Microfluidic analysis of biological samples

Abstract

The present disclosure relates to microfluidic analysis of biological samples. The present disclosure features methods and systems for processing a sample, the method including introducing a sample characterized by one or more components in a first electrolyte solution into a separation channel of a fluidic device; using an electrode for applying a potential difference across the sample to cause migration of at least one sample component toward an end of the separation channel; and adjusting at least one of a gas pressure in a reservoir comprising a second electrolyte solution and a gas pressure external to an orifice located at the end of the separation channel, such that the gas pressure in the reservoir is greater than the gas pressure external to the orifice; and directing a flow of the second electrolyte solution through a pumping channel and out of the orifice in response to the gas pressure to expel at least one sample component comprising a background electrolyte solution from the separation channel and the pumping channel through the orifice.

Description

[0001] This application is a divisional application of the application filed on March 29, 2018, with application number 201880034439.2 and invention title "Microfluidic Analysis of Biological Samples".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 478,689, filed March 30, 2017, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This disclosure relates to mass spectrometry and methods for measuring mass spectrometry information using a microfluidic sample processing system. background

[0005] Mass spectrometry is widely used for the detection of chemical substances. In a typical mass spectrometry process, molecules or particles are excited or ionized. These excited substances often decompose to form smaller ions or react with other substances to form other characteristic ions. The ion formation pattern can be interpreted by the system operator to infer the identity of the compound.

[0006] Biomolecules can sometimes be challenging to analyze using mass spectrometry. In particular, the delivery and ionization of certain large biomolecules prior to mass spectrometry analysis can present significant obstacles. Jet delivery techniques can be used to process such samples, while specialized ionization methods such as electrospray ionization have been developed to generate ions that can subsequently be analyzed.

[0007] Overview

[0008] Delivering biological samples on specially designed jet chips provides a convenient and reproducible method for processing such samples. Such chips can include one or more integrated electrospray emitters for efficiently ejecting such samples, forming a uniformly and finely dispersed sample vapor that can be injected into a mass spectrometry device. Conventional techniques for flowing such samples through jet channels involve, for example, electroosmotic pumping, in which a potential is applied at the end of the flow channel to induce fluid migration through the channel.

[0009] In conventional chips that implement electroosmotic pumping, the chemical environment of the flow channel walls is carefully controlled to ensure proper sample flow through the channels. Control of the channel wall chemical environment, along with the potential difference applied between the ends of the channels, is used to adjust the electroosmotic and electrophoretic mobility of analytes and sample matrix components within the channels.

[0010] This disclosure is characterized by systems and methods that use pressure differentials instead of electroosmotic pumping to generate sample flows and prepare for the separation of sample components in microfluidic chips and other jet delivery systems. The use of pressure differentials allows sample components to be delivered through flow channels under an applied potential difference, which would otherwise be too small for component delivery to be achieved without pressure differentials. The methods and systems disclosed herein can even allow sample delivery without any applied potential difference at either end of the flow channel.

[0011] By decoupling the applied potential difference from the flow rate of the sample components through the jet chip or system, the flow rate within the flow channel can be controlled independently of the applied potential difference across the channel. This allows the separation of sample components to be performed within the desired flow rate range through the flow channel. In effect, the flow rate and degree of separation of sample components within the flow channel can be modified independently, allowing for a significantly greater degree of control over sample separation and analysis within the mass spectrometry system.

[0012] Typically, in a first aspect, this disclosure is characterized by a method for processing a sample, the method comprising introducing a sample, characterized by one or more components, in a first electrolyte solution into a separation channel of a jet apparatus; applying a potential difference across the sample to induce migration of at least one sample component toward the end of the separation channel; adjusting at least one of a gas pressure in a reservoir comprising a second electrolyte solution and a gas pressure outside an orifice located at the end of the separation channel, such that the gas pressure in the reservoir is greater than the gas pressure outside the orifice, wherein the reservoir is connected to a pumping channel and wherein the pumping channel is connected to the separation channel near its end; and guiding a flow of the second electrolyte solution through the pumping channel and out of the orifice in response to the gas pressure in the reservoir being greater than the gas pressure outside the orifice to discharge at least one sample component through the orifice.

[0013] Embodiments of the method may include any one or more of the following features.

[0014] Adjusting the gas pressure in the reservoir may include any one or more of the following: introducing gas into the reservoir, compressing the gas in the reservoir, and heating the reservoir. Adjusting the gas pressure outside the orifice may include activating a vacuum source adjacent to the orifice. The gas pressure in the reservoir may be at least 0.5 psi greater than the gas pressure outside the orifice (e.g., at least 2.0 psi greater).

[0015] The flow rate of the second electrolyte solution in the pumping channel can be 50 nL / min or greater (e.g., 200 nL / min or greater). The first and second electrolyte solutions can have a common composition. Optionally, the composition of the second electrolyte solution can differ from that of the first electrolyte solution. The concentration of one or more organic modifiers in the second electrolyte solution can be greater than the concentration of the same one or more organic modifiers in the first electrolyte solution.

[0016] The potential difference can be between 0V and 20kV (e.g., between 0V and 10kV). At least one sample component can migrate through a dead volume of 500pL or less (e.g., 100pL or less) before being discharged from the well.

[0017] The method may include obtaining information about the discharge plume generated by the discharge of a fluid, including a second electrolyte solution, through an orifice. The method may include measuring one or more of the following to obtain the information: light transmitted through the discharge plume, light reflected from the discharge plume, light scattered by the discharge plume, and light absorbed by the discharge plume. The method may also include, based on the obtained information, diverting a portion of the flow of the second electrolyte solution into the discharge channel to adjust the discharge plume.

[0018] The method may include: adjusting the gas pressure in an auxiliary reservoir comprising a third solution such that the gas pressure in the auxiliary reservoir is greater than the pressure outside the orifice, wherein the auxiliary reservoir is connected to an auxiliary channel, and wherein the auxiliary channel is connected to the separation channel near its end; and guiding the flow of the third solution through the auxiliary channel and into the separation channel in response to the gas pressure in the auxiliary reservoir being greater than the pressure outside the orifice. The third solution may include a mass spectrometry calibration compound, wherein the calibration compound is discharged from the orifice. The third solution may include a mass spectrometry coupling agent, wherein the coupling agent is discharged from the orifice.

[0019] The method may include: obtaining information about the actual or expected migration time of at least one sample component in a separation channel; and adjusting an applied potential difference based on the actual or expected migration time, such that a first potential difference is applied during a first portion of the migration of the at least one sample component and a second potential difference is applied during a second portion of the migration of the at least one sample component, wherein the magnitudes of the first and second potential differences are different. The first potential difference may be between 10 kV and 20 kV. The second potential difference may be less than 10 kV (e.g., less than 5.0 kV). The second potential difference may be 0 V.

[0020] The method may include obtaining information about the actual migration time of at least one sample component by detecting a portion of the sample component discharged through a well. The method may include using a mass spectrometry detection system to detect this portion of the at least one sample component.

[0021] The method may include obtaining information about the expected migration time of at least one sample component from a database of reference information for at least one sample component. The at least one sample component may include multiple sample components, and the method may include, for each sample component or group of components, adjusting the applied potential difference such that different potential differences are applied during different portions of the migration of that component or group of components. The applied potential difference may alternate between continuously larger and smaller values ​​during the migration of the multiple sample components.

[0022] Embodiments of the method may also include any of the other features disclosed herein, unless otherwise expressly stated, including features disclosed with respect to different embodiments in any combination.

[0023] In another aspect, this disclosure is characterized by a jet analysis system comprising: a jet chip formed on a planar substrate, the chip being characterized by a sample reservoir connected to a separation channel at a first end of the separation channel, a background electrolyte reservoir connected to a pumping channel, wherein the pumping channel is connected to the separation channel at a second end adjacent to the separation channel opposite to the first end, an aperture extending from the second end to an outer surface of the chip, a first electrode extending from the outer surface of the chip into the sample reservoir, and a second electrode extending from the outer surface of the chip into the background electrolyte reservoir; a pressurization mechanism connected to the background electrolyte reservoir; and an electronic processor connected to the first and second electrodes and the pressurization mechanism, and configured such that during operation of the system, the electronic processor: applies a potential difference between the first and second electrodes to induce migration of at least one component of the sample disposed in the separation channel toward the second end, and activates the pressurization mechanism to adjust the gas pressure in the background electrolyte reservoir such that the gas pressure in the background electrolyte reservoir is greater than the gas pressure outside the aperture, thereby generating a flow of background electrolyte solution from the background electrolyte reservoir through the pumping channel and out of the aperture, wherein when at least one component of the sample reaches the second end, at least one component is discharged through the aperture by the flow of the background electrolyte solution.

[0024] Implementations of the system may include any one or more of the following features.

[0025] The pressurization mechanism may include at least one of a gas source, a piston, a diaphragm, and a heating device. The gas pressure in the background electrolyte reservoir may be at least 0.5 psi higher than the gas pressure outside the orifice. The flow rate of the background electrolyte solution in the pumping channel may be 50 nL / min or greater. The potential difference may be between 0 V and 20 kV.

[0026] The system may include a joint near the second end between the separation channel and the pumping channel, the joint defining the dead volume of the separation channel, wherein the dead volume may be 500 pL or less (e.g., 100 pL or less).

