironment is often missed or delayed in clinical and basic practice. (2) Methods: To address this situation, we employed a paper-based enzyme-linked immunosorbent assay method to examine acupoint environmental changes using minute volumes of easily accessible acupoint fluids. (3) Results: Our results indicated that while levels of adenosine triphosphate (ATP), interleukin-1β, interleukin-6, glutamate, substance P, and histamine were all increased in the experimental group following electroacupuncture (EA) treatment, contrary results were observed in the sham EA and transient receptor potential vanilloid 1 (Trpv1−/−) groups. Subsequently, TRPV1 and its associated molecules were augmented in mouse dorsal root ganglion, spinal cord, thalamus, and the somatosensory cortex, then examined by Western blotting and immunofluorescence techniques. Investigations revealed that these elevations were still unobserved in the sham EA or EA in the Trpv1−/− groups. Furthermore, results showed that while administering ATP could mimic EA function, it could be reversed by the ATP P2 receptor antagonist, suramin. (4) Conclusions: Our data provide novel information, indicating that changes in neurotransmitter and cytokine levels can offer insight into acupuncture mechanisms and clinical targeting.

Botulinum neurotoxins (BoNTs) produced by soil bacterium Clostridium botulinum are cause of botulism and listed as biohazard agents, thus rapid screening assays are needed for taking the correct countermeasures in a timely fashion. The gold standard method relies on the mouse lethality assay with a lengthy analysis time, i.e., 2–5 days, hindering the prompt management of food safety and medical diagnosis. Herein, we propose the first paper-based antibody-free sensor for reliable and rapid detection of BoNT/A and BoNT/C, exploiting their cleavage capability toward a synthetic peptide able to mimic the natural substrate SNAP-25. The peptide is labelled with the electroactive molecule methylene blue and immobilized on the paper-based electrode modified with gold nanoparticles. Because BoNT/A and BoNT/C can cleave the peptide with the removal of methylene blue from electrode surface, the presence of these neurotoxins in the sample leads to a signal decrease proportional to BoNT amount. The biosensor developed with the selected peptide and combined with smartphone assisted potentiostat is able to detect both BoNT/A and BoNT/C with a linearity up to 1 nM and a detection limit equal to 10 pM. The applicability of this biosensor was evaluated with spiked samples of orange juice, obtaining recovery values equal to 104 ± 6% and 98 ± 9% for 1 nM and 0.5 nM of BoNT/A, respectively.

Echocardiography is a reliable and reproducible method to assess non-invasively cardiac function in clinical and experimental research. Significant progress in the development of echocardiographic equipment and transducers has led to the successful translation of this methodology from humans to rodents, allowing for the scoring of disease severity and progression, testing of new drugs, and monitoring cardiac function in genetically modified or pharmacologically treated animals. However, as yet, there is no standardization in the procedure to acquire echocardiographic measurements in small animals. This position paper focuses on the appropriate acquisition and analysis of echocardiographic parameters in adult mice and rats, and provides reference values, representative images, and videos for the accurate and reproducible quantification of left ventricular function in healthy and pathological conditions. ispartof: CARDIOVASCULAR RESEARCH vol:117 issue:1 pages:43-59 ispartof: location:England status: published

SERS based immunoassays for point-of-care diagnostics are a promising tool to facilitate biomarker detection for early disease diagnosis and disease control. The technique is based on a sandwiched system in which antigen is first captured by a selective plasmonic paper substrate and then labeled by an extrinsic Raman label (ERL), consisting of a 60 nm gold nanoparticle (AuNP) functionalized with a mixed monolayer of detection antibody and 4-nitrobenezenethiol (NBT) as a Raman reporter molecule. Here, we report on the use of AuNP modified filter paper as a novel capture membrane in a vertical flow format. This vertical flow configuration affords reproducible flow of sample and label through the capture substrate to overcome diffusion limited kinetics and significantly reduced assay time. The filter paper was selected due to its affordability and availability, while the embedded AuNPs maximized plasmonic coupling with the ERLs and SERS enhancement. Additionally, the embedded AuNP served as a scaffold to immobilize capture antibody to specifically bind antigen. In this work, a SERS-based rapid vertical flow (SERS-RVF) immunoassay for detection of mouse IgG was developed to establish proof-of-principle. Optimization of assay conditions led to a limit of detection of 3 ng/mL, which is comparable to more traditional formats carried out in multi-well plates, and significantly reduced assay time to less than 2 min. Additionally, IgG was accurately quantified in normal mouse serum to validate the SERS-RVF assay for application to the analysis of biological samples. These results highlight the potential advantages of the SERS-RVF platform for point-of-need testing.

