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In this work, we developed a microfluidic paper-based analytical device (μPAD) for the on-line isolation of proteins and the electrochemical detection of lead ions (Pb(II)) in urine samples. The patterned filter paper was prepared through the direct printing of microchannel patterns on filter paper using an office laser printer. The paper was modified with protein precipitant and was then coupled with a detachable three-electrode system. Experimental parameters, namely, modification reagents, microchannel length and width, deposition potential, and deposition time, were optimized. Then, the maximum protein concentration under which the device can function was obtained as 300 mg L−1. The linear range was 10–500 μg L−1 with a detection limit of 9 μg L−1. The effectiveness of this device was demonstrated through the quantification of Pb(II) in urine samples and the results agreed with those of atomic absorption spectrometry (AAS).

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

Development of portable, sensitive and reliable devices for Ochratoxin A (OTA) detection is highly demanded, especially for resource-limited regions. Herein, a novel paper-based analytical device (PAD) is designed through wax printing and screen-printed technologies, which integrates sample flowing, electrode modification, cleaning and electrochemical (EC)/colorimetric signal output. To greatly enhance the detection sensitivity, we synthesized a chitosan functionalized MoS2–Au@Pt (Ch-MoS2-Au@Pt) via electrostatic self-assembly, and used it to immobilize the label aptamer (apta2) for signal regulation and amplification. Concretely, with the addition of analytes, the Ch-MoS2-Au@Pt-apta2 could be combined on the sensing interface by specific biorecognition and catalyzed reduction of H2O2, resulting in a remarkable EC response. Meanwhile, the released hydroxyl radicals (·OH) flowed to the visualization zone and promoted the oxidation of 3,3′,5,5′-tetramethylbenzidine for colorimetric detection. Consequently, the dual-mode PAD achieved acceptable prediction and accurate analysis in the range of 0.1–200 ng mL−1 and 1 × 10−4-200 ng mL−1 by matching the visual and EC signal intensity, respectively. Compared with traditional single-mode sensor for OTA, the proposed dual-mode aptasensor featuring independent signal conversion and readout, not only avoided the false-positive signal associated with detection condition and operation, but also enlarged the detection ranges and improved the sensitivity. Furthermore, the consistency of EC/colorimetric assay was validated in real OTA samples. Overall, this work provided a portable, cost-effective, sensitive and visualized aptasensor platform, which could be extended to various other mycotoxins in the field of food safety.

Bisphenol A (BPA) and its halogenated analogs tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) are common environmental contaminants and a method for their simultaneous determination is urgently needed. A paper-based analytical device (PAD) was prepared using a metal-organic framework of UiO-66-NH2 coated with molecularly imprinted polymers (MIPs) using TBBPA as a template. The maximum adsorption capacity was 120.94 mg g−1 and the imprinting factor was 4.07. The selective recognition ability of this PAD enabled the effective separation of TBBPA, TCBPA and BPA based on paper chromatography. Subsequently, the PAD cut into segments were used individually to determine the presence of target chemicals using a highly sensitive fluorescent method. Under ultraviolet light irradiation, UiO-66-NH2 acts as a photocatalyst to produce reactive oxygen species (ROS) that degrade TBBPA, TCBPA or BPA in the imprinted cavities and the fluorescent signal of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) added as a ROS probe enabled the indirect determination of target chemicals. This method could determine BPA and its halogenated analogs in dust samples simultaneously with detection limits ranging from 0.14 to 0.30 ng g−1. The intraday relative standard deviation (RSD) was ≤6.8% and interday RSD was ≤8.1%. The recoveries ranged from 91.0 to 105.6% with RSD values that were ≤7.5%. The results stemmed from this method were consistent with those obtained from LC-MS/MS. It is an environmentally-friendly approach due to the degradation of target pollutants and possesses many advantages such as high selectivity, low cost and easy-to-fabrication.

For in-situ disease markers detection, point-of-care (POC) diagnosis has great advantages in speed and cost compared with traditional techniques. The rapid diagnosis, prognosis, and surveillance of diseases can significantly reduce disease-related mortality and trauma. Therefore, increasing attention has been paid to the POC diagnosis devices due to their excellent diagnosis speed and portability. Over the past ten years, paper-based microfluidic aptasensors have emerged as a class of critical POC diagnosis devices and various aptasensors have been proposed to detect various disease markers. However, most aptasensors need further improvement before they can actually enter the market and be widely used. There is thus an urgent need to sort out the key points of preparing the aptasensors and the direction that needs to be invested in. This review summarizes the representative articles in the development of paper-based microfluidic aptasensors. These works can be divided into paper-based optical aptasensors and paper-based electrochemical aptasensors according to their output signals. Significant focus is applied to these works according to the following three parts: (1) The ingenious design of device structure; (2) Application and synthesis of nanomaterial; (3) The detection principle of the proposed aptasensor. This is a detailed and comprehensive review of paper-based microfluidic aptasensors. The accomplishments and shortcomings of the current aptasensors are outlined, the development direction and the future prospective are given. It is hoped that the research in this review can provide a reference for further development of more advanced, more effective paper-based microfluidic aptasensors for POC disease markers diagnosis.