[0027] The system may include at least one detector connected to an electronic processor and configured to obtain information about an exhaust plume generated by the discharge of a fluid, including a background electrolyte solution, through an orifice. The at least one detector may be configured to measure one or more of the following to obtain the information: light transmitted through the exhaust plume, light reflected from the exhaust plume, light scattered by the exhaust plume, and light absorbed by the exhaust plume.

[0028] The system may include a discharge channel connected to a pumping channel via a discharge valve, wherein an electronic processor is connected to the discharge valve and configured to receive information from at least one detector and, based on the obtained information, selectively activate the discharge valve to divert a portion of the flow of background electrolyte solution into the discharge channel.

[0029] The jet chip may include an auxiliary reservoir connected to an auxiliary pressurization mechanism and an auxiliary channel connected to the auxiliary reservoir and, near its second end, to a separation channel. An electronic processor may be connected to the auxiliary pressurization mechanism and configured to activate the mechanism to adjust the gas pressure in the auxiliary reservoir such that the gas pressure in the auxiliary reservoir is greater than the gas pressure outside the orifice, generating a flow of auxiliary solution from the auxiliary reservoir through the auxiliary channel and into the separation channel. The auxiliary solution may include a mass spectrometry calibration compound, wherein the calibration compound is discharged from the orifice. The auxiliary solution may also include a mass spectrometry coupling agent, wherein the coupling agent is discharged from the orifice.

[0030] The electronic processor can be configured to acquire information about the actual or expected migration time of at least one sample component in a separation channel, and to adjust the applied potential difference based on the actual or expected migration time, such that a first potential difference is applied during a first portion of the migration of the at least one sample component and a second potential difference is applied during a second portion of the migration of the at least one sample component, wherein the magnitudes of the first and second potential differences are different. The first potential difference may be between 10 kV and 20 kV. The second potential difference may be less than 10 kV (e.g., less than 5.0 kV). The second potential difference may be 0 V.

[0031] An electronic processor can be configured to obtain information about the actual migration time of at least one sample component from a detection system configured to detect a portion of the at least one sample component exiting through a well. The electronic processor can be connected to a mass spectrometry detection system and configured to receive detection information corresponding to the at least one sample component from the mass spectrometry detection system. The electronic processor can also be configured to obtain information about the expected migration time of the at least one sample component from a database of reference information for the at least one sample component.

[0032] At least one sample component may include multiple sample components, and for each sample component or group of components, the electronic processor may be configured to adjust the applied potential difference such that different potential differences are applied during different portions of the migration of that component or group of components. The electronic processor may be configured to adjust the applied potential difference such that the applied potential difference alternates between continuously larger and smaller amplitude values ​​during the migration of the multiple sample components.

[0033] Embodiments of the system may also include any of the other features disclosed herein, unless otherwise expressly stated, features disclosed with respect to different embodiments included in any combination.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While methods and materials similar to or equivalent to those described and used herein may be used in the practice or experimentation of the subject matter herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are exemplary only and are not intended to be restrictive.

[0035] Details of one or more embodiments are set forth in the accompanying drawings and the following description. Other features and advantages will become apparent from the description, drawings, and claims. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of a capillary electrophoresis system.

[0037] Figure 2 This is a schematic diagram of a pressure-based delivery electrophoretic separation system using a background electrolyte solution.

[0038] Figure 3 This is a schematic diagram of a jet chip.

[0039] Figure 4 This is a schematic diagram showing a portion of the separation channel.

[0040] Figure 5 is a schematic diagram showing a part of a jet chip Figure 3 .

[0041] Figure 6 is a schematic diagram showing another part of a jet chip Figure 3 .

[0042] Figure 7 is a schematic diagram of a jet chip connected to an electronic processor

[0043] Figure 8 is a schematic diagram of another jet chip

[0044] Figure 9 is a schematic diagram of another jet chip

[0045] Figure 10 is a flowchart showing a series of steps for separating sample components in a jet chip

[0046] Figure 11 is a schematic image of the emission of a jet chip

[0047] Figure 12 is a schematic image of a plume emitted from a jet chip

[0048] Figure 13 is a schematic diagram of a jet chip coupled to a mass spectrometry detection system

[0049] Figure 14 is a schematic diagram of a jet chip including a displacement mechanism connected to a fluid reservoir

[0050] Figure 15 is a schematic diagram of a jet chip including a vacuum source near the emitter

[0051] Like reference numerals in the various figures indicate like elements. Detailed Description

[0052] Due to the complex nature of biomolecules and sample components, their relative fragility in some cases, and the environments in which such molecules and components are present, the analysis of such molecules and components can be challenging. The bioanalytes of interest (such as proteins, nucleic acids, lipids, and carbohydrates) are often present in a sample along with various other substances of no interest (collectively referred to as the “matrix” or “matrix components”).

[0053] If the molecules and components are at least partially separated from the matrix before they are subjected to quantitative analysis techniques, then the accuracy and reproducibility of the analysis of the molecules and components can be significantly improved. This ensures that the confounding effects of the analytical signal from the matrix (which tend to obscure the signal from the analyte of interest) are reduced or eliminated.

[0054] Many techniques are available for separating a biological sample into analytes of interest and matrix components that are not intended for analysis. Among these techniques, capillary electrophoresis is particularly useful because it can be generally applied to a variety of samples and can achieve good quality separation of analytes of interest even for relatively small overall volumes of samples. Capillary electrophoresis is also a relatively gentle technique that is well suited for preserving the biochemical and stereochemical properties of analytes.

[0055] In the following sections, some general features of jet processing and sample separation via electrophoretic techniques will be discussed, and then methods and systems for achieving sample delivery and electrospray ionization via pressure-driven flow will be described.

[0056] I. Jet Sample Processing and Delivery

[0057] Capillary electrophoresis is a technique in which the components of a sample are separated from one another along the length of a separation column or channel based on differences in their migration in the presence of an electric field. The electrophoretic mobility μ of the sample components EP is determined as follows for its electrophoretic velocity ν in an electric field E EP :

[0058] ν EP = μ EP E (1)

[0059] The total velocity of component migration also depends on the velocity ν of electroosmotic flow EO , and the velocity ν of electroosmotic flow EO in turn depends on the electroosmotic mobility μ EO . In a capillary electrophoresis system,

[0060] ν EO = μ EO E (2)

[0061] The electroosmotic mobility of a component depends on the interaction of the component with the channel through which the component moves (through the zeta potential) and the relative dielectric constant of the buffer solution. The total velocity of a component migrating through the channel also depends on the electroosmotic flow of the buffer solution through the channel.

[0062] In an electrophoresis system, the electroosmotic flow of the buffer solution is toward the negatively charged cathode, and positively charged sample components will also migrate toward the cathode, moving in the same direction as the electroosmotic flow. In contrast, negatively charged sample components will tend to move toward the positively charged anode in the opposite direction to the electroosmotic flow. If the magnitude of the velocity of the electroosmotic flow is greater than the magnitude of the electrophoretic velocity, then all sample components will migrate in the same direction as the electroosmotic flow.

[0063] In traditional capillary electrophoresis systems, the separation of sample components occurs during their migration through separation channels. An electric field applied to both ends of the channel modulates the electrophoretic and electroosmotic velocities of the components within the channel. Furthermore, the surface chemistry of the channel walls can be controlled to adjust the electroosmotic migration rate of each component, and the buffer solution used can be selected to control the direction of the electroosmotic flow. By carefully selecting each of these factors, the sample component of interest (i.e., the analyte) can be separated from the matrix components, and the analytes can be separated from each other for analysis.

[0064] Figure 1 This is a schematic diagram of a capillary electrophoresis system 100 including a sample reservoir 102, a separation channel 104, a background electrolyte reservoir 106, a pumping channel 108, and an emitter 110. The emitter 110 is typically implemented as a small orifice near the intersection of the separation channel 104 and the pumping channel 108, and functions as an electrospray emitter to generate vapor containing sample components.

[0065] During operation of system 100, potential V1 is applied through electrode 116 to background electrolyte solution 112 in sample reservoir 102, which also includes the sample of interest. Potential V2 is applied through electrode 118 to background electrolyte solution 114 in background electrolyte reservoir 106. The potential difference ΔV between background electrolyte solution 112 in reservoir 102 and background electrolyte solution 114 in reservoir 106 is... sep =V1-V2 establishes an electric field between reservoirs 102 and 106. The electric field extends through the junction 120, where the separation channel 104 and the pumping channel 108 intersect.

[0066] As described above with respect to equations (1) and (2), the ionic and dipole sample components migrate at different velocities in response to the electric field established in the separation channel 104. Therefore, the different sample components arrive at the junction 120 at different times. Once separated in this manner, the different sample components can be analyzed using techniques such as mass spectrometry. To initiate mass spectrometric analysis of the sample components, the components are first ejected from the separation channel 104 via the emitter 110 and then introduced into the mass spectrometry system.

[0067] Electrospray emission via emitter 110 is particularly well-suited for introducing biological sample components into a mass spectrometry system that may be difficult to achieve using other methods. Many biological sample components include macromolecules, such as proteins, peptides, and nucleic acids, that are not well-suited for direct vaporization. Conversely, the heat required to vaporize such components can lead to undesirable side effects (e.g., protein denaturation), rendering the analysis of such components inaccurate or invalid. Electrospray emission allows many biological sample components to be converted to the gas phase under relatively mild conditions, thus avoiding undesirable alterations to the component's structure.

