Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-02-10
2001-10-02
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S052000, C436S053000, C436S172000, C436S177000, C436S178000, C436S180000, C422S081000, C422S082000, C422S082050, C210S085000, C210S094000, C210S096100, C210S511000, C210S634000, C210S739000, C210S745000, C210S746000, C210S198200, C210S243000, C210S805000
Reexamination Certificate
active
06297061
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to simultaneous diffusion based filtering and chemical reaction of analytes in streams containing both these analytes and larger particles. The invention is useful, for example, for analyzing blood to detect the presence of small particles such as antigens in a stream containing cells, or for preparing small volumes of fluid products.
BACKGROUND OF THE INVENTION
It is possible to fabricate intricate fluid systems with channel sizes as small as a micron. These devices can be mass-produced inexpensively and are expected to soon be in widespread use for simple analytical tests. However, in chemical analysis of turbid fluids, notably blood, filtering of the larger particles such as cells is generally required prior to analysis, especially optical analysis. In clinical laboratories this is generally accomplished by centrifugation. The centrifugal force generated is a function of distance from the center, and thus centrifugation is not effective in a small scale apparatus. In chemical laboratories membrane filters are used to separate the larger particles. This can be used in microscale apparatus, but clogging of the filters with use makes them impractical.
The greater diffusion of small particles relative to larger particles can be used to partially separate the species. Diffusion is a process which can easily be neglected at large scales, but rapidly becomes important at the microscale. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without any mixing other than diffusion. Moreover, due to the small lateral distances in such channels, diffusion is a powerful tool to separate molecules and small particles according to their diffusion coefficients, which is usually a function of their size.
The present invention exploits diffusion to provide simultaneous filtering and chemical reaction, which facilitates the elimination of preprocessing of specimens containing particulate constituents, thus reducing the sample size and analytical time required.
SUMMARY OF THE INVENTION
This invention provides a method and apparatus for reacting small particles in a fluid also comprising larger particles. It provides simultaneous filtering of the larger particles and reaction of the small particles. The reactor can be followed by collection of or detection of the reaction products. The reactor exploits diffusion to separate the small primary particles from the larger particles. It utilizes microscale channels wherein diffusion becomes a significant factor and wherein the fluid flow is laminar. The reactor can be simply and inexpensively manufactured and can be disposed of after use. The reactor is capable of processing a fluid volume between about 0.01 microliters and about 20 microliters within a few seconds. Operation with sub-microliter volumes of sample fluid is a particular advantage for expensive reagents or for blood analysis. Larger volumes with correspondingly longer times can be used when preferred, for example viral detection in a sample with a low viral load.
The reactor can be used for analysis, in which case the inlet fluid, termed generically the primary fluid, is a sample fluid and the small particles, termed generically the small primary particles, are analyte particles. In this case the reactor is generally coupled with a detector. Alternatively, the reactor can be used to rapidly synthesize small volumes of product fluids. In this case the primary fluid is a reagent fluid and the small primary particles are reagent particles. This has particular application to making products starting from natural substances. In the following, the reactor is described for the analysis embodiment, but the description also applies to the synthesis embodiment.
The invention uses an “H” shaped reactor. In the H-reactor the crossbar of the H is a laminar flow reaction channel. On the upstream end of the crossbar a sample (primary) stream and a reagent stream enter through separate arms of the H, and the sample stream and the reagent stream flow in adjacent laminar streams in the crossbar. Because the flow is laminar, there is no turbulent mixing of the two streams, but the analyte particles diffuse from the sample stream into the reagent stream, leaving behind the larger particles in the residual sample stream. In the reagent stream the analyte particles react with reagent particles and form product particles, thereby creating a product stream. At the downstream end of the crossbar, the residual sample stream and the product stream divide into the two downstream arms of the H. The product particles can then be detected in the product stream.
Detection of the product particles can be performed using optical, electrical, chemical, electrochemical or calorimetric analysis, or any other technique in the analytical art. More than one detection technique can be used in the same system. The preferred embodiments use optical analysis or a combination of electrochemical and optical analysis. In optical detection, the product stream can be analyzed by luminescence, fluorescence or absorbance. To increase the signal in the detection zone the product stream channel can be broadened or convoluted. The product stream can connect to a flow cytometer for analysis, particularly a flow cytometer having a microfabricated flow channel.
An example of an application of this method is in competitive immunoassays in solution. The sample stream is whole blood containing native antigens. The reagent particles are antibodies bound to a fluorescently labeled antigen. In the reaction channel, the native antibodies diffuse into the reagent stream and displace the fluorescently labeled antigens. The product stream contains both native and fluorescently labeled antigens, some of which are free and some of which remain bound to antibodies. The relative amounts of free and bound fluorescently labeled antigen, which is a function of the amount of native antigen in the blood, can be measured.
Prior to detection, the product stream can undergo further filtering or separation. In particular the product stream can join with an extraction stream in a separation channel such that the product and extraction streams flow in adjacent laminar flow streams. Smaller particles in the product stream flow into the extraction stream for detection, preferably optical detection.
An example of utilizing the separating channel, is a competitive immunoassay as above wherein the antibody-fluorescently labeled antigen complex is immobilized on a microbead. In the separation channel, the free and bound fluorescently labeled antigen can be separated by diffusion. The free antigen that enters the extraction stream can be detected by fluorescence without interference from the antigen on the beads. In lieu of differential separation, the product stream can be coupled with a flow cytometer to measure the fluorescence intensity remaining on the beads. The bead can be magnetic, and a magnetic field can be used to pin the bead in the sample stream to allow reaction with the analyte particles. Following reaction, a reverse field returns the beads to the reagent stream.
The detection process can use a second reagent stream that joins with the product stream in a “T” configuration. The two streams flow in adjacent laminar streams, and small product particles from the product stream diffuse into the second reagent stream, or small reagent particles from the second reagent stream diffuse into the product stream. Depending on the diffusion process, in either or both streams the product particles react with the second reagent particles to form secondary product particles. The secondary product particles are detected as described above for primary product particles.
First and second reagent streams are usefull, for example, for sandwich immunoassays. The first reagent is a primary antibody which binds to an antigen from the sample to form a first product. The first rege
Kenny Margaret A.
Weigl Bernhard
Wu Caicai
Yager Paul
Chin Christopher L.
Greenlee Winner and Sullivan P.C.
Pham Minh-Quan
University of Washington
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