Western blot by incorporating an affinity purification zone

• US 8,007,738 B2
• Filed: 05/21/2010
• Issued: 08/30/2011
• Est. Priority Date: 06/05/2001
• Status: Expired due to Fees

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Inventors: Murphy, Matthew B., Dubrow, Robert S.
• Primary Examiner(s): Lam; Ann
• Application Number:
  US12/784,993
• Publication Number:
  US 20100233030A1
• Time in Patent Office: 466 Days
• Field of Search: None
• US Class Current: 422/502
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing

Abstract

An upstream affinity purification region is used to bind one or more component of interest in a mixture of components prior to separating the mixture of components. Detection of the separated components and a released component of interest provide identification of the component of interest. In addition, post separation dilution is optionally used to improve detection of the mixture of components and the released component of interest. Microfluidic devices and systems suitable for performing such analyses are also provided.

Claims

1. A microfluidic device for detecting one or more component of interest in a mixture of components, the device comprising:

(i) a body structure with a plurality of microscale channels disposed therein, the plurality of microscale channels comprising;

(a) a separation channel region for separating the mixture of components; and

,

(b) a binding region upstream from and fluidly connected to the separation channel region, which binding region comprises a component-binding moiety, for binding the one or more component of interest to the component-binding moiety prior to separating the mixture of components;

(ii) a control system operably coupled to the body structure, which control system directs;

(a) flow of the mixture of components through the binding region, thereby binding the one or more component of interest to the component binding moiety, resulting in one or more bound component of interest;

(b) flow of the mixture of components through the separation channel region after flowing the mixture of components through the binding region, thereby separating the mixture of components resulting in one or more separated components;

(c) release of the one or more bound component of interest from the binding channel, resulting in one or more released component of interest; and

,

(d) flow of the one or more released component of interest through the separation channel region.

2. The device of claim 1, wherein the separation channel region comprises a polyacrylamide gel filled region.

3. The device of claim 1, wherein the separation channel region comprises a separation buffer comprising a detergent and a dye moiety.

4. The device of claim 3, wherein the dye moiety comprises a lipophilic dye.

5. The device of claim 3, wherein the separation buffer comprises a critical micelle concentration of the detergent.

6. The device of claim 1, wherein the binding region comprises a derivatized channel, which derivatized channel comprises the component-binding moiety.

7. The device of claim 1, wherein the binding region comprises a particle set, the particle set comprising a plurality of particle member types.

8. The device of claim 7, wherein the particle member types comprise a polymeric material, a silica material, a ceramic material, a glass material, a magnetic material, a metallic material, or an organic material.

9. The device of claim 7, wherein the particle member types comprise a material selected from the group consisting of:

PVDF, polyamide, nylon, and nitrocellulose.

10. The device of claim 7, wherein the particle member types are from about 0.1 mm to about 50 mm in diameter.

11. The device of claim 7, wherein one or more member of the plurality of particles comprises the component-binding moiety.

12. The device of claim 1, the plurality of microscale channels further comprising a heating zone positioned between the binding region and the separation channel region for heating the mixture of components or the released component of interest.

13. The device of claim 12, further comprising a temperature control element operably coupled to the heating zone for heating the mixture of components or the released component of interest in the heating zone.

14. The device of claim 1, further comprising a sipper capillary fluidly connected to and upstream from the binding region.

15. The device of claim 1, further comprising a source of the mixture of components fluidly connected to the binding region.

16. The device of claim 1, further comprising an injection channel fluidly connected to the binding region and the separation channel region for transferring the mixture of components or the released component of interest from the binding region to the separation channel region.

17. The device of claim 1, further comprising a detection region downstream from the separation channel region and a dilution channel fluidly coupled to the detection region.

18. The device of claim 17, wherein the control system directs flow of a diluent from the diluent channel into the detection region.

19. The device of claim 1, further comprising a detection region downstream of the separation channel region.

20. The device of claim 1, further comprising a detection system positioned proximal to a detection region, which detection region is positioned downstream of the separation channel region for detecting the mixture of components and the released component of interest.

21. The device of claim 20, wherein the detection system comprises a chemiluminescent, fluorescent, or colorimetric detector.

22. The device of claim 20, further comprising a computer operably coupled to the device and software for analyzing one or more signals produced by the detection system.

23. The device of claim 22, wherein the detection system detects the one or more separated components, resulting in a first data set and detects the released component of interest, resulting in a second data set.

24. The device of claim 23, wherein the software comprises at least a first instruction set for deconvoluting the first data set and the second data set to identify the separated components or the released component of interest.

25. The device of claim 24, wherein deconvoluting comprises determining the retention time of one or more of the separated components or the released component of interest.

26. The device of claim 24, wherein deconvoluting comprises superimposing or comparing the first data set and the second data set to identify the released component of interest.

27. The device of claim 1, further comprising a fluid direction system fluidly coupled to the body structure and operably coupled to the control system, which fluid direction system transports one or more of:

the mixture of components, the component of interest, and the released component of interest through the microscale channels as directed by the control system.

28. The device of claim 27, wherein the fluid direction system comprises one or more of:

an electrokinetic based fluid direction system and a pressure based fluid direction system.

29. The device of claim 1, wherein the component of interest comprises a protein and the component-binding moiety comprises a protein-binding moiety.

30. The device of claim 1, wherein the protein-binding moiety comprises an antibody specific to the component of interest.

31. The device of claim 1, wherein the component of interest comprises a carbohydrate and the component-binding moiety comprises a carbohydrate-binding moiety.

32. The device of claim 31, wherein the carbohydrate-binding moiety comprises a lectin specific to the carbohydrate.

33. The device of claim 1, wherein the component-binding moiety or the component of interest comprises a label moiety.