Enhanced Isotachophoresis Assays Using Additives with Spatial Gradients

Abstract

Techniques for enhanced isotachophoresis assays using additives with spatial gradients include forming a concentration gradient of an additive along a channel from an input port to an output port. The channel is used for isotachophoresis with ions of a leading electrolyte having a first mobility greater than a mobility of an analyte, and ions of a trailing electrolyte having a second mobility less than the mobility of the analyte. The additive is different from both the leading electrolyte and the trailing electrolyte; and the additive has a third mobility that assures the analyte will encounter the additive. The method further comprises introducing a mixture of the trailing electrolyte and a sample including the analyte. The method further comprises applying an electric field to the channel; and, measuring the analyte.

1 . A method comprising:
 forming a concentration gradient of each of one or more additives along a channel from an input port to an output port, wherein:
 the channel is used for isotachophoresis with ions of a leading electrolyte having a first effective mobility magnitude greater than an effective mobility of an analyte, and ions of a trailing electrolyte having a second effective mobility magnitude less than the effective mobility of the analyte; 
 each additive is different from both the leading electrolyte and the trailing electrolyte; and 
 each additive has a third mobility that assures the analyte will encounter the additive; 
   contacting a sample including the analyte to the leading electrolyte;   contacting the trailing electrolyte to the sample;   applying an electric field to the channel; and   measuring the analyte.   
     
     
         2 . A method as recited in  claim 1 , wherein each additive operates most effectively on a component of the sample along a portion of the channel where the concentration of the additive is in a particular range of concentrations. 
     
     
         3 . A method as recited in  claim 1 , wherein the channel is a microchannel. 
     
     
         4 . A method as recited in  claim 1 , wherein the one or more additives comprise a polymer that provides a sieving matrix. 
     
     
         5 . A method as recited in  claim 1 , wherein the analyte is a nucleic acid comprising a plurality of nucleotides and an additive comprises a denaturing agent. 
     
     
         6 . A method as recited in  claim 1 , wherein the one or more additives comprise a fluorescent label. 
     
     
         7 . A method as recited in  claim 1 , wherein the analyte is a nucleic acid comprising a plurality of nucleotides and the one or more additives comprise a nucleic acid probe with a fluorescent label. 
     
     
         8 . A method as recited in  claim 1 , wherein the analyte is a nucleic acid comprising a plurality of nucleotides and the one or more additives comprise a molecular beacon that comprises a nucleic acid probe with a fluorescent label at one end and a fluorescence quencher at a different end. 
     
     
         9 . A method as recited in  claim 4 , wherein forming the concentration gradient of each of one or more additives along the channel from the input port to the output port further comprises:
 disposing a photo-initiator and monomer within the channel;   covering the channel with a photo-mask that varies along the length of the channel; and   exposing the photo-mask to polymerizing light that causes the monomer to form the polymer that provides the sieving matrix.   
     
     
         10 . A method as recited in  claim 1 , wherein contacting the trailing electrolyte to the sample further comprises contacting the trailing electrolyte to the sample and a solution of spacer ions, wherein each spacer ion has a third effective mobility magnitude between the effective mobility magnitude of a product of the analyte and the effective mobility magnitude the analyte. 
     
     
         11 . A method as recited in  claim 10 , wherein the product of the analyte is a reporter molecule. 
     
     
         12 . An apparatus comprising:
 a channel connecting an input port to an output port;   a concentration gradient of each of one or more additives along the channel:   means for contacting a sample including an analyte to a leading electrolyte, wherein the leading electrolyte has a first effective mobility magnitude greater than an effective mobility of the analyte and the leading electrolyte is different from the additive;   means for contacting a trailing electrolyte to the sample, wherein the trailing electrolyte has a second effective mobility magnitude less than the effective mobility of the analyte and the trailing electrolyte is different from the additive;   means for applying an electric field to the channel; and   means for measuring the analyte.