Rapid analysis of metabolites in biofluids is of great importance for disease diagnosis or new-born disease screening. Herein, we introduce an agarose hydrogel conditioning method to enhance the performance of paper spray ionization mass spectrometry. With facile and fast hydrogel conditioning, the signal intensity of therapeutic drugs spiked in urine was 5 to 15 fold higher than that in direct paper spray ionization mass spectrometry analysis. Consequently, the sensitivity of metabolites in urine was improved via hydrogel conditioning, resulting in 9 to 15 fold decrease in the possibility of detection (POD) levels. These results show that agarose hydrogel conditioning coupled with paper spray ionization mass spectrometry could serve as a facile ionization method for ambient mass spectrometry, which might be useful in fast screening of metabolites and therapeutic drugs in raw biofluids.

The growing socio-economic burden of chronic skin wounds requires the development of new automated and non-invasive analytical systems capable of wirelessly monitoring wound status. This work describes the low-cost fabrication of single-use, omniphobic paper-based smart bandages (OPSBs) designed to monitor the status of open chronic wounds and to detect the formation of pressure ulcers. OPSBs are lightweight, flexible, breathable, easy to apply, and disposable by burning. A reusable wearable potentiostat was fabricated to interface with the OPSB simply by attaching it to the back of the bandage. The wearable potentiostat and the OPSB can be used to simultaneously quantify pH and uric acid levels at the wound site, and wirelessly report wound status to the user or medical personnel. Additionally, the wearable potentiostat and the OPSBs can be used to detect, in an in-vivo mouse model, the formation of pressure ulcers even before the pressure-induced tissue damage becomes visible, using impedance spectroscopy. Our results demonstrate the feasibility of using inexpensive single-use OPSBs and a reusable, wearable potentiostat that can be easily sterilized and attached to a new OPSB during the dressing change, to provide long term wound progression data to guide treatment decisions.

Silver nanoparticles (AgNPs) have been widely used for sterilization due to their broad-spectrum bactericidal properties. However, there exist the problems of premature releasing and accumulative toxicity when free AgNPs are applied. This study proposed a one-pot hydrothermal strategy to synthesize carbon spheres immobilized silver nanoparticles (AgNPs@CS). The synthesis involves with silver ammonia solution as Ag precursor, and corn stalk as green reducing agent and carbon precursor. Furthermore, AgNPs@CS was anchored by cellulose nanofibers (CNF) to obtain the antibacterial composite paper. The obtained CNF/AgNPs@CS paper exhibited superior antibacterial properties against E. coli and S. aureus. Notably, the accumulative release rate of AgNPs from AgNPs@CS was 10.2% in 9 days, while that from CNF/AgNPs@CS paper was only 6.7% due to the anchoring effect of both CS and CNF, which was low for avoiding the cumulative toxicity problem. In addition, the mechanical and barrier properties of CNF/AgNPs@CS paper were also improved by 29.4% (tensile index), 2.7% (tear index), 7.4% (burst index), 10% (folding endurance), 0.8% (water vapor transmission) and 9.4% (oxygen transmission rate), respectively. Therefore, the composite paper has potential application as a medical antibacterial material.

