Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus

Abstract

Towards developing rapid and portable diagnostics for detecting zoonotic diseases, we have developed microchip-based electrophoretic immunoassays for sensitive and rapid detection of viruses. Two types of microchip-based electrophoretic immunoassays were developed. The initial assay used open channel electrophoresis and laser-induced fluorescence detection with a labeled antibody to detect influenza virus. However, this assay did not have adequate sensitivity to detect viruses at relevant concentrations for diagnostic applications. Hence, a novel assay was developed that allows simultaneous concentration and detection of viruses using a microfluidic chip with an integrated nanoporous membrane. The size-exclusion properties of the in situ polymerized polyacrylamide membrane are exploited to simultaneously concentrate viral particles and separate the virus/fluorescent antibody complex from the unbound antibody. The assay is performed in two simple steps--addition of fluorescently labeled antibodies to the sample, followed by concentration of antibody-virus complexes on a porous membrane. Excess antibodies are removed by electrophoresis through the membrane and the complex is then detected downstream of the membrane. This new assay detected inactivated swine influenza virus at a concentration four times lower than that of the open-channel electrophoresis assay. The total assay time, including device regeneration, is six minutes and requires <50 microl of sample. The filtration effect of the polymer membrane eliminates the need for washing, commonly required with surface-based immunoassays, increasing the speed of the assay. This assay is intended to form the core of a portable device for the diagnosis of high-consequence animal pathogens such as foot-and-mouth disease. The electrophoretic immunoassay format is rapid and simple while providing the necessary sensitivity for diagnosis of the illness state. This would allow the development of a portable, cost-effective, on-site diagnostic system for rapid screening of large populations of livestock, including sheep, pigs, cattle, and potentially birds.

Charge of water droplets in non-polar oils

“…It is commonly used for single cell analysis [71]. For example, Reichmuth et al [86] presented a microfluidic chip of electrophoretic immunoassay, which was used to detect and concentrate viruses simultaneously. In their work, electrophoresis was used to separate and remove excessive antibodies from the antibody virus complexes.…”

Development and Applications of Portable Biosensors

“…A microfluidic filter chip 53 with a stepped microchannel configuration has been reported to capture and enable the formation of a sandwich complex of a monolayer of beads, bacteria, and peroxidase-labeled antibodies inside a microchamber. Polyacrylamide membranes with nanopores 85 for simultaneous concentration of viral particles and separation of virus-fluorescent antibody complexes was observed to improve the sensitivity and detection time when compared to the use of an electrophoretic immunoassay alone. Microfabricated pillars that were functionalized with an affinity for bacterial cells inside a PCR chip have been applied to detect E. coli in blood samples.…”

On-chip sample preparations for Point-of-Care cellular analysis of blood

“…A well-established application is the separation of WBCs and RBCs in whole blood [6][7][8][9]. Bench-top filtration concepts using fibrous membranes (nitrocellulose [7], polycarbonate [8,10], polyacrylamide [11]) can be simply down-scaled and implemented into microdevices. In such devices, whole blood flows perpendicularly to the membrane comprising a magnitude of pores of precisely controlled size, which allows the passage of cells smaller than a critical size and retains their larger counterparts.…”