Methods and Compositions for Highly Sensitive Detection of Molecules

Abstract

Disclosed are methods, kits, and compositions for the highly sensitive detection of molecules. The methods, kits, and compositions are useful in determining concentrations of molecules in samples to levels of 1 femtomolar, 1 attomolar, or lower. The methods, kits, and compositions also allow the determination of concentration over a wide range, e.g., 7-log range, without need for sample dilution.

1 - 49 . (canceled) 
     
     
         50 . A method of establishing a marker for a biological state, comprising
 i) establishing a range of concentrations for said marker in biological samples obtained from a first population, wherein said establishing comprises measuring the concentrations of said marker in said biological samples by detecting single molecules of said marker.   
     
     
         51 . The method of  claim 50  wherein said first population is a population that does not exhibit said biological state. 
     
     
         52 . The method of  claim 50  further comprising establishing a range of levels for said marker in biological samples obtained from a second population, wherein the members of said second population exhibit the biological state, and wherein said establishing comprises measuring the levels of said marker in said biological samples by detecting single molecules of said marker. 
     
     
         53 . The method of  claim 52  wherein said first and second populations are different. 
     
     
         54 . The method of  claim 52  wherein at least one member of said second population is a member of said first population, or at least one member of said first population is a member of said second population. 
     
     
         55 . The method of  claim 54  wherein substantially all the members of the second population are members of the first population who have developed the biological state. 
     
     
         56 . The method of  claim 50  wherein said detecting of single molecules of said marker is performed using a method with a limit of detection for said marker of less than about 1000, 100, 50, 20, 10, 5, 1, or 0.5 femtomolar of said marker in said samples. 
     
     
         57 . The method of  claim 50  wherein said biological state is a phenotypic states; a condition affecting the organism; a state of development; age; health; pathology; disease; disease process; disease staging; infection; toxicity; or response to chemical, environmental, or drug factors (such as drug response phenotyping, drug toxicity phenotyping, or drug effectiveness phenotyping). 
     
     
         58 . The method of  claim 57  wherein said biological state is a pathology. 
     
     
         59 . The method of  claim 50  wherein the marker is a polypeptide or small molecule. 
     
     
         60 . The method of  claim 57  wherein said state is a disease state. 
     
     
         61 . The method of  claim 60  wherein said disease state is selected from the group consisting of cancer, cardiovascular disease, inflammatory disease, autoimmune disease, neurological disease, infectious disease and pregnancy related disorders. 
     
     
         62 . The method of  claim 61  wherein said state is a disease stage state. 
     
     
         63 . The method of  claim 62  wherein said disease stage state is a cancer disease stage state. 
     
     
         64 . The method of  claim 57  wherein the state is a treatment response state. 
     
     
         65 . The method of  claim 64  wherein the treatment is a drug treatment. 
     
     
         66 . The method of  claim 65  wherein said response is selected from the group consisting of a therapeutic effect and a side effect. 
     
     
         67 . The method of  claim 66  wherein the response is a therapeutic effect. 
     
     
         68 . The method of  claim 66  wherein the response is a side effect. 
     
     
         69 . The method of  claim 68  wherein said side effect is an adverse effect. 
     
     
         70 . The method of  claim 50  wherein the detecting comprises labeling said marker with a label comprising a fluorescent moiety capable of emitting at least about 200 photons when stimulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. 
     
     
         71 . The method of  claim 70  wherein said fluorescent moiety comprises a molecule that comprises at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. 
     
     
         72 . The method of  claim 70  wherein said fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. 
     
     
         73 . The method of  claim 70  wherein said fluorescent moiety comprises AlexaFluor 647. 
     
     
         74 . The method of  claim 70  wherein said label further comprises a binding partner for said marker. 
     
     
         75 . The method of  claim 74  wherein said binding partner comprises an antibody specific for said marker. 
     
     
         76 . The method of  claim 75  wherein said antibody comprises a polyclonal antibody. 
     
     
         77 . The method of  claim 75  wherein said antibody is a monoclonal antibody. 
     
     
         78 . The method of  claim 50  further comprising establishing a threshold level for said marker based on said first and second ranges, wherein the presence of said marker in a biological sample from an individual at a level above said threshold level indicates a substantially increased probability of the presence of the biological state in said individual. 
     
     
         79 . A method for detecting the presence or absence of a biological state of an organism, comprising
 i) measuring the concentration of a marker in a biological sample from said organism, wherein said marker is a marker established by the method of  claim 50 ; and   ii) determining the presence or absence of said biological state based on said concentration of said marker in said organism.   
     
     
         80 . A method for detecting the presence or absence of a biological state in an organism, comprising
 i) measuring the concentrations of a marker in a plurality of biological samples from said organism, wherein said marker is a marker established by the method of  claim 50 ; and   ii) determining the presence or absence of said biological state based on said concentrations of said marker in said plurality of samples.   
     
