Methods, devices and systems for characterizing proteins

• US 6,475,364 B1
• Filed: 02/02/2000
• Issued: 11/05/2002
• Est. Priority Date: 02/02/1999
• Status: Expired due to Term

• Current Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Original Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Inventors: Bloxsom, Christopher, Parce, J. Wallace, Chow, Calvin Y. H., Dubrow, Robert S.
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US09/496,849
• Time in Patent Office: 1,007 Days
• Field of Search: 422/68.1, 422/99, 422/100, 204/514, 204/455, 204/601, 204/605
• US Class Current: 204/455
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

A method of characterizing a polypeptide, comprising providing a first capillary channel having a separation buffer disposed within, wherein the separation buffer comprises a non-crosslinked polymer solution, a buffering agent, a detergent, and a lipophilic dye. The separation buffer is provided such that, at the time of detection, the detergent concentration in the buffer is not above the critical micelle concentration. The polypeptide is introduced into one end of the capillary channel. An electric field is applied across a length of the capillary channel, which transports polypeptides of different sizes through the polymer solution at different rates. The polypeptide is then detected as it passes a point along the length of the capillary channel.

Claims

1. A method of performing an analytical operation on a fluid first sample material, comprising:

providing a microfluidic device that has a body having at least a first and a second channel disposed therein, the first channel comprising first and second channel segments, the first channel segment comprising a first fluid environment compatible with the performance of a first separation operation, the second channel segment comprising a detection zone for detecting the analytical operation wherein the second channel intersects the first channel at a first location substantially downstream along the first channel segment and immediately upsteam of the detection zone;

flowing the first sample material through the first channel segment to perform the first separation operation;

flowing the first sample material from the first channel segment into the second channel segment; and

introducing a first diluent consisting essentially of buffer into the first channel at the first location from the second channel, the diluent producing a second fluid environment within the second channel segment of the first channel, the second environment being more compatible than the first environment for detecting a result of the separation operation.

2. The method of claim 1, wherein the separation operation comprises an electrophoretic polypeptide separation, and the first fluid environment comprises a detergent concentration that is at or above a critical micelle concentration (CMC) for the detergent.

3. The method of claim 2, wherein the first fluid environment comprises the detergent at a concentration of greater than about 0.1%.

4. The method of claim 2, wherein detecting the analytical operation comprises detection of a lipophilic dye associated with polypeptides separated in the first operation, and the second fluid environment comprises die detergent at a concentration that is below the CMC for the detergent.

5. The method of claim 4, wherein the second fluid environment comprises the detergent at a concentration of less than 0.1%.

6. The method of claim 4, wherein the second fluid environment comprises the detergent at a concentration of about 0.05%.

7. The method of claim 2, wherein the providing step further comprises providing a separation buffer in at least the first channel segment, the separation buffer comprising a polymer matrix, a buffering agent, a first detergent and a lipophilic dye.

8. The method of claim 7, wherein the polymer matrix comprises a no-crosslinked polymer solution.

9. The method of claim 8, wherein the non-crosslinked polymer solution comprises a linear dimethylacrylamide polymer solution.

10. The method of claim 9, wherein the linear polyacrylamide polymer is present in the separation buffer at a concentration of between about 0.1 and about 20% (w/v).

11. The method of claim 7, wherein the first detergent comprises an alkylsulfonate detergent.

12. The method of claim 7, wherein the first detergent is selected from sodium octadecylsulfate, sodium decylsulfate and sodium dodecyl sulfate (SDS).

13. The method of claim 7, wherein the first detergent comprises sodium dodecyl sulfate (SDS).

14. The method of claim 7, wherein the first detergent is present in the separation buffer in the first channel segment at a concentration greater than about 0.03%.

15. The method of claim 7, wherein the diluent comprises the buffering agent with no detergent.

16. The method of claim 7, wherein the buffering agent comprises Tris-Tricine.

17. The method of claim 7, wherein the buffering agent is present in the separation buffer in the first channel segment at a concentration of between about 10 mM and about 100 mM.

18. The method of claims 7, wherein the lipophilic dye is a fluorescent lipophilic dye.

19. The method of claim 7, wherein the lipophilic dye is present in the separation buffer in the first channel segment at a concentration of from about 0.1 μ

M to about 20 μ

M.

20. A device for separating polypeptides, comprising:

a body structure having at least a first capillary channel disposed therein;

a separation buffer disposed in the first capillary channel, the separation buffer comprising;

a non-crosslinked polymer solution;

a buffering agent;

a first detergent; and

a lipophilic dye capable of binding to the polypeptide or polypeptides; and

a first port disposed in the body structure in fluid communication with the first capillary channel, for introducing polypeptides into the first capillary channel.

21. The device of claim 20, wherein the first port comprises a sample fluid disposed therein, the sample fluid comprising at least a first polypeptide of interest, and a second detergent at a concentration that is between about 0.05×

and 3×

a concentration of the first detergent in the separation buffer.

22. The device of claim 20, wherein the first port comprises a sample fluid disposed therein, the sample fluid comprising at least a first polypeptide of interest, and a second detergent at a concentration that is less than a concentration of the first detergent in the separation buffer.

23. The device of claim 20, wherein the first port comprises a sample fluid disposed therein, the sample fluid comprising at least a first polypeptide of interest, and a second detergent at a concentration that is between about 0.0025% and 1% (w/v).

24. The device of claim 20, wherein the first port comprises a sample fluid disposed therein, the sample fluid comprising at least a first polypeptide of interest, and a second detergent at a concentration that is between about 0.0025% and 0.5% (w/v).

25. The device of claim 20, wherein the first port comprises a sample fluid disposed therein, the sample fluid comprising at least a first polypeptide of interest, and a second detergent at a concentration that is less than about 0.5% (w/v).

26. The device of claim 20, wherein the body structure comprises a capillary element having the first capillary channel disposed therein, the first port comprising at least a first open end of the first capillary channel.

27. The device of claim 20, wherein the body structure comprises a planar substrate having the first capillary channel disposed in an interior portion thereof, the port being disposed in a surface of the body.

28. The device of claim 27, further comprising at least a second capillary channel disposed in the interior portion of the planar substrate, the second channel being in fluid communication with the first capillary channel.

29. The device of claim 28, wherein the second capillary channel intersects and crosses the first capillary channel.

30. The device of claim 28, wherein the first port is in fluid communication with the first capillary channel via the second capillary channel.

31. The device of claim 28, further comprising at least a second port disposed in the body structure and in fluid communication with the first capillary channel.

32. The device of claim 31, wherein each of the at least first and second ports are in fluid communication with the first capillary channel via the second capillary channel.