[0068] exist Figure 1 In this process, the electrospraying of the separated sample components by the emitter 110 is achieved by pumping the background electrolyte solution 114 from the background electrolyte reservoir 106 to the electroosmotic pump via the emitter 110. When voltages V1 and V2 are applied via electrodes 116 and 118 as discussed above, an electroosmotic flow occurs in which the background electrolyte solution 114 flows out of the background electrolyte reservoir 106 and through the pumping channel 108 and the junction 120. To facilitate the electroosmotic flow of the background electrolyte solution 114, the inner surface of the pumping channel 108 may include a surface coating that interacts with the background electrolyte solution 114 to drive the electroosmotic flow.

[0069] As the background electrolyte solution 114 passes through the junction 120, the sample components, separated from each other and conveyed through the separation channel 104, are swept by the electroosmotic flow of the background electrolyte solution 114 and discharged through the emitter 110. In this way, a fine, vapor phase spatial distribution of each component can be produced for subsequent analysis (e.g., using mass spectrometry).

[0070] The generation and rate of the electroosmotic flow through the pumping channel 108 and the subsequent electrospray through the emitter 110 depend on the potential difference applied between electrodes 116 and 118 and the chemical properties of the inner surface of the pumping channel 108. Typically, when the inner surface of the pumping channel 108 is charged and a potential difference ΔV is applied... sep =V1-V2, making electrode 116 the anode (i.e., positively charged) and electrode 118 the cathode (i.e., negatively charged), the cations in the background electrolyte solution 114 tend to migrate towards electrode 118, while the anions tend to migrate towards electrode 116. If the inner wall of channel 108 is characterized by accumulated anionic charge, then the positively charged portion of the background electrolyte solution forms two cation layers (referred to as a "double diffuse layer" or "double electric layer") on the inner wall of the channel. The first layer, closest to the channel wall, is more tightly bound than the second layer, referred to as the "mobile layer." When a potential difference ΔV is applied... sep At this time, the mobile layer is pulled in the direction of the anion cathode (i.e., electrode 118). The cation portion constituting the mobile layer becomes a solvate in the background electrolyte solution, and thus a batch transport of the background electrolyte solution occurs in the direction of electrode 118. That is, both the cation and anion portions in the background electrolyte solution 114 are transported in the direction of electrode 118 via an electroosmotic flow process.

[0071] Therefore, the volume and other physical properties of the electrospray emitted through the emitter 110 are strongly influenced by the rate of the electroosmotic flow passing through the pumping channel 108 and the junction 120. The rate of the electroosmotic flow can be adjusted by changing the applied potential difference ΔV. sepThe surface chemical properties of the inner wall of the pumping channel 108 are used to control this. Generally, a larger applied potential difference ΔV sep This results in a higher rate of electroosmotic flow.

[0072] Manipulating the surface chemistry of the inner wall of channel 108 is more complex. In the example discussed above, anionic charges can be generated on the channel wall by selecting a suitable material for forming the wall of channel 108 (or for functionalizing the wall of channel 108). In some embodiments, the amount of available anionic charges can be adjusted by flowing a solution with a suitable pH through channel 108 (e.g., to generate anionic functional groups on the channel wall via deprotonation).

[0073] Another method for controlling the rate of electroosmotic flow through pumping channel 108 and junction 120 thus involves adjusting the surface chemistry of the inner wall of channel 108. In the example discussed above, by utilizing strong anionic groups that functionalize the wall of channel 108, a relatively strong electroosmotic flow can occur, resulting in a relatively high rate of bulk delivery of background electrolyte solution 114 through pumping channel 108 and junction 120 and a relatively large volume of discharge through emitter 110. Conversely, to reduce the electroosmotic flow through channel 108, junction 120, and emitter 110, the inner wall of channel 108 can be coated with materials such as various polymers and / or surfactants to prevent the formation of charged functional groups on the wall surface and thus reduce the rate of electroosmotic flow. In this case, the rate of bulk delivery of background electrolyte solution 114 can be slowed sufficiently so that the anionic fraction in solution 114 returns to its natural migration toward junction 120 (i.e., toward anode electrode 116).

[0074] The flow rate of the background electrolyte solution 114 through the pumping channel 108 and junction 120 and exiting the emitter 110 affects not only the fluid discharge rate of the electrospray but also the rate at which sample components migrate through the separation channel 104. As the background electrolyte solution 114 passes through the junction 120, it mixes with the background electrolyte solution 112 from the separation channel 104. The mixture of solutions is discharged from the emitter 110. Therefore, as the electroosmotic flow rate of the background electrolyte solution 114 increases, the rate at which the background electrolyte solution 112 is drawn through the separation channel 104 also increases. The sample components then migrate through the separation channel 104 at an even faster rate.

[0075] In summary, electroosmotic pumping, by establishing an electroosmotic flow of the background electrolyte solution 114 in the pumping channel 108, allows for control of the electrospraying of sample components separated from each other in the separation channel 104 via the emitter 110. The electroosmotic flow rate in the pumping channel 108 can be controlled to adjust the fluid volume emitted by the electrospray and the migration time of each sample component through the channel 104.

[0076] The potentials V1 and V2 applied to electrodes 116 and 118, respectively, also affect the electroosmotic flow rate in separation channel 104. The background electrolyte solution 112 present in sample reservoir 102 and in separation channel 104 responds to the applied potential difference ΔV. sep =V1-V2 and migrate. The electroosmotic flow rate of the background electrolyte solution 112 in the separation channel 104 is also strongly affected by the surface chemical properties of the inner wall of the separation channel 104. Depending on the functionalization properties of the inner wall, the electroosmotic flow of the background electrolyte solution 112 in the separation channel 104 can occur in the direction towards the sample reservoir 102 or towards the junction 120.

[0077] As is evident from the preceding discussion, adjusting the electroosmotic flow rate in pumping channel 108 and separation channel 104 involves carefully balancing several factors. For example, the electroosmotic flow rate in pumping channel 108 and separation channel 104 depends on the potential difference ΔV. sep Potential difference ΔV sep It also depends on the potentials V1 and V2 applied to electrodes 116 and 118. However, the electrophoretic mobility of each sample component in separation channel 104 also depends on the potential difference ΔV. sep Therefore, the adjustment of the electroosmotic flow rate in the separation channel 104 and the pumping channel 108 is at least partially coupled to the adjustment of the electrophoretic mobility of the sample components in the separation channel 104.

[0078] The electroosmotic flow rate in channels 104 and 108 also depends on the surface chemistry of the inner walls of these channels, and in principle, the flow rate can be adjusted by manipulating the surface chemistry within the channels. However, in practice, applying a thin, uniform coating to small-diameter channels presents a challenging manufacturing problem. Furthermore, it is also challenging to instantaneously alter the surface chemistry of channels 104 and / or 108 by allowing the solution to flow through them, depending on the surface chemistry of the channel walls. Therefore, it may not always be possible to controllably adjust the surface chemistry of channels 104 and 108, as desired, based on the properties of the background electrolyte solutions 112 and 114.

[0079] II. Sample delivery and electrospray discharge via pressure-driven flow

[0080] Based on the foregoing discussion, it is evident that an optional fluid delivery method can be used in the pumping channel 108 to achieve greater control over the migration rate of sample components in the separation channel 104 and the fluid discharge rate through the emitter 110, which is separate from the electroosmotic flow of the background electrolyte and the electrophoretic migration of sample components present in the separation channel 104.

[0081] As an alternative to the electroosmotic flow-based pumping system discussed above, Figure 2 A schematic diagram of an electrophoresis-based separation system 200 is shown, which uses a background electrolyte solution delivered via a pump channel using pressure. The system 200 includes a sample reservoir 202, a separation channel 204, a background electrolyte reservoir 206, and a pump channel 208. A mixture of background electrolyte solution 212 and the sample is disposed in the sample reservoir 202, and a background electrolyte solution 214 is disposed in the background electrolyte reservoir 206. Electrodes 216 and 218 extend into the sample reservoir 202 and the background electrolyte reservoir 206, respectively.

[0082] As discussed above, during operation, potentials V1 and V2 are applied to electrodes 216 and 218, respectively, establishing a difference in amplitude ΔV between these electrodes. sep =V1-V2 proportional electric field. The electric field imparts electrophoretic velocity to the components of the sample, which migrate at different rates from the sample reservoir 202 to the junction 220 along the separation channel 204. Simultaneously, due to the surface chemistry of the inner wall of the pumping channel 208, the electroosmotic migration rate of the background electrolyte solution 214 in the pumping channel 208 is relatively small or even zero. That is, compared with... Figure 1 In contrast to the pumping scheme shown, as a result of the potential difference between electrodes 216 and 218, electroosmotic flow of the background electrolyte solution 214 through pumping channel 208 is largely absent.

[0083] Alternatively, in Figure 2 In this configuration, the background electrolyte reservoir 206 is connected to a gas source 230 via a conduit 232, in addition to being connected to the pump channel 208. The gas source 230 supplies gas 234 to the top space above the background electrolyte 214 in the reservoir 206. In this way, the gas source 230 is configured to pressurize the volume contained within the reservoir 206. By applying appropriate pressure to the top space (i.e., the gas-filled portion) within the reservoir 206, the gas source 230 drives the background electrolyte solution 214 from the reservoir 206 through the pump channel 208 and the junction 220. The background electrolyte solution 214 mixes with the background electrolyte solution 212 from the separation channel 204 and the sample components, and the mixture is discharged from the emitter 210.

