The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed ...for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1-7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.
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.
While a number of assays for soluble analytes have been developed using paper-based microfluidic devices, the detection and analysis of blood cells has remained an outstanding challenge. In this ...Feature, we discuss how the properties of paper determine the performance of paper-based microfluidic devices and permit the design of cellular assays, which can ultimately impact disparities in healthcare that exist in limited-resource settings.
The development of viable point-of-care diagnostic formats is integral to achieving better patient care and improved outcomes. The need for robust and low-cost tests is especially important in ...under-resourced and rural settings. Perhaps the greatest challenge is ensuring that an untrained individual is capable of operating and interpreting the test, out with a care facility. Here we present a paper-based diagnostic device capable of sensing miR-29a using both colorimetric and surface enhanced Raman scattering (SERS) analysis. Rather, than carry out the two types of analyses in tandem, we envisage that the colorimetric output is easy enough to be interpreted by the untrained-individual administering the test to provide them with qualitative feedback. If deemed positive, the test can be further validated at a centralized care facility using a handheld-Raman spectrometer to provide a semi-quantitative result. Detection of miR-29a, a microRNA associated with myocardial infarction, was achieved at a level of pg μL
−1
through the combination of three-dimensional paper-based microfluidics, colorimetric detection, and surface enhanced Raman scattering (SERS) analysis. RGB analysis of the colorimetric output generated from samples containing miR-29a at different concentrations (18-360 pg μL
−1
) showed differentiation from the control sample, however significant repeat variability indicated that it could not be used for quantifying miR-29a levels. However, the SERS analysis exhibited greater reproducibility at varying concentrations, achieving an LoD of 47 pg μL
−1
. The union of the paper-based device and the two analysis methods resulted in the production of a sensitive, reproducible and facile, point of care test (POCT), which paves the way for future implementation in the diagnosis of a range of diseases.
A paper-based microfluidics self-testing device capable of colorimetric and SERS-based sensing of cardiovascular disease associated miR-29a has been developed for improving patient care and triage.
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.
Point-of-care diagnostic tests can provide rapid and accurate information about the health of a patient without relying on the expensive equipment found in centralized laboratories. Not only does the ...development and application of these assays rely on the means of signal transduction, but also the method by which a sample is procured. In order for point-of-care tests to have real clinical utility, they must use sample types and volumes that are relatively easy to obtain ( e.g. , fingerstick volumes of blood). In this paper, we demonstrate how the design of a paper-based microfluidic device controls the transport of blood within the device and ultimately influences the development of an assay to measure the hematocrit. We show that, with directed design modifications, altering the dimensions of paper-based devices can greatly reduce the volume of blood required to initiate a hematocrit assay (from 50 μL to only 10 μL) without impacting its analytical performance.
Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, ...stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.
•Detection and differentiation of different isomers of dinitrotoluenes using visible spectroscopy.•Determination of the kinetic parameters for the reaction of dinitrotoluenes with ...hydroxide.•Elucidated the essential role of water in the reaction of dinitrotoluenes with water.•Proposing a mechanism for the reaction of dinitrotoluenes with bases supported by visible spectroscopy, NMR spectroscopy, and computations.
The reactions of dinitrotoluenes (DNT) with hydroxide ion or wet alkylamines can be used to detect and identify each isomer. The reaction products give visible spectra that can be used directly to determine the isomer speciation with sensitivities in the low micromolar range. NMR spectroscopy in dimethylformamide provides unambiguous identification of the products of each DNT when the base is hydroxide. Kinetic studies show that the initial product of this reaction is the deprotonated anion followed by subsequent formation of σ-adducts and, in some cases, a dimer, with the exception of 2,4-DNT, which does not react beyond the acid–base reaction. Alkylamines do not react with any DNT, even when the amine is acting as the solvent, unless there is water present. Water is the limiting reagent in these cases, implying that in all cases the reacting species is the hydroxide ion, not the free amine. Computational studies are consistent with the inability of the alkylamine to deprotonate the methyl group in any of the DNT isomers. A general mechanism that is applicable to hydroxide and amine bases is proposed.
The reaction of trinitrotoluene (TNT) with bases has been investigated by NMR and visible spectroscopy methods. Hydroxide ion was found to react in one of two ways, either by deprotonation of the ...methyl group or by nucleophilic attack on the aromatic ring to form a σ adduct. The rate of each mode of reaction depends upon the polarity of the solvent. In tetrahydrofuran (THF), σ adduct formation is rapid and the long-term equilibrium product is deprotonation of the methyl group. When the solvent is methanol (MeOH), the two reactions have similar rates and the σ adduct becomes the majority product. Amines were found to be ineffective in directly deprotonating TNT or in forming σ adducts. Rather, the amines react with ambient water to generate hydroxide ion, which then reacts with TNT. The solvent choice and water content are crucial to understanding the reactivity of bases with TNT. To assist in the interpretation of the experimental results, computational analysis was performed at the B3LYP/6-311+G**//HF/6-311+G** level to determine the thermodynamics of the reactions of TNT. The SM8 implicit solvation model was applied to converged geometries and suggested a strong solvation effect upon product formation. Thermodynamic analysis suggested a significant preference of alkoxide or hydroxide attack versus amine attack in any modeled dielectric, consistent with the experimental observations.
The ability to detect antibodies that are generated during an immune response is integral to the diagnosis and monitoring of infections. Assays that have been applied to the detection of antibodies ...are classically referred to as indirect immunoassays, which include three different formats: indirect, double-antigen sandwich, and total IgG capture. Each of these three approaches relies on a unique pair of reagents to capture the target antibody and transduce a detectable signal, which permits assays to be tuned for ideal performance based on the availability and quality of reagents, the resources available to the operator, and the complexity of the sample ( e.g. , serum vs. saliva). This flexibility, however, can complicate the development of diagnostic tests and increase the complexity of the devices required to perform them. A general platform that can enable the rapid development of inexpensive and simple diagnostic assays would be highly desirable, particularly for those assays intended for use in limited-resource settings. In this manuscript, we describe a paper-based microfluidic device architecture that is capable of supporting these three indirect immunoassay formats by simply changing reagents and without requiring significant alterations to the design or manufacture of the device. We compare the performances of these indirect immunoassays by developing assays for HIV antibodies in human serum. These results suggest that this device format has broad potential for the development of paper-based immunoassays.
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