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Leptospirosis is a re-emerging infectious disease that presents a diagnostic enigma for clinicians with frequent misdiagnosis due to lack of rapid and accurate diagnostic tests, as the current methods are encumbered by inherent limitations. The development of a diagnostic sensor with a sample-in-result-out capability is pivotal for prompt diagnosis. Herein, we developed a microfluidic paper-based analytical device (spin-μPAD) featuring a sample-in-result-out fashion for the detection of Leptospira specific urinary biomarker, sph2 sphingomyelinase, crucial for noninvasive point-of-care testing. Fabrication of paper devices involved precise photolithography techniques, ensuring a high degree of reproducibility and replicability. By optimizing the device's configuration and protein components, a remarkable sensitivity and specificity was achieved for detecting leptospiral sph2 in urine, even at low concentrations down to 1.5 fg/mL, with an assay time of 15 min. Further, the spin-μPAD was validated with 20 clinical samples, suspected of leptospirosis including other febrile illnesses, and compared with gold standard microscopic agglutination test, culture, Lepto IgM ELISA, darkfield microscopy, and Leptocheck WB spot test. In contrast to commercial diagnostic tools, the spin-μPAD was noninvasive, rapid, easy to use, specific, sensitive, and cost-effective. The results highlight the potential of this innovative spin-μPAD for an efficient and dependable approach to noninvasive leptospirosis diagnosis, addressing critical needs in the realms of public health and clinical settings.

Neutralizing antibodies (nAbs) produced from infection or vaccination play an important role in acquired immunity. Determining virus-specific nAb titers is a useful tool for measuring aquired immunity in an individual. The standard methods to do so rely on titrating serum samples against live virus and monitoring viral infection in cultured cells which requires high biosafety level containment. The surrogate virus neutralization test (sVNT) reduces the biohazards and it is suitable for designing rapid test device in a lateral flow assay (LFA) format. Here, we introduce the fabrication and development of a unique paper-based LFA device for determining the level of SARS-CoV-2 nAb in a sample with a semiquantitative direct colorimetric readout. A LFA-based gradient assay design was used to facilitate the sVNT, where the spike glycoprotein receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) stand in as proxies for viruses and cells, respectively. The gradient assay employed multiple test dots of ACE2 spotted in increasing concentration along the sample flow path and gold nanoparticle-conjugated RBD for readout. In this way, the number of developed spots is inversely proportional to the concentration of nAbs present in the sample. The assay was tested with both standard solutions of nAb as well as human serum samples. We have demonstrated that the device can effectively provide semiquantitative test results of nAbs by direct instrument-free colorimetric detection.

New strategies for the simultaneous and portable detection of multiple enzyme activities are highly desirable for clinical diagnosis and home care. However, the methods developed thus far generally suffer from high costs, cumbersome procedures, and heavy reliance on large-scale instruments. To satisfy the actual requirements of rapid, accurate, and on-site detection of multiple enzyme activities, we report herein a smartphone-assisted programmable microfluidic paper-based analytical device (μPAD) that utilizes colorimetric and photothermal signals for simultaneous, accurate, and visual quantitative detection of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Specifically, the operation of this μPAD sensing platform is based on two sequential steps. Cobalt-doped mesoporous cerium oxide (Co-m-CeO 2 ) with remarkable peroxidase-like activities under neutral conditions first catalytically decomposes H 2 O 2 for effectively converting colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The subsequent addition of ALP or BChE to their respective substrates produces a reducing substance that can somewhat inhibit the oxTMB transformation for compromised colorimetric and photothermal signals of oxTMB. Notably, these two-step bioenzyme-nanozyme cascade reactions strongly support the straightforward and excellent processability of this platform, which exhibit lower detection limits for ALP and BChE with a detection limit for BChE an order of magnitude lower than those of the other reported paper-based detection methods. The practicability and efficiency of this platform are further demonstrated through the analysis of clinical serum samples. This innovative platform exhibits great potential as a facile yet robust approach for simultaneous, accurate, and on-site visual detection of multiple enzyme activities in authentic samples.

Biothiols play essential roles in maintaining normal physiological functions, resisting oxidative stress, and protecting cell health. Establishing an effective and reliable sensor array for the accurate quantification and discrimination of diverse biothiols is extremely meaningful. In this work, Ag/Mn 3 O 4 , Ag 3 PO 4 , and Ag 3 Cit with excellent oxidase-mimetic activity and surface-enhanced Raman scattering (SERS)-enhanced features have been prepared and loaded onto Whatman filter paper (WFP) to build SERS paper chips as three sensing channels, which can induce 3,3',5,5'-tetramethylbenzidine (TMB) oxidation to SERS-active reporters (TMBox) and concurrently generate prominent SERS signals. Nevertheless, the addition of biothiols can suppress conversion from TMB to TMBox, which can cause the reduction of the SERS signal from TMBox. Interestingly, each SERS sensing channel can generate different TMBox signals' variations due to differences in the oxidative inhibition abilities of diverse biothiols and exclusive properties of each paper chip, which can be plotted as specific fingerprint patterns of each biothiol and further translated into intuitive two-dimensional (2D) clustering profiles through linear discriminant analysis (LDA) and hierarchical cluster analysis (HCA) techniques for precise identification of these six biothiols with the minimum concentration of 1 μM. More importantly, this SERS sensor array is exploited for the precise quantification of intracellular glutathione (GSH), and can differentiate between normal and cancer cells based on different intracellular GSH contents and even identify different types of tumor cells, demonstrating its powerful application prospects in disease diagnosis.

The patterning of hydrophilic paper with hydrophobic materials has emerged as an interesting method for the fabrication of paper-based devices (PADs). Herein, we demonstrate a digitally automated, easy, low-cost, eco-friendly, and readily available method to create highly hydrophobic barriers on paper that can be promptly employed with PADs by simply using a bioink made with rosin, a commercially available natural resin obtained from conifer trees. The bioink can be easily delivered with the use of a ballpoint pen to produce water- and organic solvent-resistant barriers, showing superior properties when compared to other methods such as wax-printing or permanent markers. The approach enables the pen to be attached to a commercially available cutting printer to perform the semiautomated fabrication of hydrophobic barriers for PADs. With the aid of digitally controlled optimization, together with features of machine learning and design of experiments, we show a thorough investigation on the barrier strength that can be further adjusted to the desired application's needs. Then, we explored the barrier sturdiness across various uses, such as wide range aqueous pH sensing and the harsh acidic/organic conditions needed for the colorimetric detection of cholecalciferol.

