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Cellulose nanopaper (CNP) has been considered as a promising material with great application potential in diverse fields. However, the hydrophilic nature of CNP significantly limits its practical application. In order to improve its water resistance, we demonstrate a facile approach to functionalize CNP by impregnating it with chitosan (CS), followed by in situ polymerization of polypyrrole (PPy). The results indicate that the obtained CNP/CS/PPy shows excellent water resistance with the wet tensile strength of up to 80 MPa, which is more than 10 times higher than that of the pure CNP. Intriguingly, new features (e.g., electrical conductivity, antibacterial activity, and so forth) are achieved at the same time. The functionalized CNP/CS/PPy shows a high conductivity of 6.5 S cm-1, which can be used for electromagnetic interference shielding applications with a high shielding performance of around 18 dB. In addition, the CNP/CS/PPy exhibits good antibacterial activity toward Staphylococcus aureus and Escherichia coli, with the bacterial reductions of 99.28 and 95.59%, respectively. Thus, this work provides a simple and versatile approach to functionalize CNP for achieving multifunctional properties.

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

Novel ways to track and verify items of a high value or security is an ever-present need. Taggants made from deoxyribonucleic acid (DNA) have several advantageous properties, such as high information density and robust synthesis; however, existing methods require laboratory techniques to verify, limiting applications. Here, we leverage DNA nanotechnology to create DNA taggants that can be validated in the field in seconds to minutes with a simple equipment. The system is driven by toehold-mediated strand-displacement reactions where matching oligonucleotide sequences drive the generation of a fluorescent signal through the potential energy of base pairing. By pooling different "input" oligonucleotide sequences in a taggant and spatially separating "reporter" oligonucleotide sequences on a paper ticket, unique, sequence-driven patterns emerge for different taggant formulations. Algorithmically generated oligonucleotide sequences show no crosstalk and ink-embedded taggants maintain activity for at least 99 days at 60 °C (equivalent to nearly 2 years at room temperature). The resulting fluorescent signals can be analyzed by the eye or a smartphone when paired with a UV flashlight and filtered glasses.

On-site detection of multiple small-molecule analytes in complex sample matrixes would be highly valuable for diverse biosensing applications. Paper electrochemical devices (PEDs) offer an especially appealing sensing platform for such applications due to their low cost, portability, and ease of use. Using oligonucleotide-based aptamers as biorecognition elements, we here for the first time have developed a simple, inexpensive procedure for the fabrication of aptamer-modified multiplex PEDs (mPEDs), which can robustly and specifically detect multiple small molecules in complex samples. These devices are prepared via an ambient vacuum filtration technique using carbon and metal nanomaterials that yields precisely patterned sensing architecture featuring a silver pseudo-reference electrode, a gold counter electrode, and three gold working electrodes. The devices are user-friendly, and the fabrication procedure is highly reproducible. Each working electrode can be readily modified with different aptamers for sensitive and accurate detection of multiple small-molecule analytes in a single sample within seconds. We further demonstrate that the addition of a PDMS chamber allows us to achieve detection in microliter volumes of biological samples. We believe this approach should be highly generalizable, and given the rapid development of small-molecule aptamers, we envision that facile on-site multi-analyte detection of diverse targets in a drop of sample should be readily achievable in the near future.

Flexible paper-based sensors may be applied in numerous fields, but this requires addressing their limitations related to poor thermal and water resistance, which results in low service life. Herein, we report a paper-based composite sensor composed of carboxylic carbon nanotubes (CCNTs) and poly-m-phenyleneisophthalamide (PMIA), fabricated by a facile papermaking process. The CCNT/PMIA composite sensor exhibits an ability to detect pressures generated by various human movements, attributed to the sensor's conductive network and the characteristic "mud-brick" microstructure. The sensor exhibits the capability to monitor human motions, such as bending of finger joints and elbow joints, speaking, blinking, and smiling, as well as temperature variations in the range of 30-90 °C. Such a capability to sensitively detect pressure can be realized at different applied frequencies, gradient sagittas, and multiple twists with a short response time (104 ms) even after being soaked in water, acid, and alkali solutions. Moreover, the sensor demonstrates excellent mechanical properties and hence can be folded up to 6000 times without failure, can bear 5 kg of load without breaking, and can be cycled 2000 times without energy loss, providing a great possibility for a long sensing life. Additionally, the composite sensor shows exceptional Joule heating performance, which can reach 242 °C in less than 15 s even when powered by a low input voltage (25 V). From the perspective of industrialization, low-cost and large-scale roll-to-roll production of the paper-based sensor can be achieved, with a formed length of thousands of meters, showing great potential for future industrial applications as a wearable smart sensor for detecting pressure and temperature, with the capability of electric heating.

The plasmonic properties of gold nanoparticles (AuNPs) are a promising tool to develop sensing alternatives to traditional, enzyme-catalyzed reactions. The need for sensing alternatives, especially in underdeveloped areas of the world, has given rise to the application of nonenzymatic sensing approaches paired with cellulosic substrates to biochemical analysis. Herein, we present three individual, low-step, wet-chemistry, colorimetric assays for three target biomarkers, namely, glucose, uric acid, and free cholesterol, relevant in diabetes control and their translation into paper-based assays and microfluidic platforms for multiplexed analysis. For glucose determination, an in situ AuNPs synthesis approach was applied into the developed μPAD, giving semiquantitative measures in the physiologically relevant range. For uric acid and cholesterol determination, modified AuNPs were used to functionalize paper with a gold-on-paper approach with the optical properties changing based on different aggregation degrees and hydrophobic properties of particles dependent on analyte concentration. These paper-based assays show sensitivity ranges and limits of detection compatible for target analyte level determination and detection limits comparable to those of similar enzymatic, colorimetric systems, relying only on plasmonic transduction without the need for enzymatic activity or other chromogenic substrates. The resulting paper-based assays were integrated into a single 3D, multiplex paper-based device using paper microfluidics, showing the capability for performing different colorimetric assays with distinct requirements in terms of sample flow and sample uptake in test zones using a combination of both horizontal and vertical flows inside the same device. The presented device allows for multiparametric, colorimetric measures of different metabolite levels from a single complex sample matrix drop using digital color analysis, showing the potential for development of low-cost, low-complexity tools for diagnostics toward the point-of-care.