Applications of electrochemical detection methods in microfluidic paper-based analytical devices (μPADs) has revolutionized the area of point-of-care (POC) testing towards highly sensitive and selective quantification of various (bio)chemical analytes in a miniaturized, low-coat, rapid, and user-friendly manner. Shortly after the initiation, these relatively new modulations of μPADs, named as electrochemical paper-based analytical devices (ePADs), gained widespread popularity within the POC research community thanks to the inherent advantages of both electrochemical sensing and usage of paper as a suitable substrate for POC testing platforms. Even though general aspects of ePADs such as applications and fabrication techniques, have already been reviewed multiple times in the literature, herein, we intend to provide a critical engineering insight into the area of ePADs by focusing particularly on the practical strategies utilized to enhance their analytical performance (i.e. sensitivity), while maintaining the desired simplicity and efficiency intact. Basically, the discussed strategies are driven by considering the parameters potentially affecting the generated electrochemical signal in the ePADs. Some of these parameters include the type of filter paper, electrode fabrication methods, electrode materials, fluid flow patterns, etc. Besides, the limitations and challenges associated with the development of ePADs are discussed, and further insights and directions for future research in this field are proposed.

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.

To date, 55% of inhabitants of the developing world still live in rural regions and they have a very urgent need to improve the level of disease diagnosis, especially in resource-limited settings. Herein, we present a microfluidic system that centrifuges whole blood and quantifies contained biomarkers in a rapid, feasible and integrated way. Such a device provides a blood-in/answer-out capability and is small enough to be carried by any individual in any environment. The successful integration a hand-powered centrifuge and immunoassay unit within a rotational paper-based device allows for diagnostic application by untrained users and in environments where access to electricity cannot be assumed. In addition, the low cost (~$ 0.5), light weight and small instrumental footprint make the device ideally suited for rapid on-site detection. To validate the applicability of the system in a clinical diagnostic testing, we successfully perform enzyme-linked immunosorbent assay (ELISA) analysis of carcinoembryonic antigen and alpha fetoprotein from human blood samples. We expect that this powerful platform technology will provide the opportunities for point-of-care diagnosis in resource-limited settings.

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.

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.

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.

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

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.

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 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.

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.

Simultaneous monitoring of electrophysiological and biochemical signals is of great importance in healthcare and fitness management, while the fabrication of highly integrated and flexible devices is crucial to these applications. Herein, we devised a multifunctional and flexible hydrogel-paper patch (HPP) that was capable of simultaneously real-time monitoring of electrocardiogram (ECG) signal and biochemical signal (glucose content) in sweat during exercise. The self-assembly of the highly porous PEDOT:PSS hydrogel on paper fiber provided the HPP with good conductivity and hydrophilic wettability for efficient electron transmission and substance diffusion, thereby enabling it to serve as a low-impedance ECG electrode and a highly sensitive glucose sensor. Additionally, the spontaneous capillary flow effect allows the paper patch to be used as microfluidic channels for the collect and analysis of sweat. Moreover, the HPP is integrated with a flexible printed circuit board (FPCB) and works as a multifunctional wearable device mounted on the chest for real-time monitoring of electrophysiological and biochemical signals during exercise.

A long-standing goal has been to create artificial enzymes with natural enzyme-like catalytic activity. Herein, a laccase-mimicking catalyst (GSH-Cu) is designed by simulating the copper active sites and spatial amino acid microenvironment of natural enzymes. In particular, the engineered GSH-Cu shows a catalytic function that conforms to Michaelis-Menten kinetics of natural laccase. The high catalytic activity of GSH-Cu can be easily inhibited by thiram through surface passivation to produce copper nanoparticles. We demonstrate that the developed GSH-Cu with high stability and recyclability can be used to fabricate effective colorimetric sensor for sensitive detection of thiram. The resulting absorption intensity can be employed to quantify thiram in the range of 2.5-250 ng mL

Gastrointestinal fistula, a complication of gastrointestinal cancer surgery, has a high mortality rate. Detection of both C-reactive protein (CRP) and prealbumin (PAB) is advantageous in the auxiliary diagnosis of postoperative complications. However, traditional detection methods are not capable of on-site rapid detection. In an attempt to overcome these challenges, a multifunctional origami-paper-based device (ePADs) was developed to simultaneously detect CRP and PAB in whole blood. After integration, functionalization, and modification, the electrochemical dual-parameter device was capable of separating blood cells and detecting target analytes. The plasma separation performance revealed a sample diffusion time of 75 s for a whole blood sample volume of 73.3 μL. The efficiency of the device in separating blood cells was 99.91%. Electrochemical results showed that the multifunctional device exhibited linearity between 5 pg mL

Graphene papers (GPs) have revolutionized the area of sensors toward low-cost, user-friendly and wearable/portable owning to their unique properties such as scalable production capacity, tunable microstructure, and extraordinary mechanical flexibility. They can be utilized as versatile building blocks by regulating their architectures to enhance various properties like electronic property, thermal conductivity and mechanical strength. Furthermore, thanks to the excellent compatibility, GPs as support materials are able to associate with other materials through various interaction forces to realize specific functionalization. With the impressive performances mentioned above, lots of smart devices like flexible sensors, advanced wearable/stretchable electronics, and high-performance point-of-care analytical devices are continually and rapidly being developed. Herein, this review aims at offering a critical engineering insight from smart architecture of macroscopic GPs to sensor application, with the emphasis on purpose-driven specific dual-mode functionalization (structural functionalization and foreign materials functionalization). We summarize the multiple possibilities to functionalize graphene and its derivative based papers/films/membranes. The sensing application of GPs in human health management, food contaminant inspection, and environmental monitoring is evaluated just before leading to our conclusions and perspectives.