     
         81 . The method of  claim 80  wherein said samples are of different types. 
     
     
         82 . The method of  claim 81  wherein said determining is based on a comparison of the concentrations of said marker in said different types of samples. 
     
     
         83 . The method of  claim 80  wherein the samples are of the same type, and wherein the samples are taken at intervals. 
     
     
         84 . The method of  claim 50  wherein the biological sample is blood, plasma, or serum. 
     
     
         85 . A business method comprising: identifying one or more markers using the system of  claim 50  that is associated with a biological state of an organism; and commercializing said one or more markers in a diagnostic to detect the presence or absence of said biological state in said organism. 
     
     
         86 . The business method of  claim 85  wherein said one or more markers are polypeptides or small molecules. 
     
     
         87 . The business method of  claim 85  wherein said one or more markers are new chemical entities. 
     
     
         88 . The business method of  claim 85  wherein said business method further comprises performing a diagnostic service for fee. 
     
     
         89 . The business method of  claim 85  wherein said business method further comprises selling a diagnostic product based on said one or more biomarkers. 
     
     
         90 . A business method comprising: identifying one or more markers using a system of  claim 50  that is associated with a biological state of an organism; and providing a diagnostic service of determining if said organism has or does not have said biological state. 
     
     
         91 . The business method of  claim 90  wherein said diagnostic service is provided by a CLIA approved laboratory. 
     
     
         92 . The business method of  claim 91  wherein said diagnostic service is provided to a health care provider, insurer or patient. 
     
     
         93 . A label for the detection of a marker, wherein the label comprises a binding partner for the marker and a fluorescent moiety, wherein the fluorescent moiety is capable of emitting at least about 200 photons when stimulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. 
     
     
         94 . The label of  claim 93  wherein the fluorescent moiety comprises a fluorescent molecule. 
     
     
         95 . The label of  claim 94  wherein the fluorescent moiety comprises a plurality of fluorescent molecules. 
     
     
         96 . The label of  claim 95  wherein the label comprises about 2-10, 2-8, 2-6, 2-4, 3-10, 3-8, 3-6 fluorescent molecules. 
     
     
         97 . The label of  claim 96  wherein the label comprises about 2-4 fluorescent molecules. 
     
     
         98 . The label of  claim 97  wherein the fluorescent dye molecules comprise at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. 
     
     
         99 . The label of  claim 97  wherein the fluorescent molecules are selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. 
     
     
         100 . The label of  claim 97  wherein the fluorescent molecules are selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 680 or AlexaFluor 700. 
     
     
         101 . The label of  claim 93  wherein the binding partner comprises an antibody. 
     
     
         102 . The label of  claim 101  wherein said antibody is a monoclonal antibody. 
     
     
         103 . The label of  claim 102  wherein said antibody is a polyclonal antibody. 
     
     
         104 . The label of  claim 101  wherein said antibody is specific for a marker of inflammation. 
     
     
         105 . The label of  claim 104  wherein said antibody is specific for a marker selected from the group consisting of chemokines, growth factors, proteases and inhibitors, cell adhesion molecules, stem cell factors, signal transduction and apoptosis molecules, neurotrophic factors, and developmental proteins. 
     
     
         106 . The label of  claim 105  wherein the antibody is specific for a growth factor. 
     
     
         107 . The label of  claim 101  wherein the antibody is specific for a cytokine. 
     
     
         108 . The label of  claim 107  wherein the cytokine is selected from the group consisting of IL-12p70, IL-10, IL-1 alpha, IL-3, IL-12 p40, IL-1ra, IL-12, IL-6, IL-4, IL-18, IL-10, IL-5, Eotaxin, IL-16, MIG, IL-8, IL-17, IL-7, IL-15, IL-13, IL-2R (soluble), IL-2, LIF/HILDA, IL-1 beta, Fas/CD95/Apo-1 and MCP-1. 
     
     
         109 . The label of  claim 101  wherein the fluorescent moiety comprises a fluorescent molecule. 
     
     
         110 . The label of  claim 109  wherein the fluorescent moiety comprises a plurality of fluorescent molecules. 
     
     
         111 . The label of  claim 110  wherein the label comprises about 2-10, 2-8, 2-6, 2-4, 3-10, 3-8, or 3-6 fluorescent molecules. 
     
     
         112 . The label of  claim 111  wherein the label comprises about 2-4 fluorescent molecules. 
     
     
         113 . The label of  claim 112  wherein the fluorescent dye molecules comprise at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. 
     
     
         114 . The label of  claim 112  wherein the fluorescent molecules are selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, or AlexaFluor 700. 
     
     
         115 . The label of  claim 112  wherein the fluorescent molecules are AlexaFluor 647 molecules.