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Point-of-care (POC) detection is crucial in clinical diagnosis in order to provide timely and specific treatment. Combining polyamidoamine (PAMAM) dendrimer, p-phenylene diisothiocyanate (PDITC) and superparamagnetic beads, a novel method to activate the surface of filter paper to bind DNA molecules has been developed. The method is based on the primary amination of the filter paper surface with PAMAM dendrimer, followed by generation of isothiocyanate groups via PDITC, and subsequent repetition of these two steps. Different parameters of the process have been optimized, including probe printing, preparation of target DNAs and detection. The result shows that, due to the highly porous structure of filter paper, high amounts of printed probes, target DNAs and magnetic beads can provide high signal intensities in the detection area via probe/target duplex formation. This method is suitable for rapid, specific and cost-efficient DNA detection on cellulose filter paper. It can be used as a POC device, in particular for diagnosis and treatment management of infectious diseases and identification of antimicrobial drug resistance genes.

Paper-based nucleic acid detection and diagnosis are currently gaining much interest in point-of-care (POC) applications. The major steps involved in any nucleic acid amplification testing (NAAT) based diagnostics are nucleic acid isolation, reverse transcription (RT) (in the case of RNA), amplification and detection. RT is an important step in quantifying the viral load in case of disease diagnosis as well as quantifying gene expression levels in other molecular studies. cDNA synthesis is routinely carried out using a thermal cycler, with the process requiring temperatures between 40ºC to 65ºC. Here we report for the first time an instrument-free RT, performed at room temperature on cellulose-based paper devices. cDNA synthesis on paper was confirmed by RT-PCR and Sanger sequencing of the PCR products. Purified RNA from varied sources such as cell lysate, tissue and blood were used to test the methodology. Synthetic hepatitis C virus (HCV) RNA and human blood RNA were used as proof-of-concept to demonstrate the use of these devices in diagnostic applications. Further, ready-to-use paper-based reverse transcription (PRT) devices have been developed, wherein only the RNA sample is added on the device and the cDNA can be eluted after 30 min of incubation at room temperature. The devices were found to be stable for 30 days at - 20ºC storage. The cellulose-based PRT devices are simple, time saving and user-friendly for a complete instrument-free cDNA synthesis at room temperature.

Flexible sensing devices have drawn tremendous attention in the past decades due to their potential applications in future hand-held, potable consumer, and wearable electronics. Here, we firstly developed an ultrasensitive wireless potentiometric aptasensor based on flexible freestanding graphene paper for kanamycin detection. Flexible graphene paper made from a simple vacuum filtration method was used as a biocompatible platform for effective immobilization of aptamer. A nuclease-assisted amplification strategy was introduced into this potentiometric biosensing system in order to significantly improve the detection sensitivity through a classic catalytic recycling reaction of target induced by the nuclease (DNase I). As expected, an ultra-low detection limit of 30.0 fg/mL for kanamycin was achieved. Furthermore, the developed potentiometric enzymatic aptasensor exhibits high selectivity, favorable flexibility, excellent stability and reproducibility, which holds great promising for its routine sensing application.

Thermophilic methane fermentation was a valid approach for handling the stillage eluted from ethanol fermentation of waste paper and kitchen waste. The wide organic loading rate (OLR) range (2-14 g VTS/(L d)) for stable performance and relatively high energy recovery efficiency (79.0%) were achieved, and OLR of 8 g VTS/(L d) was optimum for achievement of highest biogas evolution and VTS removal efficiency. Microbial community analysis revealed that hydrolysis of cellulose was the critical step for methane production from the stillage.

The synthesis and employment of volatile toxic compounds as chemical weapons with a large-scale destructive power has introduced a new insidious threat over the last century. In this framework, the development of wearable sensing tools represents a critical point within the security field, in order to provide early alarm systems. Herein, a novel wearable electrochemical biosensor was developed for the rapid and on-site detection of mustard agents. Since a chemical attack is typically carried out by spraying these volatile agents into air, the sensor was designed in order to be able to measure mustard agents directly in the aerosol phase, further than in the liquid phase. The electrodes were screen-printed onto a filter paper support, which allowed to harness the porosity of paper to pre-load all the needed reagents into the cellulose network, and hence to realise an origami-like and reagent-free device. Mustard agent detection was carried out by monitoring their inhibitory effects toward the choline oxidase enzyme, through the amperometric measurement of the enzymatic by-product hydrogen peroxide. A carbon black/Prussian blue nanocomposite was used as a bulk-modifier of the conductive graphite ink constituting the working electrode, allowing for the electrocatalysis of the hydrogen peroxide reduction. After having verified the detecting capability toward a mustard agent simulant, the applicability of the resulting origami-like biosensor was demonstrated for the rapid and real-time detection of real sulfur mustard, obtaining limits of detection equal to 1 mM and 0.019 g·min/m3 for liquid and aerosol phase, respectively.

Epoxide-bearing filter paper was first prepared by surface-initiated reversible addition-fragmentation chain transfer (RAFT) copolymerization of glycidyl methacrylate (GMA) and poly(ethylene glycol)methacrylate (PEGMA). Without the need for activation step, the capture peptide nucleic acid (PNA) probes carrying a C-terminal lysine modification can be directly immobilized on the surface-grafted poly[glycidyl methacrylate-ran-poly(ethylene glycol)methacrylate] (P(GMA-ran-PEGMA)) through ring-opening of epoxide groups in the GMA repeating units by amino groups in the PNA’s structure. The success of P(GMA-ran-PEGMA) grafting on the filter paper and subsequent PNA immobilization was confirmed by fluorescence microscopy, Fourier transform-infrared spectroscopy and X-ray photoelectron spectroscopy. Colorimetric detection with signal amplification upon DNA hybridization relies on sandwich-hybridization assay employing another biotinylated PNA strand as a reporter probe together with streptavidin-horseradish peroxidase conjugate (SA-HRP) and o-phenylenediamine (OPD) substrate. It was found that increasing ionic strength during the DNA hybridization step by addition of NaCl can increase the signal intensity, which can be visualized by naked eye. The sensing platform showed the best performance in preventing non-specific adsorption from the non-complementary DNA and discriminating between complementary and single-mismatched targets of at least 50 fmol without the requirement for stringent hybridization or washing condition. This superior ability to suppress non-specific adsorption of non-target DNA as well as other non-DNA components may be explained as a result of hydrophilic PEGMA repeating units in the surface-grafted copolymer.

