Devices and methods for sample characterization
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.
1. A method, comprising:
in a microfluidic device, introducing an analyte mixture into a separation channel;
applying an electric field across the separation channel to effect a separation of the analyte mixture via isoelectric focusing;
continuously imaging the separation and mobilization of the analyte mixture by illuminating a first side of the microfluidic device through an optical slit, which provides optical access to the separation channel and detecting light passing through the optical slit and the separation channel;
introducing an electrolyte into a separated analyte mixture after the separation;
after introducing the electrolyte, expelling via electrospray ionization into a mass spectrometer substantially all of the analyte mixture from an orifice in line with the separation channel and in electrical communication with the separation channel's electric field; and
correlating absorbance peaks of separated analytes in the separation channel with data from the mass spectrometer of the separated analytes after the expelling of the separated analytes.
2. 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.
3. The method of claim 1 , wherein the microfluidic device comprises a first separation channel and a second separation channel.
4. The method of claim 3 , 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.
5. The method of claim 1 , wherein substantially all of the separated analyte mixture is expelled from the orifice in a continuous stream.
6. The method of claim 1 , wherein:
the microfluidic device comprises a top layer, a middle layer, and a bottom layer;
the middle layer is constructed of an opaque substrate and the top layer and bottom layer are constructed of a transparent material; and
the separation channel defines the optical slit through the microfluidic device.
7. The method of claim 6 , wherein the transparent material has a transmittance to allow ultraviolet (UV) light emitted by a light source positioned on one side of the microfluidic device to be quantified by a detector positioned on the other side of the microfluidic device.
8. The method of claim 7 , wherein the transparent material has a transmissivity of at least 30%.
9. The method of claim 7 , wherein the transparent material has a transmissivity of at least 50%.
10. The method of claim 7 , wherein the transparent material has a transmissivity of at least 80%.
11. The method of claim 7 , wherein the transparent material has a transmissivity of at least 95%.
12. 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.
13. The method of claim 1 , wherein the analyte mixture comprises intact proteins.
14. The method of claim 1 , wherein the introducing of the electrolyte is performed by flowing an electrolyte solution from an electrolyte channel in fluid communication with a confluence region downstream of the separation channel.
15. The method of claim 1 , wherein replenishment of ion potential occurs on the microfluidic device.
16. The method of claim 1 , wherein the electrolyte is introduced by pressure.
17. The method of claim 1 , wherein the electrolyte is introduced by electrophoresis.
18. The method of claim 14 , wherein the microfluidic device is a cartridge comprising the separation channel, the orifice and the electrolyte introducing channel in static configuration.
19. The method of claim 18 , wherein the separation channel and the electrolyte introducing channel intersect at a confluence region.
20. The method of claim 19 , the confluence region is in the electric field of the separation channel.
21. The method of claim 19 , wherein the confluence region is in line with the separation channel and the orifice.
22. The method of claim 1 , wherein the microfluidic device comprises two electrodes generating an electric field across an electrolyte introducing channel.
23. The method of claim 18 , wherein the cartridge further comprises an anolyte introducing channel and gas delivery channels for ionization.