Multiplexed capillary electrophoresis system

• US 5,582,705 A
• Filed: 05/19/1995
• Issued: 12/10/1996
• Est. Priority Date: 05/19/1995
• Status: Expired due to Term

• Current Assignee: Iowa State University Research Foundation Incorporated (Iowa State University)
• Original Assignee: Iowa State University Research Foundation Incorporated (Iowa State University)
• Inventors: Li, Qingbo, Yeung, Edward S., Lu, Xiandan, Chang, Huan-Tsang, Fung, Eliza N.
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/444,565
• Time in Patent Office: 571 Days
• Field of Search: 204/603, 204/452, 204/601, 204/602, 204/604, 204/605, 204/451, 204/453, 204/454, 204/455, 356/344
• US Class Current: 204/603
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44782 of a plurality of samples

Abstract

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Claims

1. A multiplexed capillary electrophoresis system comprising;

(a) a capillary array of coplanar parallel capillaries, each capillary having an interior portion for placement of a target species, and an annular wall with a first transparent portion defining a transparent path extending through the capillary array perpendicular to the capillaries;

(b) a coherent light source positioned to direct a beam of coherent light along the transparent path and through the interior portion of each capillary to induce emission from the target species;

(c) a filter positioned to split the target species emission into first and second emission channels; and

(d) an image array detector positioned to detect the target species emission.

2. The system of claim 1 wherein the first transparent portion extends around the capillary.

3. The system of claim 1 wherein the transparent path comprises a plane extending through the capillaries.

4. The system of claim 1 comprising a capillary array of at least about 100 coplanar parallel capillaries.

5. The system of claim 1 wherein the first transparent portion is transparent to light having a wavelength about equal to the wavelength of the beam of coherent light.

6. The system of claim 1 wherein the first transparent portion is transparent to light having a wavelength of about 200-1500 nm.

7. The system of claim 1 wherein the parallel capillaries are substantially adjacent to each other.

8. The system of claim 1 wherein the first transparent portion is surrounded by a medium having a refractive index of about 1.3-1.5.

9. The system of claim 1 wherein the capillary array is immersed in water.

10. The system of claim 1 wherein a collimating focusing lens is interposed between the coherent light source and the capillary array.

11. The system of claim 1 wherein the beam of coherent light has a diameter of less than about 300 μ

m in the capillaries.

12. The system of claim 11 wherein the beam of coherent light has a diameter of less than about 75 μ

m in the capillaries.

13. The system of claim 1 wherein a beam expander is used to expand the beam of coherent light perpendicular to the capillary array.

14. The system of claim 1 wherein the coherent light source comprises a laser having a power output of about 0.5-50 mW.

15. The system of claim 1 wherein the first transparent portion exhibits substantially no fluorescence when exposed to the beam of coherent light.

16. The system of claim 1 wherein each annular wall has a second transparent portion for optically coupling the transparent path to a location external to the capillary array.

17. The system of claim 16 wherein the location external to the capillary array comprises a planar surface parallel to the capillary array.

18. The system of claim 16 wherein the second transparent portion of each annular wall is contiguous with the first transparent portion of the annular wall.

19. The system of claim 16 wherein the location external to the capillary array contains an optical detector.

20. The system of claim 19 wherein the optical detector is a two-dimensional image array detector.

21. The system of claim 20 wherein the optical detector is selected from a group consisting of a charge-coupled device (CCD) and a charge-injection device (CID).

22. The system of claim 19 wherein at least one capillary is in fluid communication with a sample containing a fluorescent target species so that the sample is drawn into the capillary, and wherein the optical detector is capable of detecting fluorescence emission from the target species.

23. The system of claim 1 further comprising at least one optical fiber optically coupled to the transparent path.

24. A capillary electrophoresis system comprising:

a capillary array having a plurality of coplanar parallel capillaries, each capillary having an annular wall defining an interior portion, each annular wall having a transparent portion for optically coupling the interior portion to an image array detector;

(b) a coherent light source positioned to direct a single beam of coherent light into the interior portion of each capillary;

(c) an image array detector having linearly aligned pixels located in a plane parallel to the capillary array such that at least one of the capillaries is optically coupled to a plurality of pixels; and

a filter interposed between the image array detector and the capillary array.

25. The system of claim 24 wherein at least one capillary has at least one side wall proximate to the interior portion such that at least one pixel is optically coupled to the at least one side wall.

