Glycan profiling utilizing capillary electrophoresis

Abstract

A method for glycan profiling by capillary electrophoresis (CE), and a CE system for glycan analysis (N-Glycan). The CE system uses integrated dual optical fibers for both radiation excitation and emission detection. The CE system is configured for performing a two-color detection for data analysis. A single radiation excitation source is used to excite two emission fluorophores or dyes in the sample solution to be analyzed. One emission dye is to tag the sample and the other dye is used to provide a reference marker (e.g., a Dextran Ladder) for the sample run. Two detectors (e.g., photomultipler tubes (PMTs)) are applied to simultaneously detect the fluorescent emissions from the dyes. The data collected by both detectors are correlated (e.g., synchronized, and/or super-positioned for analysis) for accurate data peak identification.

We claim: 
     
         1 . A method of glycan profiling, comprising:
 providing a separation channel having a first longitudinal axis along which a glycan sample undergoes separation into sample components, and defining a detection zone defined along the separation channel through which sample components pass;   providing a radiation source;   providing a detector;   providing an incident light guide having a second longitudinal axis, directing incident radiation from the radiation source to the detection zone, causing radiation to be emitted by sample components as they pass through the detection zone; and   providing an emission light guide having a third longitudinal axis, collecting and directing emitted radiation from the detection zone to the detector,   wherein the emission light guide and the incident light guide are positioned on opposite sides of the separation channel.   
     
     
         2 . The method of  claim 1 , wherein prior to subjecting the sample to separation, the sample is provided with a sample marker corresponding to emission of a first characteristic wavelength, wherein the sample components are labeled by the sample marker for identification as the sample undergoes separation. 
     
     
         3 . The method of  claim 2 , wherein prior to subjecting the sample separation, the sample is further provided with a reference marker corresponding to a second characteristic wavelength, wherein the reference marker provides a reference to facilitate identification of the sample components as the sample undergoes separation. 
     
     
         4 . The method of  claim 3 , further comprising subjecting the sample to high voltage to effect electrophoresis to separate the sample into the sample components along the separation channel. 
     
     
         5 . The method of  claim 4 , wherein the separation channel is a capillary channel defined in a capillary column. 
     
     
         6 . The method of  claim 5 , wherein the sample marker comprises a sample fluorophore and the reference marker comprises a reference fluorophore, wherein the sample fluorophore and the reference fluorophore are simultaneously subject to the incident radiation as they pass through the detection zone, and wherein the incident radiation induces emitted radiations in the form of fluorescence emissions of the sample marker and the reference marker as the sample components pass through the detection zone. 
     
     
         7 . The method of  claim 6 , wherein the reference fluorophore provides a Ladder as a reference marker as the sample undergoes separation. 
     
     
         8 . The method of  claim 7 , wherein the sample is N-Glycan. 
     
     
         9 . The method of  claim 8 , wherein the emission light guide and the incident light guide are positioned on opposite sides of the separation channel, and wherein the first, second and third longitudinal axis are substantially coplanar at least at or near the detection zone. 
     
     
         10 . The method of  claim 9 , wherein at least one of the second and third longitudinal axis is not perpendicular to the first longitudinal axis. 
     
     
         11 . The method of  claim 10 , wherein the emission light guide and the incident light guide are positioned on opposite sides of the separation channel, wherein the incident light guide comprises a first optical fiber having a terminating integral ball-end structure, and the emission light guide comprises a second optical fiber having a terminating integral ball-end structure, and wherein the ball-end structures do not touch exterior of the separation channel. 
     
     
         12 . An electrophoresis system for profiling glycan, comprising:
 a separation channel having a first longitudinal axis along which a glycan sample undergoes separation into sample components, and a detection zone defined along the separation channel through which sample components pass;   a radiation source;   a detector;   an incident light guide having a second longitudinal axis, directing incident radiation from the radiation source to the detection zone, causing radiation to be emitted by sample components as they pass through the detection zone;   an emission light guide having a third longitudinal axis, collecting and directing emitted radiation from the detection zone to the detector, wherein the emission light guide and the incident light guide are positioned on opposite sides of the separation channel, and at least one of the incident light guide and emission light guide comprises an optical fiber; and   a power supply providing a high voltage across ends of the separation channel to effect electrophoresis separation, wherein separated sample components pass through the detection zone.   
     
     
         13 . The system of  claim 12 , wherein prior to subjecting the sample to separation, the sample is provided with a sample marker corresponding to emission of a first characteristic wavelength, wherein the sample components are labeled by the sample marker for identification as the sample undergoes separation, and the sample is further provided with a reference marker corresponding to a second characteristic wavelength, wherein the reference marker provides a reference to facilitate identification of the sample components as the sample undergoes separation. 
     
     
         14 . The system of  claim 13 , wherein the sample maker comprises a sample fluorophore and the reference marker comprises a reference fluorophore, wherein the sample fluorophore and the reference fluorophore are simultaneously subject to the incident radiation as they pass through the detection zone, and wherein the incident radiation induces emitted radiations in the form of fluorescence emissions of the sample marker and the reference marker as the sample components pass through the detection zone. 
     
     
         15 . The system of  claim 14 , wherein the sample is N-Glycan. 
     
     
         16 . The system of  claim 15 , wherein the separation channel is a capillary channel defined in a capillary column. 
     
     
         17 . The system of  claim 16 , wherein the reference fluorophore provides a Ladder as a reference marker as the sample undergoes separation. 
     
     
         18 . The system of  claim 16 , wherein the incident light guide comprises a first optical fiber having a terminating integral ball-end structure, and the emission light guide comprises a second optical fiber having a terminating integral ball-end structure, wherein the ball-end structures do not touch exterior of the separation channel. 
     
     
         19 . The system of  claim 18 , wherein the separation channel has a first longitudinal axis along which the glycan sample undergoes separation into sample components, the incident light guide has a second longitudinal axis, and the emission light guide has third longitudinal axis, and wherein at least one of the second and third longitudinal axis is not perpendicular to the first longitudinal axis. 
     
     
         20 . The detection system of  claim 13 , wherein the emission light guide and the incident light guide are positioned on opposite sides of the separation channel, and wherein the first, second and third longitudinal axis are substantially coplanar at least at or near the detection zone.