Cyanobacterial harmful algal blooms in freshwater systems can produce cyanotoxins, such as microcystins (MCs) and nodularins (NODs), presenting serious threats to human health and ecosystems. Required routine monitoring of cyanotoxins in water samples, as posed by U.S. EPA drinking water contaminant candidate list 5 (CCL5), demands for cost-effective, reliable and sensitive MCs/NODs detection methods. We report the development of a colorimetric paper-based immunochip assisted by nanozyme catalysis with a smartphone readout system for rapid detection of cyanotoxins in water. We show that the introduction of biorthogonal click reaction enables in situ facile self-assembly of multi-layers of peroxidase-like nanozyme onto the anti-MCs/NODs monoclonal antibody. We can detect 13 variants of MCs/NODs even in the sub-microgram per liter range with detection limit of below 0.7 μg/L and satisfactory recovery percentages between 88 and 120% in different water matrices. Our technology shows a good correlation with the well-developed ELISA technology, demonstrating its great potential applications in resource-limited or less-developed regions for on-site and large-scale screening of cyanotoxins in water environment.

The research progress in the centrifugal microfluidic platform provides great opportunities for simple and effective analytical measurements in a variety of areas including biomedical engineering. In this study, we propose an optical reader that can measure the transmittance in a very sensitive and rapid manner on a hybrid paper-polymer centrifugal disc platform. This device enables real-time monitoring of multiple samples by measuring the absorbance of the light transmitted through the paper integrated on the disc between the light emitting diode (LED) and the photodiode (PD) regardless of the ambient light condition. To confirm its efficiency, we analyzed one of the blood's important indicators, glucose in a successful manner within 10 s without any additional complex image analysis. In addition, we discussed the results by comparing with the reflectance-based methods and with those of the previously reported studies by introducing a figure of merit to evaluate the performance of the assay.

We present a novel vertical flow-based paper immunosensor for the rapid and sensitive electrochemical and colorimetric detection of influenza H1N1 viruses using a different pore size (DP) sample pad. The DP sample pad consisted of two different pore size papers: larger pores (diameter: 11 µm) facing the inlet, and smaller pores (diameter: 0.45 µm) facing the conjugate pad. This sample pad offered moderate and uniform flows, and hence concentrated horseradish peroxidase tagged antibodies (HRP-Abs)-H1N1 complexes from 40 µL of sample volumes on a conjugate pad for 2 min after sample injection, thereby providing fast detection (6 min for both detection methods) with 100 µL of flushing afterwards, high sensitivity, and the simplicity of the sensor. The filtration characteristics of the DP sample pad were evaluated using fluorescent beads, indicating that only small-sized bio-particles such as viruses can pass through the sample pad. Sandwich immunoreactions of HRP-Ab-H1N1-Ab were performed on the gold paper electrode of the immunoStrip, which was determined by electrochemical impedance spectroscopy (EIS) measurements. Simultaneously, the color signal of free HRP-Ab captured on the colorimetric zone was obtained using a scanner, and the intensity was analyzed using ImageJ. This immunosensor detected H1N1 virus concentration as low as 3.3 plaque forming units (PFU)/mL (phosphate buffer saline; PBS) and 4.7 PFU/mL (saliva) by EIS, and 1.34 PFU/mL (PBS) and 2.27 PFU/mL (saliva) by the colorimetric method. Furthermore, integrating these two detection methods can reduce false results with double assurance, and this device can provide a simple and economical on-site detection platform.

A novel dual-mode cytosensor based on polyhedral AuPd alloy nanoparticles (PH-AuPd NPs) and three-dimentional reduced graphene oxide (3D-rGO) was constructed for highly sensitive detection of MCF-7 cells. The 3D-rGO was in situ synthesized on the paper working electrode (PWE) by a pollution-free hydrothermal method, increasing the specific surface area and further facilitating the modification of Au nanoparticles (AuNPs). After modified with AuNPs, the Au@ 3D-rGO/PWE was then functionalized by aptamer H1 to trap MCF-7 cells. To construct the cytosensor, PH-AuPd NPs was prepared as a novel catalytic material, and further modified with aptamer H2 for recognizing MCF-7 cells. With the occurrence of efficient recognition of MCF-7 cells, PH-AuPd NPs were bound onto the surface of the cells, and could catalyze H2O2 to generate •OH, leading to an amplified electrochemical signal. Meanwhile, as the electrolyte solution flowed, the •OH are transferred outward to the colorimetric detection zone, and catalyzed a chromogenic substrate TMB forms a colored product. The electrical signal measurement and colorimetric detection were carried out on a compatibly designed lab-on-paper device (LPD), realizing a dual-mode signal readout. This paper-based dual-mode cytosensor provided a relatively low detection limit of 20 cells mL−1 and a sensitive detection from 50 cells mL−1 to 107 cells mL−1 for MCF-7 cells, providing a reliable pathway of sensitively detecting cancer cells in clinical applications.

