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In the last decades, there is a growing search for analytical strategies to ensure clinical analysis without the need of laboratory set-up and skilled personnel. Indeed, user-friendly and low-cost devices are highly valued in the era of sustainability for their capability to be applied in low-resource contexts, such as developing countries. To address this issue, herein we report a 96-well paper-based and laboratory setup-free optical platform for the detection of butyrylcholinesterase enzyme (BChE) activity in human serum. We used chromatographic paper to realize a novel analytical tool exploiting its porous structure for reagentless synthesize Prussian Blue Nanoparticles (the sensing element), as well to load all the reagents required for the measurement. The principle of BChE activity detection relies on the reaction between the enzymatic product thiocholine and Prussian Blue, giving the Prussian White with subsequently Prussian Blue's fading, detected by a common office scanner supported by ImageJ software. Using this novel paper-based optical platform, BChE activity was linearly detected in the 2–15 U/mL range with a detection limit down to 0.8 U/mL. The accuracy was successfully demonstrated by recovery study with spiked serum and by comparing the data with the gold standard method.

In this work, we provided a novel strategy of antibody-free biomarker analysis by in-situ synthesized molecularly imprinted polymers (MIPs) on movable valve microfluidic paper-based electrochemical device (Bio-MIP-ePADs) for clinical detection of biomarkers. The newly movable valves on the device enable continuous and convenient delivery of fluid, which guarantee the performance for fabricating MIPs structure during long time electropolymerization. Moreover, this strategy can directly detect antigens by taking advantage of molecular imprinting on paper-based device, which greatly decreases the cost during clinical testing, reduces the tedious washing procedure and does not need to consider the preservation of the antibody in enzyme linked immunosorbent assay (ELISA). This feature makes the chip suitable for the on-site family treatment or commercial products. To further validate the applicability of this proposed method for clinical diagnostic testing, carcinoembryonic antigen (CEA) was applied as prototyping model target for the clinical analysis. The proposed Bio-MIP-ePADs were cheap, easy to prepare, disposable and provided reliable analysis by comparing with ELISA. We hope the application of this technology will open up a new avenue to the point-of-care testing (POCT).

This work reports a new paper-based sensing platform and its application in a label-free potentiometric immunosensor for Salmonella typhimurium detection based on the blocking surface principle. A paper-based strip electrode was integrated with a filter paper pad which acted as a reservoir of the internal solution. The design offers a convenient platform for antibody immobilization and sampling, proving also that is a simple and affordable methodology to control an ionic flux through a polymer membrane. Two different immunosensing interfaces were assembled on the developed paper-strip electrode. The simplest interface relied on direct conjugation of the antibody to the polymer membrane and the second one resorted to an intermediate layer of a polyamidoamine dendrimer, with an ethylenediamine core from the fourth generation. Electrochemical impedance spectroscopy was used to assess the successive interface modification steps and the resulting analytical performance of both immunosensors was compared. For such, the potential shift derived from the blocking effect of the ionic flux caused by antigen-antibody conjugation was correlated with the logarithm of the Salmonella typhimurium concentration in the sample. In optimized conditions, a limit of detection of 5 cells mL-1 was achieved. As a proof-of-concept, the proposed method was applied to apple juice samples, demonstrating to be a suitable prototype to be used in real scenarios in useful time (, This work received financial support from the European Union and National Funds(FCT, Fundação para a Ciência e Tecnologia) through projects UID/QUI/50006/2019 and Norte-01-0145-FEDER-000011-RL1–QUALIFOOD. N.F.D Silva is grateful to FCT grant SFRH/BD/112414/2015, financed by POPH–QREN–Tipologia 4.1–FormaçãoAvançada,subsidizedbyFSEandMCTES.

Introduction: Paper is the most important material of the printing industry and is being improved due to the increasing needs of industry. The most important process to improve the optical and physical properties of paper is the surface coating. Paper has a smoother and opaquer surface with surface coating. In addition, brightness, whiteness, and yellowness values are improved with surface coating. Ultraviolet (UV) light in sunlight causes changes in the structure of the paper and coating chemicals and accordingly causes yellowing. Para-amino benzoic acid (PABA), due to its chemical structure, is a UV-blocking agent used in sunscreen creams. Methods: The effect of PABA on paper gloss, yellowness, and lightfastness values was investigated by adding it into the surface coating formulation. For this purpose, coating formulations were prepared with cationic starch and 3%, 5%, and 7% PABA; coatings were applied to the paper according to laboratory condition standards. Coated papers were printed with an IGT-C1 lab-type offset printability device with magenta ink. The printed papers were then exposed to a light fastness test with Solarbox. The color, brightness, and yellowness values of all samples before and after lightfastness were measured with an X-Rite spectrophotometer. The gloss of coated and printed paper sample values was measured with a BYK-Gardner glossmeter. Results: The gloss values of paper to which PABA was added, increased; however, it was determined that these gloss values decreased after the lightfastness test. Conclusions: PABA-added cationic starch coatings are more affected by UV light than cationic starch coatings.

