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US-20260128270-A1 - FIXTURES AND DEVICES FOR USE IN ISOELECTRIC FOCUSING-MASS SPECTROMETRY

US20260128270A1US 20260128270 A1US20260128270 A1US 20260128270A1US-20260128270-A1

Abstract

The described and claimed technology includes a fixture for applying and/or maintaining positive pressure in an electrode reservoir and related methods for pressurizing an electrode reservoir.

Inventors

  • Greg Bogdan
  • Alexander Petrov
  • Glenn WHITE
  • Hongfeng Yin

Assignees

  • INTABIO, LLC

Dates

Publication Date
20260507
Application Date
20231013

Claims (20)

  1. 1 . A fixture comprising: at least one electrode reservoir; at least one lid, wherein when the electrode reservoir is sealed with the lid, a positive pressure is applied to and/or maintained in the electrode reservoir generating a pressure-driven flow of a fluid contained within the electrode reservoir; and a membrane disposed at a surface of the electrode reservoir, the membrane providing an electrical connection between an electrode positioned within the electrode reservoir, the fluid contained within the electrode reservoir, and at least one fluid channel in fluid communication with the electrode reservoir.
  2. 2 . A fixture comprising: at least one electrode reservoir; at least one lid, and an external pressure source, wherein the lid and/or the electrode reservoir further comprises an inlet for an external pressure source, and wherein a positive pressure is applied to and/or maintained in the electrode reservoir using the external pressure source, said positive pressure generating a pressure-driven flow of a fluid contained within the electrode reservoir; and a membrane disposed at a surface of the electrode reservoir, the membrane providing an electrical connection between an electrode positioned within the electrode reservoir, the fluid contained within the electrode reservoir, and at least one fluid channel in fluid communication with the electrode reservoir.
  3. 3 . The fixture of claim 1 or claim 2 , wherein the flow of the fluid contained within the electrode reservoir to the fluid channel is impeded by the membrane.
  4. 4 . The fixture of any one of the preceding claims , wherein a low flow rate of the fluid contained within the electrode reservoir through the membrane maintained.
  5. 5 . The fixture of claim 2 or claim 3 , wherein the external pressure source is a syringe, a pump, a gas pressure generator, or a pressure control device.
  6. 6 . The fixture of any one of claims 2 to 4 , wherein the positive pressure within the electrode reservoir is applied and/or maintained using a mechanism configured to regulate and/or control the positive pressure generated by the external pressure source.
  7. 7 . The fixture of any one of the preceding claims , wherein the membrane comprises a first surface facing the electrode reservoir and a second surface facing the fluid channel, wherein a hydrodynamic resistance between the first surface and the second surface is equal to or greater than about 40 (N/mm 2 )/(mm 3 /s).
  8. 8 . The fixture of claim 7 , wherein the hydrodynamic resistance is about 40 (N/mm 2 )/(mm 3 /s) to about 62,000 (N/mm 2 )/(mm 3 /s).
  9. 9 . The fixture of claim 8 , wherein the hydrodynamic resistance is at least about 200 (N/mm 2 )/(mm 3 /s) to about 12,000 (N/mm 2 )/(mm 3 /s).
  10. 10 . The fixture of any one of the preceding claims , wherein the low flow rate is a nL/min flow rate.
  11. 11 . The fixture of claim 10 , wherein the low flow rate is equal to or less than about 20 nL/min.
  12. 12 . The fixture of claim 11 , wherein the low flow rate is about 0.2 nL/min to about 20 nL/min.
  13. 13 . The fixture of claim 12 , wherein the low flow rate is about 1 nL/min to about 10 nL/min.
  14. 14 . The fixture of claim 13 , wherein the low flow rate is about 1 nL/min, alternatively about 2 nL/min, alternatively about 3 nL/min, alternatively about 4 nL/min, alternatively about 5 nL/min, alternatively about 8 nL/min, alternatively about 7 nL/min, alternatively about 8 nL/min, alternatively about 9 nL/min, alternatively about 10 nL/min.
  15. 15 . The fixture of any one of the preceding claims , wherein the lid comprises a top, a base, and sidewalls, and the width between the sidewalls is less than the width of the top and/or the base.
  16. 16 . The fixture of any one of the preceding claims , wherein the electrode reservoir comprises a mating bore.
  17. 17 . The fixture of claim 16 , wherein the base of the lid is configured to align with the mating bore.
  18. 18 . The fixture of any one of the preceding claims , wherein the positive pressure applied and/or maintained in the electrode reservoir is at least about 2 psi.
  19. 19 . The fixture of any one of 17, wherein the positive pressure applied and/or maintained in the electrode reservoir is about 2 psi to about 30 psi.
  20. 20 . The fixture of claim 19 , wherein the positive pressure applied and/or maintained in the electrode reservoir is about 5 psi to about 15 psi.

