Methods and devices for microfluidic point-of-care immunoassays

Abstract

Microfluidic methods and devices for heterogeneous binding and agglutination assays are disclosed, with improvements relating to mixing and to reagent and sample manipulation in systems designed for safe handling of clinical test samples.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a divisional of application Ser. No. 12 / 341,637, filed Dec. 22, 2008, which is a continuation of International PCT Patent Application No. PCT / US2007 / 014522, filed Jun. 22, 2007, which claims the benefit of U.S. Provisional Patent Application No. 60 / 816,204, filed Jun. 23, 2006. These applications are incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The invention relates to clinical assays performed in microfluidic devices and in particular to heterogeneous binding and agglutination immunoassays and to disposable devices for point-of-care immunodiagnostics.[0004]2. Description of the Related Art[0005]Detection of biomarkers at the point of care (such as, for example, in the field, in remote areas, in a doctor's office, and at the bedside in a hospital) has the potential to offer real time diagnostic information, improve patient care outcomes, decrease sa...

AI Technical Summary

Benefits of technology

[0013]Complete enzyme-linked immunosorbent assay (ELISA) systems in a microfluidic device format are provided for detection of a wide spectrum of biomarker molecules. Such devices are generally disposable and low-cost. Bioassays adapted to a robust microfluidics device format include solid phase affinity-capture assays such as those based on antibody / antigen, antigen / antibody, antibody / protein A, glycomer / lectin, and, generally, signal molecule / receptor as target:affinity-capture pairs. Preferred solid phase affinity-capture assay systems for ELISA include antibody / antigen, antigen / antibody, antibody / protein A, streptavidin / avidin, and histidine / NTA target:affinity-capture pairs. Enzyme-linked antibodies, antigens, streptavidin, and histidine-rich proteins are generally available or may be synthesized by techniques well known in the art. Detection systems for immunoaffinity capture and tagging or amplification of target biomarker signals include, for example, enzyme-linked conjugates and chromogenic substrates (immunochromogenic and ELISA-type detection), streptavidin-enzyme conjugates (again with ELISA-type or immunochromogenic detection), antibody-coupled beads, antigen-coupled beads (with immunoprecipitin or agglutination-type detection), protein A-coupled beads, streptavidin-coupled beads, and enzyme- or bead-conjugated protein-histidine-nickel chelates. Beads for tagging can be colored, fluorescent, luminescent, tagged with radio-frequency transmitters, or otherwise labeled so that binding or agglutination can be readily detected. Titrations and binding neutralization assays also provide detectable endpoints. The microfluidic device-based immunoassays disclosed here also anticipate the use of magnetic beads in affinity concentration steps or mixing.

[0014]Mixing of small volumes as required to contact the target analyte with the solid-phase affinity capture agent and with reagents is accomplished with diaphragm pumps, herein termed “bellows pumps”, useful to generate reciprocating flow across the affinity capture site, which is placed between a pair of such pumps. Stagnant liquid is broken up by use of “flow constricting” or “flow focusing” apertures positioned between the pumps and the central chamber containing the affinity capture site or sites, eliminating the need for impellers and reducing incubation time. Mean flow velocity is increased by the focusing apertures, causing exit plume microeddies characteristic of turbulent or near-turbulent flow that aid mixing, an inventive adaptation of a Penberthy eductor to a micro-scale. This mixing method is referred to herein as “micro-eductive mixing” or “eductive mixing”. By pairing bellows pumps inside the body of a microfluidic device, the system can be completely closed (without venting) during operation, a useful precaution against operator exposure to the contents of the device.

Problems solved by technology

Mixing small volumes, however, is not without unique problems.

However, these methods depend on relatively large sample volumes, large hybridization chambers and inconvenient or complicated equipment not readily adapted to the point of care.

The pressurization of an open vented system containing hazardous sample is a serious disadvantage of the teachings of U.S. Pat. No. 5,718,567.

However, in contemplating use of disposable microfluidic device-based assays of clinical specimens, design of fully closed, “single-entry” systems has not been adequately addressed.

In view of contamination hazards associated with working with potentially infectious human samples, resealable entry into the device with gasketed sealing means is often simply not acceptable.

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