Microfluidic device with gas channels for sample atomization

Abstract

Methods, devices, and systems are described for performing atomization of a sample from a fluid channel of a microfluidic device. The systems or devices disclosed herein may include a microfluidic device comprising a gas channel used for sample atomization at a fluid outlet of the microfluidic device. In some cases, the disclosed devices may be designed to perform isoelectric focusing, followed by further characterization of separated analytes using electrospray ionization coupled with atomization, and then introduce the sample into a mass spectrometer. The disclosed methods, devices, and systems provide rapid and accurate separation and characterization of protein analyte mixtures or other biological molecules by isoelectric point.

Description

[Technical Field]

[0001] This application claims priority to U.S. Patent Application No. 63 / 016,880, filed April 28, 2020, and entitled "Microfluidic Devices with Gas Channels for Sample Nebulization." The '880 application is incorporated herein by reference in its entirety. [Background technology]

[0002] Separating analyte components from a more complex analyte mixture based on the intrinsic quality of the analyte and providing a set of fractions enriched in terms of their quality status is an important part of analytical chemistry. Simplifying complex mixtures in this way reduces the complexity of downstream analysis. However, complications can arise when attempting to interface known enrichment methods and / or devices, such as mass spectrometry, with analytical instruments and / or techniques. For example, a method for introducing a sample into a mass spectrometer is electrospray ionization (ESI). However, in ESI, the formation of large droplets can introduce contaminants and adversely affect the analytical performance of mass spectrometry. Summary of the Invention [Means for solving the problem]

[0003] Recognized herein is a need for methods, devices, and systems for reducing droplet size and contamination prior to and during sample introduction into a mass spectrometer. Disclosed herein are methods, devices, and systems for sample processing and characterization, and various uses thereof. In a first aspect, the disclosure relates to methods, devices, and systems for performing separation and characterization of analytes within a mixture of analytes, and more particularly to devices (and related methods and systems) for atomizing a sample prior to or during electrospray ionization. In a second aspect, the disclosure relates to microfluidic devices (and related methods and systems) designed to perform one or more separation reactions (e.g., isoelectric focusing), followed by mobilization, atomization, and electrospray ionization of separated analytes for characterization by mass spectrometry.

[0004] Provided herein are methods, devices, and systems that enable improved quantitative performance for the separation and analysis of analytes within an analyte mixture, with potential applications in biomedical research, clinical diagnostics, and pharmaceutical manufacturing. For example, rigorous characterization of biological drugs and drug candidates (e.g., proteins) is required by regulatory agencies. The methods and devices described herein may be suitable for characterizing proteins and / or other analytes. In some cases, the methods and devices described herein may relate to characterizing analyte mixtures in which one or more enrichment steps are performed to separate the analyte mixture into enriched analyte fractions. In some cases, the methods and devices described herein may relate to performing one or more enrichment steps and separating the analyte mixture into enriched analyte fractions in a multiplexed format for high-throughput characterization of samples. In some cases, the methods and devices described herein relate to characterizing analyte mixtures in which one or more enrichment steps are performed to separate the analyte mixture into enriched analyte fractions that are subsequently introduced into a mass spectrometer via an electrospray ionization interface. In some cases, the methods and devices described herein involve the use of one or more gas channels to atomize the sample during electrospray ionization, thereby reducing the droplet size of the sample introduced into an analytical instrument (e.g., a mass spectrometer). In some cases, the methods and devices described herein involve the use of one or more microfluidic devices that include one or more gas channels to atomize the sample during electrospray ionization. The disclosed methods and devices may provide improved convenience, reproducibility, and / or analytical performance of analyte separation and characterization.

[0005] In one aspect, disclosed herein is a microfluidic chip comprising: a) a substrate comprising: i) a fluid channel having a distal end in fluid communication with an electrospray ionization orifice; and ii) a gas channel having a distal end in fluid communication with a gas exit orifice disposed adjacent the electrospray ionization orifice; wherein an angle between the distal end of the fluid channel and the distal end of the gas channel ranges from about 0 degrees to about 30 degrees.

[0006] In some embodiments, the electrospray ionization orifice is located on an edge or corner or tip of the substrate. In some embodiments, the gas exit orifice is located on the edge of the substrate adjacent to the electrospray ionization orifice. In some embodiments, the substrate comprises two or more gas channels, each having a distal end in fluid communication with a gas exit orifice. In some embodiments, the two or more gas exit orifices are located adjacent to and symmetrically about the electrospray ionization orifice. In some embodiments, the angle ranges from about 10 degrees to about 20 degrees. In some embodiments, the angle is about 15±5 degrees. In some embodiments, the gas exit orifice is configured to perform atomization of the solution exiting the electrospray ionization orifice. In some embodiments, the microfluidic device comprises three or more gas channels, each comprising a gas exit orifice located adjacent to the electrospray ionization orifice. In some embodiments, at least one of the three or more gas channels is disposed within the substrate, and at least one of the three or more gas channels is disposed within an auxiliary component of the microfluidic chip, which is positioned adjacent to the substrate such that at least one gas channel is not located in the same plane as the substrate. In some embodiments, at least one of the three or more gas channels disposed within the auxiliary component is positioned such that its gas outlet orifice is in a plane generally perpendicular to that of the substrate and is positioned symmetrically about and adjacent to the electrospray ionization orifice. In some embodiments, at least one of the three or more gas channels disposed within the auxiliary component is positioned such that its gas outlet orifice is in one or more planes rotated relative to that of the substrate and positioned in a radially symmetric pair about and adjacent to the electrospray ionization orifice. In some embodiments, the fluidic channel comprises a separation channel.In some embodiments, the microfluidic chip is configured to perform isoelectric focusing or electrophoretic separation of a sample comprising a mixture of analytes within a fluid channel. In some embodiments, the fluid channel has a width ranging from about 20 μm to about 600 μm. In some embodiments, the fluid channel has a depth ranging from about 10 μm to about 100 μm. In some embodiments, the fluid channel has a length ranging from about 0.25 cm to about 30 cm. In some embodiments, the electrospray ionization orifice has a substantially square, rectangular, circular, oval, or diamond-shaped cross-section. In some embodiments, the electrospray ionization orifice has a maximum cross-sectional dimension ranging from about 10 μm to about 100 μm. In some embodiments, the gas channel has a width ranging from about 20 μm to about 200 μm. In some embodiments, the gas channel has a depth ranging from about 10 μm to about 100 μm. In some embodiments, the gas channel has a length ranging from about 0.2 cm to about 20 cm. In some embodiments, the gas exit orifice has a substantially square, rectangular, circular, oval, or diamond-shaped cross-section. In some embodiments, the gas exit orifice has a maximum cross-sectional dimension ranging from about 10 μm to about 50 μm. In some embodiments, the gas exit orifice is positioned within 100 μm of the electrospray ionization orifice. In some embodiments, the gas exit orifice is positioned within 50 μm of the electrospray ionization orifice. In some embodiments, the gas exit orifice is positioned within 10 μm of the electrospray ionization orifice. In some embodiments, the substrate is fabricated from glass, silicon, a polymer, or any combination thereof.

[0007] In another aspect of the present disclosure, provided herein is a microfluidic chip comprising: a) a substrate comprising: i) two or more gas channels of different lengths, each configured to deliver gas to a gas exit orifice, wherein a dimension of at least one of the two or more gas channels is adjusted along a portion of its length such that each of the two or more gas channels has approximately the same hydrodynamic flow resistance.

[0008] In some embodiments, the cross-sectional area of ​​at least one of the two or more gas channels is adjusted along a portion of its length. In some embodiments, the minimum difference in the lengths of the two or more gas channels ranges from about 1 cm to about 10 cm. In some embodiments, the maximum difference in the lengths of the two or more gas channels ranges from about 1 cm to about 10 cm. In some embodiments, the substrate further comprises a fluidic channel having a distal end in fluid communication with the electrospray ionization orifice. In some embodiments, the two or more gas exit orifices are positioned symmetrically about and adjacent to the electrospray ionization orifice and configured to perform atomization of a solution exiting the electrospray ionization orifice. In some embodiments, the electrospray ionization orifice is positioned on an edge or corner of the substrate. In some embodiments, the two or more gas exit orifices are positioned on an edge of the substrate adjacent to the electrospray ionization orifice. In some embodiments, the fluidic channel comprises a separation channel. In some embodiments, the microfluidic chip is configured to perform isoelectric focusing or electrophoretic separation. In some embodiments, the gas is an atomizer gas. In some embodiments, the atomizer gas comprises air, nitrogen, oxygen, nitrous oxide, fluorourethane, helium, argon, methanol, or any combination thereof. In some embodiments, the microfluidic chip further comprises a hydrophobic coating on at least a portion of an edge of the substrate or a corner of the substrate on which the electrospray ionization orifice is disposed.

[0009] In another aspect, disclosed herein is a microfluidic chip comprising: a substrate comprising: i) a fluid channel having a proximal end in fluid communication with a fluid inlet port and a distal end in fluid communication with an electrospray ionization orifice; and ii) at least two gas channels having a proximal end in fluid communication with a gas inlet port and a distal end in fluid communication with a gas outlet orifice, respectively; wherein the at least one fluid inlet port and the at least two gas inlet ports are disposed along a first edge of the substrate.

[0010] In some embodiments, the electrospray ionization orifice is positioned on a second edge of the substrate. In some embodiments, the electrospray ionization orifice is positioned on a corner of the substrate that does not include the first edge. In some embodiments, the substrate is less than about 2.0 mm thick. In some embodiments, the fluidic channel comprises a separation channel configured to perform an electrophoretic separation. In some embodiments, the fluidic channel comprises a separation channel configured to perform an isoelectric focusing separation. In some embodiments, the substrate comprises a first separation channel and a second separation channel, wherein a distal end of the first separation channel is in fluid communication with a proximal end of the second separation channel, and a distal end of the second separation channel is in fluid communication with the electrospray ionization orifice. In some embodiments, the first separation channel is configured to perform a chromatographic separation, and the second separation channel is configured to perform an electrophoretic separation. In some embodiments, the first separation channel is configured to perform a chromatographic separation, and the second separation channel is configured to perform an isoelectric focusing separation. In some embodiments, the fluid channel comprises a separation channel configured to perform isoelectric focusing separation of a sample comprising a mixture of analytes, and the substrate further comprises a mobilizing electrolyte channel in fluid communication with a distal end of the separation channel and configured to provide electrophoretic introduction of a mobilizing electrolyte to the distal end of the separation channel.

[0011] Disclosed herein, in another aspect, is a method for performing electrospray ionization from a microfluidic chip, the method comprising: a) providing a microfluidic chip comprising a substrate, the substrate comprising: i) at least one fluid channel having a distal end in fluid communication with an electrospray ionization orifice; and ii) at least one gas channel configured to deliver gas to a gas exit orifice adjacent to the electrospray ionization orifice; b) flowing a solution through the at least one fluid channel such that the solution is expelled from the electrospray ionization orifice; and c) flowing a gas through the at least one gas channel such that the gas is expelled from the gas exit orifice, wherein the temperature of the substrate is controlled by the temperature of the gas flowing through the at least one gas channel.

[0012] In some embodiments, the temperature of the gas ranges from about 4°C to about 100°C. In some embodiments, the temperature of the substrate ranges from about 10°C to about 50°C. In some embodiments, the average temperature of the substrate is maintained at 30±5°C. In some embodiments, the at least one fluidic channel comprises a separation channel. In some embodiments, the separation channel is configured to perform isoelectric focusing separation of a sample comprising a mixture of analytes. In some embodiments, the separation channel is configured to perform electrophoretic separation of a sample comprising a mixture of analytes. In some embodiments, the electrospray ionization performance achieved when the microfluidic chip is configured to introduce a sample into a mass spectrometer is characterized by less than a 1.0% standard error variation in total mass-to-mass spectrometry signal intensity. In some embodiments, the electrospray ionization performance achieved when the microfluidic chip is configured to introduce a sample into a mass spectrometer is characterized by less than a 0.1% standard error variation in total mass-to-mass spectrometry signal intensity.

[0013] In yet another aspect, disclosed herein is a method for providing stable electrospray ionization performance, the method comprising: a) providing a microfluidic chip comprising a substrate, the substrate comprising: (i) a fluid channel having a distal end in fluid communication with an electrospray ionization orifice; and (ii) a gas channel having a distal end in fluid communication with a gas exit orifice; b) flowing a solution through the fluid channel; c) flowing a gas through the gas channel; and d) controlling the flow rates of the gas and the solution such that the ratio of the volumetric flow rates for the gas and the solution ranges from 1000:1 to 1,000,000:1. In some embodiments, the ratio of the volumetric flow rates for the gas and the solution ranges from 10,000:1 to 1,000,000:1. In some embodiments, the ratio of volumetric flow rates for gas and solution ranges from 10,000:1 to 500,000:1, more particularly, from 10,000:1 to 300,000:1.

[0014] In some embodiments, the flow of the solution is controlled by pressure, gravity, electrokinetic force, or any combination thereof. In some embodiments, the flow of the gas is provided by a compressed gas source. In some embodiments, the volumetric flow rate for the solution is less than 25 μL / min. In some embodiments, the microfluidic chip has a distal end comprising two or more gas channels in fluid communication with each gas outlet orifice, the two or more gas outlet orifices being positioned symmetrically about and adjacent to the electrospray ionization orifice. In some embodiments, the electrospray ionization orifice is positioned on an edge or corner of the substrate. In some embodiments, one or more gas outlet orifices are positioned adjacent to the electrospray ionization orifice on the edge of the substrate. In some embodiments, the electrospray ionization performance achieved when the microfluidic chip is configured to introduce a sample into a mass spectrometer is characterized by a variation in total mass spectrometry signal intensity of less than 1.0% standard error. In some embodiments, the electrospray ionization performance when the microfluidic chip is configured to introduce a sample into a mass spectrometer is characterized by less than 0.1% standard error variation in total mass spectrometric signal intensity.

[0015] In another aspect, disclosed herein is a method for providing stable electrospray ionization performance, the method comprising: a) providing a microfluidic chip comprising a substrate, the substrate comprising: (i) a fluid channel having a distal end in fluid communication with an electrospray ionization orifice; and (ii) a gas channel having a distal end in fluid communication with a gas exit orifice; b) flowing a solution through the fluid channel; c) flowing a gas through the gas channel; and d) controlling the flow rates of the gas and the solution such that the ratio of the flow rate for the gas at the gas exit orifice to the flow rate for the solution at the electrospray ionization orifice ranges from 100:1 to 1,000,000:1. In some embodiments, the ratio of the flow rate for the gas at the gas exit orifice to the flow rate for the solution at the electrospray ionization orifice ranges from 500:1 to 5,000:1.

[0016] In some embodiments, the ratio of the flow rate for the gas at the gas exit orifice and the flow rate for the solution at the electrospray ionization orifice ranges from 1,000:1 to 3,000:1.

[0017] In yet another aspect, provided herein is a microfluidic cartridge comprising: a) a microfluidic chip comprising at least one fluid port and at least two gas ports disposed on an edge of the microfluidic chip; and b) a microfluidic cartridge component in fluid communication with the microfluidic chip and configured to encompass at least a portion of the microfluidic chip, the microfluidic cartridge component comprising at least one fluid port and at least two gas ports that align with the at least one fluid port and at least two gas ports of the microfluidic chip.

[0018] In some embodiments, the microfluidic cartridge further comprises one or more elastomer components disposed between the edge of the microfluidic chip and the surface of the cartridge, the one or more elastomer components forming, in response to application of force, a substantially leak-free seal between at least one fluid port and at least two gas ports of the microfluidic chip and at least one fluid port and at least two gas ports of the microfluidic cartridge component. In some embodiments, the edge of the microfluidic chip is less than about 2.0 mm thick. In some embodiments, the edge of the microfluidic chip is about 1±0.1 mm thick.

[0019] In another aspect, disclosed herein is a system comprising: a) a microfluidic cartridge comprising two or more fluid ports and configured to be removable from the system; and b) an instrument comprising two or more fluid interconnects, each comprising two or more fluid interconnects and two or more fluid ports of the microfluidic cartridge, configured to provide a substantially leak-free fluid coupling between a fluid line of the instrument and a fluid port of the microfluidic cartridge in response to application of a force to the assembly, wherein the substantially leak-free fluid coupling is maintained when the relative fluid pressure in two of the two or more fluid lines varies by at least 10-fold.

[0020] In some embodiments, the substantially leak-free fluid coupling is maintained when the relative fluid pressure in two of the two or more fluid lines varies by at least 100-fold. In some embodiments, the two or more fluid interconnections each comprise an independently spring-loaded coupling. In some embodiments, the independently spring-loaded couplings comprise flat face seal couplings that mate with fluid ports comprising holes in the microfluidic cartridge. The present invention provides, for example, the following. (Item 1) A microfluidic chip comprising: a) a substrate, the substrate comprising: i) a fluid channel having a distal end in fluid communication with an electrospray ionization orifice; ii) a gas channel having a distal end in fluid communication with a gas exit orifice disposed adjacent the electrospray ionization orifice; A substrate comprising: Equipped with The microfluidic chip, wherein the angle between the distal end of the fluid channel and the distal end of the gas channel ranges from about 0 degrees to about 30 degrees. (Item 2) Item 2. The microfluidic chip according to item 1, wherein the electrospray ionization orifice is positioned on an edge, corner, or tip of the substrate. (Item 3) 3. The microfluidic chip of item 2, wherein the gas exit orifice is positioned on the edge of the substrate adjacent to the electrospray ionization orifice. (Item 4) 4. The microfluidic chip of any one of items 1-3, wherein the substrate comprises two or more gas channels, each of which has a distal end in fluid communication with a gas exit orifice. (Item 5) 5. The microfluidic chip according to item 4, wherein the two or more gas exit orifices are disposed adjacent to and symmetrically about the electrospray ionization orifice. (Item 6) 6. The microfluidic chip according to any one of items 1 to 5, wherein the angle ranges from about 10 degrees to about 20 degrees. (Item 7) 6. The microfluidic chip of any one of items 1 to 5, wherein the angle is about 15±5 degrees. (Item 8) 8. The microfluidic chip according to any one of items 1 to 7, wherein the gas exit orifice is configured to perform atomization of the solution discharged from the electrospray ionization orifice. (Item 9) 9. The microfluidic chip of any one of items 1-8, wherein the microfluidic device comprises three or more gas channels, each comprising a gas exit orifice positioned adjacent to the electrospray ionization orifice. (Item 10) 10. The microfluidic chip of claim 9, wherein at least one of the three or more gas channels is disposed within the substrate, and at least one of the three or more gas channels is disposed within an auxiliary component of the microfluidic chip positioned adjacent to the substrate such that the at least one gas channel is not coplanar with the substrate. (Item 11) Item 11. The microfluidic chip of item 10, wherein at least one of the three or more gas channels disposed in the auxiliary component is positioned such that its gas exit orifice is in a plane generally perpendicular to that of the substrate and is positioned symmetrically about and adjacent to the electrospray ionization orifice. (Item 12) Item 12. The microfluidic chip of item 11, wherein at least one of the three or more gas channels disposed in the auxiliary component is positioned so that its gas exit orifice lies in one or more planes that are rotated relative to that of the substrate and positioned in a radially symmetric pair manner centered on and adjacent to the electrospray ionization orifice. (Item 13) 13. The microfluidic chip of any one of items 1-12, wherein the fluid channel comprises a separation channel. (Item 14) 14. The microfluidic chip of any one of items 1-13, wherein the microfluidic chip is configured to perform isoelectric focusing or electrophoretic separation of a sample comprising a mixture of analytes in the fluid channel. (Item 15) 15. The microfluidic chip according to any one of items 1 to 14, wherein the fluid channel has a width ranging from about 20 μm to about 600 μm. (Item 16) 16. The microfluidic chip according to any one of items 1 to 15, wherein the fluid channel has a depth ranging from about 10 μm to about 100 μm. (Item 17) 17. The microfluidic chip according to any one of items 1-16, wherein the fluid channel has a length ranging from about 0.25 cm to about 30 cm. (Item 18) 18. The microfluidic chip of any one of items 1-17, wherein the electrospray ionization orifice has a substantially square, rectangular, circular, oval, or diamond-shaped cross section. (Item 19) 19. The microfluidic chip of any one of items 1-18, wherein the electrospray ionization orifice has a maximum cross-sectional dimension ranging from about 10 μm to about 100 μm. (Item 20) 20. The microfluidic chip according to any one of items 1 to 19, wherein the gas channel has a width ranging from about 20 μm to about 200 μm. (Item 21) 21. The microfluidic chip according to any one of items 1-20, wherein the gas channel has a depth ranging from about 10 μm to about 100 μm. (Item 22) 22. The microfluidic chip according to any one of items 1-21, wherein the gas channel has a length ranging from about 0.2 cm to about 20 cm. (Item 23) 23. The microfluidic chip of any one of items 1-22, wherein the gas exit orifice has a substantially square, rectangular, circular, oval, or diamond-shaped cross section. (Item 24) 24. The microfluidic chip of any one of items 1-23, wherein the gas exit orifice has a maximum cross-sectional dimension ranging from about 10 μm to about 50 μm. (Item 25) 25. The microfluidic chip of any one of items 1-24, wherein the gas exit orifice is positioned within 100 μm of the electrospray ionization orifice. (Item 26) 26. The microfluidic chip of any one of items 1-25, wherein the gas exit orifice is positioned within 50 μm of the electrospray ionization orifice. (Item 27) 27. The microfluidic chip of any one of items 1-26, wherein the gas exit orifice is positioned within 15 μm of the electrospray ionization orifice. (Item 28) 28. The microfluidic chip of any one of items 1-27, wherein the substrate is fabricated from glass, silicon, polymer, or any combination thereof. (Item 29) A microfluidic chip comprising: a) a substrate, the substrate comprising: i) two or more gas channels of different lengths, each configured to deliver gas to a gas exit orifice; A substrate comprising: Equipped with A microfluidic chip, wherein a dimension of at least one of the two or more gas channels is adjusted along a portion of its length such that each of the two or more gas channels has approximately the same hydrodynamic flow resistance. (Item 30) 30. The microfluidic chip of item 29, wherein a cross-sectional area of ​​at least one of the two or more gas channels is adjusted along a portion of its length. (Item 31) 31. The microfluidic chip of claim 29 or 30, wherein the minimum difference in length of the two or more gas channels ranges from about 1 cm to about 10 cm. (Item 32) 32. The microfluidic chip of any one of items 29-31, wherein the maximum difference in length of the two or more gas channels ranges from about 1 cm to about 10 cm. (Item 33) 33. The microfluidic chip of any one of items 29-32, wherein the substrate further comprises a fluid channel having a distal end in fluid communication with an electrospray ionization orifice. (Item 34) Item 34. The microfluidic chip according to item 33, wherein the two or more gas exit orifices are arranged symmetrically about and adjacent to the electrospray ionization orifice and are configured to perform atomization of the solution ejected from the electrospray ionization orifice. (Item 35) 35. The microfluidic chip according to any one of items 33-34, wherein the electrospray ionization orifice is positioned on an edge or corner of the substrate. (Item 36) Item 36. The microfluidic chip of item 35, wherein the two or more gas exit orifices are located on the edge of the substrate adjacent to the electrospray ionization orifice. (Item 37) 37. The microfluidic chip of any one of items 29-36, wherein the fluid channel comprises a separation channel. (Item 38) 38. The microfluidic chip of any one of items 29-37, wherein the microfluidic chip is configured to perform isoelectric focusing or electrophoretic separation. (Item 39) 39. The microfluidic chip of any one of items 29-38, wherein the gas is an atomizer gas. (Item 40) 40. The microfluidic chip of item 39, wherein the atomizer gas comprises air, nitrogen, oxygen, nitrous oxide, fluorourethane, helium, argon, methanol, or any combination thereof. (Item 41) 41. The microfluidic chip of any one of items 35-40, further comprising a hydrophobic coating on at least a portion of an edge of the substrate or a corner of the substrate on which the electrospray ionization orifice is disposed. (Item 42) A microfluidic chip comprising: a) a substrate, the substrate comprising: i) a fluid channel having a proximal end in fluid communication with the fluid inlet port and a distal end in fluid communication with the electrospray ionization orifice; ii) at least two gas channels, each having a proximal end in fluid communication with a gas inlet port and a distal end in fluid communication with a gas outlet orifice; A substrate comprising: Equipped with The microfluidic chip, wherein the at least one fluid inlet port and the at least two gas inlet ports are disposed along a first edge of the substrate. (Item 43) Item 43. The microfluidic chip of item 42, wherein the electrospray ionization orifice is positioned on a second edge of the substrate. (Item 44) Item 44. The microfluidic chip of item 43, wherein the electrospray ionization orifice is positioned on a corner of the substrate that does not include the first edge. (Item 45) 45. The microfluidic chip of any one of items 42-44, wherein the substrate is less than about 2.0 mm thick. (Item 46) 46. ​​The microfluidic chip of any one of items 42-45, wherein the fluidic channel comprises a separation channel configured to perform electrophoretic separation. (Item 47) 46. ​​The microfluidic chip of any one of items 42-45, wherein the fluidic channel comprises a separation channel configured to perform isoelectric focusing separation. (Item 48) 48. The microfluidic chip of any one of items 46-47, wherein the substrate comprises a first separation channel and a second separation channel, a distal end of the first separation channel in fluid communication with a proximal end of the second separation channel, and a distal end of the second separation channel in fluid communication with the electrospray ionization orifice. (Item 49) Item 49. The microfluidic chip of item 48, wherein the first separation channel is configured to perform a chromatographic separation and the second separation channel is configured to perform an electrophoretic separation. (Item 50) Item 49. The microfluidic chip of item 48, wherein the first separation channel is configured to perform a chromatographic separation and the second separation channel is configured to perform an isoelectric focusing separation. (Item 51) 51. The microfluidic chip of any one of items 42-50, wherein the fluidic channel comprises a separation channel configured to perform isoelectric focusing separation of a sample comprising a mixture of analytes, and the substrate further comprises a mobilizing electrolyte channel in fluid communication with a distal end of the separation channel and configured to provide electrophoretic introduction of a mobilizing electrolyte to the distal end of the separation channel. (Item 52) 1. A method for performing electrospray ionization from a microfluidic chip, comprising: a) providing a microfluidic chip comprising a substrate, the substrate comprising: i) at least one fluid channel having a distal end in fluid communication with an electrospray ionization orifice; ii) at least one gas channel configured to deliver gas to a gas exit orifice adjacent to the electrospray ionization orifice; and b) flowing the solution through the at least one fluid channel such that the solution is expelled from the electrospray ionization orifice; c) flowing the gas through the at least one gas channel such that the gas is discharged through the gas exit orifice; Including, The method wherein the temperature of the substrate is controlled by the temperature of the gas flowing through the at least one gas channel. (Item 53) Item 53. The method according to item 52, wherein the temperature of the gas ranges from about 4°C to about 100°C. (Item 54) Item 54. The method according to item 52 or item 53, wherein the temperature of the substrate ranges from about 10°C to about 50°C. (Item 55) 55. The method according to any one of items 52-54, wherein the average temperature of the substrate is maintained at 30±5°C. (Item 56) 56. The method of any one of items 52-55, wherein the at least one fluidic channel comprises a separation channel. (Item 57) 57. The method of claim 56, wherein the separation channel is configured to perform isoelectric focusing separation of a sample comprising a mixture of analytes. (Item 58) 57. The method of claim 56, wherein the separation channel is configured to perform electrophoretic separation of a sample comprising a mixture of analytes. (Item 59) 59. The method of any one of items 52-58, wherein the electrospray ionization performance achieved when the microfluidic chip is configured to introduce a sample into a mass spectrometer is characterized by less than 1.0% standard error variation in total mass spectrometry signal intensity. (Item 60) 60. The method of any one of items 52-59, wherein the electrospray ionization performance when the microfluidic chip is configured to introduce a sample into a mass spectrometer is characterized by less than 0.1% standard error variation in total mass spectrometric signal intensity. (Item 61) 1. A method for providing stable electrospray ionization performance, comprising: a) providing a microfluidic chip comprising a substrate, the substrate comprising: (i) a fluid channel having a distal end in fluid communication with an electrospray ionization orifice; and (ii) a gas channel having a distal end in fluid communication with a gas exit orifice; b) flowing a solution through said fluid channel; c) flowing a gas through the gas channel; d) controlling the flow rate of the gas and the flow rate of the solution so that the ratio of the volumetric flow rates of the gas and the solution ranges from 1000:1 to 1,000,000:1; A method comprising: (Item 62) Item 62. The method according to item 61, wherein the volumetric flow rate ratio for the gas and the solution ranges from 10,000:1 to 500,000:1. (Item 63) 63. The method of claim 61 or 62, wherein the flow of the solution is controlled by pressure, gravity, electrokinetic force, or any combination thereof. (Item 64) 64. The method of any one of items 61-63, wherein the flow of gas is provided by a compressed gas source. (Item 65) 65. The method of any one of items 61-64, wherein the volumetric flow rate for the solution is less than 25 μL / min. (Item 66) 66. The method of any one of items 61-65, wherein the microfluidic chip comprises two or more gas channels, each with a distal end in fluid communication with a gas exit orifice, the two or more gas exit orifices being positioned symmetrically about and adjacent to the electrospray ionization orifice. (Item 67) 67. The method of any one of items 61-66, wherein the electrospray ionization orifice is positioned on an edge or corner of the substrate. (Item 68) Item 68. The method of item 67, wherein the one or more gas exit orifices are positioned adjacent to the electrospray ionization orifice on the edge of the substrate. (Item 69) 69. The method of any one of items 61-68, wherein the electrospray ionization performance achieved when the microfluidic chip is configured to introduce a sample into a mass spectrometer is characterized by less than 1.0% standard error variation in total mass spectrometry signal intensity. (Item 70) 70. The method of any one of items 61-69, wherein the electrospray ionization performance when the microfluidic chip is configured to introduce a sample into a mass spectrometer is characterized by less than 0.1% standard error variation in total mass spectrometric signal intensity. (Item 71) 1. A method for providing stable electrospray ionization performance, comprising: a) providing a microfluidic chip comprising a substrate, the substrate comprising: (i) a fluid channel having a distal end in fluid communication with an electrospray ionization orifice; and (ii) a gas channel having a distal end in fluid communication with a gas exit orifice; b) flowing a solution through said fluid channel; c) flowing a gas through the gas channel; d) controlling the flow rate of the gas and the flow rate of the solution so that the ratio of the flow rate of the gas at the gas exit orifice to the flow rate of the solution at the electrospray ionization orifice ranges from 100:1 to 1,000,000:1; A method comprising: (Item 72) Item 72. The method of claim 71, wherein the ratio of the flow rate for the gas at the gas exit orifice and the flow rate for the solution at the electrospray ionization orifice ranges from 500:1 to 5,000:1. (Item 73) Item 73. The method according to item 71 or 72, wherein the ratio of the flow rate for the gas at the gas exit orifice and the flow rate for the solution at the electrospray ionization orifice ranges from 1,000:1 to 3,000:1. (Item 74) 1. A microfluidic cartridge comprising: a) a microfluidic chip comprising at least one fluid port and at least two gas ports disposed on an edge of the microfluidic chip; b) a microfluidic cartridge component in fluid communication with the microfluidic chip and configured to contain at least a portion of the microfluidic chip, the microfluidic cartridge component comprising at least one fluid port and at least two gas ports that align with the at least one fluid port and at least two gas ports of the microfluidic chip; A microfluidic cartridge comprising: (Item 75) Item 75. The microfluidic cartridge of item 74, further comprising one or more elastomer components disposed between an edge of the microfluidic chip and a surface of the cartridge, wherein the one or more elastomer components form a substantially leak-free seal between the at least one fluid port and at least two gas ports of the microfluidic chip and the at least one fluid port and at least two gas ports of the microfluidic cartridge component in response to application of a force. (Item 76) 76. The microfluidic cartridge of claim 74 or 75, wherein the edge of the microfluidic chip is less than about 2.0 mm thick. (Item 77) 77. The microfluidic cartridge of any one of items 74-76, wherein the edges of the microfluidic chip are about 1±0.4 mm thick. (Item 78) 1. A system comprising: a) a microfluidic cartridge comprising two or more fluid ports and configured to be removable from the system; b) an apparatus with two or more fluid interconnections; Equipped with each of the two or more fluid interconnects is configured to provide a substantially leak-free fluid coupling between a fluid line of the instrument and a fluid port of the microfluidic cartridge in response to application of a force to an assembly comprising the two or more fluid interconnects and the two or more fluid ports of the microfluidic cartridge; the substantially leak-free fluid coupling is maintained when the relative fluid pressure in two of the two or more fluid lines varies by at least a factor of 10; system. (Item 79) Item 79. The system of item 78, wherein the substantially leak-free fluid coupling is maintained when the relative fluid pressure within two of the two or more fluid lines varies by at least 100 times. (Item 80) 80. The system of claim 78 or 79, wherein each of the two or more fluid interconnections comprises an independently spring-loaded fitting. (Item 81) Item 81. The system of item 80, wherein the independently spring-loaded fitting comprises a flat face seal fitting that mates with a fluid port comprising a bore in the microfluidic cartridge. (Item 82) 9. The microfluidic chip of any one of items 1-8, wherein the microfluidic device comprises three or more gas channels, each comprising a gas exit orifice positioned directly adjacent to the electrospray ionization orifice. (Item 83) Item 83. The microfluidic chip of item 82, wherein at least one of the three or more gas channels is disposed within the substrate, and at least one of the three or more gas channels is disposed within an auxiliary component of the microfluidic chip that is positioned directly adjacent to the substrate such that the at least one gas channel is not coplanar with the substrate. (Item 84) Item 84. The microfluidic chip of item 83, wherein at least one of the three or more gas channels disposed in the auxiliary component is positioned so that its gas exit orifice lies in a plane generally perpendicular to that of the substrate and is positioned symmetrically about and directly adjacent to the electrospray ionization orifice. (Item 85) Item 85. The microfluidic chip of item 84, wherein at least one of the three or more gas channels disposed in the auxiliary component is positioned so that its gas exit orifice is in one or more planes that are rotated relative to that of the substrate and positioned in a radially symmetric pair about and immediately adjacent to the electrospray ionization orifice. (Item 86) Item 48. The microfluidic chip according to item 47, wherein isoelectric focusing is performed while flowing fluid from the fluid inlet port through the separation channel. (Item 87) Item 49. The microfluidic chip of item 48, wherein electrospray ionization is performed while flowing fluid from the fluid inlet port through the first and second separation channels. (Incorporated by reference)