We present a QR code paper microfluidic colorimetric assay that can exploit the hardware and software on mobile devices, and circumvent sample preparation by directly targeting volatile biomarkers. Our platform is a printable microarray of well-defined reaction regions, which outputs an instant diagnosis by directing the user to a URL containing their test result, while simultaneously storing epidemiological data for remote access and bioinformatics. To assist in the rapid identification of Escherichia coli in bloodstream infections, we employed an existing colorimetric reagent (p-dimethylaminocinnamaldehyde) and adapted its use to detect volatile indole, a biomarker produced by E. coli. Our assay was able to quantitatively detect indole in the headspace of E. coli culture after 12 h of growth (27.0 ± 3.1 ppm), assisting in species-level identification hours earlier than existing methods. Results were confirmed with headspace solid-phase microextraction (HS-SPME) two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-ToFMS), which estimated indole concentration in E. coli culture to average 32.3 ± 5.2 ppm after 12 h of growth. This QR paper microfluidic platform represents a novel development in both telemedicine and diagnostics using volatile biomarkers. We envision that our QR code platform can be extended to other colorimetric assays for real-time diagnostics in low-resource environments.

Herein, we propose the first three-dimensional origami paper-based device for the detection of several classes of pesticides by combining different enzyme-inhibition biosensors. This device was developed by integrating two different office paper-based screen-printed electrodes and multiple filter paper-based pads to load enzymes and enzymatic substrates. The versatile analysis of different pesticides was carried by folding and unfolding the filter paper-based structure, without any addition of reagents and any sample treatment (i.e. dilution, filtration, pH adjustment). The paper-based platform was employed to detect paraoxon, 2,4-dichlorophenoxyacetic acid, and atrazine by exploiting the capability of these different types of pesticides (i.e. organophosphorus insecticides, phenoxy-acid herbicides, and triazine herbicide) to inhibit butyrylcholinesterase, alkaline phosphatase, and tyrosinase, respectively. The degree of inhibition correlating to the quantity of pesticides was evaluated by chronoamperometrically monitoring the enzymatic activity in the absence and in the presence of pesticides by using a portable potentiostat. To improve the sensitivity, the paper-based electrodes were modified with carbon black alone in the case of platforms for 2,4-dichlorophenoxyacetic acid and atrazine detection, or decorated with Prussian blue nanoparticles for the detection of paraoxon. The paper-based device was applied for the detection of paraoxon, 2,4-dichlorophenoxyacetic acid, and atrazine at ppb level in both standard solutions and river water sample. The accuracy of this origami multiple paper-based electrochemical biosensor was evaluated in river water by recovery studies, obtaining satisfactory values (e.g. for paraoxon 90 ± 1% and 88 ± 2%, for 10 and 20 ppb, respectively). The proposed three-dimensional origami paper device allows for rapid, cost-effective and accurate pesticide detection in surface water as a result of combining filter and office papers, screen-printing, wax-printing and nanomaterial technology.

Pseudomonas aeruginosa is the most important opportunistic pathogen leading to serious and life-threatening infections, especially in immunocompromised patients. Because of its remarkable capacity to resist antibiotics, the selection of the right antibiotics with the exact dose for the appropriate duration is critical to effectively treat the infections and prevent antibiotic resistance. Although conventional genotypic and phenotypic antibiotic susceptibility testing (AST) methods have been dramatically advanced, they have suffered from many technical and operational issues as a generalized antibiotic stewardship program. Furthermore, given that most microbial infections are caused by their biofilms, the existing AST methods do not provide evidence-based antibiotic prescribing guidance for biofilm-based infections because the results are based on individual bacteria traditionally grown in their planktonic form. In this work, we create an innovative susceptibility testing technique for P. aeruginosa that offers clinically relevant guidelines and widely adaptable stewardship to effectively treat the infections and minimize antibiotic resistance. Our approach evaluates the antibiotic efficacy by continuously monitoring the accumulated electrical outputs from the bacterial extracellular electron transfer (EET) process in the presence of antibiotics. Our innovative paper-based culturing 3-D scaffold promotes surface-associated growth of bacterial colonies and biofilms. The platform replicates a natural habitat for P. aeruginosa where it can grow similarly to sites it infects. Our technique enables an all-electrical, real-time, easy-to-use, portable AST that can be easily translatable to clinical settings. The entire procedure takes 96 min to provide evidence-based antimicrobial prescribing guidance for biofilm-based infections.

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.

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.

The development of point-of-care bioassays for sensitive screening of protein-based cancer biomarkers would improve the opportunity for early stage diagnosis. A strategy for a fluorescence resonance energy transfer (FRET)-based bioassay has been investigated that makes use of modified cellulose paper for the detection of an epithelial cell adhesion molecule (EpCAM), which is a transmembrane glycoprotein that is overexpressed in several tumors of epithelial origin. The paper matrix was a substrate for immobilized aptamer-linked quantum dots (QDs-Apt) and Cy3 labeled complementary DNA (cDNA), which served as a donor and an acceptor, respectively. Competitive binding of EpCAM displaced the cDNA, resulting in the reduction of FRET. The paper-based bioassay was able to detect EpCAM in buffer solution as well as in 10% bovine serum solution using a reaction time of no more than 60 minutes. The dynamic range was 1-100 nM in buffer with a precision better than 4%, and the limit of detection was 250 pM in buffer and 600 pM in 10% serum.