[0084] As mentioned above Figure 2As discussed, the flow rate of the background electrolyte solution 214 through the junction 220 affects the rate at which fluid is discharged through the emitter 210, and also affects the migration rate of sample components from the reservoir 202 through the separation channel 204 due to mixing with the fluid from the separation channel 204. When the flow rate of the background electrolyte solution 214 increases, the migration rate of the sample components in the separation channel 204 also increases, resulting in a reduction in the migration time for each sample component through the separation channel 204. The flow rate of the background electrolyte solution 214 can be controlled by varying the gas pressure in the top space of the reservoir 206. Typically, increasing the gas pressure in the reservoir 206 establishes a larger pressure gradient between the reservoir 206 and the emitter 210, increasing the flow rate of the background electrolyte solution 214 through the pumping channel 208, the junction 220, and the emitter 210. As noted above, increasing the flow rate of the background electrolyte solution 214 also increases the migration rate of the sample components through the separation channel 204.

[0085] Besides the influence of the flow rate of the background electrolyte solution 214, the migration rate of the sample components through the separation channel 204 is also determined by the electrophoretic velocity, which is imparted to each sample component by the electric field established by the voltage applied at electrodes 216 and 218. Therefore, by employing pressure-based fluid delivery in the pumping channel 208, two aspects influencing the overall migration rate of the sample components through the separation channel 204 can be independently controlled: the electrophoretic mobility of the sample components (via the potential difference ΔV) sep The adjustment of the velocity of each sample component (due to the fluid emission rate through emitter 210) and the component attributable to the velocity of the background electrolyte solution 214 (due to the adjustment of the flow rate of the background electrolyte solution 214). Because these aspects can be adjusted individually, the characteristics of sample component migration within separation channel 204 and also of the electrospray plume discharged from emitter 210 are achieved better than using... Figure 1 The electroosmotic pumping scheme shown may achieve a greater degree of control.

[0086] A significant advantage of using pressure-based control of the flow rate of the background electrolyte solution 214 is that the surface chemistry of the inner wall of the pumping channel 208 does not need to be modified to generate electroosmotic flow. (See above regarding...) Figure 1In some embodiments, the rate of electroosmotic flow is controlled or modulated by adjusting the surface chemistry of the channel walls through which the flow occurs. The surface chemistry is typically matched to a specific background electrolyte solution and / or sample composition to achieve appropriate migration times for the sample components. To adapt the electrophoresis system to the processing of different samples, the surface chemistry of the channel walls may need to be altered, for example, by introducing strongly alkaline or acidic solutions into the channel to change the ionic state of the surface functional groups adhered to the wall. Depositing a thin, uniform coating on the surface of the channel walls during channel fabrication can be challenging, especially if the channel diameter is only a few micrometers.

[0087] Pressure-based control of the flow rate of the background electrolyte solution 214 eliminates the aforementioned manufacturing challenges. Because fluid flow is based on a pressure gradient rather than electroosmosis, it is generally not necessary to alter the surface chemistry of the channel walls during manufacturing or when changing samples. A variety of different background electrolyte solutions can be used for pumping because the specific properties of the background electrolyte solution do not “match” the surface chemistry of the pumping channel 208. As a result, the electrophoretic separation system can be more easily manufactured and adapted to different analytical conditions and samples of interest.

[0088] The electrophoretic sample analysis system disclosed in this paper is typically implemented, at least in part, on a jet chip. Figure 3 This is a schematic diagram showing a jet chip 300 including a sample reservoir 302, a separation channel 304, a background electrolyte solution reservoir 306, a pumping channel 308, and electrodes 316 and 318. The separation channel 304 and the pumping channel 308 intersect at a junction 320. The emitter 310 is located near the junction 320. The aforementioned components of the jet chip are generally aligned with... Figure 2 The corresponding components operate in the same manner. During operation, a vapor phase electrospray plume 340, comprising the background electrolyte solution from separation channel 304 and pumping channel 308, and optionally one or more sample components, is discharged from emitter 310.

[0089] The reservoir 306 includes a background electrolyte solution 314 and a sealed top space region 350 filled with gas. The reservoir 306 is coupled to a gas source 330 via a conduit 332 and an airtight interface 336, which supplies gas to the sealed top space of the reservoir 306.

[0090] The reservoir 302 contains the sample 360 ​​of interest for separation into components on the chip 300. For electrophoretic separation of the components of the sample 360 ​​within the separation channel 304, the reservoir 302 may also contain a background electrolyte solution 312.

[0091] Electrodes 316 and 318, and gas source 330, can be coupled to electronic processor 390 via control lines 391, 392, and 393. During operation, electronic processor 390 (if present) applies a suitable voltage to electrodes 316 and 318 to initiate electrophoretic migration of sample components from reservoir 302 through separation channel 304 toward junction 320, and activates gas source 330 to deliver gas to the top space of reservoir 306, thereby delivering background electrolyte solution 314 from reservoir 306 through pumping channel 308 and junction 320, and discharging background electrolyte solution 314 through emitter 310.

[0092] In some embodiments, chip 300 may also be configured to electrically inject a sample into separation channel 304. Chip 300 may optionally include a background electrolyte solution reservoir 370 and a waste reservoir 372 located on opposite sides of separation channel 304 to form an "injection cross" 376. Optional electrodes 374 coupled to an electron processor 390 via control lines 394 allow the electron processor 390 to apply a potential to the background electrolyte solution 312 in reservoir 370. A sample "plug" can be introduced into separation channel 304 for subsequent electrophoretic separation within the channel by alternately delivering a predetermined amount of fluid from reservoirs 302 and 370 into separation channel 304 (and directing waste fluid into reservoir 372). Additional aspects of electric sample injection are disclosed, for example, in U.S. Patent Application Publication No. 2013 / 0327936, in PCT Patent Application Publication No. WO2013 / 191908, and in U.S. Patent Application Serial No. 15 / 079,541, the entire contents of which are incorporated herein by reference.

[0093] exist Figure 4 The diagram shows a portion of the separation channel 304. The jet chip 300 is typically implemented as a planar structure extending in the x and y dimensions, and has a direction in the z direction (i.e., perpendicular to the x-axis). Figure 4The thickness of the separation channel 304 is measured in the axial direction of the xy-plane. The separation channel 304 extends in the axial direction of the xy-plane and is defined by a central axis 402 that defines the axial direction. The axis 402 can be linear or follow a non-linear (e.g., curved) path. The width w of the separation channel 304 is measured in the xy-plane in the direction perpendicular to the axis 402. The width w is typically chosen based on factors such as the desired flow rates of the sample and background electrolyte solution through the separation channel 304 and the desired interactions between the sample 360 ​​and the walls of the separation channel 304 during sample migration. Typically, w is approximately 1 micrometer or larger (e.g., 5 micrometers or larger, 10 micrometers or larger, 20 micrometers or larger, 30 micrometers or larger, 40 micrometers or larger, 50 micrometers or larger, 60 micrometers or larger, 70 micrometers or larger, 80 micrometers or larger, 90 micrometers or larger, 100 micrometers or larger, 120 micrometers or larger, 150 micrometers or larger, 200 micrometers or larger, 300 micrometers or larger, 500 micrometers or larger, 750 micrometers or larger, 1 mm or larger).

[0094] The depth of the separation channel 304, measured in the z-direction orthogonal to the xy-plane, can be selected as desired based on the thickness of the chip 300 to control the volume of sample and background electrolyte solution that can be processed by the separation channel 304 during sample separation and analysis. The depth of the separation channel 304 can also be selected to control Joule heating within the channel, adjust the flow resistance of the channel's hydrodynamics, and control the total fluid flow rate within the channel. Generally, the separation channel 304 has a depth of approximately 10 micrometers. More generally, the depth of the separation channel 304 can be 1 micrometer or greater (e.g., 5 micrometers or greater, 10 micrometers or greater, 20 micrometers or greater, 30 micrometers or greater, 40 micrometers or greater, 50 micrometers or greater, 75 micrometers or greater, 100 micrometers or greater).

[0095] Refer again Figure 3 The total length of the separation channel 304 can be selected as needed to provide a sufficient flow path along which the different components of the sample are separated before reaching the emitter 310. By selecting a suitable length for the separation channel 304, the migration time of the sample components within the separation channel 304 can be controlled. Furthermore, the peak width of the sample components at the end of the separation channel 304 (i.e., when the sample components are discharged) can be controlled. The total length corresponding to the path length defined by the axis 402 of the separation channel 304 between the reservoir 302 and the junction 320 can be 1.0 cm or greater (e.g., 5.0 cm or greater, 10 cm or greater, 20 cm or greater, 30 cm or greater, 40 cm or greater, 50 cm or greater, 75 cm or greater, 100 cm or greater).

[0096] Typically, the lateral dimensions of the pumping channel 308 are similar to those of the separating channel 304. The pumping channel 308 has a width extending in the xy-plane and a thickness measured in the z-direction orthogonal to the xy-plane. The width and depth of the pumping channel 308 can be the same as any of the widths and depths discussed above for the separating channel 304. In some embodiments, the width and / or depth of the pumping channel 308 are the same as those of the separating channel 304. However, more generally, the pumping channel 308 can have a width and / or depth different from that of the separating channel 304.

