Polymer arrays and methods of using labeled probe molecules to identify and quantify target molecule expression

Abstract

An assay system using labeled probe molecules to identify and quantify target molecules in a sample is disclosed. Where labeled probe molecules are present on a substrate such as a microarray, the identification of multiple different target molecules may be examined simultaneously. Alternatively, a known number of like probe molecules may be present on a substrate and a single target molecule may be quantified in a sample. Preferred labeled probe molecules are comprised fluorescent single stranded nucleotide analogs whose fluorescence is quenched by pairing with a homologous nucleotide target sequence.

1 - 19 . canceled  
     
     
         20 . A substrate for detecting unlabeled target molecules, comprising: 
 a surface area provided by a substrate;    a known quantity of fluorescing nucleotide probes disposed upon said substrate, wherein the fluorescing nucleotide probes has, in part or entirely, fluorescent nucleotide analogs incorporated therein and wherein fluorescing nucleotide probes of said known quantity of fluorescing nucleotide probes hybridizes to said unlabeled target molecules at a known ratio.    
     
     
         21 . The substrate of  claim 20 , wherein the fluorescing nucleotide analogs are nucleotide analogs, including 2-amino purine for adenosine or guanine; ribonucleoside or 2,6-diamino ribonucleoside, formycin A, formycin B, oxyformycin B, toyocamycin, sangivamycin, pseudoouridine, showdomycin, minimycin, pyrazomycin, 5-amino-formycin A, 5-amino-formycin B or 5-oxo-formycin A for adenosine; 4-amino-pyrazolo[3,4d]pyrimidine, 4,6-diamino-pyrazolo[3,4d]pyrimidine, 4-oxo-pyrazolo[3,4d]pyrimidine; 4-oxo-6-amino-pyrazolo[3,4d]pyrimidine, 4,6-dioxo-pyrazolo[3,4d]pyrimidine, pyrazolo[3,4d]pyrimidine, 6-amino-pyrazolo[3,4d]pyrimidine or 6-oxo-pyrazolo[3,4d]pyrimidine for cytosine or thymidine.  
     
     
         22 . The substrate of  claim 20 , wherein said fluorescing nucleotide analogs fluoresces at a wavelength of about 300 nm to about 700 nm.  
     
     
         23 . The substrate of  claim 20 , wherein said surface area is comprised of quadrants.  
     
     
         24 . The substrate of  claim 20 , wherein said fluorescing nucleotide probes are further comprised of amino acids.  
     
     
         25 . The method of  claim 20 , wherein said fluorescing nucleotide probes are further comprised of carbohydrate.  
     
     
         26 . The method of  claim 20 , wherein said surface area has from about 100 to about 10,000 fluorescing nucleotide probe molecules.  
     
     
         27 . The method of  claim 20 , wherein the substrate is a bead.  
     
     
         28 . The method of  claim 27 , wherein said bead size ranges from about 10 microns to about 20 microns.  
     
     
         29 . The method of  claim 27 , wherein the bead is formed of a ferromagnetic metal core and a polymeric coating.  
     
     
         30 . A quantification method for quantifying the effect of exposing a cellular sample to a treatment, by quantifying a target molecule in the cellular sample, comprising: 
 a. providing a first substrate;    b. affixing a known number of fluorescing nucleotide probes onto the substrate, wherein said fluorescing nucleotide probes are comprised, in part or entirety, of fluorescing nucleotide analogs;    c. detecting a first level of fluorescence from said fluorescing nucleotide probes on the substrate;    d. incubating the substrate having the known number of fluorescing nucleotide probes disposed thereon with the cellular sample;    e. providing sufficient conditions and time for unlabeled target molecules to selectively hybridize with fluorescing nucleotide probes on said substrate wherein hybridization of an unlabeled target molecule to a fluorescing nucleotide probe quenches fluorescence from said fluorescing nucleotide probe;    f. removing said substrate from the cellular sample and detecting a second level of fluorescence from said fluorescing nucleotide probes after hybridization;    g. repeating steps a. through f. with subsequent substrates, having surface areas comprising known numbers of fluorescing nucleotide probes until substantially all unlabeled target molecules are hybridized and no longer quench said fluorescing nucleotide probes;    h. quantifying the amount of target molecule in the sample solution by adding the known number of fluorescing nucleotide probes present on the first substrate and subsequent substrates contacted with and quenched by the unlabeled target molecule, whereby the amount of the target molecule is quantified;    i. exposing the cellular sample to a treatment;    j. repeating steps a. through h., in order to quantify the amount of target molecule in the cellular sample solution after said treatment;    k. quantifying the change in the amount of target molecule in the cellular sample before and after said treatment.    
     