[0021] All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term in this specification and a term in an incorporated reference, the term in this specification shall control. [Brief explanation of the drawings]

[0022] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0023] [Figure 1A] FIG. 1A provides a non-limiting schematic diagram of a microfluidic chip with multiple channels for multiple isoelectric focusing reactions, according to one aspect of the present disclosure.

[0024] [Figure 1B] FIG. 1B provides a non-limiting schematic diagram of a fluid channel network of an exemplary microfluidic chip equipped with an electrospray tip for performing separation reactions according to another aspect of the present disclosure.

[0025] [Figure 2] 2A-2B provide non-limiting schematic diagrams of a microfluidic chip comprising gas and separation channels with inlet ports located at the edge of the device. Figure 2A shows the footprint of the microfluidic chip. Figure 2B shows a top-down view of the fluid channels and gas exit orifices.

[0026] [Figure 3] Figures 3A-3D provide non-limiting schematic illustrations of modifiable aspects (e.g., design parameters) of the microfluidic chips described herein. Figure 3A shows the angle between the distal end of a fluid channel and the distal end of a gas channel. Figure 3B shows the diameter of a gas exit orifice. Figure 3C shows the proximity between the distal end of a fluid channel and the distal end of a gas channel. Figure 3D shows the angle between the edge of the microfluidic chip and the distal end of a gas channel.

[0027] [Figure 4]4A-4B provide non-limiting examples of micrographs of one design of a microfluidic chip comprising a substrate with symmetric gas channels adjacent to a fluid channel (e.g., the end of a separation channel). Figure 4A shows a micrograph of one design of the microfluidic chip. Figure 4B shows a micrograph of another design of the microfluidic chip.

[0028] [Figure 5] 5A-5C provide additional non-limiting schematic diagrams of an exemplary microfluidic chip comprising a separation channel and a gas channel. FIG. 5A shows the footprint of the microfluidic chip. FIG. 5B shows a top-down view of the fluid channels and gas exit orifices. FIG. 5C shows an isometric view of the fluid channels and gas exit orifices.

[0029] [Figure 6] Figures 6A-6C provide additional non-limiting schematic diagrams of another embodiment of a microfluidic chip comprising a separation channel and a gas channel. Figure 6A shows the footprint of the microfluidic chip. Figure 6B shows a top-down view of the fluid channels and gas exit orifices. Figure 6C shows an isometric view of the fluid channels and gas exit orifices.

[0030] [Figure 7] 7A-7C provide additional non-limiting schematic diagrams of yet another example of a microfluidic chip comprising a separation channel and a gas channel. FIG. 7A shows the footprint of the microfluidic chip. FIG. 7B shows a top-down view of the fluid channels and gas exit orifices. FIG. 7C shows an isometric view of the fluid channels and gas exit orifices.

[0031] [Figure 8] 8A-8C provide additional non-limiting schematic diagrams of yet another example of a microfluidic chip comprising a separation channel and a gas channel. FIG. 8A shows the footprint of the microfluidic chip. FIG. 8B shows a top-down view of the fluid channels and gas exit orifices. FIG. 8C shows an isometric view of the fluid channels and gas exit orifices.

[0032] [Figure 9] 9A-9B provide additional non-limiting schematic illustrations of yet another example of a microfluidic chip comprising a separation channel and a gas channel with inlet ports on opposite edges of a substrate described herein. Figure 9A shows the footprint of the microfluidic chip. Figure 9B shows a top-down view of the fluid channels and gas exit orifices.

[0033] [Figure 10] Figures 10A-10C provide additional non-limiting schematic diagrams of another embodiment of a microfluidic chip comprising a separation channel and a gas channel. Figure 10A shows the footprint of the microfluidic chip. Figures 10B and 10C show top-down close-up views of the fluid channels and gas exit orifices.

[0034] [Figure 11] 11A-11C provide additional non-limiting schematic diagrams of another embodiment of a microfluidic chip comprising a separation channel and a gas channel. FIG. 11A shows the footprint of the microfluidic chip. FIG. 11B and FIG. 11C show isometric close-up views of the gas inlet and distal outlet sections, respectively.

[0035] [Figure 12-1] Figures 12A-12D provide exemplary schematics (Figures 12A-12C) and images (Figure 12D) of various distal ends (tips) of microfluidic chips. Figure 12A shows a schematic of an unshaped tip with gas and fluid orifices. Figure 12B shows a schematic of a faceted shaped tip with gas and fluid orifices. Figure 12C shows a schematic of a rounded shaped tip with gas and fluid orifices. Figure 12D shows an image of a faceted shaped tip with gas and fluid orifices. [Figure 12-2]Figures 12A-12D provide exemplary schematics (Figures 12A-12C) and images (Figure 12D) of various distal ends (tips) of microfluidic chips. Figure 12A shows a schematic of an unshaped tip with gas and fluid orifices. Figure 12B shows a schematic of a faceted shaped tip with gas and fluid orifices. Figure 12C shows a schematic of a rounded shaped tip with gas and fluid orifices. Figure 12D shows an image of a faceted shaped tip with gas and fluid orifices.

[0036] [Figure 13] FIG. 13 provides an example image of a fluid orifice of a microfluidic chip, comprising a fluid outlet channel and a symmetric gas channel, during electrospray ionization.

[0037] [Figure 14] 14A-14B provide additional example images of a fluid orifice of a microfluidic chip comprising a fluid outlet channel and a symmetric gas channel during electrospray ionization. Fig. 14A shows an image of illumination near the electrospray ionization orifice. Fig. 14B shows an image of illumination near the electrode plate.

[0038] [Figure 15]FIG. 15 provides examples of results from numerical simulations illustrating gas flow velocities around fluid exit channel orifices of devices described herein. Panel A shows simulation results for a device described herein. Panel B shows simulation results for another device described herein. Panel C shows simulation results for another device described herein. Panel "Concentric" shows simulation results for another device described herein. Panel D shows simulation results for another device described herein. Panel E shows simulation results for another device described herein. Panel F shows simulation results for another device described herein. Panel G shows simulation results for another device described herein.

[0039] [Figure 16] FIG. 16 provides example results from a numerical simulation illustrating gas shear rates around a fluid outlet channel orifice of a device described herein. Panel A shows simulation results for a device described herein. Panel B shows simulation results for another device described herein. Panel C shows simulation results for another device described herein. Panel "Concentric" shows simulation results for another device described herein. Panel D shows simulation results for another device described herein. Panel E shows simulation results for another device described herein. Panel F shows simulation results for another device described herein. Panel G shows simulation results for another device described herein.

[0040] [Figure 17]FIG. 17 provides examples of results from numerical simulations illustrating the velocity field around a fluid outlet channel orifice of a device described herein. Panel A shows simulation results for a device described herein. Panel B shows simulation results for another device described herein. Panel C shows simulation results for another device described herein. Panel "Concentric" shows simulation results for another device described herein. Panel D shows simulation results for another device described herein. Panel E shows simulation results for another device described herein.

[0041] [Figure 18] FIG. 18 provides examples of results from numerical simulations illustrating gas pressure fields around fluid outlet channel orifices of devices described herein. Panel A shows simulation results for a device described herein. Panel B shows simulation results for another device described herein. Panel C shows simulation results for another device described herein. Panel "Concentric" shows simulation results for another device described herein. Panel D shows simulation results for another device described herein. Panel E shows simulation results for another device described herein. Panel F shows simulation results for another device described herein. Panel G shows simulation results for another device described herein.

[0042] [Figure 19] FIG. 19 shows a plot comparing gas pressure for several device designs as a function of distance from the electrospray tip.

[0043] [Figure 20A]Figures 20A-20E illustrate the design of the microfluidic cartridge / instrument interface and its components. Figure 20A shows an exploded view. Figure 20B shows a view of the assembled unit. Figure 20C shows a cross-section of the assembly in the unloaded position. Figure 20D shows a cross-section of the assembly in the contacted position. Figure 20E shows a cross-section of the assembly in the sealed configuration. [Figure 20B] Figures 20A-20E illustrate the design of the microfluidic cartridge / instrument interface and its components. Figure 20A shows an exploded view. Figure 20B shows a view of the assembled unit. Figure 20C shows a cross-section of the assembly in the unloaded position. Figure 20D shows a cross-section of the assembly in the contacted position. Figure 20E shows a cross-section of the assembly in the sealed configuration. [Figure 20C] Figures 20A-20E illustrate the design of the microfluidic cartridge / instrument interface and its components. Figure 20A shows an exploded view. Figure 20B shows a view of the assembled unit. Figure 20C shows a cross-section of the assembly in the unloaded position. Figure 20D shows a cross-section of the assembly in the contacted position. Figure 20E shows a cross-section of the assembly in the sealed configuration. [Figure 20D] Figures 20A-20E illustrate the design of the microfluidic cartridge / instrument interface and its components. Figure 20A shows an exploded view. Figure 20B shows a view of the assembled unit. Figure 20C shows a cross-section of the assembly in the unloaded position. Figure 20D shows a cross-section of the assembly in the contacted position. Figure 20E shows a cross-section of the assembly in the sealed configuration. [Figure 20E]Figures 20A-20E illustrate the design of the microfluidic cartridge / instrument interface and its components. Figure 20A shows an exploded view. Figure 20B shows a view of the assembled unit. Figure 20C shows a cross-section of the assembly in the unloaded position. Figure 20D shows a cross-section of the assembly in the contacted position. Figure 20E shows a cross-section of the assembly in the sealed configuration.

[0044] [Figure 21] 21A-21C schematically illustrate perspective views of a fitting assembly at the microfluidic cartridge / instrument interface. Fig. 21A shows a perspective view of the assembly in an unloaded position, Fig. 21B shows a perspective view of the assembly in a contacted position, and Fig. 21C shows a perspective view of the assembly in a sealed configuration.

[0045] [Figure 22] 22A-22C schematically illustrate designs for connecting microfluidic chip and cartridge components and assembling a microfluidic cartridge, in which the interface comprises an elastomeric component. FIG. 22A shows a schematic of the microfluidic chip affixed to the cartridge. FIG. 22B shows a schematic of the elastomeric component. FIG. 22C shows a schematic of a set of connected elastomeric components.

[0046] [Figure 23] FIG. 23 illustrates an example of a software architecture system as described herein.

[0047] [Figure 24] FIG. 24 shows an example block diagram of the integrated system described herein.

[0048] [Figure 25] FIG. 25 shows an example block diagram of another integrated system described herein. DETAILED DESCRIPTION OF THE INVENTION

[0049] Detailed Description Disclosed herein are methods, devices, and systems for atomizing a sample while introducing the sample into an analytical instrument (e.g., a mass spectrometer). One or more of the methods, devices, and systems disclosed herein may additionally include performing an isoelectric focusing reaction (or other separation reaction) on the analyte mixture, followed by mobilization of the separated analytes and introduction into the mass spectrometer. The introduction of the separated analytes may be performed using electrospray ionization, and sample atomization may provide greater precision, control, and improved analytical performance of downstream analytical approaches (e.g., mass spectrometry). The methods, devices, and systems disclosed herein may also enable rapid and accurate separation and characterization of protein analyte mixtures or other biological molecules by isoelectric point (or other physicochemical property).

[0050] In certain aspects, disclosed herein is a microfluidic chip comprising a substrate having a separation channel and a gas channel. In some cases, the separation channel is used to perform an isoelectric focusing reaction and comprises a distal end in fluid communication with a fluid channel outlet. The fluid channel outlet can be part of or comprise an electrospray ionization orifice, which may be used to interface a sample or separated samples into an analytical instrument (e.g., a mass spectrometer). The microfluidic chip used herein may additionally comprise an inlet port in fluid communication with the gas channel and the separation channel, which may be positioned along the edge of the substrate (i.e., the surface of the substrate defined by the maximum and minimum dimensions (e.g., length and depth) of the substrate footprint). The inlet port may be fluidically and / or electrically coupled to a channel or reservoir comprising reagents for performing one or more reactions, such as a separation reaction, a mobilization reaction, electrospray ionization, etc. In some cases, the substrate includes an electrospray ionization (ESI) tip, which is used to mobilize and eject the sample (or separated sample) via ESI. The ESI tip may be positioned on the edge of the substrate. In some cases, the ESI tip and the outlet of the gas channel (e.g., a gas outlet orifice) are positioned adjacent to each other on the edge of the substrate. In some cases, the sample (or separated sample) is introduced into an analytical instrument (e.g., a mass spectrometer).

[0051] In some cases, a gas channel is used to atomize a sample (or separated sample) from the ESI tip during ESI. Atomization is achieved by shear and inertial forces generated when a gas jet breaks a continuous liquid stream into small droplets. Sample atomization (or separated sample) may be used to improve quantitative measurement of a sample (or separated sample) under nanoflow, where the sample (or separated sample) is flowed through an ESI tip at approximately nanoliter-scale flow rates (e.g., nanoliters / minute). Sample atomization (or separated sample) may be used to improve quantitative measurement of a sample (or separated sample) under microflow, where the sample (or separated sample) is flowed through an ESI tip at approximately microliter-scale flow rates (e.g., microliters / minute). In some cases, atomization of the sample (or separated sample) reduces ion suppression, increases ionization across ion species, reduces contamination, provides more stable electrospray performance, decouples droplet formation from the ESI potential, and / or provides better accuracy. In some cases, gas channels integrated into the microfluidic chip allow for greater precision in the placement of gas for atomization relative to the separation channel or ESI tip, laminar gas flow, greater dimensional control, etc. In some cases, gas channels may be used to clean or dry the ESI tip or to direct waste products from the separation channel or ESI tip away from a downstream analysis unit (e.g., a mass spectrometer). In some cases, gas channels are used to control the temperature of the substrate (e.g., by modifying the temperature of the gas flowing through the gas channels).

[0052] The integration of gas channels into the substrate of a device can provide certain benefits and advantages. For example, greater precision in the placement of gas flows relative to fluid channel orifices can be achieved compared to external units configured to couple to the device. For example, placement of gas channel orifices relative to fluid channel orifices using standard fabrication approaches can achieve placement precision of approximately + / - 100 μm, compared to external units, which can achieve placement precision of approximately + / - 2 μm. Furthermore, the integration of gas channels into a microfluidic format can be advantageous in achieving laminar gas flow, which can help eliminate or prevent vortices or turbulence at or near the fluid channel orifices, which can provide more stable electrospray. Additionally, the proximity of the gas flow to the liquid / separation channel flow allows the gas flow to more effectively affect the liquid flow.

[0053] In another aspect of the present disclosure, provided herein is a microfluidic chip comprising a separation channel and a gas channel, wherein a portion of the gas channel is substantially parallel to a portion of the separation channel. In some cases, the gas channel and separation channel are disposed on a substrate such that the angle (also herein "convergence angle") between the distal end of the separation channel (or the distal end of the fluid outlet channel (also herein "fluid channel")) and the distal end of the gas channel connected to the separation channel ranges from about 0 degrees to about 45 degrees.

[0054] In some cases, the angle between the distal end of the gas channel and the distal end of the fluid outlet channel is about 0 degrees (parallel, non-converging), about 5 degrees, about 10 degrees, about 15 degrees, about 20 degrees, about 25 degrees, about 30 degrees, about 35 degrees, about 40 degrees, about 45 degrees, about 50 degrees, about 55 degrees, about 60 degrees, about 65 degrees, about 70 degrees, about 75 degrees, about 80 degrees, about 85 degrees, or about 90 degrees. In some cases, the angle between the distal end of the gas channel and the distal end of the fluid outlet channel is at least about 0 degrees, at least about 5 degrees, at least about 10 degrees, at least about 15 degrees, at least about 20 degrees, at least about 25 degrees, at least about 30 degrees, at least about 35 degrees, at least about 40 degrees, at least about 45 degrees, at least about 50 degrees, at least about 55 degrees, at least about 60 degrees, at least about 65 degrees, at least about 70 degrees, at least about 75 degrees, at least about 80 degrees, at least about 85 degrees, or at least about 90 degrees. In some cases, the angle between the distal end of the gas channel and the distal end of the fluid outlet channel is at most about 90 degrees, at most about 85 degrees, at most about 80 degrees, at most about 75 degrees, at most about 70 degrees, at most about 65 degrees, at most about 60 degrees, at most about 55 degrees, at most about 50 degrees, at most about 45 degrees, at most about 40 degrees, at most about 35 degrees, at most about 30 degrees, at most about 25 degrees, at most about 20 degrees, at most about 15 degrees, at most about 10 degrees, at most about 5 degrees, or at most about 0 degrees. Angles, for example, between about 10 degrees and 30 degrees, can also fall within the ranges of values ​​recited herein.

[0055] In another aspect, disclosed herein is a microfluidic chip comprising a substrate comprising a gas channel and at least one inlet port located along the edge of the substrate. In some cases, the substrate comprises a gas channel, a separation channel, and a gas channel, each separation channel comprising an inlet port located on the edge of the substrate. In some cases, the substrate comprises a fluid channel, two gas channels, at least one fluid inlet port (e.g., connected to a separation channel), and at least two gas inlet ports, wherein the ports are located along the edge of the first substrate.

[0056] In another aspect of the present disclosure, provided herein is a microfluidic chip comprising two or more gas channels, each having a different length and configured to deliver gas to a gas exit orifice (also herein, "gas channel outlet") located on an edge or, in some cases, a corner of a substrate. In some cases, the gas flow exiting the two gas exit orifices (of one or more gas channels) is configured to converge within a fluid path of a liquid exiting the fluid exit orifice (of a fluid channel). In some cases, the overall length of the gas channel may differ from the cross-sectional area of ​​all or part of each of the two or more gas channels, and each of the two or more gas channels may be adjusted to have approximately the same hydrodynamic flow resistance. In some cases, the gas channel may be narrowed at the outlet to increase the linear flow rate of the gas flow. In some cases, the gas channel may be configured to achieve supersonic (faster than sound) flow velocities at its outlet. In some cases, the configuration may consist of a narrowing gas channel section, followed by an even narrower "choke" section, and then an expanding section to achieve supersonic flow velocities.

[0057] In some cases, the microfluidic chip includes a fluid channel (e.g., a separation channel) in fluid communication (e.g., at a distal end) with a fluid outlet channel, the fluid outlet channel including a fluid outlet orifice that can function as an ESI orifice. In some cases, the fluid outlet orifice is located on an edge or corner of the substrate, and the gas channel outlet may be located adjacent to the fluid outlet orifice on the edge or corner of the substrate. In some cases, the corner on which the ESI orifice is located includes an edge on which the fluid outlet orifice is located. In other cases, the corner on which the ESI orifice is located does not include an edge on which the fluid outlet orifice is located. In some cases, the flow rate of the gas in the gas channel and / or the flow rate of the liquid in the fluid channel can be controlled or adjusted such that the ratio of the volumetric flow rates for the gas and liquid ranges from 1000:1 to 1,000,000:1. In some cases, the flow rate of the gas in the gas channel and / or the flow rate of the liquid in the fluid channel can be controlled or adjusted such that the ratio of flow rates for the gas and liquid ranges from 100:1 to 10,000:1.

[0058] In another aspect, provided herein is a cartridge component configured to interface with a microfluidic chip in an assembled microfluidic cartridge. The microfluidic chip may include two or more fluid ports disposed on the edge of the device substrate, and the cartridge component may include two or more fluid ports disposed on the surface or edge of the cartridge, the fluid ports configured to interface with the fluid ports of the microfluidic chip. In some cases, the fluid ports of the cartridge component align with those of the microfluidic chip, for example, when the chip is secured in the assembled microfluidic cartridge. The assembled microfluidic cartridge may include one or more elastomeric components positioned between the edge of the microfluidic chip and the surface of the cartridge component (e.g., at the interface of the aligned fluid ports). In some cases, application of force to an assembly including the microfluidic chip and the cartridge component is used to secure the microfluidic chip in the assembled microfluidic cartridge and establish fluid communication between the ports of the microfluidic chip and the ports of the cartridge. In some cases, the assembled microfluidic cartridge is configured to provide a substantially leak-free fluid coupling between the cartridge components and the microfluidic chip, and in some cases, the leak-free fluid coupling is maintained upon introduction of gas into the gas channels of the microfluidic chip.

[0059] In another aspect, disclosed herein is an interface design for removably connecting a microfluidic cartridge to an instrument system, the interface design configured to establish fluid communication between at least one channel of the microfluidic cartridge and a fluid line external to the microfluidic cartridge. The interface design may comprise one or more fluid interconnects, each configured to provide a substantially leak-free fluidic coupling between an external fluid line (e.g., connected to an instrument, such as a reservoir) and a fluid port of the microfluidic cartridge. In some cases in which an assembled microfluidic cartridge comprising cartridge components and a microfluidic chip is used, the interface comprises one or more fluid interconnects, each configured to provide a substantially leak-free fluidic coupling between the external fluid line and the assembled microfluidic cartridge, which in turn may provide substantially leak-free fluidic communication with the cartridge components and / or the microfluidic chip of the microfluidic cartridge. A substantially leak-free fluid coupling can be maintained even when the relative fluid pressure within two of the two or more external fluid lines varies by at least a factor of 10, as will be explained below. In some cases, the interface design comprises at least one independently spring-loaded coupling, which may be used to establish fluid communication between the microfluidic cartridge and the external fluid line. In some cases, the cartridge may simultaneously deliver gas and liquid to the chip.

[0060] In some embodiments of the present disclosure, a microfluidic chip comprises a planar substrate comprising two or more separation channels for parallel and multiplexed separation reactions and, optionally, two or more gas channels for parallel and multiplexed atomization of separated samples for downstream analysis (e.g., via ESI-MS). In a preferred aspect, the separation reaction is an isoelectric focusing reaction. In another preferred aspect, the analyte mixture consists of a protein analyte mixture, and performing two or more parallel isoelectric focusing reactions allows for rapid and accurate separation of protein components within the analyte mixture and characterization of individual protein components by their isoelectric points (pI). In some cases, the use of imaging, e.g., full-channel imaging, in combination with pI markers to visualize the position of the pI markers in the pH gradient used for isoelectric focusing, allows for more accurate determination of pI for the separated protein components of the analyte mixture.