A simple, reliable, and low-cost fabrication method is proposed here for assembling paper-based electrochemical devices (PEDs) using a commercial desktop digitally controlled plotter/cutter, together with ordinary writing tools. Permanent markers (tips of 1 mm) were used to create effective hydrophobic barriers on paper, while micromechanical pencils (mounting 4B graphite leads, 0.5 mm in diameter) were adopted for automatically drawn precise reference, counter, and working carbon electrodes. Fabrication parameters, such as writing pressure and speed, were first optimized, and the electrochemical performance of these devices was then evaluated by using potassium hexacyanoferrate(II) as redox probe. The good interdevice reproducibility (4.8%) displayed by the relevant voltammetric responses confirmed that this strategy can be profitably adopted to easily assemble paper-based electrochemical devices in a highly flexible manner. The simplicity of the instrumentation used and the low cost of each single device (about $0.04), together with the speed of fabrication (about 2 min), are other important features of the proposed strategy. Finally, to confirm the effectiveness of this prototyping method for the analysis of real samples and rapid controls, PEDs assembled by this simple approach were successfully exploited for the analysis of vitamin B6 in food supplements. [ABSTRACT FROM AUTHOR]

Human exposure to heavy metals is a concerning global problem because of its detrimental effect on our health and ecosystem. Assessing the levels of these metals is cost- and labor-intensive and nonuser friendly because current analysis approaches typically rely on heavy instrumentations like inductively coupled plasma-mass spectrometry, which is only possible in centralized labs. Hence, simple economical detection methods are in high demand in developing countries and areas with insufficient infrastructure, professional experts, and appropriate environmental treatment. Several microfluidic paper-based analytical devices have been reported as promising alternatives to conventional testing methods for on-site heavy metal detection. Paper-based microfluidics are advantageous because of their simple fabrication, biodegradability, low cost, and ability to operate without pumps. However, typical assay times for current platforms are slow, and they typically rely on pipetting a fixed volume into the assay cards. This adds complexity in actual field scenarios. Here, we report a novel, inexpensive, and straightforward capillary-driven microfluidic device combined with paper for rapid and user-friendly detection of Ni(II), Cu(II), and Fe(III) in water. A colorimetric approach was adopted to quantify these metals. The device was able to produce a homogeneous color signal within 8 s of sample insertion. The limit of detection and limit of quantification were calculated to be 2 and 6.67 ppm for nickel, 0.3 and 1 ppm for Cu, and 1.1 and 3.67 ppm for Fe, respectively. The majority (>90%) of the collected samples showed recovery in the 80-110% range with acceptable accuracy and precision (<15% RSD) for a colorimetric device. This technique can be beneficial for rapidly assessing heavy metal exposure in drinking and surface water at drastically reduced assay time and is the first example of capillary flow-driven microfluidic devices as a transport medium for heavy metal detection.

Microfluidic paper-based analytical devices (μPADs) are emerging as powerful analytical platforms in clinical diagnostics, food safety, and environmental protection because of their low cost and favorable substrate properties for biosensing. However, the existing top-down fabrication methods of paper-based chips suffer from low resolution (>200 μm). Additionally, papers have limitations in their physical properties (e.g., thickness, transmittance, and mechanical flexibility). Here, we demonstrate a bottom-up approach for the rapid fabrication of heterogeneously controlled paper-based chip arrays. We simply print a wax-patterned microchip with wettability contrasts, enabling automatic and selective assembly of cellulose microfibers to construct predefined paper-based microchip arrays with controllable thickness. This paper-based microchip printing technology is feasible for various substrate materials ranging from inorganic glass to organic polymers, providing a versatile platform for the full range of applications including transparent devices and flexible health monitoring. Our bottom-up printing technology using cellulose microfibers as the starting material provides a lateral resolution down to 42 ± 3 μm and achieves the narrowest channel barrier down to 33 ± 2 μm. As a proof-of-concept demonstration, a flexible paper-based glucose monitor is built for human health care, requiring only 0.3 μL of sample for testing.

Microfluidic paper-based analytical devices (μPADs) have attracted significant attention in the field of point-of-care (POC) diagnostics. However, the heterogeneous structure of the paper often impairs the limit of detection (LOD) for low-abundance targets when those targets are directly analyzed. One viable solution to bypass this limitation is to elevate the target concentration above the LOD on-site to reach a valid readout. Here, we developed a 3D μPADs preconcentrator (3D-μP 2 ) to increase sample concentration by electrokinetic trapping and demonstrated its application in increasing the LOD of a downstream colorimetric assay. The three-dimensional (3D) structure of this device was composed of a loading pad, a vertical fluid path formed by stacked absorbent pads, and an ion-selective membrane of PEDOT:PSS. This novel design facilitates fast preconcentration, high capacity in sample processing, and easy target retrieval. The concentration of an exemplary target, a single-stranded DNA sequence, was increased up to 170-fold within 80 s. The LOD of the colorimetric assay to verify the DNA target was increased 3 orders of magnitude with a preconcentrated sample compared to the control. The device and its analysis equipment used in this study were all cheap and portable. Thus, the 3D-μP 2 can be a powerful POC tool for sample pretreatment in resource-limited areas.

Microfluidic paper-based analytical devices (μPADs) are emerging as a prominent platform for disease detection, specifically in developing countries. This paper device offer simplicity and affordability not typically seen in centralized laboratory settings. However, detection limits in μPADs are inadequate and often require test results to be read within a specific time interval to ensure accuracy. To overcome these challenges, we are developing an on-chip mass spectrometry (MS) detection strategy for immunoassays performed on paper substrates. Herein, we present our initial results from a proof-of-concept study toward the development of μPADs capable of storing immunoassay reagents within the confinements of the 3D device, automatic splitting of biofluid into four individual test zones, immuno-capture of the disease biomarker, and on-chip MS detection of the captured species. The reported study encourages the development of point-of-care and direct-to-customer testing using disposable μPADs to collect samples, followed by sensitive analysis using portable MSs. We demonstrate this capability using malaria Plasmodium falciparum histidine-rich protein 2 ( Pf HRP2) antigen detection.

Target-responsive nanomaterials attract growing interest in the application of drug delivery, bioimaging, and sensing due to the responsive releasing of guest molecules by the smart molecule gate. However, it remains a challenge to develop smart nanomaterials with simple assembly and low nonspecific leakage starting from encapsulation strategies, especially in the sensing field. Herein, Au nanoclusters (Au NCs) were first grown on porous carbon derived from ZIF-8 (PCZIF) to be employed as nanocarriers. By employing the Au NCs as linkers and aptamer (Apta) double-strand hybrids (target Apta and SH-complementary DNA) as capping units, we reported the novel target-responsive nanomaterials of Apta/Au NCs-PCZIF/hemin through Au-S binding encapsulation for sensing assays. The Au-S binding encapsulation strategy simplified the packaging procedure and reduced non-target responsive leakage. As a proof, ochratoxin A (OTA) as a model target participates in the double-strand hybrid competitive displacement reaction and triggered Apta conformation switches from a coil to a G-quadruplex structure accompanied by the dissociation of the gatekeeper. Simultaneously, the released hemin can initiate a self-assembly to form G-quadruplex/hemin DNAzyme. Interestingly, owing to DNAzyme providing electron transfer mediators and peroxidase-like activity, we proposed an electrochemical/colorimetric dual-mode paper-based analytical device (PAD) that provided self-verification to enhance reliability and accuracy, benefiting from independent signal conversion and transmission mechanism. As a consequence, the proposed dual-mode PAD could achieve sensitive electrochemical detection and visual prediction of OTA in the range of 1 pg/mL to 500 ng/mL and 50 pg/mL to 500 ng/mL, respectively. The electrochemical detection limit for OTA was as low as 0.347 pg/mL ( S / N = 3). We believe that this work provides point-of-care testing (POCT) tools for a broad spectrum of applications.

While colorimetric-based assays are very convenient to determine biomarkers in point-of-care testing (POCT), they often suffer from pretreatment procedures for separation of plasma or serum from whole blood samples. Here, we report a simple colorimetric paper-based analytical device ( c -PAD) that is capable of performing sample-to-answer analysis by directly dropping the whole blood sample on paper. This is accomplished by utilizing sodium alginate hydrogel, which exhibits a nanometer-scale porous structure to effectively prevent the passage of large red blood cells and hemoglobin molecules, to encapsulate enzymes and chromogenic reagents. As the small targets in the blood sample enter the sensing region to trigger a chromogenic reaction, the resulting color signal is recorded by a smartphone. The interference from the red blood to the color signal can be completely avoided without the requirement of any separation process. The analytical performance of the method is evaluated by assaying glucose in real blood samples. The results show that rapid and accurate analysis can be achieved with the limit of detection as low as 0.12 mM. In addition, simultaneous detection of different targets (glucose, cholesterol, and triglycerides) in whole blood can be achieved by fabricating c -PAD with multiple sensing regions. Owing to its several essential advantages including an extremely simple procedure for fabrication, sample-to-answer analysis without tedious pretreatment, and capability to perform high-throughput analysis, the proposed c- PAD will be of great value in POCT applications of whole blood samples.

Illicit drug use causes over half a million deaths worldwide every year. Drugs of abuse are commonly smuggled through customs and border checkpoints and, increasingly, through parcel delivery services. Improved methods for detection of trace drug residues from surfaces are needed. Such methods should be robust, fieldable, sensitive, and capable of detecting a wide range of drugs. In this work, commercially produced paper with a pressure-sensitive adhesive coating was utilized for the collection and analysis of trace drug residues by paper spray mass spectrometry (MS). This modified substrate was used to combine sample collection of drug residues from surfaces with rapid detection using a single paper spray ticket. The all-in-one ticket was used to probe different surfaces commonly encountered in forensic work including clothing, cardboard, glass, concrete, asphalt, and aluminum. A total of 10 drugs (acetyl fentanyl, fentanyl, clonazolam, cocaine, heroin, ketamine, methamphetamine, methylone, U-47700, and XLR-11) were evaluated and found to be detectable in the picogram range using a benchtop mass spectrometer and in the low nanogram range using a portable ion trap MS. The novel approach demonstrates a simple yet effective sampling strategy, allowing for rapid identification from difficult surfaces via paper spray mass spectrometry.

In recent years, microfluidic paper analytical devices (μPADs) have been extensively utilized to conduct multiplex colorimetric assays. Despite their simple and user-friendly operation, the need for patterning paper with wax or other physical barriers to create flow channels makes large-scale manufacturing cumbersome. Moreover, convection of rehydrated reagents in the test zones leads to nonuniform colorimetric signals, which makes quantification difficult. To overcome these challenges, we present a device called a barrier-free μPAD (BF-μPAD) that consists of a stack of two paper membranes having different wicking rates-the top layer acting as a fluid distributing layer and the bottom layer containing reagents for colorimetric detection. Multiple analytes can be detected using this assembly without the need to pattern either layer with wax or other barriers. In one embodiment, a device is capable of delivering the sample fluid to 20 distinct dried reagent spots stored on an 8 cm × 2 cm membrane in as few as 30 s. The multiplexing feature of the BF-μPAD is demonstrated for colorimetric detection of salivary thiocyanate, protein, glucose, and nitrite. Most importantly, the device improves the limit of detection of colorimetric assays performed on conventional μPADs by more than 3.5×. To understand fluid imbibition in the paper assembly, the device geometry is modeled in COMSOL Multiphysics using the Richards equation; the results obtained provide insights into the nonintuitive flow pattern producing perfectly uniform signals in the barrier-free assembly.

This paper introduces a novel potential-resolved paper-based biosensor for simultaneous detection of multiple microRNAs (miRNAs) (taking miRNA-155 and miRNA-126 as examples) based on the bipolar electrode (BPE) electrochemiluminescence (ECL) strategy. The proposed multiple-channel paper-based sensing microfluidic platform was prepared by wax-printing technology, screen-printing method, and in situ Au nanoparticles (AuNPs) growth to form hydrophilic areas, hydrophobic boundaries, waterproof electronic bridge, driving electrode regions, and parallel bipolar electrode regions. CdTe quantum dots (QDs)-H2 and Au@g-C 3 N 4 nanosheets (NSs)-DNA1 were used as dual electrochemiluminescence signal probes, and carboxylated Fe 3 O 4 magnetic nanoparticles existed as carriers. CdTe QDs-H2/S 2 O 8 2- and Au@g-C 3 N 4 NSs-DNA1/S 2 O 8 2- could exhibit two strong and stable ECL emissions at a drive voltage of 9 and 12 V, respectively, which can be used as effective potential-resolved signal tags. In addition, the proposed three-dimensional (3D) DNA nanomachine model and the target miRNA cycle strategy were used to achieve double amplification of electrochemiluminescence intensity. More importantly, the combination of the bipolar electrode system and the potential-resolved multitarget electrochemiluminescence method can greatly reduce the spatial interference between substances. The prepared ECL biosensor showed a favorable linear response for the detection of miRNA-155 and miRNA-126 with relatively low detection limits of 5.7 and 4.2 fM, respectively. With excellent sensitivity, the strategy may provide an efficient method for clinical application, especially in detection of trace multiple targets.

One of the most important challenges in the field of microfluidics is the rapid fabrication of microchips with complex topologies. Although the processing method of microfluidic chips has made brilliant achievements in the past 20 years, almost all traditional processing methods still face huge obstacles in the production of complex topologies and three-dimensional microchannel. Nowadays, the main methods of manufacturing microfluidic chips such as numerical control microprocessing, laser ablation, inkjet printing, photolithography, dry etching, and lithography, galvanoformung and abformung (LIGA) technology are not only inapplicable to the complex topological structure and the rapid processing of three-dimensional microfluidic chips but also rely on expensive processing equipment, complex manufacturing process, and low yield. To solve the problems of these traditional processing methods, we propose a low-cost methodology to obtain a microfluidic chip by sewing the chip pipe to the substrate with an embroidery machine as low as $6. Compared with the above-mentioned traditional microprocessing technologies, the new chip processing technology proposed by us does not involve professional microprocessing equipment and professional skills. Therefore, this new chip processing technology can significantly improve the efficiency of microprocessing.

Conventional paper lateral flow assays have low sensitivity and suffer from severe interference from complex human fluid sample matrices, which prevents their practical application in the testing of whole blood samples in the point-of-care settings. To solve this problem, gold nanostar@Raman reporter@silica-sandwiched nanoparticles have been developed as the surface-enhanced Raman scattering (SERS) probes for sensing transduction; and a functionalized filter membrane assembly has been designed and constructed in the paper-based lateral flow strip (PLFS) as a built-in plasma separation unit. In this "on-strip" plasma separation unit, three layers of filter membranes are stacked and surface-modified to maximize the separation efficiency and the plasma yield. As a result, the integrated PLFS has been successfully used for the detection of carcinoembryonic antigen (CEA) in 30 μL of whole blood with the assistance of a portable Raman reader, achieving a limit of detection of 1.0 ng mL -1 . In short, this report presents an inexpensive, disposable, portable, and field-deployable paper-based device as a general point-of-care testing tool for protein biomarker detection in a drop of whole blood.

Significant efforts have been made to develop robust and reliable methods for simultaneous biothiols determination in different matrices, but there still exist the problems such as easy oxidation, tedious derivatization, and difficulty in discrimination, which brings unsatisfactory results in their accuracy and fast quantification in biological samples. To overcome these problems, a simultaneous biothiols detection method combining a "selenium signature" chemical probe and paper spray mass spectrometry (PS-MS) was proposed. In the strategy, the modified-paper substrate is used to enhance the analytical performance. Chemical probe Ebselen-NH 2 that has a specific response to biothiols was designed and covalently fixed on the surface of an oxidized paper substrate. By the identification of derivatized product with distinctive selenium isotope distribution and employment of the optimized PS-MS method, qualitative and quantitative analysis of five biothiols including glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), N-acetylcysteine (Nac), and homocysteine (Hcy) were realized. Biothiols in plasma and cell lysates were measured with satisfactory results. The established method not only provides a novel protocol for simultaneous determination of biothiols, but also is helpful for understanding the biological and clinical roles played by these bioactive small molecules.

This study is focused on identifying novel epithelial markers in circulating extracellular vesicles (EVs) through the development of a dual sandwich-type electrochemical paper-based immunosensor for Claudin 7 and CD81 determination, as well as its validation in breast cancer (BC) patients. This immunosensor allows for rapid, sensitive, and label-free detection of these two relevant BC biomarkers. Under optimum conditions, the limit of detection for Claudin 7 was 0.4 pg mL -1 , with a wide linear range of 2 to 1000 pg mL -1 , while for CD81, the limit of detection was 3 pg mL -1 , with a wide linear range of 0.01 to 10 ng mL -1 . Finally, we validated Claudin 7 and CD81 determination in EVs from 60 BC patients and 20 healthy volunteers, reporting higher diagnostic accuracy than the one observed with classical diagnostic markers. This analysis provides a low-cost, specific, versatile, and user-friendly strategy as a robust and reliable tool for early BC diagnosis.

Simple, rapid, and accurate detection methods for saccharides are potentially applicable to various fields such as clinical and food chemistry. However, the practical applications of on-site analytical methods are still limited. To this end, herein, we propose a 96-well microtiter plate made of paper as a paper-based chemosensor array device (PCSAD) for the simultaneous classification of 12 saccharides and the quantification of fructose and glucose among 12 saccharides. The mechanism of the saccharide detection relied on an indicator displacement assay (IDA) on the PCSAD using four types of catechol dyes, 3-nitrophenylboronic acid, and the saccharides. The design of the PCSAD and the experimental conditions for the IDA were optimized using a central composite design. The chemosensors exhibited clear color changes upon the addition of saccharides on the paper because of the competitive boronate esterification. The color changes were employed for the subsequent qualitative, semiquantitative, and quantitative analyses using an automated algorithm combined with pattern recognition for digital images. A qualitative linear discrimination analysis offered discrimination of 12 saccharides with a 100% classification rate. The semiquantitative analysis of fructose in the presence of glucose was carried out from the viewpoint of food analysis utilizing a support vector machine, resulting in clear discrimination of the various concentrations of fructose. Most importantly, the quantitative detection of fructose in two types of commercial soft drinks was also successfully carried out without sample pretreatments. Thus, the proposed PCSAD can be a powerful method for on-site food analyses that can meet the increasing demand from consumers for sensors of saccharides.

We introduce a new latent fingermark (LFM) development method, where compounds showing long lifetime luminescence are generated in situ by the reactions of Eu(TTA) 3 (H 2 O) 2 with LFM components. Until now, time-gated imaging could not be used to develop LFM on porous surfaces due to the difficulties with selective binding of the developing agents to the fingermark ridges. The nature of the interactions of Eu(TTA) 3 (H 2 O) 2 with the LFM material has been investigated for three model compounds commonly found in the LFM composition-oleic acid, l-serine, and squalene. The LFMs developed with the europium β-diketonate complex have been successfully photographed using a time-gated imaging scheme. The presented new approach has been demonstrated to give similar or better results than developing agents commonly used for paper samples (ninhydrin and 1,2-indanedione). Moreover, contrary to the methods mentioned above, the new approach allows for the development of amino acid-poor LFM on paper.

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

Multilayered and stacked cellulose paper has emerged as a promising platform for construction of three-dimensional (3D) cell culture because of its low cost, good biocompatibility, and high porosity. However, its poor light transmission makes it challenging to directly and clearly monitor cell behaviors (e.g., growth and proliferation) on the paper-based platform using an optical microscope. In this work, we developed a transparent microcrystalline cellulose/polyvinyl alcohol (MCC/PVA) paper with irregular pores through dissolution and regeneration of microcrystalline nanocellulose, addition of a porogen reagent (NaCl), and subsequently dipping in PVA solutions. The transparent MCC paper displays high porosity (up to 90%), adjustable pore size (between 23 and 46 μm), large thickness (from 315 to 436 μm), and high light transmission under water (>95%). Through further modification of the transparent MCC paper with PVA, the obtained transparent MCC/PVA paper shows enhanced mechanical properties (dry and wet strengths), good hydrophilicity (with a contact angle of 70.8°), and improved biocompatibility (cell viability up to 90%). By stacking and destacking multiple layers of the transparent MCC/PVA paper, it has been used for both two-dimensional and three-dimensional cell culture platforms. The transparent MCC/PVA paper under water enables both direct observation of cell morphology by an optical microscope via naked eyes and fluorescence microscope after staining. We envision that the developed transparent MCC/PVA paper holds great potential for future applications in various bioanalytical and biomedical fields, such as drug screening, tissue engineering, and organ-on-chips.

The analysis of protein antigens as biomarkers in clinical samples is particularly helpful for the early diagnosis of diseases. However, this is difficult to accomplish owing to the presence of the antigens in trace amounts as well as the complexity of the matrixes in clinical samples. In this study, a lab-on-membrane platform that can be combined with paper spray ionization mass spectrometry was developed for the in situ high-throughput sensitive detection of the prostate-specific antigen (PSA). The sensitivity of the proposed platform was enhanced via two strategies: (1) the synthesis of a biotin-streptavidin scaffold caused an increase in the capturing efficiency of PSA by a factor of 5 and (2) the immobilization of a large number of mass tag molecules on the gold nanoparticles allowed for the amplification of the mass spectrometry signals. The limit of detection was approximately 3.0 pg mL -1 . The selectivity to PSA was guaranteed by using an antibody-aptamer pairing sandwich immunoassay, and PSA detection was unaffected even when other protein antigens (carcinoembryonic antigen and carbohydrate antigen 125) were present. The modified membranes maintained their performance for at least 30 days when stored at 4 °C. Finally, analysis of human serum samples confirmed that the PSA concentration as determined using the proposed platform was consistent with that determined with a conventional chemiluminescent immunoassay. Thus, this PSA analyzing platform is suitable for prostate cancer diagnosis in clinical settings.

As the most common malignancy in humans, oral squamous cell carcinoma (OSCC) not only harms the people's health but also undermines their confidence after facial surgery. Early detection and treatment can effectively reduce these damages. The unique collateral trans-cleavage nuclease activity of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was utilized to realize the detection of nucleic acid with high sensitivity. So, in this work, we designed a point-of-care testing (POCT) platform for the detection of OSCC-associated salivary hsa-miRNA 31-5p (miR-31) via the cascade signal amplification of "invading stacking primer" (IS-primer) amplification reaction (ISAR), CRISPR/Cas12a, and dual-mode paper-based strip (dm-Strip). To amplify the detection signal of trace miR-31, the cascade signal amplification of CRISPR/Cas12a system coupling with ISAR was designed in a one-pot reaction at a constant temperature. The target miR-31 could activate the ISAR to generate numerous DNAs, which would further trigger the trans-cleavage effect of Cas12a to catalyze the nonspecific single-stranded DNA (ssDNA) cleavage. This ssDNA was labeled with digoxin and biotin at the 5' and 3' termini (digoxin/ssDNA/biotin), respectively, which led to generate the naked-eye signal and fluorescent signal of the designed dm-Strip. The whole detection time was 90 min with limit-of-detection (LOD) down to aM level. This ISAR/Cas12a-based dm-Strip (ISAR/Cas12a-dmStrip) allowed for the portable and ultrasensitive detection of miRNA, an important step in early diagnosis of OSCC and biomedical research.

Diabetes mellitus is one of the most common chronic diseases worldwide. Generally, the levels of fasting or postprandial blood glucose and other biomarkers, such as glycated albumin, glycated hemoglobin, and 1,5-anhydroglucitol, are used to diagnose or monitor diabetes progression. In the present study, we developed a sensor to simultaneously detect the glucose levels and glycation ratios of human serum albumin using a lateral flow assay. Based on the specific enzymatic reactions and immunoassays, a spiked glucose solution, total human serum albumin, and glycated albumin were measured simultaneously. To test the performance of the developed sensor, clinical serum samples from healthy subjects and patients with diabetes were analyzed. The glucose level and glycation ratios of the clinical samples were determined with reasonable correlation. The R -squared values of glucose level and glycation ratio measurements were 0.932 and 0.930, respectively. The average detection recoveries of the sensor were 85.80% for glucose and 98.32% for the glycation ratio. The glucose level and glycation ratio in our results were crosschecked with reference diagnostic values of diabetes. Based on the outcomes of the present study, we propose that this novel platform can be utilized for the simultaneous detection of glucose and glycation ratios to diagnose and monitor diabetes mellitus.

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

Exploring efficient luminophores in the electrochemiluminescence (ECL) system is highly desired to pursue a sensitive ECL sensing platform. Herein, the black phosphorus nanosheets (BP NSs) with excellent ECL properties are investigated and serve as the luminophore with the coreactant of peroxydisulfate (S 2 O 8 ) solution. Moreover, owing to the overlapping of emission and absorbance spectra, effective resonance energy transfer (RET) is realized between the BP NSs and the introduced Au nanoparticles. In order to achieve the portable and miniaturized developing trends for the paper-based ECL sensing platform, a paper-based perovskite solar cell (PSC) device is designed to act as the power source to replace the commonly utilized expensive and cumbersome electrochemical workstation. Benefiting from that, a PSC driven paper-based constant potential ECL-RET sensing platform is constructed, thereby realizing sensitive microRNAs (miRNAs) detection. What's more, to attain the preferable analytical performance, the duplex-specific nuclease (DSN) is also introduced to assist the target recycling signal amplification strategy. Based on this, highly sensitive detection of miRNA-107 with a range from 0.1 pM to 15 nM is achieved by this designed sensing platform. Most importantly, this work not only pioneers a precedent for developing a high-sensitivity PSC triggered ECL sensing platform but also explores the application prospect of BP nanomaterial in the field of bioanalysis.2- ) solution. Moreover, owing to the overlapping of emission and absorbance spectra, effective resonance energy transfer (RET) is realized between the BP NSs and the introduced Au nanoparticles. In order to achieve the portable and miniaturized developing trends for the paper-based ECL sensing platform, a paper-based perovskite solar cell (PSC) device is designed to act as the power source to replace the commonly utilized expensive and cumbersome electrochemical workstation. Benefiting from that, a PSC driven paper-based constant potential ECL-RET sensing platform is constructed, thereby realizing sensitive microRNAs (miRNAs) detection. What's more, to attain the preferable analytical performance, the duplex-specific nuclease (DSN) is also introduced to assist the target recycling signal amplification strategy. Based on this, highly sensitive detection of miRNA-107 with a range from 0.1 pM to 15 nM is achieved by this designed sensing platform. Most importantly, this work not only pioneers a precedent for developing a high-sensitivity PSC triggered ECL sensing platform but also explores the application prospect of BP nanomaterial in the field of bioanalysis.

Glass slides have been widely used for DNA immobilization in DNA microarray and numerous bioassays for decades, whereas they are faced with limitations of low probe density, time-consuming modification steps, and expensive instruments. In this work, a simple one-step surface modification method using 3-aminopropyl trimethoxysilane (APTMS) has been developed and applied to graft DNA codes on paper. Higher DNA immobilization efficiency was obtained in comparison with that in a conventional method using glass slides. Fluorescence detection, X-ray photoelectron spectroscopy (XPS), infrared spectra (FT-IR), and pH influence studies were employed to characterize the surface modification and subsequent DNA immobilization, which further reveals a mechanism in which this method lies in ionic interactions between the positively charged APTMS-modified paper surface and negatively charged DNA probes. Furthermore, an APTMS-modified paper-based device has been developed to demonstrate application in low-cost detection of a foodborne pathogen, Giardia lamblia , with high sensitivity (the detection limit of 22 nM) and high specificity. Compared with conventional methods using redundant cross-linking reactions, our method is simpler, faster, versatile, and lower-cost, enabling broad applications of paper-based bioassays especially for point-of-care detection in resource-poor settings.

Rapid and sensitive detection of metabolites and chemical residues in human tears is highly beneficial for understanding eye health. In this study, Schirmer paper was used for noninvasive microsampling of human tears, and then paper spray mass spectrometry (PSMS) was performed for direct analysis of human tears. Schirmer PSMS was successfully used for rapid diagnosis of dry-eye syndrome by detecting the volume and metabolites of human tears. Drugs of abuse, therapeutic drugs, and pharmacodynamics in human tears were also investigated by Schirmer PSMS. Furthermore, specific markers of environmental exposures in the air to human eyes, including volatile organic compounds, aerosol, and smoke, were unambiguously sampled and detected in human tears using Schirmer PSMS. Excellent analytical performances were achieved, including single-use, low-sample consumption (1.0 μL), rapid analysis (the whole analytical procedure completed within 3 min), high sensitivity (absolute limit of detection less than or equal to 0.5 pg, signal-to-noise ratio greater than or equal to 3), good reproducibility (relative standard deviation less than 10%, n = 3), and accurate quantitation (average deviation less than 3%, n = 3). Overall, our results showed that Schirmer PSMS is a highly effective method for direct tear analysis and is expected to be a convenient tool for human tear analysis in significant clinical applications.

The separation of plasma from whole blood is the first step in many diagnostic tests. Point-of-care tests often rely on integrated plasma filters, but protein retention in such filters limits their performance. Here, we investigate plasma separation on interlocked micropillar scaffolds ("synthetic paper") by the local agglutination of blood cells coupled with the capillary separation of the plasma. We separated clinically relevant volumes of plasma with high efficiency in a separation time on par with that of state of the art techniques. We investigated different covalent and noncovalent surface treatments (PEGMA, HEMA, BSA, O 2 plasma) on our blood filter and their effect on protein recovery and identified O 2 plasma treatment and 7.9 μg/cm 2 agglutination antibody as most suitable treatments. Using these treatments, we recovered at least 82% of the blood plasma proteins, more than with state-of-the-art filters. The simplicity of our device and the performance of our approach could enable better point-of-care tests.

Despite the recent developments on the construction of point-of-care testing (POCT) devices, it is still a big challenge to build portable POCT tools for simple, sensitive, selective, and quantitative determination of disease-related biomarkers. With this in mind, we developed a simple and user-friendly POCT tool for onsite analysis of DNA adenine methyltransferase (Dam) activity by using DNA tetrahedra-based hydrogel to trap glucose-producing enzymes for target recognition and signal transduction. The enzyme-encapsulated DNA hydrogel and the substrate of enzyme were separately modified on papers and then combined onto a commercial glucose test strip for the sensitive evaluation of Dam activity via using a personal glucose meter (PGM) for quantitative signal readout. Taking advantage of the great amount of enzyme entrapped in DNA hydrogel and the high signal amplification ability of enzyme, this POCT tool is capable of highly sensitive and selective determination of Dam activity with a direct detection limit down to 0.001 U/mL, which is superior to that of most previously reported biosensors. Furthermore, this device can also be applied to screen inhibitor and analyze Dam activity in spiked serum samples, indicating the great potential for clinical practice and diagnostic applications. Additionally, all the reactions for Dam assay are performed on paper, which is simple and deliverable to end-users for medical diagnostics at home or in-field.

The development of paper-based electroanalytical strips as powerful diagnostic tools has gained a lot of attention within the sensor community. In particular, the detection of nucleic acids in complex matrices represents a trending topic, especially when focused toward the development of emerging technologies, such as liquid biopsy. DNA-based biosensors have been largely applied in this direction, and currently, there are two main approaches based on target/probe hybridization reported in the literature, namely Signal ON and Signal OFF. In this technical note, the two approaches are evaluated in combination with paper-based electrodes, using a single strand DNA relative to H1047R (A3140G) missense mutation in exon 20 in breast cancer as the model target. A detailed comparison among the analytical performances, detection protocol, and cost associated with the two systems is provided, highlighting the advantages and drawbacks depending on the application. The present work is aimed to a wide audience, particularly for those in the field of point-of-care, and it is intended to provide the know-how to manage with the design and development stages, and to optimize the platform for the sensing of nucleic acids using a paper-based detection method.

Cellulose paper has strong potential as an analytical platform owing to its unique characteristics. In the present study, we investigated a procedure for functionalizing the surface of cellulose paper by dip-coating a mixture of a functional polymer and a perfluoroalkylated surfactant (surfactant 1). The functional polymer comprised a mixture of methyl methacrylate and poly(ethylene glycol) methacrylate monomers. The monomer ratio in the functional polymer affected the hydrophilicity and water absorbance of the cellulose paper after dip-coating. Furthermore, the presence of surfactant 1 during dip-coating promoted the surface segregation of poly(ethylene glycol) (PEG) moieties in the polymer, which enhanced the hydrophilicity, prevented nonspecific protein adsorption, and maintained the water absorbance of the dip-coated cellulose paper. Dip-coating with another functional polymer containing biotin groups produced a cellulose paper with a biotin-decorated surface in a one-step procedure. The displayed biotin groups immobilized avidin on the surface, and the PEG moieties in the polymer prevented nonspecific protein adsorption. We then immobilized a thrombin-binding DNA aptamer on the avidin-immobilized cellulose paper to prepare a paper-based analytical device. It is possible to visualize thrombin in model solutions and serum using the paper-based analytical device.