Pancreatic hormones such as insulin play the crucial life-supporting role of glucose metabolism. Due to rapidly growing diabetic disorders and associated health complications globally, combined with the pathogenic viral infections severely affecting people with pre-existing conditions, new user-friendly, affordable molecular diagnostic approaches that offer simplicity, cost-effectiveness, and an ultra-sensitive detection in human biofluids are necessary to improve the global health. In this regard, a low-cost paper device that enables easy monitoring of fasting blood-relevant ultra-low picomolar insulin levels is innovative, giving broader applications to any other similar critical molecular targets. The essential contribution of this lab-on-paper is the combination of label-free electrochemical insulin aptasensing with a paper electrode device as a simpler, cheaper, and reliable molecular diagnostic approach for complex serum samples with supporting independent validation methods for confirming scientific rigor and fit-for-purpose of the presented approach. Our aptasensor features a carboxylated graphene-aptamer surface design offering a clinically relevant quantitative detection of picomolar insulin present in a 10-fold diluted neat serum based on interfacial capacitance changes in proportion to the serum insulin concentration (dynamic range 5–500 pM and limit of detection 1.5 pM). Application for undiluted serum samples is demonstrated. Furthermore, analysis of an actual diabetes patient serum sample and correlation of the capacitance sensor results with peroxidase antibody label-based insulin assay approaches are presented (amperometric detection and a commercial enzyme-linked immunosorbent assay are used as validation methods).

The construction of a photoelectrochemical (PEC) strategy for multi-component detection represents a great challenge in the field of sensing. To address these challenges, herein we presented a hollow-channel paper-based PEC analytical platform based on chemiluminescence (CL) addressable strategies excited PEC strategy for multiplexed sensing application. Sandwich-structured CdS quantum dots (QDs)/reduced graphene oxide (RGO)/ZnO nanorods arrays (NRAs) heterostructure where CdS serves as visible light sensitizers, RGO acts as an electron relay between ZnO NRAs and CdS QDs, were simply assembled on the gold nanoparticles modified paper working photoelectrode (Au-PWE). The PEC performance of the CdS/RGO/ZnO can be greatly improved benefiting from the formation of type II band alignment between CdS QDs and ZnO NR as well as the super charge collection and shuttling property of RGO. Multiplexed CL emission could be achieved through controlling the CL co-reagents transport. By the virtue of CL addressable technique and the excellent PEC activity of CdS/RGO/ZnO, a highly sensitive, and selective multiple microRNAs (miRNAs) quantification method was achieved. Such a tailored strategy would break the bottleneck of the current PEC detection techniques in multiplex tracing, as well as serve as a novel concept for designing multi-channel PEC sensing method.

A paper-based electrochemical immunosensor was developed for the detection of cancer antigen 125 (CA125) by screen-printing technique. The reduced graphene oxide/thionine/gold nanoparticles (rGO/Thi/AuNPs) nanocomposites were compounded and coated onto the working electrode of immunosensor for CA125 antibody (anti-CA125) immobilization and detection signal amplification. The detection principle was based on the fact that the immunocomplex formed by specify binding of CA125 antibody and antigen could reduce the current response of thionine, which was proportional to the corresponding concentration of CA125 antigen. The immunoassay results showed that the linear range of CA125 was from 0.1 U mL-1 to 200 U mL-1 with the limit of detection (LOD) of 0.01 U mL-1 at signal to noise of 3. Quality control serum samples measured by our proposed immunosensor showed acceptable agreement with traditional ELISA method with the relative error less than 8.05%. The immunosensor exhibited good electrochemical performance with high reproducibility, reliability, stability and accuracy. The proposed immunosensor could be used for the determination of CA125 and had the potential for point-of-care testing (POCT) of other tumor marker.

Graphical Abstract Illustration of the assembly, distribution, and point-of-care use of a rapidly-deployable, cell-free COVID-19 biosensor: 1) Assemble: Assembling CFPS reagents by mixing E. coli lysate, murine RNase Inhibitor (mRI), energy sources, cofactors, and toehold switch riboregulator plasmid. 2) Print: aliquoting CFPS reagents onto paper substrates housed in a plastic test cassette. 3) Dehydrate: lyophilizing CFPS reagents on paper substrates. 4) Distribute. 5) Saliva sample: applying saliva samples onto cassette without pretreatments. 6) Reaction: bioluminescent protein expression in presence of target RNA (+), or ribosome detachment in absence of target RNA (-). 7) Visual result: bioluminescent output in the presence of target RNA and NanoLuc luciferase expression., The COVID-19 pandemic has illustrated the global demand for rapid, low-cost, widely distributable and point-of-care nucleic acid diagnostic technologies. Such technologies could help disrupt transmission, sustain economies and preserve health and lives during widespread infection. In contrast, conventional nucleic acid diagnostic procedures require trained personnel, complex laboratories, expensive equipment, and protracted processing times. In this work, lyophilized cell-free protein synthesis (CFPS) and toehold switch riboregulators are employed to develop a promising paper-based nucleic acid diagnostic platform activated simply by the addition of saliva. First, to facilitate distribution and deployment, an economical paper support matrix is identified and a mass-producible test cassette designed with integral saliva sample receptacles. Next, CFPS is optimized in the presence of saliva using murine RNase inhibitor. Finally, original toehold switch riboregulators are engineered to express the bioluminescent reporter NanoLuc in response to SARS-CoV-2 RNA sequences present in saliva samples. The biosensor generates a visible signal in as few as seven minutes following administration of 15 μL saliva enriched with high concentrations of SARS-CoV-2 RNA sequences. The estimated cost of this test is less than 0.50 USD, which could make this platform readily accessible to both the developed and developing world. While additional research is needed to decrease the limit of detection, this work represents important progress toward developing a diagnostic technology that is rapid, low-cost, distributable and deployable at the point-of-care by a layperson.

