Methods and systems for reducing background signal in assays

Abstract

Methods and systems of monitoring reactions, e.g., assays, that filter out background signal from substrate, in detecting the product. The methods and systems controllably move detectable reaction product from a first location to a second location at which the product is detected while controllably moving potentially interfering substrate materials away from or not toward the second location. Controllable movement of the different species is accomplished through the controlled combination of bulk fluid flow and differential electrophoresis of substrate and product to move the product, but not the substrate past the detection region.

1 . A method of monitoring a reaction, comprising: 
 providing a quantity of a first reaction mixture at a first location in a fluidic conduit, the first reaction mixture comprising: 
 a first reagent having a detectable label associated therewith;  
 a second reagent that reacts with the first reagent to produce a first product having the detectable label associated therewith, the first product having an electrophoretic mobility that is different from the first reagent;  
   providing a detection zone at a second location in the fluidic conduit, the second location being different from the first location;    applying an electric field to the first reaction mixture;    controlling bulk fluid flow through the fluid conduit, whereby under the electric field applied in the applying step the first product moves from the first location to the second location in a substantially greater ratio to the first reagent, as compared to the ratio of product to first reagent at the first location; and    detecting the product at the second location.    
   
   
       2 . The method of  claim 1 , wherein the ratio of product to first reagent at the second location is greater than 2 times the ratio of product to first reagent at the first location.  
   
   
       3 . The method of  claim 1 , wherein the ratio of product to first reagent at the second location is greater than 5 times the ratio of product to first reagent at the first location.  
   
   
       4 . The method of  claim 1 , wherein the ratio of product to first reagent at the second location is greater than 10 times the ratio of product to first reagent at the first location.  
   
   
       5 . The method of  claim 1 , wherein the ratio of product to first reagent at the second location is greater than 100 times the ratio of product to first reagent at the first location.  
   
   
       6 . The method of  claim 1 , wherein the ratio of product to first reagent at the second location is greater than 500 times the ratio of product to first reagent at the first location.  
   
   
       7 . The method of  claim 1 , wherein during the controlling step, the first reagent is directed from the first location substantially away from the second location.  
   
   
       8 . The method of  claim 7 , wherein at least 50% of the first reagent at the first location is directed away from the second location.  
   
   
       9 . The method of  claim 7 , wherein at least 90% of the first reagent at the first location is directed away from the second location.  
   
   
       10 . The method of  claim 7 , wherein at least 95% of the first reagent at the first location is directed away from the second location.  
   
   
       11 . The method of  claim 1 , wherein the fluid conduit comprises a microfluidic conduit.  
   
   
       12 . The method of  claim 11 , wherein the fluid conduit comprises at least first and second fluidly communicating fluid channels.  
   
   
       13 . The method of  claim 12 , wherein the first location comprises a location in the first fluid channel, and the second location comprises a location in the second fluid channel.  
   
   
       14 . The method of  claim 12 , wherein the first location comprises a zone of intersection of the first fluid channel and the second fluid channel and the second location comprises a zone in the second fluid channel.  
   
   
       15 . The method of  claim 1 , wherein the first reagent comprises a phosphorylatable compound, and the second reagent comprises a kinase enzyme.  
   
   
       16 . The method of  claim 1 , wherein the first reagent comprises a polypeptide and the second reagent comprises a protease enzyme capable of cleaving the polypeptide.  
   
   
       17 . The method of  claim 1 , wherein the first reagent comprises a phosphorylated compound, and the second reagent comprises a phosphatase enzyme.  
   
   
       18 . A method of monitoring a reaction, comprising: 
 providing a fluid channel that comprises a first channel segment and a second channel segment;    introducing into the first channel segment a substrate for the reaction having a detectable label associated therewith and a first electrophoretic mobility, and a first reagent that reacts with the substrate to produce a first product that includes the detectable label and a second electrophoretic mobility that is different from the first electrophoretic mobility;    applying an electric field between the first channel segment and the second channel segment and controlling bulk fluid flow between the first channel segment and the second channel segment, whereby the substrate, but not the product is substantially directed away from the second channel segment; and    detecting the product in the second channel segment.    
   
   
       19 . A method of monitoring a reaction, comprising: 
 providing a fluid conduit having first and second zones that are at different locations from each other;    providing a quantity of a reaction mixture in the first zone that comprises: 
 a first reagent having a detectable label and a first electrophoretic velocity in the direction of the second zone of V S  under an applied electric field;  
 a second reagent that reacts with the first reagent to produce a product that includes the detectable label and has a second electrophoretic velocity in the direction of the second zone of V P  under the applied electric field;  
   applying the electric field across a length of the fluid conduit between the first and second zones;    controlling bulk fluid movement in the direction from the first zone to the second zone (F), whereby F+V P  is greater than 0 and F+V S  is less than or equal to 0; and    detecting the product at the second zone.