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.

Filter paper provides an excellent matrix for retention of proteins containing a cellulose binding domain. To use this capability for manipulating recombinant fusion proteins, binding and elution parameters were explored and procedures developed for small scale purification, modification and assay. Proteins were tagged with the cellulose binding domain from theC thermocellumCipB gene via a cleavable linker. Filter paper disks of 6mm diameter were able to bind up to 80 μg protein although there was a substantial dependence on molecular size. Different means of introducing fusion proteins to the disks allow either binding within 20 minutes from microliter volumes or slower binding from milliliter volumes. Elution with protease in small volumes yielded greater than 10 μg amounts with concentrations in the 1-2 mg/ml range. To demonstrate their utility, disks were used for small scale protein purification, covalent modification of protein, immunoprecipitation, and in a binding assay. These versatile methods allow parallel processing of multiple samples and may find many uses when only small amounts of protein are needed.

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.

The recent outbreaks of mosquito-borne diseases (e.g., zika, dengue, and chikungunya) increased public health burden in developing countries. To control the spread of these infectious diseases, a simple, economic, reliable, sensitive, and selective diagnostic platform is required. Considering demand for affordable and accessible methods, we have demonstrated a two-step strategy for extraction and detection of viral RNAs of infectious diseases within 1 h. Ready-to-use devices for viral RNA extraction and detection were successfully fabricated using paper as a substrate. Viral RNA (e.g., zika, dengue, and chikungunya) was captured and eluted using a handheld RNA extraction paper-strip device, and another paper-chip device was used for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with a detection limit of a single copy and 10 copies of viral RNA in phosphate buffer solution (PBS) and serum, respectively. With these proposed devices, we have detected viral RNAs of zika and dengue in clinical human serum samples. The proposed paper-based extraction and detection platforms could be employed for detection of infectious viral diseases from complex clinical samples in resource-limited settings.

This paper introduces a paper-based closed Au-bipolar electrode (BPE) biosensing system for the rapid and sensitive electrochemiluminescence (ECL) detection of miRNA-155. This microfluidic paper-based sensing platform is formed by wax-printing technology, screen printing method and in-situ Au nanoparticles (NPs) growth to form hydrophilic cells, hydrophobic boundaries, water proof electronic bridge, driving electrode regions and bipolar electrode regions. For rapid and sensitive detection, the cathode of bipolar electrode was modified with the prepared DNA (S1)–AuPd NPs by hybridization chain reaction, in which the target could initiate multiple cycles reaction to load more AuPd NPs which catalyzed H2O2 reduction. In addition, a classical ECL system tris (2,2′-bipyridine) ruthenium (II)- tripropylamine (Ru(bpy)32+/TPrA) exists at the anode of the bipolar electrode. Due to the charge balance between the anode and the cathode of BPE, the ECL signal response of Ru(bpy)32+/TPrA system was enhanced in the reporting cell. The intensity of ECL was quantitatively correlated with the concentration of miRNA-155 in the range of 1 pM–10 μM with the detection limit 0.67 pM. Moreover, this method paves a novel way for highly sensitive detection of miRNA-155 in clinical application.

Rapid detection of pathogenic bacteria is extremely important for public health and safety. Here, we describe for the first time an integrated origami paper-based analytical device (PAD) incorporating cell lysis, molecular recognition, amplification and visual detection of Escherichia coli (E. coli). The device features three components: paper for its ability to extract protein molecules nonspecifically from cells, DNA superstructures for their ability to immobilize RNA-cleaving DNAzymes (RCDs) but undergo target-induced RNA cleavage on paper, and isothermal rolling circle amplification (RCA) for its ability to amplify each cleavage event into repetitive sequence units that can be detected by naked eye. This device can achieve detection of E. coli K12 with a detection limit of as low as 103 CFU&middot, mL&minus, 1 in a total turnaround time of 35 min. Furthermore, this device allowed the sensitive detection of E. coli in complex sample matrices such as juice and milk. Given that more specific RCDs can be evolved for diverse bacteria, the integrated PAD holds great potential for rapid, sensitive and highly selective detection of pathogenic bacteria in resource-limited settings.

