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 . An assay method comprising: (i) providing a planar array of particles randomly-positioned with respect to particle type, where different types of particles are differently encoded by incorporation of an optically detectable characteristic into the particles that permits said differently encoded particles to be distinguished, and wherein said differently encoded particles have different binding molecules attached thereto; (ii) determining the spatial coordinates of particular particle types within the planar array; (iii) contacting said array of particles with a solution such that, if a particular type of analyte is present in said solution, said analyte binds to the corresponding binding molecules of said particles and the binding of said analyte is indicated by an optically detectable signal; (iv) determining the presence of any such bound analyte by determining the presence of said optically detectable signal; and (v) identifying said optically detectable characteristic of particles having binding molecules bound to an analyte, without moving or separating said particles being interrogated from other particles in the array, by correlating the spatial coordinates of said particles with the location of the optically detectable signals in order to identify said binding molecules bound to an analyte. 
     
     
         31 . The method of  claim 30 , wherein the step of identifying the optically detectable characteristic of the particles types is performed before the step of contacting said array. 
     
     
         32 . The method of  claim 30 , wherein the step of identifying the optically detectable characteristic of the particles types is performed after the step of contacting said array. 
     
     
         33 . The method of performing an assay according to  claim 30 , wherein the analyte and binding molecule form a binding pair, wherein one member of the pair is a receptor and the other member is a ligand, one member is an antibody and the other member is an antigen, or one member is an enzyme and the other member is a substrate. 
     
     
         34 . The method of performing an assay according to  claim 30 , wherein the analyte and binding molecule form a binding pair, wherein the members of the pair are complementary nucleic acid strands. 
     
     
         35 . The method of  claim 34 , wherein one of the nucleic acid strands is cDNA and the complementary strands is an oligonucleotide probe, and wherein the method comprises the step of detecting hybridization between the cDNA and the oligonucleotide probe. 
     
     
         36 . The method of  claim 35 , wherein the cDNA is labeled. 
     
     
         37 . The method of  claim 30 , wherein said analyte binding molecule is an enzyme and wherein the assay comprises the steps of reacting the enzyme with a target analyte in the solution and detecting enzyme reaction products that are released into the solution. 
     
     
         38 . The method of  claim 30 , wherein optically detectable characteristic is color. 
     
     
         39 . The method of  claim 30 , wherein the solution comprises fluorescently labeled analytes, and the step of determining the presence of bound analyte comprises recording fluorescence from individual particles within the array of particles to determine the quantity of one or more associated analytes. 
     
     
         40 . The method of  claim 30 , wherein the step of contacting said array of particles comprises contacting said array of particles with a solution comprising a first analyte, such that said first analyte is capable of forming a first paired entity with a corresponding binding molecule, the method further comprising the step of contacting said array of particles, subsequent to the step of contacting said array of particles, with another solution comprising a second analyte, different from the first analyte, said second analyte capable of forming a second paired entity. 
     
     
         41 . The method of  claim 40 , wherein the first analyte and the second analyte bind to different binding molecules 
     
     
         42 . The method of  claim 30 , wherein said array of particles is chemically anchored to the substrate. 
     
     
         43 . A method of performing an assay using at least one planar array of particles, the method comprising: forming the planar array with a number of differently encoded particles, on a defined area of a substrate but wherein differently-encoded particles are randomly-positioned within said area, said differently encoded particles having different analyte binding molecules attached thereto, said particle types being encoded by incorporation of an optically detectable characteristic into the particles, that distinguishes said differently encoded particles and the binding molecules attached thereto; determining the spatial coordinates of particular particle types within the planar array; contacting said plurality of particles with a solution such that, if particular analytes are present in said solution, said analytes bind to the corresponding binding molecules attached to said particles to form a paired entity and said analytes carry an optically detectable characteristic; detecting said paired entities; and identifying, the optically detectable characteristic of the particles to which binding molecules are attached which form paired entities by correlating the spatial coordinates of said particles with the spatial coordinates of the optically detectable characteristic in order to identify said binding molecules forming said paired entities. 
     
     
         44 . The method of  claim 43 , further comprising the step of selectively marking at least one individual particle within the array of particles. 
     
     
         45 . The method of  claim 30  or  48  wherein the planar array of particles are arranged on a defined area of the substrate surface. 
     
     
         46 . The method of  claim 30  or  48  wherein the planar array of particles are permanently anchored to said substrate.