Methods for detecting target analytes and enzymatic reactions

Abstract

A microsphere-based analytic chemistry system and method for making the same is disclosed in which microspheres or particles carrying bioactive agents may be combined randomly or in ordered fashion and dispersed on a substrate to form an array while maintaining the ability to identify the location of bioactive agents and particles within the array using an optically interrogatable, optical signature encoding scheme. A wide variety of modified substrates may be employed which provide either discrete or non-discrete sites for accommodating the microspheres in either random or patterned distributions. The substrates may be constructed from a variety of materials to form either two-dimensional or three-dimensional configurations. In a preferred embodiment, a modified fiber optic bundle or array is employed as a substrate to produce a high density array. The disclosed system and method have utility for detecting target analytes and screening large libraries of bioactive agents.

1 - 29 . (canceled) 
     
     
         30 . A method of detecting target nucleic acids in a sample, said method comprising:
 (a) providing an array comprising
 (i) a population of beads comprising a first subpopulation of beads having probe nucleic acids associated therewith and a second subpopulation of beads having enzymes associated therewith, said beads of the first subpopulation being a different size from the beads of the second subpopulation; and 
 (ii) a substrate having a surface comprising a plurality of discrete sites, wherein said population of beads is randomly distributed on said surface such that sites of said plurality of discrete sites have a single bead from the first subpopulation associated therewith, and wherein said sites further comprise beads from said second subpopulation; and 
   (b) providing a sample comprising target nucleic acids;   (c) contacting said sample with said array, whereby said target nucleic acids hybridize to said probe nucleic acids; and   (d) determining the presence of said target nucleic acids hybridized to said probe nucleic acids.   
     
     
         31 . The method of  claim 30 , wherein said probe nucleic acids are attached to the beads of the first subpopulation. 
     
     
         32 . The method of  claim 30 , wherein said enzymes are attached to the beads of the second subpopulation. 
     
     
         33 . The method of  claim 30 , further comprising determining polymorphic DNA markers for said target nucleic acids. 
     
     
         34 . The method of  claim 33 , wherein said polymorphic DNA markers comprise single nucleotide polymorphisms. 
     
     
         35 . The method of  claim 30 , wherein step (d) further comprises detecting an optical signal that occurs subsequent to said target nucleic acids hybridizing to said probe nucleic acids. 
     
     
         36 . The method of  claim 35 , wherein said optical signal is transmitted through a fiber of a fiber optic bundle. 
     
     
         37 . The method of  claim 36 , wherein a first end of said fiber optic bundle is optically coupled to a surface of said array, and wherein a second end of said fiber optic bundle is optically coupled to a charge coupled device (CCD) camera. 
     
     
         38 . The method of  claim 30 , wherein said enzymes generate an optical signal. 
     
     
         39 . The method of  claim 38 , wherein the optical signal comprises a chemiluminescent signal. 
     
     
         40 . The method of  claim 39 , wherein said chemiluminescent signal is transmitted through an optical fiber. 
     
     
         41 . The method of  claim 30 , wherein sites of said plurality of discrete sites lack a single bead from said first population. 
     
     
         42 . The method of  claim 30 , wherein said plurality of discrete sites comprises a plurality of wells. 
     
     
         43 . The method of  claim 42 , wherein wells of said plurality of wells are dimensioned to accommodate a single bead from the first subpopulation. 
     
     
         44 . The method of  claim 42 , wherein wells of said plurality of wells are at a density of at least 100 wells per 1 mm 2 . 
     
     
         45 . The method of  claim 42 , wherein wells of said plurality of wells are at a density of at least 10,000 wells per 1 mm 2 . 
     
     
         46 . The method of  claim 30 , wherein said first subpopulation comprises at least 10 beads having copies of the same probe nucleic acid associated therewith. 
     
     
         47 . A method of detecting target nucleic acids in a sample, said method comprising:
 (a) providing a sample comprising target nucleic acids to an array, said array comprising a substrate, said substrate comprising a plurality of wells having a population of beads randomly distributed therein, said population of beads comprising a first subpopulation of beads having probe nucleic acids associated therewith and a second subpopulation of beads having enzymes associated therewith, said beads of the first subpopulation being a different size from the beads of the second subpopulation, and said beads of the first subpopulation being distributed such that wells of said plurality of wells contain a single bead from the first subpopulation of beads;   (b) allowing said target nucleic acids to hybridize to said probe nucleic acids; and   (c) determining the presence of said target nucleic acids hybridized to said probe nucleic acids.   
     
     
         48 . The method of  claim 47 , wherein said probe nucleic acids are attached to the beads of the first subpopulation. 
     
     
         49 . The method of  claim 47 , wherein said enzymes are attached to the beads of the second subpopulation. 
     
     
         50 . The method of  claim 47 , wherein step (c) further comprises detecting an optical signal that occurs subsequent to said target nucleic acids hybridizing to said probe nucleic acids. 
     
     
         51 . The method of  claim 50 , wherein said optical signal is transmitted through a fiber of a fiber optic bundle. 
     
     
         52 . The method of  claim 51 , wherein a first end of said fiber optic bundle is optically coupled to a surface of said array, and wherein a second end of said fiber optic bundle is optically coupled to a charge coupled device (CCD) camera. 
     
     
         53 . The method of  claim 47 , wherein said enzymes generate an optical signal. 
     
     
         54 . The method of  claim 53 , wherein the optical signal comprises a chemiluminescent signal. 
     
     
         55 . The method of  claim 54 , wherein said chemiluminescent signal is transmitted through an optical fiber. 
     
     
         56 . The method of  claim 47 , wherein wells of said plurality of wells lack a single bead from said first population. 
     
     
         57 . The method of  claim 47 , wherein wells of said plurality of wells are at a density of at least 100 wells per 1 mm 2 . 
     
     
         58 . The method of  claim 47 , wherein wells of said plurality of wells are at a density of at least 10,000 wells per 1 mm 2 . 
     
     
         59 . The method of  claim 47 , wherein said first subpopulation comprises at least 10 beads having copies of the same probe nucleic acid associated therewith.