Paper-based microfluidic devices are popular for their ability to automate multistep assays for chemical or biological sensing at a low cost, but the design of paper microfluidic networks has largely relied on experimental trial and error. A few mathematical models of flow through paper microfluidic devices have been developed and have succeeded in explaining experimental flow behavior. However, the reverse engineering problem of designing complex paper networks guided by appropriate mathematical models is largely unsolved. In this article, we demonstrate that a two-dimensional paper network (2DPN) designed to sequentially deliver three fluids to a test zone on the device can be computationally designed and experimentally implemented without experimental trial and error. This was accomplished by three new developments in modeling flow through paper networks: (i) coupling of the Richards equation of flow through porous media to the species transport equation, (ii) modeling flow through assemblies of multiple paper materials (test membrane and wicking pad), and (iii) incorporating limited-volume fluid sources. We demonstrate the application of this model in the optimal design of a paper-based signal-enhanced immunoassay for a malaria protein, PfHRP2. This work lays the foundation for the development of a computational design toolbox to aid in the design of paper microfluidic networks. ©

This paper introduces a novel potential-resolved paper-based biosensor for simultaneous detection of multiple microRNAs (miRNAs) (taking miRNA-155 and miRNA-126 as examples) based on the bipolar electrode (BPE) electrochemiluminescence (ECL) strategy. The proposed multiple-channel paper-based sensing microfluidic platform was prepared by wax-printing technology, screen-printing method, and in situ Au nanoparticles (AuNPs) growth to form hydrophilic areas, hydrophobic boundaries, waterproof electronic bridge, driving electrode regions, and parallel bipolar electrode regions. CdTe quantum dots (QDs)-H2 and Au@g-C3N4 nanosheets (NSs)-DNA1 were used as dual electrochemiluminescence signal probes, and carboxylated Fe3O4 magnetic nanoparticles existed as carriers. CdTe QDs-H2/S2O82- and Au@g-C3N4 NSs-DNA1/S2O82- could exhibit two strong and stable ECL emissions at a drive voltage of 9 and 12 V, respectively, which can be used as effective potential-resolved signal tags. In addition, the proposed three-dimensional (3D) DNA nanomachine model and the target miRNA cycle strategy were used to achieve double amplification of electrochemiluminescence intensity. More importantly, the combination of the bipolar electrode system and the potential-resolved multitarget electrochemiluminescence method can greatly reduce the spatial interference between substances. The prepared ECL biosensor showed a favorable linear response for the detection of miRNA-155 and miRNA-126 with relatively low detection limits of 5.7 and 4.2 fM, respectively. With excellent sensitivity, the strategy may provide an efficient method for clinical application, especially in detection of trace multiple targets.

Tuberculosis (TB), one of the deadliest infectious diseases, is caused by Mycobacterium tuberculosis (MTB) and remains a public health problem nowadays. Conventional MTB DNA detection methods require sophisticated infrastructure and well-trained personnel, which leads to increasing complexity and high cost for diagnostics and limits their wide accessibility in low-resource settings. To address these issues, we have developed a low-cost photothermal biosensing method for the quantitative genetic detection of pathogens such as MTB DNA on a paper hybrid device using a thermometer. First, DNA capture probes were simply immobilized on paper through a one-step surface modification process. After DNA sandwich hybridization, oligonucleotide-functionalized gold nanoparticles (AuNPs) were introduced on paper and then catalyzed the oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB). The produced oxidized TMB, acting as a strong photothermal agent, was used for the photothermal biosensing of MTB DNA under 808 nm laser irradiation. Under optimal conditions, the on-chip quantitative detection of the target DNA was readily achieved using an inexpensive thermometer as a signal recorder. This method does not require any expensive analytical instrumentation but can achieve higher sensitivity and there are no color interference issues, compared to conventional colorimetric methods. The method was further validated by detecting genomic DNA with high specificity. To the best of our knowledge, this is the first photothermal biosensing strategy for quantitative nucleic acid analysis on microfluidics using a thermometer, which brings fresh inspirations on the development of simple, low-cost, and miniaturized photothermal diagnostic platforms for quantitative detection of a variety of diseases at the point of care.

Development of selective and sensitive methods for on-site assay of tetracycline (TC) is of great significance for public health and food safety. Herein, a valid ratiometric fluorescence strategy using g-C3N4 nanosheets coupled with Eu3+ is designed for the assay of TC. In this strategy, both Eu3+ and g-C3N4 nanosheets serve as the recognition units of TC. The blue fluorescence of g-C3N4 nanosheets can be quenched by TC via the inner filter effect (IFE); meanwhile, the red fluorescence of Eu3+ can be enhanced by TC through the antenna effect (AE). The synergistic effect of AE and IFE caused by TC makes the developed ratiometric fluorescent sensor display a wide linear range for TC from 0.25 to 80 μM with a detection limit of 6.5 nM and a significant fluorescence color evolution from blue to red. Given its simplicity, free-label, excellent selectivity, high sensitivity, and recognizable color change, point-of-care testing systems, including smartphones and test paper-based assays, are developed for the visual sensing of TC. The integration of smartphones and test paper on a ratiometric fluorescent sensor greatly reduces the detection cost and time, providing a promising method for the qualitative discernment and semi-quantitative assay of TC on-site. Moreover, the potential application of the approach is also verified by detecting TC in milk.

As a global shift continues to occur in high burden diseases toward developing countries, the importance of medical diagnostics based on point-of-care testing (POCT) is rapidly increasing. However, most diagnostic tests that meet clinical standards rely on high-end analyzers in central hospitals. Here, we report the development of a simple, low-cost, mass-producible, highly sensitive/quantitative, automated, and robust paper/soluble polymer hybrid-based lateral flow biosensing platform, paired with a smartphone-based reader, for high-performance POCT. The testing architecture incorporates a polymeric barrier that programs/automates sequential reactions via a polymer dissolving mechanism. The smartphone-based reader with simple opto-mechanical parts offers a stable framework for accurate quantification. Analytical performance of this platform was evaluated by testing human cardiac troponin I (cTnI), a preferred biomarker for the diagnosis of myocardial infarction, in serum/plasma samples. Coupled with catalytic/colorimetric gold-ion amplification, this platform produced results within 20 min with a detection limit of 0.92 pg mL

Paper is emerging as a versatile platform for automated fluid handling with a broad range of applications in medical diagnostics and analytical chemistry. However, selectively controlling analyte transport in paper to achieve concentration or selection has been a challenge for functional analysis. Here, by combining paper-based microfluidics with acoustics, we present a rapid and powerful method to size dependently control movement of microparticles and cells in paper using surface acoustic waves (SAW). We demonstrate the unique capability of the paper-based SAW approach to trap and concentrate microparticles in paper and release them when required, achieving collection efficiency of over 98%. Given the correlation between collection efficiency, size, and applied power, the paper-based SAW approach is applied to isolate a mixture of microparticles (1.1, 3.2, and 5 μm in diameter) into different regions and also to trap and concentrate human prostate cancer PC3 cells at a predetermined site. This paper-based SAW approach provides opportunities to develop powerful and low-cost selection and analysis tools, capable of processing complex multicomponent samples, with potential applications in medical diagnostics.

