Methods and systems for monitoring intracellular binding reactions

Abstract

Intracellular binding reactions, and particularly DNA/DNA binding protein reactions are detected in situ, using intracellular fluorescence polarization detection. The methods comprise providing a biological cell having at least a first component of a binding reaction disposed therein. The cell is contacted with a second component of the binding reaction whereby the second component is internalized within the biological cell. At least one of the first and second components has a fluorescent label. The amount of binding between the first and second components within the cell is determined by measuring a level of polarized and/or depolarized fluorescence emitted from within the biological cell.

1 . A method of detecting intracellular binding interaction between a first component and a second component, the method comprising: 
 providing a microfluidic device that includes a body with a main analysis channel;    introducing a biological cell containing the first component of the binding reaction into the main analysis channel;    introducing the second component of the binding reaction into the main analysis channel, wherein the second component is a non-protein molecule, so that the second component contacts the biological cell whereby the second component is internalized within the biological cell;    determining an amount of binding between the first and second components within the cell by measuring a level of polarized fluorescence emitted from within the biological cell.    
     
     
         2 . The method of  claim 1 , wherein the main fluid channel is in fluid communication with an external sampling capillary or pipettor.  
     
     
         3 . The method of  claim 1 , wherein a flow controller directs fluid transport within the microfluidic device.  
     
     
         4 . The method of  claim 3 , wherein the flow controller comprises a vacuum controller.  
     
     
         5 . The method of  claim 3 , wherein the flow controller comprises a pressure controller.  
     
     
         6 . The method of  claim 3 , wherein the flow controller comprises an electrokinetic controller.  
     
     
         7 . The method of  claim 1 , wherein the second component comprises a binding fragment of a full-length protein that is capable of binding the first component.  
     
     
         8 . The method of  claim 7 , wherein the second component is between about 4 and about 100 residues in length.  
     
     
         9 . The method of  claim 7 , wherein the second component comprises a molecular weight less than about 10 kD.  
     
     
         10 . The method of  claim 1 , wherein the second component is selected from a carbohydrate, a lipid, cAMP, cGMP and diacylglycerol.  
     
     
         11 . The method of  claim 1 , wherein the first component comprises an intracellular nucleic acid binding protein and the second component comprises a nucleic acid probe.  
     
     
         12 . The method of  claim 1 , wherein the nucleic acid probe is from about 5 to about 100 bases in length.  
     
     
         13 . The method of  claim 8 , wherein the first component comprises a DNA binding protein and the second component comprises a fluorescently labeled DNA probe.  
     
     
         14 . The method of  claim 1 , wherein a level of affinity of the DNA binding protein and the DNA probe is increased or decreased upon activation of a cell signaling pathway.  
     
     
         15 . The method of  claim 11 , wherein the nucleic acid binding protein is a component of a cell signaling pathway, activation of the pathway activating or deactivating the nucleic acid binding protein.  
     
     
         16 . The method of  claim 1 , wherein the cell is selected from a mammalian cell, bacterial cell, fungal cell, yeast cell, insect cell, and a plant cell.  
     
     
         17 . The method of  claim 1 , wherein the determining step comprises: 
 directing a polarized excitation light at the cell;    measuring a level of polarized or depolarized fluorescence emitted from within the cell; and    comparing a level of polarized or depolarized fluorescence emitted from within the cell to a control, to determine a level of the intracellular binding reaction that occurred in the cell.    
     
     
         18 . The method of  claim 1 , wherein the cell is provided in a suspension of cells.  
     
     
         19 . The method of  claim 1 , further comprising: 
 exposing said cell to a test compound prior to the determining step; and    comparing an amount of binding between the first and second components within the cell in the presence of the test compound to an amount of binding between the first and second components within the cell in the absence of the test compound, the comparing step occurring after the determining step.    
     
     
         20 . A method of monitoring the expression of a gene by a cell in response to a stimulus, the method comprising: 
 providing a cell that expresses a gene, and that comprises a first component that binds to the gene;    exposing the cell to a stimulus; and    monitoring the extent of binding between the gene and the first component by fluorescently labeling at least one of the gene or the first component, and measuring a level of polarized fluorescence emitted by the cell.