Mercury contamination in the environment has reached alarming levels. Due to its persistence and bioaccumulation, mercury is one of the most widespread toxic heavy metals found in air, water and food. Thus, it is mandatory to monitor mercury and its compounds, and the availability of sensitive and rapid biosensors is highly valuable.We developed a low-cost biosensor for orthogonal detection of mercury(II) integrating three different biorecognition principles on a three-leaf paper: i) a mercury-specific bioluminescent Escherichia coli bioreporter strain expressing NanoLuc luciferase as reporter protein, ii) a purified β-galactosidase (β-gal) enzyme which is irreversibly inhibited by mercury and other metal ions, and iii) an Aliivibrio fischeri bioluminescent strain which is used to quantitatively assess sample toxicity and correct the analytical signal accordingly.Both sensory elements and substrates, Furimazine for the bioluminescent reporter strain and chlorophenol red-β-D-galactopyranoside for colorimetric detection of β-gal, were integrated in the paper sensor to provide a stable all-in-one disposable cartridge which can be easily snapped into a smartphone with a clover-shaped 3D printed housing. This is the first integration of bioluminescence and colorimetric detection on a smartphone-paper sensor, providing a readout within 15 and 60 min for the colorimetric and bioluminescent detection respectively. The biosensor was applied to water samples spiked with different concentrations of mercury, interferents and toxic chemicals providing a limit of detection for Hg(II) at the ppb levels.

SARS, a new type of respiratory disease caused by SARS-CoV, was identified in 2003 with significant levels of morbidity and mortality. The recent pandemic of COVID-19, caused by SARS-CoV-2, has generated even greater extents of morbidity and mortality across the entire world. Both SARS-CoV and SARS-CoV-2 spreads through the air in the form of droplets and potentially smaller droplets (aerosols) via exhaling, coughing, and sneezing. Direct detection from such airborne droplets would be ideal for protecting general public from potential exposure before they infect individuals. However, the number of viruses in such droplets and aerosols is too low to be detected directly. A separate air sampler and enough collection time (several hours) are necessary to capture a sufficient number of viruses. In this work, we have demonstrated the direct capture of the airborne droplets on the paper microfluidic chip without the need for any other equipment. 10% human saliva samples were spiked with the known concentration of SARS-CoV-2 and sprayed to generate liquid droplets and aerosols into the air. Antibody-conjugated submicron particle suspension is then added to the paper channel, and a smartphone-based fluorescence microscope isolated and counted the immunoagglutinated particles on the paper chip. The total capture-to-assay time was

In vitro diagnostics (IVD) are essential in healthcare systems for detection of diseases, conditions, or infections. Affordable and sensitive methods remain a challenge in the development of IVD. In this article, we give an overview of paper-based fluorogenic devices, an emerging cost-effective analytical method for IVD applications. The paper-based devices are inexpensive, sensitive, selective, user-friendly and equipment-free. Furthermore, the fluorescent detection on paper-based devices has recently attracted enormous attentions due to its high sensitivity and selectivity. We summarize and compare various fluorescent materials that are used in the paper-based fluorogenic devices, including fluorescent dyes, quantum dots, metal nanoclusters, upconversion nanoparticles, and carbon dots. We review a wide range of IVD applications of the paper-based fluorogenic devices, e.g., detection of nucleic acids, proteins, cells, and so on. It may open an avenue to improve the global healthcare systems in the developing countries at point of care settings and in emergency situations.

Waste paper, a major fraction of municipal solid waste, has a potential to serve as renewable feedstock for the biorefineries of fuels, chemicals and materials due to rich in cellulose and abundant at low cost. This study evaluates the possibility of waste office paper (WOP) to serve as a potential feedstock for the biorefinery production of poly (3-hydroxybutyrate). In this study, the WOP was pretreated, enzymatically saccharified and the hydrolysate was used for PHB production. The hydrolysate mainly consists of glucose (22.70 g/L) and xylose (1.78 g/L) and the corresponding sugar yield was about 816 mg/g. Ammonium sulphate and C/N ratio 20 were identified as most favorable for high yield of PHB. The batch fermentation of Cupriavidus necator using the pretreated WOP hydrolysate resulted in cell biomass, PHB production and PHB content of 7.74 g/L, 4.45 g/L and 57.52%, respectively. The volumetric productivity and yield achieved were 0.061 g/L/h and 0.210 g/g sugar, respectively. The results suggested that WOP could be a potential alternative feedstock for the biorefinery production of bioplastics.

Renewable and bio-based materials have gained great interest on an industrial scale owing to environmental issues. Paper industries also are constantly exploring bio-resources for intrinsic chemico-physical property enhancement of paper and paper products. These bio-resources will potentially increase their cyclability besides making paper compatible beyond traditional uses. Mechanical beating or use of chemical additives or the combination of these methods are widely used to improve critical paper characteristics such as strength, surface smoothness, density, brightness, filler retention, water and grease resistivity etc. These chemical additives as mill effluents are hazardous and have detrimental effect on environment. So, to move ahead of traditional practices, the present review discusses about the production and utility of abundantly available renewable bio-polymers and their products such as starch, cellulose, plant-based proteins, microbial biopolymers, animal-based biopolymers, and natural gums etc. They represent ample prospect in terms of research and development on their functionality and industrial applications.