This paper describes enzyme-linked immunosorbent assays (ELISAs) utilizing microfluidic thread/paper-based analytical devices (μTPAD), microfluidic fabric-based analytical devices (μFAD), and microfluidic thread-based analytical devices (μTAD). Here, the quantitative detection of biotinylated goat anti-mouse IgG (system one) and rabbit IgG (system two) antibodies via colorimetric analysis is detailed. In both systems, antibody is spotted on the detection site and subjected to a series of washes, addition of streptavidin-alkaline phosphatase (Strep-ALP) (system 1) or alkaline phosphatase (ALP)-conjugated secondary antibody (system 2), and colorimetric substrate. The devices are scanned and analyzed yielding a correlation between inverse yellow (or purple) intensity. For system one, a linear range of detection at low concentrations of streptavidin-alkaline phosphatase (Strep-ALP) was observed befire the enzyme reached a Vmax . At higher concentrations of Strep-ALP, saturation is achieved for both the μTPAD and μFAD devices. For system two, the IC50 values obtained for the non-trifurcated and trifurcated μTADs were determined to be 180.2 fmol/zone and 133.8 fmol/zone, respectively. The IC50 value was demonstrated to be 1034 fmol/zone and 208.6 fmol/zone for the μTPADs and μFADs, respectively. For all devices the lowest concentration of Strep-ALP or rabbit IgG used in the assay was 3.75 × 10-4 mg/mL and 0.7 fmol/zone, respectively. The development of this technology should further facilitate the use of these platforms for ELISA to detect and quantitate antibodies.

Bedding material is a critical component of the mouse environment and affects animal wellbeing and research integrity. Corn cob (CC) bedding has been a common bedding choice in research despite several potential negative aspects of its use. We investigated the use of compressed paper (CP) bedding as a refinement to CC bedding. CP bedding demonstrated greater total and immediate absorption, compared with CC bedding. CP-bedded cages had a reduced frequency of early cage changing prior to the Guide-recommended 2-wk interval for IVC; this reduction was proportional to room census. Intracage ammonia levels were lower in CP-bedded IVC compared with CC-bedded IVC, independent of the age, sex, and number of mice per cage. By contrast, ammonia levels were similar between CP-bedded and CC-bedded static cages. Collectively, these data support the use of CP bedding as a refinement for CC in ventilated mouse cages, in light of increased husbandry efficiency and its positive effect on the welfare of mice.

Spinocerebellar ataxias (SCAs) represent a large group of hereditary degenerative diseases of the nervous system, in particular the cerebellum, and other systems that manifest with a variety of progressive motor, cognitive, and behavioral deficits with the leading symptom of cerebellar ataxia. SCAs often lead to severe impairments of the patient's functioning, quality of life, and life expectancy. For SCAs, there are no proven effective pharmacotherapies that improve the symptoms or substantially delay disease progress, i.e., disease-modifying therapies. To study SCA pathogenesis and potential therapies, animal models have been widely used and are an essential part of pre-clinical research. They mainly include mice, but also other vertebrates and invertebrates. Each animal model has its strengths and weaknesses arising from model animal species, type of genetic manipulation, and similarity to human diseases. The types of murine and non-murine models of SCAs, their contribution to the investigation of SCA pathogenesis, pathological phenotype, and therapeutic approaches including their advantages and disadvantages are reviewed in this paper. There is a consensus among the panel of experts that (1) animal models represent valuable tools to improve our understanding of SCAs and discover and assess novel therapies for this group of neurological disorders characterized by diverse mechanisms and differential degenerative progressions, (2) thorough phenotypic assessment of individual animal models is required for studies addressing therapeutic approaches, (3) comparative studies are needed to bring pre-clinical research closer to clinical trials, and (4) mouse models complement cellular and invertebrate models which remain limited in terms of clinical translation for complex neurological disorders such as SCAs.

The frequent use of antibiotics against microbial infections may lead to the emergence of antibiotic resistant microbial strains. To overcome these microbial strains, we need to fabricate alternative materials which can handle them. It is for this reason, we have fabricated cellulose (CE) based filter paper (FP) composite scaffolds comprising of adsorbed chitosan (CS) and sliver (Ag) nanoparticles (NPs). The AgNPs are incorporated in the CS layer of the composite scaffold. Prior to evaluate the efficacy of the scaffolds against gram positive and gram negative bacterial strains, the scaffolds were characterized for the presence of the Ag NPs with field emission scanning electron microscope (FE-SEM), fourier transform infrared (FTIR) spectroscopy and x-ray diffractometer (XRD). These techniques confirmed the presence of Ag NPs in the composite scaffold. The biocompatibility of the scaffolds was assessed by subjecting pristine FP, CS adsorbed FP (CS-FP) and Ag loaded CS-FP (Ag-CS-FP) composite scaffolds to in vitro studies. From the data obtained, it was observed that NIH3T3 fibroblastic cells adhered and proliferated onto all the scaffolds. Furthermore, the scaffolds exhibited good antibacterial activity against both strains of bacteria. It is, therefore, concluded that these scaffolds could find potential application in biomedical field, particularly as a wound dressing agent.

