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 making an array, said method comprising:
 (a) providing a substrate with a surface comprising a plurality of discrete sites;   (b) providing a population of beads comprising a first subpopulation of beads having 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   (c) randomly distributing said population of beads on said surface such that sites of said plurality of discrete site have a single bead from said first subpopulation associated therewith and such that beads from said second subpopulation end up at said sites.   
     
     
         31 . The method of  claim 30 , wherein said first subpopulation and said second subpopulation are sequentially distributed on said surface. 
     
     
         32 . The method of  claim 30  further comprising making said first subpopulation of beads by attaching a digest of a prokaryotic genome to said beads. 
     
     
         33 . The method of  claim 30  further comprising making said subpopulation of beads by attaching a digest of a eukaryotic genome to said beads. 
     
     
         34 . The method of  claim 30  further comprising making said first subpopulation of beads by attaching a copies of fragments of a prokaryotic genome to said beads. 
     
     
         35 . The method of  claim 30  further comprising making said first subpopulation of beads by attaching copies of fragments of a eukaryotic genome to said beads. 
     
     
         36 . The method of  claim 30 , wherein said enzymes are attached to the beads of the second subpopulation. 
     
     
         37 . The method of  claim 30 , wherein sites of said plurality of discrete sites lack a single bead from said first population. 
     
     
         38 . The method of  claim 30  further comprising optically coupling a fiber optic bundle to said substrate. 
     
     
         39 . The method of  claim 38 , wherein said plurality of discrete sites comprises a plurality of wells. 
     
     
         40 . The method of  claim 39 , wherein wells of said plurality of wells are at a density of at least 100 wells per 1 mm 2 . 
     
     
         41 . The method of  claim 39 , wherein wells of said plurality of wells are at a density of at least 10,000 wells per 1 mm 2 . 
     
     
         42 . The method of  claim 30 , wherein said first subpopulation comprises at least 10 beads having copies of the same nucleic acid associated therewith. 
     
     
         43 . A method of making an array, said method comprising:
 (a) providing a substrate comprising a plurality of wells;   (b) randomly distributing beads of a first subpopulation of beads into wells of said plurality of wells such that the wells contain a single bead from the first subpopulation of beads, wherein beads of the first subpopulation of beads have nucleic acids associated therewith; and   (c) distributing beads of a second subpopulation of beads into the wells containing a single bead from the first subpopulation of beads, wherein beads of the second subpopulation of beads have enzymes associated therewith, and wherein beads of the second subpopulation of beads are smaller than beads of said first population of beads.   
     
     
         44 . The method of  claim 43  further comprising making said first subpopulation of beads by attaching a digest of a prokaryotic genome to said beads. 
     
     
         45 . The method of  claim 43  further comprising making said subpopulation of beads by attaching a digest of a eukaryotic genome to said beads. 
     
     
         46 . The method of  claim 43  further comprising making said first subpopulation of beads by attaching a copies of fragments of a prokaryotic genome to said beads. 
     
     
         47 . The method of  claim 43  further comprising making said first subpopulation of beads by attaching copies of fragments of a eukaryotic genome to said beads. 
     
     
         48 . The method of  claim 43 , wherein said enzymes are attached to the beads of the second subpopulation. 
     
     
         49 . The method of  claim 43 , wherein wells of said plurality of wells lack a single bead from said first population. 
     
     
         50 . The method of  claim 43  further comprising optically coupling a fiber optic bundle to said substrate. 
     
     
         51 . The method of  claim 43 , wherein wells of said plurality of wells are at a density of at least 100 wells per 1 mm 2 . 
     
     
         52 . The method of  claim 43 , wherein wells of said plurality of wells are at a density of at least 10,000 wells per 1 mm 2 . 
     
     
         53 . The method of  claim 43 , wherein said first subpopulation comprises at least 10 beads having copies of the same nucleic acid associated therewith.