Method and apparatus for detecting cancerous cells using molecules that change electrophoretic mobility

Abstract

The activity of oncogenic intracellular chemical reactions of molecules is measured by the use of fluorescently labeled substrate molecules that undergo a change in electrophoretic mobility upon a chemical reaction such as that catalyzed by an enzyme or kinase. Specificity is achieved by using labeled substrate molecules that can be acted upon only by specific oncogenic enzymes. Thus the activity of an oncogenic enzyme or class of oncogenic enzymes can be determined. Measurements are made with the intracellular presence of such substrate molecules, at some time of interest. To ensure accuracy, measurements must be made in a timely manner so as to minimize chemical reactions occurring subsequent to the time of interest. Fast controllable laser lysis is used to obtain the contents of said cell or cells into which reporter substrate molecules have been introduced. The cell contents are then subjected to capillary electrophoresis and oncogenic enzymatic activity is determined by comparing amounts of unaltered substrate molecules to the amounts of altered substrate molecules which are separated by the electrophoresis and identified by the presence of a fluorescent label.

We claim:  
     
         1 . A method for measuring the presence of oncogenic activity of intracellular chemical reactions in a cell or cells comprising: 
 providing substrate molecules for an oncoprotein containing a label, the labeled substrate molecules corresponding to chemical reactions whose activity is to be measured;    disposing said substrate molecules within said cell or cells;    allowing said substrate molecules within said cell or cells to take part in the chemical reaction to produce altered substrate molecules;    liberating said substrate molecules and said altered substrate molecules from the single cell;    detecting the label to identify the substrate molecules and/or the altered substrate molecules from said cell or cells; and    determining the presence of said chemical reaction from the presence of modified substrate.    
     
     
         2 . The method of  claim 1  further comprising quantifying the amounts of detected altered substrate molecules and/or detected unaltered substrate molecules.  
     
     
         3 . The method of  claim 1  wherein said intracellular chemical reaction in said cell comprises enzyme catalysis by a kinase.  
     
     
         4 . The method of  claim 1  wherein said altered substrate molecules exhibit a change in chemical structure as compared with the unaltered substrate molecules.  
     
     
         5 . The method of  claim 4 , wherein separating said unaltered substrate molecules and said altered substrate molecules comprises electrophoresis.  
     
     
         6 . The method of  claim 2  wherein quantifying the amounts of detected altered substrate molecules and detected unaltered substrate molecules comprises detection of the label by fluorescence following separation by electrophoresis.  
     
     
         7 . The method of  claim 1  wherein disposing said unaltered substrate molecules within said cell or cells comprises using a naturally occurring substrate molecule within said cell or cells, inducing said substrate molecule to be produced within said cell or cells, or introducing said substrate molecule into said cell or cells from outside said cell or cells.  
     
     
         8 . The method of  claim 7  wherein introducing said unaltered substrate molecules into said cell or cells from outside said cell or cells comprises microinjecting, electroporating, optoporating, vesicle fusing, pinocytic loading, or associating said substrate molecules with membrane permeant peptides.  
     
     
         9 . The method of  claim 1  further comprising stimulating said cell or cells while said unaltered substrate molecules are intracellularly present prior to liberating said unaltered substrate molecules and said altered substrate molecules from the single cell or cells.  
     
     
         10 . The method of  claim 9  further comprising comparing activity of said chemical reaction with a similar activity determined from said single cell or cells that has not been stimulated.  
     
     
         11 . The method of  claim 1 , wherein liberating said unaltered substrate molecules and said altered substrate molecules from the cell or cells comprises chemical disruption of said single cell or cells, mechanical disruption of said single cell or cells, or by electrical disruption, or by a combination thereof.  
     
     
         12 . The method of  claim 1 , wherein the label is selected from a group consisting of fluorescent labels, isotopes, labels exhibiting optical absorption, and electron spin resonance labels.  
     
     
         13 . The method of  claim 1  wherein the substrate molecules are polymers.  
     
     
         14 . The method of  claim 13  wherein the polymers are selected from a group consisting of peptides, polysaccharide, and nucleic acids.  
     
     
         15 . The method of  claim 14  wherein said polymers are modified with a fluorescent label.  
     
     
         16 . The method of  claim 14  wherein said peptides are substrates for a kinase that alters said modified peptides by the addition of a phosphate moiety to a particular amino acid within each peptide.  
     
