Isoelectric focusing devices and fixtures

Abstract

Methods, devices, and systems for performing isoelectric focusing reactions are described. The systems or devices disclosed herein may comprise fixtures that have a membrane. In some instances, the disclosed devices may be designed to perform isoelectric focusing or other separation reactions followed by further characterization of the separated analytes using mass spectrometry. The disclosed methods, devices, and systems provide for fast, accurate separation and characterization of protein analyte mixtures or other biological molecules by isoelectric point.

What is claimed is: 
     
         1 . A method for performing a plurality of isoelectric focusing reactions in parallel, the method comprising:
 providing a device comprising:
 a plurality of separation channels, each separation channel comprising a first end and a second end; and 
 wherein the first end of each separation channel is electrically coupled to a first electrode reservoir via a first membrane disposed at a surface of the first electrode reservoir; 
   introducing at least one sample comprising a mixture of analytes into at least two separation channels of the plurality of separation channels;   controlling voltage applied to the at least two separation channels to perform two parallel isoelectric focusing reactions that separate the mixture of analytes in the at least one sample; and   independently monitoring current flowing through the at least two separation channels as the two parallel isoelectric focusing reactions are performed.   
     
     
         2 . The method of  claim 1 , wherein the first electrode reservoir comprises an insert disposed within the first electrode reservoir at or adjacent to the first membrane, the insert comprising an inlet fluid path arm and an outlet fluid path arm, wherein the inlet and outlet fluid path arms are structurally coupled and in fluidic communication. 
     
     
         3 . The method of  claim 1 , wherein the second end of each separation channel is electrically coupled to a second electrode reservoir via a second membrane disposed at a surface of the second electrode reservoir. 
     
     
         4 . The method of  claim 1 , further comprising independently controlling the voltage applied to the at least two separation channels during the two parallel isoelectric focusing reactions. 
     
     
         5 . The method of  claim 1 , wherein the at least one sample comprises a first sample and a second, wherein the first sample is the same as the second sample, and the method further comprises:
 introducing the first sample into a first separation channel of the at least two separation channels and performing a first isoelectric focusing reaction using a first set of experimental conditions; and   introducing the second sample into a second separation channel of the at least two separation channels and performing a second isoelectric focusing reaction using a second set of experimental conditions, wherein the second set of experimental conditions is different from the first set of experimental conditions.   
     
     
         6 . The method of  claim 1 , wherein the at least one sample comprises a first sample and a second, wherein the first sample is different from the second sample, and the method further comprises:
 introducing the first sample into a first separation channel of the at least two separation channels and performing a first isoelectric focusing reaction; and   
       introducing the second sample into a second separation channel of the at least two separation channels and performing a second isoelectric focusing reaction. 
     
     
         7 . The method of  claim 1 , further comprising:
 recording a current trace for each of the at least two separation channels while performing the plurality of isoelectric focusing reactions.   
     
     
         8 . The method of  claim 1 , further comprising:
 flushing the at least two separation channels following completion of the isoelectric focusing reactions and automatically introducing another sample into the at least two separation channels.   
     
     
         9 . The method of  claim 1 , further comprising:
 detecting a failure in either of the at least two separation channels, wherein the failure comprises introduction of a bubble into either of the at least two separation channels, formation of a bubble within either of the at least two separation channels, an incorrectly prepared sample, an insufficiently filled first electrode reservoir, or any combination thereof.   
     
     
         10 . The method of  claim 9 , wherein when the failure is detected, the method further comprises:
 automatically re-introducing and repeating the isoelectric focusing reaction for the at least one sample.   
     
     
         11 . The method of  claim 9 , wherein detecting a failure in either of the at least two separation channels comprises monitoring the current flowing through the at least two separation channels or processing an image of the at least two separation channels. 
     
     
         12 . The method of  claim 1 , further comprising:
 measuring dynamic light scattering in at least one of the at least two separation channels while performing the isoelectric focusing reactions.   
     
     
         13 . The method of  claim 12 , further comprising:
 determining a size distribution profile, an aggregation state, or a hydrodynamic radius for one or more separated analytes from measurement of the dynamic light scattering.   
     
     
         14 . The method of  claim 2 , wherein the first membrane covers all or substantially all of an opening comprising an intersection of the inlet fluid path arm and the outlet fluid path arm, and provides a high hydrodynamic resistance, low electrical resistance connection between a high voltage electrode positioned within the first electrode reservoir and a fluid contained within the inlet fluid path arm and the outlet fluid path arm. 
     
     
         15 . The method of  claim 14 , wherein the first membrane is hydrophilic and comprises cellulose or polytetrafluoroethylene (PTFE). 
     
     
         16 . A microfluidic device comprising a planar substrate, wherein the planar substrate comprises:
 a plurality of fluid inlets, wherein all or a portion of the plurality of fluid inlets are located on one or more edges of the planar substrate; and   a plurality of separation channels, each separation channel comprising:
 i. a first end that is electrically coupled to an anolyte reservoir via a first membrane-containing high voltage electrode fixture; 
 ii. a second end that is electrically coupled to a catholyte reservoir via a second membrane-containing high voltage electrode fixture;
 wherein the first or the second end of each separation channel of the plurality of separation channels is in fluidic communication with a different fluid inlet of the plurality of fluid inlets. 
 
   
     
     
         17 . The microfluidic device of  claim 16 , wherein the plurality of separation channels is configured for UV absorbance imaging or fluorescence imaging of all or a portion of the plurality of separation channels. 
     
     
         18 . The microfluidic device of  claim 16 , further comprising a cartridge comprising the first and second membrane-containing high voltage electrode fixtures, wherein the cartridge encompasses all or a portion of the planar substrate. 
     
     
         19 . The microfluidic device of  claim 16 , wherein the cartridge is a disposable component of a system configured to perform multiplexed isoelectric focusing reactions.