Means and method for capillary zone electrophoresis with laser-induced indirect fluorescence detection

Abstract

A means and method for capillary zone electrphoresis with laser-induced indirect fluorescence detection. A detector is positioned on the capillary tube of a capillary zone electrophoresis system. The detector includes a laser which generates a laser beam which is imposed upon a small portion of the capillary tube. Fluorescence of the elutant electromigrating through the capillary tube is indirectly detected and recorded.

What is claimed is: 
     
       1. A means for capillary zone electrophoresis with laser-induced indirect fluorescence detection comprising: a capillary zone electrophoresis system including a capillary tube means having an anode and a cathode end, power means for imposing an electrical field upon the capillary tube means, .[.specimen.]. .Iadd.sample .Iaddend.means for supplying .[.specimen.]. .Iadd.sample .Iaddend.to the capillary tube means, buffer means for supplying buffer to the capillary tube means;   an indirect fluorescence detection system including a laser means for directing a laser beam upon a selected portion of the capillary tube means, a detector means for detecting fluorescence indirectly from the selected portion of the capillary tube means, the buffer containing as a principal component a fluorescing portion which fluoresces upon imposition of the laser beam and which allows indirect fluorescence detection by causing one of the set of displacement and ion-pairing of the sample and the .[.electrophoresis.]. .Iadd.fluorescing .Iaddend.portion of the buffer without labeling the sample with .[.an electrophoretic.]. .Iadd.a fluorescing .Iaddend.portion, the detector measuring a signal independent of the spectral properties of the sample but related to fluorescing changes caused by the displacement or ion-pairing which indirectly allows sensitive detection of constituent components of the .[.specimen.]. .Iadd.sample .Iaddend.during the electrophoresis process; and   an interlock and timing means associated with the power means for controlling the periods of time the electrical field is created.   
     
     
       2. The means of .[.claim 22.]. .Iadd.claim 1 .Iaddend.wherein the interlock and timing means includes safety means for disconnecting electrical power from the system. 
     
     
       3. A means for capillary zone electrophoresis with laser-induced indirect fluorescence detection comprising: a capillary zone electrophoresis system including a capillary tube means having an anode and a cathode end, power means for imposing an electrical field upon the capillary tube means, .[.specimen.]. .Iadd.sample .Iaddend.means for supplying .[.specimen.].  .Iadd.sample .Iaddend.to the capillary tube means, buffer means for supplying buffer to the capillary tube means;   an indirect fluorescence detection system including a laser means for directing a laser beam upon a selected portion of the capillary tube means, a detector means for detecting fluorescence indirectly from the selected portion of the capillary tube means, the buffer containing as a principal component a fluorescing portion which fluoresces upon imposition of the laser beam and which allows indirect fluorescence detection by causing one of the set of displacement and ion-pairing of the sample and the .[.electrophoresis.]. .Iadd.fluorescing .Iaddend.portion of the buffer without labeling the sample with .[.an electrophoretic.]. .Iadd.a fluorescing .Iaddend.portion, the detector measuring a signal independent of the spectral properties of the sample but related to fluorescing changes caused by the displacement or ion-pairing which indirectly allows sensitive detection of constituent components of the .[.specimen.]. .Iadd.sample .Iaddend.during the electrophoresis process; and   a shunting resistor means electrically connected between the power means and the anode end of a capillary tube means.   
     
     
       4. A means for capillary zone electrophoresis with laser induced indirect fluorescence detection comprising: a capillary zone electrophoresis system including a capillary tube means having an anode and a cathode end, power means for imposing an electrical field upon the capillary tube means, .[.specimen.]. .Iadd.sample .Iaddend.means for supplying .[.specimen.]. .Iadd.sample .Iaddend.to the capillary tube means, buffer means for supplying buffer to the capillary tube means;   an indirect fluorescence detection system including a laser means for directing a laser beam upon a selected portion of the capillary tube means, a detector means for detecting fluorescence indirectly from the selected portion of the capillary tube means, the buffer containing as a principal component a fluorescing portion which fluoresces upon imposition of the laser beam and which allows indirect fluorescence detection by causing one of the set of displacement and ion-pairing of the sample and the .[.electrophoresis.]. .Iadd.fluorescing .Iaddend.portion of the buffer without labeling the sample with .[.an electrophoretic.]. .Iadd.a fluorescing .Iaddend.portion, the detector measuring a signal independent of the spectral properties of the sample but related to fluorescing changes caused by the displacement or ion-pairing which indirectly allows sensitive detection of constituent components of the .[.specimen.]. .Iadd.sample .Iaddend.during the electrophoresis process; and   a power stabilizing means positioned between the laser means and the capillary tube means for stabilizing the laser beam.   
     
