Isoelectric focusing apparatus and holder

Abstract

The present invention describes methods, devices, and systems for performing isoelectric focusing reactions. The systems or devices disclosed herein can include a holder having a membrane. In some cases, the disclosed devices can be designed to perform isoelectric focusing or other separation reactions and subsequently further characterize the separated analytes using mass spectrometry. Two or more isoelectric focusing reactions can be performed in parallel. The disclosed methods, devices, and systems provide rapid and accurate separation and characterization of protein analyte mixtures or other biomolecules by their isoelectric points.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application No. 16 / 799,387, filed February 24, 2020, which claims the benefit of U.S. Provisional Patent Application No. 62 / 909,675, filed October 2, 2019, U.S. Provisional Patent Application No. 62 / 893,549, filed August 29, 2019, and U.S. Provisional Patent Application No. 62 / 885,733, filed August 12, 2019, and this application claims the benefit of U.S. Provisional Patent Application No. 16 / 799,387, filed February 24, 2020, which claims the benefit of U.S. Provisional Patent Application No. 62 / 909,675, filed October 2, 2019, No. 16 / 808,063, filed on March 3, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62 / 909,675, filed on October 2, 2019, U.S. Provisional Patent Application No. 62 / 893,549, filed on August 29, 2019, and U.S. Provisional Patent Application No. 62 / 885,733, filed on August 12, 2019, each of which is incorporated herein by reference in its entirety for all purposes. Background Art

[0003] The present disclosure relates to methods, devices, and systems for sample processing and characterization and various uses thereof. In a first aspect, the present disclosure relates to methods, devices, and systems for performing separation and characterization of analytes in analyte mixtures, and more specifically to multichannel devices (and related methods and systems) for performing multiple isoelectric focusing reactions in parallel. In a second aspect, the present disclosure relates to microfluidic devices (and related methods and systems) designed to perform one or more separation reactions (e.g., isoelectric focusing) and subsequently mobilizing and electrospraying the separated analytes for characterization by mass spectrometry. Summary of the Invention

[0004] Disclosed herein are methods, devices, and systems for achieving improved quantitative performance for separating and analyzing analytes in analyte mixtures, which have potential applications in biomedical research, clinical diagnosis, and drug manufacturing. For example, regulatory agencies require that biological drugs and drug candidates (e.g., proteins) be strictly characterized. The methods and devices described herein may be applicable to characterizing proteins and / or other analytes. In some cases, the methods and devices described herein may relate to characterizing analyte mixtures, wherein 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 to separate the analyte mixture into enriched analyte fractions in a multiplexed form for high-throughput characterization of samples. In some cases, the methods and devices described herein may relate to characterizing analyte mixtures, wherein one or more enrichment steps are performed to separate the analyte mixture into enriched analyte fractions, which are later introduced into a mass spectrometer via an electrospray ionization interface. The disclosed methods and devices may provide improvements in the convenience, reproducibility, and / or analytical performance of analyte separation and characterization.

[0005] In one aspect, the present invention discloses a holder comprising: an electrode reservoir; an inlet fluid channel comprising a first end and a second end; an outlet fluid channel comprising a first end fluidically connected to the second end of the inlet fluid channel and a second end fluidically connected to a separation channel, wherein the inlet fluid channel and the outlet fluid channel intersect with each other and are fluidically connected to each other at a plane defined by or parallel to a surface of the electrode reservoir; and a membrane arranged in the electrode reservoir at or adjacent to the plane so that the membrane covers all or substantially all openings, including the intersection of the inlet fluid channel and the outlet fluid channel; wherein the membrane provides high fluid dynamic resistance and a low resistance connection between a high voltage electrode positioned in the electrode reservoir and the fluid contained in the inlet fluid channel and the outlet fluid channel.

[0006] In some embodiments, the membrane is hydrophilic. In some embodiments, the membrane comprises a regenerated cellulose membrane. In some embodiments, the membrane comprises a woven polytetrafluoroethylene (PTFE) membrane that has been treated to be hydrophilic. In some embodiments, the cross-sectional area of ​​the membrane or opening is between about 0.001 mm 2 and 100mm 2In some embodiments, the electrode reservoir further comprises an insert disposed within the electrode reservoir and positioned at or adjacent to the membrane, wherein the insert comprises an inlet fluid path and an outlet fluid path that facilitate substantially bubble-free wetting of the surface of the membrane when the electrode reservoir is filled with an electrolyte solution.

[0007] In some embodiments, the hydrodynamic resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is greater than 0.1 ((N / mm 2 ) / (mm 3 In some embodiments, the hydrodynamic resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is greater than 1 ((N / mm 2 ) / (mm 3 / s)).

[0008] In some embodiments, the resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is less than 10,000,000 ohms. In some embodiments, the resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is less than 100,000 ohms. In some embodiments, during operation, the electrode reservoir is filled with an electrolyte solution having a concentration between about 1 millimolar (mM) and about 500 mM. In some embodiments, during operation, the electrode reservoir is filled with an electrolyte solution having a concentration between about 10 mM and about 150 mM. In some embodiments, during operation, the electrode reservoir is filled with an electrolyte solution having a pH range between about 1.5 and about 14. In some embodiments, the separation channel comprises a lumen of a capillary. In some embodiments, the separation channel comprises a fluid channel within a microfluidic device. In some embodiments, the separation channel is configured to perform electrophoresis. In some embodiments, the separation channel is configured to perform isoelectric focusing.

[0009] The present invention also discloses a fluid device, which comprises: at least one fluid inlet; at least one fluid outlet; at least one separation channel, wherein the at least one separation channel comprises a first end portion fluidically coupled to the at least one fluid inlet and a second end portion fluidically coupled to the at least one fluid outlet; wherein at least one fluid inlet or at least one fluid outlet is electrically coupled to a high-voltage electrode using a holder, wherein the holder comprises: an electrode reservoir; an inlet fluid channel, wherein the inlet fluid channel comprises a first end portion and a second end portion; an outlet fluid channel, wherein the outlet fluid channel comprises a first end portion fluidically coupled to the second end portion of the inlet fluid channel and a second end portion fluidically coupled to the at least one a second end portion fluidly connected to one of the at least one fluid inlet or the at least one fluid outlet, wherein the inlet fluid channel and the outlet fluid channel intersect and fluidically connect to each other at a plane defining or parallel to a surface of the electrode reservoir; and a membrane arranged within the electrode reservoir at or adjacent to the plane such that the membrane covers all or substantially all of the openings, including the intersection of the inlet fluid channel and the outlet fluid channel; wherein the membrane provides a high fluid dynamic resistance and a low electrical resistance connection between a high voltage electrode positioned within the electrode reservoir and the fluid contained within the inlet fluid channel and the outlet fluid channel.

[0010] In some embodiments, the device comprises at least one capillary, and wherein the at least one capillary comprises a lumen that serves as the at least one separation channel. In some embodiments, the device is a microfluidic device comprising a planar substrate, and wherein the planar substrate comprises the at least one separation channel. In some embodiments, the at least one fluid inlet or the at least one fluid outlet is arranged on at least one edge of the planar substrate. In some embodiments, the membrane is hydrophilic. In some embodiments, the membrane comprises a regenerated cellulose membrane. In some embodiments, the membrane comprises a woven polytetrafluoroethylene (PTFE) membrane that has been treated to be hydrophilic. In some embodiments, the cross-sectional area of ​​the membrane or opening is between about 0.001 mm 2 and 100mm 2In some embodiments, the electrode reservoir further comprises an insert disposed within the electrode reservoir and positioned at or adjacent to the membrane, wherein the insert comprises an inlet fluid path and an outlet fluid path, the inlet fluid path and the outlet fluid path facilitating substantially bubble-free wetting of the surface of the membrane when the electrode reservoir is filled with an electrolyte solution. In some embodiments, during operation, the electrode reservoir is filled with an electrolyte solution having a concentration between about 1 millimolar (mM) and about 500 mM. In some embodiments, during operation, the electrode reservoir is filled with an electrolyte solution having a concentration between about 10 mM and about 150 mM. In some embodiments, during operation, the electrode reservoir is filled with an electrolyte solution having a pH range between about 1.5 and about 14. In some embodiments, the hydrodynamic resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is greater than 0.1 ((N / mm 2 ) / (mm 3 In some embodiments, the hydrodynamic resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is greater than 1 ((N / mm 2 ) / (mm 3 In some embodiments, the resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is less than 10,000,000 ohms. In some embodiments, the resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is less than 100,000 ohms.

[0011] In some embodiments, the ratio of the hydrodynamic resistance to electrical resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is greater than about 0.001 ((N / mm 2 ) / (mm 3 / s)) / Ω. In some embodiments, the ratio is greater than about 0.01((N / mm 2 ) / (mm 3 In some embodiments, the ratio is greater than about 0.1 ((N / mm 2 ) / (mm 3 / s)) / Ω. In some embodiments, the ratio is greater than about 1((N / mm 2 ) / (mm 3 / s)) / Ω. In some embodiments, the ratio is greater than about 10((N / mm 2 ) / (mm 3 In some embodiments, the ratio is greater than about 100 ((N / mm 2 ) / (mm 3In some embodiments, the ratio is greater than about 1000 ((N / mm 2 ) / (mm 3 / s)) / Ω. In some embodiments, the ratio is greater than about 10000 ((N / mm 2 ) / (mm 3 / s)) / Ω.

[0012] In another aspect, disclosed herein is a method for performing multiple isoelectric focusing reactions in parallel, the method comprising: a) providing a device comprising a planar substrate, wherein the planar substrate comprises a plurality of separation channels; b) introducing a sample comprising an analyte mixture into at least two separation channels of the plurality of separation channels; c) controlling voltages applied to the at least two separation channels to perform the multiple isoelectric focusing reactions to separate the analyte mixture of the sample in the at least two separation channels; and d) independently monitoring current flowing through the at least two separation channels while performing the multiple isoelectric focusing reactions in parallel.

[0013] In some embodiments, the first ends of at least two of the plurality of separation channels are electrically coupled to an anolyte reservoir using a high-voltage electrode holder comprising a membrane. In some embodiments, the second ends of at least two of the plurality of separation channels are electrically coupled to a catholyte reservoir using a high-voltage electrode holder comprising a membrane. In some embodiments, the voltages applied to the at least two separation channels are independently controlled. In some embodiments, the samples introduced into the at least two separation channels are identical, and a first set of experimental conditions is used to perform the isoelectric focusing reaction in a first subset of the at least two separation channels, and at least a second set of experimental conditions is used to perform the isoelectric focusing reaction in at least a second subset of the at least two separation channels. In some embodiments, a different set of experimental conditions is used to perform the isoelectric focusing reaction in each of the at least two separation channels. In some embodiments, the set of experimental conditions used to perform the plurality of isoelectric focusing reactions includes at least one item from the group consisting of: buffer selection, pH gradient selection, voltage setting, current setting, electric field strength setting, a time course for varying the voltage setting, current setting, electric field strength setting, or isoelectric focusing reaction time. In some embodiments, the samples introduced into the at least two separation channels are different for at least two subsets of the at least two separation channels, and the isoelectric focusing reaction is performed in each of the at least two separation channels using the same set of experimental conditions. In some embodiments, the samples introduced into each of the at least two separation channels are different. In some embodiments, the method further comprises recording a current trace for each of the at least two separation channels while performing the multiple isoelectric focusing reactions. In some embodiments, the method further comprises flushing the at least two separation channels after completion of the isoelectric focusing reaction and automatically introducing another sample into the at least two separation channels. In some embodiments, detection of a fault in any of the at least two separation channels triggers automatic reintroduction and repetition of the isoelectric focusing reaction for the sample already introduced into that separation channel. In some embodiments, the fault comprises the introduction of bubbles, the formation of bubbles, an improperly prepared sample, an underfilled reagent reservoir, or any combination thereof. In some embodiments, the fault is detected by monitoring the current flowing through the separation channel or by processing an image of the separation channel. In some embodiments, the method further comprises measuring dynamic light scattering in at least one of the at least two separation channels while performing the isoelectric focusing. In some embodiments, the measurement of dynamic light scattering provides for the determination of a size distribution curve, the determination of an aggregation state, or the determination of a hydrodynamic radius for one or more separated analytes.In some embodiments, the membrane-containing high-voltage electrode holder comprises: a) an electrode reservoir; b) an inlet fluid channel, the inlet fluid channel comprising a first end and a second end; c) an outlet fluid channel, the outlet fluid channel comprising a first end fluidically coupled to the second end of the inlet fluid channel and a second end fluidically coupled to a separation channel, wherein the inlet fluid channel and the outlet fluid channel intersect and fluidically couple to each other at a plane defined by or parallel to a surface of the electrode reservoir; and d) a membrane disposed within the electrode reservoir at or adjacent to the plane such that the membrane covers all or substantially all of an opening, the opening comprising the intersection of the inlet fluid channel and the outlet fluid channel; wherein the membrane provides a high hydrodynamic resistance and a low electrical resistance connection between a high-voltage electrode positioned within the electrode reservoir and a fluid contained within the inlet fluid channel and the outlet fluid channel. In some embodiments, the membrane is hydrophilic and comprises cellulose or polytetrafluoroethylene (PTFE). In some embodiments, the high voltage electrode holder comprising a membrane further comprises an insert disposed within the electrode reservoir and positioned at or adjacent to the membrane, wherein the insert comprises an inlet fluid path and an outlet fluid path that contribute to wetting the surface of the membrane substantially free of bubbles when the electrode reservoir is filled with an electrolyte solution. In some embodiments, the method further comprises filling the electrode reservoir with an electrolyte solution having a concentration between about 1 millimolar (mM) and about 500 mM. In some embodiments, the method further comprises filling the electrode reservoir with an electrolyte solution having a concentration between about 10 mM and about 150 mM. In some embodiments, the electrolyte solution has a pH in the range of about 1.5 and about 14.

[0014] In another aspect, disclosed herein is a microfluidic device comprising a planar substrate, wherein the planar substrate comprises: a) a plurality of fluid inlets, wherein all or a portion of the plurality of fluid inlets are located on one or more edges of the planar substrate; b) a plurality of separation channels, wherein the plurality of separation channels comprise: i) a first end electrically coupled to an anode liquid reservoir using a high-voltage electrode holder comprising a membrane; ii) a second end electrically coupled to a cathode liquid reservoir using a high-voltage electrode holder comprising a membrane; and iii) one of the first end or the second end of each of the plurality of separation channels being fluidically connected to a different fluid inlet among the plurality of fluid inlets.

[0015] In some embodiments, the plurality of separation channels are configured for UV absorbance imaging or fluorescence imaging of all or a portion of the plurality of separation channels. In some embodiments, the microfluidic device further comprises a cartridge housing all or a portion of a substrate comprising the plurality of separation channels, wherein the cartridge comprises a plurality of high-voltage electrode holders containing membranes. In some embodiments, the cartridge is a disposable component of a system configured to perform multiplexed isoelectric focusing reactions.

[0016] Also disclosed herein is a system for performing a multiplexed isoelectric focusing reaction, the system comprising: a) a microfluidic device comprising a planar substrate, wherein the planar substrate comprises: i) a plurality of fluid inlets, wherein all or a portion of the plurality of fluid inlets are located on one or more edges of the planar substrate; and ii) a plurality of separation channels, the plurality of separation channels comprising: a first end electrically coupled to an anolyte reservoir using a high-voltage electrode holder comprising a membrane; a second end electrically coupled to a cathode reservoir using a high-voltage electrode holder comprising a membrane; and one of the first end or the second end of each of the plurality of separation channels being in fluid communication with a different fluid inlet of the plurality of fluid inlets; and b) a multiplexed power supply, wherein the multiplexed power supply is configured to: i) control a voltage applied to each of at least two separation channels; and ii) independently monitor a current flowing through each of the at least two separation channels while performing a multiplexed isoelectric focusing reaction on a plurality of samples comprising an analyte mixture.

[0017] In some embodiments, the multiplexed power supply is configured to independently control the voltage applied to each of the at least two separation channels. In some embodiments, the system further comprises an imaging unit configured to (i) acquire a UV absorbance or fluorescence image of all or a portion of each of the at least two separation channels and (ii) process the UV absorbance or fluorescence image to detect the position of one or more isoelectric point (pI) markers contained within the separation channel during operation so that pI values ​​can be determined for one or more separated analyte peaks in each of the at least two separation channels. In some embodiments, the system further comprises a dynamic light scattering unit configured to measure dynamic light scattering in at least one of the multiple separation channels. In some embodiments, the system further comprises an automated liquid handling system for loading a sample into a sample inlet port, the sample inlet port being fluidically coupled to the multiple separation channels via the multiple fluid inlets. In some embodiments, the system is configured to rinse the multiple separation channels after the multiple isoelectric focusing reaction is complete and to introduce another set of samples into the multiple separation channels in an automated manner. In some embodiments, detection of a fault in a separation channel triggers automatic reintroduction and repetition of the isoelectric focusing reaction for the sample that has been introduced into the separation channel.

[0018] Also disclosed herein is a method for performing multiple isoelectric focusing reactions in parallel, the method comprising: a) providing a plurality of sample aliquots, wherein each sample aliquot comprises a mixture of analytes; b) providing a device comprising a plurality of separation channels, wherein one sample aliquot of the plurality of sample aliquots is introduced into each separation channel; and c) providing a multiplexed power supply, wherein the multiplexed power supply is configured to independently control and monitor current flowing through each of the plurality of separation channels while performing multiple isoelectric focusing reactions to separate the analytes in each sample.

[0019] In some embodiments, the sample aliquots introduced into each separation channel are taken from the same sample, and a different set of experimental conditions are used to perform the isoelectric focusing reactions in at least two subsets of the plurality of separation channels. In some embodiments, a different set of experimental conditions are used to perform the multiple isoelectric focusing reactions in each of the plurality of separation channels. In some embodiments, at least two subsets of the plurality of sample aliquots introduced into the separation channels are taken from different samples, and the same set of experimental conditions are used to perform the multiple isoelectric focusing reactions. In some embodiments, each sample aliquot introduced into the separation channels is taken from a different sample. In some embodiments, the set of experimental conditions used to perform the multiple isoelectric focusing reactions includes buffer selection, pH gradient selection, voltage setting, current setting, electric field strength setting, a time course for varying the voltage setting, current setting, or electric field strength setting, isoelectric focusing reaction time, or any combination thereof. In some embodiments, the analyte comprises a protein. In some embodiments, the device comprises a microfluidic device. In some embodiments, the microfluidic device comprises four to eight separation channels. In some embodiments, the sample aliquots are introduced into the separation channels using an automated liquid handling system. In some embodiments, the method further includes recording a current trace for each of the plurality of separation channels while performing the plurality of isoelectric focusing reactions. In some embodiments, the method further includes monitoring the plurality of isoelectric focusing reactions using an imaging technique to detect the position of one or more separated analyte peaks in each separation channel. In some embodiments, the imaging technique includes whole-channel imaging. In some embodiments, the imaging technique includes UV absorbance imaging or fluorescence imaging. In some embodiments, fluorescence imaging includes native fluorescence imaging. In some embodiments, the method further includes using the imaging technique to detect the position of one or more pI markers so that a pI value can be determined for the one or more separated analyte peaks. In some embodiments, the method further includes flushing the separation channels after the plurality of isoelectric focusing reactions are completed and automatically introducing new sample aliquots into the separation channels. In some embodiments, the automated cycle time for introducing the plurality of sample aliquots, performing the plurality of isoelectric focusing reactions, and flushing the separation channels is between 1 minute and 30 minutes. In some embodiments, detection of an isoelectric focusing reaction failure in any of the plurality of separation channels triggers automatic reintroduction and repetition of the isoelectric focusing reaction for the sample aliquot already introduced into that separation channel. In some embodiments, the isoelectric focusing reaction failure comprises the introduction of bubbles, the formation of bubbles, or any combination thereof. In some embodiments, the isoelectric focusing reaction failure is detected by monitoring the current flowing through the separation channel or by processing an image of the separation channel.In some embodiments, the method further comprises measuring dynamic light scattering in at least one of the plurality of separation channels while performing isoelectric focusing. In some embodiments, the dynamic light scattering measurement provides a determination of a size distribution curve for one or more separated analytes. In some embodiments, the dynamic light scattering measurement provides a determination of an aggregation state for one or more separated analytes. In some embodiments, the dynamic light scattering measurement provides a determination of a hydrodynamic radius for one or more separated analytes.

[0020] Also disclosed herein is a microfluidic device comprising: a) a plurality of inlet ports; b) a substrate comprising a plurality of separation channels, wherein the proximal end of each separation channel is fluidically connected to a different inlet port; and c) a plurality of outlet ports, wherein the distal end of each separation channel is fluidically connected to a different outlet port; wherein the channels in the plurality of separation channels are configured for full-channel imaging.

[0021] In some embodiments, the microfluidic device further comprises an integrated electrode pair for each separation channel, wherein one electrode in each pair of electrodes contacts the proximal end of the separation channel and the other electrode contacts the distal end of the separation channel. In some embodiments, the microfluidic device comprises 4 to 8 separation channels. In some embodiments, the plurality of separation channels are configured for full-channel UV absorbance imaging. In some embodiments, the plurality of separation channels are configured for full-channel fluorescence imaging. In some embodiments, at least one channel within the plurality of separation channels is configured for performing dynamic light scattering measurements. In some embodiments, there are no reservoirs or wells on the device. In some embodiments, the inlet port is located on one or more edges of the device, such as Figure 1A In some embodiments, the outlet port is located on one or more edges of the device, such as Figure 1A In some embodiments, the microfluidic device is a disposable component of a system for performing an isoelectric focusing reaction. In some embodiments, the microfluidic device further comprises a cartridge comprising a portion or all of the plurality of inlet ports, the plurality of outlet ports, or the substrate. In some embodiments, the cartridge is a disposable component of a system for performing an isoelectric focusing reaction. In some embodiments, one or more of the inlet ports or outlet ports are coupled to a high voltage electrode using a membrane-containing high voltage electrode holder that provides a bubble-free electrical connection. In some embodiments, the membrane-containing high voltage electrode holder is Figure 12as shown in . In some embodiments, the membrane comprises a hydrophilic membrane. In some embodiments, the membrane comprises a regenerated cellulose membrane. In some embodiments, the membrane comprises a woven polytetrafluoroethylene (PTFE) membrane that has been treated to be hydrophilic. In some embodiments, the membrane-covered fluid port within the membrane-containing high-voltage electrode holder has a diameter in the range of about 0.5 mm to about 2 mm. In some embodiments, the electrode reservoir within the membrane-containing high-voltage electrode holder comprises an insert positioned within the electrode reservoir and at the bottom of the electrode reservoir, wherein the insert comprises an inlet fluid path and an outlet fluid path, the inlet fluid path and the outlet fluid path allowing the surface of the membrane to be wetted without bubbles when the electrode reservoir is filled. In some embodiments, the cartridge comprises at least one integrated membrane-containing high-voltage electrode holder. In some embodiments, the cartridge comprises at least one reagent reservoir. In some embodiments, the at least one reagent reservoir comprises an anolyte reservoir, a cathode reservoir, or an activated reagent reservoir. In some embodiments, the cartridge comprises at least one flow restrictor. In some embodiments, the cartridge comprises at least one valve. In some embodiments, the at least one valve comprises a shear valve. In some embodiments, the shear valve comprises as Figure 19A In some embodiments, the shear valve includes a Figure 19B In some embodiments, the cartridge has a valve design as shown. Figure 16 、 Figure 17 、 Figure 18 、 Figure 19A or Figure 19B The side manifold design shown in any of the figures above is configured to provide gaps for imaging the multiple separation channels.

[0022] Disclosed herein is a system for performing multiple isoelectric focusing reactions in parallel, the system comprising: a) a microfluidic device comprising a plurality of separation channels, wherein the device comprises: i) a plurality of inlet ports; ii) a substrate comprising the plurality of separation channels, wherein a proximal end of each separation channel is in fluid communication with a different inlet port; and iii) a plurality of outlet ports, wherein a distal end of each separation channel is in fluid communication with a different outlet port; wherein channels in the plurality of separation channels are configured for whole-channel imaging; and b) a programmable multiplexed power supply, wherein the multiplexed power supply is configured to independently control and monitor current flowing through each of the plurality of separation channels in the microfluidic device while performing multiple isoelectric focusing reactions to separate an analyte mixture into its respective components.

[0023] In some embodiments, the microfluidic device further comprises an integrated electrode pair for each separation channel, wherein one electrode in each pair of electrodes contacts the proximal end of the separation channel and the other electrode contacts the distal end of the separation channel. In some embodiments, the separation channels share a common cathode liquid. In some embodiments, the separation channels share a common anode liquid. In some embodiments, the microfluidic device comprises 4 to 8 separation channels. In some embodiments, the plurality of separation channels are configured for full-channel UV absorbance imaging. In some embodiments, the plurality of separation channels are configured for full-channel fluorescence imaging. In some embodiments, at least one channel within the plurality of separation channels is configured for performing dynamic light scattering measurements. In some embodiments, there are no reservoirs or recesses on the microfluidic device. In some embodiments, the inlet port is located on one or more edges of the microfluidic device, such as Figure 1A In some embodiments, the outlet port is located on one or more edges of the device, such as Figure 1A In some embodiments, the microfluidic device is a disposable component of the system. In some embodiments, the microfluidic device further comprises a cartridge comprising a portion or all of the plurality of inlet ports, the plurality of outlet ports, or the substrate. In some embodiments, the cartridge is a disposable component of the system. In some embodiments, one or more inlet ports or outlet ports are coupled to a high voltage electrode using a membrane-containing high voltage electrode holder that provides a bubble-free electrical connection. In some embodiments, the membrane-containing high voltage electrode holder is Figure 12as shown in . In some embodiments, the membrane comprises a hydrophilic membrane. In some embodiments, the membrane comprises a regenerated cellulose membrane. In some embodiments, the membrane comprises a woven polytetrafluoroethylene (PTFE) membrane that has been treated to be hydrophilic. In some embodiments, the membrane-covered fluid port within the membrane-containing high-voltage electrode holder has a diameter in the range of about 0.5 mm to about 2 mm. In some embodiments, the electrode reservoir within the membrane-containing high-voltage electrode holder comprises an insert positioned within the electrode reservoir and at the bottom of the electrode reservoir, wherein the insert comprises an inlet fluid path and an outlet fluid path, the inlet fluid path and the outlet fluid path allowing the surface of the membrane to be wetted without bubbles when the electrode reservoir is filled. In some embodiments, the cartridge comprises at least one integrated membrane-containing high-voltage electrode holder. In some embodiments, the cartridge comprises at least one reagent reservoir. In some embodiments, the at least one reagent reservoir comprises an anolyte reservoir, a cathode reservoir, or an activated reagent reservoir. In some embodiments, the cartridge comprises at least one flow restrictor. In some embodiments, the cartridge comprises at least one valve. In some embodiments, the at least one valve comprises a shear valve. In some embodiments, the shear valve comprises as Figure 19A In some embodiments, the shear valve includes a Figure 19B In some embodiments, the cartridge has a valve design as shown. Figure 16 、 Figure 17 、 Figure 18 、 Figure 19A or Figure 19BIn some embodiments, the system further comprises an automated liquid handling system for loading sample aliquots into each inlet port. In some embodiments, the automated liquid handling system is configured to introduce sample aliquots into each separation channel, the sample aliquots being all taken from the same sample, and the system being configured to use a different set of experimental conditions to perform the isoelectric focusing reaction in at least two subsets of the multiple separation channels. In some embodiments, a different set of experimental conditions is used to perform the isoelectric focusing reaction in each of the multiple separation channels. In some embodiments, the automated liquid handling system is configured to introduce at least two subsets of sample aliquots taken from different samples into the separation channel, and the same set of experimental conditions is used to perform the multiple isoelectric focusing reactions. In some embodiments, each sample aliquot introduced into the separation channel is taken from a different sample. In some embodiments, a set of experimental conditions for performing the multiple isoelectric focusing reactions includes buffer selection, pH gradient selection, voltage setting, current setting, electric field strength setting, a time course for changing the voltage setting, current setting or electric field strength setting, isoelectric focusing reaction time or any combination thereof. In some embodiments, the analyte includes a protein. In some embodiments, the programmable multiplexed power supply is further configured to record a current trace for each of the multiple separation channels while the isoelectric focusing reaction is being performed. In some embodiments, the system further includes an imaging unit configured to acquire images of the multiple separation channels and detect separated analyte peaks in the multiple separation channels. In some embodiments, the imaging unit is configured to perform full-channel imaging of the multiple separation channels. In some embodiments, the imaging unit is configured to acquire UV absorbance images. In some embodiments, the imaging unit is configured to acquire fluorescence images. In some embodiments, the fluorescence images include natural fluorescence images. In some embodiments, the imaging unit is further configured to detect the position of one or more pI markers so that pI values ​​can be determined for one or more separated analyte peaks. In some embodiments, the system is configured to flush the separation channel after the plurality of isoelectric focusing reactions are completed and to automatically introduce a new sample aliquot into the separation channel. In some embodiments, the automated cycle time for introducing the plurality of sample aliquots, performing the plurality of isoelectric focusing reactions, and flushing the separation channel is between 1 minute and 30 minutes. In some embodiments, detection of an isoelectric focusing reaction failure in any of the plurality of separation channels triggers automatic reintroduction and repetition of the isoelectric focusing reaction for the sample aliquot that has been introduced into the separation channel.In some embodiments, the isoelectric focusing reaction failure includes the introduction of bubbles, the formation of bubbles, or any combination thereof. In some embodiments, the isoelectric focusing reaction failure includes a sample that is not properly prepared. In some embodiments, the isoelectric focusing reaction failure includes an empty or underfilled sample well. In some embodiments, the isoelectric focusing reaction failure is detected by monitoring the current flowing through the separation channel or by processing an image of the separation channel. In some embodiments, the system further includes a dynamic light scattering measurement unit. In some embodiments, the system further includes a fluid flow controller configured to provide independently controlled pressure-driven flow through one or more separation channels, one or more mobilizer channels, or one or more auxiliary fluid channels. In some embodiments, the pressure-driven flow through the one or more separation channels, one or more mobilizer channels, or one or more auxiliary fluid channels is a pulse-free flow. In some embodiments, the system further includes a temperature controller configured to maintain the multiple separation channels at a constant temperature.

[0024] Also disclosed herein are methods comprising: a) applying an electric field across a separation channel in a microfluidic device to perform separation of an analyte mixture via isoelectric focusing; b) simultaneously and continuously imaging the separation and mobilization of the separated analyte mixture throughout the separation channel or a portion thereof; and c) expelling the separated and mobilized analytes from an orifice in the microfluidic device into a mass spectrometer via electrospray ionization; wherein the microfluidic device is oriented tilted relative to a horizontal plane such that the orifice points downwardly toward an inlet of the mass spectrometer. In some embodiments, the method further comprises correlating a separated analyte peak detected in the separation channel with mass spectrometer data for the separated analyte. In some embodiments, the separated analyte peak is detected by absorbance imaging. In some embodiments, the separated analyte peak is detected by fluorescence imaging. In some embodiments, the orifice is in electrical communication with the electric field of the separation channel. In some embodiments, the orifice is a recess in the microfluidic device such that a Taylor cone formed by electrospray ionization is completely disposed within the recess. In some embodiments, the microfluidic device comprises a first separation channel and a second separation channel. In some embodiments, the method further comprises: chromatographically enriching the analyte mixture in the first separation channel before applying the electric field to perform the isoelectric focusing separation of the analyte mixture in the second separation channel. In some embodiments, the method further comprises: introducing an amphoteric electrolyte into the separation channel to generate a pH gradient in the separation channel before the separation of the analyte mixture, introducing an isoelectric point (pi) marker into the separation channel before the separation, and continuously imaging the separation channel while the pi marker is separated. In some embodiments, the analyte mixture comprises intact proteins. In some embodiments, the mobilization is performed by introducing electrolytes into the separation channel using pressure. In some embodiments, the mobilization is performed by introducing electrolytes into the separation channel by electrophoresis. In some embodiments, the mobilization is performed by introducing electrolytes into the separation channel from an electrolyte channel that is fluidically connected to a junction area downstream of the separation channel. In some embodiments, the microfluidic device comprises two electrodes to generate an electric field across an electrolyte introduction channel. In some embodiments, there are no reservoirs or recesses on the microfluidic device. In some embodiments, the inlet port for the microfluidic device is located on one or more edges of the device, such as Figure 2 In some embodiments, the outlet port for the microfluidic device is located on one or more edges of the device, such as Figure 2In some embodiments, the microfluidic device is a disposable component of a system for performing isoelectric focusing and mass spectrometry. In some embodiments, the microfluidic device is a cartridge comprising a separation channel, an orifice, an electrolyte introduction channel, an anolyte introduction channel, and a gas delivery channel for ionization. In some embodiments, the cartridge is a disposable component of a system for performing isoelectric focusing and mass spectrometry. In some embodiments, one or more inlet ports or outlet ports of the microfluidic device are coupled to a high voltage electrode using a membrane-containing high voltage electrode holder that provides a bubble-free electrical connection. In some embodiments, the membrane-containing high voltage electrode holder is Figure 12 as shown in . In some embodiments, the membrane comprises a hydrophilic membrane. In some embodiments, the membrane comprises a regenerated cellulose membrane. In some embodiments, the membrane comprises a woven polytetrafluoroethylene (PTFE) membrane that has been treated to be hydrophilic. In some embodiments, the membrane-covered fluid port within the membrane-containing high-voltage electrode holder has a diameter in the range of about 0.5 mm to about 2 mm. In some embodiments, the electrode reservoir within the membrane-containing high-voltage electrode holder comprises an insert positioned within the electrode reservoir and at the bottom of the electrode reservoir, wherein the insert comprises an inlet fluid path and an outlet fluid path, the inlet fluid path and the outlet fluid path allowing the surface of the membrane to be wetted without bubbles when the electrode reservoir is filled. In some embodiments, the cartridge comprises at least one integrated membrane-containing high-voltage electrode holder. In some embodiments, the cartridge comprises at least one reagent reservoir. In some embodiments, the at least one reagent reservoir comprises an anolyte reservoir, a cathode reservoir, or an activated reagent reservoir. In some embodiments, the cartridge comprises at least one flow restrictor. In some embodiments, the cartridge comprises at least one valve. In some embodiments, the at least one valve comprises a shear valve. In some embodiments, the shear valve comprises as Figure 19A In some embodiments, the shear valve includes a Figure 19B In some embodiments, the cartridge has a valve design as shown. Figure 16 、 Figure 17 、 Figure 18 、 Figure 19A or Figure 19B , so as to provide clearance for imaging the plurality of separation channels. In some embodiments, the method further comprises providing an automated liquid handling system for loading a sample aliquot into each inlet port.

[0025] Disclosed herein is a microfluidic device comprising a substrate, wherein the substrate defines: a) one or more inlet ports; b) at least one separation channel configured to perform separation of an analyte mixture; and c) an orifice in fluid communication with an end of the separation channel, wherein the orifice is configured to perform electrospray ionization on separated analyte fractions eluted from the separation channel and expel them into a mass spectrometer.

[0026] In some embodiments, the microfluidic device further comprises an integrated electrode pair for the at least one separation channel, wherein one electrode in each pair of electrodes contacts the proximal end of the separation channel and the other electrode contacts the distal end of the separation channel. In some embodiments, the at least one separation channel is configured for full channel UV absorbance imaging. In some embodiments, the at least one separation channel is configured for full channel fluorescence imaging. In some embodiments, there are no reservoirs or recesses on the device. In some embodiments, the one or more inlet ports are located on one or more edges of the device, such as Figure 2 As shown. In some embodiments, the device further comprises an auxiliary fluid channel for conveying a calibration solution for calibrating mass data. In some embodiments, the microfluidic device is a disposable component of a system for performing isoelectric focusing and mass spectrometry on an analyte mixture. In some embodiments, the microfluidic device further comprises a cartridge comprising a portion or all of one or more inlet ports, orifices, or a substrate. In some embodiments, the cartridge is a disposable component of a system for performing isoelectric focusing and mass spectrometry on an analyte mixture. In some embodiments, one or more inlet ports are coupled to a high voltage electrode using a membrane-containing high voltage electrode holder that provides a bubble-free electrical connection. In some embodiments, the membrane-containing high voltage electrode holder is Figure 12As shown in . In some embodiments, the membrane comprises a hydrophilic membrane. In some embodiments, the membrane comprises a regenerated cellulose membrane. In some embodiments, the membrane comprises a woven polytetrafluoroethylene (PTFE) membrane that has been treated to be hydrophilic. In some embodiments, the membrane comprises a rigid material, for example, glass or ceramic. In some embodiments, the membrane can be treated to be hydrophilic and / or non-charged. In some embodiments, the membrane-covered fluid port in the high-voltage electrode holder containing the membrane has a diameter in the range of about 0.5 mm to about 2 mm. In some embodiments, the electrode reservoir in the high-voltage electrode holder containing the membrane comprises an insert, the insert being positioned in the electrode reservoir and at the bottom of the electrode reservoir, wherein the insert comprises an inlet fluid path and an outlet fluid path, the inlet fluid path and the outlet fluid path allowing the surface of the membrane to be wetted without bubbles when the electrode reservoir is filled. In some embodiments, the cartridge comprises at least one integrated high-voltage electrode holder containing the membrane. In some embodiments, the cartridge comprises at least one reagent reservoir. In some embodiments, the at least one reagent reservoir comprises an anolyte reservoir, a cathode reservoir, or an activated reagent reservoir. In some embodiments, the cartridge comprises at least one flow restrictor. In some embodiments, the cartridge comprises at least one valve. In some embodiments, the at least one valve comprises a shear valve. In some embodiments, the shear valve comprises Figure 19A In some embodiments, the shear valve includes a Figure 19B In some embodiments, the cartridge includes a mechanism to facilitate application of a vacuum to remove excess fluid buildup from the outer surface of the orifice. In some embodiments, the cartridge includes an atomizer mechanism to facilitate formation of a stable Taylor cone. In some embodiments, the cartridge has a Figure 16 、 Figure 17 、 Figure 18 、 Figure 19A or Figure 19B The side manifold design shown in any of the figures above is configured to provide gaps for imaging the multiple separation channels.

[0027] Disclosed herein is an apparatus comprising: a) a microfluidic device comprising a substrate, wherein the substrate defines: i) one or more inlet ports; ii) a separation channel configured to perform separation of an analyte mixture; and iii) an orifice in fluid communication with an end of the separation channel, wherein the orifice is configured to perform electrospray ionization on separated analyte fractions eluted from the separation channel and expel them into a mass spectrometer, wherein the microfluidic device is oriented at an angle relative to a horizontal plane such that the orifice points downwardly toward an inlet of the mass spectrometer; and b) an imaging device configured to simultaneously and continuously image the separation and elution of the separated analyte mixture throughout the separation channel or a portion thereof.

[0028] In some embodiments, the system further comprises a mass spectrometer. In some embodiments, the device is further configured to correlate the separated analyte peaks detected in the separation channel with mass spectrometer data for the separated analytes. In some embodiments, the separation comprises chromatographic separation. In some embodiments, the device further comprises at least two electrodes, wherein the at least two electrodes are configured to apply an electric field across the separation channel to separate the analyte mixture via isoelectric focusing. In some embodiments, the device further comprises at least two electrodes, wherein the at least two electrodes are configured to apply an electric field across the separation channel to separate the analyte mixture via electrophoresis. In some embodiments, the peak corresponding to the separated analyte fraction is detected by absorbance imaging. In some embodiments, the peak corresponding to the separated analyte fraction is detected by fluorescence imaging. In some embodiments, the orifice is electrically connected to the electric field of the separation channel. In some embodiments, the orifice is positioned in a recess on the microfluidic device so that the Taylor cone formed by electrospray ionization is completely disposed within the recess. In some embodiments, the outer surface of the orifice comprises a hydrophobic coating to prevent excessive fluid accumulation during operation. In some embodiments, the microfluidic device includes a first separation channel and a second separation channel. In some embodiments, the microfluidic device is configured to perform chromatographic enrichment of the analyte mixture in the first separation channel before applying the electric field to perform isoelectric focusing or electrophoretic separation of the analyte mixture in the second separation channel. In some embodiments, an isoelectric point (pI) marker is introduced into the separation channel before performing isoelectric focusing separation and is used to draw the pI range in the separation channel. In some embodiments, the device is also configured to determine the value of the isoelectric point of the analyte fraction for at least one separation. In some embodiments, the analyte mixture includes intact proteins. In some embodiments, mobilization is performed by introducing an electrolyte into the separation channel using pressure. In some embodiments, mobilization is performed by introducing an electrolyte into the separation channel using electrophoresis. In some embodiments, the microfluidic device further includes an additional electrolyte introduction channel, which intersects the separation channel at the junction of the separation channel and the orifice, and wherein the mobilization is performed by introducing an electrolyte from the electrolyte introduction channel into the separation channel. In some embodiments, the apparatus further comprises two electrodes configured to apply an electric field across the electrolyte introduction channel to introduce a mobilized electrolyte. In some embodiments, the microfluidic device further comprises an anolyte introduction channel and a gas delivery channel for ionization. In some embodiments, the microfluidic device further comprises an optical slit aligned with the separation channel such that light is transmitted only through the optical slit. In some embodiments, the microfluidic device is devoid of a reservoir or recessed hole.In some embodiments, the one or more inlet ports of the microfluidic device are located on one or more edges of the device, eg, . Figure 2 In some embodiments, the microfluidic device is a disposable component of the apparatus. In some embodiments, the microfluidic device further comprises a cartridge comprising a portion or all of the one or more inlet ports, the orifice, and the substrate. In some embodiments, the cartridge is a disposable component of the apparatus. In some embodiments, the one or more inlet ports are coupled to the high voltage electrode using a membrane-containing high voltage electrode holder that provides a bubble-free electrical connection. In some embodiments, the membrane-containing high voltage electrode holder is Figure 12 as shown in . In some embodiments, the membrane comprises a hydrophilic membrane. In some embodiments, the membrane comprises a regenerated cellulose membrane. In some embodiments, the membrane comprises a woven polytetrafluoroethylene (PTFE) membrane that has been treated to be hydrophilic. In some embodiments, the membrane-covered fluid port within the membrane-containing high-voltage electrode holder has a diameter in the range of about 0.5 mm to about 2 mm. In some embodiments, the electrode reservoir within the membrane-containing high-voltage electrode holder comprises an insert positioned within the electrode reservoir and at the bottom of the electrode reservoir, wherein the insert comprises an inlet fluid path and an outlet fluid path, the inlet fluid path and the outlet fluid path allowing the surface of the membrane to be wetted without bubbles when the electrode reservoir is filled. In some embodiments, the cartridge comprises at least one integrated membrane-containing high-voltage electrode holder. In some embodiments, the cartridge comprises at least one reagent reservoir. In some embodiments, the at least one reagent reservoir comprises an anolyte reservoir, a cathode reservoir, or an activated reagent reservoir. In some embodiments, the cartridge comprises at least one flow restrictor. In some embodiments, the cartridge comprises at least one valve. In some embodiments, the at least one valve comprises a shear valve. In some embodiments, the shear valve comprises as Figure 19A In some embodiments, the shear valve includes a Figure 19B In some embodiments, the cartridge includes a mechanism to facilitate application of a vacuum to remove excess fluid buildup from the outer surface of the orifice. In some embodiments, the device further includes a mechanism to facilitate application of a vacuum to remove excess fluid buildup from the outer surface of the orifice. In some embodiments, the device further includes a wiper mechanism to remove excess fluid buildup from the outer surface of the orifice. In some embodiments, the cartridge includes an atomizer mechanism to facilitate formation of a stable Taylor cone. In some embodiments, the device further includes an atomizer mechanism to facilitate formation of a stable Taylor cone. In some embodiments, the cartridge has Figure 16 、 Figure 17 、 Figure 18 、 Figure 19A or Figure 19B In certain embodiments, the side manifold design shown in any of the figures in the figure is provided to provide a gap for imaging the multiple separation channels. In certain embodiments, the device further comprises a fluid flow controller, which is configured to provide independently controlled pressure-driven flow by one or more separation channels, one or more mobilizing agent channels or one or more auxiliary fluid channels. In certain embodiments, the pressure-driven flow by the one or more separation channels, one or more mobilizing agent channels or one or more auxiliary fluid channels is pulse-free flow. In certain embodiments, the device further comprises a temperature controller, which is configured to maintain the multiple separation channels at a constant temperature. In certain embodiments, the microfluidic device includes an auxiliary fluid channel, which is used to convey a calibration solution for calibrating mass data. In certain embodiments, the imaging device can be configured to serve as a point detector during eluting analyte fractions from the separation channel, so as to provide improved time resolution for a chromatogram based on time. In certain embodiments, the imaging device pixels between 4 and 16 are sorted (binned) to serve as a point detector. In certain embodiments, the intensity data from the sorted pixels are read out at a rate of at least 1 Hz.

[0029] Disclosed herein is a holder comprising: a) an electrode reservoir; b) an inlet fluid channel and an outlet fluid channel intersecting at a plane; and c) a membrane positioned within the electrode reservoir on the plane so as to cover an opening comprising the intersection of the inlet fluid channel and the outlet fluid channel; wherein the membrane facilitates formation of a bubble-free electrical connection between a high voltage electrode and a fluid within the inlet fluid channel and the outlet fluid channel.

[0030] In some embodiments, the holder comprises Figure 12 Design shown. In some embodiments, the membrane comprises a hydrophilic membrane. In some embodiments, the membrane comprises a regenerated cellulose membrane. In some embodiments, the membrane comprises a woven polytetrafluoroethylene (PTFE) membrane that has been treated to be hydrophilic. In some embodiments, the opening covered by the membrane has a diameter in the range of about 0.5 mm to about 2 mm. In some embodiments, the electrode reservoir further comprises an insert, which is positioned in the electrode reservoir and at the bottom of the electrode reservoir and on the top of the membrane, wherein the insert comprises an inlet fluid path and an outlet fluid path, and the inlet fluid path and the outlet fluid path allow the surface of the membrane to be wetted without bubbles when the electrode reservoir is filled.

[0031] This article discloses Figure 19A or Figure 19BShear valve of the design shown.

[0032] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated as being incorporated herein by reference in its entirety. In the event of a conflict in a terminology between this document and that of an incorporated reference, the terminology in this document controls. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The novel features of the present 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 and accompanying drawings which set forth illustrative embodiments in which the principles of the present invention are utilized, in which:

[0034] Figure 1A A non-limiting schematic diagram of a microfluidic device including a four-channel isoelectric focusing design according to one aspect of the present disclosure is provided.

[0035] Figure 1B A non-limiting schematic diagram of a fluidic channel network of an exemplary microfluidic device including an electrospray tip of the present disclosure is provided.

[0036] Figure 2 A schematic top-down view of one non-limiting example of a microfluidic device for performing one or more separation reactions (eg, an isoelectric focusing reaction followed by electrospray ionization) is provided.

[0037] Figure 3 A cross-sectional view of one non-limiting example of a microfluidic device for performing one or more separation reactions (eg, an isoelectric focusing reaction followed by electrospray ionization) is provided.

[0038] Figure 4 A schematic diagram of an exemplary waste management system is provided.

[0039] Figure 5 A schematic diagram of another exemplary waste management system is provided.

[0040] Figure 6 A further example of a waste management system comprising a gripper module is schematically shown.

[0041] Figure 7 Yet another example of a waste management system comprising a tube vacuum device is schematically shown.

[0042] Figure 8 Yet another example of a waste management system using positive pressure is schematically shown.

[0043] Figure 9 An exemplary atomizer is shown schematically.

[0044] 10A to 10D Additional non-limiting examples of atomizers are schematically shown. Figure 10A : Front view of the second atomizer design. Figure 10B : Isometric view of the second atomizer design. Figure 10C : Cutaway top view of the third atomizer design. Figure 10D : Cutaway side view of the third atomizer design.

[0045] 11A to 11D A fourth example of an atomizer design is schematically shown. Figure 11A : Partial cross-sectional view as installed on the microfluidic device cartridge. Figure 11B : Isolated partial cross-sectional view. Figure 11C : Cross-sectional side view. Figure 11D : Cross-sectional top view.

[0046] Figure 12 An example of a holder is schematically shown.

[0047] 13A to 13F A non-limiting example of a holder comprising a membrane is schematically shown. Figure 13A : Partial cross-sectional view. Figure 13B : Top view of the electrode reservoir. Figure 13C : Partial cross-sectional view of the electrode reservoir. Figure 13D : Top view of the bottom of the electrode reservoir. Figure 13E : Cutaway detail of the bottom of the electrode reservoir. Figure 13F : Cut-away side view of the bottom of the electrode reservoir.

[0048] Figure 14 An exemplary method of providing reagents to one or more reservoirs of the system is schematically illustrated.

[0049] Figure 15 An exemplary method of providing reagents to one or more reservoirs is schematically illustrated.

[0050] Figure 16 An exemplary system including a cartridge as described in certain embodiments herein is schematically illustrated.

[0051] Figure 17 An exemplary embodiment of a cartridge including a microfluidic device of the present disclosure is shown.

[0052] Figure 18 Another exemplary embodiment of a cartridge including a microfluidic device of the present disclosure is shown.

[0053] Figures 19A to 19B Additional exemplary embodiments of cartridges including the microfluidic device and shear valve of the present disclosure are shown. Figure 19A: Cartridge including rotary shear valve. Figure 19B : Cartridge including spring loaded shear valve.

[0054] Figure 20 Another exemplary embodiment of a cartridge including a microfluidic device of the present disclosure is shown.

[0055] Figure 21 Schematically illustrated are non-limiting examples of securing features that may be used to secure the device to the cartridge.

[0056] Figure 22 Schematically depicts non-limiting examples of securing features that may be used to create a fluid seal of the cartridge to the device.

[0057] Figure 23 Non-limiting examples of electrical connections to a cartridge or reservoir of a device are shown schematically.

[0058] Figure 24 Schematically shown are non-limiting examples of mounting plates for coupling one or more reservoir units to a cartridge that also engages an instrument.

[0059] Figure 25 An exemplary cartridge with a reservoir structure is shown.

[0060] Figure 26 Another exemplary cartridge having a reservoir structure is shown.

[0061] Figure 27 Yet another exemplary cartridge having a reservoir structure is shown.

[0062] 28A to 28D Different perspective views of an exemplary imaging system disclosed herein are shown. Figure 28A : An imaging system including a scanning (or steering) mirror. Figure 28B : An imaging system including a mirror for full-channel imaging. Figure 28C : Figure 28B Detailed view of the imaging system. Figure 28D : Figure 28B and Figure 28C A top view of the imaging system shown in FIG.

[0063] Figure 29 An exemplary system for interfacing a microfluidic separation device / cartridge according to embodiments described herein with a mass spectrometer is schematically shown.

[0064] Figure 30 Another exemplary system according to embodiments described herein is schematically illustrated.

[0065] Figure 31An exemplary system according to embodiments described herein is schematically illustrated.

[0066] FIG. 32A to FIG. 32B Exemplary data showing mobilization reactions and mobility chromatograms. Figure 32A : Graph of UV absorbance versus number of pixels of the image sensor used to image the separation channel. Figure 32B : From e.g. Figure 32A Mobilized chromatogram (graph of UV absorbance versus time) derived from the data shown.

[0067] Figure 33 A non-limiting example of a software architecture for one embodiment of the disclosed system is shown.

[0068] Figure 34 A non-limiting example of a block diagram illustrating an integrated system in one embodiment of the present disclosure.

[0069] Figure 35 Another non-limiting example of a block diagram illustrating an integrated system in one embodiment of the present disclosure.

[0070] Figure 36 An exemplary flow chart of a computer-controlled voltage feedback loop in which the ESI tip is held at 0 V is provided.

[0071] Figure 37 An exemplary flow chart of a voltage feedback loop in which the ESI tip is held at +3000 V is provided.

[0072] Figure 38 A schematic diagram of an exemplary chip with intersecting channels is provided. DETAILED DESCRIPTION

[0073] Disclosed herein are methods, devices, and systems for performing multiple isoelectric focusing reactions (or other separation reactions) in parallel for rapid and accurate separation and characterization of protein analyte mixtures or other biomolecules by isoelectric point (or other physicochemical properties). The subject matter of this application is related to the subject matter of U.S. Patent Nos. 10,209,217B2 and 10,401,324B2 and co-pending U.S. Patent Application Nos. 15 / 363,908, 16 / 261,382, 16 / 427,767, and 16 / 532,955, each of which is incorporated herein by reference in its entirety.

[0074] In one aspect of the present disclosure, a microfluidic device is described, wherein the microfluidic device includes two or more separation channels for performing two or more separation reactions in parallel, wherein the form of microfluid can use minimal input sample volume to separate and characterize analyte mixture quickly and accurately. In certain embodiments, the microfluidic device includes a planar substrate, and the planar substrate includes two or more separation channels. In a preferred aspect, the separation reaction is an isoelectric focusing reaction. In another preferred aspect, the analyte mixture includes a protein analyte mixture, and the parallel execution of two or more isoelectric focusing reactions can quickly and accurately separate the protein components in the analyte mixture and characterize each protein component according to their isoelectric point (PI). In some cases, imaging (for example, full channel imaging) is used in combination with pI marks to allow for more accurately determining the pI of the separated protein components of analyte mixture so as to visualize the position of the pI mark in the pH gradient for isoelectric focusing.

[0075] In another aspect of the present disclosure, a method and system for operating the microfluidic device is described, wherein the separation reaction or experimental condition in each separation channel of the microfluidic device can be independently controlled using two or more high voltage power supplies (or a single multiplexed high voltage power supply). Therefore, in some cases, the microfluidic device can be used to perform the separation and characterization of two or more different samples in parallel under the same set of separation or experimental conditions. In some cases, the microfluidic device can be used to perform the separation and characterization of two or more aliquots of the same sample in parallel under two or more different reactions or experimental conditions. In some cases, a subset of the separation channels on the device can be used to perform the separation of multiple samples under the same set of separation or experimental conditions, and alternatively or in addition, different subsets of the separation channels on the device can be used to perform the separation and characterization of multiple aliquots from the same sample in parallel under multiple different reactions or experimental conditions.

[0076] Experimental conditions can be the same or different across the separation channels of the microfluidic device and can include buffer selection, electrolyte selection, pH gradient selection, voltage setting, current setting, electric field strength setting, a time course for changing the voltage setting, current setting, electric field strength setting, isoelectric focusing reaction, or a combination thereof.

[0077] In some cases, the system of the present disclosure includes one or more of the disclosed microfluidic devices and two or more high voltage power supplies (or a single multiplexed high voltage power supply allowing two or more channels to be controlled independently). In some cases, two or more high voltage power supplies (or a single multiplexed high voltage power supply allowing two or more channels to be controlled independently) are configured to monitor and / or record the current flowing through each separation channel. In some cases, the monitoring of separation channel (e.g., the current of separation channel) can be performed independently of the monitoring of other separation channels. In some cases, the current flowing through each separation channel can be used as a diagnostic tool, for example, to determine when the isoelectric focusing reaction is completed and / or to detect a fault (e.g., the introduction or formation of bubbles in the separation channel, incorrectly prepared samples, unfilled reagent reservoirs, or a combination thereof). In some cases, a fault can be detected by monitoring the current flowing through the separation channel or by processing an image of the separation channel. In some cases, the voltage source can be configured to turn off the voltage applied to the separation channel after detecting a fault. In some cases, the voltage source can be configured to restart the separation reaction after detecting a fault.

[0078] In some cases, the system can also include automatic sampler or fluid loading and unloading system, and described automatic sampler or fluid loading and unloading system are configured to be used for sample aliquot and / or other separation reaction reagent automatically and independently control to be loaded into multiple samples or reagent inlet port.In some cases, the system can also include fluid flow controller, and described fluid flow controller is configured to provide for example by the independently controlled pressure-driven flow (for example, for being used alone or for being combined with the voltage gradient being applied to two or more separation channels).In some cases, the system can also include automatic sampler or fluid flow controller, and described automatic sampler or fluid flow controller are configured to rinse, clean, flush or evacuate two or more separation channels after separation reaction (for example, isoelectric focusing reaction).In some cases, after rinsing, cleaning, flushing or evacuating two or more separation channels, automatic sampler or fluid flow controller can be configured to automatically another sample (for example, different samples, or another aliquot of identical sample) is introduced into two or more separation channels. In some cases, the autosampler or fluid flow controller can be configured to automatically reintroduce the sample, reaction reagent, or combination thereof into one or more separation channels if a fault (e.g., formation or introduction of bubbles, improperly prepared samples, unfilled reagent reservoirs, or combinations thereof) is detected (e.g., via voltage or current monitoring). In such cases, upon detecting the fault, the autosampler or fluid flow controller flushes the faulty separation channel, reintroduces the sample, reaction reagent, or combination thereof, and can reinitiate (e.g., by applying an electric field by one or more of the independently controlled voltage sources) the separation reaction.

[0079] 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. In some cases, the field of view of the image may include all or part of two or more separation channels. In some cases, imaging may include continuous imaging while performing a separation reaction. In some cases, imaging may include intermittent imaging while performing a separation reaction. In some cases, imaging may include acquiring UV absorbance images. In some cases, imaging may include fluorescence images, for example, natural fluorescence or fluorescence generated due to the presence of exogenous fluorescent labels attached to analytes. In some cases, the imaging module may be configured to, for example, determine when the isoelectric focusing reaction is completed and / or detect a fault (for example, the introduction or formation of bubbles in the separation channel).

[0080] In some cases, the system may also include a microplate handling robotic module configured to transport and replace microplates used as a source for samples and / or reagents. In some cases, the system may also include a microfluidic device handling robotic module configured to transport and replace microfluidic devices used in the system, for example, after detecting a malfunction. In some cases, microplate handling and microfluidic device handling may be performed by the same robotic module.

[0081] In another aspect of the present disclosure, a system that can include a microfluidic device is described, and the microfluidic device is designed to perform one or more separation reactions, for example, isoelectric focusing reaction, to separate a sample containing an analyte mixture into its individual components, followed by electrospray ionization of the separated analytes. In some cases, the microfluidic device can be contained in a cartridge, which also includes, for example, a high-voltage electrode connection, a reagent reservoir, a valve, etc. In some cases, the microfluidic device can include a substantially planar substrate, wherein the planar substrate includes a plurality of separation channels. In some cases, the first end of one or more separation channels of a plurality of separation channels is electrically and / or fluidically coupled to an electrode (for example, an anolyte) reservoir using a fixture, which can include a membrane. In some cases, the second end of one or more separation channels is electrically and / or fluidically coupled to an electrode (for example, a cathode) reservoir using a fixture, which can include a membrane. The membrane can be arranged in the electrode reservoir at or near a plane limiting or parallel to the surface of the electrode reservoir, and the plane can intersect with the inlet fluid channel and the outlet fluid channel. The membrane can cover all or substantially all of the opening including the intersection of the inlet fluid channel and the outlet fluid channel. In some cases, the system can also include an analytical instrument, such as a mass spectrometer. The disclosed methods, devices, and systems can improve the reproducibility and quantitative accuracy of separation data, and can also improve the correlation between separation data and downstream analytical characterization data (e.g., data obtained using a mass spectrometer or other analytical instrument).

[0082] As mentioned above, the key feature of the disclosed method, device and system is to use imaging to monitor the separation reaction in separation channel, for the purpose of detecting the existence of analyte peak and / or determining when separation reaction has been completed. In some cases, images can be collected for all or part of separation channel. In some cases, the imaging of all or part of separation channel can be performed while performing separation step and / or activation step. In some cases, image can be used to detect the position of the analyte peak enriched in separation channel. In some cases, image can be used to detect the existence of one or more marks or indicators in separation channel, for example, isoelectric point (pI) standard, and thereby determine the pI for one or more analytes. In some cases, image can be used to detect the fault (for example, bubble formation) in separation channel. In some cases, the data derived from these images can be used to determine when separation reaction is completed (for example, by monitoring peak velocity, peak position and / or peak width) and subsequently trigger the activation step.

[0083] In some cases, the mobilization step may include introducing a mobilization buffer or a mobilization electrolyte into the separation channel. In some cases, the mobilization buffer or the mobilization electrolyte can be introduced using fluid dynamic pressure. In some cases, the mobilization buffer or the mobilization electrolyte can be introduced by means of electrophoresis. In some cases, the mobilization buffer or the mobilization electrolyte can be introduced by means of a combination of electrophoresis and fluid dynamic pressure. In some cases, the mobilization of a series of one or more separated analyte bands may include prompting the separated analyte band to migrate toward the outlet or distal end of the separation channel. In some cases, the mobilization of a series of one or more separated analyte bands may include prompting the separated analyte band to migrate toward the outlet or distal end of the separation channel communicated with the downstream analytical instrument fluid. In some cases, the outlet or distal end of the separation channel can be communicated with an electrospray ionization (ESI) interface fluid so that the migrated analyte peak is injected into a mass spectrometer. In some cases, the image data for detecting the analyte peak position and determining the analyte pI can also be used to associate analyte separation data with mass spectrometry data. In some cases, image data used to detect analyte peak positions can be used to generate information about mobilization reactions and / or to correlate the mobilization information with mass spectrometry data.

[0084] In some cases, other characterization techniques can be used to monitor one or more separation reactions. In some cases, while performing separation reactions (e.g., isoelectric focusing), dynamic light scattering can be used in at least one of the separation channels. In some cases, dynamic light scattering can be used to determine size distribution curves, aggregation states or hydrodynamic radius for one or more analytes. One or more analytes can be separated using separation reactions (e.g., isoelectric focusing).

[0085] In a preferred aspect, the disclosed methods can be performed in the form of a microfluidic device, thereby allowing for processing extremely small sample volumes and integrating two or more sample processing and separation steps. In another preferred aspect, the disclosed microfluidic device includes an integrated interface for coupling to downstream analytical instruments, such as an ESI interface for performing mass spectrometry analysis on the separated analytes. In some cases, the disclosed methods can be performed in a more conventional capillary format.

[0086] Various aspects of the disclosed methods, apparatus, and systems described herein can be applied to any specific application set forth below.It will be understood that different aspects of the disclosed methods, apparatus, and systems can be taken individually, collectively, or in combination with each other.

[0087] Definitions: Unless otherwise defined, 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.

[0088] 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 herein to "or" is intended to encompass "and / or" unless otherwise stated. Similarly, the terms "include" in the singular, "includes," "including," "comprising," "includes," and "comprising" in the singular and "includes," "includes," and "comprising" in the plural are intended to be non-limiting.

[0089] As used herein, the phrases "including, but not limited to... " and "one non-limiting example is... " are meant to include variations and derivatives of the given examples as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0090] As used herein, the term "about" a number refers to the number plus or minus 10%. The term "about" when used in the context of a range refers to the range minus 10% of its minimum and plus 10% of its maximum.

[0091] As used herein, the terms "characterize" and "analyze" can be used interchangeably. "Characterize" or "analyze" can generally mean evaluating a sample, for example, to determine one or more properties of the sample or its components, or to determine the identity of the sample.

[0092] As used herein, the terms "chip" and "device" may be used interchangeably herein.

[0093] As used herein, the terms "analyte" and "species" may be used interchangeably. Analyte generally means a molecule, biomolecule, chemical, polymer, etc. that differs from another molecule, biomolecule, chemical, polymer, etc. in a measurable property. For example, two species may have slightly different mass, hydrophobicity, charge or net charge, isoelectric point, potency, or may differ in chemical modification, protein modification, etc.

[0094] Sample: The disclosed methods, devices, systems, and software can be used to separate and characterize 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 obtained from industrial enzyme or biopharmaceutical manufacturing processes, environmental samples (e.g., air samples, water samples, soil samples, surface wipe samples), and the like. In some embodiments, prior to analysis using the disclosed methods and devices for comprehensive chemical separation and characterization, the sample can be processed using any of a variety of techniques known to those skilled in the art. For example, in some embodiments, the sample can be processed to extract proteins or nucleic acids. The sample can be collected from any of a variety of sources or subjects (e.g., bacteria, viruses, plants, animals, or humans).

[0095] Sample volume: In some examples of the disclosed methods and devices, the use of a microfluidic device format enables processing of very small sample volumes. In some embodiments, the sample volume loaded into the device and used for analysis can be in the range of about 0.1 μl to about 1 ml. In some embodiments, the sample volume loaded into the device and used for analysis can 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 can 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 values ​​described in this paragraph can be combined to form a range included in the present disclosure, for example, in some embodiments, the sample volume loaded into the device and used for analysis can be in the range of about 5 μl to about 500 μl. Those skilled in the art will recognize that the sample volume used for analysis can have any value within this range, for example, about 18 μl.

[0096] Analyte: In some cases, the sample can include multiple analyte classes. In some cases, all or part of the analyte classes present in the sample can be enriched before or during analysis. In some cases, these analytes can be, for example, polysaccharides, 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 can be small molecule drugs. In some cases, these analytes can be protein molecules in a protein mixture, for example, biological protein drugs (e.g., enzyme drugs or antibody drugs) and / or lysates collected from cells isolated in culture or in vivo.

[0097] Microfluidic Device: Disclosed herein are devices designed to perform multiple analyte separation reactions in parallel, i.e., within multiple separation channels. In some cases, the disclosed device is a microfluidic device designed to perform multiple analyte separation reactions in parallel, i.e., within multiple separation channels within the device. In some cases, the microfluidic device can be designed to perform one or more different separation steps, i.e., a first separation reaction, a second separation reaction, a third separation reaction, etc., in parallel on multiple analyte samples within the same device, i.e., within multiple first separation channels, second separation channels, third separation channels, etc. within the device. In preferred embodiments, at least one of the separation steps can include isoelectric focusing, and the device can be designed to perform two or more isoelectric focusing reactions in parallel, i.e., in two or more separation channels within the device. In some cases, the number of separation channels, e.g., 4, 6, or 12, can be selected to be consistent with the layout of the microplate wells used as sample and / or reagent sources.

[0098] In some cases, the microfluidic device comprises a substantially planar substrate, wherein the planar substrate comprises: a plurality of fluid inlets, the plurality of fluid inlets or a portion thereof being located at one or more edges of the planar substrate; and a plurality of separation channels, the plurality of separation channels comprising (i) a first end electrically coupled to an electrode reservoir (e.g., an anolyte reservoir) using a first fixture, and (ii) a second end electrically coupled to another electrode reservoir (e.g., a cathode reservoir) using a second fixture, and wherein one of the first end or the second end of each of the plurality of separation channels is in fluid communication with a different fluid inlet in the plurality of fluid inlets. In some cases, the first fixture and / or the second fixture comprise a membrane. In some cases, the first fixture and / or the second fixture are high voltage electrode fixtures and optionally comprise a membrane.

[0099] Figure 1AA non-limiting schematic diagram of a microfluidic device including a four-channel isoelectric focusing design according to one aspect of the present disclosure is provided, as will be discussed in more detail in Example 1 below.

[0100] Figure 1B A non-limiting schematic diagram of a fluid channel network of an exemplary microfluidic device for performing a separation reaction and including an electrospray tip according to the second aspect of the present disclosure is provided, as will be described in more detail in Example 14 below.

[0101] In some cases, the number of separation channels within a device configured to perform each separation step (e.g., isoelectric focusing reaction) in parallel can be 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 12, at least 14, at least 16, at least 18, at least 20, or more than 20. In some cases, the number of separation channels within a device configured to perform each separation step in parallel can be at most 20, at most 18, at most 16, at most 14, at most 12, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, or at most 2. Any of the lower and upper values ​​described in this paragraph can be combined to form ranges encompassed by the present disclosure; for example, in some cases, the number of separation channels within a device configured to perform each separation step in parallel can be in the range of about 4 to about 12. One skilled in the art will recognize that the number of separation channels within a device configured to perform each separation step in parallel can have any value within this range, for example, about 5.

[0102] In some cases, the proximal end of each of the plurality of separation channels is in fluid communication with a different inlet port so that different samples or sample aliquots can be introduced into each of the separation channels. In some cases, the proximal end of each of the plurality of separation channels is in fluid communication with the same inlet port so that the same sample or sample aliquot can be introduced into the subset of separation channels. In some cases, the proximal end of each of the plurality of separation channels is in fluid communication with the distal end or outlet end of an upstream separation channel.

[0103] In some cases, the distal end of each separator channel of multiple separator channels is all communicated with different outlet port fluids. In some cases, the distal end of each separator channel in the subset of multiple separator channels is all communicated with identical outlet port fluids. This example of the shared outlet port among multiple separator channels can be used to remove content therein, for example, to promote waste collection. In some cases, the distal end of each separator channel of multiple separator channels is all communicated with the proximal end or the inlet end fluid of downstream separator channel.

[0104] In some cases, the disclosed microfluidic device may include two or more integrated electrodes configured to apply a voltage gradient along a separation channel or along an interconnecting channel intersecting the separation channel. In some cases, the device includes an integrated electrode pair for each separation channel, wherein one electrode in each pair of electrodes contacts the proximal end of the separation channel and the other electrode contacts the distal end of the separation channel. In some cases, the disclosed microfluidic device may include at least two, at least three, at least four, at least five, or at least six integrated electrodes for each separation channel. In some cases, the disclosed microfluidic device may include an integrated electrode pair for each separation channel at each separation stage, for example, a first separation channel, a second separation channel, a third separation channel, etc.

[0105] In addition to multiple separation channels (e.g., two or more first separation channels, two or more second separation channels, two or more third separation channels, etc.), the device or microfluidic device of the present disclosure may include 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.

[0106] The disclosed devices and microfluidic devices can be manufactured using any of a variety of manufacturing techniques and materials known to those skilled in the art. In some cases, these devices can be manufactured as a series of two or more separate parts and then mechanically clamped or permanently bonded together to form a complete device. In some cases, for example, a fluid channel (sometimes also referred to herein as a "microchannel") can be manufactured in a first layer (e.g., by photolithographic patterning of a glass substrate and wet chemical etching of the channel to a desired depth), and then sealed by bonding a second layer to the first layer, wherein a through hole in the second layer intersecting the fluid channel provides an external passage to the fluid channel. In some cases, the fluid channel can be manufactured in a first layer (e.g., by laser cutting a channel pattern in a suitable polymer film), and then sealed by sandwiching the first layer between a second layer and a third layer and bonding the first layer between the second layer and the third layer, wherein a through hole in the second layer and / or the third layer intersecting the fluid channel provides an external passage to the fluid channel. In the latter example, the thickness of the first layer defines the thickness (or depth) of the fluid channel.

[0107] Examples of suitable fabrication techniques include, but are not limited to, conventional machining, CNC machining, injection molding, 3D printing, alignment and lamination of one or more layers of laser or die-cut polymer films, or any of a number of micromachining techniques, such as photolithography and wet chemical etching, dry etching, deep reactive ion etching, or laser micromachining. In some embodiments, the microfluidic structure can be 3D printed from an elastomeric material.

[0108] The disclosed devices and microfluidic devices can be made using any of a variety of materials known to those skilled in the art. In general, the choice of material used depends on the choice of manufacturing technology, and vice versa. Examples of suitable materials include, but are not limited to, glass, quartz, fused quartz, silicon, any of a variety of polymers, such as polydimethylsiloxane (PDMS; elastomer), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene (PE), polyfluoroethylene, high-density polyethylene (HDPE), polyetheretherketone, polyimide, cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), polyetheretherketone (PEEK), epoxy resin, non-stick 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 in a device or microfluidic device comprising multiple layers can be made of different materials. In some cases, a given single layer in a device comprising one or more layers or a microfluidic device can be fabricated from two or more different materials.

[0109] In some cases, all or a portion of a device or microfluidic device can be optically transparent (e.g., transparent to ultraviolet (UV), visible light, 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 a separation channel is configured for imaging, e.g., full-channel imaging. For example, in some cases, a separation channel can be fabricated in a layer of optically opaque material that is sandwiched between two layers of optically transparent material, thereby forming an "optical slit" through which light can be transmitted and / or collected.

[0110] Typically, the size of the fluid channel, sample and / or reagent reservoir, etc. in the disclosed device will be optimized to (i) provide fast, accurate and reproducible separation to the sample or sample aliquot containing the analyte mixture and (ii) minimize the consumption of sample and reagent. Typically, the width of the fluid channel or reservoir can be between about 10 μm and about 2 mm. In some cases, the width of the fluid channel (or reservoir) can 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 the fluid channel (or reservoir) can 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 can be combined to form a range included in the present disclosure, for example, in some cases, the width of the fluid channel (or reservoir) can be in the range from about 100 μm to about 1 mm. Those skilled in the art will recognize that the width of the fluid channel (or reservoir) can have any value within this range, for example, about 80 μm.

[0111] Generally speaking, the depth of the fluid channel (or reservoir) will be between about 1 μm and about 1 mm. In some cases, the depth of the fluid channel (or reservoir) can 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 fluid channel (or reservoir) can 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 can be combined to form a range included in the present disclosure, for example, in some cases, the depth of the fluid channel (or reservoir) can be in the range from about 50 μm to about 100 μm. Those 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.

[0112] Cartridge: In some cases, the disclosed devices or systems can be configured to be coupled to one another or can be part of an integrated unit, such as a cartridge. The cartridge can include a microfluidic device, a substrate comprising a plurality of separation channels, a reservoir, reagents, a membrane, a valve, a fixture (e.g., those described herein, such as a high-voltage electrode fixture comprising a membrane), a fixture or feature (e.g., a screw, a pin (e.g., a pogo pin), an adhesive, a lever, a switch, a groove, a form-fitting pair, a hook and loop, a latch, a thread, a clip, a clamp, a pin, a ring, a rubber band, a rivet, a grommet, a tie, a snap, a tape, a vacuum, a seal), a gasket, an O-ring, an electrode, or a combination thereof. The cartridge can be integrally constructed or modular and include removable parts. For example, the microfluidic device can be configured to be removably coupled to the cartridge. Similarly, the reservoir, membrane, valve, etc. can all be removable from the cartridge. Where one or more components are removable, the cartridge can be configured so that each of the individual components can be aligned into place by the user with sufficient tolerance. For example, cartridge can include groove and pin so that microfluidic device can be integrated by making the device slide along cartridge until cartridge arrives for aligning pin.In some cases, the device can be configured to be flush with cartridge or its part and locate.In some cases, the device can be positioned in cartridge so that one or more inlets, outlets etc. can be connected (for example, fluidically and / or electrically) to reservoir, electrode, film and / or other useful interface unit.In some cases, the engagement of device and reservoir, electrode etc. can be performed by the user without any additional measurement or adjustment.For example, reservoir can be configured to receive electrode, and described electrode bites into place or fixes via spring pin, thus sets up electrical communication and / or fluid communication.It will be understood that these exemplary configurations of cartridge and device do not mean restrictive, and many different configurations of positioning microfluidic device or other parts of cartridge can be realized.In some cases, cartridge can be configured to the disposable component of system as herein described.

[0113] In preferred embodiments, the cartridge can include one or more reservoirs configured to hold a desired volume of fluid. In some cases, the reservoir can be capable of holding 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 of fluid. In some cases, the reservoir can be capable of holding a volume of at most about 5 mL, at most about 4.5 mL, at most about 4 mL, at most about 3.5 mL, at most about 3 mL, at most about 2.5 mL, at most about 2 mL, at most about 1.5 mL, at most about 1 mL, at most about 900 μL, at most about 800 μL, at most about 700 μL, at most about 600 μL, at most about 500 μL, at most about 400 μL, at most about 300 μL, or at most about 200 μL. Any of the lower and upper values ​​described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases, the reservoir can hold a fluid volume that can be in the range of about 200 μL to about 2 mL. Those skilled in the art will recognize that the reservoir fluid volume capacity can have any value within this range, for example, about 1.8 mL.

[0114] In some embodiments, one or more kits can be provided, which can include a cartridge, one or more reagents, and in some cases, instructions for using the kit. The reagents can be stored in a reservoir as a liquid. In some embodiments, the reagents can be dry, such as lyophilized, and can be reconstituted in a solution or buffer. In some cases, the reagents can be separated from the cartridge and can be provided in the kit.

[0115] In some cases, reservoir can be controllably coupled (for example, electrically, fluidically) to microfluidic device.For example, cartridge can include one or more valves, which can be used for flow or the flow velocity in the control device. In some cases, cartridge can include a stopcock or shear valve (for example, a slide valve or a rotary shear valve), which can allow for the controlled flow velocity during the delivery of one or more liquid reagents (for example, mobilization reagent). In some cases, cartridge can be integrated or engaged with a syringe pump, which can be used for the flow velocity in the control liquid inflow device. In some cases, piston, spring-loaded device or other mechanical methods can be used to control flow velocity.

[0116] In some cases, the cartridge can be configured to accommodate different types or models of devices. For example, the cartridge can 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 devices. In some cases, the cartridge can include a port or connector that can engage with a channel of the chip (e.g., engage with an inlet and / or outlet of the chip).

[0117] Separation and enrichment of analytes: In some cases, the disclosed devices or systems can be configured to perform one or more separation or enrichment steps, wherein multiple analytes in a mixture are separated and / or concentrated in individual fractions. For example, in some cases, the disclosed devices can be configured to perform a first enrichment step, wherein the analyte mixture in the sample is separated into and / or enriched into analyte fractions (e.g., analyte peaks or analyte bands) that contain a subset of analyte molecules from the original sample. In some cases, these separated analyte fractions can be mobilized and / or eluted, and in some cases, can then be subjected to another downstream separation and / or enrichment step. In some cases, for example, after the final separation and / or enrichment step, the separated / enriched analyte fractions can be expelled from the device for further analysis.

[0118] In some cases, the disclosed devices and systems can be configured to perform one, two, three, four, or five or more separation and / or enrichment steps. In some cases, one or more of the separation or enrichment steps can include solid phase separation techniques, e.g., reverse phase HPLC. In some cases, one or more of the separation or enrichment steps can include solution phase separation and / or enrichment techniques, e.g., capillary zone electrophoresis (CZE) or isoelectric focusing (IEF).

[0119] The disclosed devices and systems can be configured to perform any of a variety of analyte separation and / or enrichment techniques known to those skilled in the art, wherein one or more separation or enrichment steps are performed in at least a first separation channel, which is configured to be imaged in whole or in part so that the separation process can be monitored while the separation process is being performed. For example, in some cases, the imaged separation can be an electrophoretic separation, which includes, for example, isoelectric focusing, capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, electric field gradient focusing, dynamic field gradient focusing, and the like, which produces one or more separated analyte fractions from an analyte mixture. In some cases, the separation and mobilization steps can be performed in at least a first separation channel, which is configured to be imaged in whole or in part so that the separation and mobilization process can be monitored while the separation and mobilization process is being performed. In any of these cases, all or part of the imaging of the separation channel can be performed continuously or intermittently and can be performed before, during, or after the separation and / or enrichment process.

[0120] In some cases, the use of microfluidic devices can provide for rapid separation times and accurate and reproducible separation data. For example, where the 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 one to use high electric field strengths without incurring significant Joule heating, thereby enabling very rapid separation reactions without substantial dispersion and loss of separation resolution. In some cases, precise control of the fluidic channel geometry provides for precise and reproducible control over sample injection volume, electric field strength, etc., thereby enabling very precise determination of one or more parameters of the assay, such as separation resolution and / or pI determination.

[0121] The one or more parameters of the assay may include characteristics of the separation. For example, the one or more parameters may be selected from the group consisting of separation resolution, peak width, peak capacity, linearity of the pH gradient, and minimum resolvable pi difference.

[0122] In some cases, the separation time of the device and system can be in the range of about 0.1 minute to about 30 minutes. In some cases, the separation time can be at least 0.1 minute, at least 0.5 minute, 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, the separation time can be at most 30 minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, at most 10 minutes, at most 5 minutes, at most 1 minute, at most 0.5 minute or at most 0.1 minute. Any one of the lower limit value and the upper limit value described in this paragraph can be combined to form the scope included in the present disclosure, and in some cases, the separation time can be in the range of about 1 minute to about 20 minutes. One skilled in the art will recognize that the separation time can have any value within this range, for example, about 11.2 minutes.

[0123] Similarly, the separation efficiency and resolution achieved using the disclosed devices and systems can vary depending on the specific separation technology and operating parameters utilized (e.g., separation channel length, microfluidic device design, buffer composition, applied voltage, etc.) and whether one or two dimensions of separation are utilized. In some cases, for example, when performing isoelectric focusing, the use of switchable electrodes to trigger the electrophoretic introduction of mobilized 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 for analyte bands having a pI difference in the range of about 0.1 to about 0.0001 pH units. In some cases, the IEF separation resolution can allow resolution for analyte bands having a pI difference of 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.

[0124] Thus, in some cases, for example, when imaging of all or a portion of a separation channel is used to identify the position of a pI marker in an isoelectric focusing reaction and determine the pI value of the analyte for separation, the pI value can be determined to an accuracy of 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 greater than ±0.0001 pH units.

[0125] In some cases, the peak capacity achieved using the disclosed devices can be in the range of about 100 to about 20,000. In some cases, the peak capacity can 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 can 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 values ​​described in this paragraph can be combined to form ranges encompassed by the present disclosure, for example, in some cases, the peak capacity can be in the range of about 400 to about 2,000. One skilled in the art will recognize that the peak capacity can have any value within this range, for example, about 285.

[0126] Capillary Isoelectric Focusing (CIEF): In some embodiments, the separation technique may include isoelectric focusing (IEF), for example, capillary isoelectric focusing (CIEF). Isoelectric focusing (or "electric focusing") is a technique for separating molecules by differences in their isoelectric points (pi), i.e., the pH at which the molecule has zero net charge. CIEF involves adding an ampholyte (ampholyte) solution to a sample channel between reagent reservoirs containing an anode or cathode to create a pH gradient within a separation channel (i.e., a fluid channel connecting recesses containing electrodes, such as the lumen of a capillary or a channel in a microfluidic device), across which a separation voltage is applied. The ampholytes may be in solution or immobilized on the surface of the channel wall. Negatively charged molecules migrate toward the positive electrode due to the pH gradient in the medium, while positively charged molecules migrate toward the negative electrode. Proteins (or other molecules) in a pH range below their isoelectric points (pi) will be positively charged and, therefore, will migrate toward the cathode (i.e., the negatively charged electrode). The total net charge of a protein will decrease as it migrates through a gradient of increasing pH (e.g., due to protonation of carboxyl groups or other negatively charged functional groups) until the protein reaches the pH region corresponding to its pI, at which point it has no net charge and therefore migration stops. As a result, the protein mixture separates according to the relative amounts of its acidic and basic residues and becomes focused into sharp, fixed bands, with each protein located at a point in the pH gradient corresponding to its pI. This technique is capable of achieving extremely high resolution, fractionating proteins that differ by a single charge into separate bands. In some embodiments, isoelectric focusing can be performed in a separation channel that has been permanently or dynamically coated (e.g., with a neutral and hydrophilic polymer coating) to eliminate electroosmotic flow (EOF). Examples of suitable coatings include, but are not limited to, amino modifiers, hydroxypropyl cellulose (HPC), and polyvinyl alcohol (PVA), (Alco Bioseparation), linear polyacrylamide, polyacrylamide, dimethylacrylamide, polyvinylpyrrolidine (PVP), methylcellulose, hydroxyethylcellulose (HEC), hydroxypropyl methylcellulose (HPMC), triethylamine, propylamine, morpholine, diethanolamine, triethanolamine, diaminopropane, ethylenediamine, chitosan, polyethyleneimine, cadaverine, putrescine, spermidine, diethylenetriamine, tetraethylenepentamine, cellulose, dextran, polyethylene oxide (PEO), Cellulose acetate, pullulan, ethylpyrrolidino methacrylate, dimethyl methacrylate, behenyldimethylammonium bromide, Brij35, sulfobetaine, 1,2-dilauroyl n-phosphatidylcholine, 1,4-didecyl-1,4-diazobicyclo[2,2,2]octane dibromide, agarose, poly(N-hydroxyethylacrylamide), pole-323, hyperbranched polyurethane, pullulan, glycerol, adsorption coating, covalent coating, dynamic coating, etc. In some embodiments, isoelectric focusing can be performed (e.g., in an uncoated separation channel) using additives such as methylcellulose, glycerol, urea, formamide, surfactants (e.g., Triton-X100, CHAPS, digitonin) in the separation medium to significantly reduce electroosmotic flow by increasing the viscosity of the electrolyte, allowing better protein solubilization and limiting diffusion within the capillary (e.g., in the lumen of the capillary) or within the fluid channel.

[0127] As described above, the pH gradient used in capillary isoelectric focusing techniques is generated by using ampholytes, that is, ampholytes contain both acidic and basic groups and exist primarily as zwitterions within a certain pH range. The portion of the electrolyte solution on the anode side of the separation channel is referred to as the "anolyte." The portion of the electrolyte solution on the cathode side of the separation channel is referred to as the "catholate." A variety of electrolytes can be used in the disclosed methods and apparatus, 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. The electrolyte can be used in any suitable concentration, for example, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. The concentration of the electrolyte can be at least 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. The concentration of the electrolyte can be up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%. A range of electrolyte concentrations can be used, for example, 0.1% to 2%. The ampholytes can be selected from any commercial or non-commercial carrier ampholyte mixture (e.g., Servalyt pH 4-9 (Serva, Heidelberg, Germany), Beckman pH 3-10 (Beckman Instruments, Fullerton, CA, USA), Ampholine 3.5-9.5 and Pharmalyte 3-10 (both from GE Healthcare, Orsay, France), AESlytes (AES), FLUKA ampholytes (Thomas Scientific, Swedesboro, NJ, USA), Biolyte (Bio-Rad, Hercules, CA, USA)), and the like. The carrier ampholyte mixture can include a mixture of small molecules (about 300 Da to 1000 Da) containing multiple aliphatic amino and carboxylic acid groups with closely spaced pI values ​​and good buffering capacity. In the presence of an applied electric field, the carrier ampholytes separate into a smooth linear or non-linear pH gradient that gradually increases from the anode to the cathode.

[0128] In disclosed method and apparatus, can use any one in multiple pI standards to be used for calculating the isoelectric point of the analyte peak for separation.For example, can use the pI mark commonly used in CIEF application, for example, protein pI mark and synthetic small molecule pI mark.In some cases, protein pI mark can be the specific protein with generally accepted pI value.In some cases, pI mark can be can for example via imaging detection.Can use commercially available protein pI mark or synthetic small molecule pI mark multiple or combination, for example, can from Advanced Electrophoresis Solutions Co., Ltd. (Ontario, Canada, Cambridge), ProteinSimple, by the peptide library of Shimura design and the small molecule pI mark that Slais dyestuff (Alco Bioseparation) obtain.

[0129] Capillary zone electrophoresis (CZE): In some cases, the separation or enrichment technique may include capillary zone electrophoresis, a method for separating charged analytes in solution in an applied electric field. The net velocity of the charged analyte molecules is influenced by both the electroosmotic flow (EOF) μEOF exhibited by the separation system and the electrophoretic mobility μEP for each analyte (which depends on the size, shape, and charge of the molecule), so that analyte molecules exhibiting different sizes, shapes, or charges exhibit different migration velocities and separate into bands. Compared to other capillary electrophoresis methods, CZE uses a "simple" buffer or background electrolyte (the solution used for the separation).

[0130] Capillary gel electrophoresis (CGE): In some cases, the separation or enrichment technique may include capillary gel electrophoresis, a method for separating and analyzing macromolecules (e.g., DNA, RNA, and proteins) and their fragments based on size and charge. The method involves the use of a separation channel filled with a gel, wherein the gel acts as an anticonvective and / or sieving medium during the electrophoretic movement of the charged analyte molecules in an applied electric field. The gel functions to inhibit thermal convection induced by the applied electric field while also acting as a sieving medium that impedes the passage of molecules, thereby causing molecules of different size or charge to have different migration rates.

[0131] Capillary Isotachophoresis (CITP): In some cases, the separation technique may include capillary isotachophoresis, which is a method for separating charged analytes that uses a discontinuous system of two electrolytes (called a leading electrolyte and a terminating electrolyte) within a capillary or fluid channel of suitable size. The leading electrolyte contains ions with the highest electrophoretic mobility, while the terminating electrolyte contains ions with the lowest electrophoretic mobility. The analyte mixture to be separated (i.e., the sample) is sandwiched between the two electrolytes, and an applied electric field causes the charged analyte molecules within the capillary or fluid channel to separate into closely adjacent zones so as to reduce electrophoretic mobility. These zones move at a constant velocity in the applied electric field, making it possible to record their passage along the separation channel using a detector such as a conductivity detector, a photodetector, or an imaging device. Unlike capillary zone electrophoresis, it is not feasible to simultaneously determine or detect anionic and cationic analytes in a single analysis performed using capillary isotachophoresis.

[0132] Capillary Electrokinetic Chromatography (CEC): In some cases, the separation technique may include capillary electrokinetic chromatography, which is a method for separating analyte mixtures based on a combination of liquid chromatography 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 filled with HPLC packing materials, the wide variety of analyte selectivities available in HPLC are also available in CEC. The high surface area of ​​these packing materials enables CEC capillaries to accommodate relatively large amounts of sample, making the detection of subsequently eluted analytes a slightly simpler task than it is in capillary zone electrophoresis (CZE).

[0133] Micellar Electrokinetic Chromatography (MEKC): In some cases, the separation technique may include capillary electrokinetic chromatography, a method for separating analyte mixtures based on the differential partitioning between surfactant micelles (a pseudo-stationary phase) and a surrounding aqueous buffer solution (a mobile phase). The basic setup and detection methods used for MEKC are the same as those used in CZE. The difference is that the buffer solution contains a surfactant concentration above the critical micelle concentration (CMC), so that the surfactant monomers are in equilibrium with the micelles. MEKC is typically performed using alkaline conditions in an open capillary or fluid channel to generate a strong electroosmotic flow. Sodium dodecyl sulfate (SDS) is an example of a surfactant commonly used in MEKC applications. The anionic nature of the sulfate groups of SDS causes the surfactant and micelles to have electrophoretic mobilities that are opposite to the direction of the strong electroosmotic flow. As a result, the migration of the surfactant monomers and micelles is very slow, although their net movement is still in the direction of the electroosmotic flow, i.e., toward the cathode. During MEKC separation, the analyte itself is distributed between the hydrophobic interior of the micelles and the hydrophilic buffer solution. The hydrophilic analyte insoluble in the micelle flows at an electroosmotic flow rate u Oo Migrate and will be retained in the buffer at time t M The hydrophobic analyte completely dissolved in the micelle moves at the micelle velocity u C Migrate, and at the final elution time t C Wash off.

[0134] Flow Equilibrium Capillary Electrophoresis (FCCE): In some cases, the separation technique may include flow equilibrium capillary electrophoresis, a method for improving the efficiency and resolving power of capillary electrophoresis that utilizes pressure-induced countercurrent to actively delay, halt, or reverse the electrokinetic migration of analytes through the capillary. By delaying, halting, or shuddering the analytes across the detection window, the analytes of interest are efficiently confined in the separation channel for significantly longer periods than under normal separation conditions, thereby improving both the efficiency and resolving power of the separation.

[0135] Chromatography: In some cases, separation techniques can include chromatography, in which a mixture of analytes in a sample fluid (mobile phase) is passed through a column or channel packing material (stationary phase) that differentially retains the various components of the mixture, causing them to travel and separate at different rates. In some cases, a subsequent elution or mobilization step 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 reversed-phase chromatography.

[0136] Mobilization of the analyte class of separation: in some cases, provided herein is a device and system configured to perform, for example, chromatographic separation techniques (such as reversed-phase chromatography). The method implemented by the device or system can also include eluting the analyte class on the stationary phase retained in each of a plurality of separation channels (for example, by simultaneously or independently changing the buffer solution flowing through each of a plurality of separation channels), which can be referred to as "mobilization" step or reaction. In some cases, the method implemented by the device or system can also include simultaneously or independently applying pressure to each of a plurality of separation channels, or simultaneously or independently introducing electrolyte into each of a plurality of separation channels to destroy the pH gradient for isoelectric focusing, and thereby triggering the analyte peak separated to migrate out of separation channel, which can also be referred to as "mobilization" step. In some cases, the force for driving the separation reaction (for example, the pressure for reversed-phase chromatography, or the electric field for electrokinetic separation or isoelectric focusing reactions) can be cut off during the mobilization step. In some cases, the force for driving the separation reaction can be left during the mobilization step. In some examples of the disclosed methods (e.g., those that include an isoelectric focusing step), the separated analyte band can be mobilized (e.g., using hydrodynamic pressure and / or chemical mobilization techniques) such that the separated analyte band migrates toward an end of each of a plurality of separation channels connected to another fluidic channel (which can be, for example, an outlet, a waste reservoir, or a second separation channel). In some cases, for example, in those examples employing capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, or any other separation technique that separates components of an analyte mixture by differential velocity, the separation step itself can be considered a mobilization step.

[0137] In some cases, the mobilization of the analyte band can be achieved by applying fluid dynamic pressure to the end of each of the multiple separation channels simultaneously or independently. In some cases, the mobilization of the analyte band can be achieved by orienting the device so that the multiple separation channels are in a vertical position, so that gravity can be used. In some cases, the mobilization of the analyte band can be achieved using EOF-assisted mobilization. In some cases, the mobilization of the analyte band can be achieved using chemical mobilization, for example, by simultaneously or independently introducing mobilization electrolytes into each of the multiple separation channels, which change the local pH in the pH gradient used for isoelectric focusing. In some cases, any combination of these mobilization techniques can be used.

[0138] In a preferred example, the mobilization step for the analyte band of isoelectric focusing comprises chemical mobilization.Compared with the mobilization based on pressure, chemical mobilization has the following advantages, that is, by overcoming the hydrodynamic parabolic flow distribution caused by using pressure and showing minimum band broadening.Chemical mobilization can be achieved by electrolyte (that is, " mobilization electrolyte ") being introduced into separation channel to change the local pH value and / or net charge on the analyte band (or zwitterion buffer component) of separation, so that analyte band (or zwitterion buffer component and associated hydration shell) migrates in the applied electric field.In some cases, the polarity of the applied electric field for making the analyte band of separation active can make analyte migrate towards the anode that is electrically connected with the outlet of separation channel or distal end (anode mobilization).In some cases, the polarity of the applied electric field for making the analyte band of separation active can make analyte migrate towards the cathode that is electrically connected with the outlet of separation channel or distal end (cathode mobilization). The mobilization electrolyte comprises anions or cations that compete with hydroxyl groups (cathode mobilization) or hydronium ions (anode mobilization) for introduction into the separation channel or capillary. Examples of bases that can be used as cathode liquids for anode mobilization include, but are not limited to, sodium hydroxide, ammonium hydroxide ("ammonia"), diethylamine, dimethylamine, piperidine, and the like. Examples of acids that can be used as anode liquids in cathode mobilization include, but are not limited to, phosphoric acid, acetic acid, formic acid, and carbonic acid, and the like. In some cases, mobilization can be initiated by adding salt (e.g., sodium chloride) to the anode liquid or cathode liquid. In some cases, the anode can remain grounded, and a negative voltage is applied to the cathode. In some cases, the cathode can remain grounded, and a positive voltage is applied to the anode. In some cases, a non-zero negative voltage can be applied to the cathode, and a non-zero positive voltage can be applied to the anode. In some cases, a non-zero positive voltage can be applied to both the anode and the cathode. In some cases, a non-zero negative voltage can be applied to both the anode and the cathode.

[0139] In some cases, mobilization of the separated analyte bands can be initiated at a user-specified time point by triggering a switchable electrode (e.g., a cathode electrically connected to the distal end of each of a plurality of separation channels and a cathode electrically connected to the proximal end of each of a plurality of mobilization channels (e.g., a fluid channel intersecting the separation channel near the outlet or distal end of each separation channel)) between an on state and a closed state to control the electrophoretic introduction of a mobilization buffer or electrolyte into the separation channel.

[0140] In some cases, the time that is used for triggering the user of the conversion of one, two or three or more switchable electrodes independently between the open state and the closed state for each in a plurality of separation channels can be in the scope of from about 30 seconds to about 30 minutes for any activation plan.In some cases, the time that the user specifies can 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 time that the user specifies can be at most 30 minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, at most 10 minutes, at most 5 minutes, at most 4 minutes, at most 3 minutes, at most 2 minutes, at most 1 minute or at most 30 seconds.Any one in the lower limit value described in this paragraph and the upper limit value can be combined to form the scope that is included in the present disclosure, and for example, in some cases, the time that the user specifies can be in the scope of from about 2 minutes to about 25 minutes. Those skilled in the art will recognize that the user-specified time can have any value within this range, for example, approximately 8.5 minutes.

[0141] In some cases, the electric field used to achieve mobilization in any mobilization scheme disclosed herein (or to perform electrokinetic separation or isoelectric focusing reactions in those cases where such separation techniques are performed) can be in the range of from about 0 V / cm to about 1000 V / cm. In some cases, the electric field strength can 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 1000 V / cm. In some cases, the electric field strength can be at most 1000 V / cm, at most 900 V / cm, at most 800 V / cm, at most 700 V / cm, at most 600 V / cm, at most 500 V / cm, at most 450 V / cm, at most 400 V / cm, at most 350 V / cm, at most 300 V / cm, at most 250 V / cm, at most 200 V / cm, at most 150 V / cm, at most 100 V / cm, at most 80 V / cm, at most 60 V / cm, at most 40 V / cm, at most 20 V / cm, or at most 0 V / cm. Any of the lower and upper values ​​described in this paragraph can be combined to form ranges within the present disclosure, for example, in some cases, the electric field strength time can be in the 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, approximately 575 V / cm.

[0142] In some cases, mobilization of the separated analyte bands can be initiated based on data derived from independently monitoring the current (or conductivity) for each of a plurality of separation channels, where, for example, in the case of isoelectric focusing, the current through the separation channel can reach a minimum value. In some cases, detection of a minimum current value, or detection of a current value that remains constant or below a specified threshold for a specified period of time, can be used to determine whether the isoelectric focusing reaction has been completed and, thereby, can be used to trigger the initiation of a chemical mobilization step.

[0143] In some cases, the minimum or threshold current value can be in the range of from about 0 μA to about 100 μA. In some cases, the minimum or threshold current value can 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 current value or threshold current value can 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 can be combined to form a range included in the present disclosure, for example, in some cases, the minimum current value or threshold current value can be in the range from about 10 μA to about 90 μA. Those skilled in the art will recognize that the minimum current value or threshold current value can have any value within this range, for example, about 16 μA.

[0144] In some cases, the time period of appointment can 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 time period of appointment can be at most about 60 seconds, at most about 55 seconds, at most about 50 seconds, at most about 45 seconds, at most about 40 seconds, at most about 35 seconds, at most about 30 seconds, at most about 25 seconds, at most about 20 seconds, at most about 15 seconds, at most about 10 seconds or at most about 5 seconds.Any one in lower value described herein and upper value can be combined to form the scope that is included in the disclosure, for example, in some cases, the time period of appointment can be in the scope from about 5 seconds to about 30 seconds.Those skilled in the art will recognize that the time period of appointment can have any value within this scope, for example, about 32 seconds.

[0145] In some cases, the mobilization of the analyte band of separation can be initiated based on the data obtained from the image of multiple separation channels (for example, by performing automatic image processing) when performing separation reaction.Image-derived data can 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 speed of one or more analyte peaks, separation resolution, the presence of one or more analyte peaks, the rate of change of position, width or speed or its lack or any combination thereof, and can be used to determine whether the separation reaction is completed and / or trigger the initiation of the activation step in a given separation channel.In some cases, the completion of the separation step can be determined by monitoring the rate of change of separation performance parameters (for example, peak position or peak width) over a period of time (for example, in a time period of 10 seconds to 60 seconds).

[0146] In a preferred aspect of the disclosed method, the chemical activation step can be initiated in a microfluidic device, and the microfluidic device is designed to integrate CIEF with ESI-MS by changing the electric field in the device to electrophoresing the activated electrolyte into the separation channel. In some cases, the initiation of the activation step can be triggered based on the data obtained from the image of all or part of the separation channel. In some cases, the change of the electric field can be achieved by connecting or disconnecting one or more electrodes attached to one or more power supplies, wherein the one or more electrodes are positioned in the reagent recess on the device or integrated with the fluid channel of the device. In some cases, the connection or disconnection of one or more electrodes can be controlled using a method implemented by a computer and a programmable switch so that the timing and duration of the activation step can be coordinated with the separation step. In some cases, changing the electric field in the device can be used to make the activation buffer flow into the separation channel comprising the stationary phase in the form of electrophoresis or electroosmosis, so that the retained analyte is released from the stationary phase.

[0147] In some cases, three or more electrodes for each separation channel can be connected to or integrated into the device. For example, a first electrode can be electrically coupled to the proximal end of the separation channel. Similarly, a second electrode can then be coupled to the distal end of the separation channel, and a third electrode can be coupled to an activation channel, which, for example, intersects with the separation channel at its distal end and is connected to or includes a reservoir containing an activation buffer. When the separation step as determined by an image-based method is completed, the electrical coupling of the second or third electrode and its corresponding channel can be switchable between an "on" state and an "off" state. In one such example, a second electrode forming the anode or cathode of the separation circuit can be switched to an "off" mode, and a third electrode that is closed during separation can be switched to an "on" mode to initiate the introduction of the activation buffer into the channel (e.g., via electrophoresis). In some cases, the "on" state and the "off" state can respectively include the complete connection or disconnection of the electrical coupling between the electrode and the fluid channel. In some cases, the "on" state and the "off" state may include clamping the current through a given electrode to non-zero amps or zero microamps, respectively.

[0148] In some cases, triggering or initiating the activation step can include detecting no change or a change less than 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 position, peak width, peak velocity, etc.) can be used to trigger the activation step.

[0149] In some cases, triggering or initiating the activation step can include detecting no change or a rate of change less than 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 percentage changes and time periods) in one or more image-derived parameters (e.g., peak position, peak width, peak velocity, etc.) over a 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 can be used to trigger the activation step.

[0150] In some cases, a calibrator can be used to associate and / or calibrate information from a mass spectrometer during the mobilization step. In some cases, the calibrator can include a peptide, polypeptide, protein or other molecule (natural or synthetic) with a known mass. In some cases, the calibrator will be mixed with a mobilizing agent solution. The calibrator can be used to calibrate a mass spectrometer. In some cases, the calibrator can be used to associate information from a mass spectrometer to an mobilization process or a separation process. For example, the calibrator can be monitored during separation (e.g., isoelectric focusing) or mobilization.

[0151] In some embodiments, the ESI ion source on the mass spectrometer will have an adjustable power supply that can set a negative voltage on the mass spectrometer. In some embodiments, the ESI ion source on the mass spectrometer will have an adjustable power supply that can set a positive voltage on the mass spectrometer. In some embodiments, the ESI ion source on the mass spectrometer will remain grounded. In some embodiments, the ESI tip on the capillary or microfluidic device will remain grounded or close to grounded, to produce an electric field between the charged ESI ion source on the ESI tip and the mass spectrometer. In some embodiments, the ESI tip on the capillary or microfluidic device will remain under positive or negative voltage to produce an electric field between the ESI ion source of the ground on the ESI tip and the mass spectrometer.

[0152] Figure 36 An exemplary flow chart of a computer-controlled feedback loop is provided to maintain a constant voltage drop of 3000 V between the anode and cathode while holding the ESI tip voltage at 0 V during activation. In some embodiments, this feedback loop can be implemented when the mass spectrometer ESI ion source is set to a positive or negative voltage relative to ground (e.g., -3500 V). In this example, Figure 1B In the example, the ΔV between the anolyte port 108 and the mobilizer port 104 is maintained at 3000V by initially setting the anolyte port 108 to +3000V and the mobilizer port 104 to 0V. In some embodiments, different ΔVs can be set by setting the anolyte port 108 to different values. In some embodiments, anodic mobilization can be used and port 108 will be the catholyte port, which is set to, for example, -3000V. Figure 36 In the example outlined in , during mobilization, the resistance in the separation channel 112 decreases as the separating analytes and ampholytes regain charge. This causes the voltage drop across the channel 112 to decrease, thereby causing the voltage at the ESI tip 116 to increase, according to Equation 1:

[0153] V 116 =(ΔV 108-104 )*(R 105 ) / (R 109 +R 112 +R 105 )

[0154] However, by measuring or calculating the ESI tip voltage 116, the voltage settings at the anolyte port 108 and the mobilizer port 104 can be adjusted. By subtracting the ESI tip voltage 116 from both the anolyte port 108 and the mobilizer port 104 settings, ΔV 110-104It remains at 3000 V so activation is not affected, but the ESI tip 116 voltage is set to 0, according to Equation 2:

[0155] V 116 =(ΔV 108-104 )*(R 105 ) / (R 109 +R 112 +R 105 )+V 104

[0156] This feedback loop continues to operate until mobilization is complete, adjusting the ESI tip 116 voltage to zero at a regular frequency (e.g., the Nyquist rate or approximately 0.2 Hz). In some cases, the voltage at the ESI tip 116 may be adjusted to zero at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz, 1000 Hz. Maintaining a constant, stable voltage at the ESI tip 116 may be crucial for maintaining a stable electrospray during the mobilization process.

[0157] In some cases, the feedback loop operates to maintain the voltage at the ESI tip within a specified percentage of a preset value. For example, in some cases, the feedback loop operates to maintain the voltage at the ESI tip within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the preset value.

[0158] In some embodiments, the mass spectrometer ESI ion source is maintained at ground, and the ESI tip 116 will need to be maintained at a constant positive or negative voltage to generate an electric field between the ESI tip 116 and the mass spectrometer. In some embodiments, the ESI tip voltage (e.g., a preset value) can be about +5000 V, about +4000 V, about +3500 V, about +3000 V, about +2500 V, about +2000 V, about +1500 V, about +1000 V, about +500 V, or about -5000 V, about -4000 V, about -3500 V, about -3000 V, about -2500 V, about -2000 V, about -1500 V, about -1000 V, or about -500 V. Figure 37 An example flow chart of a computer controlled feedback loop is provided to maintain a constant voltage drop of 3000 V between the anode and cathode while holding the ESI tip voltage potential at 3000 V during activation. The operation of the computer controlled feedback loop is similar to Figure 36The same as in . Except that the voltage at anolyte port 108 and mobilizing agent port 104 is offset by +3000V, this will offset the voltage at ESI tip 116 to +3000V, still following equation 2. In certain embodiments, analog circuits can be used to realize the control of electric field intensity. In certain embodiments, the voltage at one or more electrodes contacted with a separation system based on a capillary or based on a microfluidic device can be controlled by using one, two, three or four or more independent high voltage power supplies. In some cases, the voltage at one or more electrodes contacted with a separation system based on a capillary or based on a microfluidic device can be controlled, for example, by using a single multiplexed high voltage power supply.

[0159] In some cases, the feedback loop operates to maintain the electric field strength within the separation channel or the voltage drop between the anode and cathode within a specified percentage of a preset value. For example, in some cases, the feedback loop operates to maintain the electric field strength within the separation channel or the voltage drop between the anode and cathode within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01% of a preset value. In some cases, the feedback loop operates to maintain the ESI tip voltage within 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V of a preset value.

[0160] In some embodiments, an alternating current (AC) signal generator such as a lock-in amplifier, a function generator, an oscillator, or other AC signal generator may be electrically coupled to a pair of electrodes. In some embodiments, the AC signal generator may be an SR8-30 (Stanford Research Systems) lock-in amplifier. In some embodiments, a plurality of AC signal generators may be electrically coupled to a plurality of pairs of electrodes. In some embodiments, the AC signal generator may be configured to superimpose an AC voltage on a DC voltage disposed between two electrodes. In some embodiments, the AC current generated by the AC signal generator may be measured. In some embodiments, the AC current generated by the AC signal generator may be used to calculate the resistance in the microfluidic channel. In some embodiments, the resistance of the microfluidic channel may vary over time. In some embodiments, the resistance of the microfluidic channel may vary over time due to isoelectric focusing. In some embodiments, the resistance of the microfluidic channel may vary over time due to chemical activation. In some embodiments, the resistance of the microfluidic channel may vary over time due to the introduction of a new reagent into the channel network between a pair of electrodes. In some embodiments, the AC signal generator may be connected to an electrode electrically connected to the distal end of the separation channel and an electrode electrically connected to the proximal end of the same separation channel. In some embodiments, the resistance change in the microfluidic channel may be measured. In some embodiments, the resistance change measured in the microfluidic channel can be used to maintain a constant voltage potential within the fluid network. In some embodiments, the frequency of the generated AC signal can be at least 0.05 Hz, at least 0.1 Hz, at least 0.5 Hz, at least 1 Hz, at least 5 Hz, at least 10 Hz, at least 50 Hz, at least 100 Hz, at least 500 Hz, at least 1000 Hz, at least 5 kHz, at least 10 kHz, at least 50 kHz, or at least 100 kHz. In some embodiments, the frequency of the lock-in amplifier AC voltage can be at most 0.05 Hz, at most 0.1 Hz, at most 0.5 Hz, at most 1 Hz, at most 5 Hz, at most 10 Hz, at most 50 Hz, at most 100 Hz, at most 500 Hz, at most 1 kHz, at most 5 kHz, at most 10 kHz, at most 50 kHz, or at most 100 kHz.

[0161] In some embodiments, the voltage of the generated AC signal can be at least 0.1 V, at least 0.5 V, at least 1 V, at least 5 V, at least 10 V, at least 20 V, at least 50 V, at least 100 V, at least 500 V, at least 1000 V, at least 5 kV, or at least 10 kV. In some embodiments, the voltage of the lock-in amplifier signal can be at most 0.1 V, at most 0.5 V, at most 1 V, at most 5 V, at most 10 V, at most 50 V, at most 100 V, at most 500 V, at most 1000 V, at most 5 kV, or at most 10 kV.

[0162] Imaging of separation channels: In some cases, the disclosed devices and systems can be configured to perform imaging on all or a portion of at least one separation channel to monitor the separation and / or mobilization reaction while performing the separation and / or mobilization reaction. In some cases, the disclosed devices and systems can be configured to perform imaging on all or a portion of multiple separation channels to monitor multiple separation and / or mobilization reactions while performing multiple separation and / or mobilization reactions. In some cases, any one of a variety of imaging techniques known to those skilled in the art can be used to image the separation and / or mobilization reaction. Examples include, but are not limited to, ultraviolet (UV) light absorption, visible light absorption, fluorescence (e.g., natural fluorescence or fluorescence caused by one or more analytes labeled with fluorophores), Fourier transform infrared spectroscopy, Fourier transform near infrared spectroscopy, Raman spectroscopy, optical spectroscopy, and the like. In some cases, multiple separation (or enrichment) channels can be the lumens of multiple capillaries. In some cases, multiple separation (or enrichment) channels can be multiple fluid channels within a microfluidic device. In some cases, a portion or all of a separation (or enrichment) channel, a joint or connecting channel, an electrospray hole or a tip connecting the end of a separation channel to a downstream analytical instrument or an electrospray hole or a tip, an electrospray hole or a tip itself, or any combination thereof, can be imaged. In some cases, a separation (or enrichment) channel can be a lumen of a capillary. In some cases, a separation (or enrichment) channel can be a fluid channel in a microfluidic device.

[0163] One or more wavelength ranges for imaging and detecting the analyte bands separated will typically depend on the selection of imaging technology and one or more materials of manufacturing device or its part. For example, in the case where UV light absorbance is used to image all or a portion of separation channel or other parts of microfluidic device, detection under about 220nm (due to the natural absorbance of peptide bonds) and / or about 280nm (due to the natural absorbance of aromatic amino acid residues) can allow people to observe protein bands during separation and / or mobilization, provided that at least a portion of the device (for example, separation channel or a portion thereof) is transparent to light at these wavelengths. In some cases, analyte to be separated can be identified by, for example, fluorophores, chromophores, chemiluminescent tags or other suitable markers before separation so that they can be imaged using fluorescence imaging, UV absorbance imaging or other suitable imaging techniques. In some cases, for example, where analyte includes a protein produced by a commercial manufacturing process, protein can be genetically modified to incorporate green fluorescent protein (GFP) domains or its variants so that they can be imaged using fluorescence. In some cases, labeling of proteins or other analyte molecules may be performed using a method that ensures that the label itself does not interfere with or perturb the characteristics of the analyte upon which the selected separation technique is based.

[0164] In some cases, imaging (or data derived therefrom) can be used to trigger, for example, an activation step or the transfer of an analyte fraction or a portion thereof from a first plurality of separation channels to another plurality of separation channels or from a first plurality of separation channels to a plurality of channels in fluid communication with the outlet end of the first plurality of separation channels. For example, in some cases, the disclosed method can include injecting an analyte mixture into a microfluidic device comprising a first plurality of separation channels and a second plurality of separation channels. The first plurality of separation channels can include a medium configured to bind an analyte from a sample analyte mixture. Therefore, when the sample analyte mixture is loaded or injected into a device such as a microfluidic device, at least one fraction of the analyte in each sample analyte mixture can be bound to a matrix and / or blocked from flowing through the first plurality of separation channels. For example, injecting an analyte mixture into a microfluidic device can achieve chromatographic separation in the first plurality of separation channels. Then, an eluent can be injected into the microfluidic device so that at least one fraction of the analyte (if present) is activated from the medium in each separation channel. In some cases, the first plurality of separation channels can be imaged while the analyte is activated. In some cases, the imaging of the first multiple separation reactions can include full column (for example, full channel) imaging and / or imaging a part for separation channel. In some cases, when imaging detects that the analyte fraction is arranged at the intersection of the first multiple separation channels and the second multiple separation channels, electric field can be applied to the second multiple separation channels so that the analyte fraction is electrically injected into the second multiple separation channels. For example, in some cases, the first multiple separation channels and the second multiple separation channels can form a series of T-junctions. In some cases, imaging can be used for detecting when the analyte fraction (for example, fraction of interest) is located at one or more of the series of T-junctions. Applying an electric field can electrically inject the analyte fraction of interest (and optionally, not other analyte fractions not located at the series of T-junctions) into the second multiple separation channels for the second separation stage. In some cases, depending on whether the analyte fraction of interest is detected at one or more of the T-junctions, electric field can be independently applied to one or more of the second multiple separation channels.

[0165] In some cases, imaging can be performed during mobilization to monitor mobilization reaction. In some cases, the imaging system for monitoring separation reaction can also be used to monitor mobilization reaction. In some cases, only a part for channel or multiple channels can be imaged to monitor mobilization reaction. In some cases, whole channel or multiple channels can be imaged, and only a part for the channel or multiple channels of imaging can be used to monitor mobilization reaction. For example, channel can be imaged with a given sampling rate, and for each image generated, the part of the image corresponding to the distal end of one or more channels can be used to generate a mobility chromatogram. The mobility chromatogram can provide information about the average absorbance of a certain pixel width (for example, 8 pixels) such as a time function. In some cases, the pixel width of the image used to generate the mobility chromatogram (e.g., which corresponds to the distal end of the channel) may include 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.

[0166] Mobility chromatogram can be used to determine the parameter of mobilization reaction.For example, mobility chromatogram can be used to calibrate mass spectrometer, to determine the time-of-flight information, peak width, peak velocity, peak activity, peak position etc. of one or more analytes.In some cases, mobility chromatogram can be generated in real time.In some cases, mobility chromatogram can be generated with sampling rate (for example, Nyquist sampling rate, 1Hz-2Hz, or the frequency that is matched with the sampling rate of mass spectrometer).In some cases, chromatogram can be used to generate the information about the absorbance of the channel fragment as a function of time.

[0167] Dynamic Light Scattering: In some cases, the systems and methods of the present disclosure include one or more detection methods including dynamic light scattering (DLS). In some cases, DLS can be used to provide information about the analyte, for example, determining the size distribution curve of the separated analytes in at least one separation channel, the aggregation of the analytes, the hydrodynamic radius of the analytes, etc. DLS can be performed before, during, or after the analyte separation. DLS can be used in conjunction with one or more methods described herein, for example, for sample separation, mobilization, and / or staggered detection of analyte size distribution curves. For example, during one or more processes described herein (e.g., separation, mobilization, injection), in addition to imaging one or more channels, DLS can also be used.

[0168] Systems and System Components: In some cases, the systems of the present disclosure may include one or more disclosed devices (e.g., microfluidic devices), one, two, three, four, or more high-voltage power supplies (or a single multiplexed high-voltage power supply that allows independent control of two or more channels), an automatic sampler and / or fluid loading and unloading system, a fluid flow controller, an imaging module, a dynamic light scattering module, a microplate loading and unloading robotic module, a waste management module (e.g., to remove or prevent droplets from accumulating on the exterior of the electrospray tip), an electrode interface unit, a processor or computer, or any combination thereof.

[0169] High Voltage Power Supply: In some cases, two or more high voltage power supplies of the disclosed system (or a single multiplexed high voltage power supply that allows independent control of two or more channels) are configured to provide simultaneous and independent electrical control of multiple separation channels, for example, to simultaneously and independently apply a specified voltage or current to each of the multiple separation channels or auxiliary fluid channels (e.g., an activation channel for delivering a chemical mobilizer to the separation channel after the isoelectric focusing reaction is completed). In some cases, two or more high voltage power supplies of the disclosed system (or a single multiplexed high voltage power supply that allows independent control of two or more channels) are configured to monitor and / or record the current (not just the total current) flowing through each of the multiple separation channels. As described herein, the separation channels can contain different samples or the same sample (e.g., aliquots of a sample). In some cases, the current flowing through each separation channel can be used, for example, to determine when the isoelectric focusing reaction is complete and / or to detect a malfunction (e.g., the introduction or formation of bubbles in the separation channel).

[0170] In some cases, the system can include two independent high voltage power supplies, three independent high voltage power supplies, four independent high voltage power supplies, five independent high voltage power supplies, six independent high voltage power supplies, seven independent high voltage power supplies, eight independent high voltage power supplies, nine independent high voltage power supplies, ten independent high voltage power supplies, eleven independent high voltage power supplies, twelve independent high voltage power supplies, thirteen independent high voltage power supplies, fourteen independent high voltage power supplies, fifteen independent high voltage power supplies, sixteen independent high voltage power supplies, seventeen independent high voltage power supplies, eighteen independent high voltage power supplies, nineteen independent high voltage power supplies, or twenty independent high voltage power supplies. In some cases, two or more high voltage power supplies can be integrated or packaged into a single multiplexed high voltage power supply that provides simultaneous and independent control of voltage and / or current for each of a plurality of separation channels or auxiliary fluid channels (e.g., a mobilization channel for delivering a chemical mobilizing agent to a separation channel after completion of an isoelectric focusing reaction).

[0171] In some cases, the two or more high voltage power supplies of the disclosed systems are programmable, for example, they can include an internal microprocessor and / or memory that allows the voltage and / or current applied to each of the plurality of separation channels or auxiliary channels to be controlled by software downloaded to the high voltage power supplies. In some cases, the two or more high voltage power supplies of the disclosed systems can be configured for control by an external processor or computer.

[0172] In some cases, two or more high voltage power supplies can be programmed or otherwise configured to operate under constant voltage mode, for example, wherein crossing each applied voltage in a plurality of separation channels and / or auxiliary channel keeps fixing in the duration of separation reaction or in the time period of appointment. In some cases, two or more high voltage power supplies can be programmed or otherwise configured to make crossing each applied voltage in a plurality of separation channels and / or auxiliary channel progressively change to at least the second specified voltage under the time of one or more appointments. In some cases, two or more high voltage power supplies can be programmed or otherwise configured to make voltage carry out two, three, four, five or more than five progressive changes on the process of separation reaction.

[0173] In some cases, two or more high voltage power supplies can be programmed or otherwise configured to operate in a constant power mode, for example, to increase the voltage applied to a given separation channel when the current drops due to conductivity changes during a separation reaction, thereby allowing one to increase the voltage to minimize separation time without causing excessive Joule heating.

[0174] As mentioned above, in some cases, the electric field for performing electrophoretic separation or isoelectric focusing reaction (or other electromotive injection or separation process) can be in the range of from about 0V / cm to about 1000V / cm. Therefore, in some cases, two or more high voltage power supplies of disclosed system can be configured to provide an adjustable voltage (for example, for a 5cm long separation channel) in the range of from about 0 volt to about 5000 volts. In some cases, two or more high voltage power supplies can be configured to provide an adjustable voltage of at least 0 volt, at least 5 volts, at least 10 volts, at least 50 volts, at least 100 volts, at least 500 volts, at least 1000 volts or at least 5000 volts. In some cases, two or more high voltage power supplies can be configured to provide an adjustable voltage of at most 5000 volts, at most 1000 volts, at most 500 volts, at most 100 volts, at most 50 volts, at most 10 volts or at most 5 volts. Any of the lower and upper values ​​described in this paragraph can be combined to form ranges encompassed by the present disclosure; for example, in some cases, two or more high-voltage power supplies can be configured to provide an adjustable voltage within a range from about 100 volts to about 1000 volts. One skilled in the art will recognize that two or more high-voltage power supplies can be configured to provide an adjustable voltage of any value within this range, for example, about 1250 volts.

[0175] Electrode Interface Unit / Holder: In some cases, the disclosed systems can include one or more holders, which can include an electrode interface unit (e.g., a high voltage electrode interface unit or holder) configured to interface one or more electrodes with one or more components of the system (e.g., one or more inlets of a microfluidic device). As described herein, the disclosed microfluidic devices can include two or more integrated electrodes configured to apply a voltage gradient along a separation channel or an interconnecting channel that intersects a separation channel. Electrode can be integrated with multiple inlet ports, outlet ports, sample and / or reagent introduction channel, interconnected channel, sample and / or reagent waste channel, reservoir (for example, sample reservoir, reagent reservoir or waste reservoir), micro pump, micro valve, vent, trap, filter, membrane and the like or any combination thereof, or electrode can be configured to engage with multiple inlet ports, outlet ports, sample and / or reagent introduction channel, interconnected channel, sample and / or reagent waste channel, reservoir (for example, sample reservoir, reagent reservoir or waste reservoir), micro pump, micro valve, vent, trap, filter, membrane and the like or any combination thereof. In some cases, fixture can include one or more membranes, which allow electricity and / or fluid communication for electrode and microfluidic device. In some cases, membrane can be in fluid and / or electrical communication with one or more reservoirs (for example, anolyte reservoir or cathode liquid reservoir) and / or microfluidic device. In some cases, membrane can be coupled to microfluidic device. In some cases, when device is set up fluid and is communicated with one or more reservoirs, film can be used to prevent bubble from being introduced into microfluidic device (for example, passage or inlet).Alternately or additionally, film can be used to prevent bubble (for example, by the bubble formed by electrolysis at electrode) from being further introduced into microfluidic device.In some cases, the volume of the electrode reservoir in the fixture (for example, any reservoir that is electrically contacted with the electrode) can be large enough to minimize or eliminate the pH change that the buffer solution contained therein causes due to the electrolysis at the electrode.In some cases, the geometry of the fixture can be configured to position film to set up fluid and / or be communicated with microfluidic device.Film and / or electrode reservoir can be positioned to be adjacent to microfluidic device (for example, be positioned on microfluidic device, be positioned next to microfluidic device, be positioned to be connected to microfluidic device, be positioned to be orthogonal to microfluidic device, etc.).In some cases, film can be coupled to microfluidic device (for example, via assembly mechanism). In some cases, the geometry of the retainer can be used to prevent bending, folding, or non-planar movement or configuration of the membrane, for example, to prevent bubble formation or application of hydrodynamic pressure when the membrane is engaged with the device. For example, the retainer can include an insert, such as a U-shaped structure, wherein the membrane can be placed at the bottom (e.g., the flat portion) of the U-shaped structure.In such an example, the U-shaped structure can be coupled to a reservoir (e.g., an electrode reservoir that can be engaged with an electrode) and allows to be communicated with film and microfluidic device fluid. The arms of the U-shaped structure can include an inlet fluid path and an outlet fluid path. In another example, the fixture can include an insert, e.g., a Y-shaped structure, in which the film can be placed at the top of the Y-shaped structure. In such an example, the Y-shaped structure can include a reservoir (e.g., an electrode reservoir) and allows to be communicated with film and microfluidic device fluid.

[0176] In some cases, the membrane is engaged with the device via an outlet fluid path and a port (e.g., a flat portion of a U-shaped structure). At least one dimension of the port can take a variety of geometric shapes and be configured to prevent bending, folding, etc. of the membrane. For example, the port can be circular and can have a diameter of at most about 5 mm, at most about 4 mm, at most about 3 mm, at most about 2 mm, at most about 1 mm, or at most about 500 μm. The membrane can cover all or a portion of the port and can include any useful size; for example, the membrane can have a diameter of about 0.001 square millimeters (mm 2 ), about 0.005mm 2 , about 0.01mm 2 , about 0.05mm 2 , about 0.1mm 2 , about 0.5mm 2 , about 1mm 2 , about 5mm 2 , about 10mm 2 , about 50mm 2 , about 100mm 2 or about 500mm 2 The membrane may include at least 0.001 (mm 2 ), at least 0.005mm 2 , at least 0.01mm 2 , at least 0.05mm 2 , at least 0.1mm 2 , at least 0.5mm 2 , at least 1mm 2 , at least 5mm 2 , at least 10mm 2 , at least 50mm 2 , at least 100mm 2 or at least 500mm 2 In some cases, the membrane may comprise up to 500 mm 2 , up to 100mm 2 , up to 50mm 2 , up to 10mm 2 , up to 5mm 2, up to 1mm 2 , up to 0.5mm 2 , up to 0.1mm 2 , up to 0.05mm 2 , up to 0.01mm 2 , up to 0.005mm 2 or at most 0.001mm 2 The membrane may include a cross-sectional area within an area range, for example, within about 0.001 mm 2 and about 100mm 2 between.

[0177] In some cases, the holder can include: an electrode reservoir; an inlet fluid channel comprising a first end and a second end; an outlet fluid channel comprising a first end fluidically coupled to the second end of the inlet fluid channel and a second end fluidically coupled to a separation channel (e.g., in a microfluidic device), the inlet fluid channel and the outlet fluid channel intersecting and fluidically coupled to each other at a plane defined or parallel to a surface of the electrode reservoir. The membrane can be disposed within the electrode reservoir at or near the plane where the inlet fluid channel and the outlet fluid channel intersect, such that the membrane covers all or substantially all of an opening comprising the intersection of the inlet fluid channel and the outlet fluid channel (see, e.g., FIG. 13A to 13F Where the holder comprises an insert comprising an inlet fluid path and an outlet fluid path, the membrane and / or the inlet fluid path and the outlet fluid path may be configured to facilitate substantially bubble-free wetting of the surface of the membrane when the electrode reservoir is filled (e.g., with a strong electrolyte, buffer, reagent, etc.).

[0178] Film can be selected by desired material properties. For example, film can be selected for desired pore size, hydrophilicity, hydrophobicity, amphipathicity, wettability, charged or uncharged, inertia, mechanical properties (for example, rigidity, compliance, flexibility, toughness) etc. In some cases, film can comprise natural or synthetic materials. Film can comprise one or more polymers. In a preferred embodiment, film comprises cellulose or regenerated cellulose and is hydrophilic. In another preferred embodiment, film comprises polymer, for example, polytetrafluoroethylene (PTFE). When using polymer, polymer (for example, PTFE) can be manufactured or processed to obtain useful characteristics (for example, weaving, processing so that surface hydrophilic etc.). In another preferred embodiment, film comprises rigid material, for example, glass or ceramics. In certain embodiments, film can be processed as hydrophilic and / or uncharged.

[0179] In some cases, the membrane provides a high hydrodynamic resistance and low electrical resistance connection between the high voltage electrode located within the electrode reservoir and the fluid (e.g., liquid, buffer, etc.) contained within the inlet and outlet fluid channels. The hydrodynamic resistance between the intersection of the inlet and outlet fluid channels and the reservoir can be about 0.01 ((N / mm 2 ) / (mm 3 / s))、about 0.1((N / mm 2 ) / (mm 3 / s)), about 1((N / mm 2 ) / (mm 3 / s)), about 10((N / mm 2 ) / (mm 3 / s)), about 100((N / mm 2 ) / (mm 3 / s)), about 1000((N / mm 2 ) / (mm 3 / s)), about 10000((N / mm 2 ) / (mm 3 / s)), about 100000((N / mm 2 ) / (mm 3 / s)) or about 1000000((N / mm 2 ) / (mm 3 The hydrodynamic resistance between the intersection of the inlet and outlet fluid channels and the reservoir may be at least 0.01 ((N / mm 2 ) / (mm 3 / s)), at least 0.1((N / mm 2 ) / (mm 3 / s)), at least 1((N / mm 2 ) / (mm 3 / s)), at least 10((N / mm 2 ) / (mm 3 / s)), at least 100((N / mm 2 ) / (mm 3 / s)), at least 1000((N / mm 2 ) / (mm 3 / s)), at least 10000((N / mm 2 ) / (mm 3 / s)), at least 100000((N / mm 2 ) / (mm 3 / s)) or at least 1000000((N / mm 2 ) / (mm 3The hydrodynamic resistance between the intersection of the inlet and outlet fluid channels and the reservoir may be at most 1,000,000 ((N / mm 2 ) / (mm 3 / s)), up to 100000((N / mm 2 ) / (mm 3 / s)), up to 10000((N / mm 2 ) / (mm 3 / s)), up to 1000((N / mm 2 ) / (mm 3 / s)), up to 100((N / mm 2 ) / (mm 3 / s)), up to 10((N / mm 2 ) / (mm 3 / s)), up to 1((N / mm 2 ) / (mm 3 / s)), up to 0.1((N / mm 2 ) / (mm 3 / s)) or up to 0.01((N / mm 2 ) / (mm 3 The hydrodynamic resistance between the intersection of the inlet and outlet fluid channels and the reservoir may be within a range of values, for example, within 1 ((N / mm 2 ) / (mm 3 / s)) and 10000((N / mm 2 ) / (mm 3 / s)).

[0180] In some cases, the hydrodynamic resistance of a portion of a membrane (e.g., a pore) can be calculated using the Hagen-Poiseuille equation, Equation 3:

[0181] R 流体动力 =8*viscosity*film thickness / (πr 孔隙 4 )

[0182] Among them, R 流体动力 is the hydrodynamic resistance of a pore, viscosity is the viscosity of the bulk liquid, r 孔隙 is the radius of a single pore. The hydrodynamic resistance across the membrane can be equal to the value given by R 流体动力 Divide by the number of pores. For example, if a membrane with pores of 3 nm diameter and a thickness of 100 μm is used to inhibit the hydrodynamic flow of an aqueous solution at 25°C (viscosity = 0.89 cP), then by this equation R 流体动力 =8*(0.89cP)*(100μm) / ((π)*(1.5nm)4 ), or 4.48*10 13 ((N / mm 2 ) / (mm 3 / s)). If the surface area of ​​the membrane is 1mm 2 And the pore area fraction is 5%, then the number of pores is equal to (1mm 2 )*(0.05) / ((π)*(1.5nm) 2 ), or 7.1*10 9 pores, and the total fluid dynamic resistance is equal to (4.48*10 13 ((N / mm 2 ) / (mm 3 / s))) / 7.1*10 9 , or 6330((N / mm 2 ) / (mm 3 / s)).

[0183] In some embodiments, the resistance of the pore can be calculated using Equation 4:

[0184] R 电 =(solution resistivity)*(film thickness) / (πr 孔隙 2 )

[0185] Among them, r 孔隙 is the radius of a single pore. For example, if the solution resistivity is 500 (Ω)(cm), the membrane thickness is 100 μm, and the pore diameter is 3 nm, then R 电 will be equal to (500Ω*cm)*(100μm) / ((π)*(1.5nm) 2 ), or 7.1*10 13 Ω. If the number of pores is 7.1*10 9 pores, the total resistance of the membrane is equal to 10000Ω in this example.

[0186] The resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir may be about 10,000,000 ohms, about 1,000,000 ohms, about 10,000 ohms, about 1,000 ohms, about 100 ohms, about 10 ohms, about 1 ohm, about 0.1 ohms, or about 0.01 ohms. The resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir may be at most 10,000,000 ohms, at most 1,000,000 ohms, at most 100,000 ohms, at most 1000 ohms, at most 10 ohms, at most 1 ohm, at most 0.1 ohms, or at most 0.01 ohms. The resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir can be within a range of values, for example, between about 100,000 ohms and 10,000,000 ohms.

[0187] In some cases, the ratio of the hydrodynamic drag to the electrical resistance (the resistance being the resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir) can be about 0.001 ((N / mm 2 ) / (mm 3 / s)) / Ω、about 0.01((N / mm 2 ) / (mm 3 / s)) / Ω、about 0.1((N / mm 2 ) / (mm 3 / s)) / Ω、about 1((N / mm 2 ) / (mm 3 / s)) / Ω、about 10((N / mm 2 ) / (mm 3 / s)) / Ω、about 100((N / mm 2 ) / (mm 3 / s)) / Ω、about 1000((N / mm 2 ) / (mm 3 / s)) / Ω、approximately 10000((N / mm 2 ) / (mm 3 In some cases, the ratio of the hydrodynamic drag to the electrical resistance (the resistance being the resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir) can be at least 0.001 ((N / mm 2 ) / (mm 3 / s)) / Ω、at least 0.01((N / mm 2 ) / (mm 3 / s)) / Ω、at least 0.1((N / mm 2 ) / (mm 3 / s)) / Ω, at least 1((N / mm 2) / (mm 3 / s)) / Ω, at least 10((N / mm 2 ) / (mm 3 / s)) / Ω、at least 100((N / mm 2 ) / (mm 3 / s)) / Ω、at least 1000((N / mm 2 ) / (mm 3 / s)) / Ω、at least 10000((N / mm 2 ) / (mm 3 / s)) / Ω or greater. In some cases, the ratio of the hydrodynamic resistance to the electrical resistance (the resistance being the resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir) can be within a range of values, for example, between 1000 ((N / mm 2 ) / (mm 3 / s)) / Ω and 1000000((N / mm 2 ) / (mm 3 / s)) / Ω.

[0188] In some cases, the electrode reservoir can be filled with an electrolyte solution having a concentration of about 0.1 millimole (mM), about 0.5mM, about 1mM, about 5mM, about 10mM, about 50mM, about 100mM, about 500mM or about 1 mole (M). Electrolyte solution concentration can be at least 0.1 millimole (mM), at least 0.5mM, at least 1mM, at least 5mM, at least 10mM, at least 50mM, at least 100mM, at least 500mM or at least 1 mole (M). Electrolyte solution concentration can be at most about 1M, at most about 500mM, at most about 100mM, at most about 50mM, at most about 10mM, at most about 5mM, at most about 1mM, at most about 0.5mM or at most about 0.1mM. Electrolyte solution concentration can be in the range of concentration, for example, between about 1 millimole (mM) and about 500mM. In some cases, during operation, the electrode reservoir is filled with an electrolyte solution having a concentration between about 10 mM and about 150 mM.

[0189] In some cases, during operation, the electrode reservoirs are filled with an electrolyte solution having a pH range between about 1.5 and about 14. For example, one electrode reservoir at the proximal end of the separation channel can contain about 1.5 pH units of electrode solution, and another electrode reservoir at the distal end of the separation channel can contain about 14 pH units of electrode solution, or one electrode reservoir at the distal end of the separation channel can contain about 1.5 pH units of electrode solution, and another electrode reservoir at the proximal end of the separation channel can contain about 14 pH units of electrode solution. It will be appreciated that the pH range or pH difference between the electrode reservoirs can be tuned based on the pH range useful for separating analyte classes. For example, if the analyte mixture includes expected pI values ​​within a narrow pH range, the pH range or difference of the electrode reservoirs can be adjusted to be narrower to achieve higher separation resolution for a given analyte mixture.

[0190] In some cases, the electrolyte solution contains a strong acid, a strong base, or a highly soluble salt. Examples of strong acids include, but are not limited to, perchloric acid, hydrochloric acid, sulfuric acid, and the like. Examples of strong bases include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, and the like. Examples of highly soluble salts include, but are not limited to, sodium chloride, potassium nitrate, magnesium chloride, and the like. In some cases, a weak acid or a weak base may be used as the electrolyte solution. Examples of weak acids include, but are not limited to, phosphoric acid, formic acid, acetic acid, carbonic acid, and the like. Examples of weak bases include, but are not limited to, ammonium hydroxide, diethylamine, dimethylamine, piperidine, and the like. In some cases, the pH of the electrolyte solution may be between approximately 1.5 pH units and approximately 14 pH units. In some cases, the pH of the electrolyte solution may be between approximately 2 pH units and approximately 11 pH units. In some cases, the pH of the electrolyte solution may be between approximately 3 pH units and approximately 9 pH units. In some cases, the pH of the electrolyte solution may be between approximately 5 pH units and approximately 8 pH units.

[0191] Fluid Flow Controllers: In some cases, the disclosed systems may include one or more programmable fluid flow controllers configured to provide, for example, independently controlled pressure-driven flows through two or more separation channels (e.g., for use alone or in combination with a voltage gradient applied to the two or more separation channels) or auxiliary channels intersecting the separation channels. In some cases, pressure-driven flows can be used to mobilize separated analyte peaks from the separation channels. In some cases, pressure-driven flows can be used, for example, to introduce chemical mobilizers into the separation channels (e.g., electrolytes that disrupt the pH gradient for isoelectric focusing), thereby mobilizing separated analyte peaks from the separation channels. In some cases, pressure-driven flows can be used, for example, to introduce chemical mobilizers into the separation channels (e.g., elution buffers for eluting analytes from a stationary phase confined within the separation channels), thereby mobilizing separated analyte peaks from the separation channels. In some cases, flow can be controlled by integrating flow restrictors into the device, for example, long capillaries or channel lengths to increase fluid dynamic resistance and provide uniform flow distribution and electrospray performance.

[0192] The control of the pressure-driven fluid flow of the disclosed device and system will typically be performed by using a pump (or other fluid actuation mechanism) and a valve. 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, the fluid flow through the system can be controlled by applying positive air pressure at one or more fluid inlets or sample or reagent reservoirs on the device. In some embodiments, the fluid flow through the system can be controlled by vacuuming 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 or (e.g., peristaltic pumps, piezoelectric pumps), microvalves (e.g., metered injection valves, piezoelectric valves, stopcocks, slide valves) can be integrated into the device. In some cases, the control or pressure-driven fluid flow of the disclosed device and system can be performed using a balloon, blister pack, piston, screw, glass frit, or a combination thereof. In some cases, the pressure-driven fluid flow can be pulseless.

[0193] In some embodiments, one or more devices or system parameters can be used to control the fluid flow through the system. In some cases, flow can be generated in the device by changing the temperature of the system (for example, changing the gas pressure in the region of the device) or by introducing a temperature gradient. In some cases, the reservoir height can be changed to drive one or more channels (for example, via liquid static pressure) flowing through the device. In some cases, a part (for example, inlet or outlet) of the device can be exposed and allowed to evaporate, thus driving the fluid flow through the channel. In some cases, fluid flow can be pulseless.

[0194] In some cases, fluid flow through the disclosed devices and systems can be performed electrically. For example, electroosmotic flow in or out of one or more channels of a device can be performed using, for example, an electroosmotic pump.

[0195] During the performance of the disclosed analyte separation methods, different modes of fluid flow control can be utilized at different points, for example, forward flow (relative to the inlet and outlet for a given device or separation channel), reverse flow, oscillating flow, or pulsating flow, or combinations thereof, can all be used. For example, in some cases, such as during a device startup step, oscillating flow or pulsating flow can be used to facilitate the movement of any bubbles that may be trapped within the device. In some cases, the device can be subjected to a vacuum (e.g., degassing) for device priming, for example, to facilitate bubble-free introduction of fluids or reagents.

[0196] During execution of disclosed analyte separation method, different fluid flow rates can be utilized at different points.For example, in some examples of disclosed device and system, volumetric flow rate can change from-100ml / s to+100ml / s.In some cases, the absolute value of volumetric flow rate can be at least 0.001ml / s, at least 0.01ml / s, at least 0.1ml / s, at least 1ml / s, at least 10ml / s or at least 100ml / s.In some cases, the absolute value of volumetric flow rate can be at most 100ml / s, at most 10ml / s, at most 1ml / s, at most 0.1ml / s, at most 0.01ml / s or at most 0.001ml / s.The volumetric flow rate at given time point can have any value within the scope of this, for example, the forward flow rate of 2.5ml / s, the reverse flow rate of-0.05ml / s or the value (that is, stop flowing) of 0ml / s. In some cases, the pressure-driven fluid flow pattern and / or fluid flow rate through each separation channel and / or auxiliary fluid channel can be programmed independently of each other to follow a specified time course.

[0197] Automatic sampler and fluid loading and unloading system: in some cases, disclosed system can also include automatic sampler or fluid loading and unloading system, and described automatic sampler or fluid loading and unloading system are configured to be used for sample aliquot and / or other separation reaction reagent automatically and independently control to be loaded into multiple samples or reagent inlet port leading to separation channel.In some cases, customized automatic sampler or fluid loading and unloading module can be incorporated into disclosed system.In some cases, commercially available automatic sampler or fluid loading and unloading module can be integrated into disclosed system.The example of suitable commercially available automatic sampler includes but is not limited to Agilent 1260 infinite dual loop automatic sampler and 1260 infinite high performance micro automatic sampler (California, USA, Santa Clara City, Agilent Technologies), HT1500L HPLC automatic sampler (Italy, Brescia City, HTA), Spark Holland Alias ​​(Netherlands, Ai Man, Spark-Holland) and SIL-20A / AC HPLC automatic sampler (Maryland, USA, Columbia City, Shimadzu Corporation). Examples of suitable commercially available fluid handling systems (or liquid handling systems) include, but are not limited to, Tecan systems (Tecan Trading Company, Switzerland), Hamilton Mylan STAR and Mylan NIMBUS systems (Hamilton, Reno, NV, USA), and Agilent Bravo automated liquid handling platform and Agilent vertical pipetting station (Agilent Technologies, Santa Clara, CA, USA).

[0198] In some cases, one or more fluid flow controllers or fluid loading and unloading systems can be used to fill or supplement one or more reservoirs. As described herein, the reservoir can be in fluid communication with a membrane (e.g., contained in a fixture and / or electrode interface unit), and the membrane can engage with the microfluidic device and prevent bubbles from forming at the interface of the device and the membrane. Various fluid controllers or fluid loading and unloading systems can be used to fill the reservoir. For example, the fluid controller can be used to distribute buffer or reagent to each reservoir. In some cases, the fluid controller can include a pipette tip (e.g., a 1000 microliter pipette tip), and the reservoir can be configured to receive the pipette tip. In some cases, the reservoir can include an entry port for filling the reservoir from the bottom. In some cases, the reservoir can include a side port for entering a pipette tip without forming bubbles. In some cases, the reservoir can include a flange that can help the integration or engagement of the fluid flow controller.

[0199] Waste management: In some embodiments, the system may further include a waste management module, which may be integrated with the microfluidic device (i.e., attached to the microfluidic device) or separate from the microfluidic device. The waste management module can be used to collect waste products from the microfluidic device. In some cases, additionally or alternatively, the waste management module can be used to manage droplet formation at the outlet or surface of the microfluidic device. For example, the waste management module can be used to prevent droplets from forming at the outlet of the device (e.g., electrospray tip) and / or prevent droplets from wicking to different segments or parts of the device (e.g., inlet, interface electrode, etc.). In some cases, the waste management module may include applying positive or negative pressure (e.g., vacuum). In this case, a vacuum can be applied to a portion of the microfluidic device (e.g., outlet or electrospray tip). For example, a flange or adapter can be applied to the chip to allow a vacuum to be engaged with the device with minimal interference with the placement of the device or with any downstream analytical units (e.g., mass spectrometer). The vacuum can then be used to aspirate the droplets or waste products as they are expelled from the outlet or electrospray tip.

[0200] Vacuum can be applied by a variety of devices, which can be formed into one or more shapes. For example, the device by which vacuum is applied can be shaped like a trumpet or a funnel. The trumpet or funnel can be configured to apply vacuum to the tip. In some cases, the device can be configured to swivel or move to different positions. In another embodiment, vacuum can be applied by a tubular device. The tube can be conical, cylindrical or any other shape. In some cases, the tube can also include an opening module, which can be used to apply vacuum and guide the waste product to a waste container. For example, the tube can be placed between the chip (for example, using a flange joint) and the mass spectrometer, and the tube can include an opening module, for example, a vacuum sleeve that guides the waste product from the electrospray tip so that the waste product does not reach the mass spectrometer. In some cases, the tube can be oriented at a certain angle, for example, perpendicular to the outlet or electrospray tip. In this case, vacuum can be applied to the tube and can aspirate the droplets when the droplets leave the device. In some cases, the tube can be transparent so that one or more imaging systems as described herein can be used to image the electrospray tip. In another embodiment, the vacuum can be applied by a modular device that can be configured to attach to the vacuum. For example, the modular device can be configured to clamp or attach to a portion of the device. Once the modular device is secured to the device, the vacuum can be applied to the modular device to direct waste products away from the microfluidic device.

[0201] In some cases, the waste management module can include the use of positive pressure. For example, an air knife can be used to direct droplets away from the electrospray tip. In such an example, the air knife can be connected to an air or nitrogen source and / or a pressurizer to generate air (or nitrogen) pressure to spray droplets or direct droplets away from the device or a portion thereof (e.g., the electrospray tip). In some cases, the waste management module can include an atomization unit. For example, the atomizer can be configured to be fixed to the chip. The atomizer can include the geometry necessary to direct air toward the chip so that droplets or waste products are directed away from the electrospray tip or outlet (e.g., to a waste container). The atomizer can include a sealing mechanism and can be connected to an air source and / or a pressurizer to generate air pressure to spray droplets or direct droplets away from the electrospray tip. In some cases, the atomizer can include a nozzle. The atomizer can be made of a polymer, metal, or ceramic material.

[0202] In some cases, the waste management module can include using mechanical methods to remove waste and / or droplets from the outlet or electrospray tip. For example, one or more wipers can be used to mechanically move (e.g., sweep) droplets from the device. Alternatively or additionally, an absorbent material can be integrated into the waste management module to suck or wick away waste material from the outlet or electrospray tip.

[0203] In some cases, the waste management module can be used in conjunction with other methods for waste management. For example, the device can include geometric shapes or chemical / material properties that allow for controlling droplet formation at the outlet and / or minimizing wicking of droplets and fluids to different segments or parts of the device (e.g., electrodes or inlets). In some cases, a coating can be used to allow droplet formation at the tip or outlet of the device and can help prevent wicking of fluids to other segments or parts of the device. In some cases, the coating can be a hydrophobic coating.

[0204] In some cases, the geometry or orientation of the device can be used to control droplet formation at the outlet and / or minimize the wicking of droplets to different segments or parts of the device. For example, the outlet or electrospray tip can be formed as a triangular tip to allow for optimal droplet formation. In some cases, the spatial orientation of the device can be used to control waste management. For example, the device can be angled so that the outlet (e.g., tip) is oriented downward, and waste can be driven by gravity out of the microfluidic device. Any suitable angle can be used to guide gravity out of the microfluidic device. For example, the angle can be about 30°, 31°, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41°, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, 50°, 51°, 52°, 53°, 54°, 55°, 56°, 57°, 58°, 59°, 60°, etc. In some cases, the system further comprises a waste container separate from the device for collecting waste products.

[0205] In some cases, the waste management module can eliminate the need for a waste reservoir on the device. For example, waste can be driven out of the device in the form of droplets or a stream and can be removed, for example, via the use of vacuum suction.

[0206] Imaging module: In some cases, the system may also include an imaging module configured to acquire a series of one or more images of two or more separation channels or a portion thereof. In some cases, the field of view of the image may include all or a portion of two or more separation channels. In some cases, imaging may include continuously imaging all or a portion of two or more separation channels while performing separation and / or mobilization reactions. In some cases, imaging may include intermittent or periodic imaging of all or a portion of two or more separation channels while performing separation and / or mobilization reactions. In some cases, imaging may include acquiring UV absorbance images. In some cases, imaging may include acquiring fluorescence images, for example, natural fluorescence or fluorescence due to the presence of exogenous fluorescent labels attached to the analyte. In some cases, the imaging module may be configured to, for example, determine when the isoelectric focusing reaction is complete and / or detect a fault (e.g., the introduction or formation of bubbles in the separation channel).

[0207] Any of a variety of imaging systems or system components can be used to achieve the purposes of 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, objectives, 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 can include an array of light sources. For example, an LED array can be used to illuminate one or more areas of the device. Depending on the imaging mode used, the light source and image sensor can be positioned on opposite sides of the microfluidic device, for example, so that an absorbance-based image can be acquired. In some cases, the light source and image sensor can be positioned on the same side of the microfluidic device, for example, so that an epifluorescence image can be acquired.

[0208] As described above, images can be acquired continuously during the separation and / or activation steps, or images can be acquired at random or specified time intervals. In some cases, a series of one or more images are acquired continuously or at random or specified time intervals. In some cases, a series of short exposure images (e.g., 10 to 20 images) are acquired quickly (e.g., millisecond time scale) and then averaged to provide a "single image" with an improved signal-to-noise ratio. In some cases, "single images" are acquired every 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, or at longer time intervals. In some cases, longer exposure times can be used to improve the signal-to-noise ratio. In some cases, a series of one or more images can include video images.

[0209] Image Processing: In some cases, as described above, the system can include a processor, controller, or computer configured to run image processing software for detecting the presence of analyte peaks, determining the positions of pI markers or separated analyte bands, determining peak width, determining 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 can be used to detect faults, such as the introduction or formation of bubbles in one of two or more separation channels. In implementing the disclosed methods and systems, any of a variety of image processing algorithms can be utilized for image pre-processing or image processing. Examples include, but are not limited to, Canny edge detection method, Canny-Deriche edge detection method, first-order gradient edge detection method (e.g., Sobel operator), second-order differential edge detection method, phase consistency (phase coherence) edge detection method, other image segmentation algorithms (e.g., intensity threshold, intensity clustering method, intensity histogram-based method, 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, eigenvalue analysis, etc.) or any combination thereof.

[0210] Microplate handling robot: In some cases, the system may also include a microplate handling robot module that is configured to transport and replace microplates used as a source for samples and / or reagents. In some cases, the system may also include a microfluidic device handling robot module that is configured to transport and replace the microfluidic device used in the system, for example, after fault detection. In some cases, microplate handling and microfluidic device handling can be handled by the same robot module. In some cases, custom robots can be incorporated into the disclosed system to perform these functions. In some cases, commercially available robot systems can be adapted and / or integrated into the disclosed system to perform these functions. Examples of suitable microplate handling robot systems include, but are not limited to, Tecan robot gripping arms (Tecan Trading Company, Switzerland) and Agilent direct drive and desktop robots (Agilent Technologies, Santa Clara, California, USA).

[0211] Failure Mode Detection and Recovery: In some cases, the disclosed systems can be configured to automatically detect system failures, such as loss of current due to bubble introduction during sample loading or bubble formation during a separation run, incorrect current distribution due to an improperly prepared sample, or absence of current due to an empty or unfilled well in the sample plate. In some cases, the disclosed systems can be configured to flag the failure and automatically rerun the sample for which the failure was detected in the corresponding separation channel. In some cases, for example, the disclosed systems can be configured to reload a particular sample from a microtiter plate or other sample source and rerun the separation reaction.

[0212] Temperature Control: In some cases, the disclosed systems and methods can be temperature controlled. In some cases, a portion of the system (e.g., a portion of the device) can be temperature controlled. In some cases, the system or one or more components of the system can be cooled using, for example, the Peltier effect, a fan or other heat sink, or an air knife. In some cases, the cooling system can be integrated with a waste management system (e.g., an air knife). In some cases, the cooling system can include a compressor for cooling. In some cases, the system can include an environment or temperature controlled chamber. In some cases, a cooling block or pre-cooling block (e.g., coupled to a stand or cartridge) can be used. In some cases, the system or components thereof can be constructed of materials that allow for heat exchange with the environment. In some cases, the system can include a liquid heat exchanger.

[0213] Applications: The disclosed methods, devices, and systems have potential applications in a variety of fields, including, but not limited to, proteomics research, cellular research, drug discovery and development, and clinical diagnostics. For example, the improved reproducibility and quantification achievable through separation-based characterization of analyte samples using the disclosed methods could be highly beneficial for the characterization of biologics and biosimilars during development and / or manufacturing.

[0214] Biological products and biosimilars are a class of drugs that include, for example, recombinant proteins, antibodies, live virus vaccines, human plasma-derived proteins, cell-based drugs, proteins of natural origin, antibody-drug conjugates, protein-drug conjugates, and other protein drugs. The FDA and other regulatory agencies require a stepwise approach to demonstrate biosimilarity, which can include comparing the proposed product and the reference product in terms of structure, function, animal toxicity, human pharmacokinetics (PK) and pharmacodynamics (PD), clinical immunogenicity, and clinical safety and efficacy (see, U.S. Department of Health and Human Services, Food and Drug Administration, “Scientific Considerations in Demonstrating Biosimilarity to a Reference Product: Guidance for Industry,” April 2015). 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 α-helical structure or a β-sheet structure), tertiary structure (i.e., the three-dimensional shape of the protein produced by folding the polypeptide backbone and secondary 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 cannot be obtained without employing laborious, time-consuming, and costly techniques such as X-ray crystallography. Therefore, for the purpose of establishing biosimilarity between candidate biopharmaceuticals and reference drugs, experimental techniques that allow for convenient, real-time, and relatively high-throughput characterization of protein structure are needed.

[0215] In some cases, the disclosed methods, apparatus, and systems can be used to provide structural comparison data for a candidate biopharmaceutical (e.g., a monoclonal antibody (mAb)) and a reference biopharmaceutical for the purpose of establishing biosimilarity. For example, in some cases, determining the isoelectric point for a candidate drug and a reference drug can provide important evidence to support the demonstration of biosimilarity. In some embodiments, isoelectric point data for a candidate drug and a reference drug that have both been treated with a site-specific protease under the same reaction conditions can provide important evidence to support the demonstration of biosimilarity. In some embodiments, the disclosed methods, apparatus, and systems can be used to monitor a biopharmaceutical manufacturing process (e.g., to monitor a bioreactor process in real time) to ensure product quality and consistency by analyzing samples drawn at different points in the production process or samples drawn from different production runs.

[0216] The disclosed apparatus and systems for performing multiple independently controlled separation reactions in parallel offer numerous advantages over currently available techniques, 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. In addition, independently monitoring and / or recording the current traces and / or voltage settings for each separation channel can be beneficial in meeting data tracking requirements for FDA submissions when attempting to demonstrate biosimilarity, etc. As previously discussed, in some cases, the disclosed apparatus and systems can be configured to identify sample run failures (e.g., the presence or formation of bubbles in a microfluidic device) and initiate a recovery step (e.g., by automatically reloading the sample from a microtiter plate or other sample source and repeating the separation reaction).

[0217] Although 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 as examples only. The present invention is not intended to be limited by the specific examples provided in this specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not intended to be interpreted as limiting. Without departing from the present invention, those skilled in the art will now appreciate many variations, modifications, and alternatives. In addition, it will be understood that all aspects of the present invention are not limited to the specific description, configuration, or relative proportions set forth herein, which depend on various conditions and variables. It will be understood that, in practicing the present invention, various alternatives to the embodiments of the present invention described herein may be adopted. Therefore, it is contemplated that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The appended claims are intended to define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are thereby covered.

[0218] Example

[0219] These examples are provided for illustrative purposes only, and are not intended to limit the scope of the claims provided herein.

[0220] Example 1 - Microfluidic device comprising four separation channels

[0221] Figure 1AProvide a non-limiting example view of the microfluidic device for performing multiple separation reactions (for example, isoelectric focusing reaction).The device includes lower substrate 101, and the lower substrate 101 can be substantially planar, including fused quartz, wherein uses for example imprinting, laser micromachining or photoetching and wet chemical etching to manufacture the fluid channel measured as the width of 210 microns and the depth of 100 microns.By bonding substrate 101 to transparent cover glass 102, seal fluid channel.In some cases, for example, when UV absorbance imaging is used to monitor separation and / or mobilization reaction, substrate 101 can be manufactured by optically transparent material.In some cases, for example, when using epifluorescence imaging to monitor separation and / or mobilization reaction, substrate 101 can be manufactured by optically opaque material.Although the device is illustrated as a rectangle, it will be understood that the device can adopt any useful shape.In certain embodiments, the microfluidic device can include tip (for example, at the distal end), which can allow for fluid guidance away from the device (for example, to be directed to a waste container or analytical unit, for example, a mass spectrometer).

[0222] Access to the fluid channels within the device is provided by a sample inlet port 103, an anode well 104, a cathode well 106, a sample outlet port 107, and a chemical mobilization agent inlet port 109. One anode well 104 and cathode well 106 are in fluid and electrical communication with the proximal end and distal end of each separation channel 105, respectively (four separation channels are shown in this non-limiting example). In some cases, electrodes can be placed in contact with the anode well 104 and cathode well 106. The separation channel extends beyond the cathode well 106 to the sample outlet port 107 (only two of the four separation channels shown in the figure are labeled). The chemical mobilization agent inlet port 109 is connected to the distal end of the separation channel 105 via a chemical mobilization channel 108 (only two of the four separation channels shown in the figure are labeled). As shown Figure 1A As shown, the inlet port 109 and outlet port 107 can be configured for loading through the side of the device, which can facilitate full channel or full device imaging.

[0223] In order to perform multiple isoelectric focusing reactions to separate protein mixtures, protein samples are premixed with an ampholyte pH gradient and pI markers, and then placed in vials and loaded onto the autosampler. The samples are sequentially loaded into the device by the autosampler via the sample inlet port 103, loaded onto the microfluidic device through the separation channel 105, and discharged from the device to waste through the sample outlet port 107.

[0224] The cathode liquid fluid (e.g., 1% N4OH in H2O) is loaded into the cathode recess 106, the anode liquid (e.g., 10 mM H3PO4) is loaded into the anode recess 104, and the mobilizing agent solution (e.g., 49% MeOH, 49% H2O, 1% acetic acid) is connected to the mobilizing agent inlet port 109.

[0225] After all reagents are loaded, an electric field of, for example, +600 V / cm is applied from one or more anode recesses 104 to the corresponding cathode recesses 106 by connecting electrodes to the anode recesses 104 and the cathode recesses 106 to initiate isoelectric focusing. As described above, the voltage and / or current applied to each separation channel 105 can be independently controlled and can also be recorded as a function of time. In some cases, the electrodes for the anode and cathode can be integrated with the device. For UV absorbance imaging, the collimated light beam provided by the UV light source is aligned with the separation channel 105, and an image sensor (e.g., a CCD camera or a CMOS camera) is placed on the other side of the separation channel 105 to measure the amount of light transmitted through each separation channel 105, thereby imaging and detecting the focused proteins (or other separated analytes) by means of their absorbance. In some cases, the focused proteins can be unlabeled and detected by their natural absorbance at 220 nm, 280 nm, or any other wavelength at which the proteins absorb light. For fluorescence imaging, i.e., epifluorescence imaging, excitation light of a suitable wavelength is delivered to the separation channel 105 by means of an optical assembly including a suitable dichroic reflector and a 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 protein (or other separated analyte) can be imaged and detected using natural fluorescence. In some cases, a non-covalently bound fluorescent tag, a chromogenic tag, a fluorogenic tag, or a chromophore tag (e.g., In some cases, portions of the device can be constructed of optically opaque materials so that light can only be transmitted 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.

[0226] As isoelectric focusing reactions are performed in multiple separation channels 105, images of the focused proteins in all or a portion of the separation channels 105 can be captured continuously and / or periodically. In some cases, positional detection of pI markers in the images of the separation channels 105 can be used to determine the local pH as a function of position along the separation channel and, by extrapolation, to make a more accurate pI determination for the separated proteins (or other analytes). In some cases, when focusing is complete, a positive pressure is applied at the sample inlet port 103 and / or the 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 electrode connected to the cathode well 106 is disconnected, and an electrode in electrical communication with the mobilizer channel 108 is used to apply an electric field of 600 V / cm from the anode well 104 to the chemical mobilizer inlet 109 to electrophoretically introduce the mobilizer into the separation channel 105. In some cases, a slight positive pressure applied to the mobilizing agent inlet 109 may be used instead of or in addition to the electrophoretic introduction of the chemical mobilizing agent.

[0227] When a mobilizing agent is introduced electrophoretically, acetic acid in the mobilizing agent solution is attracted by the electric field into the separation channel 105, where it ionizes the proteins and ampholytes and disrupts the pH gradient used for isoelectric focusing. The ionization of the enriched 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 determination of the pI for each separated protein.

[0228] Example 2: Using the disclosed apparatus and system to demonstrate biosimilarity prediction

[0229] A non-limiting example of the utility of the disclosed devices and systems is in the field of biologics and demonstration of biosimilarity. As described above, the FDA and other regulatory agencies require a stepwise approach to demonstrating biosimilarity that can include comparing a proposed product to a reference product in terms of 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 alpha helical structures or beta sheet structures), tertiary structure (i.e., the three-dimensional shape of the protein resulting from folding the polypeptide backbone and secondary domains), and quaternary structure (e.g., the number of subunits required to form an active protein complex or an aggregated state of the protein). Accurate determination of the isoelectric point of a protein can provide important data for comparison of a candidate biopharmaceutical to a reference drug in order to demonstrate biosimilarity. Aliquots of manufactured biosimilar candidates and reference drugs can be loaded into the disclosed apparatus 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 biosimilar candidate and the reference drug. In addition, monitoring and recording the current trace for each individual separation reaction (as well as other operating parameters for performing the isoelectric focusing reaction) helps comply with FDA data submission requirements.

[0230] Although 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 only as examples. Without departing from the present invention, those skilled in the art will now appreciate many variations, modifications, and alternatives. It will be understood that in practicing the present invention, various alternatives of the embodiments of the present invention described herein may be employed in any combination. The appended claims are intended to define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are thus covered.

[0231] Example 3 Microfluidic Device Including Side Ports

[0232] Figure 2A schematic top-down view of a non-limiting example of a microfluidic device for performing one or more separation reactions (for example, isoelectric focusing reaction) is provided. The device comprises substrate 201, which can be substantially planar, wherein uses for example imprinting, laser micromachining or photoetching and wet chemical etching to manufacture the fluid channel measured as the width of 210 microns and the depth of 100 microns. The fluid channel can be sealed by being bonded to a transparent cover glass (not shown) by substrate 201. In some cases, for example, when UV absorbance imaging is used to monitor separation and / or mobilization reaction, substrate 201 can be manufactured by optically transparent materials. In some cases, for example, when using epifluorescence imaging to monitor separation and / or mobilization reaction, substrate 201 can be manufactured by optically opaque materials.

[0233] Access to the fluidic channels within the device is provided by a sample inlet port 207, which can be located on the side of the chip. The chip can also include electrode reservoirs (e.g., anode wells 206, cathode wells 204), sample outlet ports 203, and chemical mobilization agent inlet ports 209. An anode well 206 and cathode well 204 are in fluid and electrical communication with the proximal and distal ends of the separation channel 205, respectively. The chemical mobilization agent inlet port 209 is connected to the distal end of the separation channel 205 via a chemical mobilization channel.

[0234] In order to perform multiple isoelectric focusing reactions to separate protein mixtures, protein samples are premixed with an ampholyte pH gradient and pI markers, and then placed in vials and loaded onto the autosampler. The samples are sequentially loaded into the device by the autosampler via the sample inlet port 207, loaded onto the microfluidic device through the separation channel 205, and discharged from the device to waste through the sample outlet port 203.

[0235] A catholyte fluid (e.g., 1% N4OH in H2O) is loaded into the cathode well 204, an anolyte (e.g., 10 mM H3PO4) is loaded into the anode well 206, and a mobilizing agent solution (e.g., 49% MeOH, 49% H2O, 1% acetic acid) is connected to the mobilizing agent inlet port 209. A membrane (not shown) can be attached to any of the anode wells or cathode wells (206 and 204) to provide electrical and fluid communication between the device and the electrodes. An isometric cross-sectional schematic of the sample outlet or ESI tip 203 is shown in FIG3.

[0236] Reference Figure 2After all reagents are loaded, an electric field of, for example, +600 V / cm is applied from one or more anode recesses 206 to the corresponding cathode recesses 204 by connecting the electrodes to the electrode reservoirs (anode recesses 206 and cathode recesses 204) to initiate isoelectric focusing. As described above, the voltage and / or current applied to each separation channel 205 can be independently controlled and can also be recorded as a function of time. In some cases, the electrodes for the anode and cathode can be integrated with the device. For UV absorbance imaging, the collimated light beam provided by the UV light source is aligned with the separation channel 205, and an image sensor (e.g., a CCD camera or a CMOS camera) is placed on the other side of the separation channel 205 to measure the amount of light transmitted through each separation channel 205, thereby imaging and detecting the focused proteins (or other separated analytes) by means of their absorbance. In some cases, the focused proteins can be unlabeled and detected by their natural absorbance at 220 nm, 280 nm, or any other wavelength at which the proteins absorb light. For fluorescence imaging, i.e., epifluorescence imaging, excitation light of a suitable wavelength is delivered to the separation channel 205 by means of an optical assembly including a suitable dichroic reflector and a bandpass filter, 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, native fluorescence can be used to image and detect the focused protein (or other separated analyte). In some cases, non-covalently bound fluorescent tags, chromogenic tags, fluorogenic tags, or chromophore tags (e.g., In some cases, portions of the device can be constructed of optically opaque materials so that light can only be transmitted through the separation channel 205, 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.

[0237] As isoelectric focusing reactions are performed in multiple separation channels 205, images of the focused proteins in all or a portion of the separation channels 205 can be captured continuously and / or periodically. In some cases, positional detection of pI markers in the images of the separation channels 205 can be used to determine the local pH as a function of position along the separation channel and, by extrapolation, to make a more accurate pI determination for the separated proteins (or other analytes). In some cases, when focusing is complete, a positive pressure is applied at the sample inlet port 207 and / or the anode well 206 to mobilize the separated protein (or other analyte) mixture toward the sample outlet 203. In some cases, when focusing is complete, the electrode connected to the cathode well 204 is disconnected, and an electrode in electrical communication with the mobilizer channel 208 is used to apply an electric field of 600 V / cm from the anode well 206 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 the electrophoretic introduction of the chemical mobilizing agent.

[0238] When a mobilizer is introduced electrophoretically, acetic acid in the mobilizer solution is attracted by the electric field into the separation channel 205, where it ionizes the proteins and ampholytes and disrupts the pH gradient used for isoelectric focusing. The ionization of the enriched protein fractions causes them to migrate out of the separation channel 205. Continued imaging of the separation channel 205 during the mobilization process can be used to refine the determination of the pI for each separated protein.

[0239] Example 4 Waste Management Using Vacuum Equipment

[0240] Figure 4 A schematic diagram of an exemplary waste management system is provided. In some cases, the waste management system can be used to direct waste away from the microfluidic device 401. In some cases, the waste management system can also be used to prevent droplets from wicking to another part of the device 401. Figure 4As shown, device 401 can be coupled to stand 405. In some cases, microfluidic device 401 can be inserted into a cartridge, which can then be coupled to stand 405. Stand and / or cartridge can include pipes and accessories to be fluidically and / or electrically connected to device 401. Waste management system can include vacuum equipment 407. Vacuum equipment 407 can have a shape like a trumpet and can be configured to apply vacuum to the tip of device 401. In some cases, device 407 can be configured to attach to a portion of device 401 using an adapter (e.g., flange 403). Flange 403 can include a slit through which a portion of device 401 (schematically shown as a pointed tip) can be assembled. Vacuum equipment 407 can be configured to swivel or move to different positions. For example, in a first configuration, device 407 can be guided toward a waste container, thereby guiding waste products to the waste container when vacuum 409 is applied. In a second configuration, the device 407 may be rotated so that the sample or analyte may be directed, for example, to a separate analysis unit, such as a mass spectrometer.

[0241] In some cases, stage 405 can be configured to move device 401. For example, the stage can allow for translation of device 401 in a direction that can be substantially parallel to one or more channels of device 401. In some cases, stage 405 can allow for translation of device 401 in one or more directions. For example, stage 405 can allow for translation of device 401 in a direction substantially parallel to one or more channels of device 401 and in a direction substantially perpendicular or orthogonal to one or more channels of device 401. Stage 405 can be configured to adjust the position of device 401 so that device 401 can be integrated with a downstream analytical unit (e.g., a mass spectrometer).

[0242] Figure 5 A schematic diagram of another exemplary waste management system is provided. Figure 4Similar to the example shown, a waste management system can be used to direct waste away from the microfluidic device 501 and / or prevent droplets from wicking to another part of the device 501. The device 501 can be coupled to a stand 505. In some cases, the microfluidic device 501 can be inserted into a cartridge, which can in turn be coupled to the stand 505. The stand and / or cartridge can include tubing and accessories to fluidically and / or electrically connect to the device 501. The waste management system can include a vacuum device 507. The vacuum device 507 can have a shape like a cylinder and can be configured to apply a vacuum to the tip of the device 501. In some cases, the device 507 can be configured to attach to a portion of the device 501 using an adapter (e.g., flange 503). The flange 503 can include a slit through which a portion of the device 501 (schematically shown as a tip) can be mounted. The vacuum device 507 can be used to apply a vacuum 509 and direct waste products to a waste container. For example, vacuum apparatus 507 can be placed between the chip (e.g., joined using flange 503) and the mass spectrometer, and vacuum apparatus 507 can include an opening module, e.g., vacuum sleeve 511, to direct waste products away from apparatus 501 so that waste products do not reach the mass spectrometer. Vacuum 509 can be applied to vacuum apparatus 507 and can aspirate droplets as they leave apparatus 501. In some cases, vacuum apparatus 507 can be transparent so that the electrospray tip can be imaged using one or more imaging systems as described herein.

[0243] In another embodiment, the vacuum can be applied by a modular device that can be configured to attach to a vacuum. Figure 6 Yet another example of a waste management system is schematically shown that includes a gripper module 615. A gripper can be secured to the device 601 and a vacuum 609 can be applied to the gripper 615 to direct waste products away from the microfluidic device. In some cases, the device 601 can be coupled to a stand 605 or inserted into a cartridge that can be coupled to the stand 605. In another example, Figure 7 Another example of a waste management system is schematically shown, including a tube vacuum device 707. A tube can be positioned adjacent to the device 701 to direct waste products away from the microfluidic device 701. A vacuum 709 can be applied to the tube vacuum device 707 to direct waste products away from the microfluidic device. In some cases, the tube can be positioned substantially orthogonal to the device 701 so that waste products can be directed away from the microfluidic device without interfering with downstream analytical units, such as a mass spectrometer. The device 701 can be coupled to or positioned on a stand 705.

[0244] Example 5 Waste Management Using Positive Pressure

[0245] In some cases, the waste management module may include the use of positive pressure. Figure 8 In the embodiment of the present invention, the air knife 807 can be used to direct the droplets away from the device 801. In such an example, the air knife 807 can be connected to an air source and / or a booster to generate air pressure to spray the droplets or direct the droplets away from the device 801. In some cases, the system can also include a vacuum unit (not shown) that can be used to collect the droplets that are directed away from the device 801. Figures 4 to 7 , the device 801 can be coupled to a stage 805 or can be coupled to a cartridge, which can be coupled to the stage 805.

[0246] In some cases, the waste management module can include an atomization unit. For example, the atomizer can be configured to be fixed to the chip. The atomizer can include the necessary geometry to direct air toward the chip so that droplets or waste products are directed away from the electrospray tip or outlet (e.g., to a waste container).

[0247] Figure 9 An exemplary atomizer 907 is schematically shown, which can be configured to be coupled to or fixed to the device 901. The atomizer 907 can include a chamber and an inlet that can direct air or nitrogen to the interior of the atomizer 907. The atomizer can additionally include a slit to direct air or nitrogen (e.g., via a nozzle, funnel, etc.) out of the atomizer. The atomizer can be configured to direct the air or nitrogen toward an outlet of the device 901, and thereby direct the waste product away from the device 901. In some embodiments, the atomizer 907 can be used to atomize the waste product.

[0248] 10A to 10D Schematically illustrates additional examples of designs for atomizer 1007. Atomizer 1007 may be configured to be coupled to or secured to device 1001. Similar to Figure 9 , the atomizer 1007 can include a chamber and an inlet that can direct air or nitrogen into the interior of the atomizer 1007. The atomizer can additionally include a slit to direct air or nitrogen out of the atomizer (e.g., via a nozzle, funnel, etc.). The atomizer can be configured to direct air toward an outlet of the device 1001 and thereby direct waste products away from the device 1001. In some embodiments, the atomizer 1007 can be used to atomize waste products.

[0249] 11A to 11D Yet another example atomizer 1107 is schematically shown. The atomizer 1107 can be configured to be coupled to or fixed to the device 1101. Similar to Figure 910 , the atomizer 1107 may include a chamber and an inlet that may direct air or nitrogen to the interior of the atomizer 1107. The atomizer may additionally include a slit to direct air or nitrogen (e.g., via a nozzle, funnel, etc.) out of the atomizer. The atomizer may be configured to direct air toward the outlet of the device 1101 and thereby direct waste products away from the device 1101. In some embodiments, the atomizer 1107 may be used to atomize waste products. The atomizer 1107 may also include a fastener 1113, such as a screw, to secure the atomizer 1107 to the stand 1105, or in some embodiments to a cartridge (not shown).

[0250] Example 6: Holder with membrane (electrode interface unit)

[0251] Figure 12 Schematically illustrate the example of fixture 1200, for example, those described herein.Fixer 1200 can be configured to engage one or more electrodes with one or more parts (for example, one or more inlets of microfluidic device) of system.Electrode can be configured to engage with multiple reservoirs 1203, and described multiple reservoirs 1203 can be via being connected by the valve 1205 that comprises device and device or cartridge fluid and be communicated with electricity.Reservoir 1203 can comprise for being used in separation reaction and / or mobilization reaction reagent and / or buffer solution.In some cases, fixture 1200 can comprise one or more membranes (not shown), and it allows to be electrically communicated for electrode and microfluidic device or cartridge via being connected by valve 1205.For example, film can be positioned at the bottom of each reservoir 1203, and described membrane can allow to be electrically communicated for reservoir and electrode via being connected by valve 1205 and device or cartridge fluid and be communicated with electricity, and via being connected by valve 1205 and reducing the incidence of forming bubble at the interface of reservoir and device or cartridge.

[0252] In some cases, the geometry of the holder can be configured to position the membrane to establish fluidic and / or electrical communication with the microfluidic device. 13A to 13F A portion of a holder 1300 including a membrane (not shown) is schematically shown. Figure 13AIn the embodiment of the present invention, the portion of the holder 1300 can include an insert, for example, a U-shaped structure 1305, which can be connected to the reservoir 1303 and allows fluid and electrical communication with the membrane and microfluidic device (not shown). The holder 1300 includes an inlet fluid channel 1304, which is fluidically coupled to an outlet fluid channel 1306, which is coupled to a separation channel (not shown). The inlet fluid channel 1304 and the outlet fluid channel 1306 intersect at a plane 1308, which defines or is parallel to the surface of the reservoir 1303. At or adjacent to the plane 1308, a membrane (not shown) can be positioned. The membrane can cover all or substantially all of an opening, and the opening includes the intersection (e.g., plane 1308) of the inlet fluid channel 1304 and the outlet fluid channel 1306.

[0253] Figure 13B A view schematically showing the bottom portion of the holder where the membrane can be positioned. Figure 13C A cross-sectional view is provided of an insert including a U-shaped structure 1305. The U-shaped structure 1305 of the insert includes an inlet fluid path 1310 and an outlet fluid path 1312, which can promote substantially bubble-free wetting of the membrane. Figure 13D A schematic diagram of the bottom of this portion of the holder is provided. The membrane can fluidically and / or electrically connect two ports 1307, which can be connected (e.g., via a fluid inlet path 1310 and / or a fluid outlet path 1312) to the reservoir 1303 of the holder 1300. Figure 13E Another view of a U-shaped structure 1305 is provided. The U-shaped structure 1305 may include or be coupled to a membrane 1309. A port 1307 may be used to establish fluid and electrical communication with the reservoir 1303 and the membrane 1309 via an inlet fluid path 1310 and / or an outlet fluid path 1312. Figure 13F Another view of the U-shaped structure 1305 is shown. The membrane 1309 can be coupled to or connected to a port 1311, which can be connected to a channel 1315. In some cases, the channel 1315 can include an inlet fluid channel (the region before the U-shaped structure) and an outlet fluid channel (the region after the U-shaped structure), which can be connected to a microfluidic device or separation channel. This connection can establish fluid and electrical communication with a reservoir (not shown) and the channel 1315.

[0254] Example 7 Reservoir filling

[0255] Figure 14Schematically illustrate the illustrative method that reagent is provided to one or more reservoirs 1403 of system described herein.The reservoir 1403 that can be a part for fixture (for example, 1200 and 1300) can be filled with damping fluid or the reagent that for example is used for separating reaction and / or mobilization reaction.In some cases, what can be expected is to fill reservoir from the bottom of reservoir.Reagent and / or damping fluid can be introduced via inlet fluid channel 1404.In some cases, reservoir 1403 can be configured to move (for example, via translation) so that reservoir 1403 can move to upward configuration, be filled and then be resealed by making reservoir 1403 move back to initial configuration.Outlet fluid channel 1406 can be connected (for example, via port and / or inlet or outlet fluid channel) with device or separation channel fluidly and / or electrically.

[0256] In some cases, the reservoir can be filled using conventional methods. Figure 15 Schematically illustrates an exemplary method of providing reagents to one or more reservoirs 1503. In such an example, a pipette (e.g., a pipette, a micropipette, etc.) 1519 can be used to introduce buffer or reagents into the reservoir 1503. In some embodiments, for example, the reservoir 1503 can also include a side port 1521. Introducing buffer or reagents via the side port 1521 can help prevent air bubbles from being trapped on top of the membrane at the bottom of the reservoir 1503.

[0257] Example 8 Cartridge Design

[0258] Figure 16Schematically illustrate the exemplary system comprising cartridge, as described in some embodiments of this paper.Cartridge 1600 can include microfluidic device 1601, which can include multiple inlet ports 1602.Port 1602 can be electrically and fluidically communicated with port 1623, and the port 1623 can be connected to a high voltage power supply (for example, being connected to a high voltage power supply via an electrode, the electrode being connected to one or more reservoirs 1603).Port can be electrically and fluidically communicated with the reservoir 1603 fluids comprising reagent or buffer (for example, anolyte, cathode liquid, mobilizing agent and background electrolyte).Valve 1625 can be used to control the flow of reagent or buffer from reservoir 1603.In some cases, one or more reservoirs 1603 also can be connected to restrictor 1627 (for example, long pipeline), which can stabilize flow rate and / or the flow distribution from reservoir 1603 to valve 1625 to port 1623. Each reservoir 1603 can include a different reagent or buffer; for example, one reservoir can contain an anolyte buffer, another reservoir can contain a catholyte buffer, and yet another reservoir can contain a mobilization buffer. The cartridge 1600 or device 1601 can also be connected to a sample line that can be used to supply a sample to the cartridge 1600 or device 1601 via valve 1625.

[0259] Figures 17 to 20 An exemplary embodiment of a cartridge is shown. Figure 17 17. In the embodiment of the present invention, cartridge 1700 may include a reservoir 1703, which may be coupled to membrane 1709; a tube 1711; and a plug 1713, which may seal the reservoir and include, for example, a hole for inserting an electrode or for filling the reservoir. Cartridge 1700 may further include a channel 1718 for inserting and / or injecting a sample. Device 1701 may be secured to cartridge 1700 using a post feature 1715. Figure 18 Another example of a cartridge is shown. An adapter 1817 can be coupled to the cartridge to easily fill the reservoir 1803. Figures 19A to 19B Yet another example of a cartridge including a valve is shown. Figure 19A In the embodiment, a stopcock valve 1921 can be integrated with each reservoir 1903, which can allow for flow rate control. Figure 19B In the embodiment, a sliding valve 1923 can be integrated with each reservoir 1903. Figure 20 Another exemplary embodiment of a cartridge is shown. Device 2001 can be fixed to cartridge 2000 and fluidically and / or electrically connected to a reservoir (not shown) and / or sample via a number of ports 2002 and / or inlet or outlet fluid channels. The fluid connection can be secured using a gasket 2030 or an O-ring.

[0260] Figure 21Schematically illustrate examples of fixing features that can be used to secure the device to the cartridge. The cartridge 2100 can include screws 2129 that can be used to secure the device 2101 to the cartridge 2100. The cartridge 2100 can include one, two, three, or more screws 2129. In some cases, nylon-tipped screws can be used. In some cases, a pressure plate (e.g., a washer) can be added between the screws 2129 and the device 2101 to evenly distribute stress and prevent stress concentration on the device 2101 or cartridge 2100.

[0261] Figure 22 An exemplary schematic diagram of a fixing feature that can be used to create a fluid-tight seal between a cartridge and a device is shown. The cartridge 2200 can include screws 2229 that can be used to secure the device 2201 to the cartridge 2200. The cartridge 2200 can include one, two, three, or more screws 2229. In some cases, the cartridge 2200 can include a gasket 2231. The gasket 2231 can engage with the device 2201 and form a seal around the inlet or outlet port 2202 of the device 2201. In some embodiments, the inlet or outlet port 2202 can be secured to the cartridge 2200 using an O-ring.

[0262] Figure 23 The electrical connection of one or more reservoirs to the device is schematically shown. Electrode 2333 may comprise platinum wire and may be secured in place using, for example, an adhesive or other fastening mechanism, as described elsewhere herein. Electrode 2333 may be in contact with reservoir 2303, thereby establishing electrical communication with the reservoir, which may be in contact with a microfluidic device, as described herein.

[0263] Figure 24 The coupling of an instrument to a cartridge is schematically shown.The cartridge 2400 may be coupled to an instrument that may be used to provide reagents to the reservoir of the cartridge via fluidic channels 2435.

[0264] Figures 25 to 27 Additional example cartridges are shown having different reservoir configurations. Figure 25 An embodiment is shown in which the cartridge comprises a plurality of oblong receptacles 2503 . Figure 26 An embodiment is shown in which the cartridge includes an oblong reservoir 2603. The reservoir 2603 can be tilted at an angle so that the reservoir 2603 is positioned sufficiently away from the outlet or tip of the device 2601. The device 2601 can be connected to the high voltage reservoir 2605 using a membrane 2609. In some cases, the membrane 2609 can be mechanically pressed between the high voltage reservoir 2605 and the device 2601. In some cases, the device 2601 can be sealed to one or more fluid channels 2631 using a gasket. Figure 27 An embodiment is shown in which the cartridge includes an additional manifold unit 2735. The manifold unit may include a reservoir 2703. The manifold unit 2735 may be coupled to the cartridge using the fastening mechanisms described herein (e.g., screws and threads).

[0265] As described herein, the cartridge can include a reservoir, reagent, membrane, valve, fixture or feature (e.g., a screw, a pin (e.g., a spring pin), an adhesive, a lever, a switch, a groove, a form-fitting pair, a hook and loop, a latch, a thread, a clip, a clamp, a pin, a ring, a rubber band, a rivet, a grommet, a tie, a snap, a tape, a vacuum, a seal), a gasket, an O-ring, an electrode, or a combination thereof. The cartridge can be integrally constructed or can be modular and include removable parts. For example, the microfluidic device can be configured to be removably coupled to the cartridge. Similarly, the reservoir, membrane, valve, etc. can all be removable from the cartridge. Where one or more components can be removable, the cartridge can be configured so that each of the various components can be aligned in place by the user with sufficient tolerance. For example, the cartridge can include grooves and pins so that the microfluidic device can be integrated by sliding the device along the cartridge until the cartridge reaches the pin for alignment. In some cases, the device can be configured to be positioned flush with the cartridge or a portion thereof. In some cases, the device can be positioned into a cartridge so that one or more inlets, outlets, etc. can be connected (e.g., fluidically and / or electrically) to a reservoir, electrodes, membrane, and / or other useful interface units. In some cases, the engagement of the device and reservoir, electrodes, etc. can be performed by the user without any additional measurements or adjustments. For example, the reservoir can be configured to receive electrodes that snap into place or are secured via spring pins to establish electrical and / or fluid communication. It will be understood that these exemplary configurations of cartridges and devices are not meant to be limiting, and many different configurations of positioning the microfluidic device or other components of the cartridge can be implemented.

[0266] Example 9 Imaging System

[0267] 28A to 28DDifferent perspective views of an exemplary imaging system disclosed herein are shown. The imaging system can be used for full channel imaging or full device imaging or imaging of multiple channels of a device. In some cases, the device can be fixed to a cartridge 2800. The cartridge can include a transparent portion. In some cases, the cartridge 2800 can be positioned near an illumination source, such as a UV illuminator 2850. The UV illuminator 2850 can be used to illuminate the device, and light can be collected via a detector 2857, such as a camera. In some cases, a mirror 2855, such as a steering mirror, can be used to direct light to the detector 2857. The imaging system can also include a second detector 2859, which can include a camera that can be used for electrospray imaging. In some cases, the imaging system can include an illumination ring 2861. The device can be configured to guide a sample or analyte to a downstream analysis unit, such as a mass spectrometer, via electrospray ionization, as described herein.

[0268] Example 10 System Configuration

[0269] Figures 29 to 31 An example of the system described herein is shown schematically. Figure 29 Examples of instruments configured to perform one or more reactions described herein are shown, for example, separation of analytes via isoelectric focusing, mobilization of analyte peaks, and downstream analysis via mass spectrometry. In certain embodiments, the system may include an automatic sampler 2901 that can be used to process and / or detect samples, which can be located in a separation unit 2903 that can include devices for isoelectric focusing, mobilization, and the like. The system may include a compliance mechanism 2905 and a downstream analysis unit 2907 that can assist the devices in the separation unit 2903 in engaging. In some cases, the downstream analysis unit 2907 is a mass spectrometer. In some cases, the system may be located on a motorized lifting arm 2909 that can be used to move any of the components described herein. For example, the lifting arm 2909 can be used to lift and lower the automatic sampler 2901, the separation unit 2903, the compliance mechanism 2905, and / or the analysis unit 2907 (e.g., a mass spectrometer interface board).

[0270] Figure 30 Another example of an instrument configured to perform one or more reactions described herein, e.g., separation of analytes via isoelectric focusing, mobilization of analyte peaks, and downstream analysis via mass spectrometry is shown. Figure 29The system may include an automatic sampler 3001, which can be used to process and / or detect samples, which can be located in a separation unit 3003, which can include devices for isoelectric focusing, mobilization, etc. The system may include a compliance mechanism 3005 and a downstream analysis unit 3007, wherein the compliance mechanism 3005 can help the devices in the separation unit 3003 to engage. In some cases, the downstream analysis unit 3007 is a mass spectrometer. In some cases, the system can be located on a motorized lifting arm 3009, which can be used to move any of the components described herein. For example, the lifting arm 3009 can be used to lift and lower the automatic sampler 3001, the separation unit 3003, the compliance mechanism 3005 and / or the analysis unit 3007 (e.g., a mass spectrometer interface board). In some cases, the system may also include one or more computers or computer processors 3011.

[0271] Figure 31 Examples of instruments configured to perform one or more reactions described herein are shown, for example, separation of analytes via isoelectric focusing, mobilization of analyte peaks, and downstream analysis via mass spectrometry. In certain embodiments, the system may include an automatic sampler 3101 that can be used to process and / or detect a sample, which may be located in a separation unit 3103 that may include devices for isoelectric focusing, mobilization, and the like. A second system may be arranged adjacent to or further away from the system and may include a separation unit 3103, a compliance mechanism 3105 that can help the devices in the separation unit 3103 engage, and a downstream analysis unit 3107. In some cases, the downstream analysis unit 3107 is a mass spectrometer. In some cases, the system may also include one or more computers or computer processors 3111 that may be coupled to the automatic sampler 3101.

[0272] Example 11 - Mobility Chromatogram

[0273] FIG. 32A to FIG. 32B Exemplary data of mobilization reaction and mobility chromatogram are shown. Full channel imaging can be performed during the separation (e.g., via isoelectric focusing) of samples comprising protein isomers. For example, biotherapeutics (e.g., antibody therapeutics) can be separated along a pH gradient (e.g., a pH 5 gradient to a pH 10.5 gradient) using isoelectric focusing. After the separation reaction, a mobilization reaction (e.g., the separated analyte is directed to a downstream analysis unit such as a mass spectrometer) can be performed. Full channel imaging of the mobilization reaction can be performed over time, and a portion of each image can be used to generate a chromatogram. Figure 32AShown are absorbance measurements for a channel of the device as a function of pixel number (or distance) along the channel. Each pixel corresponds to approximately 25 microns along the length of the separation channel. Figure 32B By plotting the Figure 32A 3-pixel-wide segment 3205 of an image or absorbance graph. In such an example, plotting the 3-pixel-wide segment 3205 can function as a point detector, thereby generating information about the activity of the analyte peak as a function of time and allowing for better correlation and / or verification of data obtained from a downstream analytical unit (e.g., a mass spectrometer).

[0274] Example 12 Computer System

[0275] Figure 33 An exemplary software architecture system is shown. The software architecture system can be integrated with the system disclosed herein and can include one or more computer processors. In some cases, one or more computer processors can be configured to collect and / or analyze data. The software architecture system can include a computer processing unit, which includes a controller service that can communicate with a first-in, first-out (FIFO) database. In some cases, the FIFO database can communicate with a second computer processor, which can include a graphical user interface and a server database. The second computer processor can, for example, communicate with a customer database via the cloud. In some cases, the computer processing unit can communicate with one or more hardware units of the system (e.g., via a wired or wireless connection). For example, the computer processing unit can be connected to a stand, one or more cameras, a high voltage power supply, an automatic sampler, a flow control system (e.g., software and hardware for microfluidic flow control, e.g., Fluigent Inc.) and / or other laboratory equipment via a USB hub.

[0276] Example 13 Integrated System

[0277] Figure 34An exemplary block diagram of an integrated system is shown. The integrated system may include one or more systems disclosed herein. The system may include an interface cartridge 3407, which may be in fluid and / or electrical communication with a plurality of reservoirs 3403. For example, the interface cartridge 3407 may be connected to an anolyte reservoir, a cathode reservoir, a mobilizing agent reservoir, and an autosampler unit. Alternatively or in addition, the interface cartridge 3407 may be in fluid and / or electrical communication with a pressure control manifold 3405, which may be coupled to a fluid drive mechanism, such as a pump. The interface cartridge 3407 may be coupled to a cartridge 3400, which may include a device 3401. The device 3401 may be in electrical and / or fluid communication with an anolyte high voltage reservoir, a cathode high voltage reservoir, a mobilizing agent high voltage reservoir, and a sample line. The anolyte high voltage reservoir, the catholyte high voltage reservoir, the mobilizing agent high voltage reservoir, and the sample line can each be in fluid and / or electrical communication with the interface cartridge 3407. The device 3401 can also be coupled to a waste management unit 3409, which can be used to direct waste away from the device 3401 and, in some cases, to direct samples to a downstream analysis unit 3411. In some embodiments, the waste management unit 3409 can include a nebulizer. In some cases, the downstream analysis unit 3411 can include a mass spectrometer.

[0278] The system can also include multiple imaging systems. For example, the system can include an imaging system 3415, which can include a camera, an illuminator, a waste container, and / or an adapter that can be used to engage with the analysis unit 3411. The system can also include an imaging system 3417, which can 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) can be connected to a cooling source, such as a fan or other temperature control platform.

[0279] Figure 35An exemplary block diagram of an integrated system is shown. The system may include: a sample 3501, a sample and reagent holder and / or a processor 3503, which may be configured to store and process samples (e.g., mix, add reagents, aspirate or dispense samples, etc.); a sample syringe 3505; and a sample tip cleaner 3507. The sample tip cleaner may include a mechanism for cleaning the sample and / or the system. The system may also include a separation unit 3509, which may include a cartridge containing the device, an imaging system (e.g., a UV illuminator and a camera). The separation unit may be coupled to a plurality of controllers 3511, which may include fluid control using negative pressure (e.g., vacuum) or positive pressure (e.g., rotary or diaphragm pumps, valves, etc.). The controller 3511 and / or the separation unit 3509 may be coupled to a fluid manifold 3513, which may include one or more reservoirs containing reagents.

[0280] The separation unit 3509 can be used to perform separation reactions (e.g., isoelectric focusing) and / or mobilization reactions. The separation unit 3509 can be connected to or coupled to a communication interface 3515 (e.g., RFID), a high voltage power supply 3517, a waste management unit 3519 (e.g., vacuum and waste container), another imaging unit 3521, and / or a downstream analysis unit 3523 (e.g., a mass spectrometer). In some cases, the separation unit 3509 can be coupled to a temperature control unit 3525. In some cases, one or more systems described herein can include a temperature control unit 3527 and / or other control units, e.g., for instrument control 3529.

[0281] Example 14: Tracking velocity of analyte as it leaves the microfluidic chip and enters the mass spectrometer

[0282] exist Figure 1BThe microfluidic channel network 100 in the device shown is manufactured in an opaque cycloolefin polymer layer of 250 microns thick. The depth of channel 112 is 250 microns, so channel 112 passes through the layer of 250 microns always. The depth of all other channels is 50 microns. The channel layer is sandwiched between two transparent cycloolefin polymer layers, to make a planar microfluidic device. Ports 102, 104, 106, 108 and 110 provide access to the channel network for introducing reagents from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, allowing channel 103 to serve as a waste channel, thereby being able to perfuse other reagents with "waste" by the channel network. Acid (e.g., 1% formic acid) is perfused into channels 109, 112, 114 and 103 and exhaust port 102 by port 108. The 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 poured into channels 107, 112, 114, and 103 through port 106 and drained into port 102. This leaves channel 112 containing the sample analyte. A base (e.g., 1% dimethylamine) is poured into channels 105, 114, and 103 through port 104 and drained into port 102. A mobilizing agent (e.g., 1% formic acid, 49% methanol) is poured into channels 111, 114, and 103 through port 110 and drained from channel 103 into port 102.

[0283] Electrophoresis of the analyte sample in channel 112 is performed by applying 4000V to port 108 and grounding port 110. The ampholytes in the analyte sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed using a 280 nm light source aligned with channel 112 and measuring the transmission of 280 nm light through channel 112 with a CCD camera. The software calculates absorbance by comparing the light transmission during the separation or activation period with a "blank" reference measurement taken without focused analyte before the analyte run, and then displays the absorbance for each pixel along the length of channel 112. Locations of the standard or focused analyte are displayed as peaks in the absorbance trace derived from the image data.

[0284] Once the focusing of the analyte has been completed, the absorbance image of the final focus is captured. The software will identify the spatial position of the pI mark and insert between the marks to 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 110 and ground port 104, and set pressure on the mobilization agent reservoir connected to port 104 to establish a flow of 100nL / min of mobilization agent solution flowing into channels 105 and 114 through port 104 and outflowing the chip at orifice 116. When the inlet voltage is -3500V to -4500V, orifice 116 is positioned 2mm away from the mass spectrometer ESI inlet.

[0285] While the pressure-driven flow directs the mobilizer from port 104 to orifice 116, some of the formic acid in the mobilizer reagent will electrophoresce as formate from channel 105 through channel 112 to the anode at port 108. As the formate travels through channel 112, the formic acid will disrupt the isoelectric pH gradient, causing the ampholytes, standards, and analyte sample to increase in charge and electrophoretically migrate out of channel 112 into channel 114, where the pressure-driven flow from port 110 will carry them into the ESI spray exiting orifice 116.

[0286] As activation occurs, the software continues to capture absorbance images and identify peaks, tracking their migration out of imaging channel 112 and into channel 114. By tracking the time taken by each peak to leave imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time taken by the peak to traverse channel 114 and be introduced into the mass spectrometer via orifice 116, allowing direct correlation between the originally focused peak and the resulting mass spectrum.

[0287] Example 15 Microfluidic Device for Electrospray and Sample Processing

[0288] Figure 2 A schematic top-down view of a non-limiting example of a microfluidic device for performing one or more separation reactions (for example, isoelectric focusing reaction) is provided. The device comprises substrate 201, wherein uses for example imprinting, laser micromachining or photoetching and wet chemical etching to manufacture the fluid channel being measured as the width of 210 microns and the depth of 100 microns. The fluid channel can be sealed by being bonded to a transparent cover glass (not shown) by substrate 201. In some cases, for example, when UV absorbance imaging is used to monitor separation and / or mobilization reaction, substrate 201 can be manufactured by optically transparent materials. In some cases, for example, when using epifluorescence imaging to monitor separation and / or mobilization reaction, substrate 201 can be manufactured by optically opaque materials.

[0289] Access to the fluidic channels within the device is provided by a sample inlet port 207, which can be located on the side of the chip. The chip can also include an anode well 206, a cathode well 204, a sample outlet port 203, and a chemical mobilization agent inlet port 209. An anode well 206 and a cathode well 204 are in fluid and electrical communication with the proximal and distal ends of the separation channel 205, respectively. The chemical mobilization agent inlet port 209 is connected to the distal end of the separation channel 205 via a chemical mobilization channel.

[0290] In order to perform multiple isoelectric focusing reactions to separate protein mixtures, protein samples are premixed with an ampholyte pH gradient and pI markers, and then placed in vials and loaded onto the autosampler. The samples are sequentially loaded into the device by the autosampler via the sample inlet port 207, loaded onto the microfluidic device through the separation channel 205, and discharged from the device to waste through the sample outlet port 203.

[0291] A catholyte fluid (e.g., 1% N4OH in H2O) is loaded into the cathode well 204, an anolyte (e.g., 10 mM H3PO4) is loaded into the anode well 206, and a mobilizing agent solution (e.g., 49% MeCN, 49% H2O, 1% formic acid) is connected to the mobilizing agent inlet port 209. A membrane (not shown) can be attached to any of the anode wells or cathode wells (206 and 204) to provide electrical and fluid communication between the device and the electrodes. An isometric schematic of the sample outlet or ESI tip 203 is shown in FIG3.

[0292] Reference Figure 2After all reagents are loaded, an electric field of, for example, +600 V / cm is applied from one or more anode recesses 206 to the corresponding cathode recesses 204 by connecting electrodes to the anode recesses 206 and the cathode recesses 204 to initiate isoelectric focusing. As described above, the voltage and / or current applied to each separation channel 205 can be independently controlled and can also be recorded as a function of time. In some cases, the electrodes for the anode and cathode can be integrated with the device. For UV absorbance imaging, the collimated light beam provided by the UV light source is aligned with the separation channel 205, and an image sensor (e.g., a CCD camera or a CMOS camera) is placed on the other side of the separation channel 205 to measure the amount of light transmitted through each separation channel 205, thereby imaging and detecting the focused proteins (or other separated analytes) by means of their absorbance. In some cases, the focused proteins can be unlabeled and detected by their natural absorbance at 220 nm, 280 nm, or any other wavelength at which the proteins absorb light. For fluorescence imaging, i.e., epifluorescence imaging, excitation light of a suitable wavelength is delivered to the separation channel 205 by means of an optical assembly including a suitable dichroic reflector and a bandpass filter, 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, native fluorescence can be used to image and detect the focused protein (or other separated analyte). In some cases, non-covalently bound fluorescent tags, chromogenic tags, fluorogenic tags, or chromophore tags (e.g., In some cases, portions of the device can be constructed of optically opaque materials so that light can only be transmitted through the separation channel 205, 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.

[0293] As isoelectric focusing reactions are performed in multiple separation channels 205, images of the focused proteins in all or a portion of the separation channels 205 can be captured continuously and / or periodically. In some cases, positional detection of pI markers in the images of the separation channels 205 can be used to determine the local pH as a function of position along the separation channel and, by extrapolation, to make a more accurate pI determination for the separated proteins (or other analytes). In some cases, when focusing is complete, a positive pressure is applied at the sample inlet port 207 and / or the anode well 206 to mobilize the separated protein (or other analyte) mixture toward the sample outlet 203. In some cases, when focusing is complete, the electrode connected to the cathode well 204 is disconnected, and an electrode in electrical communication with the mobilizer channel 208 is used to apply an electric field of 600 V / cm from the anode well 206 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 the electrophoretic introduction of the chemical mobilizing agent.

[0294] When a mobilizing agent is introduced electrophoretically, formic acid in the mobilizing agent solution is attracted by the electric field into the separation channel 205, where it ionizes the proteins and ampholytes and disrupts the pH gradient used for isoelectric focusing. The ionization of the enriched protein fractions causes them to migrate out of the separation channel 205. Continued imaging of the separation channel 205 during the mobilization process can be used to refine the determination of the pI for each separated protein.

[0295] As the protein fraction and ampholytes migrate out of separation channel 205 through cathode recess 204, the protein fraction and ampholytes mix with the mobilizing agent from channel 208 at intersection 210 (see Figure 38). The mobilizing agent is delivered to the sample outlet 203 at a defined flow rate (e.g., 7.5 nL / s), and the flow rate in the channel 208 corresponds to a linear velocity (e.g., 1.4 mm / s). As the enriched protein fraction and the ampholytes mix in the mobilizing agent, the new environment (reagent) causes their electrophoretic mobility to change. This produces a linear electrophoretic velocity for the ampholytes and protein fraction toward the electrode electrically connected to the mobilizing agent channel 208 (i.e., in a direction opposite to the direction of the mobilizing agent linear velocity toward the tip). In some cases, the chip network is designed so that the electrophoretic velocity of the enriched protein fraction will be less than the mobilizing agent flow velocity, so that the enriched protein fraction migrates out of the sample outlet (electrospray tip) 203 and enters the electrospray and enters the mass spectrometer for detection. In some embodiments, the chip network is designed so that the electrophoretic velocity of some or all of the ampholytes is greater than the mobilizing agent linear velocity, so that some or all of the ampholytes migrate toward the electrode electrically connected to the mobilizing agent channel 208 and are not introduced into the tip 203. In some cases, diluting the ampholyte concentration in this manner to reduce the amount of ionizable material in the electrospray can result in improved ionization of the enriched protein fraction. In some cases, the channel network can be designed to maximize the introduction of the enriched protein fraction into the electrospray and minimize the introduction of other sample components into the electrospray. In some cases, the channel network can be designed to maximize the introduction of the enriched protein fraction into the electrospray and minimize the introduction of ampholytes into the electrospray.

[0296] For example, the electrophoretic mobility of the NIST monoclonal antibody (NIST mAb) standard (pn 8671, NIST reference material) in 49% water, 1% formic acid, 50% MeCN mobilization agent has been measured to be 1.5 × 10 -4 cm 2 / Vs. In an electric field with a strength of 675 V / cm, this will cause a linear velocity (1.5×10 -4 cm 2 / Vs)×(675V / cm)=1.0×10 -1 cm / s, or 1 mm / s. In this example, if the mobilizer channel 208 is etched to a depth of 50 microns by a width of 110 microns, the channel 208 will have a volume of 5.5 nL / mm, so a mobilizer flow rate of 7.5 nL / s will correspond to a linear flow rate of 1.4 mm / s. This will overcome the NIST mAb electrophoretic velocity of 1 mm / s, and the NIST mAb will exit the chip through the sample outlet 203 into the electrospray.

[0297] Pharmalyte brand ampholyte gradient pH 8-10.5 has been measured to have an average of 2.7 x 10 -4 cm 2 / Vs electrophoretic mobility, which corresponds to an average linear velocity of 1.8 mm / s in our exemplary 675 V / cm electric field. In the example, the channel 208 has a depth of 50 microns and a width of 110 microns as described above, which will overcome the linear velocity of 1.4 mm / s of the mobilizer, and most of the ampholytes will migrate toward the electrode in electrical communication with the mobilizer channel 208 and will not leave the chip through the electrospray tip 203, thereby reducing the amount of ampholytes that can interfere with the ionization of the enriched protein fraction in the electrospray.

Claims

1. A fixator, comprising: electrode reservoir; an inlet fluid passage, the inlet fluid passage comprising a first end and a second end; an outlet fluid channel comprising a first end fluidically coupled to the second end of the inlet fluid channel and a second end fluidically coupled to a separation channel, wherein the separation channel is configured to perform isoelectric focusing, wherein the inlet fluid channel and the outlet fluid channel intersect and are fluidically coupled to each other at a plane that defines or is parallel to a surface of the electrode reservoir; and a membrane disposed within the electrode reservoir, wherein the membrane is disposed at or adjacent to the plane, wherein the membrane covers an opening of the electrode reservoir, the opening comprising an intersection of the inlet fluid channel and the outlet fluid channel; wherein the membrane provides a high hydrodynamic resistance and low electrical resistance connection between a high voltage electrode positioned within the electrode reservoir and a fluid contained within the inlet fluid channel and the outlet fluid channel; wherein the membrane comprises a first surface and a second surface, the first surface facing the electrode reservoir, the second surface facing the intersection of the inlet fluid channel and the outlet fluid channel, wherein the fluid dynamic resistance between the first surface and the second surface is greater than 1 ((N / mm 2 ) / (mm 3 / s)), and the resistance between the first surface and the second surface is less than 10,000,000 ohms; and The electrode reservoir further comprises an insert arranged within the electrode reservoir and positioned at or adjacent to the membrane, wherein the insert comprises an inlet fluid path and an outlet fluid path, which facilitate wetting the surface of the membrane without bubbles when the electrode reservoir is filled with a buffer solution.

2. The fastener according to claim 1, wherein: The membrane is hydrophilic.

3. The fastener according to claim 1, wherein: The membrane comprises a regenerated cellulose membrane.

4. The fastener according to claim 1, wherein: The cross-sectional area of ​​the membrane or opening is between 0.001 mm 2 and 100mm 2 between.

5. The fastener according to claim 1, wherein: The separation channel includes the lumen of a capillary tube.

6. The fastener according to claim 1, wherein The separation channel comprises a fluidic channel within a microfluidic device.

7. A fluid device, comprising: at least one fluid inlet; at least one fluid outlet; at least one separation channel, wherein the separation channel is configured to perform isoelectric focusing, the at least one separation channel comprising a first end fluidically coupled to the at least one fluid inlet and a second end fluidically coupled to the at least one fluid outlet; wherein at least one fluid inlet or at least one fluid outlet is electrically coupled to the high voltage electrode using a fixture, the fixture comprising: electrode reservoir; an inlet fluid passage, the inlet fluid passage comprising a first end and a second end; an outlet fluid channel comprising a first end fluidly coupled to the second end of the inlet fluid channel and a second end fluidly coupled to one of the at least one fluid inlet or the at least one fluid outlet, wherein the inlet fluid channel and the outlet fluid channel intersect and are fluidly coupled to each other at a plane that defines or is parallel to a surface of the electrode reservoir; and a membrane disposed within the electrode reservoir and disposed at or adjacent to the plane such that the membrane covers all of the opening, including the intersection of the inlet fluid channel and the outlet fluid channel; wherein the membrane provides a high hydrodynamic resistance and low electrical resistance connection between a high voltage electrode positioned within the electrode reservoir and a fluid contained within the inlet fluid channel and the outlet fluid channel; wherein the membrane comprises a first surface and a second surface, the first surface facing the electrode reservoir, the second surface facing the intersection of the inlet fluid channel and the outlet fluid channel, wherein the fluid dynamic resistance between the first surface and the second surface is greater than 1 ((N / mm 2 ) / (mm 3 / s)), and the resistance between the first surface and the second surface is less than 10,000,000 ohms; and The electrode reservoir further comprises an insert arranged within the electrode reservoir and positioned at or adjacent to the membrane, wherein the insert comprises an inlet fluid path and an outlet fluid path, which facilitate wetting the surface of the membrane without bubbles when the electrode reservoir is filled with a buffer solution.

8. The device according to claim 7, wherein The device comprises at least one capillary tube, and wherein the at least one capillary tube comprises a lumen that serves as the at least one separation channel.

9. The device according to claim 7, wherein The device is a microfluidic device comprising a planar substrate, and wherein the planar substrate comprises the at least one separation channel.

10. The device according to claim 7, wherein The membrane is hydrophilic.

11. The device according to claim 7, wherein The membrane comprises a regenerated cellulose membrane.

12. The device according to claim 7, wherein The cross-sectional area of ​​the membrane or opening is between 0.001 mm 2 and 100mm 2 between.

13. The device according to claim 7, wherein The ratio of the hydrodynamic resistance to electrical resistance between the intersection of the inlet and outlet fluid channels and the electrode reservoir is greater than 0.01 ((N / mm 2 ) / (mm 3 / s)) / Ω.

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