Universal readout for target identification using biological microarrays

Abstract

A method and apparatus for implementing the method is provided. The method involves performing an indirect competitive binding assay on a microarray to identify biological or chemical targets and screen for compounds of interest. The microarray comprises a common ligand located among membrane-, lipid- or protein-associated active binding sites. The method takes advantage of known or well-characterized binding kinetics, and steric interference between biological or chemicals targets of interest and universal readout units for different binding sites within the limited confines of a microspot. The biological targets, chemicals or organisms can specifically bind to target-binding sites, while the universal readout unit binds to the ligands in the microspot.

1 . A method for identifying biological targets, the method comprises:
 providing a number of biological molecules deposited and immobilized on a substrate;   providing a ligand associated with either said biological molecules or said substrate;   providing a sample containing targets of interest and universal readout units, which have an affinity to bind specifically to said ligand;   performing an assay in which said targets of interest compete indirectly with said universal readout units; and   monitoring binding-events of said universal readout units with said ligand.   
     
     
         2 . The method according to  claim 1 , wherein said biological molecules include a protein, an antibody, a peptide, an oligosaccharide, a biological membrane, a lipid, an oligonucleotide, PNA, RNA, or DNA. 
     
     
         3 . The method according to  claim 2 , wherein said biological membrane includes: a protein membrane protein, a plasma membrane, a lipid-membrane, or a cell membrane. 
     
     
         4 . The method according to  claim 1 , wherein said biological molecules are arranged in at least a microspot. 
     
     
         5 . The method according to  claim 1 , wherein said biological molecules are in an array of microspots. 
     
     
         6 . The method according to  claim 1 , wherein said ligands are co-existant with said biological molecules and common to all of said microspots. 
     
     
         7 . The method according to  claim 1 , wherein said ligands are universal adaptors. 
     
     
         8 . The method according to  claim 1 , wherein said universal readout unit is either a naturally-occurring or synthetic molecule. 
     
     
         9 . The method according to  claim 1 , wherein said universal readout unit is either labeled or conjugated with an enzyme that can produce a fluorescence, luminescent or color product. 
     
     
         10 . The method according to  claim 1 , wherein said universal readout unit and said ligands have a binding constant characterized in the order of a nanomolar or sub-nanomolar range. 
     
     
         11 . The method according to  claim 10 , wherein said binding constant is in the range of about 1×10 −5  nM to about 100 nM. 
     
     
         12 . The method according to  claim 10 , wherein said binding constant is in the range of about 0.1 nM to about 10 nM. 
     
     
         13 . The method according to  claim 1 , wherein said array involves an inhibitor that directly influences target-of-interest binding to said biological molecules. 
     
     
         14 . The method according to  claim 1 , wherein said ligands have a uniform distribution and a density for close packing of said universal readout units once binding occurs. 
     
     
         15 . The method according to  claim 1 , wherein said universal readout unit is physically larger in size than said ligand. 
     
     
         16 . The method according to  claim 1 , wherein said universal readout unit is physically similar in size with said target. 
     
     
         17 . The method according to  claim 1 , wherein a target of interest having a physical size similar to said universal readout unit is prevented from binding by steric hindrance from said universal readout unit. 
     
     
         18 . The method according to  claim 1 , wherein said substrate may comprise a ceramic substance, a glass, a metal, a metallic oxide, a crystalline material, a plastic, a polymer or co-polymer, any combinations thereof, or a coating of one material on another. 
     
     
         19 . The method according to  claim 1 , wherein said substrate has a predominantly two-dimensional (2D) surface. 
     
     
         20 . The method according to  claim 19 , wherein said substrate has a planar surface with an average surface roughness of less than about 10 nm per 1×1 μm 2 . 
     
     
         21 . The method according to  claim 19 , wherein said surface has an average surface roughness of less than about 5 nm per 1×1 μm 2 . 
     
     
         22 . The method according to  claim 19 , wherein said surface has thereon at least one kind of reactive functional group. 
     