     
         31 . The method of  claim 30 , wherein said fluorescing nucleotide probes are comprised of native and nonnative nucleotides.  
     
     
         32 . The method of  claim 30 , wherein the fluorescing nucleotide analogs are nucleotide analogs, including  2 -amino purine for adenosine or guanine; ribonucleoside or 2,6-diamino ribonucleoside, formycin A, formycin B, oxyformycin B, toyocamycin, sangivamycin, pseudoouridine, showdomycin, minimycin, pyrazomycin, 5-amino-formycin A, 5-amino-formycin B or 5-oxo-formycin A for adenosine; 4-amino-pyrazolo[3,4d]pyrimidine, 4,6-diamino-pyrazolo[3,4d]pyrimidine, 4-oxo-pyrazolo[3,4d]pyrimidine; 4-oxo-6-amino-pyrazolo[3,4d]pyrimidine, 4,6-dioxo-pyrazolo[3,4d]pyrimidine, pyrazolo[3,4d]pyrimidine, 6-amino-pyrazolo[3,4d]pyrimidine or 6-oxo-pyrazolo[3,4d]pyrimidine for cytosine or thymidine.  
     
     
         33 . The method of  claim 30 , wherein said fluorescing nucleotide analogs fluoresces at a wavelength of about 300 nm to about 700 nm.  
     
     
         34 . The method of  claim 30 , wherein said fluorescing nucleotide probes are further comprised of amino acids.  
     
     
         35 . The method of  claim 30 , wherein said fluorescing nucleotide probes are further comprised of carbohydrate.  
     
     
         36 . The method of  claim 30 , wherein said surface area has from about 100 to about 10,000 different fluorescing nucleotide probes.  
     
     
         37 . The method of  claim 30 , wherein the substrate is a bead.  
     
     
         38 . The method of  claim 37 , wherein said bead size ranges from about 10 microns to about 20 microns.  
     
     
         39 . The method of  claim 37 , wherein the bead is formed of a ferromagnetic metal core and a polymeric coating.  
     
     
         40 . The method of  claim 37 , having from about 100 to about 1,000 labeled fluorescing nucleotide probe molecules attached to the surface area of the bead.  
     
     
         41 . The method of  claim 30 , wherein said fluorescing nucleotide probes are comprised of native and nonnative nucleotides.  
     
     
         42 . The method of  claim 30 , wherein the substrate is a microarray further having the surface area divided into quadrants, wherein each different quadrant has different fluorescing nucleotide probe molecules.  
     
     
         43 . The method of  claim 42 , having from about 100 to about 10,000 different fluorescing nucleotide probe molecules located upon about 100 to about 10,000 different quadrants.  
     
     
         44 . The method of  claim 30 , wherein the substrate is a microarray further having the surface area divided into quadrants and having about 100 to about 1,000 fluorescing nucleotide probe molecules per quadrant.  
     
     
         45 . The method of  claim 30 , wherein the quantification is evaluated using a flow cytometer.  
     
     
         46 . The method of  claim 30 , wherein the second level is significantly lower than the first level and said second levels of fluorescence approach zero and/or about background levels.