17β-estradiol (17β-E2) plays a critical role in regulating reproduction in human, there is therefore an urgent need to detect it sensitively and precisely in a cost-effective and easy method. In this paper, a label-free integrated microfluidic paper-based analytical device was developed for highly sensitive electrochemical detection of 17β-E2. The microfluidic channel of the paper-based sensor was fabricated with wax printing and the three electrodes, including working, counter and reference electrode were screen-printed. Multi-walled carbon nanotubes (MWCNTs)/ thionine (THI)/ gold nanoparticles (AuNPs) Nano composites were synthesized and coated on screen-printed working electrode (SPWE) for the immobilization of anti-E2. In this electro-chemical system of paper-based immunoassay, THI molecules serving as an electrochemical mediator while MWCNTs and AuNPs, due to their excellent electrical conductivities, could accelerate electron transfer for the signal amplification. Experimental results revealed that the immunoassay is able to detect 17β-E2 as low as 10 pg mL−1, with a linear range from 0.01 to 100 ng mL−1. This microfluidic paper-based immunosensor would provide a new platform for low cost, sensitive, specific, and point-of-care diagnosis of 17β-E2.

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is β-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

Rapid and accurate clinical assessment of hemostasis is essential for managing patients who undergo invasive procedures, experience hemorrhages, or receive antithrombotic therapies. Hemostasis encompasses an ensemble of interactions between the cellular and non-cellular blood components, but current devices assess only partial aspects of this complex process. In this work, we describe the development of a new approach to simultaneously evaluate coagulation function, platelet count or function, and hematocrit using a carbon nanotube-paper composite (CPC) capacitance sensor. CPC capacitance response to blood clotting at 1.3 MHz provided three sensing parameters with distinctive sensitivities towards multiple clotting elements. Whole blood-based hemostasis assessments were conducted to demonstrate the potential utility of the developed sensor for various hemostatic conditions, including pathological conditions, such as hemophilia and thrombocytopenia. Results showed good agreements when compared to a conventional thromboelastography. Overall, the presented CPC capacitance sensor is a promising new biomedical device for convenient non-contact whole-blood based comprehensive hemostasis evaluation.

On-site multiplex enzyme detection is crucial for diagnosis, therapeutics and prognostic. To date, it is still a daunting challenge to develop portable, low-cost, and efficient multi-enzyme detection methods. Herein, a novel sample-in-result-out platform integrating ratiometric fluorescent assays with 3D origami microfluidic paper-based device (μPAD) was developed for simultaneous visual point-of-care testing (POCT) of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Cascade catalytic reaction with the same two fluorescent signal indicators was rationally designed to ratiometric fluorescent detection of ALP and BChE: substrate of ALP (pyrophosphate) and product of BChE (thiocholine) can strongly complex with Cu2+, Cu2+ oxidizes o-phenylenediamine to fluorescent 2,3-diaminophenazine (oxOPD) (emission, 565 nm), oxOPD quenches the fluorescence of carbon dots (CDs, emission at 445 nm) via inner filter effect, thus oxOPD/CDs values are relevant to ALP and BChE activities. Then 3D origami μPAD composing of four layers and two parallel channels was fabricated and simply prepared by one-step plotting with black oil-based marker and specific metal molds. After simple folding and unfolding neighboring layers to sequentially initiate reactions of pre-loaded reagents, fluorescent images on the detection zone can be captured by smartphone and analyzed by red-green-blue software for quantitative analysis. Under optimal conditions, the proposed platform was successfully performed to detect ALP and BChE with activity difference at 3 orders of magnitude in human serum samples without any pretreatment procedures. Excellent selectivity, good precision, favorable linear range, and high accuracy were exhibited. Importantly, the platform opens a promising horizon for high-throughput POCT of multiplex biomarkers.

In this work, an electrochemical biosensor based on Au nanorods (NRs) modified microfluidic paper-based analytical devices (μPADs) were constructed for sensitive detection of microRNA (miRNA) by using cerium dioxide - Au@glucose oxidase (CeO2-Au@GOx) as an electrochemical probe for signal amplification. Au NRs were synthesized by in-situ growth method in μPADs surface to enhance the conductivity and modified hairpin probe through Au-S bonds. The construction of "the signal transducer layer" was carried out by GOx catalyzing glucose to produce H2O2, which was further electrocatalyzed by CeO2. After the biosensor was constructed, an obvious electrochemical signal was observed from the reduction of H2O2. In order to make the detection more convincing, the visual detection was performed based on the oxidation of 3,3',5,5'-tetramethylbenzidine by H2O2 with the help of Exonuclease I. The electrochemical biosensor provided a wide linear range of 1.0fM to 1000fM with a relatively low detection limit of 0.434fM by the electrochemical measurement. Linear range of 10fM to 1000fM with a relatively low detection limit of 7.382fM was obtained by visual detection. The results indicated the proposed platform has potential utility for detection of miRNA.