Description

RELATED APPLICATIONS The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63/418,364, filed Oct. 21, 2022 and U.S. Provisional Patent Application Ser. No. 63/430,405, filed Dec. 6, 2022, the content of each is hereby incorporated by reference in its entirety into this disclosure. BACKGROUND In isoelectric focusing, a biologic sample, such as a protein sample, is mixed with an ampholyte solution. This sample/ampholyte solution is then pushed into a capillary or a capillary channel. One end of the channel is then connected to an anolyte solution (generally an acidic solution) and the other end is connected to a catholyte solution (a basic solution). A high voltage across the channel will cause ion migration and as a result, establishes a pH gradient across the channel. At the same time, the protein samples will also migrate toward the region along the length of the capillary channel where the local pH is the closest to the protein pI. When the protein reaches its pI, it will have net zero charge and will stop from migrating. This step is called “focusing” step. An image of the channel after all proteins in the sample reach their pI will show the relative abundance of the various protein species in the sample. The pI of the various protein species can be determined by their relative position to pI markers that are added to the sample solution. While the imaged UV trace gives pI and relative quantitation of the various proteins in the sample, there is a need to be able to identify the protein using a mass spectrometer. This is achieved by mobilizing the proteins. During mobilization, the pH at the catholyte end starts to drop and the protein will start to carry charges which causes it to migrate toward the mobilizer junction. Once it reaches the mobilizer junction, it will be carried by the flowing mobilizer out of the microfluidic chip and sprayed into a mass spec interface. Current cartridge designs do not account for the nanoliter/min flows through the membrane of the pressurized reagent reservoirs present on a microfluidic chip. If this flow is not controlled, the sample can migrate into the catholyte channel, negatively affecting the peak shape and resolutions and subsequent MS analysis. SUMMARY It has been unexpectantly discovered that adjusting, applying, and/or maintaining the pressure in the electrode reservoirs mitigates sample loss by introducing counterflows through the membrane, helps maintain peak shape and resolution, and/or ensuring that downstream analytical characterizations are accurate. This reduction of sample loss helps maintain peak shape and resolution, ensuring that downstream analytical characterizations are accurate. One aspect of the disclosure is a fixture including at least one electrode reservoir; at least one lid, wherein when the electrode reservoir is sealed with the lid, a positive pressure is applied to and/or maintained in the electrode reservoir generating a pressure-driven flow of a fluid contained within the electrode reservoir; and a membrane disposed at a surface of the electrode reservoir, the membrane providing an electrical connection between an electrode positioned within the electrode reservoir, the fluid contained within the electrode reservoir, and at least one fluid channel in fluid communication with the electrode reservoir In another aspect, the flow of the fluid contained within the electrode reservoir to the fluid channel is impeded by the membrane. In yet another aspect, a low flow rate of the fluid contained within the electrode reservoir through the membrane maintained. One aspect of the disclosure is a fixture including at least one electrode reservoir; at least one lid, wherein when the lid is compressed into the headspace of the electrode reservoir, a positive pressure is applied to and/or maintained in the electrode reservoir generating a pressure-driven flow of a fluid contained within the electrode reservoir; and a membrane disposed at a surface of the electrode reservoir, the membrane providing an electrical connection between an electrode positioned within the electrode reservoir, the fluid contained within the electrode reservoir, and at least one fluid channel in fluid communication with the electrode reservoir. In another aspect, the flow of the fluid contained within the electrode reservoir to the fluid channel is impeded by the membrane. In yet another aspect, a low flow rate of the fluid contained within the electrode reservoir through the membrane maintained. Another aspect of the disclosure is a fixture including: at least one electrode reservoir; at least one lid, and an external pressure source, wherein the lid and/or the electrode reservoir further includes an inlet for an external pressure source, and wherein a positive pressure is applied to and/or maintained in the electrode reservoir using the external pressure source, said positive pressure generating a pressure-driven flow of a