System and Method For the Separation of Analytes

Abstract

A separation module operates to fractionate or separate an analyte into fractions according to pI, i.e., pI bands, utilizing capillary isoelectric focusing (“CIEF”) within a first microchannel. The fractions are stacked to form plugs, the number of which is determined by a number of parallel second microchannels integrally connected to the first microchannel, into which the fractions are directed according to the buffer characteristics found in each of the individual microchannels. Within the microchannels the plugs are separated into proteins according to a different chemical property, i.e., “m/z,” utilizing capillary electrophoresis (“CE”).

1 . A system for separating a sample comprising:
 an analyte sampling source;   at least one microcapillary tube coupled to the analyte sampling source for introducing the sample from the analyte sampling source to a module, the module containing multiple microchannels;   a first microchannel of the multiple microchannels for receiving the sample from the at least one microcapillary tube at a first end thereof, the first microchannel having a reservoir containing an isoelectric focusing buffer having a first ionic strength at the second end thereof;   first and second electrodes that apply opposite charges to either end of the first microchannel to facilitate separating the sample into a plurality of sub-samples according to varying isoelectric points;   a plurality of second microchannels configured within the module perpendicular to a length of the first microchannel, each of the plurality of second microchannels have a first end coupled to the first microchannel at different points along the length of the first microchannel for receiving each of the plurality of sub-samples therein, each of the plurality of second microchannels having an electrophoresis buffer with an ionic strength therein, wherein the ionic strength of the electrophoresis buffer is different from the first ionic strength of the isoelectric focusing buffer; and   a third electrode that applies a charge to at least one of the plurality of second microchannels to separate a first of the plurality of sub-samples into a plurality of protein components.   
     
     
         2 . The system of  claim 1 , further comprising a translator that aligns a first detector with the first microchannel to detect the separation of the sample into the plurality of sub-samples, and aligns a second detector with one of the plurality of second microchannels. 
     
     
         3 . The system according to  claim 2 , wherein the first detector comprises UV-Visible (“UV-Vis”) and photodiode (“PDA”) arrays, wherein the module is located between the UV-Vis array and the PDA. 
     
     
         4 . The system according to  claim 3 , wherein the UV-Visible (“UV-Vis”) and photodiode (“PDA”) arrays continuously monitor the separation of the sample into the plurality of sub-samples. 
     
     
         5 . The system of  claim 4 , wherein the translator includes an X-Y translation stage for simultaneously translating the UV-Vis array and the PDA. 
     
     
         6 . The system of  claim 1 , wherein an amount of the sample received from the at least one microcapillary tube is between 1 and 500 nanoliters. 
     
     
         7 . The system of  claim 1 , wherein the analyte sampling source is selected from a group consisting of: automated sampling devices, subcutaneous microdialysis tubes, percutaneous sampling systems, air sampling stations, microinfusion pumps. 
     
     
         8 . The system of  claim 1 , wherein the module is formed from a material that is transparent to UV-Vis radiation. 
     
     
         9 . The system of  claim 8 , wherein the module is formed from a material selected from the group consisting of silicon and polydimethylsiloxane (“PDMS”). 
     
     
         10 . The system of  claim 2 , wherein the second detector is a mass spectrometer. 
     
     
         11 . A system for separating a sample comprising:
 a module formed of a material that is transparent to UV-Vis radiation, the module including a first microchannel and a plurality of second microchannels, wherein the sample is received by the first microchannel and the first microchannel is configured to separate the sample is separated into a plurality of sub-samples according to varying isoelectric points;   and further wherein the plurality of second microchannels are configured within the module perpendicular to a length of the first microchannel, each of the plurality of second microchannels have a first end coupled to the first microchannel at different points along the length of the first microchannel for receiving each of the plurality of sub-samples therein, each of the plurality of second microchannels being configured to separate each of the plurality of sub-samples into a plurality of protein components.   
     
     
         12 . The system of  claim 11 , further comprising a translator that aligns a first detector with the first microchannel to detect the separation of the sample into the plurality of sub-samples, and aligns a second detector with one of the plurality of second microchannels to detect the protein components. 
     
     
         13 . The system according to  claim 12 , wherein the first detector comprises UV-Visible (“UV-Vis”) and photodiode (“PDA”) arrays, wherein the module is located between the UV-Vis array and the PDA. 
     
     
         14 . The system according to  claim 13 , wherein the UV-Visible (“UV-Vis”) and photodiode (“PDA”) arrays continuously monitor the separation of the sample into the plurality of sub-samples. 
     
     
         15 . The system of  claim 14 , wherein the translator includes an X-Y translation stage for simultaneously translating the UV-Vis array and the PDA. 
     
     
         16 . The system of  claim 11 , wherein the module is formed from a material selected from the group consisting of silicon and polydimethylsiloxane (“PDMS”). 
     
     
         17 . The system of  claim 12 , wherein the second detector is a mass spectrometer.