[0097] The central axis extends along the length of the pumping channel 308 in the same manner as the axis 402 of the separation channel 304, and defines the path along which the pumping channel 308 extends. The length of the pumping channel 308 corresponds to the path length defined by its central axis. Typically, the length of the pumping channel 308 can be selected as desired to accommodate a desired volume of background electrolyte solution 314. In some embodiments, it is advantageous to use a pumping channel length that is significantly shorter than the length of the separation channel 304. Since the separation of sample components does not occur within the pumping channel 308, this length does not need to take component separation into account. Furthermore, by shortening the length of the pumping channel 308, the spatial pressure gradient within the pumping channel 308 can be increased, which increases the driving force applied to each volume unit of the background electrolyte solution within the pumping channel. In some embodiments, the length of the pumping channel is 0.5 cm or greater (e.g., 1.0 cm or greater, 1.5 cm or greater, 2.0 cm or greater, 2.5 cm or greater, 3.0 cm or greater, 4.0 cm or greater, 5.0 cm or greater, 7.0 cm or greater).

[0098] Without pressure applied to the top space of reservoir 306, background electrolyte solution 314 is not delivered through pump channel 308 and emitter 310. Because the flow of background electrolyte solution 314 also delivers the separated sample components from separation channel 304 through connector 320 via emitter 310, the lack of applied gas pressure in the top space of reservoir 306 ensures that sample components are not emitted through emitter 310 either.

[0099] The term "pressure," when used herein to describe the gas pressure in the top space of reservoir 306, refers to atmospheric or ambient pressure relative to the environment in which chip 300 operates, i.e., the pressure at transmitter 310. Even without any gas pressure applied to reservoir 306, the background electrolyte solution 314 is subjected to atmospheric or ambient gas pressure. However, no pressure gradient exists between reservoir 306 and transmitter 310.

[0100] Applying gas pressure to the top space of reservoir 306 establishes a pressure gradient between reservoir 306 and emitter 310, which results in fluid transport through pumping channel 308. Therefore, "gas pressure" in the top space of reservoir 306 refers to the pressure difference between the absolute pressure in reservoir 306 and the ambient pressure at emitter 310.

[0101] Typically, to initiate and maintain the delivery of background electrolyte solution 314 between reservoir 306 and transmitter 310, the applied gas pressure is selected based on the desired flow rate of the background electrolyte solution. In some embodiments, the applied gas pressure is about 2 psi. In some embodiments, the applied gas pressure is at least 0.5 psi (e.g., at least 1.0 psi, at least 1.5 psi, at least 2.0 psi, at least 2.5 psi, at least 3.0 psi, at least 5.0 psi, at least 7.0 psi, at least 10.0 psi, at least 12.0 psi, at least 15.0 psi, at least 20.0 psi).

[0102] In some embodiments, the gas pressure outside and adjacent to the transmitter 310 may differ from atmospheric pressure. Therefore, in order to initiate and maintain the delivery of the background electrolyte solution 314 between the reservoir 306 and the transmitter, the pressure difference between the gas pressure in the reservoir 306 and the gas pressure outside the transmitter 310 is at least 0.5 psi (e.g., at least 1.0 psi, at least 1.5 psi, at least 2.0 psi, at least 2.5 psi, at least 3.0 psi, at least 5.0 psi, at least 7.0 psi, at least 10.0 psi, at least 12.0 psi, at least 15.0 psi, at least 20.0 psi).

[0103] During operation, the flow rate of the background electrolyte solution 314 through the pumping channel 308 is selected to control the rate at which fluid is emitted from the emitter 310. Various different flow rates can be selected based on the desired fluid volume per unit time in the electrospray plume 340. In some embodiments, for example, the flow rate of the background electrolyte solution 314 in the pumping channel 308 is 1 nL / min or greater (e.g., 10 nL / min or greater, 20 nL / min or greater, 50 nL / min or greater, 100 nL / min or greater, 150 nL / min or greater, 200 nL / min or greater, 300 nL / min or greater, 500 nL / min or greater, 750 nL / min or greater, 1.0 μL / min or greater, 2.0 μL / min or greater, 5.0 μL / min or greater, 7.5 μL / min or greater, 10 μL / min or greater).

[0104] Experimentally, it has been observed that, in some embodiments, improved ionization of sample components can be achieved under certain conditions at relatively low flow rates of the background electrolyte solution 314. Therefore, for example, in some embodiments, more efficient component ionization can occur when the flow rate of the background electrolyte solution 314 is between 50 nL / min and 500 nL / min (e.g., between 100 nL / min and 250 nL / min).

[0105] Various background electrolyte solutions can be used in chip 300. In some embodiments, background electrolyte solutions 312 and 314 have the same composition. More generally, background electrolyte solutions 312 and 314 can have different compositions. The background electrolyte solutions used in chip 300 are typically water-based and include one or more additional components (i.e., in addition to water) to aid in the separation of sample components and / or the generation of electrospray plumes. For example, the background electrolyte solution may include one or more compounds that act as organic modifiers, such as, but not limited to, methanol and acetonitrile. The background electrolyte solution may include one or more weak acids, such as, but not limited to, formic acid.

[0106] Experimentally, it has been observed that in some embodiments, it is advantageous to ensure that the background electrolyte solution 314 comprises one or more organic modifiers at higher concentrations and / or salts at lower concentrations than the background electrolyte solution 312. Generally, efficient electrospray plume generation is promoted by using a background electrolyte solution 314 with relatively low ionic strength (e.g., where the dissolved salt concentration is less than 100 mM (e.g., less than 80 mM, less than 60 mM, less than 40 mM, less than 20 mM, less than 10 mM)). Efficient electrospray plume generation can also be promoted by using a background electrolyte solution 314 with an increased concentration of organic modifier relative to the background electrolyte solution 312, as the organic modifier reduces the surface tension of the background electrolyte solution 314, facilitating droplet formation.

[0107] Furthermore, in some embodiments, the background electrolyte solution 312 may have a composition tailored to promote favorable surface chemistry on the walls of the separation channel 304 to improve the separation of individual sample components as they migrate through the channel. The composition of the background electrolyte solution 312 may be altered when different samples are introduced onto the chip 300 for analysis.

[0108] As mentioned above, the background electrolyte solution 314 may have the same or different composition as the background electrolyte solution 312. Due to mixing and dilution occurring in the region of junction 320, the background electrolyte solution designed to promote high-quality separation in channel 304 is not necessarily optimized for promoting high-quality electrospray emission. By modifying the composition of the background electrolyte solution 314 relative to the composition of the background electrolyte solution 312 (e.g., by adjusting the concentration of one or more organic modifiers and / or one or more acids), both high-quality sample component separation and high-quality electrospray emission of the separated sample components can be achieved.

[0109] During operation of chip 300, a potential V2 can typically be applied to electrode 318 as desired. In some embodiments, V2 has a relatively large amplitude compared to the external ground voltage (which represents the nominal zero (i.e., ground) voltage). By maintaining a relatively large voltage amplitude at electrode 318, ionization of the separated sample components occurs as the components exit separation channel 304 and are discharged from emitter 310. In this way, electrospray plume 340 includes a population of ionized molecules of each sample component isolated within separation channel 304. These ionized molecules can then be directly coupled to a mass spectrometer for analysis.

[0110] Typically, the potential V2 has a positive sign relative to the external ground voltage. However, more generally, the potential V2 can also be negative relative to the external ground voltage, depending on the nature of the sample composition. The magnitude of the potential V2 is typically about 3.5 kV, but can vary depending on the sample composition being analyzed. In some embodiments, for example, the magnitude of V2 can be 0.1 kV or greater (e.g., 0.2 kV or greater, 0.5 kV or greater, 0.8 kV or greater, 1.0 kV or greater, 2.0 kV or greater, 2.5 kV or greater, 3.0 kV or greater, 3.5 kV or greater, 4.0 kV or greater, 4.5 kV or greater, 5.0 kV or greater, 6.0 kV or greater, 7.0 kV or greater, 8.0 kV or greater).

[0111] Like V2, the potential V1 can typically have a positive or negative sign relative to the external ground voltage, depending on the nature of the sample components being analyzed. The magnitude of the potential V1 can also vary depending on the sample being analyzed. In some embodiments, for example, the magnitude of V1 can be 0.1 kV or greater (e.g., 0.5 kV or greater, 1.0 kV or greater, 2.0 kV or greater, 3.0 kV or greater, 5.0 kV or greater, 7.0 kV or greater, 10.0 kV or greater, 12.0 kV or greater, 15.0 kV or greater, 17.0 kV or greater, 20.0 kV or greater, 25.0 kV or greater, 30.0 kV or greater).

[0112] Potential difference ΔV sep =V1-V2 can typically be adjusted to any value to control the electrophoretic mobility of sample components in separation channel 304. Typically, ΔV sep The larger the amplitude, the greater the electrophoretic mobility of each sample component, and the faster each component migrates through separation channel 304. When ΔV sep When ΔV = 0, no electrophoretic driving force is applied to the sample components, but some migration may still occur due to the bulk fluid transport in separation channel 304. sep When it is non-zero, ΔV is determined based on the properties of the sample being analyzed. sep The sign can be positive (i.e., V1-V2>0), or ΔV sep The sign can be negative (i.e., V1-V2<0).

[0113] Typically, ΔV sep The amplitude can be 0V or greater (e.g., 100V or greater, 200V or greater, 300V or greater, 500V or greater, 750V or greater, 1.0kV or greater, 2.0kV or greater, 3.0kV or greater, 5.0kV or greater, 7.0kV or greater, 10.0kV or greater, 12.0kV or greater, 15.0kV or greater, 17.0kV or greater, 18.0kV or greater). As discussed above, when ΔV sep When the amplitude is greater than zero, the electrophoretic velocity of each charged or dipole sample component in the separation channel 304 is greater than zero, and the sample components tend to migrate along the length of the separation channel 304.

