Methods, apparatuses, and systems for detecting and quantifying phosphoproteins

Abstract

Embodiments herein provide methods, apparatuses, and systems for detecting, monitoring, measuring, and/or characterizing the activity of phosphoproteins, such as tyrosine kinases (TKs) and downstream proteins in TK signal transduction pathways (e.g., TK pathway proteins). In various embodiments, the methods, apparatuses, and systems may use nanoparticles, such as quantum dots (QD), to detect and/or characterize the abnormally overactive TK signaling pathways that underlie tumorgenesis and tumor progression. In various embodiments, the QD-based methods, apparatuses, and systems may have a sufficiently high degree of sensitivity to enable the identification of new TK signaling pathway markers, for example for use in diagnosing, staging, monitoring, and/or prognosing cancers, or in evaluating the efficacy of cancer therapeutics.

We claim: 
     
         1 . A method for quantitating phosphoprotein activity in a biological sample comprising:
 labeling a phosphoprotein in the biological sample with a nanoparticle probe comprising a detectable nanoparticle;   providing the labeled biological sample on a solid support; and   automatically counting the nanoparticles, wherein automatically counting the nanoparticles includes:
 automatically capturing an image of the nanoparticles in each of several Z-planes; 
 automatically detecting nanoparticles that have fluoresced on the captured images; and 
 maintaining a count of discrete groups of nanoparticle probes or single nanoparticle probes. 
   
     
     
         2 . The method of  claim 1 , further comprising:
 positioning the biological sample on a stage adapted to move the biological sample in X, Y, and Z planes;   automatically moving the biological sample through multiple predetermined locations using the stage; and   capturing an image at each predetermined location to create a Z-stack.   
     
     
         3 . The method of  claim 2 , further comprising correcting for double counting of nanoparticles that appear in more than one image. 
     
     
         4 . The method of  claim 3 , further comprising binning the detected nanoparticles so that detected nanoparticles that are separated by less than a predetermined distance in any X, Y, or Z direction are counted as a single nanoparticle. 
     
     
         5 . The method of  claim 1 , wherein automatically detecting the nanoparticles comprises:
 applying an intensity threshold to a region encompassing the detected nanoparticle;   choosing a pixel within the region with a maximum intensity; and   determining a centroid of an intensity pattern around the chosen pixel.   
     
     
         6 . The method of  claim 1 , wherein the nanoparticles comprise semiconductor nanocrystals. 
     
     
         7 . The method of  claim 1 , wherein the phosphoprotein is a tyrosine kinase or a tyrosine kinase pathway protein. 
     
     
         8 . The method of  claim 7 , wherein the nanoparticle probe specifically binds to an active form of the tyrosine kinase pathway protein. 
     
     
         9 . The method of  claim 7 , wherein the phosphoprotein is pAbl, pAkt, pCrkl, pERK, pSTAT3, or pSTAT5. 
     
     
         10 . The method of  claim 1 , wherein the biological sample comprises a cancer cell. 
     
     
         11 . The method of  claim 1 , wherein the cancer cell is a non-small cell lung cancer cell, chronic myeloid leukemia cell, or a gastrointestinal stromal tumors cell. 
     
     
         12 . The method of  claim 1 , wherein the biological sample comprises tumor tissue. 
     
     
         13 . The method of  claim 1 , wherein the biological sample comprises a formalin-fixed, paraffin-embedded tissue sample. 
     
     
         14 . The method of  claim 1 , wherein the biological sample is a single cell. 
     
     
         15 . The method of  claim 14 , wherein the single cell is a blood cell. 
     
     
         16 . The method of  claim 1 , wherein the biological sample comprises a stem cell. 
     
     
         17 . The method of  claim 1 , wherein the biological sample is a sample from a subject suspected of having or known to have cancer. 
     
     
         18 . The method of  claim 1 , wherein the method is a method of diagnosing or prognosing cancer in the subject based at least upon the quantified phosphoprotein activity. 
     
     
         19 . The method of  claim 1 , wherein the nanoparticle probe comprises an antibody. 
     
     
         20 . The method of  claim 18 , wherein the antibody is an anti-Abl, anti-Akt, anti-Crkl, anti-ERK, anti-STAT3, anti-STAT5, anti-pAbl, anti-pAkt, anti-pCrkl, anti-pERK, anti-pSTAT3, or anti-pSTAT5 antibody. 
     
     
         21 . The method of  claim 1 , wherein the method is a method of determining drug effectiveness based at least upon the quantified phosphoprotein activity. 
     
     
         22 . An assay for characterizing phosphoprotein activity in a biological sample, comprising:
 one or more semiconductor nanocrystal probes comprising a binding agent that specifically binds to an activated phosphoprotein;   means for detecting the one or more semiconductor nanocrystal probes at single nanoparticle resolution in the sample;   means for quantifying phosphoprotein activity in the sample based on the detected one or more semiconductor nanocrystal probes.   
     
     
         23 . The assay of  claim 22 , wherein the means for detecting the semiconductor nanocrystal probes at single nanoparticle resolution further comprises means for quantifying the one or more semiconductor nanocrystal probes at the single nanoparticle resolution in the sample, and wherein quantifying the one or more semiconductor nanocrystal probes at the single nanoparticle resolution in the sample comprises quantifying phosphoprotein activity. 
     
     
         24 . The assay of  claim 22 , wherein the assay further includes means for localizing the semiconductor nanocrystal probes in the biological sample. 
     
     
         25 . The assay of  claim 22 , wherein the means for quantifying phosphoprotein activity in the sample comprises means for determining drug effectiveness in the sample based at least upon the quantified phosphoprotein activity. 
     
     
         26 . The assay of  claim 22 , wherein the means for quantifying phosphoprotein activity in the sample based on the detected one or more semiconductor nanocrystal probes at single nanoparticle resolution comprises determining protein biomarker activity based at least upon the quantified phosphoprotein activity. 
     
     
         27 . The assay of  claim 22 , wherein the biological sample is a single blood cell. 
     
     
         28 . The assay of  claim 22 , wherein the biological sample comprises formalin-fixed, paraffin-embedded tissue. 
     
     
         29 . The assay of  claim 22 , wherein the biological sample comprises cancer cells. 
     
     
         30 . The assay of  claim 22 , wherein the biological sample comprises tumor tissue. 
     
     
         31 . The assay of  claim 22 , wherein the biological sample is from a subject known to have or suspected of having a cancer. 
     
     
         32 . The assay of  claim 22 , wherein the activated phosphoprotein is a tyrosine kinase or a tyrosine kinase pathway protein. 
     
     
         33 . The assay of  claim 22 , wherein the activated phosphoprotein is Abl, Akt, Crkl, ERK, STAT3, or STAT5. 
     
     
         34 . The assay of  claim 22 , wherein the specific binding agent is an antibody. 
     
     
         35 . The assay of  claim 34 , wherein the antibody is an anti-Abl, anti-Akt, anti-Crkl, anti-ERK, anti-STAT3, or anti-STAT5 antibody. 
     
     
         36 . The assay of  claim 34 , wherein the antibody is a human or humanized antibody.