A novel fluorescent paper-based DNA sensor employing a highly specific pyrrolidinyl peptide nucleic acid (acpcPNA) probe was developed for the sensitive and selective detection of hepatitis C virus (HCV). The acpcPNA was covalently immobilized onto partially oxidized cellulose paper via reductive alkylation between the amine and the aldehyde groups. The fluorescence-based detection was performed by monitoring the fluorescence signal response of a fluorescent dye that selectively binds to the single-strand region of the DNA target over the PNA probe employing a custom-made portable fluorescent camera gadget in combination with a smartphone camera. Under the optimal conditions, a linear relationship between the fluorescence change in the green channel and the amount of HCV DNA from 5 to 100 pmol with a correlation coefficient of 0.9956, and the limit of detection of 5 pmol were obtained for short synthetic oligonucleotides. The acpcPNA probe exhibited very high selectivity for the complementary oligonucleotides over the single-base-mismatched, two-base-mismatched, and non-complementary DNA targets. Benefitting from the signal amplification achieved through the numerous binding sites for the dye provided by the overhanging tail of long ssDNA target sequences, this system was successfully applied to detect the HCV complementary DNA (cDNA) obtained from clinical samples with satisfactory results. The proposed fluorescent paper-based sensor demonstrated a great potential to be used as a low-cost, simple, label-free, sensitive, and selective DNA sensor for point-of-care applications.

It is very important to develop a rapid, simple, low cost point-of-care (POC) method for the early diagnosis of pathogens. In this work, a flexible paper-based electrode based on nickel metal-organic framework (Ni-MOF) composite/Au nanoparticles/carbon nanotubes/polyvinyl alcohol (Ni–Au composite/CNT/PVA) was constructed to detect target human immunodeficiency virus (HIV) DNA by DNA hybridization using methylene blue (MB) as a redox indicator. The CNT/PVA and Ni–Au composite were deposited on the cellulose membrane by vacuum filtration and drop-coating method in turn to obtain Ni–Au composite/CNT/PVA (CCP) film electrode. Compared to the CNT/PVA film electrode, CCP film electrode makes a higher loading of the probe DNA for its large specific surface area and conjugated π-electron system that can provide hydrogen bond sources to achieve interactions between MOF and single-stranded DNA, which improves the sensitivity for detecting target DNA. The variation of peak current for MB molecules adsorbed onto DNA before and after hybridization with HIV DNA was monitored. Electrochemical results proved that the CCP film maintained stable electrochemical property even after bending 200 times or stretching under different strains from 0% to 20%. The flexible paper electrode showed excellent sensing performance with a linear range of 10 nM–1 μM and a low detection limit of 0.13 nM. The target HIV DNA was successfully detected even in complex serum samples using the flexible CCP film electrode. Therefore, the simple and inexpensive flexible paper-based MOF composite film electrode can also be utilized for other pathogens POC diagnosis.

Respiratory syncytial virus (RSV) infection is the most common clinical infectious disease threatening the safety of human life. Herein, we provided a sensitive and specific method for detection and differentiation of RSV subgroups A (RSVA) and B (RSVB) with colorimetric toehold switch sensors in a paper-based cell-free system. In this method, we applied the toehold switch, an RNA-based riboswitch, to regulate the translation level of β-galactosidase (lacZ) gene. In the presence of target trigger RNA, the toehold switch sensor was activated and the expressed LacZ hydrolyzed chromogenic substrates to produce a colorimetric result that can be observed directly with the naked eye in a cell-free system. In addition, nucleic acid sequence-based amplification (NASBA) was used to improve the sensitivity by amplifying target trigger RNAs. Under optimal conditions, our method produced a visible result for the detection of RSVA and RSVB with the detection limit of 52 aM and 91 aM, respectively. The cross-reaction of this method was validated with other closely related respiratory viruses, including human coronavirus HKU1 (HCoV-HKU1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Furthermore, we used the paper-based carrier material that allows stable storage of our detection elements and rapid detection outside laboratory. In conclusion, this method can sensitively and specifically differentiate RSVA and RSVB and generate a visible colorimetric result without specialized operators and sophisticated equipment. Based on these advantages above, this method serves as a simple and portable detector in resource-poor areas and point-of-care testing (POCT) scenarios.

Herein, an environment friendly paper-based biobattery is demonstrated that yields a power of 12.5W/m3. Whatman filter papers were used not only as support for electrode fabrication but also as separator of the biobattery. To provide electrical conductivity to the paper-based cathode and anode, commercially available eyeliner containing carbon nanoparticles and Fe3O4 was directly employed as conductive ink without any binder. With an instant start-up, the as-fabricated biocompatible electrodes could hold bacteria in an active form at the anode allowing chemical oxidation of organic fuel producing current. The facile process delineated here can be employed for the tailored electrode fabrication of various flexible energy harnessing devices.