     
       5. A means for capillary zone electrophoresis with laser induced indirect fluorescence detection comprising: a capillary zone electrophoresis system including a capillary tube means having an anode and cathode end, power means for imposing an electrical field upon the capillary tube means, .[.specimen.]. .Iadd.sample .Iaddend.means for supplying .[.specimen.]. .Iadd.sample .Iaddend.to the capillary tube means, buffer means for supplying buffer to the capillary tube means;   an indirect fluorescence detection system including a laser means for directing a laser beam upon a selected portion of the capillary tube means, a detector means for detecting fluorescence indirectly from the selected portion of the capillary tube means, the buffer containing as a principal component a fluorescing portion which fluoresces upon imposition of the laser beam and which allows indirect fluorescence detection by one of the set displacement and ion-pairing of the sample and the .[.electrophoresis.]. .Iadd.fluorescing .Iaddend.portion of the buffer without labeling the sample with .[.an electrophoretic.]. .Iadd.a fluorescing .Iaddend.portion, the detector measuring a signal independent of the spectral properties of the sample but related to fluorescing changes caused by the displacement or ion-pairing which indirectly allows sensitive detection of constituent components of the .[.specimen.]. .Iadd.sample .Iaddend.during the electrophoresis process; and   an imaging means between the capillary tube means and the detector means for collecting an imaging fluorescence upon the detector means emanating from capillary tube means where the laser means has been directed.   
     
     
       6. A means for capillary zone electrophoresis with laser induced indirect fluorescence detection comprising: a capillary zone electrophoresis system including a capillary tube means having an anode and a cathode end, power means for imposing an electrical field upon the capillary tube means, .[.specimen.]. .Iadd.sample .Iaddend.means for supplying .[.specimen.]. .Iadd.sample .Iaddend.to the capillary tube means, buffer means for supplying buffer to the capillary tube means;   an indirect fluorescence detection system including a laser means for directing a laser beam upon a selected portion of the capillary tube means, a detector means for detecting fluorescence indirectly from the selected portion of the capillary tube means, the buffer containing as a principal component a fluorescing portion which fluoresces upon imposition of the laser beam and which allows indirect fluorescence detection by causing one of the set of displacement and ion-pairing of the sample and the .[.electrophoresis.]. .Iadd.fluorescing .Iaddend.portion of the buffer without labeling the sample with .[.an electrophoretic.]. .Iadd.a fluorescing .Iaddend.portion, the detector measuring a signal independent of the spectral properties of the sample but related to fluorescing changes caused by the displacement or ion-pairing which directly allows sensitive detection of constituent components of the .[.specimen.]. .Iadd.sample .Iaddend.during the electrophoresis process; and   a filtering means in position between the capillary tube means and the detector means, the filtering means including a spatial filter.   
     
     
       7. A means for capillary zone electrophoresis with laser induced indirect fluorescence detection comprising: A capillary zone electrophoresis system including a capillary tube means having an anode and a cathode end, power means for imposing an electrical field upon the capillary tube means, .[.specimen.]. .Iadd.sample .Iaddend.means for supplying .[.specimen.]. .Iadd.sample .Iaddend.to the capillary tube means, buffer means for supplying buffer to the capillary tube means;   an indirect fluorescence detection system including a laser means for directing a laser beam upon a selected portion of the capillary tube means, a detector means for detecting fluorescence indirectly from the selected portion of the capillary tube means, the buffer containing as a principal component a fluorescing portion which fluoresces upon imposition of the laser beam and which allows indirect fluorescence detection by causing one of the set of displacement and ion-pairing of the sample and the .[.electrophoresis.]. .Iadd.fluorescing .Iaddend.portion of the buffer without labeling the sample with .[.an electrophoretic.]. .Iadd.a fluorescing .Iaddend.portion, the detector measuring a signal independent of the spectral properties of the sample but related to fluorescing changes caused by the displacement or ion-pairing which indirectly allows sensitive detection of constituent components of the .[.specimen.]. .Iadd.sample .Iaddend.during the electrophoresis process; and   a filtering means in position between the capillary tube means and the detector means, the filtering means including an interference filter for selecting desired fluorescence wavelengths to be passed to detector means.   
     
     
       8. A method of improving the sensitivity of separation and detection in capillary zone electrophoresis with laser-induced indirect fluorescence detection comprising the steps of: mixing an electrophoretic solution with a fluorescent buffer solution to present a mixture;   presenting a mixture to a capillary tube of a capillary zone electrophoresis system;   directing a laser beam to a portion of the capillary tube, the laser beam being stabilized before being directed to a portion of the capillary tube;   indirectly detecting the fluorescence from the mixture passing through portions of the capillary tube upon which the laser beam is imposed by measuring a signal independent of the spectral properties of the mixture but related to the change in fluorescence of the mixture relating to displacement or ion pairing of the electrophoretic solution and the fluorescent buffer solution; and   detecting with high sensitivity, the constituent components of the electrophoretic solution by utilizing the indirect fluorescing measurements and without labeling the electrophoretic solution with a fluorescing portion.   
     