Lithium carbonate is an effective medicine for the treatment of the bipolar disorder, but the concentration of lithium in the patient's blood must be frequently monitored because of its toxicity. To date, no colorimetric methods of lithium ion detection in whole blood without pretreatment have been reported. Here, we report a colorimetric paper-based device that allows point-of-care testing in one step. This device is composed of two paper-based elements linked to each other: a blood cell separation unit and a colorimetric detection unit. After a portion of whole blood has been placed on the end of the separation unit, plasma in the sample is automatically transported to the detection unit, which displays a diagnostic color. The key feature of this device is its simple, user-friendly operation. The limit of detection is 0.054 mM and the coefficient of variance is below 6.1%, which are comparable to those of conventional instruments using the same colorimetric reaction. Furthermore, we achieved high recovery (>90%) and reproducibility (

Because of numerous inherent and unrivaled features of nanofibers made of chitin, the second most plentiful natural-based polymer (after cellulose), including affordability, abundant nature, biodegradability, biocompatibility, commercial availability, flexibility, transparency, and extraordinary mechanical and physicochemical properties, chitin nanofibers (ChNFs) are being applied as one of the most appealing bionanomaterials in a myriad of fields. Herein, we exploited the beneficial properties offered by the ChNF paper to fabricate transparent, efficient, biocompatible, flexible, and miniaturized optical sensing bioplatforms via embedding/immobilizing various plasmonic nanoparticles (silver and gold nanoparticles), photoluminescent nanoparticles (CdTe quantum dots, carbon dots, and NaYF4:Yb3+@Er3+&SiO2 upconversion nanoparticles) along with colorimetric reagents (curcumin, dithizone, etc.) in the 3D nanonetwork scaffold of the ChNF paper. Several configurations, including 2D multi-wall and 2D cuvette patterns with hydrophobic barriers/walls and hydrophilic test zones/channels, were easily printed using laser printing technology or punched as spot patterns on the dried ChNF paper-based nanocomposites to fabricate the (bio)sensing platforms. A variety of (bio)chemicals as model analytes were used to confirm the efficiency and applicability of the fabricated ChNF paper-based sensing bioplatforms. The developed (bio)sensors were also coupled with smartphone technology to take the advantages of smartphone-based monitoring/sensing devices along with the Internet of Nano Things (IoNT)/the Internet of Medical Things (IoMT) concepts for easy-to-use sensing applications. Building upon the unrivaled and inherent features of ChNF as a very promising bionanomaterial, we foresee that the ChNF paper-based sensing bioplatforms will emerge new opportunities for the development of innovative strategies to fabricate cost-effective, simple, smart, transparent, biodegradable, miniaturized, flexible, portable, and easy-to-use (bio)sensing/monitoring devices., Financial support from the Chemistry & Chemical Engineering Research Centre of Iran (Tehran, Iran), Iran’s National Elites Foundation (INEF), Nano Novin Polymer Co. (Iran), and the Nano Match program of Iran Nanotechnology Initiative Council (INIC) are gratefully acknowledged. The ICN2 is funded by the CERCA Programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa program of the Spanish Ministry of Economy, Industry and Competitiveness (MINECO; grant nos. SEV-2017-0706 and MAT2017-87202-P). The financial support of “the Czech Science Foundation (No. 19-00676S)” is also greatly appreciated.

Real-time monitoring of neonicotinoid pesticide residues is of great significance for food security and sustainable development of the ecological environment. Herein, a paper-based surface-enhanced Raman scattering (SERS) amplified approach was proposed by virtue of multilayered plasmonic coupling amplification. The unique plasmonic SERS multilayer was constructed using three-dimensional (3D) silver dendrite (SD)/electropolymerized molecular identifier (EMI)/silver nanoparticle (AgNP) sandwich hybrids with multiple hotspots and a strong electromagnetic field in nanogaps. Dendritelike 3D silver materials with remarkably high accessible surface areas and the lightning rod effect constituted the first-order enhancement of paper-based sensors. Molecular identifiers coated upon an SD layer as the interlayer were used for target capture and enrichment. Subsequently, AgNPs featuring rough surface and local plasma resonance decorated as the top layer formed the secondary enhancement of the amplification strategy. As the most brilliant part, dendritelike 3D silver coupled with AgNPs has established double Ag layers to accomplish a multistage enhancement of SERS signals based on the superposition of their electromagnetic fields. Owning to the distinctive design of the multiple coupling amplification strategy, the fabricated SERS paper chips demonstrated impressive specificity and ultrahigh sensitivity in the detection of imidacloprid (IMI), with a detection limit as low as 0.02811 ng mL-1. More importantly, the multiple SERS enhancement paper chip holds great potential for automated screening of a variety of contaminants.

Cannabinoids present a unique set of analytical challenges. An increasing number of states have voted to decriminalize recreational marijuana use, creating a need for new kinds of rapid testing. At the same time, synthetic compounds with activity similar to THC, termed synthetic cannabinoids, have become more prevalent and pose significant health risks. A rapid method capable of detecting both natural and synthetic cannabinoids would be useful in cases of driving under the influence of drugs, where it might not be obvious whether the suspect consumed marijuana, a synthetic cannabinoid, or both. Paper spray mass spectrometry is an ambient ionization technique which allows for the direct ionization of analyte from a biofluid spot on a piece of paper. Natural cannabinoids like THC, however, are labile and rapidly disappear from dried sample spots, making it difficult to detect them at clinically relevant levels. Presented here is a method to concentrate and preserve THC and synthetic cannabinoids in urine and oral fluid on paper for analysis by paper spray mass spectrometry. Sesame seed oil was investigated both as a means of preserving THC and as part of a technique, termed paper strip extraction, wherein urine or oral fluid is flowed through an oil spot on a strip of paper to preconcentrate cannabinoids. This technique preserved THC in dried biofluid samples for at least 27 days at room temperature; paper spray MS/MS analysis of these preserved dried spots was capable of detecting THC and synthetic cannabinoids at low ng/mL concentrations, making it suitable as a rapid screening technique. The technique was adapted to be used with a commercially available autosampler.

Paper spray ionization has been used as a fast sampling/ionization method for the direct mass spectrometric analysis of biological samples at ambient conditions. Here, we demonstrated that by utilizing paper spray ionization-mass spectrometry (PSI-MS) coupled with field asymmetric waveform ion mobility spectrometry (FAIMS), predictive metabolic and lipidomic profiles of routine breast core needle biopsies could be obtained effectively. By the combination of machine learning algorithms and pathological examination reports, we developed a classification model, which has an overall accuracy of 87.5% for an instantaneous differentiation between cancerous and noncancerous breast tissues utilizing metabolic and lipidomic profiles. Our results suggested that paper spray ionization-ion mobility spectrometry-mass spectrometry (PSI-IMS-MS) is a powerful approach for rapid breast cancer diagnosis based on altered metabolic and lipidomic profiles.

Owing to its critical role in the development of female reproductive tissues and as a clinical biomarker, there is an urgent need to develop a rapid and cost-effective method to sensitively detect 17β-estradiol (E2). In this work, a folding aptasensor platform with microfluidic channels for the label-free electrochemical detection of E2 is described. The platform, designed with a delicate folding structure, integrating filter holes, microfluidic channels, reaction chambers, and a three-electrode system, is extremely easy to use. To increase the detection sensitivity and immobilize the aptamer, we synthesized a novel nanoassembly consisting of amine-functionalized single-walled carbon nanotube/new methylene blue/gold nanoparticles (AuNPs) and modified the working electrode with this nanoassembly. The calibration curve obtained from the experimental results exhibited a linear range between 10 pg mL-1 and 500 ng mL-1 (R2 = 0.993), and a detection limit of 5 pg mL-1 was achieved (S/N = 3). Furthermore, experiments to detect E2 in clinical serum were conducted, and the results were highly similar to those obtained using a large electrochemical luminescence apparatus. By integrating multiple functional components, adopting novel nanoassemblies, and using a folding structure, this paper-based platform not only has great potential as a simple and convenient integrated device for point-of-care testing of E2, but also as a portable, low-cost, and highly sensitive aptasensor platform capable of detecting many diagnostic biomarkers with the appropriate aptamers.