     
         23 . The method according to  claim 22 , wherein said reactive functional group includes: amino, carboxyl, hydroxyl, thiol groups, amine-reactive groups, thiol-reactive groups, Ni-chelating groups, or anti-His-antibody groups. 
     
     
         24 . The method according to  claim 1 , wherein said substrate is a well-bottom in a microplate having a number of wells. 
     
     
         25 . The method according to  claim 1 , wherein said method involves a protein-capture reagent microarray. 
     
     
         26 . The method according to  claim 1 , wherein said method involves a glycolipid microarray. 
     
     
         27 . The method according to  claim 1 , wherein said method involves a glycoprotein microarray. 
     
     
         28 . The method according to  claim 1 , wherein said method detects toxins using a ganglioside microarray. 
     
     
         29 . The method according to  claim 1 , wherein said method identifies PIP-binding proteins using a phosphoinositol microarray. 
     
     
         30 . The method according to  claim 1 , wherein said method identifies lipid-raft-binding proteins using a sphingolipid microarray. 
     
     
         31 . The method according to  claim 1 , wherein said method involves screening for ligands using a ligand-gated ion channel microarray. 
     
     
         32 . A method for identifying biological targets or screening for chemical compounds, the method comprises:
 providing a number of probes and biological-molecule-associated ligands on a support;   providing a number of targets of interest and a number of labeled receptors in a sample solution, wherein said targets have an affinity to bind with said probes and said receptors have an affinity to bind with said ligands;   performing an indirect competitive binding assay in which said targets of interest compete with said labeled receptors for space on said support; and, monitoring binding-events of said receptors with said ligands.   
     
     
         33 . The method according to  claim 32 , wherein said method further comprises performing an assay with an inhibitor present in said sample solution. 
     
     
         34 . The method according to  claim 33 , wherein said inhibitor is a chemical compound or biological molecule. 
     
     
         35 . The method according to  claim 34 , wherein said chemical compound includes pharmaceutical compounds. 
     
     
         36 . The method according to  claim 32 , wherein said receptors have a known dissociation constant (K d ) with respect to said ligands. 
     
     
         37 . The method according to  claim 32 , wherein said receptors provides an indication of binding affinity of said targets of interest. 
     
     
         38 . The method according to  claim 32 , wherein said ligands function as universal adaptors. 
     
     
         39 . The method according to  claim 32 , wherein said biological molecules include a biological membrane, a lipid, a protein, an antibody, a peptide, an oligosaccharide, an oligonucleotide, PNA, RNA, or DNA. 
     
     
         40 . The method according to  claim 37 , wherein said biological membrane is selected from the group consisting of cell membranes, membrane protein, lipid-membranes, and plasma membranes. 
     
     
         41 . The method according to  claim 32 , wherein said receptor each has a size of about 2-100 nm. 
     
     
         42 . The method according to  claim 32 , wherein said receptor has a size of at least about 4-20 nm. 
     
     
         43 . The method according to  claim 32 , wherein said target of interest is an antibody, a protein, a hormone, a toxin, or a nucleic acid. 
     
     
         44 . The method according to  claim 43 , wherein said nucleic acid is RNA or DNA 
     
     
         45 . The method according to  claim 44 , wherein said nucleic acid is mRNA, tRNA, gDNA, or cDNA. 
     
     
         46 . The method according to  claim 32 , further comprising determining the relative orientation of said biological molecule-associated ligand on said support. 
     
     
         47 . A method for identifying biological or chemical targets, the method comprising:
 providing on a solid support a number of biological probes and co-mingled ligands in a microspot; providing a sample containing a biological or chemical target of interest and a universal readout unit, which is a binding partner for said ligands; performing an assay in which said biological or chemical target of interest competes with said universal readout unit for limited space on said microspot; and,   monitoring binding-events of said universal readout unit.   
     
     
         48 .- 52 . (canceled) 
     
     
         53 .- 84 . (canceled)