Spatially-encoded analyte detection

Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals

Reexamination Certificate

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C435S006120

Reexamination Certificate

active

06294392

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable
FIELD OF THE INVENTION
This invention relates to the field of diagnostics. In particular this invention provides devices and methods that allow rapid detection and/or quantitation of multiple analytes and yet does not require the use of labels or labeling steps.
BACKGROUND OF THE INVENTION
Immunoassays and nucleic acid hybridization chemistries are rapidly being developed towards the goal of detecting genetic defects, performing disease diagnostics, and performing prognostic evaluations (Sosnowski et al. (1997)
Proc. Natl. Acad. Sci. USA,
94: 1119-1123). Antibodies, nucleic acid binding proteins, receptor ligands, and nucleic acids are known to bind very specifically and with high efficiency to their congnate “binding partner” under suitable conditions. This phenomenon is frequently used for the recognition and diagnosis of disease-causing organisms (e.g., HIV), pathological conditions (e.g. cancer, liver disease, kidney disease, degenerative joint disease, etc.), substance abuse (e.g. detection of products such as cotinine, etc.), and the like.
Numerous disease markers, and pathogen markers (e.g. proteins and/or nucleic acids) are well known and have been thoroughly characterized. Thus, binding partners (e.g. nucleic acids, antibodies, and the like), that specifically bind such markers can be synthesized and/or isolated and used as markers for recognition of the disease state, or disease-causing agent (Landegren et al. (1988)
Science,
242: 229, Mikkelson (1996)
Electroanalysis,
8: 15-19). Various assays are carried out routinely in microbiology laboratories or pathology laboratories using such methodologies.
Nucleic acid hybridization, antibody binding reactions, protein binding reactions, and lectin binding reactions are generally detected through the use of labels that either intercalate into the molecule (e.g. into the double helix of a DNA) or are covalently attached to either the target or the probe molecule (see, e.g., Sosnowski et al. (1997)
Proc. Natl. Acad. Sci. USA,
94: 1119-1123, LePecq and Paoletti (1966)
Anal. Biochem.,
17: 100-107, Kapuscinski and Skoczylas (1977)
Anal. Biochem.,
83: 252-257). In some cases, electrogenerated chemiluminescence has also been utilized to detect an intercalated electroactive luminescent marker (Pollard-Knight et al. (1990)
Anal. Biochem.,
185: 84-89, Pollard-Knight et al.(1990)
Anal. Biochem.,
185: 353-358, Tizard et al. (1990)
Proc. Natl. Acad. Sci. USA,
12: 4514-4518). All of these detection strategies require the derivatization of the target or probe molecule, either before (e.g. for covalent labeling) or after (e.g. for intercalation or indirect labeling) the binding reaction between the probe and target molecule. This introduces contamination problems. In addition, where multiple analytes are analyzed simultaneously multiple labels must be employed. In addition, tedious sample handling is required which further enhances the risk of contamination and/or leads to false analysis. These and other problems are overcome by the present invention.
SUMMARY OF THE INVENTION
This invention provides novel devices and methods for detecting and/or quantifying a plurality of analytes in a sample. This invention provides a flow-through microfluidic (e.g., capillary) biosensor for detecting different target analytes (e.g. nucleic acids) in a sample after binding to their cognate “binding partners” (e.g. nucleic acids, antibodies, lectins, etc.). In general, binding partner “probes”, specific to various analytes are immobilized in different sections of a capillary channel, e.g. using photolabile biotin/avidin technology. The sample is then flushed through the capillary, so that the target analytes are bound to the binding partners (capture agents) immobilized on the capillary wall and the rest of the sample is eluted from the capillary. Finally, the complexed (bound) analyte is released along the entire length of the channel and flushed past a detector. In a preferred embodiment, the desorbed, target-analytes are detected at a copper electrode poised downstream using sinusoidal voltammetry (Singhal and Kuhr (1997)
Anal. Chem.,
69: 3552-3557, Singhal et al. (1997)
Anal. Chem.,
69: 1662-1668). The time from the elution of the target analyte(s) to detection is used to determine the identity of each analyte. Multiple analytes, of the same species of molecule (e.g., all nucleic acids), or of different species (e.g. proteins and nucleic acids), can be diagnosed by using a single biosensor in this manner. The sensor is highly specific due to the use of specific binding partners, and extremely sensitive due to electrochemical detection.
Thus, in one embodiment, this invention provides devices for detecting a two or more analytes in a sample. The devices comprise a channel having affixed therein a binding partner for each of the two or more analytes, where the binding partners for each of the two or more analytes are located in different regions of the channel and the channel has a cross-sectional area small enough such that when analytes are released from the two or more binding partners into a fluid flowing through the channel, the analytes remain spatially segregated until they reach a detection point along, or at the end of, the channel downstream from the binding partners; and a detector that detects the analytes at the detection point.
The channel can be any convenient channel, e.g. a capillary tube, a capillary electrophoresis tube, a channel etched in a surface, a channel formed by hydrophobic agents printed onto a surface, etc. The channel can have essentially any dimension(s) as long as the analytes remain sufficiently segregated to be distinguished when they reach a detection region in the channel or at the channel end. Preferred channels have a cross-sectional area that provides a Renold's number (Re) of less than about 1. Preferred channels have a cross-sectional diameter or width less than or equal to about 500 &mgr;m, more preferably less than or equal to about 100 &mgr;m, and most preferably less than or equal to about 50 &mgr;m. In particularly preferred devices the two or more target analytes comprise at least three, preferably at least 4, more preferably at least 5, and most preferably at least 10, at least 50, at least 100, or at least 500 different analytes (and hence that many different binding partners). A wide variety of binding partners are suitable including, but not limited to antibodies, binding proteins, and nucleic acids. Similarly many detectors are suitable and include spectrophotometers (e.g. absorbance spectrophotometers), and electroanalytic detectors (including essentially any amperometric and/or voltammetric and/or potentiometric and/or coulometric detectors). Preferred detectors include voltammeters, especially a sinusoidal voltammeters.
In another embodiment, this invention provides methods of detecting two or more target analytes in a sample. The methods involve providing a detection device as described herein; ii) passing a fluid comprising a sample through the channel under conditions where the target analytes present in the fluid bind to their respective binding partners thereby spatially encoding the analytes along the channel; iii) releasing the analytes from the binding partners into fluid flow passing along the channel; and iv) detecting the analytes at a position along the channel downstream from the binding partners. In preferred methods, the analytes are not labeled. In particularly preferred embodiments the analytes are not labeled. In particularly preferred devices the two or more target analytes comprise at least three, preferably at least 4, more preferably at least 5, and most preferably at least 10, at least 50, at least 100, or at least 500 different analytes (and hence that many different binding partners are present in the channel(s) comprising the detection device). In some preferred embodiments, the fluid flow induced by a pressure difference and/or by electroosmotic flow. fluid flow. Preferred “sample” fluids fo

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