Mushroom cultivation is an economically feasible bio-technological process for conversion of various lignocellulosic wastes. The aim of this study was to evaluate the suitability of waste paper supplemented with corn stalk and wheat bran for oyster mushroom cultivation. Pure culture of Oyster mushroom (Pleurotus ostreatus (Jacq.: Fr.) Kummer) was purchased from YB Plant Micropropagation Plc; Mekelle, Ethiopia. Then, the pure culture was used as inoculum for spawn preparation using sorghum prepared in Microbiology laboratory, Department of Biology, Aksum University. Waste paper supplemented with corn stalk and wheat bran with 0%, 25% and 50% were prepared. The substrates were mixed with the spawn that has been inoculated with pure culture of oyster mushroom aseptically for their productivity and biological efficiency (BE) for cultivation of P. ostreatus mushroom. Data were analyzed using SPSS version 20. Higher (26.20 ± 19.36) mean weight, pileus diameter (7.90 ± 2.66 cm), total yield (646.4 ± 273.1 g) and BE (64.64 ± 273%) were obtained from waste paper (50%) supplemented with cornstalk (25%) and wheat bran (25%). And lower (17.92 ± 81.95%) BE were obtained from waste paper (100%). Moreover, the highest (3.88 ± 0.32 cm) mean stalk length was obtained from waste paper (50%) supplemented with corn stalk (50%). This study revealed that waste paper supplemented with corn stalk and wheat bran resulted in high BE and total yield. Thus, utilization of waste paper appears to be a promising alternative for the cultivation of oyster mushroom when supplemented with other substrates.

Paper-based analytical devices (PADs) are very popular for point-of-care diagnostics, which provide a fast, cost-effective and possible multiplexed detection of a spectrum of molecules. They have been matching forward proudly to contribute to the modern analytical science and life science. Accompanying with their advantages and huge potentials, low detection sensitivity is continuing to challenge the application of PADs from bench to bedside. In order to improve the sensitivity and enhance the signal readout, variable signal amplification strategies have been investigated and applied for PADs. In this review, we have firstly classified formats of PADs according to the engineering design. Advances for improving sensitivity of PADs in recent five years are then summarised according to three popular types of signal amplification strategies (nanomaterial based, nucleic acid based, and engineering of PADs based). Pros and cons of each signal amplification approach have been discussed accordingly. Finally, the future perspectives of PADs are proposed.

In this work, we present a multiplexed (eight simultaneous measurements) paper-based electrochemical device developed in a very simple way and using low-cost materials, such as paper, carbon ink and multifunctional connector headers. Meanwhile, we have also combined the paper-based electrochemical platform with a glass-fiber strip in order to integrate easily a sampling step. Both approaches, simultaneous measuring and sampling, have been applied to the determination of glucose using bienzymatic biosensors. They are fabricated by adsorbing the mixture of enzymes (glucose oxidase and horseradish peroxidase), as well as the ferrocyanide, mediator of the electron transfer, on the paper-based electrode. After drying, the measuring solution (containing either glucose standards or samples) is added and the eight corresponding chronoamperograms are recorded. In the case of the microfluidic approach for sampling purposes, the glass-fiber pad (sampler) is immersed in a container with the solution, which flows by capillarity until it reaches the working electrode. The integration of one more step of the analytical process advances towards real and useful lab-on-a-chip devices. With these designs, a linear range comprised between 0.5 and 15 mM was achieved for glucose determination, with an excellent precision. If the sampler is employed, it is not necessary to use micropipettes and, nevertheless, precise measurements are obtained. The RSD of the slopes obtained for different calibrations performed in different days, with different arrays of electrochemical cells and different solutions is ca. 1%. Accurate results are obtained in the determination of glucose in real samples (orange fruit and cola beverages).

Background Until now, environmental sustainability issues are almost entirely unsolved for packaging materials. With the final aim of finding materials with a single recycling channel, cellulose fiber/poly(vinyl)alcohol composites were investigated. Methods After extrusion and injection molding, samples of composite with different cellulose fiber content (30%, 50% and 70% w/w) were tested. Results Tensile mechanical tests exhibited an improvement in composite stiffness when the reinforcement content was increased together with a decrease in composite elongation. Solubility tests performed at room temperature and 45°C showed different behavior depending on the water-resistant film applied on the composite (50% cellulose fiber content). In particular, the uncoated composite showed complete solubility after 2 hours, whereas at the same time point, no solubility occurred when a non-water-soluble varnish was used. Conclusions The proposed composites, named Poly-paper, appear to warrant further investigation as highly sustainable packaging.

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.

Convenient biosensor for simultaneous multi-analyte detection was increasingly required in biological analysis. A novel flower-like silver (FLS)-enhanced fluorescence/visual bimodal platform for the ultrasensitive detection of multiple miRNAs was successfully constructed for the first time based on the principle of multi-channel microfluidic paper-based analytical devices (µPADs). Fluorophore-functionalized DNA1 (DNA1-N-CDs) was combined with FLS, which was hybridized with quencher-carrying strand (DNA2-CeO2) to form FLS-enhanced fluorescence biosensor. Upon the addition of the target miRNA, the fluorescent intensity of DNA1-N-CDs within the proximity of the FLS was strengthened. The disengaged DNA/CeO2 complex could result in color change after joining H2O2, leading to real-time visual detection of miRNA firstly. If necessary, then the fluorescence method was applied for a accurate determination. In this strategy, the growth of FLS in µPADs not only reduced the background fluorescence but also provided an enrichment of "hot spots" for surface enhanced fluorescence detection of miRNAs. Results also showed versatility of the FLS in the enhancement of sensitivity and selectivity of the miRNA biosensor. Remarkably, this biosensor could detect as low as 0.03fM miRNA210 and 0.06fM miRNA21. Interestingly, the proposed biosensor also possessed good capability of recycling in three cycles upon change of the supplementation of DNA2-CeO2 and visual substitutive device. This method opened new opportunities for further studies of miRNA related bioprocesses and will provide a new instrument for simultaneous detection of multiple low-level biomarkers.

