Methods and/or Use of Oligonucleotide-Bead Conjugates for Assays and Detections

Abstract

The present disclosure is directed to methods and/or uses of oligonucleotide-bead conjugates for assays and detections and related systems and/or kits. Certain methods are directed to a method for detecting one or more biological targets of a sample in a detection assay, comprising: providing a molecular probe, comprising a binding moiety and an oligonucleotide sequence, to a sample comprising one or more biological targets; binding the one or more biological targets with the binding moiety; providing a detectable component to the sample, wherein the detectable component comprises a signal generating moiety conjugated to an oligonucleotide sequence complementary to the oligonucleotide sequence of the molecular probe; hydridizing the oligonucleotide sequence of the target-bound molecular probe to the detectable component; and detecting a signal generated from the hydridized detectable component. Various other embodiments, applications etc. are disclosed herein.

What is claimed is: 
     
         1 . A method for assaying a target of a sample, comprising:
 i) providing to the sample:
 1) a first molecular probe, comprising a first binding moiety conjugated to a first oligonucleotide sequence; and 
 2) a first bead conjugate, comprising a first bead conjugated to a second oligonucleotide sequence that is complementary to the first oligonucleotide sequence of the first molecular probe, wherein the first bead comprises or is encoded with one or more signal generating moieties; 
   ii) binding the target in the sample with the first binding moiety of the first molecular probe;   iii) hybridizing the first oligonucleotide sequence of the first molecular probe with the second oligonucleotide sequence of the first bead conjugate; and   iv) providing to the sample a second binding moiety comprising one or more signal generating moieties;   v) further binding the target of the first binding moiety-bound target with the second binding moiety to form a sandwich-complex;   vi) detecting a signal generated from the sandwich complex;   
       wherein the method is characterized by one or more of the following:
 a) the conjugation between the first oligonucleotide sequence and the first binding moiety and conjugation between the complementary second oligonucleotide sequence and the first bead conjugate, comprises one or more covalent bond linkages, comprising a hydrazone, oxime, triazine, or other covalent bond, wherein the formation of the conjugates are at least 90% efficient; and 
 b) the first binding moiety and the second binding moiety comprise strong binding affinities for the target. 
 
     
     
         2 . The method of  claim 1 , wherein the mode of addition comprises:
 i) the first molecular probe and the first bead conjugate are combined together and hybridized prior to contacting the sample;   ii) the first molecular probe is combined with the sample prior to the addition of the first bead conjugate; or   iii) the first bead conjugate is combined with the sample prior to the addition of the first molecular probe.   
     
     
         3 . The method of  claim 1 , wherein the method comprises:
 i) the first molecular probe binding the target prior to hybridizing with the first bead conjugate; or   ii) the first molecular probe hybridizing with the first bead conjugate prior to binding the target.   
     
     
         4 . A method for assaying a target of a sample, comprising:
 i) providing to the sample:
 1) a first molecular probe, comprising a first binding moiety conjugated to a first oligonucleotide sequence; 
 2) a first bead conjugate, comprising a first bead conjugated to a second oligonucleotide sequence, wherein the first bead comprises or is encoded with one or more signal generating moieties; and 
 3) a first universal adapter, comprising an oligonucleotide sequence having a first sequence segment complementary to the first oligonucleotide sequence of the first molecular probe and a second sequence segment complementary to the second oligonucleotide sequence of the first bead conjugate; 
   ii) binding the target in the sample with the first binding moiety of the first molecular probe;   iii) hybridizing the first oligonucleotide sequence of the first molecular probe to the first oligonucleotide sequence segment of the first universal adapter;   iv) hybridizing the second oligonucleotide sequence of the first bead conjugate to the complementary second oligonucleotide sequence segment of the first universal adapter; and   v) providing to the sample a second binding moiety comprising one or more signal generating moieties;   iv) further binding the target of the first binding moiety-bound target with the second binding moiety to form a sandwich-complex;   vi) detecting a signal generated from the sandwich complex;   
       wherein the method is characterized by one or more of the following:
 a) the conjugation between the first oligonucleotide sequence and the first binding moiety and conjugation between the complementary second oligonucleotide sequence and the first bead conjugate, comprises one or more covalent bond linkages, comprising a hydrazone, oxime, triazine, or other covalent bond, wherein the formation of the conjugates are at least 90% efficient; and 
 b) the first binding moiety and the second binding moiety comprise strong binding affinities for the target. 
 