Demand on the quick screening of gastric cancer (GC) has significantly stimulated the development of biomarker sensing techniques. Herein, we report the novel fluorescent paper-based analytical devices (PADs) for detections of GC-related microRNA-21 (miRNA-21) and circular RNA from Hippocampus Abundant Transcript 1 gene (circRNA-HIAT1) with prominent reliability and sensitivity. The PADs, constructed by in-situ synthesis of blue-emissive carbon dots (CDs) and conjugations of probe DNAs, exhibit the superior uniformity and stability. In the presence of targets, rolling circle amplifications (RCA) are triggered to generate long DNA strands for the assemblies of green-/red-emissive labels. Consequently, remarkable blue-to-green and blue-to-red emission color transitions of the PADs are achieved, implementing the color-analysis of miRNA-21 and circRNA-HIAT1, respectively. Benefited from the efficient RCA, coupled with the drastic ratiometric fluorescent changes, the limit of detections (LODs) of PADs are found to be several fM with the upper limit of the linear detection range at 1 nM. More importantly, the fluorescent PADs possess excellent specificity, as well as anti-interference capability in biological settings, enabling their applications in accurate GC screening with plasma samples. Overall, the proposed fluorescent PADs are featured with robust sensing platform, facile signal readout, and exceptional dual-type RNA sensing performance, holding high potential in point-of-care testing (POCT).

Treatment for cancer depends on the type of cancer, and the stage or its development, and thus the need for point-of-care technology that can allow rapid and precise detection of biomarkers is increasing. Here, we present a simple on chip electrical detection of Alpha-fetoprotein (AFP). We rely on using a novel peptide modified plastic-paper microfluidic chips to perform efficient and specific impedimetric detection of AFP in human serum. The chips are prepared from a lower sheet of plastic and upper layer of cellulose chromatography paper modified with silver-20 wt% graphene printed electrodes. Diphenylalanine (FF) was proposed to involve in detection zone of the fabricated microchips in order to improve the sensing performance and the stability of immobilized antibodies according to amine-aldehyde reaction. The target protein is captured on the surface of microchips using specific monoclonal antibodies and the electrical response of the chip is monitored in the presence and absence of different concentrations of AFP. The influence of several parameters including the material types for screen printing of electrodes, FF concentrations, solvent and pH of FF solution on electrical response and cellulose fibers morphology was explored. The impedance measurements of AFP on the fabricated microchip in the optimized parameters exhibited a detection limit of 1 and 10 ng ml-1 in PBS and plasma, respectively. This platform developed here can be adopted to develop systems for rapid detection of biomarkers using portable electric devices.

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.

Although significant technological advancements have been made in the development of analytical biosensor chips for detecting bacterial strains (E. coli, S. Mutans and B. Subtilis), critical requirements i.e. limit of detection (LOD), fast time of response, ultra-sensitivity with high reproducibility and good shelf-life with robust sensing capability have yet to be met within a single sensor chip. In order to achieve these criteria, we present an electrically-receptive thermally-responsive (ER-TR) sensor chip comprised of simple filter paper used as substrate coated with composite of poly(N-isopropylacrylamide) polymer (PNIPAm) - graphene nanoplatelet (GR) followed by evaporation of Au electrodes for capturing both Gram-positive (S. mutans and B. subtilis) and Gram-negative (E. coli) bacterial cells in real-time. Autoclave water, tap water, lake water and milk samples were tested with ER-TR chip with and without bacterial strains at varying concentration range 101-105 cells/mL. The sensor was integrated with in-house built printed circuit board (PCB) to transmit/receive electrical signals. The interaction of E. coli, S. mutans and B. subtilis cells with fibers of PNIPAm-GR resulted in a change of electrical resistance and the readout was monitored wirelessly in real-time using MATLAB algorithm. Finally, prepared ER-TR chip exhibited the reproducibility of 85-97% with shelf-life of up to four weeks after testing with lake water sample.

A visual colorimetric microfluidic paper-based analytical device (μPAD) was constructed following the direct synthesis of enzyme-inorganic hybrid nanomaterials on the paper matrix. An inorganic solution of MnSO4 and KH2PO4 containing a diluted enzyme (glucose oxidase, GOx) was subsequently pipetted onto cellulose paper for the in situ growth of GOx@Mn3(PO4)2 hybrid functional materials. The characterization of the morphology and chemical composition validated the presence of hybrid materials roots in the paper fiber, while the Mn3(PO4)2 of the hybrid provided both a surface for enzyme anchoring and a higher peroxidase-like catalytic activity as compared to the Mn3(PO4)2 crystal that was synthesized without enzyme modulation. This new approach for the in situ growth of an enzyme-inorganic hybrid on a paper matrix eliminates centrifugation and the dry process by casting the solution on paper. The sensing material loading was highly reproducible because of the accuracy and stability of pipetting, which eventually contributed to the reliability of the μPAD. The self-assembled natural and artificial enzyme hybrid on the μPADs specifically detected glucose from a group of interferences, which shows great specificity using this method. Moreover, the colorimetric signal exhibited detection limitation for glucose is 0.01mM, which lies in the physiological range of glucose in biological samples.

The fluorescent paper for colorimetric detection of metal ions has been widely fabricated using various sensing probes, but it still remains an elusive task to design a test paper with multicolor variation with target dosages for accurate determination. Herein, we report a profuse color-evolution-based fluorescent test paper sensor for rapid and visual monitoring of Cu2+ in human urine by printing tricolor probe onto filter paper. The tricolor probe consists of blue-emission carbon dots (bCDs), green-emission quantum dots (gQDs) and red-emission quantum dots (rQDs), which is based on the principle that the fluorescence of gQDs and rQDs are simultaneously quenched by Cu2+, whereas the bCDs as the photostable internal standard is insensitive to Cu2+. Upon the addition of different amounts of Cu2+, the ratiometric fluorescence intensity of the tricolor probe continuously varied, leading to color changes from shallow pink to blue with a detection limit of 1.3nM. When the tricolor probe solution was printed onto a sheet of filter paper, as-obtained test paper displayed a more profuse color evolution from shallow pink to light salmon to dark orange to olive drab to dark olive green to slate blue to royal blue and to final dark blue with the increase of Cu2+ concentration compared with dual-color probe-based test paper, and dosage scale as low as 6.0nM was clearly discriminated. The sensing test paper is simple, rapid and inexpensive, and serves as a visual platform for ultrasensitive monitoring of endogenous Cu2+ in human urine.