[0061] In certain aspects of the present disclosure, methods and systems for operating microfluidic devices or cartridges use two or more high-voltage power supplies (or a single multiplexed high-voltage power supply), which allows for independent control of separation reactions or experimental conditions within each separation channel of the microfluidic chip. Thus, in some cases, a microfluidic chip may be used to perform separation and characterization of two or more different samples under the same set of separation or experimental conditions in parallel. In some cases, a microfluidic chip may be used to perform separation and characterization of two or more aliquots of the same sample under two or more different reaction or experimental conditions in parallel. In some cases, a subset of separation channels on a device may be used to perform separation of multiple samples under the same set of separation or experimental conditions; alternatively, or in addition, different subsets of separation channels on a device may be used to perform separation and characterization of multiple aliquots from the same sample under multiple different reaction or experimental conditions in parallel. In some cases, the device includes one or more gas channels for each subset of separation channels that are used to atomize the sample within each of the separation channels for introduction of the separated sample into the mass spectrometer (e.g., via atomization during ESI).

[0062] The conditions may be the same or may vary across separation channels of the microfluidic chip and may comprise buffer selection, electrolyte selection, pH gradient selection, voltage setting, current setting, field strength setting, time course for varying voltage setting, current setting, field strength setting, isoelectric focusing reaction time, or any combination thereof.

[0063] In some cases, the system may further include an autosampler or fluid handling system configured for automatic, independently controlled loading of sample aliquots and / or other reagents (e.g., for separation reagents, gases for mobilization, electrospray ionization, nebulization) into one or more inlet ports. In some cases, the system may further include a fluid flow controller configured to provide, for example, independently controlled pressure-driven flow through two or more channels (e.g., to deliver reagents to the fluidic and / or gasic channels). In some cases, the system may further include an autosampler or fluid flow controller configured to flush, wash, rinse, or drain the fluidic channels following a separation reaction (e.g., an isoelectric focusing reaction). In some cases, following the flushing, washing, rinsing, or draining of the separation channel, the autosampler or fluid flow controller may be configured to automatically introduce another sample (e.g., a different sample or another aliquot of the same sample) into two or more separation channels. In some cases, the autosampler or fluid flow controller may be configured to automatically reintroduce sample, reaction reagents, or a combination thereof into one or more separation channels if a disturbance (e.g., air bubble formation or introduction, an incorrectly prepared sample, an underfilled reagent reservoir, or a combination thereof) is detected (e.g., via voltage or current monitoring). In such cases, following detection of the disturbance, the autosampler or fluid flow controller may flush the disturbed separation channel and reintroduce sample, reaction reagents, or a combination thereof, and the separation reaction may be resumed (e.g., via application of an electric field by one or more independently controlled voltage sources).

[0064] In some cases, the system may further include an imaging module configured to obtain a series of one or more images of the separation channel and / or the gas channel or an outlet of either of the channels. In some cases, the image field of view may include all or a portion of the separation channel or the gas channel. In some cases, the image field of view may include all or a portion of the fluid channel or the fluid channel outlet. In some cases, the imaging may include continuous imaging while the separation reaction, the mobilization reaction, and / or the electrospray ionization is performed. In some cases, the imaging may include intermittent imaging while the separation reaction, the mobilization reaction, and / or the electrospray ionization is performed.

[0065] In some cases, imaging may comprise continuous or intermittent imaging while electrospray ionization and / or atomization is performed. In some cases, imaging may include obtaining UV absorbance images. In some cases, imaging may include fluorescence images, for example, of either intrinsic fluorescence or fluorescence due to the presence of exogenous fluorescent labels attached to the analyte. In some cases, imaging may be used to determine parameters of ESI or the Taylor cone formed during ESI. In some cases, parameters include Taylor cone shape, ESI jet, ESI plume, atomization efficiency, flow rate, droplet size, gas pressure, liquid pressure, ESI stability, ESI emitter contamination, and bubbles in the fluid stream.

[0066] In another aspect of the present disclosure, a system is described that may include a microfluidic chip designed to perform one or more separation reactions, e.g., isoelectric focusing reactions, to separate a sample comprising a mixture of analytes into its individual components, followed by electrospray ionization of the separated analytes, with or in parallel with sample atomization. In some cases, the microfluidic chip may be housed within a cartridge, further comprising, e.g., high-voltage electrode connections, reagent reservoirs, valves, anchoring mechanisms, fittings, channels, etc. In some cases, the microfluidic chip may comprise a generally planar substrate, the planar substrate comprising at least one gas channel and a separation channel separation reaction configured to perform, e.g., an isoelectric focusing reaction. In some cases, the gas channel is used for sample atomization during electrospray ionization. In some cases, the gas channel is used to move liquid in the separation channel away from the separation channel (e.g., away from an analytical instrument, e.g., a mass spectrometer, toward a waste receptacle, etc.). In some cases, the substrate further comprises an electrospray ionization tip at the distal end of the separation channel, and the gas channel may be used to clean or dry the electrospray tip.

[0067] In some cases, a first end of one or more separation channels of the plurality of separation channels is electrically and / or fluidically coupled to an electrode (e.g., anolyte) reservoir using a fixture, which may comprise a membrane. In some cases, a second end of one or more separation channels is electrically and / or fluidically coupled to an electrode (e.g., catholyte reservoir) using a fixture, which may comprise a membrane. The membrane may be an electrode positioned within the reservoir in a plane that defines or is parallel to the surface of the electrode reservoir, or adjacent to it, which plane may intersect the inlet and outlet fluid channels. In some cases, the system may further comprise an analytical instrument, such as a mass spectrometer. The disclosed methods, devices, and systems enable improved reproducibility and quantitative accuracy of separation data and also enable improved correlation between separation data and downstream analytical characterization data, such as that obtained using a mass spectrometer or other analytical instrument.

[0068] Another feature of the disclosed methods, devices, and systems, as set forth above, is the use of images to monitor a separation reaction within a separation channel for the purpose of detecting the presence of an analyte peak and / or to determine when the separation reaction has reached completion. In some cases, images may be obtained for all or a portion of the separation channel. In some cases, imaging of all or a portion of the separation channel may be performed while the separation step and / or mobilization step is being performed. In some cases, images may be used to detect the presence of one or more markers or indicators, e.g., isoelectric point (pI) standards, within the separation channel and thus determine the pI for one or more analytes. In some cases, images may be used to detect obstructions (e.g., bubble formation) within the separation channel. In some cases, data derived from such images may be used to determine when the separation reaction is complete (e.g., by monitoring peak velocity, peak position, and / or peak width) and subsequently trigger a mobilization step.

[0069] In some cases, the mobilization step may include introducing a mobilization buffer or mobilization electrolyte into the separation channel. In some cases, the mobilization buffer or mobilization electrolyte may be introduced using hydrodynamic pressure. In some cases, the mobilization buffer or mobilization electrolyte may be introduced using electrophoresis. In some cases, the mobilization buffer or mobilization electrolyte may be introduced using a combination of electrophoresis and hydrodynamic pressure. In some cases, mobilizing a series of one or more separated analyte bands may include migrating the separated analyte bands toward an outlet or distal end of the separation channel. In some cases, mobilizing a series of one or more separated analyte bands may include migrating the separated analyte bands toward an outlet or distal end of the separation channel that is in fluid communication with a downstream analytical instrument. In some cases, the outlet or distal end of the separation channel may be in fluid communication with an electrospray ionization (ESI) interface such that the migrating analyte peaks are injected into a mass spectrometer. In some cases, the image data used to detect analyte peak positions and determine analyte pIs may also be used to correlate analyte separation data with mass spectrometry data. In some cases, the image data used to detect analyte peak positions may be used to generate information about the recruitment reaction and / or to correlate the recruitment information with mass spectrometry data.

[0070] Another important feature of the disclosed methods, devices, and systems as described above is the use of imaging to monitor sample atomization (e.g., during the electrospray ionization reaction). Imaging may be used to detect the presence of a Taylor cone. In some cases, images may be obtained of all or a portion of the separation channel, the electrospray ionization tip, or the region between the device (e.g., a microfluidic chip) and the analytical instrument (e.g., a mass spectrometer or a grounded electrode plate). In some cases, imaging of all or a portion of the ESI tip may be performed while ESI is being performed. In some cases, imaging may be used to detect the presence of a Taylor cone. In some cases, imaging may be used to determine parameters of the Taylor cone, such as droplet size, Taylor cone shape, Taylor cone size, ESI jet shape, ESI jet size, ESI plume shape, ESI plume size, flow rate, gas pressure, and liquid pressure.

[0071] In preferred aspects, the disclosed methods may be implemented in a microfluidic device format, thereby enabling the processing of extremely small sample volumes and the integration of two or more sample processing and separation steps. In another preferred aspect, the disclosed microfluidic devices and cartridges include an integrated interface for coupling to downstream analytical instruments, such as an ESI interface for performing mass spectrometry on the separated analytes. In some cases, the disclosed methods may be implemented in a more traditional capillary format.

[0072] Various aspects of the disclosed methods, devices, and systems described herein may be applied to any of the specific applications described below. It should be understood that different aspects of the disclosed methods, devices, and systems may be understood individually, collectively, or in combination with one another.

[0073] Definitions: Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0074] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to encompass "and / or" unless stated otherwise. Similarly, the terms "comprise," "comprises," "comprising," "include," "includes," and "including" are not intended to be limiting.

[0075] As used herein, the phrases "including but not limited to" and "one non-limiting example is" are intended to include variations and derivatives of a given example, as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0076] As used herein, the term "about" a number refers to that number plus or minus 10%. When used in connection with a range, the term "about" refers to a range of -10% from the lowest value to +10% from the highest value.

[0077] As used herein, the terms "characterization" and "analysis" may be used interchangeably. "Characterizing" or "analyzing" may generally mean assessing a sample, for example, determining one or more properties of the sample or its components, or determining the identity of the sample.

[0078] As used herein, the terms "chip" and "device" may be used interchangeably herein.

[0079] As used herein, the terms "analyte" and "species" may be used interchangeably. An analyte generally refers to a molecule, biomolecule, substance, macromolecule, etc. that differs in a measurable property from another molecule, biomolecule, chemical, macromolecule, etc. For example, two species may have slightly different masses, hydrophobicity, charge or net charge, isoelectric points, potencies, or may differ in terms of chemical modifications, protein modifications, etc.

[0080] As used herein, a "fluid channel" generally refers to a channel of a device (e.g., a microfluidic chip) configured to transport a fluid, e.g., a gas or a liquid (such as a solution), within the channel. In some cases, the fluid is transported from a proximal end of the channel toward a distal end of the channel.

[0081] As used herein, a "gas channel" generally refers to a fluid channel configured to transport a gas within the channel. In some cases, the gas is transported from a proximal end of the channel toward a distal end of the channel.

[0082] As used herein, a "microfluidic device" generally refers to a microfluidic chip, e.g., a glass or polymer substrate, comprising one or more fluid channels. In some cases, a "microfluidic device" may further comprise additional components, such as a holder, into which the microfluidic chip is mounted to facilitate ease of handling. In some cases, a "microfluidic device" may refer to a microfluidic chip that is attached to or mounted within a more complex "cartridge component" that may comprise additional functional features, such as reagent reservoirs, valves, fluid connectors, etc., to create a "microfluidic cartridge." In some cases, an assembly comprising a microfluidic chip and a cartridge component may be referred to as a "microfluidic device" or a "microfluidic cartridge."

[0083] Sample: The disclosed methods, devices, systems, and software may be used for the separation and characterization of analytes obtained from any of a variety of biological or non-biological samples. Examples include, but are not limited to, tissue samples, cell culture samples, whole blood samples (e.g., venous, arterial, or capillary blood samples), plasma, serum, saliva, interstitial fluid, urine, sweat, tears, protein samples derived from industrial enzyme or biological drug manufacturing processes, environmental samples (e.g., air samples, water samples, soil samples, surface swipe samples), and the like. In some embodiments, samples may be processed using any of a variety of techniques known to those skilled in the art prior to analysis using the disclosed methods and devices for integrated chemical separation and characterization. For example, in some embodiments, samples may be processed to extract proteins or nucleic acids. Samples may be collected from any of a variety of sources or subjects, such as bacteria, viruses, plants, animals, or humans.

[0084] Sample Volume: In some instances of the disclosed methods and devices, the use of a microfluidic device format may enable the processing of very small sample volumes. In some embodiments, the sample volume loaded into the device and used for analysis may range from about 0.1 μl to about 1 ml. In some embodiments, the sample volume loaded into the device and used for analysis may be at least 0.1 μl, at least 1 μl, at least 2.5 μl, at least 5 μl, at least 7.5 μl, at least 10 μl, at least 25 μl, at least 50 μl, at least 75 μl, at least 100 μl, at least 250 μl, at least 500 μl, at least 750 μl, or at least 1 ml. In some embodiments, the sample volume loaded into the device and used for analysis may be at most 1 ml, at most 750 μl, at most 500 μl, at most 250 μl, at most 100 μl, at most 75 μl, at most 50 μl, at most 25 μl, at most 10 μl, at most 7.5 μl, at most 5 μl, at most 2.5 μl, at most 1 μl, or at most 0.1 μl. Any of the lower and upper limits described in this paragraph may be combined to form a range included within the present disclosure; for example, in some embodiments, the sample volume loaded into the device and used for analysis may range from about 5 μl to about 500 μl. One of skill in the art will recognize that the sample volume used for analysis may have any value within this range, for example, about 18 μl.

[0085] Analytes: In some cases, a sample may consist of multiple analyte species. In some cases, all or a portion of the analyte species present in a sample may be enriched prior to or during analysis. In some cases, these analytes may be, for example, glycans, carbohydrates, nucleic acid molecules (e.g., DNA, RNA), peptides, polypeptides, recombinant proteins, intact proteins, protein isoforms, digested proteins, fusion proteins, antibody-drug conjugates, protein-drug conjugates, metabolites, or other biologically relevant molecules. In some cases, these analytes may be small molecule drugs. In some cases, these analytes may be protein molecules within protein mixtures, such as biological protein preparations (e.g., enzyme or antibody preparations) and / or lysates collected from cells isolated from culture or in vivo.

[0086] Microfluidic Device: Disclosed herein are devices designed to perform atomization of a sample or separated sample (e.g., a mixture of analytes separated via isoelectric focusing) at or near a fluidic orifice in a substrate of the device. In some cases, the disclosed devices are microfluidic devices comprising a substrate having a separation channel and one or more gas channels, where the gas channels are used to atomize the sample, e.g., a sample comprising analytes separated using a separation reaction such as isoelectric focusing. Sample atomization may be used to break up a liquid into small liquid droplets or the like (e.g., via breaking the surface tension of the droplets) at or near the fluidic orifice (e.g., at or near the distal end of the separation channel or the distal end of a fluidic outlet channel fluidically coupled to the separation channel) to achieve nanoflow or substantially nanoscale volumetric ejection of the sample. In some cases, the fluidic orifice comprises or is configured to be an electrospray tip, and sample atomization may be used to achieve nanoflow during electrospray ionization.

[0087] In some cases, the device may include a substrate having a plurality of gas channels. The substrate may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more gas channels. The substrate may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 gas channels. The substrate may comprise up to 20, up to 19, up to 18, up to 17, up to 16, up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 channel. The substrate may have varying and different gas channels, for example, ranging from 2 to 4 gas channels.

[0088] As described herein, the location of one or more gas channel outlets (also herein referred to as "gas outlet orifices") may be located adjacent to the exit orifice of a fluid channel (also herein referred to as "fluid channel orifice"), which may comprise or be configured to serve as an electrospray ionization orifice. In some cases, the gas channel outlet is positioned about 0 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, or more, from the fluid channel orifice. In some cases, the gas channel outlet is positioned at least about 0 μm, at least about 1 μm, at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 6 μm, at least about 7 μm, at least about 8 μm, at least about 9 μm, at least about 10 μm, at least about 15 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 150 μm, at least about 200 μm, at least about 250 μm, at least about 300 μm, at least about 350 μm, at least about 400 μm, or more, from the fluid channel orifice.In some cases, the gas channel outlet is positioned at most about 400 μm, at most about 350 μm, at most about 300 μm, at most about 250 μm, at most about 200 μm, at most about 150 μm, at most about 100 μm, at most about 90 μm, at most about 80 μm, at most about 70 μm, at most about 60 μm, at most about 50 μm, at most about 40 μm, at most about 30 μm, at most about 20 μm, at most about 15 μm, at most about 10 μm, at most about 9 μm, at most about 8 μm, at most about 7 μm, at most about 6 μm, at most about 5 μm, at most about 4 μm, at most about 3 μm, at most about 2 μm, at most about 1 μm, or at most about 0 μm from the fluid channel orifice. The gas channel outlet may be positioned within a range of values, for example, 10 μm to 100 μm, from the fluid channel orifice.

[0089] In some embodiments, the separation and gas channels may exit the chip in a generally coplanar orientation. In some embodiments, the separation and gas channels may be generally non-coplanar. In some embodiments, the separation channel may protrude from the plane formed by the gas channel by a distance of 0-500 μm. In some embodiments, the intersection between the fluidic separation channel and the gas channel may be shaped such that the gas channel exit plane is recessed into the microfluidic device relative to the separation channel. In some preferred embodiments, the separation channel nominally exits from a corner of the chip, bisecting the corner with the adjacent edges. In some embodiments, the gas channel exits, which terminate along each of the edges of the adjacent substrate, form a plane at the separation channel orifice that is necessarily recessed (by geometry) when viewed along the axis of the separation channel.

[0090] As described herein, the gas exit orifice or fluid channel orifice may be located at an edge or a corner or tip of the substrate, respectively. The edge of the substrate may, in some cases, be defined by the surface of the substrate with the longest and shortest dimensions (e.g., the surface of the substrate defined by the length and thickness of the device, see, e.g., FIG. 2A ). The substrate may have features in the shape of a trapezoid or tip. In a preferred embodiment, the substrate comprises a fluid channel orifice and two gas exit orifices fluidly coupled to two gas channels. In some cases, the two gas exit orifices are positioned symmetrically from the fluid channel orifice or axis defined by the fluid flow path exiting the fluid channel orifice. In other cases, the gas exit orifice is positioned asymmetrically from the fluid channel orifice or axis defined by the fluid flow path exiting the fluid channel orifice.

[0091] The substrate footprint may have any useful geometric shape, such as, for example, a rectangle, a circle, an oval, a triangle, a square, a diamond, a pentagon, etc. In preferred aspects, the substrate may have a generally rectangular footprint. In some cases, the longest dimension of the generally rectangular footprint ranges from about 10 to about 100 mm. In some cases, the shortest dimension of the generally rectangular footprint ranges from about 2 to about 50 mm. In some cases, the thickness of the generally rectangular footprint ranges from about 0.5 mm to about 2 mm. In some embodiments, the thickness of the generally rectangular footprint can be 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2 mm. In some embodiments, the substrate exit orifice may be further shaped into a wedge, pyramid, cone, or other three-dimensional shape. The shape may include flat features where some or all of the channels (gas or fluid) exit. In some embodiments, the substrate surface may be chemically modified to alter its surface energy or to make it more hydrophobic or hydrophilic. In some embodiments, the surface may maintain a predefined contact angle with the fluid. In some embodiments, the surface may be prepared to be micro-roughened by laser treatment, co-deposition of nanoparticles, or other means known in the art to enhance hydrophobicity or hydrophilicity as part of the modification.

[0092] The cross-section of any of the channels and / or orifices (e.g., fluid channels, gas channels) described herein may take any useful geometric shape, e.g., circular, rectangular, oval, triangular, square, diamond, etc. In certain preferred embodiments, the cross-sectional shape of the electrospray ionization orifice is approximately square or rectangular.

[0093] In some cases, it may be useful to use more than one gas channel for atomization. For example, it may be useful to have two or more gas channels for atomization, particularly with gas outlets on opposite sides of the fluid outlet orifice. In some cases, the substrate may include one or more pairs of gas channels flanking the fluid outlet orifice. In some cases, the substrate may include four or more gas channels. In such cases, at least two of the four or more gas channels may be disposed within an auxiliary component and positioned such that the gas outlet orifices lie in one or more planes rotated relative to that of the substrate. For example, a pair of gas channels and their gas outlet orifices may be positioned relative to the fluid outlet orifice such that the gas channels are radially symmetric from the fluid channel and fluid outlet orifice. In one such example, the fluid channel orifice may be surrounded by two orthogonal or perpendicular planes of the gas channel orifice, and the gas outlets are each radially symmetric from the fluid channel orifice. In another example, the fluid channel orifice may be surrounded by two planar surfaces of the gas channel orifice, one or more of which are rotated relative to the substrate and positioned in a radially symmetrical pair about and adjacent to the electrospray ionization orifice. In an embodiment, the gas channel may have an annular cross-section such that the gas channel orifice is concentric with the outlet of the fluid orifice.

[0094] The gas flow from one or more gas channels may be configured to atomize the sample at the fluid orifice or at a distance from the fluid orifice. For example, the gas may be atomized at about 1 micrometer (μm), about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm from the fluid orifice (e.g., in an axial distance, where the axis is the axis of the direction of the fluid exiting the fluid orifice). The sample may be atomized at a distance of about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, or about 1000 μm. The gas may atomize the sample at a distance of less than about 1000 μm, about 950 μm, about 900 μm, about 850 μm, about 800 μm, about 750 μm, about 700 μm, about 650 μm, about 600 μm, about 550 μm, about 500 μm, about 450 μm, about 400 μm, about 350 μm, about 300 μm, about 250 μm, about 200 μm, about 150 μm, about 100 μm, about 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, about 15 μm, about 10 μm, about 5 μm, about 1 μm, or less from the fluid orifice. The gas may atomize the sample at a distance within the range of values ​​described herein, for example, about 50 μm to 300 μm. In some embodiments, the gas stream for atomizing the sample is directly adjacent to the fluid orifice.

[0095] A microfluidic chip may include a substrate having multiple inlet ports that can be used to provide reagents to the channels. Reagents, as described elsewhere herein, may include anolyte solution, catholyte solution, electrolyte solution, buffer, mobilization reagent, sample or sample reagent, air, or gas (for gas channels), etc. Each channel of the substrate may have its own inlet port, or in some cases, two or more channels may be connected and the connected channels may share an inlet port. In some cases, the inlet ports are positioned along the edge of the substrate (see, e.g., Figures 2A, 5A, 6A, 7A, and 8A). In some cases, the substrate may include at least four inlet ports positioned along the edge of the substrate. The substrate may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more inlet ports. The substrate may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, or more inlet ports. The substrate may comprise up to 50, up to 40, up to 30, up to 20, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 inlet port. The substrate may comprise a range of inlet ports, for example, from about 2 to 8 inlet ports, each or a subset of which may be located along the edge of the substrate.

[0096] In some cases, the disclosed device is a microfluidic chip comprising multiple separation channels and gas channels, hi such cases, the microfluidic chip is designed to perform multiple analyte separation reactions in parallel, i.e., in multiple separation channels within the device, followed by (i) mobilization and (ii) electrospray ionization combined with nebulization.

[0097] FIG. 1A provides a non-limiting schematic diagram of a microfluidic chip with a four-channel isoelectric focusing design according to one aspect of the present disclosure, as will be discussed in more detail in Example 1 below.

[0098] FIG. 1B provides a non-limiting schematic diagram of the fluid channel network of an exemplary microfluidic chip for performing separation reactions, including an electrospray tip, according to a second aspect of the present disclosure, as will be described in more detail in Example 3 below.

[0099] 2A-2B provide a non-limiting schematic diagram of a microfluidic chip comprising gas channels and separation channels with inlet ports located at the edge of the device, as will be discussed in more detail in Example 4 below.

[0100] 3A-3D provide non-limiting schematic diagrams of modifiable aspects (eg, design parameters) of the microfluidic chips described herein, as will be discussed in more detail in Example 5 below.

[0101] 4A-4B provide a non-limiting example of a micrograph of one design of a microfluidic chip comprising a substrate with symmetric gas channels adjacent to a fluid channel (e.g., at the end of a separation channel).

[0102] 5A-5C provide additional non-limiting schematic diagrams of exemplary microfluidic chips comprising separation channels and gas channels.

[0103] 6A-6C provide additional non-limiting schematic illustrations of another exemplary microfluidic chip comprising a separation channel and a gas channel.

[0104] 7A-7C provide additional non-limiting schematic illustrations of yet another exemplary microfluidic chip comprising a separation channel and a gas channel.

[0105] 8A-8C provide additional non-limiting schematic illustrations of yet another exemplary microfluidic chip comprising a separation channel and a gas channel.

[0106] 9A-9B provide additional non-limiting schematic illustrations of yet another exemplary microfluidic chip comprising a separation channel and a gas channel with inlet ports on opposite edges of a substrate described herein.

[0107] FIG. 10 provides an example image of a fluid orifice of a microfluidic chip, comprising a fluid outlet channel and a symmetric gas channel, during electrospray ionization.

[0108] 11A-11B provide additional example images of a fluid orifice of a microfluidic chip, comprising a fluid outlet channel and a symmetric gas channel, during electrospray ionization.

[0109] In addition to gas channels and / or inlet ports located on the edge of the substrate of the microfluidic chip, the substrate may comprise multiple separation channels (e.g., two or more first separation channels, two or more second separation channels, two or more third separation channels, etc.) and one or more gas channels. A device or microfluidic chip of the present disclosure (or its substrate) may comprise multiple inlet ports, outlet ports, sample and / or reagent introduction channels, interconnecting channels, sample and / or reagent waste channels, reservoirs (e.g., sample reservoirs, reagent reservoirs, or waste reservoirs), micropumps, microvalves, vents, traps, filters, membranes, and the like, or any combination thereof.

[0110] The disclosed microfluidic chips and microfluidic cartridges may be fabricated using any of a variety of fabrication techniques and materials known to those skilled in the art. In some cases, devices may be fabricated as a series of two or more separate parts, which are then either mechanically crimped or permanently bonded together to form a completed device. In some cases, for example, fluid channels (sometimes also referred to herein as "microchannels") may be fabricated in a first layer (e.g., by photolithographic patterning of a glass substrate and wet chemical etching of the channels to a desired depth), and then sealed by bonding a second layer to the first layer, with through-holes in the second layer that intersect with the fluid channels providing external access to the fluid channels. In some cases, the fluid channels may be machined into a first layer (e.g., by laser cutting a channel pattern in a suitable polymer or ceramic film) and then sealed by sandwiching and bonding the first layer between a second layer and a third layer, with through-holes in the second and / or third layers that intersect the fluid channels providing external access to the fluid channels. In the latter example, the thickness of the first layer defines the thickness (or depth) of the fluid channels.

[0111] Examples of suitable fabrication techniques include, but are not limited to, conventional machining, CNC machining, injection molding, 3D printing, laser cutting or die cutting, alignment and lamination of one or more layers of polymer or ceramic films, or any of several microfabrication techniques such as photolithography and wet chemical etching, dry etching, deep reactive ion etching, or laser micromachining. In some embodiments, the microfluidic structures may be 3D printed from elastomeric, polymeric, or ceramic materials.

[0112] The disclosed microfluidic chips and microfluidic cartridges may be fabricated using any of a variety of materials known to those skilled in the art. Generally, the choice of material used will depend on the choice of fabrication technique, and vice versa. Examples of suitable materials include, but are not limited to, glass, quartz, fused silica, silicon, any of a variety of polymers, such as polydimethylsiloxane (PDMS, an elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene (PE), polyfluorinated polyethylene, high-density polyethylene (HDPE), polyetheretherketone, polyimide, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), polyetheretherketone (PEEK), epoxy resins, anti-adhesion materials such as Teflon (polytetrafluoroethylene (PTFE)), various photoresists such as SU8 or any other thick-film photoresist, or any combination of these materials. In some cases, different layers within a microfluidic chip or microfluidic cartridge comprising multiple layers may be fabricated from different materials, and in some cases, a given single layer within a device or microfluidic chip comprising one or more layers may be fabricated from two or more different materials.