Many potential chemotherapeutics fail to reach patients. One of the key reasons is that compounds are tested during the drug discovery stage in two-dimensional (2D) cell cultures, which are often unable to accurately model in vivo outcomes. Three-dimensional (3D) in vitro tumor models are more predictive of chemotherapeutic effectiveness than 2D cultures, and thus, their implementation during the drug screening stage has the potential to more accurately evaluate compounds earlier, saving both time and money. Paper-based cultures (PBCs) are an emerging 3D culture platform in which cells suspended in Matrigel are seeded into paper scaffolds and cultured to generate a tissue-like environment. In this study, we demonstrate the potential of matrix-assisted laser desorption/ionization-mass spectrometry imaging with PBCs (MALDI-MSI-PBC) as a drug screening platform. This method discriminated regions of the PBCs with and without cells and/or drugs, indicating that coupling PBCs with MALDI-MSI has the potential to develop rapid, large-scale, and parallel mass spectrometric drug screens.

A digital fluorescence detector (DFD), a handheld fluorescence detection device, can convert the fluorescence signal of samples into the corresponding fluorescer concentration. Herein, by adopting a DFD as the readout, a novel intelligent platform was developed based on a ratiometric paper-based device (RPD) for multiple aminoglycoside detection. There are five layers and four parallel channels contained in the designed RPD, functioning as reagent storage, fluidic path control and signal processing, respectively. The rationale of this design lies in the fact that aptamer/graphitic carbon nitride nanosheet (Apt/g-C3N4 NS) modified layers can catalyze o-phenylenediamine to fluorescent 2,3-diaminophenazine (DAP) in the presence of H2O2. When Apt was removed from nanosheets via the Apt-target reaction, the peroxidase-like activity would be decreased, thus decreasing the production of DAP. All the changes of the fluorescence DAP signal can be read out using a portable DFD. Based on the DFD signal change related to the concentration of the target, a quantitative reaction platform was established. Furthermore, the sample flow and Apt-target reaction time can be reasonably regulated using the H2O2-cleavable hydrophobic compound modified layer placed between the target recognition region and detection region. Then, the practicality of this platform was verified through realizing sensitive analysis of streptomycin, tobramycin, and kanamycin simultaneously. Overall, with merits including portability and ease of operation, the platform shows great potential in on-site simultaneous detection of multiple targets, especially in resource-limited settings.

In this work, we introduce our recent finding that the carbon fiber paper (CFP) treated by simple air annealing (OCFP) could be used for exceptionally high-performance electrochemical nitrite sensing. The air-annealing process endows the pristine CFP with higher defective edge/plane sites, more oxygen-containing functional groups, higher roughness, and improved wettability. The electrochemical studies show that the OCFP exhibits excellent sensing performance for nitrite, with an ultralow determination limit of 0.1 μM and a detection limit of 0.07 μM, an ultrawide linear determination range of 0.1-3838.5 μM, a fast current response of 1 s, and a high sensitivity of 930.4 μA mM

Salmon fish farmers face remarkable problems in fish rearing and handling due to the spread of disease by infectious pancreatic necrosis virus (IPNV). Therefore, we developed a straightforward and sensitive technique to detect IPNV-based on recombinant human apoferritin heavy chain (hAFN-H) protein nanoparticles. In this study, the 24 subunits of the hAFN-H were genetically modified to express 6×His-tag and protein-G at their C-terminal site using Escherichia coli. We thus achieved a two-step signal amplifying strategy that utilizes a recombinant hAFN-H nanoprobe having a protein-G-binding site that targets the Fc region of monoclonal antibodies and a 6×His-tag that actively interacts with the functionalized Ni-NTA derivatives. In this study, we report a considerable advancement in magnetic bead-based detection systems that use Ni-NTA-Atto 550, reliably exhibiting detection limits of 1.02 TCID50/mL (50% tissue culture infective dose). Additionally, we propose a lateral flow chip-based detection method that uses the hAFN-H surface functionalized with 5 nm of the Ni-NTA-nanogold complex as a nanoprobe; the limit of detection towards IPNV was 0.88 TCID50/mL. The detection of IPNV by this recombinant hAFN-H nanoprobe was linear to virus titers in the range of 101-103 TCID50/mL.

The development of low cost, portable, and disposable biosensors for equipment-free and naked-eye biosensing is in eager demand for their widespread application in biomedical field, but it is still a challenge. Herein, we propose a novel paper analytical device (PAD) for truly equipment-free and naked-eye biosensing using dopamine as the chromogenic agent based on target-initiated catalyzed oxidation reaction. The dopamine-functionalized PAD (DPAD) possesses a significant three-dimensional net structure, excellent hydrophilicity, and unique response toward G-quadruplex DNAs against other DNAs, benefiting the bio/chemo reaction occurrence to assay target biomolecules. In light of the exceptional properties, the fabricated DPAD was applied in the analysis of Dam MTase through target-triggered exponential isothermal amplification. The recognition and methylation of H1 by Dam MTase contribute to formation of abundant hemin/G-quadruplexes, which catalyze oxidation of dopamine into dopachrome and reduce the dopamine amount on the DPAD surface. In comparison with the case in which Dam MTase is absent, an evident deep pink signal originating from dopachrome is observed directly by the naked eye and relied on Dam MTase concentrations. Therefore, truly equipment-free and naked-eye detection of Dam MTase is achieved with a detection limit of 1.46 U/mL. The fabricated DPAD not only achieves Dam MTase-visualized detection but also permits the accurate determination of other analytes by varying recognizable DNA's sequences, thus offering a universal biosensor and depicting significant potential for widespread applications in biomedical field.

Chemiluminescence (CL) immunoassays for simultaneous detection of early acute myocardial infarction (AMI) biomarkers, including copeptin, heart-type fatty acid binding protein (h-FABP), and cardiac troponin I (cTnI), were developed by using Co2+/N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized magnetic carbon composite (Co2+-ABEI-Fe3O4@void@C) as an interface and a three-dimensional microfluidic paper-based device (3D μPAD) as a detection system. For CL immunoassays, Co2+-ABEI-Fe3O4@void@C was assembled with chitosan (CS) and gold nanoparticle-conjugated antibody (Au-Ab) sequentially to form the sensing platform (Co2+-ABEI-Fe3O4@void@C-CS/Au-Ab). In the presence of antigen (Ag), Ag was captured by the sensing interface to form an immunocomplex, leading to an increase in CL intensity due to the catalysis of -COO- existing in Ag. A 3D μPAD with three detection zones for simultaneous detection of copeptin, h-FABP, and cTnI was designed and fabricated to obtain time-resolved CL signals. Three kinds of immunocomplexes formed with copeptin, h-FABP, and cTnI were added to three detection zones of 3D μPAD, respectively. After injecting H2O2, three time-resolved CL signals were generated in one CL detection run by virtue of time-delayed transport of H2O2 to different detection zones. The three time-resolved CL signals were used for the simultaneous determination of copeptin, h-FABP, and cTnI. The detection limit of copeptin, h-FABP, and cTnI was 0.40 pg/mL, 0.32 pg/mL, and 0.50 pg/mL, respectively, which is at least 1 order of magnitude lower than most of the reported immunoassays. The immunoassays could be directly used for the detection of copeptin, h-FABP, and cTnI in human serum samples. The proposed immunoassays are simple, fast, sensitive, and selective, and are of great application potential in early diagnosis and treatment of AMI.