l-dopa is a catecholamine neurotransmitter used to treat Parkinson's disease. This paper presents a low-cost paper-based biosensor aimed at enhancing the convenience of monitoring l-dopa concentrations. ZnO nanorods (ZnO-NRs) were synthesized on papers in less than 90 min using a microwave-assisted hydrothermal method. The ZnO-NRs amplify green fluorescence signals to enhance the detection sensitivity of l-dopa, best measured at excitation/emission wavelengths of 475/537 nm. We systematically characterized the effect of reaction conditions on the corresponding fluorescence enhancements. The proposed ZnO NRs-paper biosensor presented a ∼3-fold increase in green fluorescence compared to unmodified papers. The linear range of detection for l-dopa was 25-2000 nM, with a limit of detection of 24 nM, which meets the clinical requirements for the monitoring of l-dopa in Parkinson's patients.

The development of low-cost analytical devices for on-site water quality monitoring is a critical need, especially for developing countries and remote communities in developed countries with limited resources. Microbial electrochemical cell-based (MXC) biosensors have been quite promising for quantitative and semi-quantitative (often qualitative) measurements of various water quality parameters due to their low cost and simplicity compared to traditional analytical methods. However, conventional MXC biosensors often encounter challenges, such as the slow establishment of biofilms, low sensitivity, and poor recoverability, making them unable to be applied for practical cases. In response, MXC biosensors assembled with paper-based materials demonstrated tremendous potentials to enhance sensitivity and field applicability. Furthermore, the paper-based platforms offer many prominent features, including autonomous liquid transport, rapid bacterial adhesion, lowered resistance, low fabrication cost (

Paper-based microfluidic biosensors have recently attracted increasing attentions in point-of-care testing (POCT) territories benefiting from their affordable, accessible and eco-friendly features, where technologies for fabricating such biosensors are preferred to be equipment free, easy-to-operate and capable of rapid prototyping. In this work, we developed a pen-on-paper (PoP) strategy based on two custom-made pens, i.e., a wax pen and a conductive-ink pen, to fully write paper-based microfluidic biosensors through directly writing both microfluidic channels and electrodes. Particularly, the proposed wax pen is competent to realize one-step fabrication of wax channels on paper, as the melted wax penetrates into paper during writing process without any post-treatments. The practical applications of the fabricated paper-based microfluidic biosensors are demonstrated by both colorimetric detection of Salmonella typhimurium DNA with detection limit of 1 nM and electrochemical measurement of glucose with detection limit of 1 mM. The developed PoP strategy for making microfluidic biosensors on paper characterized by true simplicity, prominent portability and excellent capability for rapid prototyping shows promising prospect in POCT applications.

Graphical abstract, Highlights • A new and rigorous approach for assessing the effect of enzymatic pretreatment on AD is proposed. • Enzymes can improve anaerobic digestion increasing biogas yields by up to 26%. • First study to isolate the effect of catalytic activity and organic load from enzymes to evaluate enzymatic pretreatment. • Enzymes did not appear to be inhibited or denatured in the presence of biosludge., Anaerobic digestion of biosludge has not yet been implemented in pulp mills due to low biogas yields. Enzymatic pretreatment of biosludge has shown improvements in biogas yields but results are varied. A key limitation of previous studies is that they fail to consider the COD contribution from the enzyme solutions. The aim of this study was to systematically investigate the potential for enzymatic pretreatment on the anaerobic digestibility of pulp mill biosludge. Out of the six enzymes tested, four enhanced the anaerobic digestibility of biosludge. At the end of the BMP, a maximum improvement of 26% in biogas yield was observed with protease from B. licheniformis. There was no correlation between enzymatic activities on standard substrates and/or on biosludge and the effect of enzymes on biogas yields. Enzymes have potential for improving biosludge anaerobic digestibility but more research on optimal conditions and potential synergies with other pretreatment is needed.

Microfluidic paper-based analytical devices (μPADs) have attracted much attention over the past decade. They embody many advantages, such as abundance, portability, cost-effectiveness, and ease of fabrication, making them superior for clinical diagnostics, environmental monitoring, and food safety assurance. Despite these advantages, μPADs lack the high sensitivity to detect many analytes at trace levels than other commercial analytical instruments such as mass spectrometry. Therefore, a preconcentration step is required to enhance their sensitivity. This review focuses on the techniques used to separate and preconcentrate the analytes onto the μPADs, such as ion concentration polarization, isotachophoresis, and field amplification sample stacking. Other separations and preconcentration techniques, including liquid-solid and liquid-liquid extractions coupled with μPADs, are also reviewed and discussed. In addition, the fabrication methods, advantages, disadvantages, and the performance evaluation of the μPADs concerning their precision and accuracy were highlighted and critically assessed. Finally, the challenges and future perspectives have been discussed.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays are the gold standard for virus diagnosis. Point-of-care (POC) technologies have shown great progress during this period. Herein, we propose a novel fuchsine dye-loaded polymersome for a colorimetric paper-based dot blot spike protein diagnostic assay for COVID-19 via smartphone-assisted sensing. The prepared platform aimed to create an adaptable tool that competes with traditional nanoparticle-based assays employing gold and silver. Analytical characterization and application of the testing platform showed high sensitivity (10 times better than gold nanoparticles), stability, fast turnaround, and reproducibility. The potential and possibilities demonstrated by the current platform could be observed in its adaptability for different markers and pathologies. In addition, smartphone-assisted sensing emphasizes the ability to use the tool at home by common peoples which can lower the burden on the healthcare facilities and reach more underdeveloped regions., Research Foundation of Ege University, Turkey [TOA-2020-21862]; Republic of Turkey, Ministry of Development [2010K120810/2020K12150700, 2016K121190], The research project was supported by the Research Foundation of Ege University, Turkey (Project number: TOA-2020-21862) . Republic of Turkey, Ministry of Development provided the support for the infra-structure of EGE MATAL (Ege University/Izmir) via 2010K120810/2020K12150700 and 2016K121190 grants.