     
     
         5 . The method of  claim 4 , wherein the mode of addition comprises:
 i) the first molecular probe, the first universal adapter, and the first bead conjugate are combined together and hybridized prior to contacting the sample;   ii) the first molecular probe and the first universal adapter are combined together and hybridized prior to contacting the sample;   iii) the first bead conjugate and the first universal adapter are combined together and hybridized prior to contacting the sample;   iv) the first molecular probe, alone or in combination with the first bead conjugate, is combined with the sample prior to the addition of the first universal adapter; or   v) the first universal adapter is combined with the sample prior to the addition of the first molecular probe and/or the first bead conjugate.   
     
     
         6 . The method of  claim 4 , wherein the method comprises:
 i) the first molecular probe hybridizing with the first universal adapter prior to said first molecular probe binding the target;   ii) the first molecular probe hybridizing with the first universal adapter after said first molecular probe binds the target;   iii) the first bead conjugate hybridizing with the first universal adapter prior to the first molecular probe binding the target;   iv) the first bead conjugate hybridizing with the first universal adapter after the first molecular probe binds the target;   v) the first universal adapter hybridizing with the first molecular probe and hybridizing with the first bead conjugate prior to said first molecular probe binding the target; or   vi) the first universal adapter hybridizing with the first molecular probe and hybridizing with the first bead conjugate after said first molecular probe binds the target.   
     
     
         7 . A method for crosslinking, comprising:
 i) introducing to a sample comprising one or more targets:
 a) one or more first antibody-oligonucleotide conjugates, comprising a first antibody conjugated to a first oligonucleotide sequence; and 
 b) one or more second antibody-oligonucleotide conjugates, comprising a second antibody conjugated to a second oligonucleotide sequence; 
   ii) binding at least a first target of the one or more targets with the first antibody of the one or more first antibody-oligonucleotide conjugates and with the second antibody of the one or more second antibody-oligonucleotide conjugates to form one or more sandwich-complexes;   iii) contacting the one or more sandwich-complexes with:
 a) one or more first bead-oligonucleotide conjugate, comprising a first bead conjugated to a complementary first oligonucleotide sequence; and 
 b) one or more second bead-oligonucleotide conjugate, comprising a second bead conjugated to a complementary second oligonucleotide sequence; 
   iv) crosslinking the one or more sandwich-complexes by:
 a) hybridizing the first oligonucleotide sequences of the one or more sandwich-complexes with the complementary first oligonucleotide sequences of the one or more first bead-oligonucleotide conjugates; and 
 b) hybridizing the second oligonucleotide sequences of the one or more sandwich-complexes with the complementary second oligonucleotide sequences of the one or more second bead-oligonucleotide conjugates. 
   
     
     
         8 . The crosslinking method of  claim 7 , wherein the formation of the crosslinked one or more sandwich-complexes forms an agglutination. 
     
     
         9 . The crosslinking method of  claim 7 , wherein the method further comprises detecting, measuring, and/or quantifying the degree of the formed agglutination to determine the amount of the one or more targets in the sample. 
     
     
         10 . The crosslinking method of  claim 7 , wherein the first antibody or the second antibody comprise a monoclonal antibody or a polyclonal antibody. 
     
     
         11 . The crosslinking method of  claim 7 , wherein:
 i) the first antibody comprises a first polyclonal antibody and the second antibody comprises a second polyclonal antibody;   ii) the first antibody comprises a first monoclonal antibody and the second antibody comprises a second monoclonal antibody;   iii) the first antibody comprises a first monoclonal antibody and the second antibody comprises a first polyclonal antibody; or   iv) the first antibody comprises a first polyclonal antibody and the second antibody comprises a first monoclonal antibody.   
     
     
         12 . The crosslinking method of  claim 7 , wherein the first antibody comprises a first polyclonal antibody and the second antibody comprises a second polyclonal antibody. 
     
     
         13 . The crosslinking method of  claim 7 , wherein the first antibody comprises a first monoclonal antibody and the second antibody comprises a second monoclonal antibody. 
     