26. The system of claim 24 wherein the at least one capillary has first and second side walls on opposite sides of the interior portion and the less than about six pixels comprises a group of pixels having a leading pixel, a middle group of pixels, and a trailing pixel, such that the leading pixel is optically coupled to the first side wall, the middle group of pixels is optically coupled to the interior portion, and the trailing pixel is optically coupled to the second side wall.

27. The system of claim 26 wherein the middle group of pixels comprises two pixels.

28. The system of claim 26 wherein the middle group of pixels comprises one pixel.

29. The system of claim 24 further comprising an imaging lens for optically coupling the at least one capillary to the at least about six pixels.

30. The system of claim 24 wherein the image array detector is a two-dimensional image array detector.

31. The system of claim 30 wherein the two-dimensional image array detector is selected from a group consisting of a charge-coupled device (CCD) and a charge-injection device (CID).

32. The system of claim 30 wherein the two-dimensional image array detector is a charge-injection device (CID).

33. The system of claim 24 wherein the parallel capillaries are substantially adjacent to each other.

34. The system of claim 24 wherein when a target species is placed in the interior portion of each capillary and emission is induced by the coherent light, the filter is positioned to filter only a portion of the induced emission.

35. A capillary electrophoresis system comprising:

(a) at least one capillary having an annular wall defining an interior portion for placement of a fluorescent target species wherein said annular wall has a transparent portion for optically coupling the interior portion to a detector;

(b) a coherent light sourer positioned to direct a single beam of coherent light having a wavelength of about 200-1500 nm so as to contact the interior portion and induce fluorescence emission from the target species;

(c) first and second long-pass filters positioned to split the fluorescence emission into first and second emission channels, respectively; and

(d) a detector for simultaneously detecting the fluorescence emission in the first and second emission channels.

36. The system of claim 35 wherein the transparent portion extends around the capillary.

37. The system of claim 36 wherein the first long-pass filter is interposed between the detector and the transparent portion of an annular wall such that fluorescence emission passes through the first filter, and the second long-pass filter is interposed between the first filter and the transparent portion of the annular wall at an angle of about 1°

-89°

relative to the first filter, such that a portion of the fluorescence emission passes through the second filter before passing through the first filter.

38. The system of claim 37 wherein the angle is about 20°

-40°

.

39. The system of claim 37 wherein the portion of fluorescence passing through both the first filter and the second filter is about 25%-75% of the fluorescence emission passing through the first filter.

40. The system of claim 36 wherein the second long-pass filter is interposed between the detector and the transparent portion of an annular wall such that fluorescence emission passes through the second filter, and the first long-pass filter is interposed between the second filter and the transparent portion of the annular wall at an angle of between about 1°

-89°

relative to the second filter, such that a portion of the fluorescence emission passes through the first filter before passing through the second filter.

41. The system of claim 35 wherein at least one capillary is in fluid communication with a sample containing a fluorescent target species such that the sample is drawn into the capillary and brought into contact with the beam of coherent light.

42. The system of claim 35 wherein the at least one capillary comprises a capillary array of coplanar parallel capillaries, the annular wall of each capillary having a first transparent portion defining a transparent path extending through the capillary array perpendicular to the capillaries, and wherein the coherent light source comprises a coherent light source positioned to direct the single beam of coherent light along the transparent path.

43. The system of claim 42 wherein each annular wall has a second transparent portion for optically coupling the transparent path to the detector.

44. The system of claim 43 wherein the detector comprises a first linear array detector for detecting fluorescence emission in the first emission channel and a second linear array detector for detecting fluorescence emission in the second emission channel.

45. The system of claim 43 wherein the detector comprises a two dimensional image array detector.

46. The system of claim 45 wherein the detector is selected from a group consisting of a charge-coupled device (CCD) and a charge-injection device (CID).

47. The system of claim 35 wherein the first long-pass filter has a wavelength cutoff value such that it transmits less than about 0.1% of light having a wavelength about equal to the wavelength of the beam of coherent light, and wherein the second long-pass filter has a wavelength cutoff value higher than the wavelength cutoff value of the first long-pass filter.

48. The system of claim 35 wherein the first long-pass filter comprises a Raman long-pass filter having a wavelength cutoff value about equal to the wavelength of the beam of coherent light.

49. The system of claim 35 wherein the single beam of coherent light has a wavelength of about 488 nm, and wherein the first long-pass filter is a Raman long-pass filter having a wavelength cutoff value of 488 nm and the second long-pass filter is a standard long-pass filter having a wavelength cutoff value higher than the wavelength cutoff value of the first long-pass filter.