Method and apparatus for precise selection and extraction of a focused component in isoelectric focusing performed in micro-channels

Abstract

An apparatus and method are disclosed for the precise selection and extraction of a selected analyte in a focused zone produced by isoelectric focusing performed in micro-channels. A cross-channel microfluidic device comprises a sample mixture introduction and separation channel and an extraction channel, which are in fluid communication with each other at a point of intersection. Means are provided for selectively moving the pattern of separated zones following cIEF to the intersection point, and means are provided for applying an extraction pressure to direct a single zone containing a selected analyte into and then out of the extraction channel for collection.

1 . Apparatus for selective extraction of an analyte from a mixture of analytes using capillary isolelectric focusing comprising: 
 a capillary separation channel filled with a medium containing a mixture of ionic components in which target analytes are separated into zones in stationary or near stationary equilibrium;    a capillary extraction channel intersecting with and angularly displaced from said capillary separation channel and in fluid communication therewith at the location of intersection;    means for causing selected zones of analytes separated by capillary isoelectric focusing to move to said intersection of the separation and extraction channels in a time-independent manner;    optical whole column imaging detection apparatus for monitoring the isoelectric focusing process and observing the positions of the separated zones of analyte; and    means for applying an extraction force to direct a single zone containing a selected analyte into and then out of the extraction channel in a time-independent manner.    
   
   
       2 . Apparatus according to  claim 1 , wherein said intersecting separation channel and said extraction channel are coplanar and are formed in a monolithic microfluidic device or microchip as elongate separation and extraction channels.  
   
   
       3 . Apparatus according to  claim 2 , wherein said separation channel is made of a UV-transparent plastic.  
   
   
       4 . Apparatus according to  claim 3 , wherein said UV transparent plastic is selected from the group consisting of polycarbonate, polyfluorinated polyethylene and polyethylmethacrylate.  
   
   
       5 . Apparatus according to  claim 1 , wherein said intersecting separation channel and said extraction channel intersect at right angles and are formed of four pieces of silica, glass or quartz tubing coated with linear or cross-linked polyacrylamide.  
   
   
       6 . Apparatus according to  claim 5 , wherein said means for causing selected zones or analytes to move to said intersection of the separation and extraction channels in a time-independent manner is operable to tilt said separation channel from a horizontal orientation to allow gravitational force to move said zones along the channel.  
   
   
       7 . Apparatus according to  claim 5 , wherein said means for applying an extraction force is operable to raise or lower slightly one end of the extraction channel relative to the other end, thereby causing the zone to move into the extraction channel under the microforce of gravity.  
   
   
       8 . Apparatus according to  claim 1 , wherein said means for selectively causing separated zones to move to the intersection of the separation and extraction channels and said means for applying an extraction pressure each comprises a microfluidic delivering system.  
   
   
       9 . Apparatus according to  claim 6 , wherein said means for applying an extraction pressure comprises a microfluidic delivering system.  
   
   
       10 . Apparatus for the precise selection and extraction of an analyte from a mixture of ionic analytes, separated by capillary isoelectric focusing comprising: 
 a capillary separation channel having a sample inlet end and an outlet end and having at each end an electrolyte holder in fluid communication with the separation channel through a semi-permeable membrane;    a capillary extraction channel angularly displaced from said capillary separation channel and in fluid communication therewith at a location of intersection and having an extraction outlet end and an inlet end;    means for filling the capillary separation channel with a mixture of ionic analytes and for causing different analytes to separate and focus into stationary or near stationary zones by capillary isoelectric focusing electrophoresis;    means for causing a selected analyte zone of the analyte zones separated by capillary isoelectric focusing to move to said intersection of the separation and extraction channels in a time-independent manner;    an optical image detection apparatus including a light source for irradiation of the whole or at least a large portion of the separation channel with ultraviolet (UV) light or other light and capable of acquiring real-time, or at least very rapid, digital images of ultraviolet absorption or fluorescence detection of focused analyte zones simultaneously in the whole, or at least a large portion, of the separation channel; and    means for applying an extraction force to direct the selected analyte zone or a portion thereof into and then out of the extraction channel for collection or interface to a second analytical apparatus.    
   
   
       11 . A method for fractionating and extracting analytes capable of resolution by capillary isoelectric focusing comprising the steps of: 
 providing a capillary separation channel and a capillary extraction channel in fluid communication therewith;    introducing an analyte sample containing a mixture of ionic analytes prepared for electrophoresis into the separation channel;    separating and focusing components of the analyte mixture into discrete zones in the separation channel using capillary isoelectric focusing;    monitoring the process of capillary isoelectric focusing and the positions of the separated analyte zones using whole column imaging detection;    causing the separated zones to move selectively to the intersection of the separation and extraction channels; and    applying an extraction force to direct a zone containing desired analytes into and then out through the extraction channel.    
   
   
       12 . A method according to  claim 11 , wherein the separated zones are caused to move selectively to the intersection of the separation and extraction channels by orientation of the separation channel so that gravity causes the analyte sample to move along the separation channel.  
   
   
       13 . A method according to  claim 11 , wherein microfluidic delivery is used to move the separated zones to the intersection of the separation and extraction channels.  
   
   
       14 . A method according to  claim 11 , wherein said extraction zone is applied using a microfluidic delivery pump.  
   
   
       15 . A method according to  claim 11 , wherein said analytes comprise a mixture of proteins.  
   
   
       16 . Method for precise selection and extraction of an analyte from a mixture of ionic analytes separated by capillary isoelectric focusing comprising the steps of: 
 providing a capillary separation channel and a capillary extraction channel in fluid communication therewith;    filling to overflow the extraction channel with a matrix buffer such as water or a selected pH and ionic strength buffer and then sealing the ends of the extraction channel;    filling to overflow the separation channel with a sample containing a mixture of ionic analytes prepared for isoelectric focusing electrophoresis;    separating and focusing components of the analyte mixture into discrete zones in the separation channel using capillary isoelectric focusing;    monitoring in real-time the process of capillary isoelectric focusing and positions of the separated analyte zones until the focusing is complete and focused zones are stationary or near stationary, using whole separation channel imaging detection;    using real-time, whole separation channel imaging detection, selecting an analyte zone, causing said selected analyte zone to move and align with the intersection of the separation and extraction channels and detecting said alignment;    sealing the ends of the separation channel; and    unsealing the ends of the extraction channel and applying an extraction force to direct the selected analyte zone or a portion thereof into and then out of the extraction channel for collection or interface to a second analytical apparatus.