Multicolored particles

Abstract

The present invention relates to a set of polymer particles stained with at least two fluorescent dyes, wherein at least 16 subsets of particles can be resolved on the basis of variable emission from the at least two fluorescent dyes wherein emission from at least one dye derives from a fluorescent dye covalently attached to the particle surface, and wherein all particles in said set of polymer particles can bind a uniform amount of a capture reagent. The invention also relates to a method for the preparation of said set of polymer particles as well as a kit comprising said set of polymer particles. The invention further relates to methods and uses of said set of polymer particles.

1 . A set of polymer particles stained with at least two fluorescent dyes, wherein at least 16 subsets of particles can be resolved on the basis of variable emission from the at least two fluorescent dyes, wherein emission from at least one dye of the at least two fluorescent dyes derives from a fluorescent dye covalently attached to a particle surface of at least one particle in the set of polymer particles, wherein all particles in said set of polymer particles can bind a uniform amount of a capture reagent, and wherein the at least two fluorescent dyes are not dyed nanoparticles. 
     
     
         2 . The set of polymer particles of  claim 1 , wherein said dyes are attached in defined concentrations. 
     
     
         3 . The set of polymer particles according to  claim 1 , wherein said at least one fluorescent dye that is covalently attached to the particle surface is covalently attached via a bifunctional crosslinker or directly to each particle. 
     
     
         4 . The set of polymer particles according to  claim 1 , wherein all particles in said set of polymer particles contain sufficient binding sites for uniform covalent attachment of capture reagent. 
     
     
         5 . The set of polymer particles of  claim 4 , wherein said binding sites are free SH- or amino-groups. 
     
     
         6 . The set of polymer particles according to  claim 1 , wherein all particles in said set of polymer particles are uniformly labeled with said capture reagent. 
     
     
         7 . The set of polymer particles of  claim 6 , wherein said capture reagent is covalently attached via a bifunctional crosslinker or directly to each particle. 
     
     
         8 . The set of polymer particles according to  claim 1 , wherein the capture reagent optionally has attached fluorescent dyes. 
     
     
         9 . The set of polymer particles according to  claim 1 , wherein said capture reagent is a protein or a nucleic acid. 
     
     
         10 . The set of polymer particles according to  claim 9 , wherein said protein is an antibody-based molecule. 
     
     
         11 . The set of polymer particles according to  claim 3 , wherein the bifunctional crosslinker is based on maleimide-thiol chemistry. 
     
     
         12 . The set of polymer particles according to  claim 11 , wherein the bifunctional crosslinker is SPDP. 
     
     
         13 . The set of polymer particles according to  claim 1 , wherein the fluorescent dyes are UV/violet excitable, 488 nm excitable, 532 nm (YAG) excitable, 595 nm (Krypton) excitable, 633 nm excitable, Infrared excitable, 488 nm excitable, 633 nm excitable Tandem conjugates of PE and APC, Tandem conjugates of reactive dyes, 488 nm excitable and/or quantum dots. 
     
     
         14 . The set of polymer particles according to  claim 1 , wherein the fluorescent dyes are hydrophilic forms of cyanine dyes such as reactive forms of Alexa 488 and 647. 
     
     
         15 . The set of polymer particles according to  claim 1 , wherein the size of each particle is less than 100 μm in diameter. 
     
     
         16 . The set of polymer particles according to  claim 1 , wherein the particles are monodispersed particles. 
     
     
         17 . A method for the preparation of the set of polymer particles according to  claim 1  comprising, in either sequence:
 a. attaching the at least two fluorescent dyes in defined concentrations to the set of polymer particles, wherein at least one dye is covalently attached to the particle surface; and 
 b. optionally attaching different capture reagents of interest to different subsets of polymer particles, wherein said capture reagent may be covalently attached directly to each particle or via a bifunctional crosslinker or biotin-streptavidin and optionally has attached fluorescent dyes. 
 
     
     
         18 . A set of polymer particles prepared according to the method of  claim 17 . 
     
     
         19 . A kit comprising a set of polymer particles according to  claim 1 . 
     
     
         20 . The set of polymer particles according to  claim 1 , wherein said set of polymer particles is stained with two fluorescent dyes.