Microcystin-LR (MC-LR) is a cyanobacterial toxin produced as a result of eutrophication in polluted water in warm weather conditions. The MC-LR could cause health problems in mammal organs such as the liver, heart, and muscle. Therefore, the World Health Organization (WHO) has stipulated a limit of1.0 ng/mL in drinking water. Thus, detection and quantification are vital, but current techniques require complex and expensive offsite analysis. We have developed an inexpensive, sensitive, and field-deployable sensor based on bioactivated multiwalled carbon nanotubes (MWCNTs, diameter 20 nm) and micropore filter paper (0.45-μm pore size) for the detection of MC-LR. A specially designed DNA oligonucleotide (5-NH

In this study, a paper-based sensor combined with visual distance-readout technique for point of-care testing (POCT) of urea was developed by urease-mediated chitosan viscosity change. A series of factors that affect the performance of the sensor were investigated, including the type of filter paper, chitosan concentration, acetic acid concentration and enzymatic reaction conditions. Under optimal conditions, the proposed method for urea determination has good linearity between 3.8–15.1 mM. The limit of quantitation is 3.8 mM. Finally, the paper-based sensor was successfully applied to the determination of urea in two diesel exhaust fluid (DEF) samples. The recoveries of urea were 91.4 % and 109.9 % in DEF-1 and DEF-2, respectively. The present study provides a novel approach, which integrates paper-based sensor and visual distance-readout technique, for monitoring urea in POCT application, especially in remote or resource-limited regions.

In this work, a novel time-resolved ratiometric fluorescent probe based on dual lanthanide (Tb: terbium, and Eu: europium)-doped complexes (Tb/DPA@SiO2-Eu/GMP) has been designed for detecting anthrax biomarker (dipicolinic acid, DPA), a unique and major component of anthrax spores. In such complexes-based probe, Tb/DPA@SiO2 can serve as a stable reference signal with green fluorescence and Eu/GMP act as a sensitive response signal with red fluorescence for ratiometric fluorescent sensing DPA. Additionally, the probe exhibits long fluorescence lifetime, which can significantly reduce the autofluorescence interferences from biological samples by using time-resolved fluorescence measurement. More significantly, a paper-based visual sensor for DPA has been devised by using filter paper embedded with Tb/DPA@SiO2-Eu/GMP, and we have proved its utility for fluorescent detection of DPA, in which only a handheld UV lamp is used. In the presence of DPA, the paper-based visual sensor, illuminated by a handheld UV lamp, would result in an obvious fluorescence color change from green to red, which can be easily observed with naked eyes. The paper-based visual sensor is stable, portable, disposable, cost-effective and easy-to-use. The feasibility of using a smartphone with easy-to-access color-scanning APP as the detection platform for quantitative scanometric assays has been also demonstrated by coupled with our proposed paper-based visual sensor. This work unveils an effective method for accurate, sensitive and selective monitoring anthrax biomarker with backgroud-free and self-calibrating properties.

A novel paper-based potentiometric sensor with an enhanced response for the detection of glucose in biological fluids is presented. The electrode consists on platinum sputtered on a filter paper and a Nafion membrane to immobilize the enzyme glucose oxidase. The response obtained is proportional to the logarithm of the concentration of glucose, with a sensitivity of -119 +/- 8 mV.decade(-1), a linear range that spans from 10(-4)M to 10(-2.5) M and a limit of detection of 10(-4.5) M of glucose. It is shown that Nafion increases the sensitivity of the technique while minimizing interferences. Validation with human serum samples shows an excellent agreement when compared to standard methods. This approach can become an interesting alternative for the development of simple and affordable devices for point of care and home-based diagnostics.

Although nucleic acid amplification test (NAT) is widely used for pathogen detection, rapid NAT systems that do not require special and expensive instruments must be developed in order to enable point of care (POC)-NATs, which contribute to early initiation of treatment. As a POC-NAT system, Kaneka DNA chromatography chip (KDCC), developed using DNA tag-bound primer through modified substance, was shown to be suitable for POC testing, due to the rapid detection time, simple procedures, and low manufacturing costs. However, owing to some modifications in primer, the detection performance and amplification speed were shown to be reduced when using KDCC, counteracting the increased speed of detection. To solve these issues and improve the speed of this NAT system, we investigated a better modification substance for KDCC. Here, azobenzene-modified primers were shown to have the highest amplification speed and detection performance in KDCC, of all modifications tested in this study, showing 10-100-fold lower detection limit but maintaining the same reaction time. Additionally, rapid herpes simplex virus detection system with azobenzene modified primers was developed. We believed that this breakthrough will contribute toward enabling greater utilization of POC-NATs for medical care, especially in developing countries and clinics.

Since the inception of the first electrochemical devices on paper substrates, many different reports of microfluidic paper-based electroanalytical devices (μPEDs), innovative hydrophobic barriers and electrode fabrication processes have allowed the incorporation of diverse materials, resulting in different applications and a boost in performance. These advancements have led to the creation of paper-based devices with comparable performance to many standard conventional devices, with the added benefits of pumpless fluidic transport, component separation and reagent storage that can be exploited to automate and handle sample preprocessing. Herein, we review μPEDs, summarize the characteristics and functionalities of μPEDs, such as separation, fluid flow control and storage, and outline the conventional and emerging fabrication and modification approaches for μPEDs. We also examine the recent application of μPEDs in biomedicine, the environment, and food and water safety, as well as some limitations and challenges that must be addressed.