[0113] In some cases, all or a portion of the microfluidic chip or microfluidic cartridge may be optically transparent (e.g., transparent to ultraviolet (UV), visible, and / or near-infrared light) to facilitate imaging of the separation channel and / or other portions of the device. In some cases, all or a portion of the separation channel is configured for imaging, e.g., full-channel imaging. For example, in some cases, the separation channel may be fabricated in a layer of optically opaque material sandwiched between two layers of optically transparent material, thereby forming an "optical slit" through which light may be transmitted and / or collected. In some cases, all or a portion of the fluidic orifice may be configured for imaging, e.g., during electrospray ionization, to determine parameters of the Taylor cone (e.g., droplet size, Taylor cone shape, etc., as described elsewhere herein).

[0114] Generally, the dimensions of fluid channels, gas channels, sample, and / or reagent reservoirs, etc., within the disclosed devices will be optimized to (i) provide fast, accurate, and reproducible separation of samples or sample aliquots comprising analyte mixtures, and (ii) minimize sample and reagent consumption. Generally, the width of a fluid or gas channel may be from about 10 μm to about 2 mm. In some cases, the width of a fluid or gas channel may be at least 10 μm, at least 25 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 750 μm, at least 1 mm, at least 1.5 mm, or at least 2 mm. In some cases, the width of a fluid or gas channel may be at most 2 mm, at most 1.5 mm, at most 1 mm, at most 750 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any of the lower and upper limits described in this paragraph may be combined to form a range included within the present disclosure; for example, in some cases, the width of a fluid channel (or reservoir) may range from about 100 μm to about 1 mm. One of ordinary skill in the art will recognize that the width of a fluid channel (or reservoir) can have any value within this range, for example, about 80 μm.

[0115] Generally, the length of a fluid or gas channel may be from about 0.5 cm to about 10 cm. In some cases, the length of a fluid or gas channel may be at least 0.1 cm, at least 0.5 cm, at least 1 cm, at least 2 cm, at least 3 cm, at least 4 cm, at least 5 cm, at least 6 cm, at least 7 cm, at least 8 cm, at least 9 cm, at least 10 cm, or more. In some cases, the length of a fluid or gas channel may be up to 10 cm, up to 9 cm, up to 8 cm, up to 7 cm, up to 6 cm, up to 5 cm, up to 4 cm, up to 3 cm, up to 2 cm, up to 1 cm, up to 0.5 cm, or up to 0.1 cm. Any of the lower and upper limits described in this paragraph may be combined to form ranges included within the present disclosure; for example, in some cases, the length of a fluid channel (or reservoir) may range from about 5 cm to about 10 cm. Those skilled in the art will recognize that the length of the fluid channel (or reservoir) can have any value within this range, for example, about 8 cm.

[0116] Generally, the depth of the fluid channel (or reservoir) will be from about 1 μm to about 1 mm. In some cases, the depth of the fluid channel (or reservoir) may be at least 1 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, or at least 1 mm. In some cases, the depth of a fluid channel (or reservoir) may be at most 1 mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, at most 40 μm, at most 30 μm, at most 20 μm, at most 10 μm, at most 5 μm, or at most 1 μm. Any of the lower and upper limits described in this paragraph may be combined to form a range included within the present disclosure; for example, in some cases, the depth of a fluid channel (or reservoir) may range from about 50 μm to about 100 μm. One of ordinary skill in the art will recognize that the depth of a fluid channel (or reservoir) can have any value within this range, for example, about 55 μm.

[0117] In some embodiments, the depth of the gas channels will match that of the fluid channels. The depth of the gas channels may be from about 1 μm to about 1 mm. In some cases, the depth of the gas channels may be at least 1 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, or at least 1 mm. In some cases, the depth of the gas channel may be at most 1 mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, at most 40 μm, at most 30 μm, at most 20 μm, at most 10 μm, at most 5 μm, or at most 1 μm. Any of the lower and upper limits described in this paragraph may be combined to form a range included within the present disclosure; for example, in some cases, the depth of the gas channel may range from about 50 μm to about 100 μm. One skilled in the art will recognize that the depth of the fluid channel (or reservoir) can have any value within this range, for example, about 55 μm.

[0118] The cross-sectional dimension of the gas exit orifice will generally be within about 10 μm to about 100 μm. In some cases, the cross-sectional dimension of the gas exit orifice may be at least 10 μm, at least 25 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 750 μm, at least 1 mm, at least 1.5 mm, or at least 2 mm. In some cases, the cross-sectional dimension of the gas exit orifice may be at most 2 mm, at most 1.5 mm, at most 1 mm, at most 750 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any of the lower and upper limits set forth in this paragraph may be combined to form a range within the present disclosure, e.g., in some cases, the cross-sectional dimension of the gas exit orifice may range from about 10 μm to about 100 μm. One of ordinary skill in the art will recognize that the cross-sectional dimension of the exit orifice can have any value within this range, e.g., about 80 μm.

[0119] Cartridge: In some cases, the disclosed microfluidic devices or chips may be configured to be coupled to one another or may be part of an integrated unit such as a microfluidic cartridge. The cartridge may comprise a microfluidic chip, which may comprise a substrate comprising a separation channel, at least one gas channel, and other reservoirs, reagents, membranes, valves, fixtures (e.g., membrane-containing high-voltage electrode fixtures), anchoring devices or features (e.g., screws, pins (e.g., pogo pins), adhesives, levers, switches, grooves, form-fitting pairs, hooks and loops, latches, threads, clips, clamps, tines, rings, rubber bands, rivets, grommets, knots, snaps, tape, vacuums, seals), gaskets, O-rings, electrodes, or combinations thereof. The cartridge may be monolithically constructed or may be modular and comprise removable parts. For example, the microfluidic chip may be configured to removably couple to the cartridge. Similarly, each of the reservoirs, membranes, valves, etc. may be removable from the cartridge. In cases where one or more components may be removable, the microfluidic cartridge may be configured so that each individual component can be aligned in place with sufficient tolerance by the user. For example, the microfluidic cartridge may include grooves and pins so that a microfluidic chip can be integrated by sliding the chip along the cartridge until it reaches the pins for alignment. In some cases, the chip may be configured to be positioned flush with the cartridge or a portion thereof. In some cases, the chip may be positioned within the cartridge so that one or more inlets, outlets, etc. can be connected (e.g., fluidically and / or electrically) to reservoirs, electrodes, membranes, and / or other useful interface contact units. In some cases, interface contact between the chip and reservoirs, electrodes, etc. may be performed by the user without any additional measurements or adjustments from the user.For example, the reservoirs may be configured to receive electrodes that snap into place or are secured via pogo pins, thereby establishing electrical and / or fluid communication. These exemplary configurations of the cartridge and chip are not intended to be limiting, and it should be understood that many different configurations of positioning of the microfluidic chip or other components of the microfluidic cartridge can be achieved. In some cases, the microfluidic cartridge may be configured to be a removable and / or disposable component of the systems described herein.

[0120] In a preferred embodiment, the cartridge component interfaces with the microfluidic chip at the edge of the microfluidic chip. The microfluidic chip may include two or more fluid ports, and the cartridge component may include an edge that mirrors the number of fluid ports of the chip. In some cases, the edge of the cartridge includes two or more fluid ports that match the two or more fluid ports of the microfluidic chip. The cartridge may also include one or more elastomeric components (e.g., gaskets, O-rings, etc.) used to form a substantially leak-free seal between the two or more fluid ports of the microfluidic chip and the two or more fluid ports of the cartridge component upon application of a force to an assembly including the microfluidic chip and the cartridge component. For example, the assembly may include screws, clamps, or other fastening mechanisms used to apply a force and form a leak-free seal between the ports of the microfluidic chip and the ports of the cartridge component.

[0121] In some cases, the cartridge includes one or more reservoirs configured to contain a desired volume of fluid. In some cases, the reservoirs may be capable of containing at least about 200 microliters (μL), at least about 300 μL, at least about 400 μL, at least about 500 μL, at least about 600 μL, at least about 700 μL, at least about 800 μL, at least about 900 μL, at least about 1 milliliter (mL), at least about 1.5 mL, at least about 2 mL, at least about 2.5 mL, at least about 3 mL, at least about 3.5 mL, at least about 4 mL, at least about 4.5 mL, or at least about 5 mL. In some cases, the reservoir may be capable of containing up to about 5 mL, up to about 4.5 mL, up to about 4 mL, up to about 3.5 mL, up to about 3 mL, up to about 2.5 mL, up to about 2 mL, up to about 1.5 mL, up to about 1 mL, up to about 900 μL, up to about 800 μL, up to about 700 μL, up to about 600 μL, up to about 500 μL, up to about 400 μL, up to about 300 μL, or up to about 200 μL. Any of the lower and upper limits described in this paragraph may be combined to form a range included within the present disclosure; for example, in some cases, the reservoir may contain a volume of fluid ranging from about 200 μL to about 2 mL. One of skill in the art will recognize that the reservoir fluid volume capacity can have any value within this range, for example, about 1.8 mL.

[0122] In such cases where the microfluidic cartridge comprises a reservoir, the reservoir may be controllably coupled (e.g., electrically, fluidically) to the microfluidic chip. For example, the cartridge may comprise one or more valves that can be used to control flow volumes or rates within the chip. In some cases, the cartridge may comprise stopcock valves or shear valves (e.g., sliding or rotary shear valves) that can enable controlled flow rates during delivery of one or more liquid reagents (e.g., mobilization reagents). In some cases, the cartridge may be integrated with or interfaced with a syringe pump that can be used to control the flow rate of liquid into the chip. In some cases, the flow rate may be controlled using a piston, spring-loaded device, or other mechanical approach.

[0123] In some cases, the cartridge may be configured to accommodate different types or models of chips. For example, the cartridge may be configured to accommodate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 different types or models of chips. In some cases, the cartridge may include ports or connections that can interface with channels of the chip (e.g., interface with inlets and / or outlets of the chip).

[0124] Instrument Interface Design: In preferred embodiments, the cartridge is configured to couple to the instrument system through an interface design used to interface other units (e.g., reservoirs, electrodes, fluid handling units) with the microfluidic cartridge and / or microfluidic chip. In some cases, the interface design comprises two or more fluid interconnects, each configured to provide a substantially leak-free fluid coupling between an external fluid line or reservoir and a fluid port of the microfluidic cartridge in response to application of a force to an assembly comprising the interface device and the microfluidic cartridge. In some cases, the fluid coupling remains substantially leak-free even as the relative fluid pressures within two of the two or more external fluid lines at the point of that fluid coupling to the two or more fluid ports of the cartridge vary. For example, the fluid coupling can remain leak-free even when the relative fluid pressure in the two external fluid lines varies by a factor of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100. The fluid coupling can remain leak-free even when the relative fluid pressure in the two external fluid lines varies by a factor of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100. In some cases, the fluid coupling can remain leak-free even when the relative fluid pressure in the two external fluid lines varies by up to 100 times, up to 90 times, up to 80 times, up to 70 times, up to 60 times, up to 50 times, up to 40 times, up to 30 times, up to 20 times, up to 10 times, up to 9 times, up to 8 times, up to 7 times, up to 6 times, up to 5 times, up to 4 times, up to 3 times, up to 2 times, or up to 1 time.

[0125] In some cases, two or more fluid interconnections comprise independently spring-loaded fittings, which can be useful in generating a repeatable sealing force. In some cases, the instrument interface comprises a component configured to couple to the microfluidic cartridge assembly. For example, the instrument interface and cartridge can have components (e.g., a conical fitting assembly, a flat face seal assembly) configured to mate. In some cases, the spring-loaded fitting comprises a conical fitting that mates with a fluid port comprising a bore in the microfluidic cartridge. In some cases, the spring-loaded fitting comprises a flat face seal fitting that mates with a fluid port comprising a bore in the microfluidic cartridge. In some aspects, the bore in the microfluidic cartridge is tapered. In some cases, the instrument interface may be mechanically coupled to the microfluidic cartridge assembly using one or more fastening mechanisms. In some cases, the instrument interface and / or the microfluidic cartridge assembly may comprise magnets that enable removable coupling, or may be mechanically coupled using, for example, interlocking geometries of the instrument interface and cartridge assembly. For example, the instrument interface may include threads (e.g., screw threads, internal threads, etc.), and the assembly may include complementary threads that can engage the threads of the interface. Additionally or alternatively, the interface device and / or assembly may include a snap-fit ​​joint (e.g., a cantilever snap fit, an annular snap fit, etc.) that allows for interlocking of the instrument interface and the microfluidic cartridge assembly. Alternatively or additionally, the instrument interface and / or the microfluidic cartridge assembly may include components that allow for an interference fit, a pressure fit, a shrink fit, a clearance fit, etc. Other examples of fastening mechanisms may include, by way of non-limiting example, form-fit pairs, hook and loop, latches, threads, screws, staples, clips, clamps, prongs, rings, headless nails, rubber bands, rivets, grommets, pins, tethers, snaps, Velcro®, adhesives (e.g., glue), tape, vacuum, seals, combinations thereof, or any other type of fastening mechanism.

[0126] Analyte Separation and Concentration: In some cases, the disclosed devices or systems may be configured to perform one or more separation or concentration steps in which multiple analytes in a mixture are separated and / or concentrated in individual fractions. For example, in some cases, the disclosed devices (e.g., microfluidic chips or microfluidic cartridges) may be configured to perform a first concentration step in which a mixture of analytes in a sample is separated into and / or concentrated as analyte fractions (e.g., analyte peaks or analyte bands) containing a subset of analyte molecules from the original sample. In some cases, these separated analyte fractions may be mobilized and / or eluted, and in some cases, may then undergo another downstream separation and / or concentration step. In some cases, for example, following a final separation and / or concentration step, the separated / concentrated analyte fractions may be ejected from the device for further analysis.

[0127] In some cases, the disclosed devices and systems may be configured to perform one, two, three, four, or five or more separation and / or concentration steps. In some cases, one or more of the separation or concentration steps may include a solid-phase separation technique, e.g., reverse-phase HPLC. In some cases, one or more of the separation or concentration steps may include a solution-phase separation and / or concentration technique, e.g., capillary zone electrophoresis (CZE) or isoelectric focusing (IEF).

[0128] The disclosed devices and systems may be configured to perform any of a variety of analyte separation and / or concentration techniques known to those skilled in the art, with the separation or concentration step being performed in at least a first separation channel configured to be imaged, in whole or in part, so that the separation process can be monitored as it occurs. For example, in some cases, the imaged separation may be an electrophoretic separation, including, for example, isoelectric focusing, capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow-balanced capillary electrophoresis, electric field gradient focusing, dynamic field gradient focusing, and the like, that produces one or more separated analyte fractions from an analyte mixture. In some cases, the separation and mobilization steps may be performed in at least a first separation channel configured to be imaged, in whole or in part, so that the separation and mobilization process can be monitored as it occurs. In any of these cases, imaging of the entire or partial separation channel may be performed continuously or intermittently, and may be performed prior to, during, or following the separation and / or concentration process.

[0129] In some cases, the use of a microfluidic device format can provide fast separation times and accurate, reproducible separation data. For example, in cases where a microfluidic device is configured to perform electrophoretic separations and / or isoelectric focusing reactions, the high surface area-to-volume ratio of the microfluidic channels can allow the use of high electric field strengths without incurring significant Joule heating, thereby enabling very fast separation reactions without substantial dispersion and loss of separation resolution. In some cases, precise control of fluid channel geometry provides accurate and reproducible control of sample injection volume, electric field strength, etc., thereby enabling highly accurate determination of one or more parameters of an assay, such as separation resolution and / or pI determination.

[0130] The one or more parameters of the assay may comprise a property of the separation. For example, the one or more parameters may be selected from the group consisting of separation resolution, peak width, peak capacity, pH gradient linearity, and minimum resolvable pI difference.

[0131] In general, the separation time required to achieve complete separation will vary depending on the specific separation technique and operating parameters employed (e.g., separation channel length, microfluidic device design, buffer composition, applied voltage, etc.). In some cases, separation times achieved using the disclosed devices and systems may range from about 0.1 minutes to about 30 minutes. In some cases, separation times may be at least 0.1 minutes, at least 0.5 minutes, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, or at least 30 minutes. In some cases, separation times may be up to 30 minutes, up to 25 minutes, up to 20 minutes, up to 15 minutes, up to 10 minutes, up to 5 minutes, up to 1 minute, up to 0.5 minutes, or up to 0.1 minutes. Any of the lower and upper limits described in this paragraph may be combined to form a range included within the present disclosure; for example, in some cases, separation times may range from about 1 minute to about 20 minutes. Those skilled in the art will recognize that the separation time can have any value within this range, for example, about 11.2 minutes. In some cases, the separation time may be longer than 20 minutes.

[0132] Similarly, the separation efficiency and resolution achieved using the disclosed devices and systems can vary depending on the specific separation technique and operating parameters employed (e.g., separation channel length, microfluidic device design, buffer composition, applied voltage, etc.), as well as whether one or two dimensions of separation are employed. In some cases, for example, when performing isoelectric focusing, the use of switchable electrodes to trigger the electrophoretic introduction of mobilizing electrolytes into the separation channel can result in improved separation resolution. For example, in some cases, the separation resolution of IEF performed using the disclosed methods and devices can provide resolution of analyte bands whose pIs differ by about 0.1 to about 0.0001 pH units. In some cases, the IEF separation resolution can enable resolution of analyte bands whose pIs differ by less than 0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001, less than 0.0005, or less than 0.0001 pH units.

[0133] Thus, in some cases, for example, when using an image of all or a portion of a separation channel to identify the location of pI markers in an isoelectric focusing reaction and determine pI values ​​for separated analytes, the accuracy to which the pI values ​​can be determined may be less than ±0.1 pH units, less than ±0.05 pH units, less than ±0.01 pH units, less than ±0.005 pH units, less than ±0.001 pH units, less than ±0.0005 pH units, or less than ±0.0001 pH units.

[0134] In some cases, the peak capacity achieved using the disclosed devices may range from about 100 to about 20,000. In some cases, the peak capacity may be at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 15,000, or at least 20,000. In some cases, the peak capacity may be at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, or at most 100. Any of the lower and upper limits described in this paragraph may be combined to form a range within the present disclosure; for example, in some cases, the peak capacity may range from about 400 to about 2,000. One of ordinary skill in the art will recognize that the peak capacity can have any value within this range, for example, about 285.

[0135] Capillary Isoelectric Focusing (CIEF): In some embodiments, the separation technique may include isoelectric focusing (IEF), e.g., capillary isoelectric focusing (CIEF). Isoelectric focusing (or "electrofocusing") is a technique for separating molecules by their isoelectric point (pI), i.e., the difference in pH at which the molecules have a net zero charge. CIEF involves adding an ampholyte (ampholyte) solution to a sample channel between reagent reservoirs containing an anode or cathode, and generating a pH gradient within the separation channel (i.e., the lumen of a fluid channel connecting electrode-containing wells, e.g., a capillary or channel in a microfluidic device), across which a separation voltage is applied. The ampholyte can be in the solution phase or immobilized on the surface of the channel wall. Negatively charged molecules migrate through the pH gradient within the medium toward the positive electrode, while positively charged molecules migrate toward the negative electrode. Proteins (or other molecules) within a pH range below their isoelectric point (pI) are positively charged and will therefore migrate toward the cathode (i.e., the negatively charged electrode). The protein's overall net charge will decrease as it migrates through an increasing pH gradient (e.g., due to protonation of carboxyl or other negatively charged functional groups) until it reaches the pH range corresponding to its pI, at which point it has no net charge and therefore ceases migration. As a result, the mixture of proteins separates based on their relative content of acidic and basic residues, with each protein becoming focused into a sharp, stationary band located at the point in the pH gradient corresponding to its pI. This technique is capable of extremely high resolution, where proteins differ by a single charge and are fractionated into distinct bands. In some aspects, isoelectric focusing may be performed while flowing a fluid (e.g., catholyte or mobilization reagent) from a fluid inlet, through a capillary or channel, and out the distal end of the capillary or channel. In some embodiments, isoelectric focusing may be performed in a separation channel that is permanently or dynamically coated to eliminate electroosmotic flow (EOF), for example, with a neutral and hydrophilic polymer coating.Examples of suitable coatings include, but are not limited to, amino modifiers, hydroxypropyl cellulose (HPC) and polyvinyl alcohol (PVA), Guarant® (AlcorBioseparations), linear polyacrylamide, polyacrylamide, dimethylacrylamide, polyvinylpyrrolidine (PVP), methylcellulose, hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), triethylamine, propylamine, morpholine, diethanolamine, triethanolamine, diaminopropane, ethylenediamine, chitosan, polyethyleneimine, cadaverine, putrescine, spermidine, diethylenetriamine, tetraethylenepentamine, cellulose, dextran, These include polyethylene oxide (PEO), cellulose acetate, amylopectin, ethylpyrrolidine methacrylate, dimethyl methacrylate, didodecyldimethylammonium bromide, Brij® 35, sulfobetaine, 1,2-dilauroyl sn-phosphatidylcholine, 1,4-didecyl-1,4-diazoniabicyclo[2.2.2]octane dibromide, agarose, poly(N-hydroxyethylacrylamide), POL-323, hyperbranched polyaminoesters, pullulan, glycerol, adsorbent coatings, covalent coatings, dynamic coatings, and the like. In some embodiments, isoelectric focusing may be performed (e.g., in an uncoated separation channel) using additives such as methylcellulose, glycerol, urea, formamide, detergents (e.g., Triton®-X100, CHAPS, digitonin) in the separation medium to significantly reduce electroosmotic flow, allow for better protein solubilization, and limit diffusion inside the capillary (e.g., within the lumen of the capillary) or fluidic channel by increasing the viscosity of the electrolyte.

[0136] As noted above, the pH gradient used for capillary isoelectric focusing techniques is generated through the use of ampholytes, i.e., amphoteric molecules that contain both acidic and basic groups and exist mostly as zwitterions within a certain range of pH. The portion of the electrolyte solution on the anode side of the separation channel is known as the "anolyte." That portion of the electrolyte solution on the cathode side of the separation channel is known as the "catholyte." A variety of electrolytes may be used in the disclosed methods and devices, including, but not limited to, phosphoric acid, sodium hydroxide, ammonium hydroxide, glutamic acid, lysine, formic acid, dimethylamine, triethylamine, acetic acid, piperidine, diethylamine, and / or any combination thereof. Electrolytes may be used at any suitable concentration, such as 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. The electrolyte concentration may be at least 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The electrolyte concentration may be up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, 0.01%, 0.001%, or 0.0001%. A range of electrolyte concentrations, for example, 0.1% to 2%, may be used. The ampholyte may be selected from any commercial or non-commercial carrier ampholyte mixture (e.g., Servalyt pH 4-9 (Serva, Heildelberg, Germany), Beckman pH 3-10 (Beckman Instruments, Fullerton, CA, USA), Ampholine 3.5-9.5 and Pharmalyte 3-10 (both General Electric Healthcare, Orsay, France), AESlytes (AES), FLUKA ampholytes (Thomas Scientific, Swedesboro, NJ), Biolyte (Bio-Rad, Hercules, CA)), and the like.The carrier ampholyte mixture may consist of a mixture of small molecules (approximately 300-1,000 Da) containing multiple aliphatic amino and carboxylate groups, with closely spaced pI values ​​and good buffering capacity. In the presence of an applied electric field, the carrier ampholytes partition into a smooth linear or nonlinear pH gradient that gradually increases from the anode to the cathode.

[0137] Any of a variety of pI standards may be used in the disclosed methods and devices to calculate the isoelectric point for separated analyte peaks. For example, pI markers generally used in CIEF applications, such as protein pI markers and synthetic small molecule pI markers, may be used. In some cases, the protein pI marker may be a specific protein with a generally accepted pI value. In some cases, the pI marker may be detectable, for example, via imaging. Various commercially available protein pI markers or synthetic small molecule pI markers, or combinations thereof, may be used, such as small molecule pI markers available from Advanced Electrophoresis Solutions, Ltd. (Cambridge, Ontario, Canada), ProteinSimple, peptide libraries designed by Shimura, and Slais dyes (Alcor Biosepartions).

[0138] Capillary Zone Electrophoresis (CZE): In some cases, a separation or concentration technique may include capillary zone electrophoresis, a method for the separation of charged analytes in solution within an applied electric field. The net velocity of charged analyte molecules is affected by both the electroosmotic flow (EOF), or μEOF, exhibited by the separation system, and the electrophoretic mobility, or μEP, for individual analytes (depending on the size, shape, and charge of the molecules), such that analyte molecules exhibiting different sizes, shapes, or charges exhibit differential migration velocities and separate into zones. In contrast to other capillary electrophoresis methods, CZE uses a "simple" buffer or background electrolyte as the separation solution.

[0139] Capillary Gel Electrophoresis (CGE): In some cases, a separation or concentration technique may include capillary gel electrophoresis, a method for the separation and analysis of macromolecules (e.g., DNA, RNA, and proteins) and their fragments based on their size and charge. The method involves the use of a gel-filled separation channel in which the gel acts as a counterflow and / or sieving medium during the electrophoretic migration of charged analyte molecules in an applied electric field. The gel functions to suppress thermal convection caused by the application of an electric field and also acts as a sieving medium to retard the passage of molecules, thereby resulting in differential migration rates for molecules of different size or charge.

[0140] Capillary isotachophoresis (CITP): In some cases, separation techniques may include capillary isotachophoresis, a method for the separation of charged analytes that uses a discontinuous system of two electrolytes (known as a leading electrolyte and a terminating electrolyte) within a capillary or fluid channel of suitable dimensions. The leading electrolyte contains ions with the highest electrophoretic mobility, while the terminating electrolyte contains ions with the lowest electrophoretic mobility. The analyte mixture (i.e., sample) to be separated is sandwiched between these two electrolytes, and application of an electric field results in the division of the charged analyte molecules within the capillary or fluid channel into closely adjacent zones in order of decreasing electrophoretic mobility. The zones move at a constant speed within the applied electric field so that a detector, such as a conductivity detector, photodetector, or imaging device, can be used to record their passage along the separation channel. Unlike capillary zone electrophoresis, simultaneous determination or detection of anionic and cationic analytes is not feasible in a single analysis performed using capillary isotachophoresis.

[0141] Capillary Electrokinetic Chromatography (CEC): In some cases, separation techniques may include capillary electrokinetic chromatography, a method for the separation of analyte mixtures based on a combination of liquid chromatographic and electrophoretic separation methods. CEC offers both the efficiency of capillary electrophoresis (CE) and the selectivity and sample capacity of packed-capillary high-performance liquid chromatography (HPLC). Because the capillaries used in CEC are packed with HPLC-packed materials, the wide variety of analyte selectivities available in HPLC are also available in CEC. The large surface area of ​​these packing materials allows CEC capillaries to accommodate relatively large sample volumes, making subsequent detection of eluted analytes a somewhat simpler task than in capillary zone electrophoresis (CZE).

[0142] Micellar electrokinetic chromatography (MEKC): In some cases, separation techniques may include capillary electrokinetic chromatography, a method for the separation of analyte mixtures based on differential partitioning between surfactant micelles (pseudostationary phase) and a surrounding aqueous buffer solution (mobile phase). The basic setup and detection methods used for MEKC are identical to those used in CZE. The difference is that the buffer solution contains the surfactant at a concentration above the critical micelle concentration (CMC), so that the surfactant monomers are in equilibrium with the micelles. MEKC is typically performed in open capillaries or fluid channels using alkaline conditions to generate a strong electroosmotic flow. Sodium dodecyl sulfate (SDS) is an example of a commonly used surfactant in MEKC applications. The anionic nature of the sulfate group in SDS causes the surfactant and micelles to have an electrophoretic mobility that is opposite to the direction of the strong electroosmotic flow. As a result, surfactant monomers and micelles migrate very slowly, but their net movement is still in the direction of electroosmotic flow, i.e., toward the cathode. During MEKC separation, analytes partition themselves between the hydrophobic interior of the micelles and the hydrophilic buffer solution. Hydrophilic analytes that are insoluble inside the micelles undergo electroosmotic flow at a rate u o The buffer migrates at tM Hydrophobic analytes that are completely solubilized within the micelles will be detected at a micellar velocity u c The migration time is t c It elutes at .