Understanding how differences in animal husbandry practices affect the reproducibility of research results is critical. We sought to understand how different beddings might influence dietary obesity studies. We compared the effects of paper and corncob bedding on weight gain, metabolism, and gut microbiome (GM) of mice fed a high-fat diet (HFD) or a normal diet (ND) and evaluated effects on fecal and cecal microbiomes collected from these cohorts after euthanasia. Male C57BL/6J mice at 5 wk age were allowed to acclimate to the facility and the assigned bedding for one week before being placed on HFD or remaining on the ND for 12 wk. Fecal pellets and cecal samples were collected and frozen for batched 16S sequencing. Mice had similar body weight, visceral gonadal white adipose tissue (GWAT), subcutaneous inguinal white adipose tissue (IWAT), liver and spleen weights and metabolic changes regardless of the bedding type. Baseline microbiota differences were detected one week after bedding assignment. After 12 wk, the GM showed significant differences depending on both bedding and diet. The effects of the bedding were not significantly different between endpoint fecal and cecal GM, despite the inherent differences in microbiota in fecal and cecal samples. A correlation was detected between diet and the relative abundance of Bacteroidetes and Verrucomicrobia: Akkermansia. In conclusion, this study demonstrates the importance of considering bedding type when performing dietary experiments.

Publishing a primary research article is typically the result of a collaborative effort between a variety of researchers across differing career stages. STAR Protocols can complement a research article and empower authors to share the expertise they contributed to the larger study. In this Backstory, we interview members of the Gennarino lab, who published a Cell paper and four protocols, covering bioinformatics, culturing of patient-derived cell lines, neuroimaging from mouse brain sections and primary neurons, and mouse seizure recordings. For more information on the protocols related to this backstory, please refer to (Gennarino et al., 2018).

The 3D architecture of the uterus plays a key role in determine pregnancy outcome. A new paper in Development reveals the dynamic changes in luminal folding pattern that occur during the preimplantation period in mice. To hear more about the story, we caught up with first author Manoj Madhavan and corresponding author Ripla Arora, Assistant Professor at Michigan State University.

The combination of bedding substrate and nesting material within the microenvironment of mice is an important consideration for animal care programs in regard to optimizing animal wellbeing. We used 3 general or breeding mouse colonies in our institution to evaluate the effects of bedding substrate on nest building, breeding performance, and recognition of animal health concerns. A scoring system was developed to assess the incorporation of bedding into the nest cup base and walls (nest base incorporation, NBI) in a controlled study with mice bedded on either compressed paper (CP) or corncob (CC) bedding. Compared with CC cages, CP cages had higher NBI scores. To determine the influence of bedding type on the recognition of animal health concerns in an animal facility, cages bedded with CC followed by CP were evaluated for the overall frequency of health-concern reports during a 2-mo time frame for each bedding type in a single-subject A-B study design. The frequency of animal health-concern reports was similar in cages using CC or CP bedding. The animal health condition, rather than bedding type, was associated with the severity of the health problem at the initial report. Breeding performance was compared for 6 mo in matched CC and CP cages containing one of 13 genetically modified mouse lines. NBI scores were higher for breeders housed on CP compared with CC bedding. Monogamous breeder pairs housed on CP had significantly higher indexes of breeding performance (measured as the number of pups per dam per week on study) than did CC cages. This report supports the use of CP bedding in the mouse microenvironment to improve general wellbeing by supporting nesting behavior and reproductive performance without hindering the detection of animal health concerns.

The study was focused on assessment of potential health risks of paper-based food contact materials (FCMs) in a step-wise approach using three toxicological bioassays in vitro and chemical analyses of migrating contaminants. 3T3 NRU cytotoxicity test showed high sensitivity to detect basal toxicity of FCMs extracts and served as a first-line test for selection of samples for further testing. The reconstructed human intestine model EpiIntestinal showed more realistic tissue response than cell culture monolayer and higher resistance despite prolonged exposure to the selected 6 samples, i.e. negligible decrease of viability and intestinal penetration, nevertheless an increase of IL-8 after exposure to black printed sample extract. Yeast based assays identified weak agonistic/antagonostic activity to human androgen receptor of the black printed sample. In accordance with the biological effects, the targeted LC and GC analytical methods confirmed the presence of high amounts of phthalates, photoinitiators and PAHs that could justify the hazard of the black printed sample. Heavily printed uncoated FCMs are recognized not to be suitable for direct contact with food. The selected bioassays and chemical analyses might be useful tools to detect targeted biological effects of xenobiotics suspected to contribute to human exposure from food.

