Methods and arrays for target analyte detection and determination of target analyte concentration in solution

Abstract

Arrays of single molecules and methods of producing an array of single molecules are described. Arrays with defined volumes between 10 attoliters and 50 picoliters enable single molecule detection and quantitation.

1 . A method of producing an array of single molecules, comprising:
 a) providing a sample and an array, said sample comprising a molecules of target analyte in a concentration, said array comprising at least 1000 sites, each site having a defined volume between 10 attoliters and 50 picoliters; and   b) contacting said array with said sample such that the ratio of molecules of target analyte to each site of said array is less than 1:1, so as to produce an array comprising sites containing single molecules.   
     
     
         2 . The method of  claim 1 , wherein said target analyte comprises biomolecules. 
     
     
         3 . The method of  claim 2 , wherein said biomolecule is selected from the group consisting of proteins, nucleic acids, lipids, and carbohydrates. 
     
     
         4 . The method of  claim 2 , wherein said biomolecule is selected from the group consisting of hormones, cytokines and cellular antigens. 
     
     
         5 . The method of  claim 2 , wherein said biomolecule is a receptor. 
     
     
         6 . The method of  claim 5 , wherein said receptor is selected from the group consisting of neural receptors, hormonal receptors, and nutrient receptors. 
     
     
         7 . The method of  claim 2 , wherein said biomolecules are labeled. 
     
     
         8 . The method of  claim 1 , wherein said target analyte is an environmental pollutant. 
     
     
         9 . The method of  claim 1 , wherein said target analyte is selected from the group consisting of pesticides, insecticides and toxins. 
     
     
         10 . The method of  claim 1 , wherein said target analyte is a drug. 
     
     
         11 . The method of  claim 1 , wherein said ratio is less than 1:5. 
     
     
         12 . The method of  claim 1 , wherein said ratio is 1:10. 
     
     
         13 . The method of  claim 11 , wherein said ratio is between 1:5 and 1:500. 
     
     
         14 . The method of  claim 1 , wherein the percentage of sites containing a single molecule provide a measure of the concentration of molecules in said sample. 
     
     
         15 . An array produced according to the method of  claim 1 . 
     
     
         16 . The method of  claim 1 , further comprising, after step b), sealing the array such that the contents of each site cannot escape said site. 
     
     
         17 . An array produced according to the method of  claim 16 . 
     
     
         18 .- 25 . (canceled) 
     
     
         26 . A method of detecting a target analyte in a sample, the method comprising:
 a) providing a sample and an array, said sample comprising a biomolecule target analyte, said array comprising at least 1000 sites, each site having a defined volume between 10 attoliters and 50 picoliters;   b) contacting said array with said sample such that the ratio of biomolecule target analyte to each site of said array is less than 1:1; and   c) determining the number of sites of said array which contain a single biomolecule.   
     
     
         27 .- 44 . (canceled) 
     
     
         45 . A method of detecting a target analyte in a sample, the method comprising:
 a) providing a sample and an array, said sample comprising a biomolecule target analyte in a first concentration, said array comprising at least 10,000 sites, each site having a defined femtoliter volume;   b) diluting said sample to create a diluted sample, said diluted sample comprising a biomolecule target analyte in a second concentration;   c) contacting said array with said diluted sample such that the ratio of biomolecule target analyte to each site of said array is between 1:5 and 1:500; and   d) determining the number of sites of said array which contain a single biomolecule.   
     
     
         46 .- 61 . (canceled)