Paper-based microfluidic analytical devices (μPADs) have recently attracted attention as a point-of-care test kit because of their low cost and nonrequirement for external forces. To directly detect biomarkers in whole blood, however, they need to be assembled with a filter such as a plasma separation membrane (PSM) because the color of the blood cells interferes with the colorimetric assay. However, this assembly process is rather complicated and cumbersome, and the fluid does not uniformly move to the detection zone when the adhesion between the paper and PSM is not perfect. In this study, we report a simple three-dimensional (3D) printing method for fabricating PSM-integrated 3D-μPADs made of plastics without the need for additional assembly. In detail, PSM was coated with parylene C to prevent its dissolution from organic solvent during 3D printing. Then, the coated PSM was superimposed on the paper. Detection zones and a reservoir were printed on the paper and PSM via liquid photopolymerization, using a digital light processing printer. The limit of detection of the PSM-integrated 3D-μPADs for glucose in whole blood was 0.3 mM, and these devices demonstrated clinically relevant performance on diabetes patient blood samples. Our 3D-μPADs can also simultaneously detect multiple metabolic disease markers including glucose, cholesterol, and triglycerides in whole blood. Our results suggest that our printing method is useful for fabricating 3D-μPADs integrated with PSM for the direct detection of biomarkers in whole blood.

Flexible pressure sensors have attracted considerable attention because of their potential applications in healthcare monitoring and human-machine interactions. However, the complicated fabrication process and the cos of sensing materials limit their widespread applications in practice. Herein, a flexible pressure sensor with outstanding performances is presented through an extremely simple and cost-efficient fabrication process. The sensing materials of the sensor are based on low-cost carbon black (CB)@airlaid paper (AP) composites, which are just prepared by drop-casting CB solutions onto APs. Through simply stacking multiple CB@APs with an irregular surface and a fiber-network structure, the obtained pressure sensor demonstrates an ultrahigh sensitivity of 51.23 kPa-1 and an ultralow detection limit of 1 Pa. Additionally, the sensor exhibits fast response time, wide working range, good stability, as well as excellent flexibility and biocompatibility. All the comprehensive and superior performances endow the sensor with abilities to precisely detect weak air flow, wrist pulse, phonation, and wrist bending in real time. In addition, an array electronic skin integrated with multiple CB@AP sensors has been designed to identify spatial pressure distribution and pressure magnitude. Through a biomimetic structure inspired by blooming flowers, a sensor with the open-petal structure has been designed to recognize the wind direction. Therefore, our study, which demonstrates a flexible pressure sensor with low cost, simple preparation, and superior performances, will open up for the exploration of cost-efficient pressure sensors in wearable devices.

Polymerization-based signal amplification (PBA) is a material-based approach to improving the sensitivity of paper-based diagnostic tests. Eosin Y is used as an assay label to photo-initiate free-radical polymerization to produce colored hydrogels in the presence of target analytes captured by bioactive paper. PBA achieves high-contrast and time-independent signals, but its nanomolar detection limit makes it impractical for early diagnosis of many diseases. In this work, we demonstrated efficient localization of large quantities of eosin Y per captured target analyte by incorporating eosin Y-loaded liposomes into PBA. This new "materials approach" allowed 30-fold signal enhancement compared to conventional PBA. To further improve the detection limit of liposome-enhanced PBA, we used a continuous flow-through assay format with 100 μL of analyte solution, achieving sub-nanomolar detection limits with high-contrast signals that were easily discernible to the unaided eye.

Minimally invasive devices to detect molecules in dermal interstitial fluid (ISF) are desirable for point-of-care diagnostic and monitoring applications. In this study, we developed a microneedle (MN) patch that collects ISF for on-patch biomarker analysis by surface-enhanced Raman scattering (SERS). The micrometer-scale MNs create micropores in the skin surface, through which microliter quantities of ISF are collected onto plasmonic paper on the patch backing. The plasmonic paper was prepared by immobilizing poly(styrenesulfonate) (PSS) coated gold nanorods (AuNRs) on a thin strip of filter paper using plasmonic calligraphy. Negatively charged PSS was used to bind positively charged rhodamine 6G (R6G), which served as a model compound, and thereby localize R6G on AuNR surface. R6G bound on the AuNR surface was detected and quantified by acquiring SERS spectra from the plasmonic paper MN patch. This approach was used to measure pharmacokinetic profiles of R6G in ISF and serum from rats in vivo. This proof-of-concept study indicates that a plasmonic paper MN patch has the potential to enable on-patch measurement of molecules in ISF for research and future medical applications.

Paper-based optoelectronic noses (OENs) are being developed based on printing of organic and organometallic reagents on hydrophilic substrates that can visualize the odor of volatiles. In this work, we report for the first time the use of nanoparticles for fabrication of novel paper-based OENs, which represent much higher sensitivity and produce simple but discriminant colorimetric signature of volatile metabolomes. This nano-optoelectronic nose (NOEN) system, which is fabricated by dropping of gold and silver nanoparticles (each synthesized by 8 chemical species) on the paper, gives obvious colorimetric signatures for chemicals having individual or combined functional groups. Owning to their ultrasensitivity, these simple devices need very small amounts of analytes. These devices could detect and discriminate 45 volatile organic compounds in 9 chemical families including phenols, alchohols, ketones, aldehydes, amines, acids, esters, arenes, and hydrocarbons. In addition to excellent discrimination ability, this NOEN sensor shows ultrahigh sensitivity such that could determine volatile compounds with detection limits around or lower than 10 ppb. Moreover, it can be combined with multivariate calibration methods for quantitative analysis of a metabolite in a complex mixture.

This paper describes an approach for colorimetric detection of salivary α-amylase, one of the potential biomarkers of autonomic nervous system (ANS) activity, for enabling assessment of fatigue. The ability of α-amylase to cleave α-bonds of polysaccharides is utilized for developing a colorimetric assay. In the proposed approach, 2-chloro-4-nitrophenyl-α-d-maltotrioside as substrate releases a colored byproduct upon cleavage by salivary α-amylase. Introduction of maltose as a noncompetitive inhibitor yields desirable linear responses in the physiologically relevant concentration range (20–500 μg/mL) with a limit of detection (LOD) of 8 μg/mL (in aqueous solution). The concentrations of substrate and noncompetitive inhibitor are subsequently optimized for colorimetric detection of salivary α-amylase. A facile paper-based “strip” assay is proposed for analysis of human saliva samples with marginal interference from saliva components. The proposed assay is rapid, specific, and easy-to-implement for colorimetric detection of salivary α-amylase between 20 and 500 μg/mL. Complementary RGB (red, green, blue components) analysis offers quantitative detection with a LOD of 11 μg/mL. The two assay formats are benchmarked against the Phadebas test, a state of the art method for spectrophotometric detection of α-amylase. The reported paper-based methodology possesses a high potential for estimation of altered ANS responses toward stressors that possibly could find applications in assessment of fatigue and for monitoring onset of fatigue. Nanyang Technological University Accepted version This work is supported by Provost’s office and Institute for Sports Research (ISR), School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore.