Materials used for biosensor development normally include silicon, glass, and synthetic polymers, however, paper is a practical and cheap option for the reduction of manufacturing costs with a wide range of applications. Paper-based biosensors have been widely produced, yet poorly characterized on the interaction of different type of molecules with its intricate microstructure. In this work, five ink solutions were prepared as model samples to examine their diffusion and imbibition behavior on grade 3MM chromatography paper. Different mathematical models, previously reported for porous matrices, were fitted and results revealed that upward wicking (r2 ≥ 0.90) equations described the experimental data during the initial stage (< 5 s) and yielded similar permeability values to those calculated from the matrix structural properties. The diffusion coefficient was determined up to attaining equilibrium using the diffusion equation in a cylinder element (r2 ≥ 0.90). This study enabled the characterization of the performance from 3MM chromatography paper, by using ink as a surrogate model of small molecules (e.g. mycotoxins) or small colloidal particles.

Personalized health care (PHC) is being appreciated globally to combat clinical complexities underlying various metabolic or infectious disorders including diabetes, cardiovascular, communicable diseases etc. Effective diagnoses majorly depend on initial identification of the causes which are nowadays being practiced in disease-oriented approach, where personal health profile is often overlooked. The adoption of PHC has shown significantly improved diagnoses in various conditions including emergency, ambulatory, and remote area. PHC includes personalized health monitoring (PHM), which is its integral part and may provide valuable information's on various clinical conditions. In PHC, bio-fluids are analyzed using various diagnostic devices including lab based equipment and biosensors. Among all types of biosensing systems, paper based biosensors are commercially attracted due to its portability, easy availability, cheaper manufacturing cost, and transportability. Not only these, various intrinsic properties of paper has facilitated the development of paper based miniaturized sensors, which has recently gained ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment free, Deliverable to all end-users) status for point of care diagnosis in miniaturized settings. In this review, importance of paper based biosensors and their compatibility for affordable and low cost diagnostics has been elaborated with various examples. Limitations and strategies to overcome the challenges of paper biosensor have also been discussed. We have provided elaborated tables which describe the types, model specifications, sensing mechanisms, target biomarkers, and analytical performance of the paper biosensors with their respective applications in real sample matrices. Different commercial aspects of paper biosensor have also been explained using SWOT (Strength, Weakness, Opportunities, Threats) analysis.

Nanocomposite (NC) papers using bifunctional nanoparticles are prepared for 3D cell growth experiments. Cells show better affinity to NC papers than to paper itself and differentiate the chemical group on the external surface of the nanoparticles. Cells possess spherical shaped morphology and form agglomerates onto the (NC) papers like as observed by cell growth in 3D materials. Furthermore, the preparation of “Janus” paper and the differentiation of its two distinct faces by cells is presented.

An electrochemical immunofiltration analysis was introduced into microfluidic paper-based analytical devices (μPADs) for the first time, which was based on photolithography and screen-printing technology. The hydrophilic test zones of the aldehyde-functionalized screen-printed electrodes (SPEs) were biofunctionalized with capture antibodies (Ab1). A sensitive immune detection method was developed by using primary signal antibody functionalized gold nanoparticles (GNPs/Ab2) and alkaline phosphatase conjugated secondary antibody (ALP-IgG). Differential pulse voltammetry (DPV) was performed to detect the electrochemical response. The microfluidic paper-based electrochemical immunosensor (μ-PEI) was optimized and characterized for the detection of human chorionic gonadotropin (HCG), a model analyte, in a linear range from 1.0mIUmL-1 to 100.0 IU mL-1 with a detection limit of 0.36mIUmL-1. Additionally, the proposed μ-PEI was used to test HCG in real human serum and obtained satisfactory results. The disposable, efficient, sensitive and low-cost μ-PEI has exhibited great potential for the development of point-of-care testing (POCT) devices that can be applicated in healthcare monitoring.

This work describes the fabrication and evaluation of an electroanalytical paper-based platform based on the combination of both, reusable and disposable materials in order to generate simple, versatile and low-cost microfluidic devices. With this aim, a holder containing metal wires that act as reusable reference and counter electrodes has been developed. The gold-sputtered paper electrode is disposable and easily interchangeable, meanwhile the platform that includes reference and counter electrodes can be reused. The detection zone in the paper is delimited by drawing a hydrophobic line with an inexpensive permanent marker. The effect of experimental variables such as adding solutions through the face where the gold was sputtered (upwards) or through the opposite one (downwards) as well as of other working parameters were studied by cyclic and differential pulse voltammetry with potassium ferrocyanide as a common redox probe and indicator species for enzymatic, immune and DNA biosensing. Enzymatic determination of glucose in real food samples prove the feasibility of the developed system for the construction of electrochemical biosensors.

The present study was aimed to develop an ultrasensitive technique for electroanalysis of ketamine; a date rape drug. It involved the fabrication of nano-hybrid based electrochemical micro fluidic paper-based analytical device (EμPADs) for electrochemical sensing of ketamine. A paper chip was developed using zeolites nanoflakes and graphene-oxide nanocrystals (Zeo–GO). EμPAD offers many advantages such as facile approach, economical and potential for commercialization. Nanocrystal modified EμPAD showed wide linear range 0.001–5 nM/mL and a very low detection limit of 0.001 nM/mL. The developed sensor was tested in real time samples like alcoholic and non-alcoholic drinks and found good correlation (99%). The hyphenation of EμPAD integrated with nanocrystalline Zeo–GO for detection of ketamine has immense prospective for field-testing platforms. An extensive development could be made for industrial translation of this fabricated device.