[0114] Figure 5 A schematic diagram of a portion of the jet chip 300 in the region of the transmitter 310 is shown. Specifically, Figure 5 The intersection of the separation channel 304 and the pumping channel 308 at the junction 320 is shown. Although various geometries can be used to achieve the junction between the separation channel and the pumping channel, Figure 5 For the purposes of discussion, one possible geometry is shown. Channels 304 and 308 intersect at an inner point 386. Extending from the inner point 386 are two imaginary lines 382 and 384. Line 382 extends in the xy-plane in a direction perpendicular to the central axis of channel 304 and indicates the end of channel 304. Similarly, line 384 extends in the xy-plane in a direction perpendicular to the central axis of channel 308 and indicates the end of channel 308. The enclosed volume includes lines 382 and 384, the emitter 310, and portions of the walls of channels 304 and 308. Figure 5Region 380 in the middle corresponds to the “dead volume” of the jet chip 300, through which the separated sample components are transported after leaving the separation channel 304 and before they are discharged from the emitter 310.

[0115] A key advantage of the pressure-based fluid delivery method disclosed herein is the relatively small dead volume on chip 300. By maintaining a small dead volume, diffusion (i.e., spatial broadening) of the separated sample components can be reduced, ensuring that the concentration of each component in the electrospray plume 340 is not significantly diluted by the background electrolyte solution 314. In some embodiments, for example, the dead volume 380 may be 500 pL or less (e.g., 400 pL or less, 300 pL or less, 200 pL or less, 100 pL or less, 50 pL or less, 30 pL or less, 20 pL or less, 10 pL or less, 3 pL or less, 1 pL or less).

[0116] Figure 6 A schematic diagram of a portion of a jet chip 300, including a separation channel 304, a pumping channel 308, and a transmitter 310, is shown. A reservoir 306 at the end of the pumping channel 308 opposite the transmitter 310 is coupled to a conduit 332 via an interface 336. Typically, the interface 336 is a hermetically sealed interface that can be implemented in various ways. For example, in some embodiments, the interface 336 includes one or more sealing members, such as an O-ring connecting the conduit 332 to the reservoir 306.

[0117] The conduit 332 is connected to the gas source 330 via valve 335, which in turn is connected to the electronic processor 390 via communication line 395. During operation of the chip 300, the electronic processor 390 can regulate the gas pressure in the reservoir 306 by opening and closing valve 335, and thus regulate the flow rate of the background electrolyte solution 314 through the pump channel 308. In some embodiments, a pressure detector 337 is located within or coupled to the reservoir 306 and is also connected to the electronic processor 390 via communication line 396. The pressure detector 337 can transmit a measurement signal to the processor 390, which includes information about the gas pressure within the sealed top space of the reservoir 306. Based on the measurement information, the processor 390 can then adjust the gas pressure in the reservoir 306 by opening or closing valve 335.

[0118] In some embodiments, chip 300 includes another valve 341 connected to a sealed top space of reservoir 306 and also connected to electronic processor 390 via communication line 397. Processor 390 can reduce the flow rate of background electrolyte solution 314 in pumping channel 308 by reducing the gas pressure in reservoir 306. The reduction in gas pressure can be achieved by opening valve 341 to release excess gas from reservoir 306. For example, based on gas pressure measurements from pressure detector 337, electronic processor 390 can adjust the gas pressure in reservoir 306 by opening valve 335 to allow more gas to enter from gas source 330 (e.g., to increase the gas pressure in reservoir 306) or by opening valve 341 to release excess gas pressure from the reservoir (e.g., to reduce the gas pressure in the reservoir). Therefore, electronic processor 390 can have complete control over the flow rate of background electrolyte solution 314 on chip 300. Since the flow of the background electrolyte solution 314 also largely determines the spatial range and fluid volume of the electrospray plume, the electronic processor 390 can adjust the characteristics of the plume by activating valves 335 and 341.

[0119] In some embodiments, to further control the electrospray plume, chip 300 may include a valve 343 that separates the pumping channel 308 from the fluid discharge channel 345. Valve 343 is connected to electronic processor 390 via communication line 398. For certain applications where a high flow rate of background electrolyte solution 314 is desired, but a relatively small total volume of fluid flow is also desired to generate an electrospray plume, electronic processor 390 may open valve 343 to divert a certain amount of background electrolyte solution 314 from pumping channel 308 to fluid discharge channel 345. In this way, the volume of background electrolyte solution reaching emitter 310 per unit time is reduced, but the flow rate of background electrolyte solution 314 in pumping channel 308 (and through emitter 310) is maintained.

[0120] Figure 7 A schematic diagram of a jet chip 300, including many of the features discussed above, is shown. (As in...) Figure 7 The diagram also shows a detector 704 connected to the electronic processor 390 via a communication line 705. During operation, the detector 704 can be used to measure one or more characteristics of the electrospray plume 304 and transmit measurement signals, including information about the electrospray plume, to the electronic processor 390. Based on this information, the electronic processor 390 can adjust the flow rate and / or fluid volume in the pumping channel 308, and / or adjust the voltages V1 and / or V2 applied to electrodes 316 and 318, respectively, as discussed above.

[0121] For example, in some embodiments, detector 704 is an imaging detector, such as a CCD chip or a CMOS-based detector. Information obtained from one or more images of the electrospray plume acquired by detector 704 (e.g., information about the spatial distribution, shape, and color of the plume) can be used by electronic processor 390 to adjust the operation of chip 300. In some embodiments, detector 704 is a non-imaging detector, such as a light intensity detector (e.g., a photodiode) and / or a spectral detector (e.g., a grating-based spectrometer or other dispersion-based spectrometer). Measurement information, including any one or more of the following, can be used by electronic processor 390 to adjust the operation of chip 300: light transmitted through the plume, light reflected from the plume, optical scattering by the plume, absorption at different wavelengths by the plume, and / or refraction / diffraction of light by the plume.

[0122] In some embodiments, detector 704 may include a detection device configured to measure the voltage, current, and / or another electrical characteristic of the electrospray plume. Information obtained from the measurement of the plume's electrical characteristics may also be used by electronic processor 390 to adjust the operation of chip 300, for example, to achieve a stable plume for discharging sample components into the mass spectrometry detection system.

[0123] Therefore, both optical and non-optical measurements can be used to adjust the operation of chip 300. As an example, in some embodiments, detector 704 can be used to obtain images of chip 300, and in particular, images of transmitter 310 and the electrospray plume 340 emitted from the transmitter. Electronic processor 390 can then analyze the images to determine information about the electrospray plume and adjust the operation of chip 300 based on that information.

[0124] Figure 11 This is a schematic diagram showing a representative image 1100 of the chip 300 acquired by detector 704. In the image, multiple droplets 1104 forming the electrospray plume 340 are visible. Droplets 1102 formed on the emitter 310 are also visible. Electronic processor 390 is configured to analyze image 1100 to measure the maximum size (i.e., maximum cross-sectional size or diameter) of the droplets 1104. Electronic processor 390 can then use this maximum size information to adjust the operation of chip 300.

[0125] For example, the electronic processor 390 can determine the average maximum size of the droplet 1104 and compare the average maximum size to a threshold. It has been observed that when the droplet 1104 becomes too large (i.e., the average maximum size exceeds the threshold), the voltage applied by the processor 390 to ionize the electrospray plume may be too low. Alternatively or additionally, when the droplet 1104 becomes too large, the flow rate of the ejected sample components and background electrolyte solution through the emitter 310 may be too high.

[0126] Therefore, in some embodiments, the electronic processor 390 is configured to increase the amplitude of the voltage applied to ionize the electrospray plume. Alternatively or additionally, in some embodiments, the electronic processor 390 is configured to reduce the flow rate of the background electrolyte solution and sample components through the emitter 310 by adjusting the pressure applied to the top space of the reservoir 306 (i.e., by reducing the pressure applied to the top space) such that the pressure gradient between the reservoir 306 and the ambient pressure outside the chip 300 is reduced.

[0127] The electronic processor 390 can also be configured to acquire one or more additional images of the chip 300 and the electrospray plume 340 after the operation of adjusting the chip 300 as discussed above. The processor 390 analyzes these additional images to identify plume droplets in the images, repeatedly determining the maximum size for each identified plume droplet, determining a new average maximum size for the droplet, and then comparing the new average maximum size with a threshold to determine whether the electrospray ionization voltage and / or the flow rate through the emitter 310 should be further adjusted. This process is repeated until the maximum size of the droplets forming the electrospray plume 340 is determined by the processor 390 to fall below the threshold.

[0128] When the electrospray ionization voltage is very low, the electrospray plume 340 can completely disappear from image 1100 (i.e., droplets 1104 are not visible in image 1100 and are not recognized by processor 390). When electronic processor 390 determines that no droplets are visible in image 1100 (or more generally, when the number of recognized droplets is less than a threshold percentage of the expected number of droplets, such as 50%, 60%, 70%, 80%, 90%, or even less), electronic processor 390 can be configured to increase the magnitude of the applied electrospray ionization voltage. This change can be performed when electronic processor 390 recognizes that the low electrospray ionization voltage is the cause of the absence of plume 340, while keeping the flow rate through emitter 310 approximately constant.