Herein, a ratiometric electrochemiluminescence (ECL) lab-on-paper platform, based on enhancing effect of the N-(4-aminobutyl)-N-ethylisoluminol-functionalized glutathione-coated cobalt/gold bimetallic nanoclusters (GCAL) and quenching effect of carbon quantum dots (CQDs), as well as benefiting from high conductivity copper paper electrode, was developed to achieve accurate and sensitive analysis of DNA methylation. Concretely, due to one-to-multiple amplification effect, one target DNA could initiate the formation of supersandwich structure with multiple signal probes labeled with GCAL. Thus, the GCAL signal enhancement effect was realized. Simultaneously, multiple signal probes are connected to hinder the electron conduction rate on the electrode surface, which results in the quenching of CQDs signal. With the method proposed here, wide linear relationship in the range of 1 fM to 10 pM with a low detection limit of 0.27 fM for sensitive detecting methylated DNA was achieved. It is believed that this work provided a rapid, low-cost and stabilized method for specific determination of methylated DNA, and the immediate detection of cancer in the clinic will become possible in the future.

Various strategies have been proposed for the sensing of acetylcholinesterase (AChE) activity and organophosphorus pesticides (OPs). However, the practical application of most methods is restricted by their intrinsic drawbacks such as complexity, long analysis time, and high cost. Thus, it is highly desirable to develop simple, fast and sensitive approaches for AChE activity and OPs detection. Herein, we reported a simple paper-based fluorescent sensor (PFS) based on the aggregation induced emission (AIE) effect of tetraphenylethylene (TPE) and the addition reaction capability of maleimide, which has been used as a powerful tool for rapid naked-eye detection of AChE activity and OPs. The introduction of TPE provides the probe with unique fluorescence property in solid state and is of great importance for improving the sensitivity of PFS. The hydrolysis product of acetylthiocholine catalyzed by AChE induced the maleimide ring destruction and activated the fluorescence performance of TPE. Given that AChE activity can be specifically inhibited by OPs, the as-proposed PFS can also be utilized for sensitive detection of OPs. Meanwhile, the variation of fluorescence signal can be readily detected by naked eyes, and low detection limits of 2.5mUmL(-1) and 0.5ngmL(-1) for AChE activity and OPs are obtained, respectively. Moreover, it has been successfully applied for AChE activity and OPs detection in diluted human serum samples, showing its great potential to be applied in real samples. Thus, this strategy possesses considerable advantages of simplicity, rapid detection, portability, cost efficiency and visualization.

In recent years, it has been proposed that unrealistic requirements for academics and medical doctors to publish in scientific journals, combined with monetary publication rewards, have led to forms of contract cheating offered by organizations known as paper mills. Paper mills are alleged to offer products ranging from research data through to ghostwritten fraudulent or fabricated manuscripts and submission services. While paper mill operations remain poorly understood, it seems likely that paper mills need to balance product quantity and quality, such that they produce or contribute to large numbers of manuscripts that will be accepted for publication. Producing manuscripts at scale may be facilitated by the use of manuscript templates, which could give rise to shared features such as textual and organizational similarities, the description of highly generic study hypotheses and experimental approaches, digital images that show evidence of manipulation and/or reuse, and/or errors affecting verifiable experimental reagents. Based on these features, we propose practical steps that editors, journal staff, and peer reviewers can take to recognize and respond to research manuscripts and publications that may have been produced with undeclared assistance from paper mills.

This World Federation for Ultrasound in Medicine and Biology position paper reviews the diagnostic potential of ultrasound contrast agents for clinical decision-making and provides general advice for optimal contrast-enhanced ultrasound performance in musculoskeletal issues. In this domain, contrast-enhanced ultrasound performance has increasingly been investigated with promising results, but still lacks everyday clinical application and standardized techniques; therefore, experts summarized current knowledge according to published evidence and best personal experience. The goal was to intensify and standardize the use and administration of ultrasound contrast agents to facilitate correct diagnoses and ultimately to improve the management and outcomes of patients.

In this note we describe a four-channel paper microfluidic device (μPAD) that utilizes gold nanoparticles (AuNPs) as colorimetric sensors and specifically tailored aptamers to capture a set of targeted drugs, including cocaine, codeine and methamphetamine. The design utilizes salt-induced aggregation of AuNPs to produce a color change that indicates the presence of target analytes. In the absence of the targets, the aptamers bind to AuNPs, preventing aggregation. This μPAD was developed by optimizing the concentrations of aggregating agents, AuNPs and aptamers. The resultant device is sensitive and specific, producing a positive result upon detection of the target analyte.