     
         17 . The method of  claim 16 , wherein said peptide has been modified by covalent addition of a fluorescent group.  
     
     
         18 . The method of  claim 1 , said substrate molecules comprise carbohydrates, phospholipids, entire proteins, or organic compounds not ordinarily found within the cell.  
     
     
         19 . The method of  claim 1  wherein detecting the label comprises performing voltammetry or mass spectrometry.  
     
     
         20 . The method of  claim 1  further comprising simultaneously performing each of said steps with a plurality of different substrate molecules, each reporting on a specific chemical reaction within said cell or cells.  
     
     
         21 . A method for measuring oncogenic activity of a chemical reaction in a minute volume of tens of pl or less comprising: 
 providing substrate molecules for an oncoprotein containing a label;    disposing said substrate molecule into said minute volume where said chemical reactions occurs producing altered substrate molecules within said minute volume;    terminating said chemical reactions;    detecting the label to identify unaltered substrate molecules and/or the altered substrate molecules to determine activity of the chemical reaction corresponding to the oncoprotein.    
     
     
         22 . The method of  claim 21  further comprising quantifying changes in the amounts of the unaltered substrate molecules and/or the altered substrate molecules.  
     
     
         23 . An apparatus for measuring an activity of oncogenic chemical reactions of intracellular molecules comprising: 
 means for disposing labeled substrate molecules into said cell or cells to form labeled altered substrate molecules therein;    means for liberating said substrate and/or altered substrate molecules from said cell or cells;    means for separating said substrate and/or altered substrate molecules from each other;    means for detecting said unaltered substrate molecules and said altered substrate molecules from a cell or cells before any substantial alteration of said unaltered substrate molecules and said altered substrate molecules has occurred.    
     
     
         24 . The apparatus of  claim 23  further comprising means for quantifying changes in the amounts of said unaltered substrate molecules and/or said altered substrate molecules.  
     
     
         25 . The apparatus of  claim 23  wherein said means for detecting detects enzyme catalysis by a kinase.  
     
     
         26 . The apparatus of  claim 23  wherein said means for detecting detects a change in electrophoretic mobility of said unaltered substrate molecules versus said altered substrate molecules.  
     
     
         27 . The apparatus of  claim 23  wherein said means for separating comprises capillary electrophoresis.  
     
     
         28 . The apparatus of  claim 24  wherein said means for quantifying changes in the amounts of said unaltered substrate molecules and/or said altered substrate molecules comprises means for quantifying fluorescence of said unaltered substrate molecules and/or said altered substrate molecules following separation of said unaltered substrate molecules and said altered substrate molecules by capillary electrophoresis.  
     
     
         29 . The apparatus of  claim 23  wherein said means for disposing comprises means for inducing said substrate molecules to be produced within said cell or cells, or means for introducing said substrate molecule into said cell or cells from outside said cell or cells.  
     
     
         30 . The apparatus of  claim 29  wherein said means for introducing said substrate molecules into said cell or cells from outside said cell or cells comprises means for microinjecting, means for electroporating, means for optoporating, means for vesicle fusing, means for pinocytic loading, or means for associating said substrate molecules with membrane permeant peptides.  
     
     
         31 . The apparatus of  claim 23  wherein said means for disposing said substrate molecules comprises means for providing said substrate molecules from naturally occurring compounds or synthetically derived compounds.  
     
     
         32 . The apparatus of  claim 23  further comprising means for stimulating said cell or cells while said substrate molecules are present intracellularly prior to detecting said unaltered substrate molecules and said altered substrate molecules.  
     
     
         33 . The apparatus of  claim 32  wherein said means for detecting comprises means for obtaining the contents of said cell or cells, and means for separating part or all of said contents by capillary electrophoresis  
     
     
         34 . The apparatus of  claim 33 , wherein said means for obtaining the contents comprises chemical means for disruption, physical means for disruption, electrical means for disruption, or a combination thereof.  
     
     
         35 . The apparatus of  claim 23  wherein said means for disposing provides substrate molecules that correspond to oncogenic intracellular chemical reactions.  
     
     
         36 . The apparatus of  claim 35  wherein said means for disposing provides substrate molecules which are fluorescent.  
     
     
         37 . The apparatus of  claim 36  wherein said means for disposing provides substrate molecules which are modified peptides.  
     