[0143] Flow-balanced capillary electrophoresis (FCCE): In some cases, separation techniques may include flow-balanced capillary electrophoresis, a method for increasing the efficiency and resolution of capillary electrophoresis that utilizes pressure-induced reverse flow to actively slow, stop, or reverse the electrokinetic migration of analytes through a capillary. By slowing, stopping, or moving the analytes back and forth across the detection window, the analytes of interest are effectively confined to the separation channel for a much longer period of time than under normal separation conditions, thereby increasing both the efficiency and resolution of the separation.

[0144] Chromatography: In some cases, separation techniques may include chromatographic techniques in which an analyte mixture within a sample fluid (mobile phase) is passed through a column or channel-filled material (stationary phase) that differentially retains various constituents of the mixture, thereby causing them to travel at different rates and separate. In some cases, a subsequent step of elution or mobilization may be required to displace analytes with high binding affinity for the stationary phase. Examples of chromatographic techniques that can be incorporated into the disclosed methods include, but are not limited to, ion-exchange chromatography, size-exclusion chromatography, and reverse-phase chromatography.

[0145] Mobilization of Separated Analyte Species: In some cases, provided herein are devices and systems configured to perform chromatographic separation techniques, such as, for example, reversed-phase chromatography. A method implemented by a device or system may further include elution of analyte species retained on the stationary phase in each of the multiple separation channels (e.g., by simultaneously or independently varying the buffer flowing through each of the multiple separation channels), which may be referred to as a "mobilization" step or reaction. In some cases, a method implemented by a device or system may further include simultaneously or independently applying pressure to each of the multiple separation channels, or simultaneously or independently introducing electrolyte into each of the multiple separation channels to disrupt the pH gradient used for isoelectric focusing and thus trigger migration of separated analyte peaks out of the separation channels, which may also be referred to as a "mobilization" step. In some cases, the force used to drive the separation reaction (e.g., pressure for reversed-phase chromatography, or an electric field for electrokinetic separation or isoelectric focusing reactions) may be turned off during the mobilization step. In some cases, the force used to drive the separation reaction may remain on during the mobilization step. In some cases of the disclosed methods, e.g., those involving an isoelectric focusing step, the separated analyte bands may be mobilized (e.g., using hydrodynamic pressure and / or chemical mobilization techniques) so that the separated analyte bands migrate toward each end of a plurality of separation channels that are connected to another fluidic channel (e.g., which may be an outlet, a waste reservoir, or a second separation channel). In some cases, e.g., capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow-balanced capillary electrophoresis, or any other separation technique that separates components of an analyte mixture by velocity difference is employed; in those cases, the separation step itself may be considered a mobilization step.

[0146] In some cases, mobilization of the analyte zone may be implemented by simultaneously or independently applying hydrodynamic pressure to one or both ends of each of the multiple separation channels. In some cases, mobilization of the analyte zone may be implemented by orienting the device so that the multiple separation channels are in a vertical position so that gravity can be employed. In some cases, mobilization of the analyte zone may be implemented using EOF-assisted mobilization. In some cases, mobilization of the analyte zone may be implemented using chemical mobilization, for example, by simultaneously or independently introducing into each of the multiple separation channels a mobilizing electrolyte that shifts the local pH in the pH gradient used for isoelectric focusing. In some cases, any combination of these mobilization techniques may be employed.

[0147] In one preferred case, the mobilization step for the isoelectrically focused analyte zone includes chemical mobilization. Compared to pressure-based mobilization, chemical mobilization has the advantage of exhibiting minimal zone expansion by overcoming the hydrodynamic parabolic flow profile induced through the use of pressure. Chemical mobilization may be implemented by introducing an electrolyte (i.e., a "mobilizing electrolyte") into the separation channel to alter the local pH and / or net charge on the separated analyte zone (or zwitterionic buffer components and associated hydration shells) so that they migrate within the applied electric field. In some cases, the polarity of the applied electric field used to mobilize the separated analyte zone may be such that the analyte migrates toward an anode in electrical communication with the outlet or distal end of the separation channel (anodic mobilization). In some cases, the polarity of the applied electric field used to mobilize the separated analyte zone may be such that the analyte migrates toward a cathode in electrical communication with the outlet or distal end of the separation channel (cathodic mobilization). The mobilization electrolyte consists of either anions or cations that compete with hydroxyl (cathodic mobilization) or hydronium ions (anodic mobilization) for introduction into the separation channel or capillary. Examples of bases that can be used as catholyte for anodic mobilization include, but are not limited to, sodium hydroxide, ammonium hydroxide ("ammonia"), diethylamine, dimethylamine, piperidine, etc. Examples of acids that can be used as anolyte in cathodic mobilization include, but are not limited to, phosphoric acid, acetic acid, formic acid, and carbonic acid, etc. In some cases, mobilization may be initiated by the addition of a salt (e.g., sodium chloride) to the anolyte or catholyte. In some cases, the anode may be held at ground and a negative voltage is applied to the cathode. In some cases, the cathode may be held at ground and a positive voltage may be applied to the anode. In some cases, a non-zero negative voltage may be applied to the cathode and a non-zero positive voltage may be applied to the anode. In some cases, a non-zero positive voltage may be applied to both the anode and the cathode.In some cases, a non-zero negative voltage may be applied to both the anode and the cathode.

[0148] In some cases, mobilization of the separated analyte bands may be initiated at a user-defined time point by triggering electrodes (e.g., cathodes in electrical communication with the distal ends of each of a plurality of separation channels and cathodes in electrical communication with the proximal ends of each of a plurality of mobilization channels (e.g., fluidic channels that intersect the separation channels near the outlet or distal end of each separation channel)) that are switchable between on and off states to control the electrophoretic introduction of mobilization buffer or electrolyte into the separation channels.

[0149] In some cases, the user-defined time for independently triggering the transition of one, two, or three or more switchable electrodes between an ON state and an OFF state for each of a plurality of separation channels may range from about 30 seconds to about 30 minutes for any of the recruitment schemes. In some cases, the user-defined time may be at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, or at least 30 minutes. In some cases, the user-defined time may be up to 30 minutes, up to 25 minutes, up to 20 minutes, up to 15 minutes, up to 10 minutes, up to 5 minutes, up to 4 minutes, up to 3 minutes, up to 2 minutes, up to 1 minute, or up to 30 seconds. Any of the lower and upper limits described in this paragraph may be combined to form a range included within the present disclosure; for example, in some cases, the user-defined time may range from about 2 minutes to about 25 minutes. Those skilled in the art will recognize that the user-defined time can have any value within this range, for example, about 8.5 minutes.

[0150] In some cases, the electric field used to effect mobilization in any of the mobilization scenarios disclosed herein (or to perform an electrokinetic separation or isoelectric focusing reaction in those cases in which such separation techniques are performed) may range from about 0 V / cm to about 1,000 V / cm. In some cases, the electric field strength may be at least 0 V / cm, at least 20 V / cm, at least 40 V / cm, at least 60 V / cm, at least 80 V / cm, at least 100 V / cm, at least 150 V / cm, at least 200 V / cm, at least 250 V / cm, at least 300 V / cm, at least 350 V / cm, at least 400 V / cm, at least 450 V / cm, at least 500 V / cm, at least 600 V / cm, at least 700 V / cm, at least 800 V / cm, at least 900 V / cm, or at least 1,000 V / cm. In some cases, the electric field strength may be up to 1,000 V / cm, up to 900 V / cm, up to 800 V / cm, up to 700 V / cm, up to 600 V / cm, up to 500 V / cm, up to 450 V / cm, up to 400 V / cm, up to 350 V / cm, up to 300 V / cm, up to 250 V / cm, up to 200 V / cm, up to 150 V / cm, up to 100 V / cm, up to 80 V / cm, up to 60 V / cm, up to 40 V / cm, up to 20 V / cm, or up to 0 V / cm. Any of the lower and upper limits described in this paragraph may be combined to form ranges included within the present disclosure; for example, in some cases, the electric field strength may range from about 40 V / cm to about 650 V / cm. Those skilled in the art will recognize that the electric field strength can have any value within this range, for example, about 575 V / cm.

[0151] In some cases, mobilization of separated analyte bands may be initiated based on data derived from independently monitoring the current (or conductivity) for each of multiple separation channels, e.g., in the case of isoelectric focusing, the current passing through the separation channel may reach a minimum value. In some cases, detection of a minimum current value, or a current value that remains constant or falls below a specified threshold for a specified time period, may be used to determine whether the isoelectric focusing reaction has reached completion and, therefore, may be used to trigger the initiation of a chemical mobilization step.

[0152] In some cases, the minimum or threshold current value may range from about 0 μA to about 100 μA, in some cases the minimum or threshold current value may be at least 0 μA, at least 1 μA, at least 2 μA, at least 3 μA, at least 4 μA, at least 5 μA, at least 10 μA, at least 20 μA, at least 30 μA, at least 40 μA, at least 50 μA, at least 60 μA, at least 70 μA, at least 80 μA, at least 90 μA, or at least 100 μA. In some cases, the minimum or threshold current value may be at most 100 μA, at most 90 μA, at most 80 μA, at most 70 μA, at most 60 μA, at most 50 μA, at most 40 μA, at most 30 μA, at most 20 μA, at most 10 μA, at most 5 μA, at most 4 μA, at most 3 μA, at most 2 μA, at most 1 μA, or at most 0 μA. Any of the lower and upper limits described in this paragraph may be combined to form a range included within the present disclosure; for example, in some cases, the minimum or threshold current value may range from about 10 μA to about 90 μA. One of ordinary skill in the art will recognize that the minimum or threshold current value can have any value within this range, for example, about 16 μA.

[0153] In some cases, the prescribed time period may be at least 5 seconds, at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 25 seconds, at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds, or at least 60 seconds. In some cases, the prescribed time period may be up to about 60 seconds, up to about 55 seconds, up to about 50 seconds, up to about 45 seconds, up to about 40 seconds, up to about 35 seconds, up to about 30 seconds, up to about 25 seconds, up to about 20 seconds, up to about 15 seconds, up to about 10 seconds, or up to about 5 seconds. Any of the lower and upper limits described herein may be combined to form ranges encompassed within the present disclosure, and in some cases, the prescribed time period may range from about 5 seconds to about 30 seconds. One skilled in the art will recognize that the prescribed time period may have any value within this range, for example, about 32 seconds.

[0154] In some cases, mobilization of separated analyte bands may be initiated based on data derived from images of the separation channel (e.g., by performing automated image processing) as the separation reaction is carried out. The image-derived data may be used to monitor the presence or absence of one or more analyte peaks, the position of one or more analyte peaks, the width of one or more analyte peaks, the velocity of one or more analyte peaks, the separation resolution, the rate of change or lack thereof of the presence, position, width, or velocity of one or more analyte peaks, or any combination thereof, and may be used to determine whether the separation reaction is complete and / or to trigger the initiation of a mobilization step within a given separation channel. In some cases, the completion of a separation step may be determined by monitoring the rate of change of a separation performance parameter (e.g., peak position or peak width) over a period of time (e.g., over a period of 10 to 60 seconds).

[0155] In some embodiments, a chemical mobilization step may be initiated in a microfluidic device designed to integrate CIEF with ESI-MS by varying the electric field within the device and electrophoretically driving a mobilization electrolyte into the separation channel. In some cases, the initiation of the mobilization step may be triggered based on data derived from an image of all or a portion of the separation channel. In some cases, the change in the electric field may be implemented by connecting or disconnecting one or more electrodes attached to one or more power supplies, where the one or more electrodes are positioned in a reagent well on the device or integrated with the fluidic channels of the device. In some cases, the connection or disconnection of one or more electrodes may be controlled using computer-implemented methods and programmable switches so that the timing and duration of the mobilization step can be coordinated with the separation step. In some cases, varying the electric field within the device may be used to electrophoretically or electroosmotically flow a mobilization buffer into a separation channel comprising a stationary phase such that retained analytes are released from the stationary phase.

[0156] In some cases, three or more electrodes per separation channel may be connected to or integrated into the device. For example, a first electrode may be electrically coupled to the proximal end of the separation channel, an electrode reservoir coupled to the separation channel, or another channel in electrical and / or fluid communication with the separation channel. Similarly, a second electrode may then be coupled to the distal end of the separation channel, an electrode reservoir coupled to the distal end of the separation channel, or another channel in electrical and / or fluid communication with the distal end of the separation channel, and a third electrode may be coupled to a mobilization channel (or a channel or reservoir connected thereto), for example, that intersects the separation channel at the distal end of the separation channel and connects to or comprises a reservoir containing a mobilization buffer. Depending on the completion of the separation step as determined by an image-based method, the electrical coupling of the second or third electrode to their respective channels may be switchable between an "on" state and an "off" state. In one such example, a second electrode, which may form the anode or cathode of the separation circuit, may be switched to an "off" mode and may be turned off during separation, and a third electrode may be switched to an "on" state to initiate the introduction of mobilization buffer into the channel (e.g., via electrophoresis). In some cases, the "on" and "off" states may include complete connection or disconnection of electrical coupling between the electrodes and the fluidic channel, respectively. In some cases, the "on" and "off" states may include fixing the current passing through a defined electrode at non-zero or zero microamperes, respectively.

[0157] In some cases, triggering or initiating a recruitment step may involve detecting no change or a change below a specified threshold for one or more image-derived separation parameters as described above. For example, in some cases, a change of less than 20%, 15%, 10%, or 5% in one or more image-derived parameters (e.g., peak location, peak width, peak velocity, etc.) may be used to trigger a recruitment step.

[0158] In some cases, triggering or initiating a recruitment step may involve detecting no change or a rate of change below a specified threshold for one or more image-derived separation parameters as described above. For example, in some cases, a change of less than 20%, 15%, 10%, or 5% (or any combination of these rates of change and time periods) in one or more image-derived parameters (e.g., peak location, peak width, peak velocity, etc.) over a time period of at least 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, or 60 seconds may be used to trigger a recruitment step.

[0159] In some cases, a calibrant may be used during the mobilization step to correlate and / or calibrate information from the mass spectrometer. In some cases, the calibrant may consist of a peptide, polypeptide, protein, or other molecule (either natural or synthetic) with a known mass. In some cases, the calibrant will be mixed with the mobilization agent solution. The calibrant may be used to calibrate the mass spectrometer. In some cases, the calibrant may be used to correlate information from the mass spectrometer to the mobilization or separation process. For example, the calibrant may be monitored during separation (e.g., isoelectric focusing) or mobilization.

[0160] Electrospray Ionization (ESI) and Mass Spectrometry: In preferred embodiments, the disclosed methods, devices, and systems are configured for performing electrospray ionization of separated analyte mixtures and for injecting the separated analyte mixtures into a mass spectrometer. In ESI, droplets of sample and solution are ejected from the distal end of a capillary or microfluidic device, which includes an electrospray feature, such as an emitter tip or orifice, by application of an electric field between the capillary tip or emitter and the mass spectrometer source plate. In some embodiments, the voltage between the capillary or emitter tip and the mass spectrometer may be 500 to 6,000 V or -500 to -6,000 V. The droplets are stretched and expanded within this induced electric field, forming a cone-shaped ejection (i.e., a "Taylor cone") with smaller and smaller droplets that vaporize and produce gas-phase ions that are introduced into the mass spectrometer for further separation and detection. In preferred embodiments, the methods, devices, and systems of the present disclosure include performing an atomization process (e.g., using a gas channel) during ESI. In some cases, atomization may be performed to achieve nanoflow or nanoscale droplet generation, which may aid in reduced ion suppression, increased ionization, reduced contamination, more stable electrospray performance, better detection accuracy, or higher detection signal during mass spectrometry. For example, ESI performance, including atomization, may be characterized by a standard error variation of less than 1.0% in the total mass spectrometry signal. In some cases, ESI may be performed while flowing fluid from a fluid inlet, through a capillary or channel, and out the distal end of the capillary or channel.

[0161] As described herein, in some cases, a microfluidic device (e.g., a microfluidic chip or microfluidic cartridge) includes a fluid orifice that serves as an emitter tip (e.g., an ESI orifice). The emitter tip may be sharpened to provide a small surface and droplet volume using a lapping wheel, file, machining tool, CNC machining tool, water-jet cutting, or other tool or process for forming an ESI tip to provide a small surface volume, and the like. In some cases, the emitter tip may be positioned at an edge or corner of the microfluidic device. In other cases, the tip may be extended by heating and stretching the distal portion of the chip. In some cases, the tip may then be cut to a desired length or diameter. In some cases, the electrospray tip may be coated with a hydrophobic coating, which may minimize the size of droplets formed on the tip. In some embodiments, the system may electrospray a mobilizing agent, catholyte, or any other liquid during a separation step when the analyte is not eluting from the device. In some embodiments, the substrate exit orifice may be further shaped into a wedge, pyramid, cone, or other three-dimensional shape. In some embodiments, the shape may include flat features where some or all of the channels (gas or fluid) exit. In some embodiments, the substrate may be hydrophobic or hydrophilic, have a chemically modified surface, or maintain a predefined contact angle with the fluid, i.e., water, organic solvents, etc.

[0162] The mass-to-charge ratio (or "mass") for analytes ejected from a microfluidic device (e.g., a biologic or biosimilar) and introduced into a mass spectrometer can be measured using any of a variety of different mass spectrometer designs. Examples include, but are not limited to, time-of-flight mass spectrometry, quadrupole mass spectrometry, ion trap or orbitrap mass spectrometry, distance-of-flight mass spectrometry, Fourier transform ion cyclotron resonance, resonance mass measurement, and nanomechanical mass spectrometry.

[0163] In some embodiments, the electrospray feature of the microfluidic device may be aligned with the separation channel. In some embodiments, the electrospray feature of the microfluidic device may be oriented perpendicular to the separation channel or at an intermediate angle. In some embodiments of the disclosed methods, substantially all of the separated and / or concentrated analyte fraction from the final separation or concentration step performed in the capillary or microfluidic device is ejected from the electrospray tip or feature in a continuous flow. In some embodiments, a portion of the analyte mixture (e.g., a fraction of interest) may be ejected from the microfluidic device through an outlet or fluid orifice configured to interface with an analytical instrument, such as a mass spectrometer, or another device configured to fractionate and / or concentrate at least a portion of the sample.

[0164] In some embodiments, ejection from the capillary or microfluidic device is performed using pressure, electrical force, ionization, or any combination thereof. In some embodiments, ejection coincides with the mobilization step as described above. In some embodiments, the sheath liquid used for electrospray ionization is used as the electrolyte for electrophoretic separation. In preferred embodiments, an atomizing gas (e.g., flowing through a gas channel in the microfluidic device) is provided to atomize the sample and / or reduce droplet size during introduction of the sample into an analytical instrument such as a mass spectrometer. During atomization, a gas (e.g., air, oxygen, nitrogen, etc.) flow is directed toward the sample at a sufficiently high velocity to aerosolize the sample. The resulting sample comprises smaller droplets, which then evaporate more rapidly and may allow for improved ionization of the sample introduced into the mass spectrometer.

[0165] Imaging electrospray ionization performance: Disclosed herein are devices, methods, and systems for improving electrospray ionization performance and, therefore, the quality of mass spectrometry data collected for capillary-based or microfluidic-based ESI-MS systems. In some cases, imaging of the Taylor cone in an electrospray ionization setup may be used to assess the performance of the atomization process during ESI. For example, imaging of the Taylor cone may provide data regarding the size, shape, or other characteristics (e.g., droplet size, uniformity, etc.) of the Taylor cone or the atomization process. In some cases, imaging may be used in a computer-implemented method to provide feedback control of one or more operating parameters so that the shape, density, or other characteristics of the Taylor cone are maintained within specified ranges. In some embodiments, operating parameters that can be controlled through such a feedback process include, but are not limited to, the alignment of the electrospray tip or orifice with the mass spectrometer inlet, the distance between the electrospray tip and the mass spectrometer inlet (e.g., by mounting a capillary tip or microfluidic device with integrated electrospray features on a programmable precision XYZ translation stage), the flow rate of the analyte sample through the electrospray tip (e.g., by adjusting the pressure, electric field strength, or a combination thereof used to drive the ejection of the analyte sample), the voltage applied, e.g., at the proximal end of the channel, between the electrospray tip or orifice and the mass spectrometer inlet, the volumetric flow rate of the sheath liquid or sheath gas or nebulizer gas surrounding the ejected analyte sample, or any combination thereof.

[0166] Imaging of Separation Channels: In some cases, the disclosed devices and systems may be configured to image all or a portion of at least one separation channel to monitor a separation and / or mobilization reaction while it is occurring. In some cases, the disclosed devices and systems may be configured to image all or a portion of multiple separation channels to monitor multiple separation and / or mobilization reactions in parallel while they are occurring. In some cases, the separation and / or mobilization reactions may be imaged using any of a variety of imaging techniques known to those skilled in the art. Examples include, but are not limited to, ultraviolet (UV) light absorbance, visible light absorbance, fluorescence (e.g., intrinsic fluorescence or fluorescence resulting from labeling one or more analytes with fluorophores), Fourier transform infrared spectroscopy, Fourier transform near-infrared spectroscopy, Raman spectroscopy, optical spectroscopy, and the like. In some cases, the multiple separation (or concentration) channels may be the lumens of multiple capillaries. In some cases, the multiple separation (or concentration) channels may be multiple fluid channels within a microfluidic device. In some cases, all or a portion of the separation (or concentration) channel, a junction or connecting channel connecting the separation channel and the end of a downstream analytical instrument or the electrospray orifice or tip, the electrospray orifice or tip itself, or any combination thereof, may be imaged. In some cases, the separation (or concentration) channel may be the lumen of a capillary. In some cases, the separation (or concentration) channel may be a fluid channel within a microfluidic device.

[0167] The wavelength range used for imaging and detection of separated analyte bands will typically depend on the imaging technique and the choice of material from which the device, or portions thereof, is fabricated. For example, if UV light absorbance is used to image all or a portion of a separation channel or other portion of a microfluidic device, detection at about 220 nm (due to the intrinsic absorbance of peptide bonds) and / or about 280 nm (due to the intrinsic absorbance of aromatic amino acid residues) may allow visualization of protein bands during separation and / or mobilization, provided that at least a portion of the device, e.g., the separation channel or portions thereof, is transparent to light at these wavelengths. In some cases, the analytes to be separated may be labeled with, for example, a fluorophore, chromophore, chemiluminescent tag, or other suitable label prior to separation so that they can be imaged using fluorescence imaging, UV absorbance imaging, or other suitable imaging techniques. For example, in some cases where the analytes comprise proteins produced by a commercial manufacturing process, the proteins may be genetically engineered to incorporate a green fluorescent protein (GFP) domain or a variant thereof so that they can be imaged using fluorescence. In some cases, labeling of proteins or other analyte molecules may be performed using approaches to ensure that the label itself does not interfere with or perturb the analyte properties on which the chosen separation technique is based.

[0168] In some cases, the image (or data derived therefrom) may be used to trigger a mobilization step or other transfer of separated analyte fractions or portions thereof, for example, from a first separation channel or channels to a second separation channel or channels, or from a first separation channel or channels to a second separation channel or channels in fluid communication with the outlet end of the first separation channel or channels. For example, in some cases, the disclosed methods may include injecting an analyte mixture into a microfluidic device containing a first plurality of separation channels and a second plurality of separation channels. The first plurality of separation channels may contain a medium configured to bind analytes from the sample analyte mixture. Thus, when the sample analyte mixture is loaded or injected into a device, e.g., a microfluidic device or microfluidic cartridge, at least a fraction of the analytes in each sample analyte mixture may be bound to a substrate and / or prevented from flowing through the first plurality of separation channels. For example, injecting an analyte mixture into a microfluidic device can cause chromatographic separation within a first plurality of separation channels. An eluent can then be injected into the microfluidic device such that at least a fraction of the analytes, if present, are mobilized from the medium in each separation channel. In some cases, the first plurality of separation channels can be imaged while the analytes are being mobilized. In some cases, imaging the first plurality of separation reactions can include imaging the entire column (e.g., all channels) and / or imaging a portion of the separation channels. In some cases, an electric field can be applied to the second plurality of separation channels when the imaging detects that the analyte fraction is located at the intersection of the first plurality of separation channels and the second plurality of separation channels, such that the analyte fraction is electrokinetically injected into the second plurality of separation channels. For example, in some cases, the first plurality of separation channels and the second plurality of separation channels can form a series of T-junctions.In some cases, imaging may be used to detect when an analyte fraction (e.g., a fraction of interest) is located at one or more of the series of T-junctions. Applying an electric field can electrokinetically inject the analyte fraction of interest (optionally, but not other analytes not located at the series of T-junctions) into a second plurality of separation channels for a second stage of separation. In some cases, an electric field may be applied independently to one or more of the second plurality of separation channels depending on whether the analyte fraction of interest is detected at one or more of the T-junctions.

[0169] In some cases, imaging may be performed during recruitment to monitor the recruitment reaction. In some cases, the imaging system used to monitor the dissociation reaction may also be used to monitor the recruitment reaction. In some cases, only a channel or a portion of multiple channels may be imaged to monitor the recruitment reaction. In some cases, the entire channel or multiple channels may be imaged, and only the imaged channel or portions of multiple channels may be used to monitor the recruitment reaction. For example, channels may be imaged at a given sampling rate, and for each image generated, a portion of the image corresponding to the distal end of one or more channels may be used to generate a mobility chromatogram. The mobility chromatogram may provide information about the average absorbance, for example, over a certain pixel width (e.g., 8 pixels), as a function of time. In some cases, the pixel width of an image used to generate a mobility chromatogram (e.g., corresponding to the distal end of a channel) may comprise at least 1 pixel, at least 2 pixels, at least 3 pixels, at least 4 pixels, at least 5 pixels, at least 6 pixels, at least 7 pixels, at least 8 pixels, at least 9 pixels, at least 10 pixels, at least 15 pixels, at least 20 pixels, at least 25 pixels, at least 30 pixels, at least 35 pixels, at least 40 pixels, at least 50 pixels, at least 60 pixels, at least 70 pixels, at least 80 pixels, at least 90 pixels, or at least 100 pixels.

[0170] Mobility chromatograms may be used to determine parameters of the mobilization reaction. For example, mobility chromatograms may be used to calibrate a mass spectrometer and determine time-of-flight information, peak width, peak velocity, peak mobility, peak position, etc., of one or more analytes. In some cases, mobility chromatograms may be generated in real time. In some cases, mobility chromatograms may be generated at a sampling rate (e.g., Nyquist sampling rate, 1-2 Hz, or a frequency matching the sampling rate of the mass spectrometer). In some cases, chromatograms may be used to generate information about the absorbance of a segment of a channel as a function of time.

[0171] Systems and System Components: In some cases, a system of the present disclosure may comprise one or more of the disclosed devices (e.g., a microfluidic device), one or more high-voltage power supplies (or in the case of multiple parallel separations, a single multiplexed high-voltage power supply allowing independent control of two or more channels), an autosampler and / or fluid handling system, a fluid flow controller, an imaging module, a dynamic light scattering module, a microplate handling robotic module, a waste management module (e.g., to remove or prevent accumulation of fluid droplets outside of the electrospray tip), an electrode interface contact unit, a processor or computer, or any combination thereof.

[0172] High-voltage power supply: In some cases, one or more high-voltage power supplies (or a single multiplexed high-voltage power supply allowing independent control of two or more channels) of the disclosed systems are configured to provide simultaneous, independent electrical control of multiple separation channels, e.g., simultaneously and independently apply a defined voltage or current to each of multiple separation channels or auxiliary fluid channels (e.g., mobilization channels used to deliver chemical mobilization agents to the separation channels following completion of an isoelectric focusing reaction). In some cases, two or more high-voltage power supplies (or a single multiplexed high-voltage power supply allowing independent control of two or more channels) of the disclosed systems are configured to monitor and / or record the current flowing through each separation channel of the multiple separation channels (rather than just the total current). As described herein, the separation channels may comprise different samples or the same sample (e.g., aliquots of a sample). In some cases, the current flowing through each separation channel may be used, for example, to determine when the isoelectric focusing reaction is complete and / or to detect disturbances (e.g., the introduction or formation of air bubbles within the separation channel).

[0173] In some cases, two or more high-voltage power supplies may be programmed or otherwise configured to operate in a constant voltage mode, for example, where the voltage applied across each of a plurality of separation channels and / or auxiliary channels is held fixed for the duration of a separation reaction or for a predetermined period of time. In some cases, two or more high-voltage power supplies may be programmed or otherwise configured to produce a step change in the voltage applied across each of a plurality of separation channels and / or auxiliary channels from a first predetermined voltage to at least a second predetermined voltage over one or more predetermined periods of time. In some cases, two or more high-voltage power supplies may be programmed or otherwise configured to produce two, three, four, five, or more than five step changes in voltage over the course of a separation reaction.

[0174] In some cases, two or more high voltage power supplies may be programmed or otherwise configured to operate in a constant power mode, increasing the voltage applied to a given separation channel as the current drops during the separation reaction due to, for example, conductivity changes, thereby allowing the voltage to increase and minimize separation times without inducing excessive Joule heating.

[0175] As noted above, in some cases, the electric field used to conduct an electrophoretic separation or isoelectric focusing reaction (or other electrokinetic injection or separation process) may range from about 0 V / cm to about 1,000 V / cm. Thus, in some cases, two or more high-voltage power supplies of the disclosed systems may be configured to provide an adjustable voltage ranging from about 0 volts to about 5,000 volts (e.g., for a 5 cm long separation channel). In some cases, the two or more high-voltage power supplies may be configured to provide an adjustable voltage of at least 0, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, or at least 5,000 volts. In some cases, the two or more high-voltage power supplies may be configured to provide an adjustable voltage of up to 5,000, up to 1,000, up to 500, up to 100, up to 50, up to 10, or up to 5 volts. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges within the present disclosure, e.g., in some cases, two or more high-voltage power supplies may be configured to provide an adjustable voltage ranging from about 100 volts to about 1,000 volts. One skilled in the art will recognize that two or more high-voltage power supplies may be configured to provide an adjustable voltage of any value within this range, e.g., about 1,250 volts.

[0176] Fluid Flow Controller: In some cases, the disclosed systems may include one or more programmable fluid flow controllers configured to provide, for example, independently controlled pressure-driven flow through one or more separation channels or auxiliary channels intersecting the separation channels (e.g., for use alone or in combination with a voltage gradient applied to one or more separation channels). In some cases, pressure-driven flow may be used to mobilize a separated analyte peak out of the separation channel. In some cases, pressure-driven flow may be used, for example, to introduce a chemical mobilization agent (e.g., an electrolyte that disrupts a pH gradient used for isoelectric focusing) into the separation channel, thereby mobilizing the separated analyte peak out of the separation channel. In some cases, pressure-driven flow may be used, for example, to introduce a chemical mobilization agent (e.g., an elution buffer for eluting the analyte from a stationary phase confined within the separation channel) into the separation channel, thereby mobilizing the separated analyte peak out of the separation channel. In some cases, flow may be controlled by the integration of flow restrictors into the device, for example, long capillary or channel lengths to increase hydrodynamic resistance and provide uniform flow profiles and electrospray performance.

[0177] Pressure-driven fluid flow control through the disclosed devices and systems will typically be implemented through the use of pumps (or other fluid actuation mechanisms) and valves. Examples of suitable pumps include, but are not limited to, syringe pumps, programmable syringe pumps, peristaltic pumps, diaphragm pumps, piston pumps, and the like. In some embodiments, fluid flow through the system may be controlled by applying positive air pressure at one or more fluid inlets or sample or reagent reservoirs on the device. In some embodiments, fluid flow through the system may be controlled by drawing a vacuum at one or more fluid outlets or waste reservoirs. Examples of suitable valves include, but are not limited to, check valves, electromechanical two-way or three-way valves, pneumatic two-way and three-way valves, and the like. In some cases, one or more micropumps (e.g., peristaltic pumps, piezoelectric pumps), microvalves (e.g., metered injection valves, piezoelectric valves, stopcock valves, slide valves) may be integrated into the device. In some cases, controlled or pressure-driven fluid flow through the disclosed devices and systems may be implemented using bladders, blister packs, pistons, screws, glass frits, or combinations thereof. In some cases, the pressure-driven fluid flow may be pulse-free.

[0178] In some embodiments, fluid flow through the system may be controlled using one or more device or system parameters. In some cases, flow may be generated within the device by modifying the temperature of the system (e.g., to change the gas pressure within an area of ​​the device) or by introducing a temperature gradient. In some cases, reservoir height may be changed to drive flow through one or more channels of the device (e.g., via hydrostatic pressure). In some cases, a portion of the device (e.g., an inlet or outlet) may be exposed and allowed to evaporate, thereby driving fluid flow through a channel. In some cases, fluid flow may be pulseless.

[0179] In some cases, fluid flow through the disclosed devices and systems may be electrically implemented, for example, electroosmotic flow within or outside one or more of the channels of the device may be implemented using, for example, an electroosmotic pump.

[0180] Gas Flow Controller: In some cases, the disclosed systems may comprise one or more programmable gas flow controllers configured to provide independently controlled pressure-driven gas flow, e.g., through one or more gas channels or auxiliary channels that intersect with the fluidic channels. In some cases, the flow rate may be controlled by the integration of flow restrictors into the device, e.g., long capillaries or channel lengths to increase hydrodynamic resistance, variable geometries, etc., to provide a uniform gas flow profile.

[0181] Pressure-driven gas flow control through the disclosed devices and systems can comprise the use of pumps (or other fluid / gas actuation mechanisms) and valves. In some embodiments, gas flow through the system may be controlled using positive pneumatic pressure applied to one or more gas inlets of the device. In some embodiments, gas flow through the system may be controlled using a vacuum applied to one or more gas outlets. Examples of suitable valves include, but are not limited to, check valves, electromechanical two-way or three-way valves, pneumatic two-way and three-way valves, and the like. In some cases, one or more micropumps (e.g., peristaltic pumps, piezoelectric pumps) or microvalves (e.g., metered injection valves, piezoelectric valves, stopcock valves, slide valves) may be integrated into the device. In some cases, controlled or pressure-driven fluid flow through the disclosed devices and systems may be implemented using bladders, blister packs, pistons, screws, glass frits, or combinations thereof. In some cases, pressure-driven fluid flow may be pulse-free.

[0182] In some embodiments, gas flow through the system may be controlled using one or more device or system parameters. In some cases, flow may be generated within the device by modifying the temperature of the system (e.g., to change the gas pressure within the area of ​​the device) or by introducing a temperature gradient. In some cases, reservoir height may be altered to drive flow through one or more channels of the device (e.g., via hydrostatic pressure). In some cases, fluid flow may be pulseless.

[0183] Gas flow through one or more gas channels may be effected using a motive force, such as pressure-driven flow, electrokinetic force, gravity, centrifugal force, etc., or a combination thereof. In some cases, the gas flow is driven using a compressed gas source. In some preferred cases, the inlet gas pressure upstream of the gas exit orifice ranges from 100 to 110 pounds per square inch (PSI). The inlet gas pressure upstream of the gas exit orifice may be about 50 PSI, about 60 PSI, about 70 PSI, about 80 PSI, about 90 PSI, about 100 PSI, about 110 PSI, about 120 PSI, about 130 PSI, about 140 PSI, about 150 PSI, or more. In some cases, the inlet gas pressure upstream of the gas exit orifice may be at least about 50 PSI, at least about 60 PSI, at least about 70 PSI, at least about 80 PSI, at least about 90 PSI, at least about 100 PSI, at least about 110 PSI, at least about 120 PSI, at least about 130 PSI, at least about 140 PSI, at least about 150 PSI, or more. In some cases, the inlet gas pressure upstream of the gas exit orifice may be up to about 150 PSI, up to about 140 PSI, up to about 130 PSI, up to about 120 PSI, up to about 110 PSI, up to about 100 PSI, up to about 90 PSI, up to about 80 PSI, up to about 70 PSI, up to about 60 PSI, up to about 50 PSI, or less. The inlet gas pressure upstream of the gas exit orifice can fall within the range of, for example, about 50 PSI to about 110 PSI. The gas pressure at the gas exit orifice can be about 0 PSI, about 5 PSI, about 10 PSI, about 15 PSI, or about 20 PSI. In particular, the gas pressure at the lateral gas exit orifice can be about 0 PSI.

[0184] The gas flow rate may fall within a range of values ​​and may be adjusted according to a particular geometry or utility (e.g., for atomization to dry an ESI tip, etc.) The gas flow rate may be about 10 m / s, about 20 m / s, about 30 m / s, about 40 m / s, about 50 m / s, about 60 m / s, about 70 m / s, about 80 m / s, about 90 m / s, about 100 m / s, about 150 m / s, about 200 m / s, about 300 m / s, about 400 m / s, about 500 m / s, or more. The gas flow rate may be at least about 10 m / sec, at least about 20 m / sec, at least about 30 m / sec, at least about 40 m / sec, at least about 50 m / sec, at least about 60 m / sec, at least about 70 m / sec, at least about 80 m / sec, at least about 90 m / sec, at least about 100 m / sec, at least about 150 m / sec, at least about 200 m / sec, at least about 300 m / sec, at least about 400 m / sec, at least about 500 m / sec, or more. The gas flow rate may be up to about 500 m / s, up to about 400 m / s, up to about 300 m / s, up to about 200 m / s, up to about 100 m / s, up to about 90 m / s, up to about 80 m / s, up to about 70 m / s, up to about 60 m / s, up to about 50 m / s, up to about 40 m / s, up to about 30 m / s, up to about 20 m / s, up to about 10 m / s, or less. The gas flow rate may fall within a range of values, for example, from about 50 m / s to 150 m / s. In some aspects, the gas flow rate may be at sonic velocities (e.g., about 350 m / s), depending on ambient conditions. In some aspects, the gas flow rate may be at supersonic velocities, depending on ambient conditions.

[0185] The gas source may comprise air, nitrogen, oxygen, a noble gas (e.g., helium, argon, etc.), an electron carrier gas (e.g., nitrous oxide, or a fluoropolymer, e.g., fluorourethane). Combinations of gases, such as nitrogen and oxygen, may also be used. In some cases, a solvent (e.g., methanol) may be added to the gas line, which may help drive charge onto the molecules when the gas flow converges with the fluid flow path of the liquid at or near the fluid channel orifice. In some cases, the gas source may be obtained from an analytical instrument (e.g., a mass spectrometer) and may be integrated into the devices, systems, and methods described herein.

[0186] Different modes of fluid flow control may be utilized at different times during the performance of the disclosed analyte separation methods; for example, forward flow (relative to the inlets and outlets for a given device or fluid or gas channel), reverse flow, oscillating or pulsatile flow, or combinations thereof may all be used. For example, in some cases, oscillating or pulsatile flow may be used during a device priming step, e.g., to facilitate the release of any air bubbles that may be trapped within the device. In some cases, the device may be subjected to a vacuum (e.g., evacuated) for device priming, e.g., to facilitate bubble-free introduction of fluids or reagents.

[0187] Different fluid flow rates may be utilized at different times during the implementation of the disclosed analyte separation methods. For example, in some instances of the disclosed devices and systems, the volumetric flow rate may range from -100 mL / sec to +100 mL / sec. In some instances, the absolute value of the volumetric flow rate may be at least 0.001 mL / sec, at least 0.01 mL / sec, at least 0.1 mL / sec, at least 1 mL / sec, at least 10 mL / sec, or at least 100 mL / sec. In some instances, the absolute value of the volumetric flow rate may be at most 100 mL / sec, at most 10 mL / sec, at most 1 mL / sec, at most 0.1 mL / sec, at most 0.01 mL / sec, or at most 0.001 mL / sec. The volumetric flow rate at a given time may have any value within this range, for example, a forward flow rate of 2.5 mL / sec, a reverse flow rate of -0.05 mL / sec, or a value of 0 mL / sec (i.e., stopped flow). In some cases, the pressure-driven fluid flow mode and / or fluid flow rate through each separation channel and / or auxiliary fluid channel may be programmed independently of one another to follow a defined time course.

[0188] During ESI, the flow rate of the sample (or separated sample) from the ESI orifice can be adjusted to obtain near-nanovolume flow. For example, the flow rate of the sample as it is expelled to form the Taylor cone may be about 1 nanoliter / minute (nL / min), 5 nL / min, 10 nL / min, 20 nL / min, 30 nL / min, 40 nL / min, 50 nL / min, 60 nL / min, 70 nL / min, 80 nL / min, 90 nL / min, 100 nL / min, 200 nL / min, 300 nL / min, 400 nL / min, 500 nL / min, 600 nL / min, 700 nL / min, 800 nL / min, 900 nL / min, 1000 nL / min (1 μL / min), 2 μL / min, 3 μL / min, 4 μL / min, 5 μL / min, 10 μL / min, or more. The flow rate of the sample as it is expelled to form the Taylor cone can be at least about 1 nanoliter per minute (nL / min), at least about 5 nL / min, at least about 10 nL / min, at least about 20 nL / min, at least about 30 nL / min, at least about 40 nL / min, at least about 50 nL / min, at least about 60 nL / min, at least about 70 nL / min, at least about 80 nL / min, at least about 90 nL / min, at least about 100 nL / min, at least about The flow rate may be 200 nL / min, at least about 300 nL / min, at least about 400 nL / min, at least about 500 nL / min, at least about 600 nL / min, at least about 700 nL / min, at least about 800 nL / min, at least about 900 nL / min, at least about 1000 nL / min (1 μL / min), at least about 2 μL / min, at least about 3 μL / min, at least about 4 μL / min, at least about 5 μL / min, at least about 10 μL / min, or more.The flow rate of the sample as it is expelled to form the Taylor cone is up to about 10 μL / min, up to about 5 μL / min, up to about 4 μL / min, up to about 3 μL / min, up to about 2 μL / min, up to about 1 μL / min, up to about 900 nL / min, up to about 800 nL / min, up to about 700 nL / min, up to about 600 nL / min, up to about 500 nL / min, up to about 400 The flow rate may be up to about 300 nL / min, up to about 200 nL / min, up to about 100 nL / min, up to about 90 nL / min, up to about 80 nL / min, up to about 70 nL / min, up to about 60 nL / min, up to about 50 nL / min, up to about 40 nL / min, up to about 30 nL / min, up to about 20 nL / min, up to about 10 nL / min, up to about 9 nL / min, up to about 8 nL / min, up to about 7 μL / min, up to about 6 μL / min, up to about 5 μL / min, up to about 4 μL / min, up to about 3 μL / min, up to about 2 μL / min, or up to about 1 μL / min. The flow rate of the sample as it is expelled to form the Taylor cone can fall within a range of values, for example, from about 500 nL / min to about 1 μL / min.

[0189] Autosamplers and Fluid Handling Systems: In some cases, the disclosed systems may further include an autosampler or fluid handling system configured for automated, independently controlled loading of sample aliquots and / or other separation reaction reagents into multiple sample or reagent inlet ports on the separation channel. In some cases, a custom-made autosampler or fluid handling module may be incorporated into the disclosed systems. In some cases, a commercially available autosampler or fluid handling module may be integrated into the disclosed systems. Examples of suitable commercially available autosamplers include, but are not limited to, the Agilent 1260 Infinity Dual Loop Autosampler and 1260 Infinity High Performance Micro Autosampler (Agilent Technologies, Santa Clara, CA), the HT1500L HPLC Autosampler (HTA, Brescia, Italy), Spark Holland Alias ​​(Spark-Holland, Emmen, Netherlands), and the SIL-20A / AC HPLC Autosampler (Shimadzu, Columbia, MD). Examples of suitable commercially available fluid handling systems (or liquid handling systems) include, but are not limited to, the Tecan Fluent® system (Tecan Trading AG, Switzerland), the Hamilton Microlab STAR and Microlab NIMBUS systems (Hamilton, Reno, NV), and the Agilent Bravo automated liquid handling platform and Agilent vertical pipetting station (Agilent Technologies, Santa Clara, CA).

[0190] In some cases, one or more fluid flow controllers or fluid handling systems may be used to fill or replenish one or more reservoirs. The reservoirs may be in fluid communication with a cartridge, microfluidic device, or assembly as described herein, or may be connected to a fluid line that is in interface contact with the cartridge or assembly, e.g., via an interface contact unit (see, e.g., Figures 17A-17B and Example 9 below). The reservoirs may comprise, for example, a compressed gas unit, reagents for performing a separation reaction (e.g., catholyte, anolyte, carrier ampholyte, etc.), reagents for performing a mobilization reaction, or reagents for performing an electrospray ionization reaction.

[0191] Waste Management: In some cases, a gas channel or multiple gas channels in a microfluidic device are used to manage waste within or adjacent to a fluidic (e.g., separation) channel. The gas channel may be used, for example, to direct fluid away from a fluidic orifice that is in fluid communication with the separation channel. For example, the gas channel may be used to direct fluid toward a waste receptacle or generally away from a downstream analytical instrument (e.g., a mass spectrometer). In some cases, the gas channel exhausts air at a gas exit orifice adjacent to the fluidic orifice, and the airflow is used to remove excess liquid from the fluidic orifice (which may comprise or serve as an electrospray tip). In some cases, the gas channel may be used to clean the electrospray tip. For example, higher pressure may be applied to direct excess fluid or waste products away from the electrospray tip.

[0192] In certain embodiments, the system includes a waste management module, which can be either integrated with (i.e., attached to) or separate from the microfluidic device. The waste management module may be used to collect waste products from the microfluidic device. In some cases, the waste management module may additionally or alternatively be used to manage droplet formation at an outlet or surface of the microfluidic device. For example, the waste management module may be used to prevent droplets from forming at the outlet (e.g., electrospray tip) of the device and / or from wicking up to a different segment or portion of the device (e.g., an inlet, an interfaced electrode, etc.). In some cases, the waste management module may include application of positive or negative pressure (e.g., vacuum). In such cases, a vacuum may be applied to a portion of the microfluidic device (e.g., an outlet or electrospray tip). For example, a flange or adapter may be applied to the chip, thereby allowing a vacuum to interface with the device with minimal interference to the device installation or any downstream analysis unit (e.g., a mass spectrometer). A vacuum may then be used to suck the droplets or waste products as they exit the outlet or electrospray tip. In some cases, the waste management module uses positive pressure. For example, an airflow (e.g., from the atomizer module) may be used to direct the droplets away from the electrospray tip. In such examples, the airflow may be connected to an air or nitrogen gas source and / or a pressurizer to generate air (or nitrogen gas) pressure to expel the droplets or direct them away from the device or a portion thereof (e.g., the electrospray tip). In some cases, the waste management module may include a spray unit. For example, the atomizer may be configured to be affixed to the chip.The atomizer may have the necessary geometry to direct air toward the tip so that droplets or waste products are directed away from the Electospray tip or outlet (e.g., to a waste receptacle). The atomizer may have a sealing mechanism and may be connected to an air source and / or pressurizer to generate air pressure to eject or direct droplets away from the Electospray tip. In some cases, the atomizer may include a nozzle. The atomizer may be made of a polymer, metal, or ceramic material.

[0193] In some cases, the waste management methods described herein may be used in conjunction with other approaches for waste management. For example, the device may have a geometry or chemical / material properties that allow for control of droplet formation at the fluid orifice or outlet and / or minimize droplet and fluid wicking to different segments or portions of the device (e.g., electrodes or inlets). In some cases, a coating may be used to allow droplet formation at the tip or outlet of the device and may help prevent fluid wicking to other segments or portions of the device. In some cases, the coating may be a hydrophobic coating.

[0194] In some cases, the geometry or orientation of the device may be used to control droplet formation at the outlet and / or minimize siphoning of droplets into different segments or portions of the device. For example, the outlet or electrospray tip may be shaped with a triangular tip to enable optimal droplet formation. In some cases, the geometry of the device may be used to control waste management. For example, in cases where a microfluidic device comprises a gas channel and a fluid channel, the geometry of the gas channel may be optimized to allow high gas pressure to direct fluid away from the fluid orifice or outlet. High gas pressure may also be used, for example, to remove debris or other undesired products or by-products from the fluid orifice (e.g., an ESI tip).

[0195] Imaging Module: In some cases, the system may further include an imaging module configured to acquire a series of one or more images of two or more separation channels or portions thereof. In some cases, the image field of view may include all or a portion of two or more separation channels. In some cases, the imaging may include continuous imaging of all or a portion of two or more separation channels while a separation and / or mobilization reaction is being performed. In some cases, the imaging may include intermittent or periodic imaging of all or a portion of two or more separation channels while a separation and / or mobilization reaction is being performed. In some cases, the imaging may include acquiring UV absorbance images. In some cases, the imaging may include acquiring fluorescence images, e.g., of intrinsic fluorescence or fluorescence due to the presence of exogenous fluorescent labels attached to the analytes. In some cases, the imaging module may be configured, e.g., to determine when an isoelectric focusing reaction is complete and / or to detect obstructions (e.g., the introduction or formation of bubbles in the separation channel).

[0196] Any of a variety of imaging systems or system components may be utilized for purposes of implementing the disclosed methods, devices, and systems. Examples include, but are not limited to, one or more light sources (e.g., light-emitting diodes (LEDs), diode lasers, fiber lasers, gas lasers, halogen lamps, arc lamps, etc.), focusing lenses, objective lenses, mirrors, filters, beam splitters, prisms, image sensors (e.g., CCD image sensors or cameras, CMOS image sensors or cameras), and the like, or any combination thereof. In some cases, the one or more light sources may comprise an array of light sources. For example, an LED array may be used to illuminate one or more regions of the device. Depending on the imaging mode utilized, the light source and image sensor may be positioned on opposite sides of the microfluidic device, e.g., so that absorbance-based images can be obtained. In some cases, the light source and image sensor may be positioned on the same side of the microfluidic device, e.g., so that epifluorescence images can be obtained.

[0197] As described above, images may be acquired continuously during the separation and / or recruitment steps, or may be acquired at random or predetermined time intervals. In some cases, a series of one or more images is acquired continuously, or at random or predetermined time intervals. In some cases, a series of short-exposure images (e.g., 10-20 images) is acquired rapidly (e.g., on a millisecond time scale) and then averaged to provide a "single image" with improved signal-to-noise ratio. In some cases, a "single image" is acquired every 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, or at longer intervals. In some cases, longer exposure times may be used to improve signal-to-noise ratio. In some cases, the series of one or more images may comprise a video image.

[0198] Image Processing: In some cases, as described above, the system may include a processor, controller, or computer configured to run image processing software to detect the presence of analyte peaks, determine the location of pI markers or separated analyte bands, determine peak widths, and determine peak shape (e.g., Gaussian fit or other curve-fitting algorithms) or changes in any of these parameters over time. In some cases, image processing may be used to detect obstructions, such as the introduction or formation of air bubbles, in one of the two or more separation channels. Any of a variety of image processing algorithms may be utilized for image pre-processing or image processing when implementing the disclosed methods and systems. Examples include, but are not limited to, Canny edge detection methods, Canny-Delicie edge detection methods, first-order gradient edge detection methods (e.g., Sobel operator), second-order difference edge detection methods, phase congruence edge detection methods, other image segmentation algorithms (e.g., intensity thresholding, intensity clustering methods, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., generalized Hough transform for detecting arbitrary shapes, circular Hough transform, etc.), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation, Savitzky-Golay smoothing, eigenanalysis, etc.), or any combination thereof.

[0199] Microplate Handling Robot: In some cases, the system may further include a microplate handling robot module configured to transport and exchange microplates that serve as sources for samples and / or reagents. In some cases, the system may further include a microfluidic device handling robot module configured to transport and exchange microfluidic devices used in the system, for example, after a fault is detected. In some cases, microplate handling and microfluidic device handling may be handled by the same robot module. In some cases, a custom robot may be incorporated into the disclosed system to perform these functions. In some cases, commercially available robotic systems may be adapted and / or integrated into the disclosed system to perform these functions. Examples of suitable microplate handling robotic systems include, but are not limited to, Tecan Robotic Gripper Arms (Tecan Trading AG, Switzerland) and Agilent Direct Drive and BenchBot Robots (Agilent Technologies, Santa Clara, CA).

[0200] Temperature Control: In some cases, the disclosed systems and methods may be temperature controlled. In some cases, a gas channel or multiple gas channels of a device may be used to adjust or control the temperature of the substrate. For example, the temperature of the gas may be varied so that heat can be dissipated into or from the gas channel, heating or cooling the device. In some cases, a portion of the system (e.g., a portion of the device) may be temperature controlled. In some cases, the system or one or more components of the system may be cooled using, for example, a Peltier, fan or other heat sink, air knife. In some cases, the cooling system may be integrated with a waste management system (e.g., an air knife). In some cases, the cooling system may include a compressor for cooling. In some cases, the system may include an environmental or temperature control chamber. In some cases, a cooling block or pre-cooled block may be used (e.g., coupled to the stage or cartridge). In some cases, the system of its components may be constructed from materials that allow heat exchange with the environment. In some cases, the system may include a liquid heat exchanger. In some cases, the system may include a liquid heat exchanger. In some embodiments, the system will control the temperature within a range of about 15-35° C. In some embodiments, the system will control the temperature within about + / - 5° C. In some embodiments, the system will control the temperature within about + / - 1° C.

[0201] Applications: The disclosed methods, devices, and systems have potential applications in a variety of fields, including, but not limited to, proteomics research, cell research, drug discovery and development, and clinical diagnostics. For example, the improved reproducibility and quantitation that can be achieved for separation-based characterization of analyte samples using the disclosed methods can be highly beneficial for the characterization of biological and biopharmaceutical products during development and / or manufacturing.

[0202] Biologics and biosimilars are types of drugs that include, for example, recombinant proteins, antibodies, live viral vaccines, human plasma-derived proteins, cell-based drugs, naturally occurring proteins, antibody-drug conjugates, protein-drug conjugates, and other protein drugs. The FDA and other regulatory agencies require the use of a tiered approach to demonstrating biosimilarity, which may include comparison of the proposed product and a reference product with respect to structure, function, animal toxicity, human pharmacokinetics (PK) and pharmacodynamics (PD), clinical immunogenicity, and clinical safety and efficacy (see, e.g., “Scientific Considerations in Demonstrating Biosimilarity to a Reference Product: Guidance for Industry,” US Department of Health and Human Services, Food and Drug Administration, April 2015). Examples of structural characterization data that may be required for a protein product include primary structure (i.e., amino acid sequence), secondary structure (i.e., the degree of folding to form an alpha helix or beta sheet structure), tertiary structure (i.e., the three-dimensional shape of the protein produced by folding of the polypeptide backbone and secondary structural domains), and quaternary structure (e.g., the number of subunits required to form an active protein complex or an aggregated state of the protein). In many cases, this information may not be available without employing laborious, time-intensive, and expensive techniques such as X-ray crystallography. Thus, there is a need for experimental techniques that allow for convenient, real-time, and relatively high-throughput characterization of protein structure for the purpose of establishing biosimilarity between candidate biological drugs and reference drugs.

[0203] In some cases, the disclosed methods, devices, and systems may be used to provide structural comparison data for a biological drug candidate (e.g., a monoclonal antibody (mAb)) and a reference biological drug for the purpose of establishing biosimilarity. For example, in some cases, determination of the isoelectric points for a drug candidate and a reference drug may provide important evidence in support of demonstrating biosimilarity. In some embodiments, isoelectric point data for a drug candidate and a reference drug, both treated with a site-specific protease under the same reaction conditions, may provide important evidence in support of demonstrating biosimilarity. In some embodiments, the disclosed methods, devices, and systems may be used to monitor a biological drug manufacturing process (e.g., monitor a bioreactor process in real time) and ensure product quality and consistency by analyzing samples taken at different times in the production process or from different production steps.