Neisseria meningitidis (N. meningitidis), Streptococcus pneumoniae (S. pneumoniae), and Haemophilus influenzae type b (Hib) are three most common pathogens accounting for most bacterial meningitis, a serious global infectious disease with high fatality, especially in developing nations. Because the treatment and antibiotics differ among each type, the identification of the exact bacteria causing the disease is vital. Herein, we report a polymer/paper hybrid microfluidic biochip integrated with loop-mediated isothermal amplification (LAMP) for multiplexed instrument-free diagnosis of these three major types of bacterial meningitis, with high sensitivity and specificity. Results can be visually observed by the naked eye or imaged by a smartphone camera under a portable UV light source. Without using any specialized laboratory instrument, the limits of detection of a few DNA copies per LAMP zone for N. meningitidis, S. pneumoniae and Hib were achieved within 1 hour. In addition, these three types of microorganisms spiked in artificial cerebrospinal fluid (ACSF) were directly detected simultaneously, avoiding cumbersome sample preparation procedures in conventional methods. Compared with the paper-free non-hybrid microfluidic biochip over a period of three months, the hybrid microfluidic biochip was found to have a much longer shelf life. Hence, this rapid, instrument-free and highly sensitive microfluidic approach has great potential for point-of-care (POC) diagnosis of multiple infectious diseases simultaneously, especially in resource-limited settings.

Accurate, rapid, and low-cost molecular diagnostics is essential in managing outbreaks of infectious diseases, such as the pandemic of coronavirus disease (COVID-19). Accordingly, microfluidic paper-based analytical devices (μPADs) have emerged as promising diagnostic tools. Among the extensive efforts to improve the performance and usability of μPADs, electrochemical impedance spectroscopy (EIS) based sensing mechanisms have shown great promise, because of their high sensitivity and label-free operations. However, the method to improve EIS biosensing on μPADs is less explored. Here, we present an experimental approach to enhancing the biosensing performance of paper-based EIS nanobiosensors with working electrodes (WEs) decorated with vertically grown zinc oxide nanowires (ZnO NWs). Through a comparison among different EIS settings and an examination of ZnO-WE effects on EIS measurements, we show that Faradaic processes with Fe-based electron mediators are more reliable for ZnO-NW-enhanced working electrodes. We calibrate sensors featuring varied morphologies of ZnO NWs and achieve a low limit of detection (0.4 pg ml−1) for detecting p24 antigen as a marker for human immunodeficiency virus (HIV). Through theoretical analysis, imaging, and electrochemical characterizations, we reveal that the total surface area and electrochemical surface area indicate the sensitivity and sensing range of the EIS nanobiosensors. Finally, we demonstrate that the nanobiosensors are capable of differentiating the concentrations (blank, 10 ng ml−1, 100 ng ml−1, and 1 μg ml−1) of IgG antibody (CR3022) to SARS-CoV-2 in human serum samples, thus confirming the feasibility of applying the devices to COVID-19 diagnosis. This work provides a methodology that can inform the rational design of high-performance EIS-based μPADs and has the potential to facilitate rapid diagnosis in pandemics., Highlights • Paper-based impedance biosensors can provide accurate, rapid, and low-cost diagnostics of infectious diseases. • Nanowires can be directly synthesized on paper-based electrodes for biosensing in a facile process. • Electrochemical surface areas of nanowires affect the sensitivity and detection range of impedance-based biosensors. • Testing of a protein marker specific to COVID-19 has been demonstrated.

Fluorescent test papers are promising for the wide applications in the assays of diagnosis, environments and foods, but unlike classical dye-absorption-based pH test paper, they are usually limited in the qualitative yes/no type of detection by fluorescent brightness, and the colorimetry-based quantification remains a challenging task. Here, we report a single dual-emissive nanofluorophore probe to achieve the consecutive color variations from blue to red for the quantification of blood glucose on its as-prepared test papers. Red quantum dots were embedded into silica nanoparticles as a stable internal standard emission, and blue carbon dots (CDs) were further covalently linked onto the surface of silica, in which the ratiometric fluorescence intensity of blue to red is controlled at 5:1. While the oxidation of glucose induced the formation of Fe(3+) ions, the blue emission of CDs was thus quenched by the electron transfer from CDs to Fe(3+), displaying a serial of consecutive color variations from blue to red with the dosage of glucose. The high-quality test papers printed by the probe ink exhibited a dosage-sensitive allochromatic capability with the clear differentiations of ~5, 7, 9, 11mM glucose in human serum (normal: 3-8mM). The blood glucose determined by the test paper was almost in accordance with that measured by a standard glucometer. The method reported here opens a window to the wide applications of fluorescent test paper in biological assays.

A novel colorimetric/fluorescence bimodal lab-on-paper cyto-device was fabricated based on concanavalin A (Con A)-integrating multibranched hybridization chain reaction (mHCR). The product of mHCR was modified PtCu nanochains (colorimetric signal label) and graphene quantum dot (fluorescence signal label) for in situ and dynamically evaluating cell surface N-glycan expression. In this strategy, preliminary detection was carried out through colorimetric method, if needed, then the fluorescence method was applied for a precise determination. Au-Ag-paper devices increased the surface areas and active sites for immobilizing larger amount of aptamers, and then specifically and efficiently captured more cancer cells. Moreover, it could effectively reduce the paper background fluorescence. Due to the specific recognition of Con A with mannose and the effective signal amplification of mHCR, the proposed strategy exhibited excellent high sensitivity for the cytosensing of MCF-7 cells ranging from 100 to 1.0×10(7) and 80-5.0×10(7) cellsmL(-1) with the detection limit of 33 and 26 cellsmL(-1) for colorimetric and fluorescence, respectively. More importantly, this strategy was successfully applied to dynamically monitor cell-surface multi-glycans expression on living cells under external stimuli of inhibitors as well as for N-glycan expression inhibitor screening. These results implied that this biosensor has potential in studying complex native glycan-related biological processes and elucidating the N-glycan-related diseases in biological and physiological processes.

With the increasing concerns of food safety and environmental protection, it is desirable to develop reliable, effective, and portable sensors for detection of organophosphorus pesticides (OPs). Here, a cascade reaction system integrated with threaded 3D microfluidic paper analytical device (3D μPAD) was firstly developed for background-free and visual detection of OPs in agricultural samples. Butyrylcholinesterase (BChE) hydrolyzed acetylcholine into thiocholine (TCh), which reduced MnO

A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 4 min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1-80.0 ng/mL. The detection limit was 0.03 ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring.