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We report for the first time a highly sensitive and rapid quantitative method for the detection of Salmonella Typhimurium (S. Typhimurium) using a conductive immunosensor on a paper-based device (PAD). S. Typhimurium monoclonal antibodies (MA) were first immobilized on a paper-based device and then captured by S. Typhimurium. After an immunoreaction on the device, the polyclonal antibody-colloidal gold conjugate (PA-AuNPs) was dropped to bind with S. Typhimurium. After a complete sandwich reaction, a dark red color appeared on the paper-based device, which can be observed by the naked eye for a rapid screening test. The electrical conductivity of PA-AuNPs between the screen-printed electrodes on the paper-based device was also measured for an accurate quantitative analysis. The electrical conductivity correlated well with the concentration of S. Typhimurium, which was controlled by the amount of S. Typhimurium attached to the polyclonal antibody-colloidal gold conjugate. The device showed a linear correlation for the concentration of the S. Typhimurium in the range of 10-10 8 CFU mL -1 in a logarithmic plot, with an R 2 value of 0.9882 and a limit of detection (LOD) as low as 10 CFU mL -1 . This simple, highly sensitive, and rapid method for the S. Typhimurium detection was successfully performed within 30 min, and it can be developed into small portable measuring devices in order to facilitate preliminary screening tests.

In this paper, a paper-supported analytical device based on a sandwich immunoreaction and luminescence resonance energy transfer (LRET) was reported for the visual and quantitative determination of a cancer biomarker, in which upconversion nanoparticles (UCNPs) were located on the surface of the paper as energy donors and gold nanoparticles (AuNPs) were used as energy acceptors. Upon the recognition of the cancer biomarker by two rationally selected antibodies, the upconversion luminescence was quenched by the AuNPs in a biomarker concentration-dependent manner. As a model target, CEA was detected using this immunosensor, and a linear relationship within 0.5-30 ng mL-1 was obtained in buffer solution, with a detection limit of 0.21 ng mL-1. The immunosensor was also applicable in 20-fold diluted human serum with a linear range of 0.5-30 ng mL-1 and a detection limit of 0.36 ng mL-1. This technique also realized the qualitative judgment of the critical concentration of CEA in serum samples by the naked eye. This approach displays great application potential for point-of-care testing in clinical applications, as well as the potentiality to be extended to other kinds of disease-related biomolecules.

Binding protein scaffolds, such as rcSso7d, have been investigated for use in diagnostic tests; however, the functional performance of rcSso7d has not yet been studied in comparison to antibodies. Here, we assessed the analyte-binding capabilities of rcSso7d and antibodies on cellulose with samples in buffer and 100% human serum.

In the emergency diagnosis of patients, acute myocardial infarction (AMI) is always time-consuming to diagnose, and the process requires multiple laboratory procedures, expensive equipment and skilled workers. Herein, we developed an easy-to-use, low-cost and portable fluorescent lateral flow immunoassay based on paper microfluidics for the point-of-care diagnostics of non-communicable diseases. The fluorescent lateral flow immunoassay can produce results in less than 10 minutes, and the limit of detection (LOD) is 0.019 ng ml -1 . The slope was linear from 0 to 100 ng ml -1 ; the equation is y = 0.0342e 2.1181x and R 2 = 0.9618, which are distinctive features that ensure maximum amplification of the signal and recording of quantitative values by an analyser. The detection sensitivity showed an exceptional increase to 0.01 ng ml -1 . Compared with conventional bioassay readers, our analyser shows some advantages to easily, clearly and effectively read data. The present point-of-care test for cardiac troponin I decreases the turnaround time and has a high coefficient of variation even at lower concentrations of troponin. So, the development of lateral flow assay-based point-of-care assays with higher analytical performance for real world samples can decrease the rule-out time for AMI in emergency departments and other fields.

We developed a multiplexed point-of-care immunodiagnostic assay for antibody detection in human sera made through the vertical stacking of functional paper layers. In this multiplexed vertical flow immunodiagnostic assay (xVFA), a colorimetric signal is generated by gold nanoparticles captured on a spatially-multiplexed sensing membrane containing specific antigens. The assay is completed in 20 minutes, following which the sensing membrane is imaged by a cost-effective mobile-phone reader. The images are sent to a server, where the results are rapidly analyzed and relayed back to the user. The performance of the assay was evaluated by measuring Lyme-specific antibodies in human sera as model target antibodies. The presented platform is rapid, simple, inexpensive, and allows for simultaneous and quantitative measurement of multiple antibodies and/or antigens making it a suitable point-of-care platform for disease diagnostics.

In this study, we demonstrate a three-dimensional surface-modified origami-paper-based analytical device (3D-soPAD) for immunoassay applications. The platform enables the sequential steps of immunoassays to be easily performed using a folded, sliding paper design featuring multiple pre-stored reagents, allowing us to take advantage of the vertical diffusion of the analyte through the different paper layers. The cellulose substrate is composed of carboxymethyl cellulose modified with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, which provide covalent bonding sites for bio-recognition molecules. After the optimization of the operation parameters, we determined the detection limit of the 3D-soPAD for human immunoglobulin G (HIgG) which can be as low as 0.01 ng mL-1, with a total turnaround time of 7 min. In order to study the long-term storage of the platform, anti-HIgG horseradish peroxidase (aHIgG-HRP) conjugates were stored by freeze-drying in sugar matrices composed of 10% sucrose/10% trehalose (w/w%) on the paper device, retaining 80% of their activity after 75 days of storage at 4 °C. To evaluate the performance of the paper device using real samples, we demonstrated the detection of protein A (a biomarker for Staphylococcus aureus infection) in highly viscous human synovial fluid. These results show that the proposed 3D-soPAD platform can provide sensitive, high-throughput, and on-site prognosis of infection in resource-limited settings.

Microfluidic platforms are an attractive option for incorporating complex fluid handling into low-cost and rapid diagnostic tests. A persistent challenge for microfluidics, however, is the mismatch in the "world-to-chip" interface - it is challenging to detect analytes present at low concentrations in systems that can only handle small volumes of sample. Here we describe a new technique termed pre-concentration by liquid intake by paper (P-CLIP) that addresses this mismatch, allowing digital microfluidics to interface with volumes on the order of hundreds of microliters. In P-CLIP, a virtual microchannel is generated to pass a large volume through the device; analytes captured on magnetic particles can be isolated and then resuspended into smaller volumes for further processing and analysis. We characterize this method and demonstrate its utility with an immunoassay for Plasmodium falciparum lactate dehydrogenase, a malaria biomarker, and propose that the P-CLIP strategy may be useful for a wide range of applications that are currently limited by low-abundance analytes.

The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (μPADs) is reported. The μPADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) was deposited at the control and test zones. μPAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody to travel past the capture zone and into the test zone via capillary action. The developed competitive immunoassay system was successfully demonstrated on the μPAD detection of biotin as a model compound with a detection limit of 0.10 μg mL -1 . The applicability of the proposed immunoassay system for point-of-need testing was further demonstrated using aflatoxin B 1 , a highly toxic foodborne substance, with a detection limit of 1.31 ng mL -1 . The μPAD with the competitive immunoassay format showed promising results for practical applications in point-of-need testing involving small molecular weight targets in food and water safety and quality monitoring, environmental analysis, and clinical diagnostics.

Colorimetric detection methods that produce results readable by eye are important for diagnostic tests in resource-limited settings. In this work, we have compared three main types of colorimetric methods - enzymatic reactions, silver deposition catalyzed by gold nanoparticles, and polymerization-based amplification - in a paper-based immunoassay for detection of Plasmodium falciparum histidine-rich protein 2, a biomarker of malarial infection. We kept the binding events in the immunoassay constant in order to isolate the effect of the detection method on the outcome of the test. We have highlighted that the optimal readout time in a test can vary significantly - ranging from immediately after addition of a visualization agent to 25 minutes after addition of a visualization agent - depending on the colorimetric method being used, and accurate time keeping is essential to prevent false positives in methods where substantial color develops over time in negative tests. We have also shown that the choice of a colorimetric method impacts the calculated limit-of-detection, the ease of visual perception of the readout, and the total cost of the assay, and therefore directly impacts the feasibility and the ease-of-use of a test in field settings.

We report a paper-based assay platform for the detection of the kidney disease marker Trefoil Factor 3 (TFF3) in human urine. The sensor is based on a quantitative metalloimmunoassay that can determine TFF3 concentrations via electrochemical detection of environmentally stable silver nanoparticle (AgNP) labels attached to magnetic microbeads via a TFF3 immunosandwich. The paper electroanalytical device incorporates two preconcentration steps that make it possible to detect concentrations of TFF3 in human urine at the low end of the target TFF3 concentration range (0.03-7.0 μg mL(-1)). Importantly, the paper device provides a level of accuracy for TFF3 determination in human urine equivalent to that of a commercial kit. The paper sensor has a dynamic range of ∼2.5 orders of magnitude, only requires a simple, one-step incubation protocol, and is fast, requiring only 10 min to complete. The cost of the materials at the prototypic laboratory scale, excluding reagents, is just US$0.42.

This study reports a simple, rapid, low-cost, robust, and multiplexed barcoded paper-based assay (BPA) compatible with mobile devices. An inkjet printer and an XYZ dispensing platform were used to realize mass-manufacturing of barcoded paper-based analytical devices (BPADs) with high precision and efficiency. We designed a new group of barcodes and developed an application (APP) for the reading of the new code. The new barcodes possess a 16 times higher coding capacity than the standard Codabar code in our experiment on drug residue detection. The BPA system allows applications in the assays of blood-transmitted infections, drug residues in milk and multiplex nucleic acids. The whole detection process and the readout of the results can be completed within 10 minutes. The limit of detection for enrofloxacin (ENR) (8 ng mL -1 ) satisfies the requirements of drug residue monitoring. Its high rapidity, simplicity, efficiency and selectivity make the BPA system extremely suitable to be applied in rapid and on-site detection.

Diagnostic tests in resource-limited settings require technologies that are affordable and easy to use with minimal infrastructure. Colorimetric detection methods that produce results that are readable by eye, without reliance on specialized and expensive equipment, have great utility in these settings. We report a colorimetric method that integrates a paper-based immunoassay with a rapid, visible-light-induced polymerization to provide high visual contrast between a positive and a negative result. Using Plasmodium falciparum histidine-rich protein 2 as an example, we demonstrate that this method allows visual detection of proteins in complex matrices such as human serum and provides quantitative information regarding analyte levels when combined with cellphone-based imaging. It also allows the user to decouple the capture of analyte from signal amplification and visualization steps.

The lateral-flow immunoassay (LFA) is an inexpensive point-of-care (POC) paper-based diagnostic device with the potential to rapidly detect disease biomarkers in resource-poor settings. Although LFA is inexpensive, easy to use, and requires no laboratory equipment, it is limited by its sensitivity, which remains inferior to that of gold standard laboratory-based assays. Our group is the only one to have previously utilized various aqueous two-phase systems (ATPSs) to enhance LFA detection. In those studies, the sample was concentrated by an ATPS in a test tube and could only be applied to LFA after it had been extracted manually. Here, we bypass the extraction step by seamlessly integrating a polyethylene glycol-potassium phosphate ATPS with downstream LFA detection in a simple, inexpensive, power-free, and portable all-in-one diagnostic device. We discovered a new phenomenon in which the target biomarkers simultaneously concentrate as the ATPS solution flows through the paper membranes, and our device features a 3-D paper well that was designed to exploit this phenomenon. Studies with this device, which were performed at room temperature in under 25 min, demonstrated a 10-fold improvement in the detection limit of a model protein, transferrin. Our next-generation LFA technology is rapid, affordable, easy-to-use, and can be applied to existing LFA products, thereby providing a new platform for revolutionizing the current state of disease diagnosis in resource-poor settings.

Smartphone-based optical detection is a potentially easy-to-use, handheld, true point-of-care diagnostic tool for the early and rapid detection of pathogens. Paper microfluidics is a low-cost, field-deployable, and easy-to-use alternative to conventional microfluidic devices. Most paper-based microfluidic assays typically utilize dyes or enzyme-substrate binding, while bacterial detection on paper microfluidics is rare. We demonstrate a novel application of smartphone-based detection of Salmonella on paper microfluidics. Each paper microfluidic channel was pre-loaded with anti-Salmonella Typhimurium and anti-Escherichia coli conjugated submicroparticles. Dipping the paper microfluidic device into the Salmonella solutions led to the antibody-conjugated particles that were still confined within the paper fibers to immunoagglutinate. The extent of immunoagglutination was quantified by evaluating Mie scattering from the digital images taken at an optimized angle and distance with a smartphone. A smartphone application was designed and programmed to allow the user to position the smartphone at an optimized angle and distance from the paper microfluidic device, and a simple image processing algorithm was implemented to calculate and display the bacterial concentration on the smartphone. The detection limit was single-cell-level and the total assay time was less than one minute.

The lateral-flow immunoassay (LFA) is a well-established diagnostic technology that has recently seen significant advancements due in part to the rapidly expanding fields of paper diagnostics and paper-fluidics. As LFA-based diagnostics become more complex, it becomes increasingly important to quantitatively determine important parameters during the design and evaluation process. However, current experimental methods for determining these parameters have certain limitations when applied to LFA systems. In this work, we describe our novel methods of combining paper and radioactive measurements to determine nanoprobe molarity, the number of antibodies per nanoprobe, and the forward and reverse rate constants for nanoprobe binding to immobilized target on the LFA test line. Using a model LFA system that detects for the presence of the protein transferrin (Tf), we demonstrate the application of our methods, which involve quantitative experimentation and mathematical modeling. We also compare the results of our rate constant experiments with traditional experiments to demonstrate how our methods more appropriately capture the influence of the LFA environment on the binding interaction. Our novel experimental approaches can therefore more efficiently guide the research process for LFA design, leading to more rapid advancement of the field of paper-based diagnostics.

A potential-resolution strategy for multiplex electrochemiluminescence (ECL) immunoassay on a microfluidic paper-based analytical device (μ-PAD) was demonstrated for the first time, using tris-(bipyridine)-ruthenium(ii) (Ru(bpy)(3)(2+)) and carbon nanodots (CNDs) as the ECL labels for high-throughput ECL immunoassay on μ-PAD. Based on this strategy, simultaneous detection of four tumor markers using only two screen-printed carbon working electrodes (one electrode for two analytes) on an immunodevice consisting of a piece of patterned paper (denoted as μ-PECLI in this work) was realized, which is a simplification of the configuration of traditional μ-PADs. As a further development of μ-PECLI in low-cost and disposable applications, battery-triggered (constant-potential) ECL detection on μ-PECLI was developed, allowing the traditional electrochemical workstation to be abandoned. In addition, to exactly control the output voltage of the battery, a low-cost and simple voltage-controller was designed and fabricated for the first time. The battery-triggered ECL immunoassay principle on μ-PECLI is explained. We found that the simultaneous determination of two analytes in one paper working zone could be achieved by controlling the operational constant-potential (+1.2 V for Ru(bpy)(3)(2+) labels and -1.2 V for CNDs labels (vs. Ag/AgCl auxiliary electrode)) by simply reversing the connection mode. Finally, four tumor markers in human serum samples from the Tumor Hospital were assayed and the results obtained were in acceptable agreement with the reference values from parallel single-analyte test. This battery-triggered μ-PECLI provides a new strategy for high-throughput, low-cost, sensitive, automated and simultaneous multiplex immunoassay and point-of-care diagnosis.

A novel addressable electrode array based on paper was assembled on the crossing points of the row/column electrodes to form a 4 × 6 sensor array by a facile home-made device-holder, one paper layer contained the sensing sites, the other paper layer the printed counter electrode and reference electrode.

A novel 3D microfluidic paper-based immunodevice, integrated with blood plasma separation from whole blood samples, automation of rinse steps, and multiplexed CL detections, was developed for the first time based on the principle of origami (denoted as origami-based device). This 3D origami-based device, comprised of one test pad surrounded by four folding tabs, could be patterned and fabricated by wax-printing on paper in bulk. In this work, a sandwich-type chemiluminescence (CL) immunoassay was introduced into this 3D origami-based immunodevice, which could separate the operational procedures into several steps including (i) folding pads above/below and (ii) addition of reagent/buffer under a specific sequence. The CL behavior, blood plasma separation, washing protocol, and incubation time were investigated in this work. The developed 3D origami-based CL immunodevice, combined with a typical luminuol-H(2)O(2) CL system and catalyzed by Ag nanoparticles, showed excellent analytical performance for the simultaneous detection of four tumor markers. The whole blood samples were assayed and the results obtained were in agreement with the reference values from the parallel single-analyte test. This paper-based microfluidic origami CL detection system provides a new strategy for a low-cost, sensitive, simultaneous multiplex immunoassay and point-of-care diagnostics.

In this paper, chemiluminescence immunoassay (CLIA) was introduced into the recently proposed microfluidic paper-based analytical devices (μPADs) through covalent fabrication strategy for the first time. This novel paper-based CLIA, with high-throughput, rapid, stable and reusable CL response to trace amounts of analyte in real biological samples, combines the simplicity and low-cost of the μPADs with the high sensitivity and selectivity of CLIA. Periodate oxidation, which can form covalent bonds between polysaccharides and proteins, was used for activation of μPADs to covalently immobilize antibodies on μPADs in this work for the first time. Thus, the paper-based sandwich CLIA and regeneration of it can be easily realized for further development of this technique in sensitive, specific and low-cost applications. The application test of this paper-based CLIA was successfully performed, as a model, through the determination of biomarkers in human serum on paper microzone plate. The paper-based CLIA will be very useful when the level of an analyte in real biological sample is important for point-of-care testing, public health and environmental monitoring in remote regions, developing or developed countries.

A high-throughput, simple, fast, low-cost and sensitive paper-based electrochemical immunodevice has been demonstrated based on a functionalized 3D paper-based device for point-of-care diagnosis., (This journal is © The Royal Society of Chemistry 2012)

A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.

ZnO nanorods inorganic-organic heterostructured light-emitting diodes have been demonstrated on a cheap/disposable paper substrate and applied in multiplexed photoelectrochemical immunoassay.

A photoelectrochemical immunoassay was introduced for the first time into a microfluidic paper-based analytical device equipped with a paper supercapacitor for low-cost, simple, portable, and disposable point-of-care testing, based on a chemiluminescence light source and a digital multi-meter.

Rapid, sensitive, and quantitative detection of biomarkers is needed for early diagnosis of disease and surveillance of infectious outbreaks. Here, we exploit a plasmonic syringe filter and surface-enhanced Raman spectroscopy (SERS) in the development of a rapid detection system, using human IgG as a model diagnostic biomarker. The novel assay design facilitates multiple passages of the sample and labeling solution through the detection zone enabling us to investigate and maximize sampling efficiency to the capture substrate. The vertical flow immunoassay process in this study involves the utilization of filter paper embedded with gold nanoparticles (AuNPs) to form a plasmonic substrate. Capture antibody (anti-human IgG) is then immobilized onto the prepared plasmonic paper and inserted into a vertical flow device (syringe filter holder). Sample solution is passed through the filter paper and the target antigen (human IgG) is selectively captured by the immobilized antibody to form an antibody–antigen complex. Next, functionalized AuNPs as extrinsic Raman labels (ERLs) are passed through the filter paper to label the captured biomarker molecules forming a layered structure. This sandwiched geometry enhances plasmonic coupling and SERS signal to provide highly sensitive detection of biomolecules. Systematic studies to investigate the impact of multiple infuse/withdraw cycles of the sample and labeling solutions reveal that antigen and ERL binding are maximized with 10 and 20 cycles, respectively. The optimized assay achieves a detection limit of ∼0.2 ng mL−1 for human IgG with a total assay time of less than 5 minutes, meeting the demands for rapid point of care diagnostics. Additionally, the optimized platform was implemented in the quantitative analysis of the SARS-CoV-2 nucleocapsid protein, the typical target in commercial, FDA-approved rapid antigen tests for COVID-19. [ABSTRACT FROM AUTHOR]

Lateral flow immunochromatographic assays (LFIAs) are analytical devices used to detect the presence of one or more target analytes in a liquid sample. While LFIAs are one of the simplest and inexpensive types of immunoassays, they consist of multiple components (sample pad, conjugate pad, membrane, absorbent pad, backing card) and materials, requiring time-consuming device assembly. Here, we report a unique lateral flow immunochromatographic assay constructed from a single piece of cellulose paper, which is fabricated via laser cutting. Compared with conventional lateral flow immunochromatographic devices, this single-layer immunoassay enables simpler and faster fabrication, while minimizing material consumption and overall device costs. For proof-of-concept, this device was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a biomarker for malaria infection, which could be detected at concentrations as low as 4 ng mL−1 by the naked eye with no cross reactivity with other common Plasmodium protein biomarkers. While offering similar speed and ease-of-use as conventional LFIAs with a higher detection sensitivity than existing LFIAs for PfHRP2 detection, this single-layer lateral flow immunoassay has the potential to improve malaria testing, as well as the detection of other important protein biomarkers for point-of-care testing. [ABSTRACT FROM AUTHOR]

In this study, a novel paper-based SERRS immunoassay based on magnetic separation and alkaline phosphatase (ALP) enzyme catalyzed hydrolysis reaction was developed. By using modified antibodies conjugated to magnetic beads, capture of mouse IgG followed by addition of ALP-labeled antibodies would form a sandwich-like immunoconjugate. After magnetic separation, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), a low SERRS active compound, was added as the substrate for ALP to generate a high SERRS response. Detection was conducted on a silver colloid/PVP/filter paper SERS substrate by spotting a pre-aggregated silver colloid sol onto polyvinyl pyrrolidone (PVP) modified filter paper using a semi-automatic TLC sample applicator. The optimization of the highly SERS active paper-based substrate, dynamic hydrolysis process of BCIP, quantitative detection of IgG, and selectivity of the assay was studied in detail. By taking advantage of magnetic separation in order to decrease the background interference, the selective enzyme reaction involved in producing a highly SERRS active product could detect mouse IgG from 1 to 500 ng mL−1 with a LOD of 0.33 ng mL−1. [ABSTRACT FROM AUTHOR]

The quantification of blood cells provides critical information about a patient's health status. Sophisticated analytical equipment, such as hematology analyzers, have been developed to perform these measurements, but limited-resource settings often lack the infrastructure required to purchase, operate, and maintain instrumentation. To address these practical challenges, paper-based microfluidic devices have emerged as a platform to develop diagnostic assays specifically for use at the point-of-care. To date, paper-based microfluidic devices have been used broadly in diagnostic assays that apply immunoassay, clinical chemistry, and electrochemistry techniques. The analysis of cells, however, has been largely overlooked. In this communication, we demonstrate a paper-based microfluidic device that enables the controlled transport of red blood cells (RBCs) and the measurement of the hematocrit―the ratio of RBC packed cell volume to total volume of whole blood. The properties of paper, device treatment, and device geometry affect the overall extent and reproducibility of transport of RBCs. Ultimately, we developed an inexpensive (US$0.03 per device) thermometer-styled device where the distance traveled by RBCs is proportional to the hematocrit. These results provide a foundation for the design of paper-based microfluidic devices that enable the separation and detection of cells in limited-resource settings. [ABSTRACT FROM AUTHOR]

The document is a contents list for the journal "Lab on a Chip," published by the Royal Society of Chemistry. It includes a range of papers on topics such as microfluidics, nanotechnology, and biomedical research. Some of the specific papers mentioned include a programmable and automated optical electrowetting-on-dielectric (oEWOD) driven platform for single cell assay operations, an on-chip phytofluidic device for studying nutrient flow in plant roots, and optimized microfluidic formulation for improved lipid nanoparticle mediated genome editing. The journal covers a variety of scientific disciplines and offers valuable research for those interested in these fields. [Extracted from the article]

In this issue we highlight three recent papers that demonstrate new strategies to extend the capabilities of paper microfluidics. Paper (a mesh of porous fibers) has a long history as a substrate to perform biomolecular assays. Traditional lateral flow immunoassays (LFAs) are widely used for rapid diagnostic tests, and perform well when a yes or no answer is required and the analyte of interest is at relatively high concentrations. High concentrations are required because usually only a small volume of analyte-containing fluid flows past the detection region, leading to a limited signal. Further, the small pores within paper matrices prevent the use of paper to control the flow of larger particles and cells, limiting the use of paper microfluidics for cell-based diagnostics. The work we highlight addresses these important unmet challenges in paper microfluidics: enriching low concentration analytes to a higher concentration in a smaller volume that can be processed effectively, and using paper to pump flows in larger channels amenable to cells. Applying these new approaches may allow diagnosis of disease states currently technically unachievable using current LFA systems, while maintaining many of the “un-instrumented” advantages of an assay on self-wicking paper. [ABSTRACT FROM AUTHOR]

The complexity of current paper-fluidic diagnostic devices is limited by their imperfect control of reagent incubation and delivery. Valves providing complete fluid obstruction and multiple actuation steps within a single paper-fluidic diagnostic would increase the range of diagnostic bioassays in these porous membranes. Here, we report the rapid fabrication of tunable wax-ink valves that are thermally actuated within porous membranes. Varying width bands of wax-ink were printed onto strips of nitrocellulose and cellulose membranes and characterized by their triggered obstruction and release of fluid wicking through the membranes. To demonstrate the utility of these wax-ink valves, we have transformed a traditional lateral flow immunoassay into a multi-step, semi-autonomous assay that enhances bacterial detection signal intensity 6-fold. The wax-ink valves are applicable to other paper fluidic assays by fully obstructing fluid flow for a sustained reaction time and are able to be actuated multiple times for sequential fluid delivery with minimal user involvement. These easily fabricated wax-ink valves are a versatile addition to the toolkit of fluidic control mechanisms required to develop more sensitive paper-based diagnostics. [ABSTRACT FROM AUTHOR]

The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (μPADs) is reported. The μPADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was deposited at the control and test zones. μPAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody to travel past the capture zone and into the test zone via capillary action. The developed competitive immunoassay system was successfully demonstrated on the μPAD detection of biotin as a model compound with a detection limit of 0.10 μg mL−1. The applicability of the proposed immunoassay system for point-of-need testing was further demonstrated using aflatoxin B1, a highly toxic foodborne substance, with a detection limit of 1.31 ng mL−1. The μPAD with the competitive immunoassay format showed promising results for practical applications in point-of-need testing involving small molecular weight targets in food and water safety and quality monitoring, environmental analysis, and clinical diagnostics. [ABSTRACT FROM AUTHOR]

The article focuses on controlled transport of reagents within paper devices using two dimensional (2D) paper networks. The geometry of the paper network and dissolvable barriers makes them able to transport multiple analytes and reagents. It states these paper-based lateral flow immunoassay strips analyse only a single analyte per strip but their utility can be increased using highly sensitive particle labels like quantum dots. It also offers information on an experiment and its result.

Diagnostic assays can provide valuable information about the health status of a patient, which include detection of biomarkers that indicate the presence of an infection, the progression or regression of a disease, and the efficacy of a course of treatment. Critical healthcare decisions must often be made at the point-of-care, far from the infrastructure and diagnostic capabilities of centralized laboratories. There exists an obvious need for diagnostic tools that are designed to address the unique challenges encountered by healthcare workers in limited-resource settings. Paper, a readily-available and inexpensive commodity, is an attractive medium with which to develop diagnostic assays for use in limited-resource settings. In this article, we describe a device architecture to perform immunoassays in patterned paper. These paper-based devices use a combination of lateral and vertical flow to control the wicking of fluid in three-dimensions. We provide guidelines to aid in the design of these devices and we illustrate how patterning can be used to tune the duration and performance of the assay. We demonstrate the use of these paper-based devices by developing a sandwich immunoassay for human chorionic gonadotropin (hCG) in urine, a biomarker of pregnancy. We then directly compare the qualitative and quantitative results of these paper-based immunoassays to commercially available lateral flow tests (i.e., the home pregnancy test). Our results suggest paper-based devices may find broad utility in the development of immunoassays for use at the point-of-care. [ABSTRACT FROM AUTHOR]

ZnO nanorods inorganic-organic heterostructured light-emitting diodes have been demonstrated on a cheap/disposable paper substrate and applied in multiplexed photoelectrochemical immunoassay. [ABSTRACT FROM AUTHOR]

A novel microplate-compatible chemiluminescence (CL) immunoassay has been developed for the determination of human immunoglobulin G (IgG) based on the luminol–AgNO3–gold nanoparticles CL system. Polystyrene microtiter plates were used for both immunoreactions and CL measurements. The primary antibody, goat-anti-human IgG, was first immobilized on polystyrene microwells. Then the antigen (human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex. The gold label could trigger the reaction between luminol and AgNO3, accompanied by light emission. Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng mL−1, with a detection limit of 12.8 ng mL−1(∼80 pM) at a signal to noise ratio of three (S/N= 3). Compared with other reported CL immunoassay method based on gold labels, the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes, making the method more simple, time-saving and easily automated. The present CL method is promising for the determination of clinically important bioactive analytes. [ABSTRACT FROM AUTHOR]

A method for detecting glutathione selectively in whole blood deposited on filter paper is described. GSH is fractionated from proteins, hemoglobin and other potentially interfering components and determined using a resorufin-acrylate fluorescent probe. The relative standard deviation is lower than 5% (n = 5). Recoveries of GSH from whole blood are between 94% and 108.6%. [ABSTRACT FROM AUTHOR]

We report a method for converting the colorimetric results of an enzyme-linked immunosorbent assay (ELISA) into length based on the coffee-ring effect, so that the quantitative detection of analytes can be carried out simply using a ruler. The influence of the shape and lamination of the paper strip on the test results is studied. As a demonstration, human IgG is quantitatively analyzed. It is found that the width of the colored stains correlates with the concentration of the analyte which can be measured for quantitative analysis. The method is promising for quantitative point-of-care detection of biomarkers under resource-limited settings. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (P. aeruginosa) is a pathogen that persistently colonizes the respiratory tract of patients with chronic lung diseases. The risk of acquiring a chronic P. aeruginosa infection can be minimized by rapidly detecting the pathogen in the patient's airways and promptly administrating adequate antibiotics. However, the rapid detection of P. aeruginosa in the lungs involves the analysis of sputum, which is a highly complex matrix that is not always available. Here, we propose an alternative diagnosis based on analyzing breath aerosols. In this approach, nanoparticle immunosensors identify bacteria adhered to the polypropylene layer of a surgical facemask that was previously worn by the patient. A polypropylene processing protocol was optimized to ensure the efficient capture and analysis of the target pathogen. The proposed analytical platform has a theoretical limit of detection of 105 CFU mL−1 in aerosolized mock samples, and a dynamic range between 105 and 108 CFU mL−1. When tested with facemasks worn by patients, the biosensors were able to detect chronic and acute P. aeruginosa lung infections, and to differentiate them from respiratory infections caused by other pathogens. The results shown here pave the way to diagnose Pseudomonas infections at the bedside, as well as to identify the progress from chronic to acute infection. [ABSTRACT FROM AUTHOR]