[0204] The disclosed devices and systems for performing multiple, independently controlled separation reactions in parallel offer several advantages over currently available technologies, such as the ability to perform different isoelectric focusing reactions (or other separation reactions) in different channels (e.g., using different pH gradients, different focusing times, different focusing voltages, etc.) for more detailed and accurate sample characterization (e.g., more accurate determination of pI), or the ability to simultaneously process multiple samples in parallel using the same set of separation reaction conditions for higher-throughput sample characterization. Additionally, independent monitoring and / or recording of the current traces and / or voltage settings used per separation channel can be advantageous in meeting data tracking requirements for FDA submissions when attempting to demonstrate biosimilarity, etc. As noted, in some cases, the disclosed devices and systems may be configured to identify sample process failures, e.g., the presence or formation of air bubbles within a microfluidic device, and to initiate a recovery step, e.g., by automatically reloading sample from a microtiter plate or other sample source and repeating the separation reaction. (Example)

[0205] These examples are provided for illustrative purposes only, and not to limit the scope of the claims provided herein. [Example]

[0206] Example 1 - Microfluidic device with multiple separation channels FIG. 1A provides a diagram of one non-limiting example of a microfluidic device for performing multiple separation reactions, e.g., isoelectric focusing reactions. The device includes a lower substrate 101, which may be substantially planar, made of fused silica, in which fluidic channels 210 μm wide and 100 μm deep are fabricated using, for example, embossing, laser micromachining, or photolithography and wet chemical etching. The fluidic channels are sealed by bonding the substrate 101 to a transparent coverslip 102. In some cases, e.g., when UV absorbance imaging is used to monitor separation and / or recruitment reactions, the substrate 101 may be fabricated from an optically transparent material. In some cases, e.g., when epifluorescence imaging is used to monitor separation and / or recruitment reactions, the substrate 101 may be fabricated from an optically opaque material. While illustrated as a rectangle, it should be understood that the device may take any useful shape. In some embodiments, a microfluidic device may comprise a tip (e.g., at the distal end) that may allow fluid to be directed away from the device (e.g., to a waste receptacle or an analytical unit, e.g., a mass spectrometer).

[0207] Access to the fluidic channels within the device is provided through a sample inlet port 103, an anode well 104, a cathode well 106, a sample outlet port 107, and a chemical mobilizer inlet port 109. One anode well 104 and one cathode well 106 are in fluid and electrical communication with the proximal and distal ends of each separation channel 105, respectively (four separation channels are shown in this non-limiting example). Electrodes may, in some cases, be placed in contact with the anode well 104 and the cathode well 106. The separation channels extend beyond the cathode well 106 to a sample outlet port 107 (labeled for only two of the four separation channels shown in the figure). The chemical mobilizer inlet port 109 is connected to the distal end of the separation channel 105 via a chemical mobilization channel 108 (labeled for only two of the four separation channels shown in the figure). As illustrated in FIG. 1A, the inlet port 109 and outlet port 107 may be configured to be loaded through the side of the device, which may facilitate full channel or full device imaging.

[0208] For use in performing multiple isoelectric focusing reactions and separating mixtures of proteins, protein samples are placed into vials and premixed with an ampholyte pH gradient and pI marker before being loaded into an autosampler. Samples are loaded sequentially into the device by the autosampler via sample inlet port 103, onto the microfluidic device, through separation channel 105, and out of the device through sample outlet port 107 to waste.

[0209] A catholyte fluid (e.g., 1% NN4OH in HO) is loaded into the cathode well 106, an anolyte (e.g., 10 mM H3PO4) is loaded into the anode well 104, and a mobilizer solution (e.g., 49% MeOH, 49% HO, 1% acetic acid) is connected to the mobilizer inlet port 109.

[0210] After all reagents are loaded, an electric field of, for example, +600 V / cm is applied from one or more of the anode wells 104 to the corresponding cathode wells 106 by connecting electrodes to the anode wells 104 and cathode wells 106 and initiating isoelectric focusing. As described above, the voltage and / or current applied to each of the separation channels 105 may be independently controlled and recorded as a function of time. In some cases, the electrodes used for the anodes and cathodes may be integrated with the device. For UV absorbance imaging, a collimated beam of light provided by a UV light source is aligned with the separation channels 105, and an image sensor (e.g., a CCD or CMOS camera) is placed on the opposite side of the separation channels 105 to measure the amount of light transmitted through each of the separation channels 105, thereby imaging and detecting proteins (or other separated analytes) focused by their absorbance. In some cases, the focused proteins may be unlabeled and detected through intrinsic absorbance at 220 nm, 280 nm, or any other wavelength at which the proteins will absorb light. For fluorescence imaging, i.e., epifluorescence imaging, excitation light of a suitable wavelength is delivered to the separation channel 105 using an optical assembly comprising a suitable dichroic reflector and bandpass filter, and the emitted fluorescence is collected from the separation channel 105 by the same optical assembly and imaged onto an image sensor. In some cases, the focused proteins (or other separated analytes) may be imaged and detected using intrinsic fluorescence. In some cases, the focused proteins may be detected using non-covalently bound fluorescent, chromogenic, fluorescent, or chromogenic labels, such as SYPRO® Ruby, Coomassie Blue, and the like. In some cases, portions of the device may be constructed from optically opaque materials so that light is transmitted only through the separation channel 105, thereby preventing any stray light from reaching the image sensor without passing through the separation channel 105 and increasing the sensitivity of the UV absorbance measurement.

[0211] Images of focused proteins within all or a portion of the separation channels 105 can be continuously and / or periodically captured as isoelectric focusing reactions are performed within the multiple separation channels 105. In some cases, detection of the location of pI markers within the images of the separation channels 105 may be used to determine the local pH as a function of position along the separation channel and, by extrapolation, make a more accurate determination of the pI for the separated proteins (or other analytes). In some cases, when focusing is complete, positive pressure is applied at the sample inlet port 103 and / or anode well 104 to mobilize the separated protein (or other analyte) mixture toward the sample outlet 107. In some cases, when focusing is complete, the electrodes connected to the cathode well 106 are disconnected, and electrodes in electrical communication with the mobilizer channel 108 are used to apply a 600 V / cm electric field from the anode well 104 to the chemical mobilizer inlet 109 to electrophoretically introduce the mobilizer into the separation channels 105. In some cases, a slight positive pressure applied to the mobilizing agent inlet 109 may be used instead of, or in addition to, electrophoretic introduction of the chemical mobilizing agent.

[0212] In the case of electrophoretic introduction of the mobilizing agent, acetic acid in the mobilizing agent solution is drawn by the electric field into the separation channel 105, where it ionizes proteins and ampholytes, disrupting the pH gradient used for isoelectric focusing. The ionization of the concentrated protein fractions causes them to migrate out of the separation channel 105 toward the sample outlet 107. Continued imaging of the separation channel 105 during the mobilization process can be used to refine the pI determination for each separated protein. [Example]

[0213] Example 2 - Prophetic Example of Use of the Disclosed Devices and Systems for Demonstration of Biosimilarity One non-limiting example of the utility of the disclosed devices and systems is in the field of biologics and biosimilarity demonstration. As noted above, the FDA and other regulatory agencies require the use of a tiered approach to demonstrating biosimilarity, which may include comparison of the proposed product and a reference product with respect to structure, function, animal toxicity, human pharmacokinetics (PK) and pharmacodynamics (PD), clinical immunogenicity, and clinical safety and efficacy. Examples of structural characterization data that may be required for protein products include primary structure (i.e., amino acid sequence), secondary structure (i.e., the degree of folding to form an alpha-helical or beta-sheet structure), tertiary structure (i.e., the three-dimensional shape of the protein produced by folding of the polypeptide backbone and secondary structural domains), quaternary structure (e.g., the number of subunits required to form an active protein complex or an aggregated state of the protein), and post-translational modifications. Accurate determination of protein isoelectric point can provide important data for the comparison of a biological drug candidate to a reference drug to demonstrate biosimilarity. Sample aliquots of the manufactured biofollow-on candidate and reference drug may be loaded into the disclosed device or system and characterized under one or more sets of isoelectric focusing reaction conditions (e.g., using different buffers, pH gradients, applied voltages, and / or currents, etc.) to determine accurate pI values ​​under one or more sets of reaction conditions and provide valuable comparative data for the biofollow-on candidate and reference drug. Additionally, monitoring and recording of current traces (and other operating parameters used to perform the isoelectric focusing reactions) for each individual separation reaction facilitates compliance with FDA data submission requirements. [Example]

[0214] Example 3 - Tracking the Velocity of Analyte Peaks as They Exit the Microfluidic Chip and Enter the Mass Spectrometer FIG. 1B shows another non-limiting example of a microfluidic device described herein. Microfluidic channel network 110 is fabricated in a 250 μm thick layer of opaque cyclic olefin polymer. Channel 122 is 250 μm deep, cutting all the way through the 250 μm layer. All other channels are 50 μm deep. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer, fabricating a planar microfluidic device. Ports 112, 114, 116, 118, and 120 provide access to the channel network for reagent introduction and electrical contact from external reservoirs. Port 112 is connected to a vacuum source, allowing channel 113 to act as a waste channel, allowing priming of other reagents through the channel network to "waste." Acid (e.g., 1% formic acid) is primed through port 118 into channels 119, 122, 124, and 113 and up to port 112. A sample (e.g., peptide or protein diluted in 4% Pharmalyte 3-10, 12.5 mM pI standard 3.38 (purified peptide, sequence: Trp-Asp-Asp-Asp), 12.5 mM pI standard 10.17 (purified peptide, sequence: Trp-Tyr-Lys-Arg)) is primed through port 118 into channels 119, 122, 124, and 113 and to port 112. This leaves channel 122 containing the sample analyte. A base (e.g., 1% dimethylamine) is primed through port 114 into channels 115, 124, and 113 and to port 112. A mobilizing agent (e.g., 1% formic acid, 49% methanol) is primed through port 120 into channels 121, 124, and 113 and out of channel 113 to port 112.

[0215] Electrophoresis of the analyte sample in channel 122 is performed by applying 4,000 V to port 118 and connecting port 120 to ground. Ampholytes in the analyte sample establish a pH gradient in channel 122. Absorbance imaging of the separation is performed using a 280 nm light source aligned with channel 122 and measuring the transmission of 280 nm light through channel 122 with a CCD camera. The software calculates absorbance by comparing the light transmission during separation or mobilization to a "blank" reference measurement obtained in the absence of focused analyte before the analyte is flowed, and then displays the absorbance per pixel over the length of channel 122. Locations where the standard or analyte is focused appear as peaks in the absorbance trace derived from the image data.

[0216] Once the analyte has completed focusing, a final focused absorbance image is captured. The software will identify the spatial location of the pI markers, interpolate between them, and calculate the pI of the focused analyte fraction peak. At this point, the control software will trigger a relay to disconnect the ground at port 120, connect port 114 to ground, and set the pressure on the mobilizer reservoir connected to port 114 to establish a flow of 100 nL / min of mobilizer solution through port 114 into channels 115 and 124 and out of the chip at orifice 126. Orifice 126 is positioned 2 mm away from the mass spectrometer ESI inlet with an inlet voltage of -3,500 V to -4,500 V.

[0217] Pressure-driven flow will direct the mobilizer from port 114 to orifice 126, while a portion of the formic acid in the mobilizer reagent will electrophorese in the form of formate from channel 115 through channel 122 to the anode at port 118. As the formate progresses through channel 122, it disrupts the isoelectric pH gradient, increasing the charge on the ampholytes, standards, and analyte sample, causing them to electrophoretically migrate from channel 122 into channel 124, and pressure-driven flow from port 120 will carry them into the ESI spray and out orifice 126.

[0218] As recruitment occurs, the software continues to capture absorbance images, identifying peaks and tracking their migration from imaging channel 122 into channel 124. By tracking the time each peak exits imaging channel 122, its velocity, and the flow rate within channel 124, the software can calculate the time the peak traverses channel 124 and is introduced into the mass spectrometer through orifice 126, allowing direct correlation between the original focused peaks and the resulting mass spectrum. [Example]

[0219] Example 4 - Microfluidic device with gas channels for liquid atomization 2A-2B show top-down schematic views of an exemplary microfluidic device described herein, comprising a separation channel and two gas channels. Referring to FIG. 2A, the microfluidic device comprises a substrate 200, which is approximately 1 millimeter (mm) thick and comprises fused silica. The channels are chemically etched to a depth of 40 microns and a width of 86-600 microns. In some embodiments, the microfluidic device may comprise a tip (e.g., at the distal end), which may allow fluid to be directed away from the device (e.g., to a waste receptacle or an analytical unit, e.g., a mass spectrometer).

[0220] Access to the fluidic channels within the device is provided through a sample inlet port 203, an anode port 204, a cathode port 206, a sample outlet port 207 (also referred to herein as a "fluidic orifice"), and a chemical mobilizer inlet port 209. The anode port 204 and the cathode port 206 are in fluid and electrical communication with the proximal and distal ends of the separation channel 205, respectively. Electrodes can, in some cases, be placed in contact with the anode port 204 and the cathode port 206. In other cases, the anode port 204 and the cathode port 206 are in fluid and / or electrical communication with electrode reservoirs (not shown), which connect to the anode port 204 and the cathode port 206, for example, via channels. The separation channel 205 extends beyond the cathode port 206 to the sample outlet port 207. Chemical mobilizer inlet port 209 is connected to the distal end 205 of the separation channel via chemical mobilization channel 208. The device also includes two gas channels 211 and 213, which have gas orifices or outlets adjacent to sample outlet port 207. Gas inlet ports 215 and 217 allow gas (e.g., air, nitrogen, etc.) to enter gas channels 211 and 213. In some cases, the gas orifices or outlets of gas channels 211 and 213 may be positioned symmetrically from sample outlet port 207. The inlet ports, including anode port 204, cathode port 206, sample inlet port 203, chemical mobilizer inlet port 209, and gas inlet ports 215 and 217, may be configured to be loaded through a side or edge 220 of the device, which may facilitate various processes such as reagent loading, imaging of the entire channel, or the entire device, etc.

[0221] For use in performing isoelectric focusing reactions and separating mixtures of proteins, protein samples are premixed with an ampholyte pH gradient and pI marker before being placed in a vial and loaded into an autosampler. Samples are loaded sequentially into the device by the autosampler via sample inlet port 203, onto the microfluidic device, through separation channel 205, and out of the device through sample outlet port 207 to waste.

[0222] A catholyte fluid (e.g., 1% NN4OH in HO) is loaded into the cathode port 206, an anolyte (e.g., 10 mM H3PO4) is loaded into the anode port 204, and a mobilizer solution (e.g., 49% MeOH, 49% HO, 1% acetic acid) is connected to the mobilizer inlet port 209.

[0223] After all reagents are loaded, an electric field of, for example, +600 V / cm is applied from the anode port 204 to the corresponding cathode port 206 by connecting electrodes to the anode and cathode reservoirs (not shown) and initiating isoelectric focusing. In some cases, the electrodes used for the anode and cathode may be integrated with the device. For UV absorbance imaging, a collimated beam of light provided by a UV light source is aligned with the separation channels 205, and an image sensor (e.g., a CCD camera or CMOS camera) is placed on the opposite side of the separation channels 205 to measure the amount of light transmitted through each of the separation channels 205, thereby imaging and detecting the focused proteins (or other separated analytes) using absorbance. In some cases, the focused proteins may be unlabeled and detected through their intrinsic absorbance at 220 nm, 280 nm, or any other wavelength at which the proteins will absorb light. For fluorescence imaging, i.e., epifluorescence imaging, excitation light of a suitable wavelength is delivered to the separation channel 205 using an optical assembly comprising suitable dichroic reflectors and bandpass filters, and the emitted fluorescence is collected from the separation channel 205 by the same optical assembly and imaged onto an image sensor. In some cases, the focused proteins (or other separated analytes) may be imaged and detected using intrinsic fluorescence. In some cases, the focused proteins may be detected using non-covalently bound fluorescent, chromogenic, fluorescent, or chromogenic labels, such as SYPRO® Ruby, Coomassie Blue, and the like. In some cases, portions of the device may be constructed from optically opaque materials so that light is transmitted only through the separation channel 205, thereby preventing any stray light from reaching the image sensor without passing through the separation channel 205 and increasing the sensitivity of the UV absorbance measurement.

[0224] Images of focused proteins within all or a portion of the separation channel 205 can be continuously and / or periodically captured as isoelectric focusing reactions are performed within the multiple separation channels 205. In some cases, detection of the location of pI markers within the separation channel 205 images may be used to determine the local pH as a function of position along the separation channel and, by extrapolation, make a more accurate determination of pI for the separated proteins (or other analytes). In some cases, when focusing is complete, positive pressure is applied at the sample inlet port 203 and / or anode port 204 to mobilize the separated protein (or other analyte) mixture toward the sample outlet 207. In some cases, when focusing is complete, the electrodes connected to the cathode port 206 are disconnected, and electrodes in electrical communication with the mobilizer channel 208 are used to apply a 600 V / cm electric field from the anode port 204 to the chemical mobilizer inlet 209 to electrophoretically introduce the mobilizer into the separation channel 205. In some cases, a slight positive pressure applied to the mobilizing agent inlet 209 may be used instead of, or in addition to, electrophoretic introduction of the chemical mobilizing agent.

[0225] In the case of electrophoretic introduction of the mobilizing agent, acetic acid in the mobilizing agent solution is drawn by the electric field into the separation channel 205, where it ionizes proteins and ampholytes, disrupting the pH gradient used for isoelectric focusing. The ionization of the concentrated protein fractions causes them to migrate out of the separation channel 205 toward the sample outlet 207. Continued imaging of the separation channel 205 during the mobilization process can be used to refine the pI determination for each separated protein.

[0226] 2B shows an enlarged schematic view of the outlet portion of the microfluidic device illustrated in FIG. 2A. Gas channels 211 and 213 each have a gas orifice 219 and 221, respectively, from which gas is discharged. Gas orifices 219 and 221 are positioned adjacent to sample outlet port 207 and are used to atomize the sample near sample outlet port 207. In some cases, gas channels 211 and 213, or portions thereof, are positioned symmetrically from sample outlet 207. In some cases, gas channel orifices 219 and 221 are positioned symmetrically from sample outlet 207. In some cases, gas channels 211 and 213 each comprise a region parallel to a portion of separation channel 205. As shown in FIG. 2A, gas channels 211 and 213 have different lengths. However, gas channels 211 and 213 can be configured to provide substantially similar hydrodynamic flow resistances to gas exit orifices 219 and 221, respectively. For example, gas channels 211 and 213 may have different cross-sectional areas along portions of the channels but approximately the same cross-sectional area near gas exit orifices 219 and 221. In some cases, the gas channels may narrow at their distal ends to increase the flow rate or velocity at the gas exit orifices. For devices in which gas exit orifices 219 and 221 are positioned symmetrically from sample outlet 207, similar hydrodynamic flow resistances at each of gas exit orifices 219 and 221 may be beneficial in achieving a steady airflow for sample nebulization near sample outlet 207. In other cases, gas exit orifices 219 and 221 may not be positioned symmetrically from sample outlet 207. In such cases, the hydrodynamic flow resistance at each of the gas outlet orifices 219 and 221 may be different so that the volume, amount, or flow rate of air reaching the sample outlet 207 is approximately the same.

[0227] During atomization, which may be performed in parallel with ESI, the sample is dispersed or broken into smaller droplets. In some cases, the atomized droplets are then exposed to a complementary drying gas, resulting in evaporation of the liquid and production of gas-phase ions that are introduced into a mass spectrometer (not shown). Continuous imaging of the sample outlet 207 or the area surrounding the sample outlet 207 during the electrospray process can be used to determine ESI or Taylor cone characteristics, such as droplet size, Taylor cone shape, etc. [Example]

[0228] Example 5 - Design parameters for optimizing sample atomization during ESI Atomization of a sample as disclosed herein is achieved by shear and inertial forces generated by a gas jet to break up a continuous liquid stream into small droplets. Atomization of a sample (or separated sample) may be used to improve quantitative measurement of a sample (or separated sample) under microflow, where the sample (or separated sample) is flowed through an ESI tip at approximately microliter-scale flow rates (e.g., microliters / minute). Atomization is particularly useful for higher flow rates in the microliter / minute range (several, tens, or hundreds of μl / minute, where additional solvent must be removed from the sample to achieve single ions that are collected in a mass spectrometer for analysis). Various parameters of the gas channels disclosed herein can be modified and configured to optimize atomization. Figures 3A-3D schematically show non-limiting examples of modifiable parameters of a microfluidic device comprising a fluid outlet channel and two gas channels that can be used to atomize a sample within the fluid outlet channel. In some cases, the fluid output channel is fluidly coupled (eg, at the proximal end) to a separation channel (eg, at the distal end) for use in a separation reaction such as isoelectric focusing.

[0229] FIG. 3A shows a schematic diagram of a variable convergence angle between the distal end of the gas channel and the distal end of the fluid ejection channel, which includes an orifice (also referred to herein as the "sample outlet," "fluid orifice," or "fluid exit orifice"). In such a configuration, gas channels 311 and 313 are positioned symmetrically from the outlet of fluid ejection channel 307. The convergence angle between the distal section of gas channel 313 and the distal section of the fluid ejection channel can range, for example, from about 0 degrees to about 45 degrees. The convergence angle can be modified to achieve useful atomization properties. For example, a 15-degree convergence angle can be used to substantially replicate a coaxial flow, such as that used in a capillary-based sheath flow system. Furthermore, the convergence of the two gas jets generates more inertial and shear forces, which aid in the breakup of the liquid sample into smaller droplets. In some cases, a lower convergence angle can be useful, for example, in reducing sample backpressure or backflow at or near sample outlet 307. Reduced backpressure or backflow of sample at or near the sample outlet 307 can help reduce the amount of sample that is reintroduced into the fluidic discharge channel. Optimal atomization can occur when gas flow-induced backpressure immediately prior to the sample outlet is minimized, while maintaining high shear forces and flow rates at or near the fluidic (liquid) discharge channel orifice and providing a steady gas flow to efficiently atomize the sample.

[0230] Figure 3B shows a schematic diagram of the variable diameter of the gas channel or gas exit orifice. In addition to the geometry and convergence angle of the gas channel and fluid ejection channel, the diameter of one or both of the gas channels 311 and 313 or gas exit orifices can also be varied. For example, the diameter of the gas exit orifice 311 can be 40 to 400 microns. In some cases, it is preferable to have a narrower gas exit orifice to maximize the local velocity of the gas toward or near the exit of the fluid ejection channel. Figure 3C shows a schematic diagram of the variable positioning of the gas channel relative to the gas channel or fluid ejection channel. An additional variable aspect can be the positioning of the gas exit orifice relative to the exit or orifice of the fluid ejection channel. Figure 3D shows a schematic diagram of the variable angle of the exit of the gas exit orifice. Referring to Figures 3A-3D, optimal atomization can occur when a steady nanoflow or microflow is achieved without substantial backpressure at the exit of the fluid channel.

[0231] 4A-4B show non-limiting examples of micrographs of microfluidic devices described herein. FIG. 4A shows a microfluidic device with two gas channels 411a and 413a with exit orifices positioned symmetrically from fluid exit channel orifice 407a. The gas exit orifices converge with the fluid exit channel orifice. The diameter of each of the gas exit orifices is approximately 114 micrometers (μm), and the diameter of the fluid exit channel is approximately 98 μm. The angle of convergence of gas channel 413a and the fluid exit channel is approximately 15 degrees. FIG. 4B shows a microfluidic device with two gas channels 411b and 413b with exit orifices positioned symmetrically from fluid exit channel orifice 407b. The gas exit orifices converge slightly outside the fluid exit channel orifice and are positioned adjacent to the fluid exit channel orifice with a gap of 0-200 microns between them. The diameter of each of the gas outlet orifices is about 119 μm and the diameter of the fluid outlet channel is about 94 μm The angle of convergence of the gas channel 413 and the fluid outlet channel is about 15 degrees. [Example]

[0232] Example 6 - Microfluidic device with atomizing gas channel As described in Example 5, various parameters of the gas channel and fluid outlet channel are variable. Figures 5A-9B schematically illustrate non-limiting examples of microfluidic devices comprising a gas channel and a separation channel. Referring to Figure 5A, access to the fluid channels within device 500 is provided through sample inlet port 503, anode port 504, cathode port 506, sample outlet port 507 (also referred to herein as "fluid orifice"), and chemical mobilizer inlet port 509. Anode port 504 and cathode port 506 are in fluid and electrical communication with the proximal and distal ends, respectively, of separation channel 505. Electrodes can be placed in contact with anode port 504 and cathode port 506 in some cases. In other cases, the anode port 504 and the cathode port 506 are in fluid and / or electrical communication with an electrode reservoir (not shown), which connects to the anode port 504 and the cathode port 506, for example, via a channel. The separation channel 505 extends beyond the cathode port 506 to a sample outlet port 507. A chemical mobilizer inlet port 509 is connected to the distal end of the separation channel 505 via a chemical mobilizer channel 508. The device also includes two gas channels 511 and 513, which have gas orifices or outlets adjacent to the point where they converge with the sample outlet port 507. The angle of convergence of the gas channel 513 and the fluid outlet channel is approximately 30 degrees. Gas inlet ports 515 and 517 allow for the entry of gas (e.g., air, nitrogen, etc.) into the gas channels 511 and 513. In some cases, the gas orifices or outlets of gas channels 511 and 513 may be positioned symmetrically from sample outlet port 507. Inlet ports, including anode port 504, cathode port 506, sample inlet port 503, chemical mobilizer inlet port 509, and gas inlet ports 515 and 517, may be configured to load through the side or edge of the device, which may facilitate various processes such as reagent loading, imaging of the entire channel, or the entire device.

[0233] FIG. 5B shows an enlarged schematic view of the outlet portion of the microfluidic device illustrated in FIG. 5A. Gas channels 511 and 513 each have gas orifices 519 and 521, respectively, from which gas exits. Gas channels 511 and 513 converge with sample outlet port 507 and are used to nebulize the sample near sample outlet port 507. Similar to the embodiment shown in FIGS. 2A-2B, gas channels 511 and 513, or portions thereof, are positioned symmetrically from sample outlet 507. In some cases, gas channel orifices 519 and 521 are positioned symmetrically from sample outlet 507. In some cases, gas channels 511 and 513 each comprise a region parallel to a portion of separation channel 505. Gas channels 511 and 513 have different lengths. However, gas channels 511 and 513 can be configured to provide substantially similar hydrodynamic flow resistances to gas outlet orifices 519 and 521, respectively. For example, gas channels 511 and 513 may have different cross-sectional areas along portions of the channels but approximately the same cross-sectional area near gas exit orifices 519 and 521. For devices in which gas exit orifices 519 and 521 are positioned symmetrically from sample exit 507, similar hydrodynamic flow resistances at each of gas exit orifices 519 and 521 may be beneficial in achieving a steady airflow for sample nebulization near exit 507. In other cases, gas exit orifices 519 and 521 may not be positioned symmetrically from sample exit 507. In such cases, the hydrodynamic flow resistances at each of gas exit orifices 519 and 521 may be different such that the volume, amount, or flow rate of air reaching sample exit 507 is approximately the same.

[0234] FIG. 5C shows an isometric view of the device depicted in FIGS. 5A-5B.

[0235] 6A-6C schematically show another example of a microfluidic device comprising a gas channel and a separation channel. FIG. 6A shows the layout of the device. FIGS. 6A-6B show enlarged and isometric views, respectively, of the outlet portion of the device shown in FIG. 6A. The device is similar to that shown in FIGS. 5A-5C, with gas channels 611 and 613 converging with sample outlet port 607 and used to atomize the sample near sample outlet port 607. Gas channels 611 and 613 converge with the fluid outlet channel at an angle of approximately 15 degrees. Gas channels 611 and 613 are positioned asymmetrically with respect to fluid channel outlet orifice 607. Although gas channels 611 and 613 are positioned asymmetrically with respect to fluid outlet channel orifice 607, the outlet flow path of the gas within each of the channels is symmetrical with respect to the axis of the fluid flow path of the fluid from fluid outlet orifice 607.

[0236] 7A-7C schematically illustrate another example of a microfluidic device comprising a gas channel and a separation channel. FIG. 7A shows the layout of the device. FIGS. 7A-7B show enlarged and isometric views, respectively, of the outlet portion of the device illustrated in FIG. 7A. The distal ends of gas channels 711 and 713 are parallel to the fluid outlet channel. Gas channels 711 and 713 are positioned such that orifices 719 and 721, respectively, are spaced approximately 30 μm from fluid channel outlet orifice 707. Gas channels 711 and 713 are used to atomize the sample near sample outlet port 707. Gas channels 711 and 713 are positioned symmetrically with respect to fluid channel outlet orifice 707, and the outlet flow path of the gas within each of the channels is symmetric with respect to the axis of the fluid flow path of the fluid from fluid outlet orifice 707.

[0237] 8A-8C schematically show another embodiment of a microfluidic device comprising a gas channel and a separation channel. FIG. 8A shows the layout of the device. FIGS. 8A-8B show enlarged and isometric views, respectively, of the outlet portion of the device shown in FIG. 8A. Gas channels 811 and 813 are positioned such that orifices 819 and 821, respectively, are spaced approximately 40 μm from fluid channel outlet orifice 807. Gas channels 811 and 813 are used to atomize the sample near sample outlet port 807. Gas channels 811 and 813 are positioned symmetrically with respect to fluid channel outlet orifice 807, and the outlet flow path of the gas within each of the channels is symmetric with respect to the axis of the fluid flow path of the fluid from fluid outlet orifice 807. The distal ends of gas channels 811 and 813 converge with the fluid outlet channel at an angle of approximately 15 degrees.

[0238] 9A-9B schematically illustrate another example of a microfluidic device comprising a gas channel and a separation channel. FIG. 9A illustrates the layout of device 900. Referring to FIG. 9A, access to the fluidic channels within the device is provided through a sample inlet port 903, an anode port 904, a cathode port 906, a sample outlet port 907 (also referred to herein as a "fluidic orifice"), and a chemical mobilizer inlet port 909. The anode port 904 and the cathode port 906 are in fluid and electrical communication with the proximal and distal ends of a separation channel 905, respectively. Electrodes can be placed in contact with the anode port 904 and the cathode port 906 in some cases. In other cases, the anode port 904 and the cathode port 906 are in fluid and / or electrical communication with electrode reservoirs (not shown), which connect to the anode port 904 and the cathode port 906, for example, via channels. The separation channel 905 extends beyond the cathode port 906 to a sample outlet port 907. A chemical mobilizer inlet port 909 is connected to the distal end of the separation channel 905 via a chemical mobilizer channel 908. The device also includes two gas channels 911 and 913, which have gas orifices or outlets adjacent to the point where they converge with the sample outlet port 907. Gas inlet ports 915 and 917 allow gas (e.g., air, nitrogen, etc.) to enter the gas channels 911 and 913. The gas orifices or outlets of the gas channels 911 and 913 are positioned symmetrically from the sample outlet port 907. One of the inlet ports, including the anode port 904 and the gas inlet port 917, is positioned along one side or edge of the device, while the cathode port 906, the sample inlet port 503, the chemical mobilizer inlet port 509, and the gas inlet port 915 are configured to be loaded through the opposite side or edge of the device.

[0239] 9B shows an enlarged view of the outlet portion of the device illustrated in FIG. 9A. The distal ends of gas channels 911 and 913 are parallel to the fluid outlet channel. Gas channels 911 and 913 are positioned such that orifices 919 and 921, respectively, are spaced approximately 30 μm from fluid channel outlet orifice 907. Gas channels 911 and 913 are used to atomize the sample near sample outlet port 907. Gas channels 911 and 913 are positioned symmetrically with respect to fluid channel outlet orifice 907, and the outlet flow path of the gas within each of the channels is symmetric with respect to the axis of the fluid flow path of the fluid from fluid outlet orifice 907. The distal ends of gas channels 911 and 913 converge with the fluid outlet channel at an angle of approximately 15 degrees.

[0240] 10A-10C schematically illustrate another example of a microfluidic device comprising a gas channel and a separation channel. FIG. 10A illustrates the layout of the device. FIGS. 10B and 10C illustrate enlarged views of the outlet portion of the device illustrated in FIG. 10A. The distal ends of gas channels 1111 and 1113 are parallel to the fluid outlet channel. Gas channels 1111 and 1113 are positioned such that orifices 1119 and 1121, respectively, are spaced approximately 15 μm from fluid channel outlet orifice 1107. Gas channels 1111 and 1113 are used to atomize the sample near sample outlet port 1107. Gas channels 1111 and 1113 are positioned symmetrically with respect to fluid channel outlet orifice 1107, and the outlet flow path of the gas within each of the channels is symmetric with respect to the axis of the fluid flow path of the fluid from fluid outlet orifice 1107.

[0241] 11A-11C schematically show another example of a microfluidic device comprising a gas channel and a separation channel. The device may be similar to that shown in FIGS. 10A-10C. FIG. 11A shows the layout of the device. FIGS. 11B-11C show isometric enlarged views of the gas inlet and distal outlet portions, respectively, of the device shown in FIG. 11A. Gas inlet port 1215 features a roughly elliptical cross-section. The distal end of the gas channel is parallel to the fluid outlet channel. The gas channels are positioned such that orifices 1219 and 1221 are each spaced approximately 15 μm from fluid channel outlet orifice 1207. The gas channels are used to atomize the sample near sample outlet port 1207. The gas channels are positioned symmetrically with respect to fluid channel outlet orifice 1207, and the outlet flow path of the gas within each of the channels is symmetric with respect to the axis of the fluid flow path of the fluid from fluid outlet orifice 1207.

[0242] 12A-12D provide exemplary schematics (FIGS. 12A-12C) and images (FIG. 12D) of various distal ends (tips) of a microfluidic chip. The distal ends of the gas channels are parallel to the fluid outlet channel. The gas channels are positioned such that orifices 1319 and 1321 are each spaced approximately 15 μm from fluid channel outlet orifice 1307. The gas channels are used to atomize the sample near sample outlet port 1307. The gas channels are positioned symmetrically with respect to fluid channel outlet orifice 1307, and the outlet flow path of the gas within each of the channels is symmetric with respect to the axis of the fluid flow path of the fluid from fluid outlet orifice 1307.

[0243] FIG. 12A shows a schematic diagram of an unshaped tip with gas orifices 1319 and 1321 and fluid orifice 1307. FIG. 12B shows a schematic diagram of a faceted shaped tip with gas orifices 1319 and 1321 and fluid orifice 1307. The faceted shaped tip includes chamfered surfaces on top surface 1325 and bottom surface 1327, which are absent on the unshaped tip as shown in FIG. 12A. FIG. 12C shows a schematic diagram of a rounded shaped tip with gas orifices 1319 and 1321 and fluid orifice 1307. The rounded shaped tip is uniformly chamfered, forming a rounded tip without sharp angles, as shown on FIGS. 12A-12B. Figure 12D shows an image, similar to that shown in schematic Figure 12B, of a faceted shaped tip with gas orifices 1319 and 1321 and fluid orifice 1307. The faceted shaped tip includes chamfered surfaces on top surface 1325 and bottom surface 1327, which are absent on the unshaped tip as shown in Figure 12A. [Example]

[0244] Example 7 - Imaging the Taylor Cone during Atomization and ESI The performance of ESI as a function of atomization can be monitored using imaging. Figure 13 shows a non-limiting example of a fluorescent image of the orifice of a microfluidic device in a fluid outlet channel during ESI. The fluid outlet channel is filled with a fluorescent dye. Each panel in Figure 13 illustrates the sample flow rate out of the fluid outlet channel (1.3 μL / min, 4.6 μL / min, 2.5 μL / min, and 4.7 μL / min) and the applied gas pressure of 70 PSI in gas channels 1011 and 1013, respectively. The upper panel demonstrates a device with a shaped tip, and the lower panel demonstrates a device without a defined tip shape. The tip shaping creates a pyramidal shape around the exit orifice. This generally requires chamfering the top and bottom corners of the tip near the orifice using mechanical grinding and / or polishing at a well-controlled angle (preferably 30 degrees) using any of several means known in the art.

[0245] Figures 14A-14B show non-limiting examples of fluorescent images of the orifice in the outlet (orifice) portion of a microfluidic device during ESI combined with atomization. The fluid outlet channel is filled with a fluorescent dye, and the tip of the device is positioned approximately 8 millimeters (mm) from the grounded plate (counter electrode). A voltage potential of 4,000 V is applied between the tip and the grounded plate. The sample containing the fluorescent dye is ejected from the fluid outlet channel at a rate of approximately 2-3 μL / min. A gas pressure of approximately 100 PSI is applied through each of the gas channels to atomize the sample. Figure 14A shows an image of the device when a UV light source is placed near the device orifice, demonstrating the spray plume surrounding the orifice resulting from atomization and the voltage drop. Figure 14B shows an image of the device when a UV light source is placed near the grounded plate, demonstrating that the spray plume virtually reaches the grounded plate under given ESI and atomization conditions. [Example]

[0246] Example 8 - Numerical simulation of flow profile in variable tip design The gas shear rate at and around the orifice of a fluid outlet channel of a microfluidic device can be characterized using numerical simulations (e.g., 3D simulations of the entire microfluidic chip, 2D axisymmetric models of concentric cylindrical implementations in COMSOL). Figure 15 shows an example of results from a finite element analysis of gas flow velocity over and around the fluid outlet channel orifice as a function of variable parameters (e.g., convergence angle, gas outlet orifice diameter, proximity of the gas outlet orifice to the fluid outlet channel orifice, etc.) of a microfluidic device comprising a fluid outlet channel and two gas channels. All simulations, except for the "concentric" panel, use a gas flow rate of 11 standard cubic centimeters per minute (sccm) per channel, while the "concentric" model uses a gas flow rate of 370 sccm.

[0247] Panel A of Figure 15 shows gas flow velocities for a device with an exit orifice positioned symmetrically from the orifice of the fluid exit channel. The gas exit orifice converges with the fluid exit channel orifice. The diameter of each of the gas exit orifices is approximately 114 micrometers (μm), and the diameter of the fluid exit channel is approximately 98 μm. The angle of convergence of the gas channel and fluid exit channel is approximately 15 degrees. Panel B of Figure 15 shows gas flow velocities for the same device as in Panel A of Figure 15, but with a truncated tip. Panel C of Figure 15 shows gas flow velocities for the device shown in Figures 6A-6C, in which the gas exit orifice converges with the fluid exit channel orifice at a 15-degree angle. The exit angle is symmetric, and the lower channel includes a bend near the orifice. Panel D of Figure 15 shows gas flow velocities for the device shown in Figures 2A-2B, in which the gas channel is spaced approximately 115 μm from the fluid exit channel. Panel E of Figure 15 shows gas flow velocities for the device shown in Figures 5A-5C, in which the gas outlet orifices converge with the fluid channel outlet orifices and the gas channels are positioned adjacent to the fluid channel outlets. Panel F of Figure 15 shows gas flow velocities for the device shown in Figures 7A-7C, in which the gas channels bend outward near the gas outlet orifices so that gas exits the fluid outlet channels parallel to the outward fluid flow path. The gas channel orifices are each positioned approximately 30 μm from the fluid outlet channel orifices. Panel G of Figure 15 shows gas flow velocities for the device shown in Figures 8A-8C, in which the gas channels bend near the orifices so that the gas and fluid outlet channels converge at an angle of approximately 15 degrees. The gas channel orifices are each positioned approximately 40 μm from the fluid outlet channel orifices. Panel "Concentric" of Figure 15 shows the results of a simulation of a fluid outlet channel radially surrounded by an annular concentric gas channel.

[0248] Numerical simulations may also be used to determine or model gas shear rates from gas channels. Figure 16 shows example results from a finite element analysis of gas shear rates above and around a fluid outlet channel orifice as a function of variable parameters (e.g., convergence angle, gas outlet orifice diameter, proximity of the gas outlet orifice to the fluid outlet channel orifice, etc.) for a microfluidic device comprising a fluid outlet channel and two gas channels. All simulations use a gas flow rate of 11 standard cubic centimeters per minute (sccm) per channel, except for the "concentric" panel, which uses a gas flow rate of 370 sccm.

[0249] Panel A of Figure 16 shows the gas shear rate for a device with an exit orifice positioned symmetrically from the orifice of the fluid exit channel. The gas exit orifice converges with the fluid exit channel orifice. The diameter of each of the gas exit orifices is approximately 114 micrometers (μm), and the diameter of the fluid exit channel is approximately 98 μm. The angle of convergence of the gas channel and fluid exit channel is approximately 15 degrees. Panel B of Figure 16 shows the gas shear rate for the same device as in Panel A of Figure 16, but with a truncated tip. Panel C of Figure 16 shows the gas shear rate for the device shown in Figures 6A-6C, in which the gas exit orifice converges with the fluid exit channel orifice at a 15-degree angle. The exit angle is symmetric, and the lower channel includes a bend near the orifice. Panel D of Figure 16 shows the gas shear rate for the device shown in Figures 2A-2B, in which the gas channel is spaced approximately 115 μm from the fluid outlet channel. Panel E of Figure 16 shows the gas shear rate for the device shown in Figures 5A-5C, in which the gas outlet orifice converges with the fluid channel outlet orifice and the gas channel is positioned adjacent to the fluid channel outlet. Panel F of Figure 16 shows the gas shear rate for the device shown in Figures 7A-7C, in which the gas channel bends outward near the gas outlet orifice so that gas exits the fluid outlet channel parallel to the fluid flow path out. The gas channel orifices are each positioned approximately 30 μm from the fluid outlet channel orifice. Panel G of Figure 16 shows the gas shear rate for the device shown in Figures 8A-8C, in which the gas channel bends near the orifice so that the gas channel and fluid outlet channel converge at an angle of approximately 15 degrees. The orifices of the gas channels are each positioned approximately 40 μm from the orifices of the fluid outlet channel. Panel "Concentric" of Figure 16 models a fluid outlet channel that is radially surrounded by annular concentric gas channels.

[0250] Figure 17 shows an example of results from a finite element analysis illustrating the velocity field at and around the fluid outlet channel orifice as a function of variable parameters (e.g., convergence angle, gas outlet orifice diameter, proximity of the gas outlet orifice to the fluid outlet channel orifice, etc.) of a microfluidic device comprising a fluid outlet channel and two gas channels. Figure 17 shows a perspective view of the device. All simulations use a gas flow rate of 11 standard cubic centimeters per minute (sccm) per channel, except for the "concentric" panel, which uses a gas flow rate of 370 sccm.

[0251] Panel A of Figure 17 shows gas flow rates for the device shown in Figures 2A-2B, in which the gas channel is spaced approximately 115 μm from the fluid outlet channel. Panel B of Figure 17 shows gas flow rates for the device shown in Figures 5A-5C, in which the gas outlet orifice converges with the fluid channel outlet orifice and the gas channel is positioned adjacent to the fluid channel outlet. Panel C of Figure 17 shows gas flow rates for the device shown in Figures 6A-6C, in which the gas outlet orifice converges with the fluid outlet channel orifice at a 15-degree angle. The outlet angle is symmetric, and the lower channel includes a bend near the orifice. Panel D of Figure 17 shows gas flow rates for the device shown in Figures 7A-7C, in which the gas channel bends outward near the gas outlet orifice so that gas exits the fluid outlet channel parallel to the outward fluid flow path. The orifices of the gas channels are each positioned approximately 30 μm from the orifices of the fluid outlet channels. Panel E of Figure 17 shows the gas flow rates for the device shown in Figures 8A-8C, in which the gas channels bend near the orifices so that the gas and fluid outlet channels converge at an angle of approximately 15 degrees. The orifices of the gas channels are each positioned approximately 40 μm from the orifices of the fluid outlet channels.

[0252] 18 shows example results from a finite element analysis illustrating the gas pressure field from atomization on and around the fluid outlet channel orifice as a function of variable parameters (e.g., convergence angle, gas outlet orifice diameter, proximity of the gas outlet orifice to the fluid outlet channel orifice, etc.) of a microfluidic device comprising a fluid outlet channel and two gas channels. All simulations use a gas flow rate of 11 standard cubic centimeters per minute (sccm) per channel, except for the "concentric" panel, which uses a gas flow rate of 370 sccm.

[0253] Panel A of Figure 18 shows the gas pressure field for a device with an exit orifice positioned symmetrically from the orifice of the fluid exit channel. The gas exit orifice converges with the fluid exit channel orifice. The diameter of each of the gas exit orifices is approximately 114 micrometers (μm), and the diameter of the fluid exit channel is approximately 98 μm. The angle of convergence of the gas channel and fluid exit channel is approximately 15 degrees. Panel B of Figure 18 shows the gas pressure field for the same device as in Panel A of Figure 18, but with a truncated tip. Panel C of Figure 18 shows the gas pressure field for the device shown in Figures 6A-6C, in which the gas exit orifice converges with the fluid exit channel orifice at a 15-degree angle. The exit angle is symmetric, and the lower channel includes a bend near the orifice. Panel D of Figure 18 shows the gas pressure field for the device shown in Figures 2A-2B, in which the gas channel is spaced approximately 115 μm from the fluid exit channel. Panel E of Figure 18 shows the gas pressure field for the device shown in Figures 5A-5C, in which the gas outlet orifices converge with the fluid channel outlet orifices and the gas channels are positioned adjacent to the fluid channel outlets. Panel F of Figure 18 shows the gas pressure field for the device shown in Figures 7A-7C, in which the gas channels bend outward near the gas outlet orifices so that gas exits the fluid outlet channels parallel to the fluid flow path. The gas channel orifices are each positioned approximately 30 μm from the fluid outlet channel orifices. Panel G of Figure 18 shows the gas pressure field for the device shown in Figures 8A-8C, in which the gas channels bend near the orifices so that the gas and fluid outlet channels converge at an angle of approximately 15 degrees. The gas channel orifices are each positioned approximately 40 μm from the fluid outlet channel orifices. Panel "Concentric" of Figure 18 models a fluid outlet channel that is radially surrounded by an annular concentric gas channel.

[0254] Figure 19 shows a plot comparing gas pressure for several device designs as a function of distance (mm) from the tip (fluid channel exit orifice). "TSTKC4" represents the device shown in Figure 4A, "TSTKC6" represents the device shown in Figure 4A with a truncated tip, "E5E1" represents the device shown in Figure 2A, "E5E2" represents the device shown in Figure 5A, "E5E3" represents the device shown in Figure 6, "E5E4" represents the device shown in Figure 7A, and "E5E5" represents the device shown in Figure 8A. [Example]

[0255] Example 9 - Microfluidic Chip-Cartridge Interface and Cartridge-Instrument Interface As described herein, the system may include a cartridge configured to interface with the microfluidic chip, and in some cases, the cartridge (which interfaces with the microfluidic chip) is configured to interface with the instrument.

[0256] Figure 20A shows a schematic of an exploded view of the interface between the microfluidic chip, cartridge, and instrument interface. A microfluidic device 1702 interfaces with or is inserted into a cartridge 1704. The microfluidic device has six ports located on the edge of the device that interface with the edge of the cartridge (see, e.g., Figures 2A, 5A, 6A, 7A, 8A). The cartridge includes six fluid ports that align with the six ports of the microfluidic device and seal to the ports using an elastomeric material (e.g., gaskets or O-rings, not shown).

[0257] The cartridge 1704 is configured to interface with an instrument, which may be accomplished via an interface device 1706. The interface device 1706 includes six independently spring-loaded fitting assemblies (also referred to herein as "interconnects") 1710, which are configured to fluidly and / or electrically communicate with the cartridge and the microfluidic device via ports. The spring-loaded fitting assemblies couple to external fluid lines providing, for example, sample, anolyte, catholyte, mobilization reagent, gas (e.g., for nebulization), etc. Each spring-loaded fitting assembly can be, for example, a conical fitting or a flat face seal fitting that mates with a fluid port. In such an example, the independently spring-loaded fittings include conical fittings or flat face seal fittings that mate with six fluid ports (only four of the six are indicated by arrows) via holes 1712 in the microfluidic cartridge. In some aspects, the holes 1712 can be tapered. The interface device 1706 may be configured to mate with the cartridge 1704 in a precise manner using locating pins 1708 .

[0258] 20B shows a schematic of the interface between the microfluidic device, cartridge, and interface device in a "sealed" configuration. A crimping force 1712 may be applied to contact all three components, thereby establishing a substantially leak-free fluid communication.

[0259] 20C-20E schematically show cross-sectional views of a spring-loaded fitting assembly in unloaded, contacted, and sealed configurations. FIG. 20C shows the spring-loaded fitting assembly in the unloaded configuration, in which the fluid interconnect is not in contact with the elastomeric component 1714 of the cartridge. FIG. 20D shows the spring-loaded fitting assembly in the contacted configuration, in which the fluid interconnect is in contact with the elastomeric component 1714 of the cartridge. FIG. 20E shows the spring-loaded fitting assembly in the sealed configuration, in which the fluid interconnect is in contact with the elastomeric component 1714 of the cartridge (e.g., in response to application of a crimping force such as 1612). In the sealed configuration, the cartridge is crimped to the interface device via the spring-loaded fitting, and a sealing force is generated between the tubing and the cartridge. The spring 1716 of the spring-loaded fitting helps establish a repeatable sealing force.

[0260] 21A-21C diagrammatically show perspective views of a spring-loaded coupling assembly in an unloaded, contacted, and sealed configuration, as demonstrated in FIGS. 20C-20E.

[0261] 22A-22C schematically show the interface between a microfluidic device and a cartridge. Referring to FIGS. 22A-22B, the interface between the device's ports and the cartridge is an elastomeric component 1901 (e.g., an O-ring or gasket), which helps create a substantially leak-free seal between the cartridge and the device. In some embodiments, a set of connected gaskets may be used (e.g., in cases where the pitch or spacing between the device's ports is identical), as shown in FIG. 22C. [Example]

[0262] Example 10 - Computer System FIG. 23 shows an exemplary software architecture system. The software architecture system may be integrated with a system disclosed herein and may include one or more computer processors. In some cases, the one or more computer processors may be configured to collect and / or analyze data. The software architecture system may include a computer processing unit including a controller service that may communicate with a first-in, first-out (FIFO) database. In some cases, the FIFO database may communicate with a second computer processor that may include a graphical user interface and a server database. The second computer processor may communicate with a customer database, for example, via the cloud. In some cases, the computer processing unit may communicate with one or more hardware units of the system (e.g., via a wired or wireless connection). For example, the computer processing unit may be connected via a USB hub to a stage, one or more cameras, a high-voltage power supply, an autosampler, a flow control system (e.g., software and hardware for microfluidic flow control, e.g., Fluigent Inc.), and / or other laboratory equipment. [Example]

[0263] Example 11 - Integrated System 24 shows an exemplary block diagram of an integrated system. The integrated system may include one or more systems disclosed herein. The system may include an interface contact cartridge 2107, which may be in fluid and / or electrical communication with multiple reservoirs 2103. For example, the interface contact cartridge 2107 may be connected to an anolyte reservoir, a catholyte reservoir, a mobilizer reservoir, and an autosampler unit. Alternatively, or in addition, the interface contact cartridge 2107 may be in fluid and / or electrical communication with a pressure control manifold 2105, which may be coupled to a fluid driver, such as a pump. The interface contact cartridge 2107 may be coupled to a cartridge 2100, which may include a device 2101. The device 2101 may be in electrical and / or fluid communication with an anolyte high-voltage reservoir, a catholyte high-voltage reservoir, a mobilizer high-voltage reservoir, and a sample line. The anolyte high-voltage reservoir, catholyte high-voltage reservoir, mobilizer high-voltage reservoir, and sample line may each be in fluid and / or electrical communication with interface contact cartridge 2107. Device 2101 may also be coupled to a waste management unit 2109, which is used to direct waste away from device 2101 and in some cases may also be used to direct sample to a downstream analysis unit 2111. In some embodiments, waste management unit 2109 may comprise an atomizer. In some cases, downstream analysis unit 2111 may comprise a mass spectrometer.

[0264] The system may also include multiple imaging systems. For example, the system may include imaging system 2115, which may include a camera, an illuminator, a waste receptacle, and / or an adapter, which may be used to interface with the analysis unit 2111. The system may also include imaging system 2117, which may include an illuminator (e.g., a UV illumination source), a mirror, and / or a camera or other suitable detector. In some cases, the detector (e.g., a camera) may be connected to a cooling source, e.g., a fan or other temperature control platform.

[0265] 25 shows an exemplary block diagram of an integrated system. The system may include a sample 2201, a sample and reagent holder and / or processor 2203 that may be configured to store the sample and process the sample (e.g., mix the sample, add reagents, aspirate or dispense, etc.), a sample injector 2205, and a sample tip washer 2207. The sample tip washer may include mechanisms for washing the sample and / or the system. The system may also include a separation unit 2209, which may include cartridges and an imaging system (e.g., a UV illuminator and camera) that make up the device. The separation unit may be coupled to a plurality of controllers 2211, which may include fluid control using negative pressure (e.g., vacuum) or positive pressure (e.g., rotary or diaphragm pumps, valves, etc.). The controller 2211 and / or the separation unit 2209 may be coupled to a fluid manifold 2213, which may include one or more reagent-containing reservoirs.

[0266] The separation unit 2209 may be used to perform a separation reaction (e.g., isoelectric focusing) and / or a mobilization reaction. The separation unit 2209 may be connected or coupled to a communication interface 2215 (e.g., RFID), a high-voltage power supply 2217, a waste management unit 2219 (e.g., vacuum and waste receptacle), another imaging unit 2221, and / or a downstream analysis unit 2223 (e.g., mass spectrometer). In some cases, the separation unit 2209 may be coupled to a temperature control unit 2225. In some cases, one or more systems described herein may include a temperature control unit 2227 and / or other control units, for example, for instrument control 2229.

[0267] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention also covers any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and methods and structures within the scope of these claims and their equivalents are intended to be covered thereby.

Claims

1. A microfluidic chip comprising: The microfluidic chip comprises: a) a substrate; The substrate is i) a fluidic channel having a distal end in fluid communication with an electrospray ionization orifice, the fluidic channel comprising a separation channel configured to perform isoelectric focusing or electrophoretic separation of a sample comprising a mixture of analytes in the fluidic channel; ii) a gas channel having a distal end in fluid communication with a gas exit orifice disposed adjacent to the electrospray ionization orifice, the gas exit orifice configured to effect atomization of a solution exiting the electrospray ionization orifice; and Equipped with The microfluidic chip, wherein an angle between the distal end of the fluid channel and the distal end of the gas channel ranges from about 0 degrees to about 30 degrees.

2. The microfluidic chip of claim 1 , wherein the gas exit orifice is located on an edge of the substrate adjacent to the electrospray ionization orifice.

3. 3. The microfluidic chip of claim 2, wherein the substrate comprises two or more gas exit orifices, the two or more gas exit orifices being arranged adjacent to and symmetrically about the electrospray ionization orifice.

4. The microfluidic chip of any one of claims 1 to 3, wherein the angle ranges from about 10 degrees to about 20 degrees.

5. 5. The microfluidic chip of claim 1, wherein the microfluidic chip comprises three or more gas channels, each of the three or more gas channels comprising a gas exit orifice positioned adjacent to the electrospray ionization orifice, a first subset of the three or more gas channels being positioned within the substrate, and a second subset of the three or more gas channels being positioned within an auxiliary component of the microfluidic chip that is positioned adjacent to the substrate such that the second subset of the three or more gas channels is not positioned in the same plane as the substrate.

6. The second subset of the three or more gas channels disposed within the auxiliary component comprises: (a) the gas exit orifice is positioned such that it lies in a plane substantially perpendicular to the plane of the substrate and is positioned symmetrically about and adjacent to the electrospray ionization orifice; or 6. The microfluidic chip of claim 5, wherein (b) its gas exit orifices are positioned in one or more planes oriented at an angle to the plane of the substrate and are positioned equidistant from the electrospray ionization orifice.

7. The fluid channel comprises: (a) a width ranging from about 20 μm to about 600 μm; (b) a depth ranging from about 10 μm to about 100 μm; or (c) a length ranging from about 0.25 cm to about 30 cm and / or The gas channel (a) a width ranging from about 20 μm to about 200 μm; (b) a depth ranging from about 10 μm to about 100 μm; or (c) a length ranging from about 0.2 cm to about 20 cm The microfluidic chip according to any one of claims 1 to 6, having one or more dimensions of:

8. The microfluidic chip of any one of claims 1 to 7, wherein the electrospray ionization orifice has a maximum cross-sectional dimension ranging from about 10 μm to about 100 μm.

9. The microfluidic chip of any one of claims 1 to 8, wherein the gas exit orifice has a maximum cross-sectional dimension ranging from about 10 μm to about 50 μm.

10. The microfluidic chip of any one of claims 1 to 9, wherein the gas exit orifice is located within 100 μm, 50 μm, or 15 μm of the electrospray ionization orifice.

Images