ABSTRACT: Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.
Project description:Sensitive, single volume detections of multiple diabetes antibodies can provide immunoprofiling and early screening of at-risk patients. To advance the state-of-the-art suspension assays for diabetes antibodies, porous hydrogel droplets are leveraged in microfluidic serpentine arrays to enhance reagent transport. This spatially multiplexed assay is applied to the detection of antibodies against insulin, glutamic acid decarboxylase, and insulinoma-associated protein 2. Optimization of assay protocol results in a shortened assay time of 2 h, with better than 20 pg mL Supporting Information detection limits across all three antibodies. Specificity and cross-reactivity tests show negligible background, nonspecific antibody-antigen, and nonspecific antibody-antibody bindings. Multiplexed detections are able to measure within 15% of target concentrations from low to high ranges. The technique enables quantifications of as little as 8000 molecules in each 500 µm droplet in a single volume, multiplexed assay format, a breakthrough necessary for the adoption of diabetes panels for clinical screening and monitoring in the future.
Project description:Isolating small objects, such as particles, cells, and molecules, in individual aqueous droplets is useful for chemical and biological assays. We have developed a simple microfluidic platform to immobilize (park) microparticles at defined locations, and isolate particles in monodisperse droplets surrounded by immiscible oil. While conventional methods can only achieve stochastic encapsulation of objects within larger droplets, our in situ method ensures that a single particle is entrapped in a similar-sized droplet, with ?95% yield for parking and isolation. This enables time-lapse studies of reactions in confined volumes and can be used to perform enzymatic amplification of a desired signal to improve the sensitivity of diagnostic assays. To demonstrate the utility of our technique, we perform highly sensitive, multiplexed microRNA detection by isolating encoded, functional hydrogel microparticles in small aqueous droplets. Non-fouling hydrogel microparticles are attractive for microRNA detection due to favorable capture kinetics. By encapsulating these particles in droplets and employing a generalizable enzyme amplification scheme, we demonstrate an order of magnitude improvement in detection sensitivity compared to a non-amplified assay.
Project description:Highly sensitive and multiplexed detection of clinically relevant proteins in biologically complex samples is crucial for the advancement of clinical proteomics. In recent years, aptamers have emerged as useful tools for protein analysis due to their specificity and affinity for protein targets as well as their compatibility with particle-based detection systems. In this study, we demonstrate the highly sensitive detection of human ?-thrombin on encoded hydrogel microparticles functionalized with an aptamer capture sequence. We use static imaging and microfluidic flow-through analysis techniques to evaluate the detection capabilities of the microgels in sandwich-assay formats that utilize both aptamers and antibodies for the reporting of target-binding events. Buffers and reagent concentrations were optimized to provide maximum reaction efficiency while still maintaining an assay with a simple workflow that can be easily adapted to the multiplexed detection of other clinically relevant proteins. The three-dimensional, nonfouling hydrogel immobilization scaffold used in this work provides three logs of dynamic range, with a limit of detection of 4 pM using a single aptamer capture species and without the need for spacers or signal amplification.
Project description:The integration of semiconductor nanoparticle quantum dots (QDs) into a modular, microfluidic biosensor for the multiplexed quantitation of three important cancer markers, carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and Her-2/Neu (C-erbB-2) was achieved. The functionality of the integrated sample processing, analyte capture and detection modalities was demonstrated using both serum and whole saliva specimens. Here, nano-bio-chips that employed a fluorescence transduction signal with QD-labeled detecting antibody were used in combination with antigen capture by a microporous agarose bead array supported within a microfluidics ensemble so as to complete the sandwich-type immunoassay. The utilization of QD probes in this miniaturized biosensor format resulted in signal amplification 30 times relative to that of standard molecular fluorophores as well as affording a reduction in observed limits of detection by nearly 2 orders of magnitude (0.02 ng/mL CEA; 0.11 pM CEA) relative to enzyme-linked immunosorbent assay (ELISA). Assay validation studies indicate that measurements by the nano-bio-chip system correlate to standard methods at R(2)=0.94 and R(2)=0.95 for saliva and serum, respectively. This integrated nano-bio-chip assay system, in tandem with next-generation fluorophores, promises to be a sensitive, multiplexed tool for important diagnostic and prognostic applications.
Project description:Point-of-care tests (POCT) for pathogens are considered important for low-resource countries and facilities. Although lateral flow immunoassays (LFIA) have many advantages including speed and ease of use, their sensitivity is limited without specific equipment. Furthermore, their response cannot be enhanced through enzymatic reactions. Owing to these limitations, LFIAs have not yet been generally adopted as the standard protocol for in vitro analysis of infectious pathogens. We aimed to develop a novel pipetting-based immunoassay using a removable magnetic ring-coupled pipette tip. The "magnetic bead-capture antibody-targeted protein complex" was simply purified by pipetting and quantified by enzymatic colour development or using a lateral flow system. This pipetting-based immunoassay was applied to detect the nucleoprotein (NP) of the influenza A virus. Using an HRP-conjugated monoclonal antibody as a probe, the assay allowed for specific and sensitive detection. Furthermore, when this assay was applied exclusively for antigen capture in the lateral flow system, the limit of detection improved 100-fold and displayed greater sensitivity than the lateral flow system alone. Therefore, the pipetting-based immunoassay may be potentially used as a sensitive POCT to clinically detect a target antigen.
Project description:The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.
Project description:High affinity capture agents against protein targets are essential components for immunoassays, regardless of specific analysis format. Here, we describe the use of DNA-encoded antibodies for rapidly screening the kinetic and equilibrium binding properties of twelve commercial antibodies in a parallel analysis format using a multiplexed array of microring optical resonators. We show that DNA-encoding offers advantages in terms of antigen binding capacity, compared to covalently tethered antibodies; we also demonstrate that this linkage modality facilitates the rapid self-assembly of multiplexed arrays on account of complementarity between the DNA sequences on the antibodies and sensor array, respectively. Furthermore, DNA-encoded antibodies also allow for sensor array regeneration and reprogramming, as chaotropic agents can be used to disrupt the DNA-DNA duplexes that link the capture agents to the sensor without harming the underlying DNA on the surface, which can subsequently be reloaded with antibodies either targeting the same or different antigens.
Project description:Diverse assays spanning from immunohistochemistry (IHC), to microarrays (protein, DNA), to high-throughput screens rely on probe-target hybridization to detect analytes. These large-format 'chips' array numerous hybridization sites across centimeter-scale areas. However, the reactions are prone to intra-assay spatial variation in hybridization efficiency. The mechanism of spatial bias in hybridization efficiency is poorly understood, particularly in IHC and in-gel immunoassays, where immobilized targets are heterogeneously distributed throughout a tissue or hydrogel network. In these systems, antibody probe hybridization to a target protein antigen depends on the interplay of dilution, thermodynamic partitioning, diffusion, and reaction. Here, we investigate parameters governing antibody probe transport and reaction (i.e., immunoprobing) in a large-format hydrogel immunoassay. Using transport and bimolecular binding theory, we identify a regime in which immunoprobing efficiency (?) is sensitive to the local concentration of applied antibody probe solution, despite the antibody probe being in excess compared to antigen. Sandwiching antibody probe solution against the hydrogel surface yields spatially nonuniform dilution. Using photopatterned fluorescent protein targets and a single-cell immunoassay, we identify regimes in which nonuniformly distributed antibody probe solution causes intra-assay variation in background and ?. Understanding the physicochemical factors affecting probe-target hybridization reduces technical variation in large-format chips, improving measurement precision.
Project description:There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally responsive poly(N-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-co-N,N-dimethylaminoethylacrylamide was prepared by reversible addition-fragmentation chain transfer polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, coaggregation of the AuNP half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity.
Project description:In recent years, species identification in herbs has attracted considerable attention due to several cases of fraud; hence inexpensive high-throughput authentication methods are highly welcomed. Species authentication is often performed through DNA analysis and several specific regions (barcodes) are considered suitable. Each barcode (Bar) possesses different qualities in terms of universality and discrimination power. A multiplexed format where information can be extracted simultaneously from several barcode regions is seemingly appropriate to ensure the power of both universality and discrimination. In this approach, we amplified DNA from five different barcode regions in a multiplexed PCR format followed by high-resolution melting (HRM). This multiplexed Bar-HRM approach was first applied to plants spanning the plant kingdom and then gradually narrowing down the genetic variability within the Lamiaceae and the Solanaceae families to finally reach closely related cultivars. Universality was demonstrated through distinct melting profiles obtained for species originating from 29 different families spanning the angiosperms, gymnosperm, mosses, and liverwort (Marchantiophyta). Discrimination power was retained for species, sub-species, and a few cultivars through the application of multivariate statistics to the high-resolution melting profiles. This preliminary investigation has shown the potential to discriminate a vast amount of species within the whole plant kingdom. It requires no a priori knowledge of the species' DNA sequence and occurs in a closed system within 2.5?h at a reduced cost per sample compared to other DNA based approaches.