Recently, surface enhanced Raman scattering (SERS) has attracted much attention in medical diagnosis applications owing to better detection sensitivity and lower limit of detection (LOD) than colorimetric detection. In this paper, a novel calibration-free SERS−based μPAD with multi-reaction zones for simultaneous quantitative detection of multiple cardiac biomarkers — GPBB, CK−MB and cTnT for early diagnosis and prognosis of acute myocardial infarction (AMI) are presented. Three distinct Raman probes were synthesised, subsequently conjugated with respective detecting antibodies and used as SERS nanotags for cardiac biomarker detection. Using a conventional calibration curve, quantitative simultaneous measurement of multiple cardiac biomarkers on SERS-based μPAD was performed based on the characteristic Raman spectral features of each reporter used in different nanotags. However, a calibration free point-of-care testing device is required for fast screening to rule-in and rule-out AMI patients. Partial least squares predictive models were developed and incorporated into the immunosensing system, to accurately quantify the three unknown cardiac biomarkers levels in serum based on the previously obtained Raman spectral data. This method allows absolute quantitative measurement when conventional calibration curve fails to provide accurate estimation of cardiac biomarkers, especially at low and high concentration ranges. Under an optimised condition, the LOD of our SERS-based μPAD was identified at 8, 10, and 1 pg mL−1, for GPBB, CK−MB and cTnT, respectively, which is well below the clinical cutoff values. Therefore, this proof-of-concept technique shows significant potential for highly sensitive quantitative detection of multiplex cardiac biomarkers in human serum to expedite medical decisions for enhanced patient care.

This study presents a slidable paper-embedded plastic microdevice fully integrated with DNA extraction, loop-mediated isothermal amplification (LAMP), and colorimetric detection functionalities. The developed microdevice consists of three layers that allow a sliding movement to mix the sample and reagents for DNA purification, amplification, and detection in a sequential manner. An FTA card was employed in the main chamber for DNA extraction and purification from intact bacterial cells. Subsequently, LAMP reagents and fuchsin-stored chambers were pulled toward the main chambers for DNA amplifications at 65 °C. After 30 min, the detection reagents-stored chambers were then moved to main chambers for result analysis. For the detection of LAMP amplicons, a novel colorimetric fuchsin-based method was employed. The wide applicability of the integrated microdevice was demonstrated by successfully screening three major foodborne pathogens, namely Salmonella spp., Staphylococcus aureus, and Escherichia coli O157:H7 in food, enabling highly sensitive detection of 3.0 × 101 CFU/sample of Gram-negative bacteria (Salmonella spp. and Escherichia coli O157:H7) and 3.0 × 102 CFU/sample of Gram-positive bacteria (Staphylococcus aureus) within 75 min. The portable and integrated microdevice presented in this study holds significant promise for point-of-care applications to accurately and rapidly diagnose and control diseases.

A novel paper-based colorimetric glucose sensor was proposed employing Prussian blue nanoparticles (PB NPs) as mimic peroxidase. The sensor was manufactured by spraying solution containing PB NPs, glucose oxidase and chromogenic agents into a paper, then coating the filter layer and spreading layer on the top. The layer-by-layer structure enabled the sensor detect glucose in whole blood, as well as elimination of the coffee-ring effect which ensure the performance. As a powerful alternative to natural peroxidase, PB NPs showed the mimic enzymatic activity well preserved in dry environment. The manufacture process of the sensor is easy to be industrialized. Under optimal conditions, the sensor exhibited a linear range from 2.5 mM to 25 mM for glucose in blood with satisfactory reproducibility (the coefficient of variant4%), great storage stability (1 month at 45 °C) and excellent linearity compared with those commercial kits (R 0.99). Coupled with a handhold device, the PB NPs-based test strip realized the goal of personal operation, user-friendly control, automatic readouts, and data storage, and able to link the Cloud, showing unique potential in clinical application, especially in community-level medical scenarios.

Analysis of cytokines levels in human serum is critical as it can be a "symptom diagnostic biomarker" in COVID-19, giving real-time information about human health status. Here, we present the construction and performance of a low-price immunosensor (∼US$0.428 per test) based on microfluidic paper-based system to detect cytokine for predicting the health status of COVID-19 patients. Interleukin-6 (IL-6) was selected as the detection model for the close relationship between IL-6 and COVID-19. The assay, which we integrated into foldable paper system, leverages the magnetic immunoassay, the streptavidin-horseradish peroxidase (HRP) associated with tetramethyl benzidine/hydrogen peroxide (TMB/H

Electronic devices with multifunctional capabilities is forever an attractive area with diverse scope including towards developing solutions to sustainable energy technology. Microbial biofuel cells (MiBFCs) are one such sustainable energy technology based electronic device which can not only harvest energy, but can perform biosensing leading to bioremediation. However, low energy yield, costly fabrication procedures and bulky devices are some of the limitations of such MiBFCs. In this work, for the first time a simple vacuum filtration fabrication technique is used for making thin and conductive electrodes with homogeneous CNT solution for MiBFC application. The fully paper-based MiBFC is integrated into a compact micro device with 3D printed components which adds novelty to the work. The MiBFC is capable of maintaining a stable open circuit voltage of 410 mV for more than 1 h and can deliver a maximum power density of 192 μW/cm