[0129] Even very low electrospray ionization voltages can cause droplets 1102 to appear on the edge of chip 300, such as... Figure 11 As shown. When the electronic processor 390 analyzes image 1100 and detects the presence of droplets 1102 overlapping a portion of chip 300 near emitter 310, the electronic processor 390 can be configured to increase the magnitude of the applied electrospray ionization voltage. This change can be performed when the electronic processor 390 recognizes that a low electrospray ionization voltage is the cause of the presence of droplets 1102 on the edge of chip 300, while maintaining a substantially constant flow rate through emitter 310.

[0130] When the electrospray ionization voltage is too high, it has been observed that the electrospray plume 340 can “twitch” or emerge from different points within the emitter 310 over time. In some embodiments, the electronic processor 390 is configured to recognize movement in the spatial position of the electrospray plume 340 and adjust the operation of the chip 300 based on the movement.

[0131] Figure 12 This is a schematic diagram showing a representative image 1200 obtained by detector 704, illustrating an electrospray plume 340 emerging from emitter 310 of chip 300. Electronic processor 390 is configured to analyze image 1200 to determine whether the plume 340 appears from different spatial locations over time. For example, processor 390 may acquire a first image of the plume 340 using detector 704 at a first time t1 and analyze the first image to determine a boundary region 1202 surrounding the plume 340 in the image and a point of origin 1206 of the plume 340. Processor 390 may then acquire a second image of the plume 340 via detector 704 at a later time t2 and analyze the second image to determine a second boundary region 1204 surrounding the plume 340 in the image and a second point of origin 1208 of the plume 340 in the second image.

[0132] Based on boundary regions 1202 and 1204 and / or starting points 1206 and 1208, processor 390 can then determine whether the plume 340 has "twitched" over time. For example, if boundary regions 1202 and 1204 have moved more than a threshold amount relative to each other in the two (registered) images, as in Figure 12 As schematically shown, processor 390 can then determine that the plume 340 is twitching over time. Alternatively or additionally, if the starting points 1206 and 1208 have moved relative to each other in the two images by more than a threshold amount, such as Figure 12 As shown, processor 390 can then determine that the plume 340 is twitching over time. When processor 390 determines that the plume 340 is twitching, as will be explained in more detail below, processor 390 can be configured to adjust operation by reducing the electrospray voltage applied to expel sample components from emitter 310 into the mass spectrometry detection system.

[0133] In some embodiments, a mass spectrometry detection system oriented to receive sample components discharged from emitter 310 can measure one or more characteristics of the plume 340, and these measured characteristics can be used by processor 390 to adjust the operation of chip 300. For example, the mass spectrometry detection system can be used to determine the flow rate of particles within the plume 340 by detecting particles as they are introduced into the mass spectrometry detection system. If the flow rate is less than a threshold, processor 390 can be configured to increase the flow rate through emitter 310. For example, processor 390 can apply increased pressure to the top space 350 of reservoir 306 via gas source 330, increasing the rate at which fluid is discharged through emitter 310.

[0134] In some embodiments, detector 704 may be an electrical detector that measures one or more electrical characteristics of the plume 340 for the purpose of adjusting the operation of chip 300. For example, detector 704 may measure the current of the plume 340, and the current measurement may be used by processor 390 to estimate the flow rate of the plume 340. Processor 390 may also determine the rate of change of current and / or the change in current over time. If the rate of change or change in current is too large, processor 390 may adjust the operation by increasing the electrospray voltage (if the plume 340 sputters) or decreasing the electrospray voltage (if the plume 340 twitches).

[0135] As is apparent from the foregoing, in some embodiments, multiple measurement results can be used by the electronic processor 390 to adjust the operation of the chip 300. For example, the detector 704 may include both an imaging detector such as a camera and an electrical detector, and each of these detectors can perform any of the measurements discussed above. Furthermore, the plume 340 may be directed to an inlet of a mass spectrometry detection system communicating with the electronic processor 390. The electronic processor 390 can then adjust various operating parameters of the chip 300 based on any combination of the measured values ​​discussed above.

[0136] Optionally, a light source 702 may be present and connected to an electronic processor 390 via a communication line 703. During operation, the light source 702 can provide illumination radiation that interacts with the electrospray plume 340. Light transmitted by, reflected from, scattered from, or otherwise emitted from the electrospray plume 340 through any of the various processes can be detected by a detector 704, generating a measurement signal in the detector. The light source 702 can typically include any of a variety of different light sources, including laser-based sources, diode sources, incandescent sources, and other light-emitting elements. In the absence of the light source 702, the signal measured by the detector 704 is obtained from the interaction of ambient light in the environment of the chip 300 with the electrospray plume 340.

[0137] In some embodiments, regarding Figure 2 At least some components of the separation system 200 discussed are implemented in a modular housing that interfaces with existing mass spectrometry systems. Figure 7 In this configuration, any one or more of the electronic processor 390, gas source 330, valve 335, light source 702, and detector 704 can be housed within the modular housing 710. The housing 710 also includes a support structure for securing the chip 300. Figure 7 (Not shown in the image), so that various electrical and fluid connections can be established between the chip 300 and components within the housing. The housing 710 may also include a communication interface 712 connected to the electronic processor 390 via a communication line 707, the communication interface 712 being configured to connect to a mass spectrometry system (in...). Figure 7 (Not shown in the image) and connected to the mass spectrometry system via an interface. During operation, the electronic processor 390 can exchange information with components of the mass spectrometry system via interface 712, enabling various adaptive analysis and processing methods based on detection signals from the mass spectrometry system.

[0138] Figure 8 A schematic diagram of a jet chip 300 including many of the features discussed above is shown. Generally, the chip 300 may also include various other sample and fluid handling and delivery elements. For example, the chip 300, implemented or connected within or to reservoirs 302, separation channels 304, reservoirs 306, and / or pumping channels 308, may include one or more valves, conduits, gates, liquid reservoirs, gas reservoirs, electrodes, and other components. Activated elements such as valves, gates, and electrodes may be connected to an electronic processor 390 via communication lines. Alternatively, a gas source connected to the electronic processor 390 may be coupled to a gas reservoir in the manner discussed above to induce fluid delivery by pressurizing a sealed top space within the gas reservoir.

[0139] In some embodiments, such as Figure 8 As shown, additional channels and reservoirs can be coupled to pump channel 308 to deliver additional compounds into the mixture of fluids (e.g., background electrolyte solution and sample components) discharged through emitter 310. Figure 8 In this configuration, auxiliary reservoir 802 is coupled to pumping channel 308 via channel 804, and auxiliary reservoir 806 is coupled to pumping channel 304 via channel 808. Generally, any number of auxiliary reservoirs and channels can be coupled to pumping channel 308.

[0140] Various methods can be used to initiate fluid transport from reservoirs 802 and / or 806 through channels 804 and 808, respectively. For example, in some embodiments, reservoirs 802 and / or 806 and channels 804 and / or 808 may include electrodes connected to an electronic processor 390. The processor 390 applies a voltage to the electrodes, and the electric field established within channels 804 and / or 808 causes fluid transport within the channels. In some embodiments, reservoirs 802 and / or 806 may be connected to a gas source as discussed above, and the electronic processor 390 regulates fluid transport within the channels by opening and closing valves coupled to the reservoirs and / or channels.

[0141] Auxiliary reservoirs and channels are available for delivering various formulations to the background electrolyte solution 314 within the pump channel 308. Because the pump channel 308 is very close to the transmitter 310 and intersects with the separation channel 304, the interaction between the separated sample components and the formulation occurs just before the components are expelled into the electrospray plume, ensuring that the added formulation does not modify the sample components in a way that could adversely affect subsequent analysis of the components in the mass spectrometry system. Furthermore, because the formulation is introduced just before being expelled through the transmitter 310, it does not migrate upwards into the separation channel 304 in a direction away from the transmitter 310.

[0142] Various formulations can be introduced via auxiliary reservoirs and channels connected to pump channel 308. For example, in some embodiments, one or more coupling agents can be added to background electrolyte solution 314. Coupling agents improve the accuracy of subsequent mass spectrometry characterization. Suitable coupling agents are disclosed, for example, in the following publications, the entire contents of which are incorporated herein by reference: Remsburg et al., J. Am. Soc. Mass Spectrom 19:261 (2008); and Soukup-Hein et al., Anal. Chem. 80:2612 (2008).

[0143] In some embodiments, one or more calibration compounds can be introduced via auxiliary reservoirs and channels. The calibration compound, added to the background electrolyte solution 314, is emitted in an electrospray plume and introduced into the mass spectrometry analysis system. The calibration compound provides a reference marker for calibrating the mass spectrometric analysis of sample components. Multiple calibration compounds can be used. One such example is glutamate fibrin peptides, which are useful for calibrated peptide analysis.

[0144] In some embodiments, the jet chip may include multiple emitters from which sample components may be discharged. Figure 9This is a schematic diagram of a chip 300 including three sample reservoirs 302a-c connected to three separation channels 304a-c, respectively, with the separation channels 304a-c terminating at emitters 310a-c. The chip 300 also includes three background electrolyte solution reservoirs 306a-c connected to three pumping channels 308a-c, each pumping channel terminating at one of the emitters 310a-c. During operation, the voltage difference ΔV sep Separation voltages can be applied to both ends of one or more of the separation channels 304a-c. Typically, the separation voltage applied to both ends of each channel can be the same or different. By selectively guiding the driving background electrolyte solution from one of the reservoirs 306a-c through one of the corresponding channels 308a-c and out of one of the emitters 310a-c, an electrospray plume can be selectively generated from one of the emitters. Thus, under the control of the electronic processor 390, sample components from any of the separation channels 304a-c can be selectively discharged from the chip 300.

