Amplified bioassay

Abstract

A method for assaying a biological sample includes forming sensitized microcapsules filled with unique oligomarkers, capturing sensitized microcapsules in the presence of analytes, releasing oligomarkers from microcapsules and detecting and measuring oligomarkers to detect and quantify presence of analyte in biological sample. Using encapsulated oligomarkers provides for an amplified high sensitivity assay and using plurality of oligomarker types provides for a multiplexed assay.

1 . A kit for assaying a biological sample potentially containing two or more analytes, said kit comprising:
 (a) a stationary solid phase support with immobilized receptors for binding thereto said analytes and   (b) two or more different types of sensitized microcapsules, each different type of said sensitized microcapsules having a surface membrane and an interior volume, said surface membrane of said each different type of said sensitized microcapsules having an incorporated receptor that specifically binds to a particular one of said analytes and said interior volume of said each different type of said sensitized microcapsules includes a unique first member of a binding pair, each said unique first member of a binding pair in said each different type of said sensitized microcapsules is different and specifically binds a second member of the binding pair.   
     
     
         2 . The kit according to  claim 1  further comprising:
 a disrupting agent suitable to disrupt said sensitized microcapsules and release each said unique first member of the binding pair from each different type of said sensitized microcapsules when said analytes are present in the biological sample. 
 
     
     
         3 . The kit according to  claim 1  further comprising:
 a device comprising:
 means for immobilizing each said unique first member of the binding pair released from said each different type of said sensitized microcapsules in spatially separated locations; and 
 means for detecting each said unique first member of the binding pair released from said each different type of said sensitized microcapsules immobilized in said spatially separated locations. 
 
 
     
     
         4 . The kit according to  claim 1 , wherein said unique first member of the binding pair comprises a tag. 
     
     
         5 . The kit according to  claim 4 , wherein said tag is an electrochemically detectable species, a colorimetrically detectable species, a fluorescent species, an oligomarker, an oligonucleotide molecule, or an oligonucleotide molecule conjugated to a fluorophore tag, an electrochemical tag, a quantum dot tag, or a dye tag. 
     
     
         6 . The kit according to  claim 3 , wherein said means for detecting each said unique first member of the binding pair are colorimetric, fluorescent, or electrochemical means of detection. 
     
     
         7 . The kit according to  claim 1 , wherein said unique first member of the binding pair is an antigen, a hapten, an antibody, a cytokine, a chemokine, an enzyme, a drug, a protein, a biomarker, a DNA, or an RNA. 
     
     
         8 . The kit according to  claim 1 , wherein said second member of the binding pair is an antigen, a hapten, an antibody, a cytokine, a chemokine, an enzyme, a drug, a protein, a biomarker, a DNA, or an RNA. 
     
     
         9 . The kit according to  claim 3 , wherein said unique first member of the binding pair is an oligonucleotide molecule. 
     
     
         10 . The kit according to  claim 1 , wherein said microcapsules are liposomes or polymer based microspheres. 
     
     
         11 . The kit according to  claim 3 , wherein said means for immobilizing each said unique first member of the binding pair released from said each different type of said microcapsules comprises the second members of the binding pair which are immobilized. 
     
     
         12 . The kit according to  claim 9 , wherein said means for immobilizing each said unique first member of the binding pair released from said each different type of said sensitized microcapsules comprises an array of immobilized oligonucleotides complementary to said oligonucleotide molecules, wherein each said oligonucleotide molecule released from said each different type of said sensitized microcapsules is brought into contact with said array and hybridizes with said immobilized oligonucleotides. 
     
     
         13 . A kit for assaying a biological sample potentially containing at least one analyte, said kit comprising:
 (a) a first sensitized microcapsule, said first sensitized microcapsule having a surface membrane and an interior volume, said surface membrane of said first sensitized microcapsule having an incorporated receptor that specifically binds to said analyte and said interior volume of said first sensitized microcapsule includes a unique first member of a binding pair that specifically binds a second member of the binding pair;   (b) an immobilized binder of said analyte;   (c) a second sensitized microcapsule filled with tags, said second sensitized microcapsule having a surface membrane and an interior volume, said surface membrane of said second sensitized microcapsule having an incorporated second member of the binding pair for specifically binding to said first member of the binding pair; and   (d) an immobilized second member of the binding pair for specifically binding to said first member of the binding pair.   
     
     
         14 . The kit according to  claim 13  further comprising:
 a disrupting agent suitable to disrupt said first sensitized microcapsule and release said first member of the binding pair from said first sensitized microcapsule when said analyte is present in the biological sample and 
 a disrupting agent suitable to disrupt said second sensitized microcapsule and releasing said tags when said analyte is present in the biological sample. 
 
     
     
         15 . The kit according to  claim 13  further comprising:
 release said first member of the binding pair from the first sensitized microcapsule when said analyte is present in the biological sample and to disrupt said second sensitized microcapsule and release said tags when said analyte is present in the biological sample. 
 
     
     
         16 . The kit according to  claim 13 , wherein said tags are electrochemically detectable species, colorimetrically detectable species, fluorescent species, oligomarkers, oligonucleotide molecules, or oligonucleotide molecules conjugated to a fluorophore tag, an electrochemical tag, a quantum dot tag, or a dye tag. 
     
     
         17 . The kit according to  claim 13 , wherein said first member of the binding pair is an antigen, a hapten, an antibody, a cytokine, a chemokine, an enzyme, a drug, a protein, a biomarker, a DNA, or an RNA. 
     
     
         18 . The kit according to  claim 13 , wherein said second member of the binding pair is an antigen, a hapten, an antibody, a cytokine, a chemokine, an enzyme, a drug, a protein, a biomarker, a DNA, or an RNA. 
     
     
         19 . The kit according to  claim 13 , wherein said first member of the binding pair is an oligonucleotide molecule. 
     
     
         20 . The kit according to  claim 13 , wherein said microcapsules are liposomes or polymer based microspheres. 
     
     
         21 . The kit according to  claim 19 , wherein said immobilized second member of the binding pair comprises oligonucleotides immobilized on an array which are complementary to said oligonucleotide molecules.