Three-dimensional visualization of cellular and subcellular-structures in histological-tissues is essential for understanding the complexities of biological-phenomena, especially with regards structural and spatial relationships and pathologlical-diagnosis. Recent advancements in tissue-clearing technology, such as Magnified Analysis of Proteome (MAP), have significantly improved our ability to study biological-structures in three-dimensional space; however, their wide applicability to a variety of tissues is limited by long incubation-times and a need for advanced imaging-systems that are not readily available in most-laboratories. Here, we present optimized MAP-based method for paper-thin samples, Paper-MAP, which allow for rapid clearing and subsequent imaging of three-dimensional sections derived from various tissues using conventional confocal-microscopy. Paper-MAP successfully clear tissues within 1-day, compared to the original-MAP, without significant differences in achieved optical-transparency. As a proof-of-concept, we investigated the vasculature and neuronal-networks of a variety of human and rodent tissues processed via Paper-MAP, in both healthy and diseased contexts, including Alzheimer’s disease and glioma.

Luminescent chemosensors for hydrogen sulphide (H2S) are of great interest because of the close association of H2S with our health. However, current probes for H2S detection have problems such as low sensitivity/selectivity, poor aqueous-solubility or interference from background fluorescence. This study reports an ultrasensitive and time-gated "switch on" probe for detection of H2S, and its application in test paper for visualization of exhaled H2S. The complex probe is synthesized with a luminescent Tb(3+) centre and three ligands of azido (-N3) substituted pyridine-2,6-dicarboxylic acid, giving the probe high hydrophilicity and relatively fast reaction dynamics with H2S because there are three -N3 groups in each molecule. The introduced -N3 group as a strong electron-withdrawing moiety effectively changes the energy level of ligand via intramolecular charge transfer (ICT), and thus breaks the energy transferring from ligand to lanthanide ion, resulting in quenching of Tb(3+) luminescence. On addition of H2S, the -N3 group can be reduced to an amine group to break the process of ICT, and the luminescence of Tb(3+) is recovered at a nanomolar sensitivity level. With a long lifetime of luminescence of Tb(3+) centre (1.9 ms), use of a time-gated technique effectively eliminates the background fluorescence by delaying fluorescence collection for 0.1 ms. The test paper imprinted by the complex probe ink can visualize clearly the trace H2S gas exhaled by mice.

Paper-based analytical devices (PADs) are widely used in point-of-care testing (POCT) as they are cost-effective, simple and straightforward. However, poor sensitivity hinders their use in detecting diseases with low abundance biomarkers. The poor detection limit of PADs is mainly attributed to the low concentration of analytes, and the complexity of biological fluid, leading to insufficient interactions between analytes and capture antibodies. This study aims to overcome these difficulties by developing a paper-based cationic isotachophoresis (ITP) approach for simultaneously detecting pico-molar levels of two essential cardiac protein markers: acidic troponin T (cTnT) and basic troponin I (cTnI) spiked into human serum samples. The approach utilizes 3-aminopropyltrimethoxysilane (APTMS) treated glass fiber papers with decreasing cross-sectional area assembled on a 3D printed cartridge device. Our results showed that in the presence of cTnT monoclonal antibody (mAb), fluorescently labeled cTnI and cTnT could be effectively enriched in cationic ITP. Each individual target was captured subsequently by a test line in the detection zone where the capture mAb was immobilized. Detailed analysis suggests that the technology is capable of simultaneous on-board depletion of abundant plasma proteins and enrichment of cTnI/cTnT by ~1300-fold with a sensitivity of 0.6 pmol/L for cTnT and a sensitivity of 1.5 pmol/L for cTnI in less than 6 min. The results demonstrate the potential of this technology for rapid, ultra-sensitive and cost-effective analysis of multiplex protein markers in clinical serum samples at point of care.

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.

Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis.