COMPOUNDS, COMPOSITIONS, AND METHODS FOR IMPROVING ASSAYS
Provided are compounds, compositions, kits, systems, devices, and methods for improving an assay such as, for example, a multiplexed PCR assay (e.g., a multiplexed immuno-PCR assay). A solid support (e.g., a bead) may be provided according to some embodiments of the present invention. The solid support may comprise an encoding agent (e.g., a dye), a nucleic acid sequence (e.g., an oligonucleotide and/or primer); and a molecular recognition element (e.g., an antibody). A detection reagent may be provided according to some embodiments of the present invention. The detection reagent may comprise a molecular recognition element (e.g., an antibody) and a nucleic acid tag. In some embodiments, at least a portion of the nucleic acid sequence of the solid support and at least a portion of the nucleic acid tag of the detection reagent are configured to participate in a nucleic acid amplification process. A solid support and detection reagent may bind to the same target, thereby forming a reagent pair.
1 . A plurality of detection reagents comprising:
a first plurality of detection reagents and each detection reagent in the first plurality of detection reagents comprises:
a first molecular recognition element and
a first nucleic acid sequence; and a second plurality of detection reagents and each detection reagent in the second plurality of detection reagents comprises:
a second molecular recognition element and
a second nucleic acid sequence;
wherein the first and second nucleic acid sequences each include a first portion and a second portion; and
wherein the nucleic acid sequence of the first portion for the first nucleic acid sequence is different than the nucleic acid sequence of the first portion for the second nucleic acid sequence, and the nucleic acid sequence of the second portion for the first and second nucleic acid sequences are the same.
2 . The plurality of detection reagents of claim 1 , wherein the first portion for the first and second nucleic acid sequences is in the 5′region of the respective sequence and second portion is in the 3′region of the respective sequence.
3 . A method of detecting a target in a sample, the method comprising:
combining a solid support and a sample comprising the target to form a first composition comprising a target-bound solid support, wherein the solid support comprises:
(a) an encoding agent;
(b) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a primer that hybridizes to the complement of a second nucleic acid sequence; and
(c) a first molecular recognition element,
wherein the first molecular recognition element comprises an antibody or fragment thereof, aptamer, modified aptamer, peptide, protein, carbohydrate, and/or combinations thereof wherein the solid support does not comprise a primer that hybridizes to the second nucleic acid sequence;
optionally washing the target-bound solid support;
combining a detection reagent and the target-bound solid support to form a complex comprising the solid support, target, and detection reagent, wherein the detection reagent comprises a second molecular recognition element, wherein the second molecular recognition element comprises an antibody or fragment thereof, aptamer, modified aptamer, peptide, protein, carbohydrate, and/or combinations thereof, and the second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleic acid tag comprising at least a first portion and a second portion, wherein the first portion of the second nucleic acid sequence is substantially identical to the primer in the first nucleic acid sequence, wherein the first nucleic acid sequence and second nucleic acid sequence comprise single stranded-DNA; and wherein the primer in the first nucleic acid sequence and at least the first portion of the second nucleic acid sequence are configured to participate in a nucleic acid amplification process, wherein the nucleic acid amplification process is polymerase chain reaction (PCR) configured to amplify the nucleic acid tag;
optionally washing the complex ;
optionally combining an amplification agent and the complex;
optionally releasing the first nucleic acid sequence from the solid support; and
amplifying the nucleic acid tag using PCR, thereby detecting the target in the sample.
4 . The method of claim 3 , wherein the method comprises washing the target-bound solid support.
5 . The method of claim 3 , wherein the method comprises washing the complex.
6 . The method of claim 3 , wherein the method comprises combining the amplification agent and the complex.
7 . The method of claim 3 , wherein the method comprises releasing the first nucleic acid sequence from the solid support.
8 . The method of claim 3 , wherein the method comprises combining the amplification agent and the complex and releasing the first nucleic acid sequence from the solid support.
9 . The method of claim 3 , wherein the method comprises washing the target-bound solid support and comprises washing the complex.
10 . The method of claim 3 , wherein the method comprises washing the target-bound solid support and combining the amplification agent and the complex.
11 . The method of claim 3 , wherein the method comprises washing the target-bound solid support and releasing the first nucleic acid sequence from the solid support.
12 . The method of claim 3 , wherein the method comprises washing the complex and combining the amplification agent and the complex.
13 . The method of claim 3 , wherein the method comprises washing the complex and releasing the first nucleic acid sequence from the solid support.
14 . The method of claim 3 , wherein the method comprises washing the target-bound solid support, washing the complex, combining the amplification agent and the complex, and releasing the first nucleic acid sequence from the solid support.
15 . The method of claim 3 , wherein the detection reagent is present in the first composition.
16 . The method of claim 3 , wherein the amplification agent is present in the first composition.
17 . The method of claim 3 , further comprising detecting the target by detecting an assay signal.
18 . The method of claim 3 , wherein the method is a multiplexed assay that is performed in a microfluidic device comprising a plurality of wells and the method further comprises isolating the solid support from one or more additional solid supports and in at least one well of the plurality wells.
19 . A method of improving the specificity and/or reducing the background signal for an assay, the method comprising:
providing a solid support comprising an encoding agent, a nucleic acid sequence, and a molecular recognition element; and
performing the assay in the presence of the solid support or the plurality of solid supports, thereby improving the specificity and/or reducing the background signal for the assay.