Surface-enhanced Raman scattering (SERS) spectroscopy is an effective approach for trace-level detection of molecular substance. Plasmonic metallic nanostructures with high distribution densities and small gap widths are always expected for constructing SERS substrates. We report here a paper-based SERS substrate, where the three-dimensional (3D) network of paper fibers was used as the platform for supporting the gold nanoparticle clusters. Such a 3D arrangement of plasmonic porous clusters supplies high-density hotspots with large total volume and large surface area for the interaction with molecules. Comparison between different papers found that the filter papers commonly available in labs are the best choice. An enhancement factor higher than 104 has been achieved in the detection of R6G molecules. The preparation of such SERS substrates is very simple and convenient, implying low-cost, disposable, and environment-friendly SERS techniques. Furthermore, the paper-based flexible SERS substrates can be easily tailored into different shapes and sizes for fitting different applications.

A variety of routine methods are available for the detection of silver (I) (Ag+) ions, but most of them rely on expensive, sophisticated and desktop instruments. Herein, a low-cost, instrument-free and portable Ag+ biosensor was described by initially designing a new class of 3D origami microfluidic paper-based analytical devices (μPADs) into each of which one piece of reagent-loaded nanoporous membrane was integrated. It combines analyte-triggered self-growing of silver nanoparticles to block the membrane's pores in situ for rapid yet efficient signal amplification with a handheld personal glucose meter for a portable and sensitive quantitative readout based on the biocatalytic reactions between the glucose oxidase and glucose. Its utility is well demonstrated with the specific detection of the analyte with a limit of detection as low as ∼58.1 pM (3σ), which makes this new biosensing method one of the most sensitive Ag+ assays in comparison with many other typical methods recently reported. Moreover, the satisfactory recovery of analyzing several types of real water examples, i.e., tap water, drinking water, pond water and soil water, additionally validates its feasibility for practical applications.

A versatile paper-based immunosensor with dual fluorescence (FL) and chemiluminescence (CL) detection strategy is established for sensitive detection of prostate specific antigen (PSA). A multifunctional and stable NH2-MIL-53(Fe), which shows a strong FL and catalytic capacity for luminol-H2O2 CL system, is conjugated to detection antibodies by simple glutaraldehyde cross-linking method to fabricate the immunosensor. PSA is detected by this dual detection strategy with the detection limits of 0.3 ng/mL for CL and 0.2 ng/mL for FL. The dual-mode detection is achieved by FL and CL methods at one detection area for the first time with high sensitivity and accuracy. This simple, and low-cost detection strategy can expand the development and application of MOFs in paper-based immunoassay.

COVID-19 has evolved into a global pandemic. Early and rapid detection is crucial to control of the SARS-CoV-2 transmission. While representing the gold standard for early diagnosis, nucleic acid tests for SARS-CoV-2 are often complicated and time-consuming. Serological rapid antibody tests are characterized by high rates of false-negative diagnoses, especially during early infection. Here, we developed a novel nanozyme-based chemiluminescence paper assay for rapid and sensitive detection of SARS-CoV-2 spike antigen, which integrates nanozyme and enzymatic chemiluminescence immunoassay with the lateral flow strip. The core of our paper test is a robust Co–Fe@hemin-peroxidase nanozyme that catalyzes chemiluminescence comparable with natural peroxidase HRP and thus amplifies immune reaction signal. The detection limit for recombinant spike antigen of SARS-CoV-2 was 0.1 ng/mL, with a linear range of 0.2-100 ng/mL. Moreover, the sensitivity of test for pseudovirus could reach 360 TCID50/mL, which was comparable with ELISA method. The strip recognized SARS-CoV-2 antigen specifically, and there was no cross reaction with other coronaviruses or influenza A subtypes. This testing can be completed within 16 min, much shorter compared to the usual 1-2 h required for currently used nucleic acid tests. Furthermore, signal detection is feasible using the camera of a standard smartphone. Ingredients for nanozyme synthesis are simple and readily available, considerably lowering the overall cost. In conclusion, our paper test provides a high-sensitive point-of-care testing (POCT) approach for SARS-CoV-2 antigen detection, which should greatly facilitate early screening of SARS-CoV-2 infections, and considerably lower the financial burden on national healthcare resources., Highlights • Point-of-care antigen tests for screening of SARS-CoV-2 infection were established. • Chemiluminescence improves sensitivity of paper test and benefits early screening. • Nanozyme with high peroxidase activity and stability catalyzes chemiluminescence. • Nanozyme chemiluminescence paper test brings high efficiency and low cost.

