Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-19
2003-04-01
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S052000, C436S053000, C436S514000, C436S518000, C436S172000, C436S177000, C436S180000, C422S081000, C422S082000, C422S082080, C210S085000, C210S094000, C210S096100, C210S511000, C210S634000, C210S739000, C210S745000, C210S748080, C210S198200, C210S243000
Reexamination Certificate
active
06541213
ABSTRACT:
BACKGROUND OF THE INVENTION
The immunoassay is the workhorse of analytical biochemistry. It allows the unique binding abilities of antibodies to be widely used in selective and sensitive measurement of small and large molecular analytes in complex samples. The driving force behind developing new immunological assays is the constant need for simpler, more rapid, and less expensive ways to analyze the components of complex sample mixtures. Current uses of immunoassays include therapeutic drug monitoring, screening for disease or infection with molecular markers, screening for toxic substances and illicit drugs, and monitoring for environmental contaminants.
Flow injection immunoassays have taken advantage of specific flow conditions. (U. de Alwis and G. S. Wilson,
Anal. Chem
. 59, 2786-9 (1987)), but also use high Reynolds number effects for mixing. Micro-fabricated capillary electrophoresis devices, which are truly microfluidic, have been used for rapidly separating very small volumes of immunoreagents following binding reactions (N. Chiem and D. J. Harrison,
Anal. Chem
. 69, 373-8 (1997)). One of the unique features of microfluidic devices that has yet to be exploited for immunoassay development is the presence of laminar flow under low Reynolds number conditions. Laminar flow allows quantitative diffusional transport between adjacent flowing streams, while retaining the relative positions of non-diffusing components such as cells and larger microspheres. While these conditions are impediments to application of some macro-scale techniques, they allow creation of new types of analyses that are uniquely well suited to microfluidic systems, such as the H-Filter for extraction of solutes (J. P. Brody, P. Yager, R. E. Goldstein, R. H. Austin,
Biophysical Journal
71(6), 3430-3441 (1996); U.S. Pat. No. 5,932,100; J. P. Brody and P. Yager,
Sensors and Actuators A
(
Physical
) A58(1), 13-18 (1997); the V-Groove device for low-volume flow cytometry; U.S. Pat. No. 5,726,751, the T-Sensor for detection of diffusable analytes (A. E. Kamholz, B. H. Weigl, B. A. Finlayson, P. Yager, [1999
] Anal. Chem
., 71(23):5340-5347; U.S. Pat. No. 5,716,852; U.S. Pat. No. 5,972,710; B. H. Weigl and P. Yager,
Science
283, 346-347 [1999]; R. B. Darling, J. Kriebel, K. J. Mayes, B. H. Weigl, P. Yager, Integration of microelectrodes with etched microchannels for in-stream electrochemical analysis, &mgr;TAS '98, Banff, Canada [1998]; B. H. Weigl and P. Yager,
Sensors and Actuators B
(
Chemical
) B39 (1-3), 452-457 [1996]; B. H. Weigl, M. A. Holl, D. Schutte, J. P. Brody, P. Yager,
Anal. Methods & Instr
., 174-184 [1996]; B. H. Weigl, et al., Simultaneous self-referencing analyte determination in complex sample solutions using microfabricated flow structures (T-Sensors), &mgr;TAS '98, Banff, Canada [1998]) and others as described in U.S. Pat. No. 5,922,210; U.S. Pat. No. 5,747,349; U.S. Pat. No. 5,748,827; U.S. Pat. No. 5,726,404; U.S. Pat. No. 5,971,158; U.S. Pat. No. 5,974,867 and U.S. Pat. No. 5,948,684; WO 98/43066 published Oct. 1, 1998; U.S. Ser. No. 08/938,584 filed Sep. 26, 1997; WO 99/17100 published Apr. 8, 1999; WO 99/17119 published Apr. 8, 1999; U.S. Ser. No. 09/196,473 filed Nov. 19, 1998; U.S. Ser. No. 09/169,533 filed Oct. 9, 1998; WO 99/60397 published Nov. 25, 1999; U.S. Ser. No. 09/404,454 filed Sep. 22, 1999; and Ser. No. 09/464,379, filed Dec. 15, 1999 for “Magnetically-Actuated Fluid Handling Devices for Microfluidic Applications.”
All publications referred to herein are hereby incorporated by reference in their entirety to the extent not inconsistent herewith.
SUMMARY
This invention provides a method for detecting the presence of analyte particles comprising providing binding particles capable of binding with said analyte particles; providing a system in which at least one of said binding particles and said analyte particles can diffuse toward the other; providing means for detecting any of said particles or complexes between them, or a diffusion front of said binding particles, said analyte particles, or said complexes in said system, and detecting said particles or complexes or said diffusion front. When said analyte particles and said binding particles meet and bind to each other, a slowing of the particles or a diffusion front may be detected as an indication of the presence of said analyte particles. The binding particles, or the analyte particles, or complexes between them must be visible or detectable, e.g. by optical or electrical detection means or other detection means known to the art, or must be labeled to become visible or detectable.
This invention also provides a device for determining the presence or concentration of sample analyte particles in a medium comprising: means for contacting a first medium containing analyte particles with a second medium containing binding particles capable of binding to said analyte particles; wherein at least one of said analyte or binding particles is capable of diffusing into the medium containing the other of said analyte or binding particles; and means for detecting the presence of diffused particles. One or both of the analyte and binding particles may be labeled or unlabeled.
The “diffusion front” (also referred to as “diffusion profile” herein) is a detectable edge or line created by diffusing particles. It may be more or less sharp or diffuse depending on system parameters such as relative amounts of analyte and binding particles, relative diffusion coefficients of both, amount of labeling, viscosities of the system, and other parameters known to the art. The term “slowing” with reference to the diffusion front includes stopping, as well as any detectable amount of slowing. The “diffusion front” may include a detectably more intense area or line closer to the point(s) from which diffusion of particles begins caused by complexing of labeled particles to form slower-diffusing complexes, with relatively less intense areas further from said points caused by uncomplexed labeled particles; or the “diffusion front” may be the absolute border of the area into which particles have diffused.
Systems allowing diffusion of analyte or binding particles toward each other can be systems in which fluids containing analyte particles (referred to herein as analyte fluids) are placed in contact with fluids containing binding particles (referred to herein as “diffusion fluids”), or fluids containing analyte particles, are placed in contact with solids containing binding particles capable of diffusing into the analyte fluid. Or, the system may be one in which fluids containing binding particles are placed in contact with solids containing analyte particles capable of diffusing into the diffusion fluids. Such systems can be flowing or stationary systems as described below, or can comprise fluids separated by membranes capable of allowing diffusion of analyte and/or binding particles therethrough, or can comprise two fluids containing analyte and binding particles respectively separated by a removable barrier, which is removed to allow diffusion to take place.
Slowing of the diffusion front may be observed or detected; or the position of the diffusion front after a predetermined time from when the particles begin diffusing may be observed or otherwise detected and compared with a similar calibration or control system or systems containing known amounts of analyte particles, e.g. from 0 to any typical concentration. In this way, concentration as well as presence of analyte particles can be determined.
Concentration may also be calculated based on the principles and algorithms described in the Examples below, and determinable without undue experimentation by those skilled in the art.
This invention also provides methods for detecting the presence of at least first and second analyte particles in a first fluid comprising: providing a second fluid comprising first and second binding particles for said first and second analyte particles, respectively; flowing said first and secon
Hatch Anson
Kamholz Andrew
Weigl Bernhard H.
Yager Paul
Chin Christopher L.
Do Pensee T.
University of Washington
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