Steroid hormones play important roles in metabolism and metabolic diseases. Currently, various detection methods are employed in clinical labs, mainly immunoassays and LC-MS/MS, but these methods suffer from antibody cross-reactivity or the need for complex LC processes, respectively. Here, we utilized single antibody to capture and separate multiple hormones from samples to avoid LC procedures and used MS/MS to analyze multiple molecules in a single run. In our strategy, testosterone (T), androstenedione (4-AD), and androsterone (ADT) were affinity-captured simultaneously using only T antibody. The qualitative and quantitative analysis of three androgens was realized through MS/MS spectra using testosterone-D3 (T-D3) as an internal standard. Standard curves for standard solution or spiked serum samples were realized in the range of 0.01–2 μg L−1. The LODs for the three androgens were 2.3 × 10−3 μg L−1 for testosterone, 4.6 × 10−3 μg L−1 for androstenedione, and 2.8 × 10−3 μg L−1 for androsterone. The recovery results verified the reliability and stability of our method. This strategy has widespread potential for advancing the combination of immunoassay and MS methods in the analysis of small molecules, with high through-put and low cost. [ABSTRACT FROM AUTHOR]

Dye-encoded bead-based assays are widely used for diagnostics. Multiple bead populations are required for multiplexing and can be produced using different dye colors, labeling levels, or combinations of dye ratios. Ready-to-use multiplex bead populations restrict users to specific targets, are costly, or require specialized instrumentation. In-house methods produce few bead plexes or require many fine-tuning steps. To expand bead encoding strategies, we present a simple, safe, and cost-effective bench-top system for generating bead populations using photobleaching. By photobleaching commercially available dye-encoded magnetic beads for different durations, we produce three times as many differentiable bead populations on flow cytometry from a single dye color. Our photobleaching system uses a high-power LED module connected to a light concentrator and a heat sink. The beads are photobleached in solution homogeneously by constant mixing. We demonstrate this photobleaching method can be utilized for cross-testing antibodies, which is the first step in developing immunoassays. The assay uses multiple photobleached encoded beads conjugated with capture antibodies to test many binding pairs simultaneously. To further expand the number of antibodies that can be tested at once, several antibodies were conjugated to the same bead, forming a pooled assay. Our assay predicts the performance of antibody pairs used in ultrasensitive Simoa assays, narrowing the number of cross-tested pairs that need to be tested by at least two-thirds and, therefore, providing a rapid alternative for an initial antibody pair screening. The photobleaching system can be utilized for other applications, such as multiplexing, and for photobleaching other particles in solution. [ABSTRACT FROM AUTHOR]

Paper-based immunoassays, usually in the form of lateral flow tests, are currently the standard platform for home diagnostics. However, conventional lateral tests are often complicated by severe non-specific adsorption of detector particles when applied to test samples containing salivary fluid. It is believed that a high concentration of proteinaceous substances in salivary fluid causes particle aggregation and adhesion. In this study, we developed a stacking flow platform for single-step detection of a target antibody in salivary fluid. Stacking flow circumvents the need for separate sample pre-treatments, such as filtration or centrifugation, which are often required prior to testing saliva samples using paper-based immunoassays. This is achieved by guiding the samples and reagents to the test strip through different paths. By doing so, salivary substances that interfere with the particle-based sensing system are removed before they come into contact with the detection reagents, which greatly reduces the background. In addition, the stacking flow configuration enables uniform flow with a unique flow regulator, which leads to even test lines with good quantification capability, enabling the detection of ~20 ng mL−1α-fetoprotein in the serum. We have successfully applied the stacking flow device to detect dengue-specific immunoglobulins that are present in salivary fluid. [ABSTRACT FROM AUTHOR]

The development of sensitive breast cancer biomarker (CA 15-3) measurement techniques is of great significance for the early diagnosis and therapeutic monitoring of breast cancer worldwide. Herein, we have designed a split-type photoelectrochemical (PEC) immunoassay platform for the ultrasensitive determination of CA 15-3 using In2O3/ZnIn2S4 heterojunctions with high photovoltaic conversion performance as a photoactive substrate. Efficient photocurrent-responsive In2O3/ZnIn2S4 was formed by surface loading narrow-bandgap ZnIn2S4 on MOF-derived wide bandgap In2O3 nanomaterials. Subsequently, a sandwich immunoreaction was performed using an alkaline phosphatase (ALP)-labeled secondary antibody as a signaling indicator to produce large amounts of ascorbic acid by enzymatic biocatalysis. Since ascorbic acid is an excellent electron donor capable of promoting photogenerated electron separation in semiconductor materials, it enhances the anodic photocurrent of In2O3/ZnIn2S4-modified electrodes. Under the optimal catalyst loading and immunoreaction time, the In2O3/ZnIn2S4-based PEC immunoassay showed a good photocurrent response to CA 15-3 over a wide detection range from 1.0 mU mL-1 to 10000 mU mL-1 with a limit of detection of 0.45 mU mL-1. Owing to the excellent photocurrent response of In2O3/ZnIn2S4 to ascorbic acid and the split-type immunoassay protocol, the constructed PEC immunosensing platform has the advantages of high precision, anti-interference, and acceptable accuracy, and the results are equivalent to those of commercial enzyme-linked immunosorbent assay (ELISA) kits. [ABSTRACT FROM AUTHOR]

Integrating electrochemistry into capillary-flow driven immunoassay devices provides unique opportunities for quantitative point-of-care testing. Although custom electrodes can be inexpensive and are tunable, they require skilled fabrication. Here, we report the incorporation of a commercial electrode into a capillary-flow driven immunoassay (iceCaDI) device for a single end-user step sandwich electrochemical enzyme-linked immunosorbent assay (ELISA). The iceCaDI device is a pump-free portable microfluidic device with an integrated commercial screen-printed electrode and flow driven by capillary action. The iceCaDI device is composed of alternating polyester transparency film and double-sided adhesive film layers that are patterned with a laser cutter. This platform was designed to address known limitations of laminated device fabrication methods and operation. First, we developed a foldable laminated device fabrication using hinges for easy assembly and precise alignment. Second, reagent dispersing was achieved by incorporating a 1 mm wide arrow-shaped notch in the middle of the channel that trapped an air bubble and formed a baffle that facilitated reagent spreading to cover the detection area. Third, small vent holes were added to the top layer of the channels to prevent air bubbles from blocking flow. Finally, we fabricated a CRP immunosensor with a detection range of 0.625 to 10.0 μg mL−1 as a proof-of-concept to demonstrate an automatically driven sandwich electrochemical ELISA using the iceCaDI device. Three concentrations of CRP were successfully measured under flow conditions within 8 min. Our proposed device is a promising approach and a step forward in the development of point-of-care (POC) devices for techniques that traditionally require multiple user steps. [ABSTRACT FROM AUTHOR]

Developing an efficient signal amplification strategy is very important to improve the sensitivity of bioanalysis. In this paper, a liposome-assisted enzyme catalysis signal amplification strategy was developed for electrochemiluminescence (ECL) immunoassay of prostate specific antigen (PSA) in a split-type mode. The sandwich immunoreaction occurred in a 96-well plate, and glucose oxidase (GOx) encapsulated and antibody-modified liposomes were used as labels. The ECL detection was carried out using a rGO-Au NP modified glassy carbon electrode (GCE). The large amount of generated H2O2, i.e. the coreactant of the luminol system, and the excellent catalytic behavior of rGO-Au NPs greatly boosted the ECL signal, resulting in the signal amplification. The developed ECL immunosensor for detecting PSA achieved a wider linear range from 1.0 × 10−13 to 1.0 × 10−8 g mL−1 and a detection limit of 1.7 × 10−14 g mL−1. The application of the proposed strategy was demonstrated by analyzing PSA in human serum samples with recoveries from 89.0% to 113.0%, and relative standard deviations (RSDs) were less than 6.6%. This work provides a new horizon to expand the application of liposomes for ECL bioanalysis. [ABSTRACT FROM AUTHOR]

An impedimetric biosensor is used to measure electrical impedance changes in the presence of biomolecules from sinusoidal input voltages. In this paper, we present a new portable impedance-based biosensor platform to improve the sensitivity of immunoassays with microparticles as a label. Using a 2 × 4 interdigitated electrode array with a 10/10 μm electrode/gap and a miniaturized impedance analyzer, we performed immunoassays with microparticles by integrating a microfluidic channel to evaluate signal enhancement. First, to understand the material dependency of microparticles on the sensor array, magnetic, silica, and polystyrene microparticles were tested. Among these microparticles, magnetic microparticles presented a high signal enhancement with relevant stability from the sensor array. With the magnetic microparticles, we demonstrate a series of immunoassays to detect human tumor necrosis factor (TNF-α) and compare the level of signal enhancement by measuring the limit of detection (LOD). With the microparticles, we achieved over ten times improvement of LOD from sandwich immunoassays. By incorporating with sample preparation and flow manipulation systems, this impedance sensor array can be utilized for digital diagnostics for a real sample-in answer-out system. [ABSTRACT FROM AUTHOR]

Excessive release of neutrophil extracellular traps (NETs) has been reported in various human pathologies, including COVID-19 patients. Elevated NET levels serve as a biomarker, indicating increased coagulopathy and immunothrombosis risks in these patients. Traditional immunoassays employed to quantify NET release focus on bulk measurements of released chromatin in simplified microenvironments. In this study, we fabricated a novel NET-array device to quantify NET release from primary human neutrophils with single-cell resolution in the presence of the motile bacteria Pseudomonas aeruginosa PAO1 and inflammatory mediators. The device was engineered to have wide chambers and constricted loops to measure NET release in variably confined spaces. Our open NET-array device enabled immunofluorescent labeling of citrullinated histone H3, a NET release marker. We took time-lapse images of primary healthy human neutrophils releasing NETs in clinically relevant infection and inflammation-rich microenvironments. We then developed a computer-vision-based image processing method to automate the quantification of individual NETs. We showed a significant increase in NET release to Pseudomonas aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α [20 ng mL−1] and interleukin-6 [50 ng mL−1], but not leukotriene B4 [20 nM], compared to the infection alone. We also quantified the temporal dynamics of NET release and differences in the relative areas of NETs, showing a high percentage of variable size NET release with combined PAO1 – inflammatory mediator treatment, in the device chambers. Importantly, we demonstrated reduced NET release in the confined loops of our combined infection–inflammation microsystem. Ultimately, our NET-array device stands as a valuable tool, facilitating experiments that enhance our comprehension of the spatiotemporal dynamics of NET release in response to infection within a defined microenvironment. In the future, our system can be used for high throughput and cost-effective screening of novel immunotherapies on human neutrophils in view of the importance of fine-tuning NET release in controlling pathological neutrophil-driven inflammation. [ABSTRACT FROM AUTHOR]