Cell culture-based intestinal models are important to evaluate nanoformulations intended for oral drug delivery. We report the use of a floating structured paper chip as a scaffold for Caco-2 cells and HT29-MTX-E12 cells that are two established cell types used in intestinal cell models. The formation of cell monolayers for both mono- and cocultures in the paper chip are confirmed and the level of formed cell-cell junctions is evaluated. Further, cocultures show first mucus formation between 6-10 days with the mucus becoming more pronounced after 19 days. Hybrid vesicles (HVs) made from phospholipids and the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) in different ratios are used as a representative soft nanoparticle to assess their mucopenetration ability in paper chip-based cell cultures. The HV assembly is characterized, and it is illustrated that these HVs cross the mucus layer and are found intracellularly within 3 h when the cells are grown in the paper chips. Taken together, the moist three-dimensional cellulose environment of structured paper chips offers an interesting cell culture-based intestinal model that can be further integrated with fluidic systems or online read-out opportunities.

Pesticides are largely used at worldwide level to improve food production, fulfilling the needs of the global population which is increasing year by year. Although pesticides are beneficial for crop production, their extensive use has serious consequences for the pollution of the produced food as well as for soil and groundwaters. Indeed, it is reported that 50% of sprayed pesticides reach different destinations other than their target species, including soil, surface waters, and groundwaters. For this reason, we developed a flower-like origami paper-based device for pesticides detection in aerosol phase for precision agriculture. In detail, the paper-based electrochemical platform detects paraoxon, 2,4-dichlorophenoxyacetic acid, and glyphosate at ppb levels by measuring their inhibitory activity towards three different enzymes namely butyrylcholinesterase, alkaline phosphatase, and peroxidase enzyme, respectively. This integrated electrochemical device is composed of three office paper-based screen-printed electrodes and filter paper-based pads loaded with enzymes and enzymatic substrates. The pesticide detection is carried out by measuring through chronoamperometric technique the initial and residual enzymatic activity by using a smartphone-assisted potentiostat and evaluating the percentage of inhibition, proportional to the amount of aerosolized pesticides. This paper-based device was able to detect the three classes of pesticides in aerosol phase with limits of detection equal to 30 ppb, 10 ppb, and 2 ppb, respectively for 2,4-D, glyphosate, and paraoxon.

Surging interests in point-of-care diagnostics have led to the development of many lightweight and cost-effective paper-based sensors. Particularly, sensors using colorimetric readouts are considered highly advantageous because no additional detector or device is required for signal display. Herein, we introduce an electrochemically operated colorimetric sensor that can compensate for the disadvantages of traditional colorimetry, hence enhancing response time, reusability and color uniformity. On a single paper substrate, carbon/graphite paste was screen printed to form the working and counter electrodes, and Ag/AgCl ink was applied for the reference electrode. Prussian blue and Glucose oxidase were employed on the one of the carbon electrodes for the detection of analytes, hydrogen peroxide and glucose. For the colorimetric readout, indium tin oxide nanoparticles and polyaniline were consecutively introduced on the other carbon electrode, which is used as the counter electrode. The color change of electrochromic polyaniline could be clearly observed, and its application as a colorimetric sensor was demonstrated by the quantitative analyses of hydrogen peroxide and glucose. This paper-based electrochromic glucose sensor showed a short response time of 30 s and exhibited a detection limit of 126 μM for glucose. Along with its rapid and easy detection by incorporating the merits of electrochemical sensing and colorimetry, the paper-based electrochromic sensor could potentially contribute to the development of point-of-care devices by combination with portable power sources.

Heavy metal pollution has shown great threat to the environment and public health worldwide. Current methods for the detection of heavy metals require expensive instrumentation and laborious operation, which can only be accomplished in centralized laboratories. Various microfluidic paper-based analytical devices have been developed recently as simple, cheap and disposable alternatives to conventional ones for on-site detection of heavy metals. In this review, we first summarize current development of paper-based analytical devices and discuss the selection of paper substrates, methods of device fabrication, and relevant theories in these devices. We then compare and categorize recent reports on detection of heavy metals using paper-based microfluidic devices on the basis of various detection mechanisms, such as colorimetric, fluorescent, and electrochemical methods. To finalize, the future development and trend in this field are discussed.

We have explored AuNPs (13 nm) both as a catalyst and as a core for synthesizing water-dispersible and highly stable core-shell structural gold@Prussian blue (Au@PB) nanoparticles (NPs). Systematic characterization by transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) disclosed AuNPs coated uniformly by a 5 nm thick PB layer. Au@PB NPs were attached to single-layer graphene oxide (GO) to form Au@PB decorated GO sheets. The resulting hybrid material was filtered layer-by-layer into flexible and free-standing GO paper, which was further converted into conductive reduced GO (RGO)/Au@PB paper via hydrazine vapour reduction. High-resolution TEM images suggested that RGO papers are multiply sandwich-like structures functionalized with core-shell NPs. Resulting sandwich functionalized graphene papers have high conductivity, sufficient flexibility, and robust mechanical strength, which can be cut into free-standing electrodes. Such electrodes, used as non-enzymatic electrochemical sensors, were tested systematically for electrocatalytic sensing of hydrogen peroxide. The high performance was indicated by some of the key parameters, for example the linear H2O2 concentration response range (1-30 μM), the detection limit (100 nM), and the high amperometric sensitivity (5 A cm(-2) M(-1)). With the advantages of low cost and scalable production capacity, such graphene supported functional papers are of particular interest in the use as flexible disposable sensors.