     
       9. A method of improving the sensitivity of separation and detection in capillary zone electrophoresis with laser-induced indirect fluorescence detection comprising the steps of: mixing an electrophoretic solution with a fluorescent buffer solution to present a mixture;   presenting a mixture to a capillary tube of a capillary zone electrophoresis system;   directing a laser beam to a portion of the capillary tube, the laser beam being stabilized before being directed to a portion of the capillary tube;   indirectly detecting the fluorescence from the mixture passing through portions of the capillary tube upon which the laser beam is imposed by measuring a signal independent of the spectral properties of the mixture but related to the change in fluorescence of the mixture relating to displacement or ion pairing of the electrophoretic solution and the fluorescent buffer solution;   detecting with high sensitivity, the constituent components of the electrophoretic solution by utilizing the indirect fluorescing measurements and without labeling the electrophoretic solution with a fluorescing portion; and   the laser-induced fluorescence from the mixture being filtered spatially before detection.   
     
     
       10. A method of improving the sensitivity of separation and detection in capillary zone electrophoresis with laser-induced indirect fluorescence detection comprising the steps of: mixing an electrophoretic solution with a fluorescent buffer solution to present a mixture;   presenting a mixture to a capillary tube of a capillary zone electrophoresis system;   directing a laser beam to a portion of the capillary tube, the laser beam being stabilized before being directed to a portion of the capillary tube;   indirectly detecting the fluorescence from the mixture passing through portions of the capillary tube upon which the laser beam is imposed by measuring a signal independent of the spectral properties of the mixture but related to the change in fluorescence of the mixture relating to displacement or ion pairing of the electrophoretic solution and the fluorescent buffer solution;   detecting with high sensitivity, the constituent components of the electrophoretic solution by utilizing the indirect fluorescing measurements and without labeling the electrophoretic solution with a fluorescing portion; and   the laser-induced fluorescence of the mixture being interface filtered before detection. .Iadd.11. A method of capillary zone electrophoresis comprising the steps of:   (a) providing a capillary tube with a selected portion through which fluorescence can be detected;   (b) providing a sample of analyte ions in solution;   (c) providing a buffer solution with a selected concentration of fluorescing ions;   (d) applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) supplying the sample to the capillary tube filled with the buffer solution;   (f) using a concentration of the fluorescing ions in the buffer at a level which generates detectable levels of displacement or ion-pairing of the analyte ions and the fluorescing ions, but which is diluted to increase the efficiency of detection;   (g) irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions; and   (h) detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution. .Iaddend. .Iadd.12. The method of claim 11 wherein the sample contains analyte ions that are naturally non-fluorescing. .Iaddend. .Iadd.13. The method of claim 11 wherein the concentration of the fluorescing ions in the buffer is substantially less than 400 μM. .Iaddend. .Iadd.14. The method of claim 11 wherein the concentration of the fluorescing ions in the buffer is approximately 50 μM. .Iaddend. .Iadd.15. A method of capillary zone electrophoresis comprising the steps of:   (a) providing a capillary tube with a selected portion through which fluorescence can be detected;   (b) providing a sample of analyte ions in solution;   (c) providing a buffer solution with a selected concentration of fluorescing ions;   (d) applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) supplying the sample to the capillary tube filled with the buffer solution, wherein the ions interact with the wall of the capillary tube to produce background noise;   (f) irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions;   (g) detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution; and   (h) prior to steps (d) through (g), silating the capillary tube for the purpose of reducing interaction of ions with the capillary tube wall, thereby decreasing the background noise, peak-broadening and long term drift of the separation process. .Iaddend. .Iadd.16. A method of capillary zone electrophoresis comprising the steps of:   (a) providing a capillary tube with a selected portion through which fluorescence can be detected;   (b) providing a sample of analyte ions in solution;   (c) providing a buffer solution with a selected concentration of fluorescing ions;   (d) applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) supplying the sample to the capillary tube filled with the buffer solution;   (f) using a concentration of the fluorescing ions in the buffer which is substantially lower than 400 μM to increase the efficiency of detection;   (g) irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions; and   (h) detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution. .Iaddend.   
     