One of the concerns of parents in the first days of their baby's birth is the baby's risk of jaundice/hyperbilirubinemia. This is because more than 60% of babies are born with jaundice that, if not timely diagnosed and subsequently treated, can lead to serious damage to their health. On the other hand, despite recent progress in sensor technology for clinical applications, the development of easy-to-use, cost-effective, sensitive, specific, and portable diagnostic devices, which use nontoxic and biodegradable materials in their design and fabrication, is still in high demand. Herein we present an easy-to-use, cost-effective, selective, nontoxic, and disposable photoluminescent nanopaper-based assay kit with a smartphone readout for easy diagnosis of neonatal jaundice through visual determination of Bilirubin (BR) in infants' blood samples. The developed BR assay kit comprises highly photoluminescent carbon dot (CD) sensing probes embedded in a bacterial cellulose (BC) nanopaper substrate (CDBN). The photoluminescence (PL) of the developed BR sensor is quenched in the presence of BR as a PL quencher and then selectively recovered upon blue light (λ = 470 nm) exposure, due to conversion of the unconjugated BR to the colorless oxidation products (non-PL quencher) through BR photoisomerization and photooxidation, that subsequently leads to selective PL enhancement of CDBN. The recovered PL intensity of the developed BR assay kit, which was monitored by integrated smartphone camera, was linearly proportional to the concentration of BR in the range of 2-20 mg dL

In this study, a novel photoluminescence material for the detection of tributyltin (TBT) was developed by using a paper-based nanocomposite system. For this purpose, molecularly imprinted polymeric nanoparticles (MIN) were synthesized with mini-emulsion polymerization technique. Graphene quantum dots obtained by the hydrothermal pyrolysis were immobilized to the nanoparticle surface via EDC-NHS coupling. The fabrication of sensing platform for TBT can be divided into two steps that are the preparation of nanocomposite and the applying the nanocomposite onto nitrocellulose membrane. The selectivity constant and association kinetics were calculated to analyze the interaction of TBT with immobilized MINs. The results proved that the developed nanosensor is promising for the determination of TBT with high selectivity and sensitivity reaching a detection limit of 0.23 ppt in seawater. This novel photoluminescent nanosensor has the potential to pave the way for further studies and applications., Recep Üzek thanks to TUBITAK for the given scholarship. We acknowledge the support from European Commission through the Graphene Flagship Core 2 project and from Ministerio de Economıa, Industria y Competitividad (MINECO), Agencia Estatal de Investigacion (AEI) for the project MAT2017-87202-P. The ICN2 is funded by the CERCA Programme/Generalitat de Catalunya. ICN2 is supported by the Severo Ochoa program from Spanish MINECO (Grant No. SEV2017-0706).

Multiclass chemical contamination of food has aroused ever-increasing attention due to the increasingly common findings of the co-occurrence of multiclass contamination, such as mycotoxin (aflatoxin M1, AFM1) and illegal additive (melamine, MEL). In the present study, a rapid, ultrasensitive detection paper was developed on the basis of a unique bridge-antibody label to realize on-site simultaneous detection of AFM1 and MEL in milk. This detection paper used the bridge-antibody label on fluorescent particles (i.e., the fluorescent Eu nanoparticles were first conjugated with polyclonal antibodies and then with monoclonal antibodies). Dramatically enhanced sensitivity was found, probably due to the increase in immobilization of efficient monoclonal antibodies onto microspheres. Under optimal conditions, the lower limits of detection were 0.009 and 0.024 ng/mL for AFM1 and MEL in milk, respectively, in comparison with similar works. Moreover, the cutoff values were 0.4 and 150 ng/mL for AFM1 and MEL, respectively. The recoveries ranged from 88.7% to 105.0% for AFM1 and from 84.6% to 117.7% for MEL, with relative standard deviations (RSDs) of 0.5-9.9% during the intraday and interday experiments. Comparison experiments conducted using the detection paper, HPLC, and UPLC-MS/MS found excellent agreement in the simultaneous detection of AFM1 and MEL in milk. This proposed method can be extensively employed for simultaneous monitoring of multiclass chemical contaminants to ensure food safety.

This paper focuses on one of the most commonly encountered materials in our society, namely paper. Paper is an inherently complex material, yet its use provides for chemical analysis approaches that are elegant in their simplicity of execution. In the first half of the previous century, paper in scientific research was used mainly for filtration and chromatographic separation. While its use decreased with the rise of modern elution chromatography, paper remains a versatile substrate for low-cost analytical tests. Recently, we have seen renewed interest to work with paper in (bio)analytical science, a result of the growing demand for inexpensive, portable analysis. Dried blood spotting, paper microfluidics, and paper spray ionization are areas in which paper is (re)establishing itself as an important material. These research areas all exploit several properties of paper, including stable sample storage, passive fluid movement and manipulation, chromatographic separation/extraction, modifiable surface and/or volume, easily altered shape, easy transport, and low cost. We propose that the real, and to date underexploited, potential of paper lies in utilizing its combined characteristics to add new dimensions to paper-based (bio)chemical analysis, expanding its applicability. This article provides the reader with a short historical perspective on the scientific use of paper and the developments that led to the establishment of the aforementioned research areas. We review important characteristics of paper and place them in a scientific context in this descriptive, yet critical, assessment of the achieved and the achievable in paper-based analysis. The ultimate goal is the exploration of integrative approaches at the interface between the different fields in which paper is or can be used.

Blood is the most intensely studied human biofluid and is used as the main indicator (so-called “gold standard”) of the health status of the individual. Blood is also a very complex fluid, containing a multiplicity of components, including cells and cell fragments (red and white blood cells, platelets), proteins, and many other organic molecules. Laboratory-based blood analysis is a standard procedure that provides a wealth of information about the concentration of key components and enables medical personnel to establish diagnoses and treatments. To date, options for point-of-care (POC) or point-of-use (POU) blood analysis are limited and very low-cost options even more so. One analytical platform that contains the promise to fill this need utilizes paper-based microfluidic devices. This is a research area that has exhibited significant growth in the past few years, primarily because of its simplicity, fundamental low cost, and versatility. This article reviews the basic properties of blood, how these pr...

Heavy metals are highly toxic at trace levels and their pollution has shown great threat to the environment and public health worldwide where current detection methods require expensive instrumentation and laborious operation, which can only be accomplished in centralized laboratories. Herein, we report a low-cost, paper-based microfluidic analytical device (μPAD) for facile, portable, and disposable monitoring of mercury, lead, chromium, nickel, copper, and iron ions. Triple indicators or ligands that contain ions or molecules are preloaded on the μPADs and upon addition of a metal ion, the colorimetric indicators will elicit color changes observed by the naked eyes. The color features were quantitatively analyzed in a three-dimensional space of red, green, and blue or the RGB-space using digital imaging and color calibration techniques. The sensing platform offers higher accuracy for cross references, and is capable of simultaneous detection and discrimination of different metal ions in even real water samples. It demonstrates great potential for semiquantitative and even qualitative analysis with a sensitivity below the safe limit concentrations, and a controlled error range.