The development of smartphone–based biosensors for point-of-care testing (POCT) applications allows realizing “all in one” instruments, with large potential distribution among the general population. With this respect, paper color-based detection performed by reflectance measurement is the most popular, simple, inexpensive and straightforward method. Despite the large number of scientific publications related to these biosensors, they still suffer from a poor detectability and reproducibility related to inhomogeneity of color development, which leads to low assay reproducibility. To overcome these problems, we propose a smartphone paper-based biosensor, in which all the reagents necessary to complete the analysis are co-entrapped on paper in a “wafer”-like bilayer film of polyelectrolytes (Poly (allyl amine hydrochloride/poly(sodium 4-styrene sulfonate)). Using a 3D printing low-cost technology we fabricated the smartphone-based device that consists in a cover accessory attached to the smartphone and incorporating a light diffuser over the flash to improve the image quality, a mini dark box and a disposable analytical cartridge containing all the reagents necessary for the complete analysis. The biosensor was developed exploiting coupled enzyme reactions for quantifying L-lactate in oral fluid, which is considered a biomarker of poor tissue perfusion, a key element in the management of severe sepsis, septic shock and in sports performance evaluation. The developed method is sensitive, rapid, and it allows detecting L-lactate in oral fluid in the relevant physiological range, with a limit of detection of 0.1mmolL−1. The extreme simplicity of assay execution (no reagents need to be added) and flexibility of fabrication of the device, together with the high assay versatility (any oxidase can be coupled with HRP-based color change reaction) make our approach suitable for the realization of smartphone-based biosensors able to non-invasively detect a large variety of analytes of clinical interest.

In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL-1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1-100ngmL-1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA.

Hydrogen peroxide (H2O2), an important reactive oxygen species (ROS), is related to the oxidative stress in organisms, and plays important roles in a variety of cellular activities as well. So it is of crucial importance to develop sensitive and accurate sensing strategies to detect H2O2 in biological systems. Herein, by taking advantage of the unique emission characteristics of aggregation induced emission (AIE) fluorogens, we proposed a non-enzymatic fluorescence platform for facile and sensitive detection of H2O2, both in solution state using fluorescence spectrometer and on paper-based sensor via visual inspection. Through the reaction between L-cysteine and H2O2, the fluorescence of TPE-M-L, an AIE fluorogen formed between maleimide-functionalized tetraphenylethene (TPE-M) and L-cysteine, is quenched, and highly sensitive non-enzymatic H2O2 assay is readily carried out. The limit of detection (LOD) of 10nM in solution state and 2.5μM on paper-based sensor were obtained for H2O2 detection, which were superior or comparable to those previously reported in literature. Moreover, by integrating glucose oxidase with the AIE fluorogen of TPE-M-L, highly sensitive and selective glucose detection was also conveniently achieved both in solution state and on paper-based sensor by the as-proposed strategy, with the LODs of 50nM in solution state and 10μM via visual observation, much better than those obtained by other fluorescence methods. The as-proposed sensing strategy was also successfully applied to assay glucose in human serum samples. Therefore, the paper-based fluorescence sensor exhibits the advantages of simple fabrication, high sensitivity and portability, and has great potential to be applied in on-site assay of H2O2 and glucose in real samples.

We introduce for the first time a paper-based analytical device (PAD) for the non-enzymatic detection of glucose by modifying a screen-printed carbon electrode with cobalt phthalocyanine, graphene and an ionic liquid (CoPc/G/IL/SPCE). The modifying composite was characterized by UV-visible spectroscopy, energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The disposable devices show excellent conductivity and fast electron transfer kinetics. The results demonstrated that the modified electrode on PADs had excellent electrocatalytic activity towards the oxidation of glucose with NaOH as supporting electrolyte (0.1M). The oxidation potential of glucose was negatively shifted to 0.64V vs. the screen-printed carbon pseudo-reference electrode. The paper-based sensor comprised a wide linear concentration range for glucose, from 0.01 to 1.3mM and 1.3-5.0mM for low and high concentration of glucose assay, respectively, with a detection limit of 0.67µM (S/N = 3). Additionally, the PADs were applied to quantify glucose in honey, white wine and human serum. The disposable, efficient, sensitive and low-cost non-enzymatic PAD has great potential for the development of point-of-care testing (POCT) devices that can be applied in healthcare monitoring.

In order to achieve high predictive value of cell chemosensitivity test for clinical efficacy, cancer cells were suggested to be encapsulated and cultured in hydrogel to mimic the natural microenvironment of tumors. However, handling 3D cells/hydrogel culture construct is tedious and cellular response is difficult to be quantitatively analyzed. In the current study, a novel platform for conducting 3D cell culture and analyzing cell viability has been developed towards a high throughput drug screening. Cells encapsulated in the hydrogel were cultured in the microwells of a paper substrate. The substrate was then immersed in the culture medium containing drug for 2 days. At 24 and 48h during the culture course, the paper substrate was placed on the measurement electrodes for conducting the impedance measurement in order to quantify the cell viability in the hydrogel. Cell viability of two human hepatoma cell lines (Huh7 and Hep-G2) was quantitatively investigated under the treatment of two drugs (doxorubicin and etoposide). The results represented by IC50 values revealed that Huh7 cells had a higher drug resistance than Hep-G2 cells and doxorubicin had a higher efficacy than etoposide for treating hepatocellular carcinoma. The current work has demonstrated a high throughput, convenient, and quantitative platform for the investigation of chemosensitivity of cells cultured in the 3D environment.

