Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-08-17
2003-09-02
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C435S007210, C435S006120, C435S007200, C436S514000, C422S091000, C422S068100, C422S050000, C210S222000
Reexamination Certificate
active
06613581
ABSTRACT:
BACKGROUND OF THE INVENTION
Analytic detection of biomolecules, e.g., proteins, nucleic acids, and the like, is fundamental to molecular biology. In many applications, it is desirable to detect the presence of one or more particular molecules in a sample. For example, identification of a particular DNA sequence within a mixture of restriction fragments is used to determine the presence, position, and number of copies of a gene in the genome. It is also an integral technique in DNA typing. Analytic detection is also used, e.g., in disease diagnosis and drug development, to determine the presence of a particular antibody or protein, e.g., in a blood sample or large chemical library. Detection of biomolecules is therefore of fundamental value in, e.g., diagnostic medicine, archaeology, anthropology and modem criminal investigation. To meet these needs many techniques, e.g., DNA blotting, RNA blotting, protein blotting, and ELISA assays, have been developed to detect the presence of a particular molecule or fragment in the midst of a complex sample containing similar molecules.
For example, western blotting is useful for detecting one or more specific proteins in a complex protein mixture, such as a cell extract. The procedure involves fractionating the protein mixture, generally by denaturing polyacrylamide gel electrophoresis, and transferring and immobilizing the mixture onto a solid membrane of either nitrocellulose or nylon by electroblotting. The loaded membrane is then incubated with an antibody raised against the protein of interest. The antibody-antigen complex so formed on the membrane is then detected by a procedure that typically involves the application of a second antibody, raised against the first antibody, and to which an enzyme has been covalently linked. The insoluble reaction product generated by the enzyme action can then be used to indicate the position of the target protein on the membrane. The sensitivity of detection can be increased by amplification of the signal using either the biotin-streptavidin system or by chemiluminescence detection.
This classical procedure is very time consuming and labor intensive. For example, transferring the proteins to a membrane is generally a time consuming step and is typically done by capillary blotting or by the faster and more efficient methods of vacuum blotting or electrophoretic blotting.
More recently, new and faster microfluidic methods of performing biological assays in microfluidic systems have been developed, such as those described by the pioneering applications of Parce et al., “High Throughput Screening Assay Systems in Microscale Fluidic Devices” WO 98/00231 and in Knapp et al., “Closed Loop Biochemical Analyzers” (WO 98/45481; PCT/US98/06723). For example, high throughput methods for analyzing biological reagents, including proteins, are described in these applications.
Improved methods for performing western blot and affinity assays are, accordingly desirable, particularly those which take advantage of high-throughput, low cost microfluidic systems. The present invention provides these and other features by providing high throughput microscale systems for analyte detection, western blots, and the like, and many other features that will be apparent upon complete review of the following disclosure.
SUMMARY OF THE INVENTION
The present invention provides methods, devices, systems, and kits for detecting a component of interest in a complex mixture. Typically, the method comprises separating a mixture of components, which mixture of components contains the component of interest. To detect the component of interest, the mixture of components or the separated components are contacted with a component-binding moiety specific to the component of interest. The component-binding moiety binds to the component of interest and is detected, thereby detecting the component of interest.
In one embodiment, the component of interest and the various components of the mixture are labeled with two detectably different labels so that both the component of interest and the mixture of components are concurrently detected.
In another embodiment, the separated components are bound to or adsorbed to a particle set. The particle set is optionally stacked in a detection region and a component-binding moiety specific to the component of interest is directed into the region of the device containing the particle set with the bound components. The component-binding moiety thereby binds to the component of interest, thus providing detection of the component of interest.
The devices, systems, and methods of the invention are useful in a variety of detection systems, e.g., western assays, biotin-avidin systems, lectin/carbohydrate systems, and in other applications that will be apparent upon further review.
In one aspect, the method comprises providing a body structure having a plurality of microscale channels disposed therein, the plurality comprising a microfluidic separation channel and at least one side channel intersecting the separation channel, wherein the separation channel and the side channel are fluidly coupled. A mixture of components is flowed through the separation channel, resulting in separated components. A labeled component-binding moiety is then flowed through a side channel and into the separation channel, wherein it binds to the component of interest. The component-binding moiety is then detected, thereby detecting the component of interest.
The separated components are typically labeled components that are optionally detected simultaneously with the component-binding moiety. This embodiment optionally includes deconvoluting the detection signal to identify the separated components and the component of interest. This embodiment includes two detectably different label moieties having detectably different spectral characteristics, such as different excitation or emission maximum. The different labels include, but are not limited to fluorescent labels, chemiluminescent labels and colorimetric labels. For example, the separated components are optionally labeled with a first fluorescent dye and the component-binding moiety is labeled with a second fluorescent dye. These two dyes are typically detectably different. In another embodiment, the component of interest and the component-binding moiety are optionally labeled with detectably different colorimetric labels. In another embodiment, the component of interest is labeled with one type of label, e.g., chemiluminescent, and the component-binding moiety is labeled with a second type of label, e.g., fluorescent.
In another aspect, a microfluidic system comprising a particle set is provided. A body structure having at least one microfluidic channel disposed therein is provided, and a mixture of components is flowed through the microfluidic channel, separating the mixture of components and producing separated components. The separated components are then bound to a particle set comprising a plurality of particle member types. The separated components bound to the particle set are then contacted with a component-binding moiety specific to the component of interest, thereby binding the moiety to the component of interest. The component-binding moiety is then detected, thus detecting the component of interest. After being bound to the separated components, the particle set is flowed into a detection channel downstream of the separation and binding events. The particle set is optionally stacked or fixed in the detection channel. In one embodiment, stacking occurs against a barrier located in the detection channel.
The particle set is comprised of a plurality of particle member types, that optionally comprise a polymeric material, a silica material, a ceramic-material, a glass material, a magnetic material, a metallic material, an organic material, or a combination of these materials. In one embodiment, binding comprises adsorbing the separated components onto the members of the particle set. In these embodiments, the particle member types optionally comprise PVDF, nitrocellulose, or
Murphy Matthew B.
Wada H. Garrett
Caliper Technologies Corp.
Davis Deborah A
Filler Andrew L.
Le Long V.
Murphy Matthew B.
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