Traditional manufacturing methods and materials used to fabricate epidermal electronics for physiological monitoring, transdermal stimulation, and therapeutics are complex and expensive, preventing their adoption as single-use medical devices. This work describes the fabrication of epidermal, paper-based electronic devices (EPEDs) for wearable and implantable applications by combining the spray-based deposition of silanizing agents, highly conductive nanoparticles, and encapsulating polymers with laser micromachining. EPEDs are inexpensive, stretchable, easy to apply, and disposable by burning. The omniphobic character and fibrous structure of EPEDs make them breathable, mechanically stable upon stretching, and facilitate their use as electrophysiological sensors to record electrocardiograms, electromyograms, and electrooculograms, even under water. EPEDs can also be used to provide thermotherapeutic treatments to joints, map temperature spatially, and as wirelessly powered implantable devices for stimulation and therapeutics. This work makes epidermal electronic devices accessible to high-throughput manufacturing technologies and will enable the fabrication of a variety of wearable medical devices at a low cost.

A novel strategy for fabricating the paper-based valves on microfluidic paper-based analytical devices (μPADs) was described to control fluid in a user-friendly way. Initial prototypes of 3D μPADs manipulate the spatial distribution of fluid within the device. The movable paper channel in a different layer could be achieved using the channel's connection or disconnection to realize the valve function using plastic comb binding spines (PCBS). The entire valve manipulation process was similar to a desk calendar that can be flipped over and turned back. It is notable that this kind of PCBS valve can control a fluid in a simple and easy way without the timing setting or any trigger, and this advantage makes it user-friendly for untrained users to carry out the complex and high throughput operations. The reusable plastic comb binding spines greatly reduce the cost of fabricating paper-based valves. To evaluate the performance, the actual samples of Fe (II) and nitrite were successfully analyzed. We hope this method will introduce a new approach to fabrication of paper-based valves on μPADs in the future.

A paper chip device-based recombinase polymerase amplification (RPA) method was developed for highly sensitive and selective single-step detection of foodborne pathogens. A paper chip was manufactured by simply stacking functional papers. RPA reagents and fluorescent probe were dried on the reaction zone of a patterned poly(ether sulfone) membrane. The RPA reaction was initiated by adding pathogen DNAs into an injection hole. Paper chip-based analysis of pathogens showed optimal performance at 37 °C for 20 min and the results were comparable to those obtained with solution-based RPA reactions. Based on the paper chip-based fluorescence signal, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium were simultaneously detected with detection limits of 102 cfu/mL. The diagnostic utility of the device was demonstrated by the reliable detection of E. coli and S. aureus present in spiked milk. This ready-to-use device could be integrated with simple nucleic acid extraction for food pathogen detect...

In this work, we characterize the impact of large-volume processing upon the analytical sensitivity of flow-through paper-based immunoassays. Larger sample volumes feature greater molar quantities of available analyte, but the assay design principles which would enable the rapid collection of this dilute target are ill-defined. We developed a finite-element model to explore the operating conditions under which processing large sample volumes via pressure-driven convective flow would yield an improved binding signal. Our simulation results underscore the importance of establishing a high local concentration of the analyte-binding species within the porous substrate. This elevated abundance serves to enhance the binding kinetics, matching the time scale of target capture to the period during which the sample is in contact with the test zone (i.e., the effective residence time). These findings were experimentally validated using the rcSso7d-cellulose-binding domain (CBD) fusion construct, a bifunctional binding protein which adsorbs to cellulose in high abundance. As predicted by our modeling efforts, the local concentration achieved using the rcSso7d-CBD species is uniquely enabling for sensitivity enhancement through large-volume processing. The rapid analyte depletion which occurs at this high surface density also permits the processing of large sample volumes within practical time scales and flow regimes. Using these findings, we present guidance for the optimal means of processing large sample volumes for enhanced assay sensitivity.

We report the development of a surface-enhanced Raman spectroscopy sensor chip by decorating gold nanoparticles (AuNPs) on ZnO nanorod (ZnO NR) arrays vertically grown on cellulose paper (C). We show that these chips can enhance the Raman signal by 1.25 × 107 with an excellent reproducibility of 92% clinical sensitivity and specificity. Our technology has the potential to be used for the early detection of prenatal diseases and can be adapted for point-of-care applications.

This paper describes, for the first time, the use of oxidized buckypaper (BP) as a sorbent membrane of a stir-disc solid phase extraction module. The original device, consisting of a BP disc (d = 34 mm) enveloped in a polypropylene mesh pouch, was designed to extract organic micropollutants (OMPs) from environmental water samples in dynamic mode. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to analyze the extracts. Several classes of pesticides and pharmaceuticals were chosen as model compounds to evaluate key parameters affecting the recovery rates. To this end, the effects of adsorption time, desorption time, stirring speed, type and volume of solvent, and sample volume were thoroughly examined. After optimization, a novel and in-depth study was conducted to find a correlation between physicochemical properties of the analytes and extraction yields. Recoveries were mainly governed by a combination of logP and pKa values. As indicated, hydrophilic compounds with l...

Aptamer-based paper strip sensor for detecting Vibrio fischeri was developed. Our method was based on the aptamer sandwich assay between whole live cells, V. fischeri and DNA aptamer probes. Following 9 rounds of Cell-SELEX and one of the negative-SELEX, V. fischeri Cell Aptamer (VFCA)-02 and -03 were isolated, with the former showing approximately 10-fold greater avidity (in the subnanomolar range) for the target cells when arrayed on a surface. The colorimetric response of a paper sensor based on VFCA-02 was linear in the range of 4 × 101 to 4 × 105 CFU/mL of target cell by using scanning reader. The linear regression correlation coefficient (R2) was 0.9809. This system shows promise for use in aptamer-conjugated gold nanoparticle probes in paper strip format for in-field detection of marine bioindicating bacteria.

Highly selective and sensitive detection of guanosine 3′-diphosphate-5′-diphosphate (ppGpp), namely, the stringent in plants or microorganisms responding to strict or extreme environmental conditions such as stress and starvation, which plays an important role in gene expression, rRNA and antibiotics production, regulations of virulence of bacteria, and growth of plants, faces a great challenge owing to its extreme similarity to normal nucleotides. By modifying the surface groups of a facile two-step hydrothermal route prepared carbon dots (CDs) with terbium ions (Tb3+) in this contribution, a novel fluorescent probe with excellent properties such as highly physical and chemical stability, narrow emission and excitation wavelength-independent emission was prepared. The Tb3+ ions on the surface of CDs cannot only preserve the intrinsic fluorescence (FL) of CDs but also keep its own coordination capacity with rare earth complex, and thus the clamp structure (four phosphate groups) of ppGpp can specific bind...