A paper-based microfluidic device based on unconventional principle was developed and used to detect lead ions through a two-step process including heated incubation and subsequent mixing. The device was made by generating a superhydrophobic pattern, which defines channel and reservoir barriers, on a water-impermeable paper substrate, followed by loading and drying the reagents in the defined reservoirs. Different from the conventional paper-based devices that are made of water-permeable paper, the as-prepared device holds water drops in discrete reservoirs, and the water drops will not move unless the device is titled along the direction of the predefined channels. In this way, the liquid samples applied onto the device are handled as individual drops and could be stored, transported, and mixed on demand. Different from the conventional paper-based devices that use capillary force to drive liquid, our new device uses wetting and gravity as driving force. We name this operation principle suspending-droplet mode paper-based device (SD-μPAD). The use of a Teflon contact-printing stamp makes the production of such devices rapid, cost efficient, and mass productive. Utilizing a G-quadruplex-based luminescence switch-on assay, we demonstrated rapid, convenient, highly sensitive, and low cost detection of lead(II) ions in water samples, using a custom made battery-powered portable device, and a smart phone as the detector.

In this article, we describe a facile method named as Rubik's Cube stamping (RCS) for equipment-free fabrication of microfluidic paper-based analytical devices (μPADs). RCS is inspired by the worldwide ubiquitous RC toy and requires no specialized electric equipment other than a classical six-faced RC that is assembled with home-made small iron components. It can pattern various rosin microstructures in paper simply by either using different functional faces of the modified RC or applying its internal pivot mechanism to adjust the components' patterning forms on one functional face. Such a versatile stamping method is quite simple and inexpensive, and thus holds potential for producing rosin-patterned μPADs by untrained users in resource-limited environments such as small laboratories and private clinics, or even at home and in the field. Moreover, a set of one-channel devices are fabricated to design a point-of-care aptamer-based assay with near sample-in-answer-out capability that integrates enzymatic reactions for robust yet efficient signal amplification and a personal glucometer for portable, user-friendly, rapid and quantitative readout. Its utility is well demonstrated with the sensitive and specific detection of adenosine as a model target in buffer samples and undiluted human urine within several minutes. With the advantages of low cost, simplicity, portability, rapidity, and aptamer variety, this general point-of-care assay system reported here may find broad applications including home healthcare, field-based environmental monitoring or food analysis and emergency situations.

In this work, aiming at the construction of a disposable, wireless, low-cost and sensitive system for bioassay, we report a closed bipolar electrode electrochemiluminescence (BPE-ECL) sensing platform based on graphite paper as BPE for the first time. Graphite paper is qualified as BPE due to its unique properties such as excellent electrical conductivity, uniform composition and ease of use. This simple BPE-ECL device was applied to the quantitative analysis of oxidant (H2O2) and biomarker (CEA) respectively, according to the principle of BPE sensing-charge balance. For the H2O2 analysis, Pt NPs were electrodeposited onto the cathode through a bipolar electrodeposition approach to promote the sensing performance. As a result, this BPE-ECL device exhibited a wide linear range of 0.001-15mM with a low detection limit of 0.5µM (S/N=3) for H2O2 determination. For the determination of CEA, chitosan-multi-walled carbon nanotubes (CS-MWCNTs) were employed to supply a hydrophilic interface for immobilizing primary antibody (Ab1); and Au@Pt nanostructures were conjugated with secondary antibody (Ab2) as catalysts for H2O2 reduction. Under the optimal conditions, the BPE-ECL immunodevice showed a wide linear range of 0.01-60ngmL-1 with a detection limit of 5.0pgmL-1 for CEA. Furthermore, it also displayed satisfactory selectivity, excellent stability and good reproducibility. The developed method opened a new avenue to clinical bioassay.

Loop-mediated isothermal amplification (LAMP) has been widely used for detecting pathogens. However, power-free and clear visualization of results still remain challenging. In this study, we developed a paper device integrated with power-free DNA detection strategy realized by polydopamine aggregation. In the presence of DNA amplicons, the polymerization of dopamine into aggregated polydopamine was hindered, while in the absence of DNA amplicons, polydopamine aggregation is facilitated. The porosity of the paper enabled the capillary flow of dispersed polydopamine for positive sample, while aggregated polydopamine remained at the bottom of the paper strip due to large size of the aggregates for negative sample. Based on this mechanism, we fabricated a slidable paper device integrating LAMP with dopamine polymerization for the naked-eye detection, operated in a seamless manner. Moreover, the introduced paper device was successfully used to detect DNA extracted from Escherichia coli O157:H7 and SARS-CoV-2 within 25 min, as well as Enterococcus faecium within 35 min. The detection limits of both Escherichia coli O157:H7 and SARS-CoV-2 were 10−4 ng/μL. The introduced paper device can be used as a simple and sensitive tool for detecting multiple infectious pathogens, making it an ideal tool particularly for resource-limited environment.