     
        .Iadd.17.  A system for capillary zone electrophoresis comprising: (a) a capillary tube with a selected portion through which fluorescence can be detected;   (b) a sample of analyte ions in solution;   (c) a buffer solution with fluorescing ions at a concentration which generates detectable levels of displacement or ion-pairing of the analyte ions and the fluorescing ions, but which fluorescing ion concentration is diluted to increase the efficiency of detection;   (d) means for applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) means for supplying the sample to the capillary tube filled with the buffer solution;   (f) laser means for irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions; and   (g) means for detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution. .Iaddend. .Iadd.18. The system of claim 17 wherein the concentration of the fluorescing ions in the buffer is substantially less than 400 μM. .Iaddend. .Iadd.19. The system of claim 17 wherein the concentration of the fluorescing ions in the buffer is approximately 50 μM. .Iadd.20. A system for capillary zone electrophoresis comprising:   (a) a capillary tube with a selected portion through which fluorescence can be detected;   (b) a sample of analyte ions in solution;   (c) a buffer solution with fluorescing ions wherein the concentration of the fluorescing ions used is substantially less than 400 μM, to increase the efficiency of detection;   (d) means for applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) means for supplying the sample to the capillary tube filled with the buffer solution;   (f) laser means for irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions; and   (g) means for detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution. .Iaddend. .Iadd.21. A method of capillary zone electrophoresis for detecting a biochemical substance comprising the steps of:   (a) providing a capillary tube with a selected portion through which fluorescence can be detected;   (b) providing a sample comprising biochemical analyte ions in solution;   (c) providing a buffer solution with a selected concentration of fluorescing ions;   (d) applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) supplying the sample to the capillary tube filled with the buffer solution;   (f) irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions; and   (g) detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution. .Iaddend.   
     
     
        .Iadd.22.  The method of claim 21 wherein the concentration of the fluorescing ions in the buffer used is at a level which generates detectable levels of displacement or ion-pairing of the analyte ions and the fluorescing ions, but which is minimized to increase the efficiency of detection. .Iaddend. .Iadd.23. The method of claim 21 wherein the biochemical substance is selected from a group consisting of nucleic acids, peptides, nucleotides, fatty acids, sugars, glycolytic intermediates, as well as combinations and compounds thereof. .Iaddend. .Iadd.24. The method of claim 21 wherein the biochemical substance is 
     
     
        naturally non-fluorescing. .Iaddend. .Iadd.25.  A system for detecting a biochemical substance using capillary zone electrophoresis comprising: (a) a capillary tube with a selected portion through which fluorescence can be detected;   (b) a sample comprising biochemical analyte ions in solution;   (c) a buffer solution with a selected concentration of fluorescing ions;   (d) means for applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) means for supplying the sample to the capillary tube filled with the buffer solution;   (f) laser means for irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions; and   (g) means for detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution. .Iaddend. .Iadd.26. The system of claim 25 wherein the concentration of the fluorescing ions in the buffer is used at a level which generates detectable levels of displacement or ion-pairing of the analyte ions and the fluorescing ions, but which is minimized to increase the efficiency of detection. .Iaddend. .Iadd.27. The system of claim 25 wherein the concentration of the fluorescing ions in the buffer is used at a level which is substantially lower than 400 μM to increase the efficiency of detection. .Iaddend. .Iadd.28. A method of capillary zone electrophoresis for detecting a cell comprising the steps of:   (a) providing a capillary tube with a selected portion through which fluorescence can be detected;   (b) providing a sample comprising analyte ions of the cell in solution;   (c) providing a buffer solution with a selected concentration of fluorescing ions;   (d) applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) supplying the sample to the capillary tube filled with the buffer solution;   (f) irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions; and   (g) detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution. .Iaddend.   
     
     
        .Iadd.29.  The method of claim 28, wherein the concentration of the fluorescing ions in the buffer is used at a level which generates detectable levels of displacement or ion-pairing of the analyte ions and the fluorescing ions, but which is diluted to increase the efficiency of detection. .Iadd.30. The method of claim 28, wherein the cells are analyzed in vivo. .Iaddend. .Iadd.31. A method of capillary zone electrophoresis for detecting a biotechnical material selected from the group consisting of bacterium and virus, the method comprising the steps of: (a) providing a capillary tube with a selected portion through which fluorescence can be detected;   (b) providing a sample comprising analyte ions of the biotechnical material in solution;   (c) providing a buffer solution with a selected concentration of fluorescing ions;   (d) applying a high voltage through the capillary tube to move the fluorescing ions of said buffer solution through said capillary tube;   (e) supplying the sample to the capillary tube filled with the buffer solution;   (f) irradiating the fluorescing ions in the capillary tube selected portion with a coherent radiation of a wavelength effective to excite fluorescence in the fluorescing ions; and   (g) detecting a change in the fluorescence emitted through the selected portion of the capillary tube, caused by displacement or ion-pairing of the analyte and the fluorescing ions in the buffer solution. .Iaddend.   
     
     
        .Iadd.32.  The method of claim 31 wherein the concentration of the fluorescing ions in the buffer is used at a level which generates detectable levels of displacement or ion-pairing of the analyte ions and the fluorescing ions, but which is minimized to increase the efficiency of detection. .Iaddend.