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

Water is one of the most widespread impurities and contaminants for pharmaceuticals and chemical products. A simple, fast and reliable water assay method is of high significance for the pharmaceutical and chemical manufacturing industries. In this work, lanthanide metal-organic framework Eu-dipicolinic acid/2-aminophthalic acid (Eu-DPA/PTA-NH 2 ) was prepared as a double-color ratiometric fluorescent water sensor, which shows ultrasensitive (limit of detection 0.01% v/v) and linear broad-range (0-100% v/v) response to water. An innovative analytical device based on a one-to-two logic gate was constructed by using the water content and the two fluorescence responses as the input and output signals, respectively. The direct analysis of water content can be achieved by the four output types, NOT (0, 1), PASS 0 (0, 0), PASS 1 (1, 1), and YES (1, 0). Importantly, the metal-organic framework (MOF)-loaded fiber paper was prepared as a microsensor, and a water assay tube was designed for rapid water detection of solid pharmaceuticals via ratiometric chromaticity, coupled with a portable visual determination device. The paper-based microsensor is response-rapid (20 s), long-term stable (at least 30 days), and can achieve the accurate (relative error <9.8%) visual assay of trace water in solid pharmaceuticals. To our knowledge, this is the first time to report an MOF-based double-color ratiometric fluorescent water sensor, a water analytical logic device, and a paper-based water microsensor for point-of-care visual water assay in solid samples via ratiometric chromaticity.

Exposure to trace metals in airborne particulate matter (PM) has been linked to various adverse health effects. Quantifying metals in PM is important; however, current analytical tools tend to be bulky and expensive. A need therefore exists for more rapid, low-cost, portable tools for multiplexed determination of metals in PM. Electrochemical paper-based devices (ePADs) have been used for detecting metals in PM but require different devices and methods for different metals, making the systems more complicated than desired. Recently reported Janus ePADs offer a solution to this problem by allowing for multiple electrochemical experiments from a single sample. Here, we sought to determine if a Janus ePAD containing four independent channels and working electrodes could be used for simultaneous detection of multiple metals in PM. Online sample pretreatment in each channel during sample delivery yielded optimal conditions for each experiment. The design allows the device to conduct square-wave anodic stripping voltammetry (SWASV) and square-wave cathodic stripping voltammetry (SWCSV) for simultaneous detection of Cd, Pb, Cu, Fe, and Ni from a single sample. Two detection zones each with shared reference and counter electrodes were used for SWASV and SWCSV, respectively. The proposed sensors reached LODs down to 0.5, 0.5, 1.0, 0.5, and 1.0 μg L -1 , for Cd(II), Pb(II), Cu(II), Fe(II), and Ni(II), respectively. The linear working ranges were 0.5-400.0 μg L -1 for Cd(II), Pb(II), and Fe(II), 1.0-400.0 μg L -1 for Cu(II), and 0.5-200.0 μg L -1 for Ni(II). The devices were applied for Cd(II), Pb(II), Cu(II), Fe(II), and Ni(II) determination in PM samples, and the results agreed with those using traditional ICP-MS analyses at 95% confidence.

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

We present a paper-based system that integrates bioluminescence resonance energy transfer (BRET) and isothermal amplification for the analysis of tumor-associated circulating microRNAs (miRNAs) in clinical serum samples. The analysis procedure could be easily accomplished with two pieces of functionalized paper and a low-cost smartphone-based device, which enables sequence-specific quantification of femtomolar miRNAs, without the need for tedious handling of aqueous reactions and operation of sophisticated equipment. Furthermore, the analytical performance of the proposed paper-based system was highly stable at room temperature, demonstrating its capability for cold-chain-free and remote deployment. These qualities highlight the practical utility of our method for the portable and field-ready miRNA diagnostic tests in resource-limited settings.

Integrating ratiometric photoelectrochemical (PEC) techniques with paper microfluidics to construct a ratiometric PEC paper analytical device for practical application is often restricted by the grave dependence of ratiometric assay on photoactive materials and low mass-transfer rates of the paper channel. Herein, a universal donor/acceptor-induced ratiometric PEC paper analytical device with a hollow double-hydrophilic-walls channel (HDHC) was fabricated for high-performance microRNA-141 (miRNA-141) quantification. Concretely, a photoanode and photocathode were integrated on the paper-based sensing platform in which the photocathode served as a biosensing site for the pursuit of higher selectivity. For formulation of a cascading signal amplification strategy, a unique duplex-specific nuclease-induced target recycling reaction was engineered for the output of a double amount of all useful DNA linkers instead of conventional output of only one available DNA product, which could guarantee the output of abundant DNA linkers with the initiation of a cascade of hybridization chain reaction on both the trunk and branch in the presence of miRNA-141. Then the formed dendriform polymeric DNA duplex structures were further decorated with glucose oxidase (GOx)-mimicking gold nanoparticles by the electrostatic interaction to form a branchy gold tree (BGT). Profiting from the perfect GOx-mimicking activity of BGT and high mass-transfer rates of HDHC, the cathodic photocurrent from Ag 2 S/Cu 2 O hybrid structure was in a "signal off" state while the anodic photocurrent from graphene quantum dots (GQDs) and Ag 2 Se QDs cosensitized ZnO nanosheets was in a "signal on" state because BGT-catalyzed glucose oxidation reaction evoked the consumption of dissolved O 2 as an electron acceptor and the generation of H 2 O 2 as an electron donor. With calculation of the ratio of two photocurrent intensities, the quantitative detection of miRNA-141 was achieved with high sensitivity, accuracy, and reliability.

Chemiluminescence (CL) immunoassays for simultaneous detection of early acute myocardial infarction (AMI) biomarkers, including copeptin, heart-type fatty acid binding protein (h-FABP), and cardiac troponin I (cTnI), were developed by using Co 2+ / N -(aminobutyl)- N -(ethylisoluminol) (ABEI) functionalized magnetic carbon composite (Co 2+ -ABEI-Fe 3 O 4 @void@C) as an interface and a three-dimensional microfluidic paper-based device (3D μPAD) as a detection system. For CL immunoassays, Co 2+ -ABEI-Fe 3 O 4 @void@C was assembled with chitosan (CS) and gold nanoparticle-conjugated antibody (Au-Ab) sequentially to form the sensing platform (Co 2+ -ABEI-Fe 3 O 4 @void@C-CS/Au-Ab). In the presence of antigen (Ag), Ag was captured by the sensing interface to form an immunocomplex, leading to an increase in CL intensity due to the catalysis of -COO - existing in Ag. A 3D μPAD with three detection zones for simultaneous detection of copeptin, h-FABP, and cTnI was designed and fabricated to obtain time-resolved CL signals. Three kinds of immunocomplexes formed with copeptin, h-FABP, and cTnI were added to three detection zones of 3D μPAD, respectively. After injecting H 2 O 2 , three time-resolved CL signals were generated in one CL detection run by virtue of time-delayed transport of H 2 O 2 to different detection zones. The three time-resolved CL signals were used for the simultaneous determination of copeptin, h-FABP, and cTnI. The detection limit of copeptin, h-FABP, and cTnI was 0.40 pg/mL, 0.32 pg/mL, and 0.50 pg/mL, respectively, which is at least 1 order of magnitude lower than most of the reported immunoassays. The immunoassays could be directly used for the detection of copeptin, h-FABP, and cTnI in human serum samples. The proposed immunoassays are simple, fast, sensitive, and selective, and are of great application potential in early diagnosis and treatment of AMI.