This paper describes the development of a novel paper-based capillary electrophoresis (pCE) microdevice using mineral paper, which is durable, oil and tear resistant, and waterproof. The pCE device is inexpensive (~$1.6 per device for materials), simple to fabricate, lightweight, and disposable, so it is more adequate for point-of-care (POC) pathogen diagnostics than a conventional CE device made of glass, quartz, silicon or polymer. In addition, the entire fabrication process can be completed within 1h without using expensive clean room facilities and cumbersome photolithography procedures. A simple cross-designed pCE device was patterned on the mineral paper by using a plotter, and assembled with an OHP film via a double-sided adhesive film. After filling the microchannel with polyacrylamide gel, the injection, backbiasing, and separation steps were sequentially operated to differentiate single-stranded DNA (ssDNA) with 4 bp resolution in a 2.9cm-long CE separation channel. Furthermore, we successfully demonstrated the identification of the PCR amplicons of two target genes of Escherichia coli O157:H7 (rrsH gene, 121 bp) and Staphylococcus aureus (glnA gene, 225 bp). For accurate assignment of the peaks in the electropherogram, two bracket ladders (80 bp for the shortest and 326 bp for the longest) were employed, so the two amplicons of the pathogens were precisely identified on a pCE chip within 3min using the relative migration time ratio without effect of the CE environments. Thus, we believe that the pCE microdevice could be very useful for the separation of nucleic acids, amino acids, and ions as an analytical tool for use in the medical applications in the resource-limited environments as well as fundamental research fields.

Laccases are important multicopper oxidases that are involved in many biotechnological processes; however, they suffer from poor stability as well as high cost for production/purification. Herein, we found that DNA-copper hybrid nanoflowers, prepared via simple self-assembly of DNA and copper ions, exhibit an intrinsic laccase-mimicking activity, which is significantly higher than that of control materials formed in the absence of DNA. Upon testing all four nucleobases, we found that hybrid nanoflowers composed of guanine-rich ssDNA and copper phosphate (GNFs) showed the highest catalytic activity, presumably due to the affirmative coordination between guanine and copper ions. At the same mass concentration, GNFs had similar Km but 3.5-fold higher Vmax compared with those of free laccase, and furthermore, they exhibited significantly-enhanced stability in ranges of pH, temperature, ionic strength, and incubation period of time. Based on these advantageous features, GNFs were applied to paper microfluidic devices for colorimetric detection of diverse phenolic compounds such as dopamine, catechol, and hydroquinone. In the presence of phenolic compounds, GNFs catalyzed their oxidation to react with 4-aminoantipyrine for producing a colored adduct, which was conveniently quantified from an image acquired using a conventional smartphone with ImageJ software. Besides, GNFs successfully catalyzed the decolorization of neutral red dye much faster than free laccase. This work will facilitate the development of nanoflower-type nanozymes for a wide range of applications in biosensors and bioremediation.

Neuron-specific enolase (NSE) had clinical significance on diagnosis, staging, monitoring effect and judging prognosis of small cell lung cancer. Thus, there had a growing demand for the on-site testing of NSE. Here, a wireless point-of-care testing (POCT) system with electrochemical measurement for NSE detection was developed and verified. The wireless POCT system consisted of microfluidic paper-based analytical devices (μPADs), electrochemical detector and Android's smartphone. Differential pulse voltammetry (DPV) measurement was adopted by means of electrochemical detector which including a potentiostat and current-to-voltage converter. μPADs were modified with nanocomposites synthesized by Amino functional graphene, thionine and gold nanoparticles (NH2-G/Thi/AuNPs) as immunosensors for NSE detection. Combined with μPADs, the performance of the wireless POCT system was evaluated. The peak currents showed good linear relationship of the logarithm of NSE concentration ranging from 1 to 500ngmL-1 with the limit of detection (LOD) of 10pgmL-1. The detection results were automatically stored in EEPROM memory and could be displayed on Android's smartphone through Bluetooth in real time. The detection results were comparable to those measured by a commercial electrochemical workstation. The wireless POCT system had the potential for on-site testing of other tumor markers.

Drug detection in biofluids has always been great importance for clinical diagnosis. Many detection technologies such as chromatography-mass spectrometry, have been applied to the detection of drugs. However, these technologies required multi-step operations, including complicated and time-consuming pretreatment processes and operations of bulky detection instruments, significantly limiting development of drug detection in clinical diagnosis. Herein, a portable 3D-printed paper cartridge was fabricated for fast sample preconcentration and direct drugs quantitative detection in biofluids by a portable Raman spectrometer. This cartridge contained both paper tip with silver nanowires to preconcentrate samples and achieve surface-enhanced Raman Scattering (SERS) measurement, and 3D-printed cartridge to build enclosed environment for the improvement of detection, which cost only one dollar. The preconcentration ability of the cartridge was up to 18.13-fold fluorescence enhancement and compared to the non-preconcentration method, it achieved 9.93-fold improvement of SERS performance. The anticancer drug of epirubicin hydrochloride, cyclophosphamide and their mixtures were quantitatively detected in the bovine serum or artificial urine. The integrated detection procedure required only 1 h, including sample pretreatment and preconcentration, drying, SERS measurements, and quantification analysis. This 3D-printed paper cartridge constituted a portable detection platform that would be potentially a practical and point-of-care detection tool for drug preconcentration and quantification on the clinical diagnosis.