Devices and methods for sample characterization

• US 10,401,324 B2
• Filed: 12/18/2018
• Issued: 09/03/2019
• Est. Priority Date: 11/30/2015
• Status: Active Grant

• Current Assignee: Intabio LLC (Danaher Corporation)
• Original Assignee: Intabio, Inc. (Danaher Corporation)
• Inventors: Gentalen, Erik
• Primary Examiner(s): Huang, Ryan B
• Application Number:
  US16/224,558
• Publication Number:
  US 20190128843A1
• Time in Patent Office: 259 Days
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 3/0268 using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 2223/40 Imaging
• CPC Class Codes: G01N 2223/50 Detectors
• CPC Class Codes: G01N 2550/00 Electrophoretic profiling, e.g. for proteome analysis
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 27/44795 Isoelectric focusing
• CPC Class Codes: H01J 49/04 Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
• CPC Class Codes: H01J 49/167 : Capillaries and nozzles specially adapted therefor; electrostatic spraying per se B05B5/00

Abstract

Devices and methods for characterization of analyte mixtures are provided. Some methods described herein include performing enrichment steps on a device before expelling enriched analyte fractions from the device for subsequent analysis. Also included are devices for performing these enrichment steps.

Claims

1. A method, comprising:

applying an electric field across a separation channel in a microfluidic device to effect a separation of an analyte mixture via isoelectric focusing;

simultaneously and continuously imaging the separation and mobilization of the separated analyte mixture in the whole separation channel or a portion thereof; and

expelling separated and mobilized analytes via electrospray ionization from an orifice on the microfluidic device into a mass spectrometer.

2. The method of claim 1, further comprising correlating separated analyte peaks detected in the separation channel with mass spectrometer data for the separated analytes.

3. The method of claim 2, wherein the separated analyte peaks are detected by absorbance imaging.

4. The method of claim 2, wherein the separated analyte peaks are detected by fluorescence imaging.

5. The method of claim 1, wherein the orifice is in electrical communication with the separation channel'"'"'s electric field.

6. The method of claim 1, wherein the orifice is a recess on the microfluidic device, such that a Taylor cone formed by electrospray ionization is disposed entirely within the recess.

7. The method of claim 1, wherein the microfluidic device comprises a first separation channel and a second separation channel.

8. The method of claim 7, further comprising:

chromatographically-enriching the analyte mixture in the first separation channel before applying the electric field to effect the isoelectric focusing separation of the analyte mixture in the second separation channel.

9. The method of claim 1, further comprising introducing ampholytes into the separation channel before the separation of the analyte mixture to generate a pH gradient in the separation channel, introducing isoelectric point (pI) markers into the separation channel before the separation, and continuously imaging the separation channel while the pI markers are separated.

10. The method of claim 1, wherein the analyte mixture comprises intact proteins.

11. The method of claim 1, wherein the mobilization is effected by introducing an electrolyte into the separation channel using pressure.

12. The method of claim 1, wherein the mobilization is effected by introducing an electrolyte into the separation channel by electrophoresis.

13. The method of claim 1, wherein the mobilization is effected by introducing an electrolyte into the separation channel from an electrolyte channel in fluid communication with a confluence region downstream of the separation channel.

14. The method of claim 13, wherein the microfluidic device is a cartridge comprising the separation channel, the orifice, the electrolyte introducing channel, an anolyte introducing channel, and gas delivery channels for ionization.

15. The method of claim 1, wherein replenishment of ion potential occurs on the microfluidic device.

16. The method of claim 1, wherein the microfluidic device comprises two electrodes generating an electric field across an electrolyte introducing channel.

17. A method, comprising:

performing a separation of an analyte mixture in a separation channel in a microfluidic device;

simultaneously and continuously imaging the separation and mobilization of the separated analyte mixture in the whole separation channel or a portion thereof;

expelling separated and mobilized analytes via electrospray ionization from an orifice on the microfluidic device into a mass spectrometer; and

correlating separated analyte peaks detected in the separation channel with mass spectrometer data for the separated analytes.

18. The method of claim 17, wherein the separation is performed using isoelectric focusing.

19. The method of claim 17, wherein the separation is performed using chomatography.

20. The method of claim 17, wherein the separated analyte peaks are detected by absorbance imaging.

21. The method of claim 17, wherein the separated analyte peaks are detected by fluorescence imaging.

22. The method of claim 17, wherein the orifice is a recess on the microfluidic device, such that a Taylor cone formed by electrospray ionization is disposed entirely within the recess.

23. The method of claim 17, wherein the microfluidic device comprises a first separation channel and a second separation channel.

24. The method of claim 23, further comprising:

chromatographically-enriching the analyte mixture in the first separation channel before applying an electric field to effect an isoelectric focusing separation of the analyte mixture in the second separation channel.

25. The method of claim 24, further comprising introducing ampholytes into the second separation channel before the separation of the analyte mixture to generate a pH gradient in the second separation channel, introducing isoelectric point (pI) markers into the second separation channel before the separation, and continuously imaging the second separation channel while the pI markers are separated.

26. The method of claim 17, wherein the analyte mixture comprises intact proteins.