To achieve personalized healthcare, a quick, accurate, and high-throughput method to detect disease biomarkers is essential. In the traditional practice, mass spectrometry is one of the most powerful tools and is widely studied. However, the test of human serum usually requires complicated sample pretreatment, tedious operations, and precise condition control, especially for the detection of enzymes as biomarkers. As butyrylcholinesterase (BuChE) has an indicative significance in detecting degenerative disease, liver injury, and organophosphate poisoning, the quick quantification of BuChE is of vital importance to the clinic. In this paper, we report the design and fabrication of a portable 3D-printed enzyme reactor paper spray cartridge (3D ER-PS) with integrated functions: temperature control, enzyme reaction, analyte transfer, and paper spray ionization. Coupled with mass spectrometry, quantitative testing of BuChE activity in human serum was realized conveniently and accurately. While it only requires very simple sample preparation, the results from current 3D ER-PS approach are well consistent with those obtained using Ellman's method. This 3D ER-PS platform not only provides a novel solution for the liquid biopsy of BuChE in clinics but also contributes to the development of quick and targeted medical approaches for analyzing other types of serum biomarker molecules in the field of disease diagnosis.

Infectious diseases remain one of the leading causes of deaths in developing countries because of a lack of basic sanitation, healthcare clinics, and centralized laboratories. Paper-based rapid diagnostic tests, such as the lateral-flow immunoassay (LFA), provide a promising alternative to the traditional laboratory-based tests; however, they typically suffer from having a poor sensitivity. Biomarker preconcentration and signal enhancement are two common methods to improve the sensitivity of paper-based assays. While effective, these methods often require multiple liquid handling steps which are not ideal for use by untrained personnel in a point-of-care setting. Our lab previously discovered the phenomenon of an aqueous two-phase system (ATPS) separating on paper, which allowed for the seamless integration of concentration and detection of biomarkers on the LFA. In this work, we have extended the functionality of an ATPS separating on paper to automate the sequential delivery of signal enhancement reagents in addition to concentrating biomarkers. The timing of reagent delivery was controlled by changing the initial composition of the ATPS. We applied this technology to automate biomarker concentration and nanozyme signal enhancement on the LFA, resulting in a 30-fold improvement in detection limit over the conventional LFA when detecting Escherichia coli , all while maintaining a single application step.

Traditional fingerprints are usually obtained by pressing an inked finger on a paper. The inks would contaminate fingers and more importantly, these fingerprints are visible and able to be photocopied. In order to develop a smart membrane for fingerprint recording and document security, microrod assemblies of carbon quantum dots (CQDs)-Eu (III) are embedded in a electrospun nanofibrous (NFs) membrane which has strong red emission under UV irradiation owing to aggregation induced Dexter energy transfer from CQDs to Eu (III) ions. A clear blue emission fingerprint could be recorded on the membrane after a finger touch because the phosphate (Pi) secreted through sweat glands blocks the solid-state Dexter energy transfer, recovering the UV-irradiated blue emissions of CQDs. The Pi-based fingerprint on the membrane, which is invisible under daylight and could not be photocopied, greatly improves the security of the fingerprint and, furthermore, has the capability to identify the people who touched the secret document through the fingerprint analysis, showing that the intelligent NFs membrane can be applied for both fingerprint security and document counterspy.

We report herein the colorimetric identification of live cells based on a nucleic acid amplification testing (NAAT) methodology using an all-in-one origami paper microdevice integrated with DNA purification, loop-mediated isothermal amplification (LAMP), and on-site colorimetric detection. First, origami paper was partially embossed to create microchannel networks and chambers. Subsequently, hydrophobic polydimethylsiloxane prepolymer was coated onto the embossed paper to stabilize the structures on paper and provide fluid barriers. The paper microdevice was composed of splitting, purification, wicking, reaction, and dye pads folded alternatively to accomplish sensitive and specific NAAT. For the viability assay, propidium monoazide (PMA) was employed to penetrate dead cells and form covalent bonds with necrotic cell DNA; thus, amplification can be solely performed with DNA obtained from live bacterial cells. Purification functionality was implemented into the microdevice using chitosan to electrostatically capture DNA. Herein, methylene blue, which is typically used for electrochemical detection, is introduced for the first time for colorimetric detection of LAMP amplicons. This origami paper microdevice was successfully applied to determine the viability of foodborne pathogens, such as Escherichia coli O157:H7 and Salmonella spp., in which amplification was performed for 30 min followed by the execution of the colorimetric method for 10 min, thereby demonstrating tremendous potential for multiplexing and versatility for point-of-care applications. The introduced origami paper microdevice could be an attractive substitute as an instantaneous and convenient screening tool for the identification of viable pathogens in the control and monitoring of foodborne outbreaks in low-resource environments.

Paper-based scaffolds support the three-dimensional culture of mammalian cells in tissue-like environments. These Tissue Papers, a name that highlights the use of materials obtained from (plant) tissue to generate newly functioning (human) tissue structures, are a promising analytical tool to quantify cellular responses in physiologically relevant extracellular gradients and coculture architectures. Here, we highlight current examples of Tissue Papers, commonly used methods of analysis, and current measurement challenges.

Herein, a novel paper-based chemiluminescence (CL) device has been reported for the estimation of total phenolic content of food samples. The CL system implemented on the paper was based on a hydrogen peroxide (H 2 O 2 )-rhodamine b (RhoB)-cobalt metal organic framework (CoMOF) reaction. It was found that the reaction of H 2 O 2 with RhoB molecules, loaded into the nanopores of CoMOF (R@CoMOF), can produce an intensive CL emission. The experiments on the paper indicated that in the presence of CoMOF, the CL emission was greatly increased. In addition to this strong catalyzing effect, application of CoMOF on the paper improved the stability of the CL system for several days. As a useful analytical application for the obtained paper-based CL device (PCD), it was examined for the detection of phenolic antioxidants. It was observed that the addition of 5 μL of phenolic compounds (PC) on the paper containing the CL reagents can remarkably decrease the CL intensity. This effect was applied to design a simple analytical assay for PC. After the optimization process, the best sensitivity was obtained for gallic acid, quercetin, catechin, kaempferol, and caffeic acid with detection limits of 0.98, 1.36, 1.48, 1.81, and 2.55 ng mL -1 , respectively. The relative standard deviations (RSD%) were also less than 5%. This study is the first report on the practical application of PCD using a nanomaterial assisted CL reaction. It is simple, portable, and low-cost and consumes a very low amount of reagents and sample solution. The device was successfully applied in the investigation of total antioxidant capacity of molasses and honey samples.