     
         38 . The apparatus of  claim 37 , wherein said modified peptides are substrates for a kinase that alters said modified peptides by the addition of a phosphate moiety to a particular amino acid within each said peptide.  
     
     
         39 . The apparatus of  claim 37  wherein said peptides have been modified by covalent addition of a fluorescent group to allow detection by fluorescence.  
     
     
         40 . The apparatus of  claim 23  wherein said means for disposing provides substrate molecules comprising nucleic acids, carbohydrates, phospholipids, entire proteins, or compounds not ordinarily found within cells.  
     
     
         41 . The apparatus of  claim 23  wherein said means for detecting comprises means for performing voltammetry or means for mass spectrometry on said unaltered substrate molecules and said altered substrate molecules.  
     
     
         42 . The apparatus of  claim 23  further comprising means for simultaneously disposing and detecting a plurality of different substrate molecules, each different substrate molecule reporting on chemical reactions within said cell or cells.  
     
     
         43 . An apparatus for measuring an activity of chemical reactions of molecules in a minute volume of the order of 100 pl or less comprising: 
 means for disposing substrate molecules having a label into said minute volume for oncogenic chemical reactions to occur producing altered substrate molecules; and    means for detecting the label to identify the unaltered substrate molecules and the altered substrate molecules to determine activity of the oncogenic chemical reaction.    
     
     
         44 . The apparatus of  claim 43  further comprising means for quantifying changes in the amounts of said unaltered substrate molecules and said altered substrate molecules.  
     
     
         45 . An apparatus for measuring an activity of oncogenic intracellular chemical reactions of molecules in a cell or cells in which labeled substrate molecules have been disposed to allow for an in vivo reaction wherein labeled altered substrate molecules are formed, comprising: 
 a detector of said labeled unaltered substrate molecules and said labeled altered substrate molecules; and    a cell sampling device communicating with said detector, which cell sampling device extracts said unaltered substrate and altered substrate molecules from said cell, and which cell sampling device collects and transfers said unaltered substrate and altered substrate molecules into said detector before any further substantial alteration occurs.    
     
     
         46 . The apparatus of  claim 45  further comprising a data processor coupled to said detector to quantify changes in the amounts of said unaltered substrate molecules and said altered substrate molecules.  
     
     
         47 . The apparatus of  claim 23  further comprising a data processor coupled to said detector to quantify changes in the amounts of said unaltered substrate molecules and said altered substrate molecules.  
     
     
         48 . The apparatus of  claim 43  further comprising a data processor coupled to said detector to quantify changes in the amounts of said unaltered substrate molecules and said altered substrate molecules.  
     
     
         49 . The method of  claim 1  where oncogenic activity of intracellular chemical reactions in a cell is related to a bcr-abl oncoprotein and where said providing substrate molecules for a kinase provides substrates for bcr-abl tyrosine kinase.  
     
     
         50 . The method of  claim 21  where said oncogenic activity is related to a bcr-abl oncoprotein and where providing substrate molecules for a kinase comprises providing substrates molecules for bcr-abl tyrosine kinase.  
     
     
         51 . The apparatus of  claim 23  where said activity of oncogenic chemical reactions of intracellular molecules is related to a bcr-abl oncoprotein, where said means for disposing labeled substrate molecules into said cell disposed substrate molecules for bcr-abl tyrosine kinase, and where said means for detecting said unaltered substrate molecules and said altered substrate molecules detects phosphorylated substrate molecules for bcr-abl tyrosine kinase.  
     
     
         52 . The apparatus of  claim 43  where said activity of oncogenic intracellular chemical reactions is related to a bcr-abl oncoprotein, where said detector of said labeled unaltered substrate molecules and said labeled altered substrate molecules detects phosphorylated and/or nonphosphorylated substrate molecules for bcr-abl tyrosine kinase.  
     
     
         53 . The apparatus of  claim 45  where said activity of oncogenic chemical reactions of intracellular molecules is related to a bcr-abl oncoprotein, where said means for disposing labeled substrate molecules into said cell disposed substrates molecules for bcr-abl tyrosine kinase, and where said means for detecting said unaltered substrate molecules and said altered substrate molecules detects phosphorylated and/or nonphosphorylated substrate molecules for bcr-abl tyrosine kinase.