Rapid magnetic flow assays

This application is a continuation of International PCT Patent Application No. PCT/US2007/006585, filed Mar. 15, 2007, now pending, which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 60/782,649, filed Mar. 15, 2006, and U.S. Provisional Patent Application No. 60/844,811, filed Sep. 14, 2006. These applications are incorporated herein by reference in their entireties.

This invention was made with government support under Contract No. UO1 AI061187, awarded by the National Institutes of Health. The government has certain rights in this invention.

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 660115_—455C1_SEQUENCE_LISTING.txt. The text file is 6 KB, was created on Sep. 3, 2008, and is being submitted electronically via EFS-Web.

1. Field of the Invention

The present invention relates to the general fields of molecular biology and medical science, and more particularly to improved methods for nucleic acid and immunological bioassays.

2. Description of the Related Art

There has been a dramatic transition in clinical laboratory diagnostic assays from the macroscale to the microscale, with specimen volume requirements decreasing from milliliters to microliters, and continuing reduction of assay times from hours to minutes.

These improvements are due in part to advances in materials and fabrication, to the rapidity of mass and heat transfer at the microscale, and to increases in detection sensitivity, but also represent a continuing effort at innovation.

Numerous heterogeneous binding assay systems are known in the prior art and need not be reviewed here. Particles, beads and microspheres, impregnated with color or having a higher diffraction index, are widely used to speed target isolation. The sensitivity of these assays can be improved with ELISA, with fluorescent dyes, by fluorescence quenching, with QDots as tags, for example, thereby achieving higher sensitivity, smaller test pads and larger arrays. Increasingly, nucleic acid assay targets have replaced serological testing. Conventional means for nucleic acid amplification have had a dramatic effect on assay sensitivity and robustness.

However, more can be accomplished to improve sensitivity and accelerate detection of the assay endpoint. Typically the problem is one of diffusion kinetics and mass transfer.

The zeta-potential at the shear boundary layer around particles in solution can slow the close approach needed for binding and immobilization of an assay target. In U.S. Pat. No. 6,720,411, particle colloids such as gold colloids coated with oligomers are aggregated in such a way that the color changes with the state of aggregation. As illustrated in Example 2B of U.S. Pat. No. 6,720,411, the color changes noted are reported to occur over several hours. The endpoint is temperature and salt sensitive and thus represents Brownian motion as path length, the counterion layer as a diffusion barrier, and reduction of interfacial tension as a driving potential. Antibodies can be detected by similar methods, as illustrated in U.S. Pat. No. 6,974,669. However, these detection methods are inherently slow.

Target migration and complexation rates in a solid chromatographic matrix also limit the sensitivity and velocity of endpoint detection in lateral flow assays. Immunochromatographic tests, commonly referred to as Lateral Flow Assays, have been widely used for qualitative and semi-quantitative assays relying on visual detection. One advantage is the wide variety of analytes that can be detected using this type of test. Consequently, a large industry exists for commercialization of this methodology. See, e.g., U.S. Pat. No. 5,120,643, U.S. Pat. No. 4,943,522, U.S. Pat. No. 5,770,460, U.S. Pat. No. 5,798,273, U.S. Pat. No. 5,504,013, U.S. Pat. No. 6,399,398, U.S. Pat. No. 5,275,785, U.S. Pat. No. 5,504,013, U.S. Pat. No. 5,602,040, U.S. Pat. No. 5,622,871, U.S. Pat. No. 5,656,503, U.S. Pat. No. 4,855,240, U.S. Pat. No. 5,591,645, U.S. Pat. No. 4,956,302, U.S. Pat. No. 5,075,078, and U.S. Pat. No. 6,368,876 and U.S. Pat. No. 648,982. Techniques for lateral flow assays are discussed in TechNotes #303 “Lateral Flow Tests” by Bang\'s Laboratories, Inc. (Fishers, Ind.), which is incorporated herein in full by reference.

As another example, in U.S. Pat. No. 5,989,813, amplicons are prepared by amplification of target nucleic acid sequences in the presence of forward and reverse primers conjugated with biotin and digoxigenin, respectively, for use in lateral flow assays. The amplicons are bound to particles with streptavidin and agglutinate in the presence of antibody to digoxigenin. By lateral flow in a sorbent, bifunctional amplicon complexes are detected as trapped aggregates excluded from the fibrous matrix. Other solids are interferences in the assay. In a second variant of the lateral flow format, the avidin conjugates are wicked into a membrane and migrate until encountering a detection strip coated with a capture agent. Accumulation of dyed particles at the detection strip is detected. The assays are generally dependent on flow rate in the materials, particle size and pore dimensions as well as laminar barriers to diffusion.

In Lateral Flow Assays, it is well known that capillary flow rate and adequate contact between the analyte and its corresponding capture antibody immobilized within the membrane are critical to the assay sensitivity. This demands careful membrane selection to optimize dwell time and flow rates. Contact between capture antibody and target analyte again involves convective and diffusional barriers to endpoint detection. These and other limitations of lateral flow assays are discussed in co-assigned US Patent Application 2007/0042427, “Microfluidic Laminar Flow Detection Strip”, herein incorporated in full by reference.

It is not uncommon that magnetic beads are used to concentrate bioanalytes before or during assay (see for example US 2003/0032028). Beads have several advantages over arrays because beads have a higher specific surface area, move through the liquid sample matrix, and hence have more encounters per unit time with an assay target than the corresponding array. Conceptually, use of magnetic microspheres is generally regarded as a concentration step, substituting for centrifugation or filtration.