Multiplexed Analyses of Test Samples
First Claim
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1. A method comprising:
- providing an aptamer that is immobilized on a first solid support, the aptamer having a specific binding affinity for the target molecule and bearing a first tag having an affinity to a first capture element, the first solid support comprising a first capture element, and the first tag being associated with the first capture element to immobilize the aptamer on the first solid support,said first solid support having been washed with one or more solutions that dissociate aggregated aptamers;
contacting said immobilized aptamer with the test sample, wherein an aptamer-target affinity complex is formed if said target molecule is present in said test sample;
removing one or more components of the mixture not associated with said first solid support;
attaching a second tag with an affinity to a second capture element to said target molecule in the aptamer-target affinity complex;
releasing the aptamer-target affinity complex from said first solid support;
exposing the released aptamer-target affinity complex to a second solid support comprising a second capture element and allowing the second tag to associate with said second capture element;
removing any components of the mixture not associated with said second solid support; and
eluting aptamers from said second solid support with one or more solutions comprising a chaotropic salt that disrupts aptamer/analyte interactions.
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Abstract
The present disclosure describes methods, devices, reagents, and kits for the detection of one or more target molecules that may be present in a test sample. The described methods, devices, kits, and reagents facilitate the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer) where the aptamer-aptamer interactions are significantly reduced or eliminated while maintaining the aptamer-target interaction.
5 Citations
32 Claims
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1. A method comprising:
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providing an aptamer that is immobilized on a first solid support, the aptamer having a specific binding affinity for the target molecule and bearing a first tag having an affinity to a first capture element, the first solid support comprising a first capture element, and the first tag being associated with the first capture element to immobilize the aptamer on the first solid support, said first solid support having been washed with one or more solutions that dissociate aggregated aptamers; contacting said immobilized aptamer with the test sample, wherein an aptamer-target affinity complex is formed if said target molecule is present in said test sample; removing one or more components of the mixture not associated with said first solid support; attaching a second tag with an affinity to a second capture element to said target molecule in the aptamer-target affinity complex; releasing the aptamer-target affinity complex from said first solid support; exposing the released aptamer-target affinity complex to a second solid support comprising a second capture element and allowing the second tag to associate with said second capture element; removing any components of the mixture not associated with said second solid support; and eluting aptamers from said second solid support with one or more solutions comprising a chaotropic salt that disrupts aptamer/analyte interactions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method of detecting the presence of, or determining the amount of, a target molecule in a sample, the method comprising:
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providing a plurality of immobilized aptamers, wherein each of said aptamers is specific to a target molecule, and wherein each of said plurality of aptamers comprises a first cleavable capture tag, said aptamers being immobilized on a solid support having probes adhered to the surface thereof, the probes binding to the first tag, such that the aptamers are immobilized onto the solid support through binding of the first tag and the probe; contacting the immobilized aptamers with a sample containing target molecules to form a mixture containing aptamer-target molecule complexes bound to the solid support; partitioning aptamer-target molecule complexes bound to the solid support from the remainder of the mixture; introducing a second capture tag to the target molecule component of the aptamer-target molecule complexes; dissociating the aptamer-target molecule complexes from the surface of the solid support by cleaving the first cleavable capture tag; providing a second solid support having probes adhered to the surface of the support, wherein the probes are capable of binding to the second capture tag on target molecules; contacting the dissociated aptamer-target molecule complexes with the second solid support such that the aptamer-target molecule complexes become bound to the second support through binding of the second capture tag and probe; eluting aptamers from said second solid support with one or more buffered solutions comprising a chaotropic salt that disrupts aptamer/analyte interactions but supports aptamer/aptamer interactions and DNA hybridization; dissociating the aptamer-target molecule; and detecting the free aptamers. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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Specification