Isoelectric focusing arrays and methods of use thereof

Patent metadata

Field	Value
Publication number	US-10768141-B2
Application number	US-201615749039-A
Country	US
Kind code	B2
Filing date	Sep 8, 2016
Priority date	Sep 11, 2015
Publication date	Sep 8, 2020
Grant date	Sep 8, 2020

Abstract

Isoelectric focusing devices configured for multiplex separation of sample components of interest in a polymeric separation medium are provided. Also provided are methods of using the devices as well as systems and kits that include the devices. The devices, systems and methods find use in a variety of different applications, including diagnostic and validation assays.

First claim

That which is claimed is:

1. A device comprising:
a polymeric separation medium comprising two or more isoelectric focusing regions each comprising an acidic boundary sub-region, a basic boundary sub-region, and a focusing sub-region positioned between the acidic boundary sub-region and the basic boundary sub-region,
wherein each isoelectric focusing region is configured for multiplex separation of sample components of interest from two or more samples in the polymeric separation medium,
wherein the two or more isoelectric focusing regions are arranged in series along an axis of the polymeric separation medium, and
wherein each isoelectric focusing region comprises a pH gradient along the axis of the polymeric separation medium.

2. The device of claim 1 , wherein the isoelectric focusing region comprises a polymeric gel.

3. The device of claim 2 , wherein the pH gradient is an immobilized pH gradient.

4. The device of claim 1 , wherein the polymeric separation medium further comprises functional groups that covalently bond to the sample components of interest in the polymeric separation medium upon application of an applied stimulus.

5. The device of claim 1 , wherein the polymeric separation medium comprises:
a plurality of microwells arranged for parallel separations of the sample components of interest along the axis from each microwell.

6. The device of claim 5 , wherein each microwell is dimensioned to accommodate a single cell.

7. The device of claim 5 , further comprising a polymeric gel cover configured to contact a top surface of the polymeric separation medium.

8. The device of claim 7 , wherein the polymeric gel cover contacts one or more electrodes configured to apply an electric field to the polymeric separation medium.

9. The device of claim 7 , wherein the polymeric gel cover comprises a pH gradient along an axis of the polymeric gel cover substantially parallel to the axis of the polymeric separation medium.

10. The device of claim 7 , wherein the polymeric gel cover comprises a lysis reagent.

11. The device of claim 5 , wherein the polymeric separation medium comprises two or more isoelectric focusing regions arranged in series along the axis of the polymeric separation medium.

12. The device of claim 5 , wherein the polymeric separation medium further comprises functional groups that covalently bond to the sample components of interest in the polymeric separation medium upon application of an applied stimulus.

13. A method comprising:
contacting a sample to the polymeric separation medium of the device according to claim 1 ; and
applying an electric field to the polymeric separation medium in a manner sufficient to perform a multiplex isoelectric focusing separation of the sample components of interest in the polymeric separation medium.

14. The method of claim 13 , wherein the polymeric separation medium comprises two or more isoelectric focusing regions arranged in series along an axis of the polymeric separation medium.

15. The method of claim 13 , wherein the polymeric separation medium comprises:
a pH gradient along an axis of the polymeric separation medium; and
a plurality of microwells arranged for parallel separations of the sample components of interest along the axis from each microwell; and
wherein, prior to applying the electric field, the method comprises contacting a top surface of the polymeric separation medium with a polymeric gel cover comprising a pH gradient along an axis of the polymeric gel cover substantially parallel to the axis of the polymeric separation medium.

16. The method of claim 15 , wherein the sample comprises cells and/or cellular components.

17. The method of claim 16 , further comprising lysing the cells to produce cellular components of interest in the sample.

18. The method of claim 13 , further comprising immobilizing the separated sample components in the polymeric separation medium.

19. The method of claim 13 , further comprising detecting the separated sample components.

20. A system comprising:
the device according to claim 1 ; and
an electric field source.

21. A kit comprising:
the device according to claim 1 ; and
a packaging containing the device.

22. The device of claim 1 , wherein the acidic boundary sub-region comprises immobilines that buffer at an acidic pH and the basic boundary sub-region comprises immobilines that buffer at a basic pH.

23. The device of claim 1 , wherein the acidic boundary sub-region is positioned on a same side of the focusing sub-region as a positive electrode, and the basic boundary sub-region is positioned on a same side of the focusing sub-region as a negative electrode.

Assignees

• Univ California

Inventors

• Herr Amy E
• Tentori Augusto M
• Yamauchi Kevin A

Classifications

• G01N27/44747 Primary

  Composition of gel or of carrier mixture · CPC title

• G01N27/44795

  Isoelectric focusing · CPC title

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• B01D57/02

  by electrophoresis (treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26) · CPC title