Self-assembled micrometer-scale vesicles composed of lamellar phase forming amphiphiles are useful as chemical microreactors, as minimal artificial cells, as protocell mimics for studies of the origins of life, and as vehicles for the targeted delivery of drugs. Given their varied uses, discovery of a universal mechanism that is simple, rapid, and that produces vesicles from a large variety of amphiphiles with different chemical and physical properties at high yield is extremely desirable. Here we show that cellulose, in the form of cellulose paper, facilitates the assembly of membranous vesicles 5-20 μm in diameter from scientifically and technologically important amphiphiles of diverse chemical structures and functionality such as fatty acids (fatty acid vesicles), amphiphilic diblock copolymers, and amphiphilic triblock copolymers (polymersomes). Assembly of vesicles occurred within 90 min of placing the amphiphile-coated cellulose paper into aqueous solutions. Varying thermal and chemical conditions, however, are required for the high-yield assembly of vesicles from the different amphiphiles. The vesicles, when attached to cellulose fibers, have membranes that remain unsealed. This topological characteristic of the vesicles grown on paper allowed the scalable separation of the process of growth from the process of loading cargo (temporally decoupled growth and loading). We demonstrate a temporally decoupled process to rapidly produce large quantities of protein-loaded polymersomes on the benchtop by using high temperatures to accelerate the growth of the polymersomes and subsequently milder temperatures during diffusive loading of the protein cargo.

The health and well-being of cattle is a significant concern for global agricultural output. In dairy production within low and middle income countries (LMICs), there is a significant biosensing challenge in detecting sexually transmitted infection (STI) pathogens during animal husbandry, due in part to difficulties associated with the limited infrastructure for veterinary medicine. Here we demonstrate low-cost, multiplexed and sample-to-answer paper-origami tests for the detection of three bovine infectious reproductive diseases in semen samples, collected at a test site in rural India. Pathogen DNA from one viral pathogen, Bovine Herpes virus-1 (BoHV-1) and two bacteria (Brucella and Leptospira) was extracted, amplified (using loop-mediated isothermal amplification, LAMP) and detected fluorescently, enabling

Paper-based cultures are an emerging platform for preparing 3D tissue-like structures. Chemical gradients can be imposed upon these cultures, generating microenvironments similar to those found in poorly vascularized tumors. There is increasing evidence that the tumor microenvironment is responsible for promoting drug resistance and increased invasiveness. Acidosis, or the acidification of the extracellular space, is particularly important in promoting these aggressive cancer phenotypes. To better understand how cells respond to acidosis there is a need for 3D culture platforms that not only model relevant disease states but also contain sensors capable of quantifying small molecules in the extracellular environment. In this work, we describe pH-sensing optodes that are capable of generating high spatial and temporal resolution maps of pH gradients in paper-based cultures. This sensor was fabricated by suspending microparticles containing pH-sensitive (fluorescein) and pH-insensitive (diphenylanthracene) dyes in a polyurethane hydrogel, which was then coated onto a transparent film. The pH-sensing films have a fast response time, are reversible, stable in long-term culture environments, have minimal photobleaching, and are not cytotoxic. These films have a pKa of 7.61 ± 0.04 and are sensitive in the pH range corresponding to normal and tumorigenic tissues. With these optodes, we measured the spatiotemporal evolution of pH gradients in paper-based tumor models.

As a cost-effective approach for detecting trace amounts of pesticides, filter paper-based SERS sensors have been the subject of intensive research. One of the hurdles to overcome is the difficulty of retaining nanoparticles on the surface of the paper because of the hydrophilic nature of the cellulose fibers in paper. This reduces the sensitivity and reproducibility of paper-based SERS sensors due to the low density of nanoparticles and short retention time of analytes on the paper surface. In this study, filter paper was treated with alkyl ketene dimer (AKD) to modify its property from hydrophilic to hydrophobic. AKD treatment increased the contact angle of the aqueous silver nanoparticle (AgNP) dispersion, which consequently increased the density of AgNPs. The retention time of the analyte was also increased by preventing its rapid absorption into the filter paper. The SERS signal was strongly enhanced by the increased number of SERS hot spots owing to the increased density of AgNPs on a small contact area of the filter surface. The reproducibility and sensitivity of the SERS signal were optimized by controlling the distribution of AgNPs on the surface of the filter paper by adjusting the concentration of the AgNP solution. Using this SERS sensor with a hydrophobicity-modified filter paper, the spot-to-spot variation of the SERS intensity of 25 spots of 4-aminothiophenol was 6.19%, and the limits of detection of thiram and ferbam as test pesticides were measured to be 0.46 nM and 0.49 nM, respectively. These proof-of-concept results indicate that this paper-based SERS sensor can serve for highly sensitive pesticide detection with low cost and easy fabrication.

This work reports a microfabricated electrochemical paper-based analytical device (ePAD) for the voltammetric determination of DNA. The device is patterned by wax-printing on paper and features a circular assay zone connected to an inlet zone and a sink via grooved microfluidic channels for accelerated flow rate. An electrochemical cell with integrated electrodes is formed on the reverse side of the paper by sputtering of thin metal films (Sn, Pt and Ag as the working, counter and reference electrode, respectively). Proof-of-principle of the ePAD for biosensing is demonstrated for a DNA assay involving attachment of capture DNA, hybridization with biotinylated target oligonucleotide and labeling with streptavidin-conjugated CdSe/ZnS quantum dots (QDs). After the acidic dissolution of the QDs, the released Cd(II) is quantified by anodic stripping voltammetry (ASV) at the Sn-film working electrode. Thanks to the synergistic effects of QDs amplification, the inherent sensitivity of ASV and the excellent detection capabilities of the Sn-film working electrode for Cd(II), the target DNA can be detected at levels as low as 0.11 pmol L

Porous media made of nitrocellulose and glass fiber are common "paper" substrates for lateral flow assays, microfluidic paper analytical devices and other point-of-care diagnostic assays. Such assays commonly use optical labels such as gold nanoparticles, latex beads, or fluorescent nanoparticles to visualize the presence of analytes. Fluorescent labels are commonly used in bioassays to enhance sensitivity, but autoluminescence of the paper substrate worsens signal-to-noise ratios of fluorescence-based assays. To date, there exists no systematic investigation of autoluminescence wavelengths or lifetimes of porous membranes used in lateral flow assays. In response, we quantified the autoluminescence of commonly used porous materials across the visible spectrum via excitation-emission spectroscopy and time-resolved fluorescence spectroscopy, and demonstrate that autoluminescence is solely due to autofluorescence with lifetimes of about 5 ns in the visible spectrum. Counterintuitively, we found that spectroscopy alone does not provide sufficient information to select candidate paper substrates for fluorophore-labeled assays. Therefore, we developed a simple quantitative framework to select a low-fluorescence substrate that minimizes both the overlap of paper and fluorophore emission spectra and the fluorescence intensity on an imaging system of interest (such as a gel imager). Use of this framework was shown to lower the limit of detection of an influenza A nucleoprotein immunoassay by over 50%. The tools developed in this manuscript enable assay developers to screen appropriate, low-fluorescence porous substrates and enhance the sensitivity of membrane-based fluorescence assays.