Color-encoding and in-situ interrogation of matrix-coupled chemical compounds

Abstract

A method and apparatus for the physico-chemical encoding of a collection of beaded resin (“beads”) to determine the chemical identity of bead-anchored compounds by in-situ interrogation of individual beads. The present invention provides method and apparatus to implement color-coding strategies in applications and including the ultrahigh-throughput screening of bead-based combinatorial compounds libraries as well as multiplexed diagnostic and environmental testing and other biochemical assays.

1 . A method for encoding and decoding solid supports comprising: 
 (a) providing a population of solid supports comprising M batches thereof, wherein M is an integer greater than 1;    (b) attaching to one or more of said batches one or more fluorophore tag(s), each tag being identifiable by optical interrogation, wherein said one or more tag(s) constitutes a binary code or extended binary code; and    (c) decoding the code composed of the one or more tag(s) on said solid supports by optical interrogation of the tag(s) carried out without isolating the solid supports from each other and without detaching the tags(s) from the solid supports.    
     
     
         2 . The method of  claim 1 , wherein the optical interrogation of each fluorophore tag comprises determining its relative abundance.  
     
     
         3 . The method of  claim 1 , wherein each fluorophore tag is attached to a solid support by covalent bonding.  
     
     
         4 . The method of  claim 1 , wherein the fluorophore tag is a dye selected from the group consisting of the following dyes, which are activated as an active ester selected from the group consisting of succinimidyl, sulfosuccinimidyl, p-nitrophenol, pentafluorophenol, HOBt and N-hydroxypiperidyl esters thereof: 3-(ε-carboxypentyl)-3′-ethyl-oxacarbocyanine-6,6′-disulfonic acid; 1-(ε-carboxypentyl)-1′-ethyl-3,3,3,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid; 1-(ε-carboxypentyl)-1′-ethyl-3,3,3,3′-tetramethyl-3H-benz(e)indocarbocyanine-5,5′,7,7′-tetrasulfonic acid; and 1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid.  
     
     
         5 . The method of  claim 1 , wherein the fluorophore tag is a dye selected from the group consisting of the following dyes, which are activated as an active ester selected from the group consisting of succinimidyl, sulfosuccinimidyl, p-nitrophenol, pentafluorophenol, HOBt and N-hydroxypiperidyl esters thereof: 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoic acid; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoic acid; 6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoic acid; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid; 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3 a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid; and 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid.  
     
     
         6 . The method of  claim 1 , wherein the fluorophore tag is a dye selected from the group consisting of compounds with the following chemical structures:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         7 . The method of  claim 1 , wherein the decoding is carried by multi-color fluorescence imaging in combination with spectral analysis.  
     
     
         8 . The method of  claim 1 , wherein M is an integer from 2 to 25.  
     
     
         9 . The method of  claim 1 , wherein the fluorophore tag is optically distinguishable by emission wavelength.  
     
     
         10 . The method of  claim 1 , wherein the fluorophore tag is optically distinguishable by emission intensity, by adjusting the ratio of the relative quantities of the fluorophore tags.  
     
     
         11 . The method of  claim 13 , wherein the ratio of the relative quantities of the fluorophore tags is from about 1:1 to 4:1.  
     
     
         12 . The method of  claim 1 , wherein the fluorophore tag is optically distinguishable by excited-state lifetime.  
     
     
         13 . The method of  claim 1 , wherein the fluorophore tag is optically distinguishable by emission wavelength, excited-state lifetime and emission intensity.  
     
     
         14 . The method of  claim 1  wherein the solid support comprises a bead.  
     
     
         15 . The method of  claim 14 , wherein the decoding is carried out while the beads are on a planar substrate.  
     
     
         16 . The method of  claim 15  wherein the optical interrogation is carried out using multi-color fluorescent imaging in combination with spectral analysis.  
     
     
         17 . The method of  claim 14 , wherein the decoding is carried out while the beads are arranged in a planar bead array.  
     
     
         18 . The method of  claim 14  wherein the optical interrogation is carried out using multi-color fluorescent imaging in combination with spectral analysis.  
     
     
         19 . The method of  claim 14 , wherein the bead is composed of a material selected from the group consisting of polystyrene, polyethylene, cellulose, polyacrylate, polyacrylamide, silica and glass.  
     
     
         20 . The method of  claim 1 , wherein the decoding step comprises the steps of: 
 (a) collecting spectral fluorescence data for each respective solid support so as to determine the respective abundance of the tag(s) bound thereto; and    (b) analyzing the collected spectral fluorescence data by comparing the respective relative abundances of the tag(s) determined in (a).    
     
     
         21 . The method of  claim 20  wherein the solid support is a bead.  
     
     
         22 . The method of  claim 21  wherein spectral fluorescence data is collected by: 
 (a) forming a static planar array or a dynamic planar array of beads; and    (b) obtaining a fluorescence image for each bead.    
     
     
         23 . The method of  claim 22 , wherein the planar array of beads is formed adjacent to the planar walls of a sandwich flow cell and controlled by light-controlled electrokinetic means.  
     
     
         24 . The method of  claim 23 , wherein spectral fluorescence data are collected for the bead array by initially forming a spatially encoded array of beads at an interface between an electrode and an electrolyte solution, comprising the following steps: 
 (a) providing an electrode and an electrolyte solution;    (b) providing multiple types of beads, each type being stored in accordance with chemically or physically distinguishable bead characteristics in one of a plurality of reservoirs, each reservoir containing a plurality of like-type beads suspended in said electrolyte solution;    (c) providing said reservoirs in the form of an m×n grid arrangement;    (d) patterning said electrode to define m×n compartments corresponding to said m×n grid of reservoirs;    (e) depositing m×n droplets from said m×n reservoirs onto said corresponding m×n compartments, each said droplet originating from one of said reservoirs and remaining confined to one of said m×n compartments and each said droplet containing at least one bead;    (f) positioning a top electrode above said droplets so as to simultaneously contact each said droplet;    (g) generating an electric field between said top electrode and said m×n droplets;    (h) using said electric field to form a bead array in each said m×n compartments, each said bead array remaining spatially confined to one of said m×n droplets;    (i) illuminating said m×n compartments on said patterned electrode with a predetermined light pattern to maintain the position of said bead arrays in accordance with said predetermined light pattern and the pattern of m×n compartments; and    (j) positioning said top electrode closer to said electrode thereby fusing said m×n droplets into a continuous liquid phase, while maintaining each of said m×n bead arrays in one of the corresponding m×n compartments.    
     
     
         25 . The method of  claim 24 , wherein said compartments are hydrophilic and the remainder of said electrode surface is hydrophobic.