     
         14 . The crosslinking method of  claim 7 , wherein the first antibody comprises a first monoclonal antibody and the second antibody comprises a first polyclonal antibody. 
     
     
         15 . The method of  claim 1 , wherein:
 i) the assay comprises a singleplex or multiplex assay; and   ii) the assay detects, measures, or quantifies the level of binding and/or amount of the target present in the sample with one or more of the following:
 immunoturbidity, latex agglutination, gold particle agglutination, visual inspection, a change in light transmittance through said sample, increased light transmittance through said sample, flow cytometry, immunomagnetic cellular depletion, immunomagnetic cell capture, array, bead array, multiplex bead array, microarray, antibody array, cellular array, chemiluminescence, infrared, microscopy, imaging, high content screening (HCS), mass cytometry, lateral flow immunoassay, immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, a blotting method, a Western blot, a Southern blot, a Southwestern blot, labeling inside an electrophoresis system, labeling on a surface, labeling on an array, PCR amplification, elongation followed by PCR amplification, immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, pretargeting imaging, therapeutic agent, or combinations thereof. 
   
     
     
         16 . The method of  claim 1 , wherein the method further comprises preparing and isolating the first molecular probe, comprising:
 i) providing the first binding moiety;   ii) conjugating the first binding moiety with at least one first oligonucleotide sequence at greater than 90% efficiency to form first binding moiety-oligonucleotide conjugates; and   iii) isolating the first binding moiety-oligonucleotide conjugates from the conjugation mixture by binding, retaining, and/or retarding a substantial portion of:
 a) the conjugates, removing a substantial portion of the unconjugated first oligonucleotide sequence in a wash step followed by release of the bound, retained, and/or retarded conjugates; or 
 b) the unconjugated first oligonucleotide sequences, followed by collecting a substantial portion of the non-bound, non-retained, and/or non-retarded conjugates in a wash step. 
   
     
     
         17 . The method of  claim 1 , wherein the isolation step utilizes an immobilized binder, chromatography, affinity chromatography, size exclusion chromatography, HPLC, reverse-phase chromatography, electrophoresis, capillary electrophoresis, polyacrylamide gel electrophoresis, agarose gel electrophoresis, free flow electrophoresis, differential centrifugation, thin layer chromatography, immunoprecipitation, hybridization, solvent extraction, dialysis, filtration, diafiltration, tangential flow filtration, ion exchange chromatography, hydrophobic interaction chromatography, or combinations thereof. 
     
     
         18 . The method of  claim 1 , wherein the binding moiety comprises an antibody, a monoclonal antibody, a polyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a small molecule, an aptamer, a chelator, or combinations or derivatives thereof. 
     
     
         19 . The method of  claim 1 , wherein the signal generating moiety or the one or more signal generating moieties of the first bead conjugate, the hybridized first bead conjugate, the second binding moiety, or of the scaffold, comprises one or more of the following:
 a directly detectable signal generating moiety, an indirectly detectable signal generating moiety, a fluorescent dye, a fluorophore, a fluorochrome, a chromophore, a biofluorescent protein, a luminescent species, a chemiluminescent compound, a electrochemiluminescent label, a bioluminescent label, a phosphorescent species, a fluorophore labeled DNA dendrimer, Quantum Dot, a tandem dye, a FRET dye, a heavy atom, a spin label, a radioactive isotope, a nanoparticle, a light scattering nanoparticle or microsphere, a diffracting particle, a polymer, a polymer particle, a bead, a solid surface, a Raman particle, a metal particle, a stable isotope, a heavy metal chelate, a magnetic particle, an RFID tag, a microbarcode particle, an enzyme, an enzyme substrate, a molecule specifically recognized by another substance carrying a label or reacts with a substance carrying a label, an antibody, an antibody fragment, an antigen, a nucleic acid, a nucleic acid analog, oligonucleotide, oligonucleotide analog, complementary oligonucleotide, complementary oligonucleotide analog, a ligand, a protein, a peptide ligand, a protein substrate, a receptor; a substrate, a secondary reporter, a hapten, or combinations or derivatives thereof.   
     
     
         20 . The method of  claim 1 , wherein the method comprises an automated system or robotic system.