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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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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

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.

Paper-based point-of-care (POC) devices exhibit the advantages of simplicity, rapidity, trim sizes, and low cost, which are of particular importance for food safety, biological analysis, and medical diagnosis. However, the materials utilized to make paper-based POC rarely produce multiple signals, hampering further applications in diverse situations. Herein, we present an appealing approach, namely Colorimetric-Temperature Dual-Signal Output Sensor (CTDSS), and construct a CTDSS based on coordinative self-assembly biomimetic nanozymes Fe-GMP-L-His CPNs as a proof of concept. These CPNs mimic the structure of horseradish peroxidase (HRP), in which Fe (II) is the center, nucleotide GMP and histidine are chosen as ligands to simulate metal coordination of the pyrrole ring and protein function in HRP, respectively. This strategy allows CPNs to show an excellent peroxidase-like activity, efficiently converting H

Paper is increasingly recognized as a user-friendly and ubiquitous substrate for construction of microfluidic devices. Microfluidic paper-based analytical devices (μPADs) provide an alternative technology for development of affordable, portable, disposable and low-cost diagnostic tools for improving point of care testing (POCT) and disease screening in the developing world, especially in those countries with no- or low-infrastructure and limited trained medical and health professionals. We in this review present fabrication techniques for microfluidic devices and their respective applications for biological detection as reported to date. These include: (i) fabrication techniques: examples of devices fabricated by using two-dimensional (2D) and three-dimensional (3D) methods; (ii) detection application: biochemical, immunological and molecular detection by incorporating efficient detection methods such as, colorimetric detection, electrochemical detection, fluorescence detection, chemiluminescence (CL) detection, electrochemiluninescence (ECL) detection, photoelectrochemi (PEC) detection and so on. In addition, main advantages, disadvantages and future trends for the devices are also discussed in this review.

There is a critical and urgent demand for a simple, rapid and specific qualitative and quantitative colorimetric biosensor for the detection of the food contaminant Escherichia coli O157:H7 (E. coli O157:H7) in complex food products due to the recent outbreaks of food-borne diseases. Traditional detection techniques are time-consuming, require expensive instrumentation and are labour-intensive. To overcome these limitations, a novel, ultra-rapid visual biosensor was developed based on the ability of E. coli O157:H7 proteases to change the optical response of a surface-modified, magnetic nanoparticle-specific (MNP-specific) peptide probe. Upon proteolysis, a gradual increase in the golden color of the sensor surface was visually observed. The intensification of color was correlated with the E. coli O157:H7 concentration. The color change resulting from the dissociation of the self-assembled monolayer (SAM) was detected by the naked eye and analysed using an image analysis software (ImageJ) for the purpose of quantitative detection. This biosensor demonstrated high sensitivity and applicability, with lower limits of detection of 12 CFU mL−1 in broth samples and 30–300 CFU mL−1 in spiked complex food matrices. In conclusion, this approach permits the use of a disposable biosensor chip that can be mass-produced at low cost and can be used not only by food manufacturers but also by regulatory agencies for better control of potential health risks associated with the consumption of contaminated foods.

Nucleic acid testing (NAT), as a molecular diagnostic technique, including nucleic acid extraction, amplification and detection, plays a fundamental role in medical diagnosis for timely medical treatment. However, current NAT technologies require relatively high-end instrumentation, skilled personnel, and are time-consuming. These drawbacks mean conventional NAT becomes impractical in many resource-limited disease-endemic settings, leading to an urgent need to develop a fast and portable NAT diagnostic tool. Paper-based devices are typically robust, cost-effective and user-friendly, holding a great potential for NAT at the point of care. In view of the escalating demand for the low cost diagnostic devices, we highlight the beneficial use of paper as a platform for NAT, the current state of its development, and the existing challenges preventing its widespread use. We suggest a strategy involving integrating all three steps of NAT into one single paper-based sample-to-answer diagnostic device for rapid medical diagnostics in the near future.

We report a paper-based surface-enhanced Raman spectroscopy (SERS) biosensor integrating the enrichment capability, namely enPSERS biosensor, for the sensitive, label-free detection of free bilirubin in blood serum for the accurate diagnosis of jaundice and its related diseases. This biosensor comprises multifunctional graphene oxide-plasmonic gold nanostar (GO-GNS) hybrids decorated on the filter paper, which integrates the high sensitivity of SERS detection, enrichment for serum bilirubin and fluorescence superquenching capability of GO-GNS hybrids for sensitive detection of serum bilirubin. The study of adsorption kinetics reveals that both electrostatic and π-π interactions between the GO-GNS hybrids and targets are responsible for the enrichment of bilirubin, and the adsorption process follows the pseudo-second-order kinetic model. The results of SERS detection of bilirubin in blood serum show two differential linear response ranges from 5.0 to 150 μM and 150–500 μM with the detection limit as low as 0.436 μM. The comparison of the results obtained from our present enPSERS biosensor with the commercial diazo reaction method for determination of free bilirubin in blood serum reveals the clinical effectiveness and suitability of the developed paper-based SERS biosensor. We believe that this sensitive and label-free SERS biosensor holds considerable promise for clinical translation in accurate diagnosis of jaundice.

Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis.

In this work, we demonstrate a novel low-cost, self-powered paper-based biosensor for glucose monitoring. The device operating mechanism is based on a glucose/oxygen enzymatic fuel cell using an electrochemical energy conversion as a transducing element for glucose monitoring. The self-powered glucose biosensor features (i) a 3D origami paper-based structure for easy system integration onto paper, (ii) an air-cathode on paper for low-cost production and easy operation, and (iii) a screen printed chitosan/glucose oxidase anode for stable current generation as an analytical signal for glucose monitoring. The sensor showed a linear range of output current at 1–5 mM glucose (R2=0.996) with a sensitivity of 0.02 µA mM−1. The advantages offered by such a device, including a low cost, lack of external power sources/sophisticated external transducers, and the capacity to rapidly generate reliable results, are well suited for the clinical and social settings of the developing world.