Method for calibrating fluorescent plate readers

Abstract

The present invention relates to a fluorescent polymer cast on the surface of wells of a plate and a plate comprising the fluorescent polymer. The plate is used in a method for calibrating read-out values of plate readers. The method for calibrating the plate readers can effectively reduce the deviation of read-out values among different plate readers.

What is claimed is: 
     
         1 . A fluorescent polymer comprising a pre-determined amount of a fluorescent dye imbedded in a polymer, wherein the fluorescent polymer is transparent, dry and solvent- and porogen-free. 
     
     
         2 . A plate comprising a fluorescent polymer of  claim 1  which is casted on the wells of the plate, wherein the fluorescent polymer is transparent, dry and solvent- and porogen-free. 
     
     
         3 . The plate of  claim 2 , wherein the plate is a microplate. 
     
     
         4 . The plate of  claim 3 , wherein the microplate has 6, 12, 24, 48, 96, 384 or more wells. 
     
     
         5 . The plate of  claim 2 , wherein the amounts of the fluorescent dye imbedded in the fluorescent polymer on wells of the plate are present as a concentration gradient. 
     
     
         6 . The plate of  claim 2 , wherein the fluorescent dye is Alexa Fluor, 7-Aminoactinomycin D, 8-Anilinonaphthalene-1-sulfonic acid, ATTO dyes, Auramine-rhodamine stain, Benzanthrone, Bimane, 9,10-Bis(phenylethynyl)anthracene, 5,12-Bis(phenylethynyl)naphthacene, Bisbenzimide, Blacklight paint, Brainbow, Calcein, Carboxyfluorescein, Carboxyfluorescein diacetate succinimidyl ester, Carboxyfluorescein succinimidyl ester, 1-Chloro-9,10-bis(phenylethynyl)anthracene, 2-Chloro-9,10-bis(phenylethynyl)anthracene, 2-Chloro-9,10-diphenylanthracene, Coumarin, DAPI, Dark quencher, DiOC6, DyLight Fluor, Epicocconone, FlAsH-EDT2, Fluo-3, Fluo-4, FluoProbes, Fluorescence image-guided surgery, Fluoro-Jade stain, FMN-binding fluorescent proteins, Fura-2, Fura-2-acetoxymethyl ester, Green fluorescent protein (GFP), Heptamethine dyes, Hoechst stain, Iminocoumarin, Indian yellow, Indo-1, Laurdan, Lucifer yellow, Luciferin, MCherry, Merocyanine, Nile red, Optical brightener, Perylene, Phloxine, Phycobilin, Phycoerythrin, Phycoerythrobilin, Pyranine, Rhodamine, Rhodamine 123, Rhodamine 6G, RiboGreen, RoGFP, Rubrene, (E)-Stilbene, (Z)-Stilbene, Sulforhodamine 101, Sulforhodamine B, SYBR Green I, SYBR Safe, Synapto-pHluorin, Tetraphenyl butadiene, Tetrasodium tris(bathophenanthroline disulfonate)ruthenium(II), Texas Red, Titan yellow, TSQ, Umbelliferone, Violanthrone, Yellow fluorescent protein or YOYO-1. 
     
     
         7 . The plate of  claim 2 , wherein the fluorescent dye is Nile red. 
     
     
         8 . The plate of  claim 2 , wherein the polymer is styrene-butadiene copolymer, acrylonitrile-styrene copolymer (SAN), general purpose polystyrenre (GPPS) such as high impact polystyrene (HIPS), acrylonitrile-styrene copolymer (AS resin), acrylonitrile-butadiene-styrene copolymer (ABS resin), polymethyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polyvinyl butyral (PVB), polylactic acid (PLA) or styrene ethylene/butylene styrene copolymer (SEBS). 
     
     
         9 . The plate of  claim 2 , wherein the polymer is PMMA. 
     
     
         10 . A method of producing a fluorescent polymer comprising: mixing a monomer and a pre-determined amount of fluorescent dye; and forming the fluorescent polymer by polymerizing the monomer with a polymerization initiator, wherein the fluorescent polymer is transparent, dry and solvent- and porogen-free; provided that no liquid solvent is added in the method. 
     
     
         11 . A method of producing a fluorescent polymer plate, comprising: (a) mixing a monomer solution and a pre-determined amount of fluorescent dye; (b) adding the resulting mixture of (a) to wells of the plate; and polymerizing the monomer with a polymerization initiator on the wells, wherein the fluorescent polymer is transparent, dry and solvent- and porogen-free; provided that no liquid solvent is added in the method. 
     
     
         12 . The method of  claim 10 , wherein the polymerization initiator is a free radical initiator, a cationic initiator, or an anionic initiator. 
     
     
         13 . The method of  claim 12 , wherein the free radical initiator is peroxide or azo compound. 
     
     
         14 . The method of  claim 13 , wherein the peroxide is acyl peroxide, acetyl peroxide, benzoyl peroxide, alkyl peroxide, hydroperoxide or perester. 
     
     
         15 . The method of  claim 13 , wherein the azo compound is 2,2′-Azobisisobutyronitrile (AIBN), 2,2′-azobis(2,4-dimethylpentanenitrile), 4,4′-azobis(4-cyanovaleric acid), or 1,1′-azobis(cylohexanecarbonitrile). 
     
     
         16 . A method for calibrating a vendor plate reader, comprising: providing a plate reader calibrated by a certified reference material (CRM); placing the plate of  claim 2  in the plate reader to calibrate the plate reader; obtaining a set of original values from the plate readers; and recording the set of values in a software. 
     
     
         17 . A method for calibrating read-out values of a user plate reader, comprising providing the plate and software according to  claim 16 ; placing the plate in a plate reader; obtaining a set of read-out values from the plate reader; calibrating the set of read-out values based on the software and a linear regression anylasis. 
     
     
         18 . A method for calibrating read-out values of plate readers comprising: providing a master plate reader calibrated by a certified reference material (CRM); placing the plate according to  claim 2  in the master plate reader; and obtaining a first set of read-out values from the master plate reader. 
     
     
         19 . The method of  claim 18 , which further comprises: placing the plate according to  claim 2  in a second plate reader; obtaining a second set of read-out values from the second plate reader; obtaining a linear regression formula based on the first set of read-out values and the second set of read-out values; and calibrating the second set of read-out values based on the linear regression formula. 
     
     
         20 . The method of  claim 16 , wherein the CRM is standard reference material (SRM). 
     
     
         21 . The method of  claim 17 , wherein the linear regression is a Weighted Least Squares (WLS) method. 
     
     
         22 . A kit comprising the plate according to  claim 2 , and a protocol for using the kit to calibrate a plate reader.