[0145] Figure 9 The multi-emitter configuration shown may be advantageous, for example, because it allows for the separation of voltage ΔV. sep Simultaneous application to multiple separation channels. Therefore, for example, a separation voltage can be used to analyze a sample regardless of when it is applied. For a common sample in each of the sample reservoirs and separation channels, different process conditions (e.g., separation voltage, background electrolyte solution) can be used in each combination of sample reservoirs and separation channels so that a single sample can be analyzed under different sets of conditions. When different samples are located in at least some sample reservoirs, Figure 9 The multi-channel configuration allows for the multiplexing of different samples, which may have significantly different separation conditions and / or times. For example, components of samples that separate and migrate relatively quickly through their respective separation channels can be ejected first from their respective emitters, while components of samples that separate and migrate more slowly can be ejected later when they reach the end of their respective separation channels. In this way, the total time for analyzing multiple samples can be reduced compared to the sequential analysis of samples in a chip with a single separation channel.

[0146] Although Figure 9 The chip 300 shown has three sample reservoirs, three separation channels, three background electrolyte solution reservoirs, and three pumping channels, but more generally, the chip 300 may include two or more of any of these components (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, or even more).

[0147] Figure 10This is a flowchart 1000 illustrating a series of steps for performing pressure-based electrospraying and / or injection to separate sample components. In a first step 1002, a sample containing the components to be separated and / or analyzed is introduced into a sample reservoir 302. The introduction can be performed by injection via a port connected to the reservoir, through a fluid channel in communication with the reservoir, or by any of a variety of other methods. Generally, the sample is introduced in a background electrolyte solution (i.e., in a form partially or completely converted to a solvate).

[0148] Next, in step 1004, the electronic processor 390 applies a potential difference between the sample reservoir 302 and the ends of the separation channel 304 via electrodes 316 and 318. Typically, for example, the electronic processor 390 activates one or more power sources (not shown in the figure) to apply the potential difference. The application of the potential difference causes the components of the sample to migrate electrophoretically from the sample reservoir 302 through the separation channel 304 toward the emitter 310. The migration rate of each component varies depending on its respective electrophoretic mobility and, in some embodiments, on its interaction with the walls of the separation channel 304.

[0149] In step 1006, the electronic processor 390 pressurizes the headspace region of the background electrolyte solution reservoir 306 to cause the background electrolyte solution to flow from the reservoir 306 through the pumping channel 308 and out of the emitter 310. Generally, for example, the electronic processor opens and closes valve 335 to control the gas pressure in the headspace region using gas supplied from gas source 330.

[0150] After the components separate from each other during electrophoretic migration through separation channel 304, background electrolyte solution pressure-driven flow from reservoir 306 through pump channel 308 and from emitter 310 results in the respective discharge of sample components from emitter 310 in step 1008. Due to the different electrophoretic mobilities of the sample components, each component is discharged from emitter 310 at different times. If the components are subsequently injected or received into a mass spectrometry system (or into another type of analytical system) in step 1010, they are thus introduced at different times, simplifying component handling and analysis. The process then terminates in step 1012.

[0151] In some embodiments, it may be advantageous to ensure, in steps 1004 and 1006, that the start of the flow of the background electrolyte solution through the pumping channel 308 and out of the emitter 310 occurs simultaneously or nearly simultaneously with the application of a potential difference between the sample reservoir 302 and the end of the separation channel 304. Experimentally, it has been found that generating a high-quality electrospray plume from the emitter 310 can be difficult if the start of the flow of the background electrolyte solution and the application of the potential difference do not occur within a short time interval. Therefore, in some embodiments, the time difference between the start of the flow of the background electrolyte solution and the application of the potential difference (either of which may occur first) can be 25 ms or less (e.g., 15 ms or less, 10 ms or less, 5 ms or less, 2 ms or less, 1 ms or less, 500 microseconds or less, 200 microseconds or less, 100 microseconds or less).

[0152] As discussed above, the process of generating an electrospray plume by using pressure-driven flow of the background electrolyte solution to expel sample components from the emitter 310 can be decoupled from the process of separating sample components within the separation channel. The generation of a suitable electrospray plume is controlled by pressure-driven flow of the background electrolyte solution. The generation of ions from the separated sample components is controlled by the difference between the potential at the emitter 310 and the ground potential or reference potential outside the chip 300.

[0153] Therefore, the potential difference applied between electrodes 316 and 318 is used to control the electrophoretic separation of sample components only in separation channel 304. "Separation" can be performed with a zero-volt potential difference between electrodes 316 and 318. Under these conditions, sample components will not migrate significantly, but discharge from emitter 310 still occurs (in the form of background electrolyte solution), which allows for the measurement, verification, and adjustment of the electrospray plume characteristics by adjusting one or more operating parameters, as discussed above.

[0154] More generally, the electronic processor 390 is configured to implement various adaptive processing techniques involving the adjustment of the applied potential difference to improve sample component separation and analysis performance. For example, in some embodiments, the electronic processor 390 is configured to adjust the migration rate of sample components through the separation channel 304 by changing the potential difference applied between electrodes 316 and 318. Because the discharge from the emitter 310 occurs via pressure-driven flow of the background electrolyte solution, the applied potential difference can generally be adjusted as desired to control component migration time without adversely affecting component discharge.

[0155] For example, before the separated sample components initially reach the end of separation channel 304, the electronic processor 390 can apply a relatively high first potential difference between electrodes 316 and 318. Ejection from emitter 304 is coupled to or allowed into a mass spectrometry system, which analyzes the ejection to determine the presence of the component of interest. Information regarding the presence or absence of the component of interest is determined or received by processor 390. When no component of interest is detected, processor 390 maintains a high first potential difference between electrodes 316 and 318 to keep the migration time of the sample components through separation channel 304 short.

[0156] When one or more components of interest are detected in the electrospray plume, the electronic processor 390 can reduce the applied potential difference to a lower second potential difference, effectively reducing the migration rate of the sample components through the separation channel 304. Then, as the individual components reach the end of channel 304 and are discharged from the emitter 310, they can be analyzed individually in time sequence. Changing the applied potential difference in this way effectively alters the time interval between sample components arriving at the mass spectrometry system for analysis.

[0157] For complex samples with many components of interest, the electronic processor 390 can be configured to cycle multiple times between high and low applied potential differences to adjust the flow rate of the components within the separation channel 304. The processor 390 is not limited to alternating between only two applied potential differences; more generally, at any point, the processor 390 can select any potential difference to be applied between electrodes 316 and 318 to control the migration time of sample components within the separation channel 304.

[0158] In some embodiments, the electronic processor 390 can be configured to execute an analysis procedure to identify one or more specific components in a sample. That is, the processor is configured to specifically search for the presence or absence of a specific component, regardless of the presence or absence of other components in the sample. The electronic processor 390 may receive configuration information regarding the migration time of a specific component (e.g., as part of a procedure, or from another source such as a database or system user). To reduce the total analysis time for a specific sample, the electronic processor 390 may apply a relatively high potential difference between electrodes 316 and 318 until the first of the specific components is expected to reach the end of separation channel 304 based on the configuration information. The electronic processor 390 then reduces the applied potential difference so that if the first specific component is present in the sample and is expelled from emitter 310, it can be accurately detected.

[0159] Next, the electronic processor 390 increases the applied potential difference again until the next expected component of the specific composition, based on configuration information, reaches the end of the separation channel 304. After this time, the applied potential difference is again decreased by the processor 390 to facilitate the detection of the second specific component if it is present in the sample. The processor 390 can alternately increase and decrease the applied potential difference in this way to reduce the total analysis time of the sample while ensuring that the specific component of interest is well separated in time for detection.

[0160] Adjusting the applied potential difference within separation channel 304 is also advantageous when there is a time-limited window for the analysis of a particular sample. For example, in proton exchange mass spectrometry, for labeling purposes, a sample can be processed to replace some hydrogen atoms with deuterium atoms. After sample digestion, the deuterium-labeled protein is then analyzed to locate the deuterium-labeled sites in the structure.

[0161] However, deuterium atoms remain relatively volatile in sample proteins and readily undergo exchange with normal hydrogen atoms again. Therefore, analysis of deuterated proteins is performed relatively quickly before this re-exchange occurs to a large extent. To analyze sample components with time-limited lifetimes or time analysis windows, such as deuterated proteins, the electronic processor 390 can adjust the applied potential difference between electrodes 316 and 318 as discussed above to reduce the total analysis time of the sample. In particular, the processor 390 can maintain a relatively high potential difference as the component of interest migrates through channel 304 to reduce the amount of time consumed traveling along the length of the channel. When the component of interest is expected to reach the end of channel 304 (or when it is detected from emission from emitter 310), the processor 390 reduces the potential difference between electrodes 316 and 318 to allow each component to be analyzed. After a particular component is emitted, the processor 390 again increases the potential difference until the next component of interest reaches the end of channel 304 or is detected in emission from emitter 310. In this way, the electronic processor 390 can, for example, implement various procedures / sequences suitable for the analysis of time-constrained samples in proton exchange mass spectrometry.

[0162] In the embodiments discussed above, the pressure-driven flow of the background electrolyte solution occurs under the control of processor 390, which directs gas source 330 to supply gas to the top space 350 of reservoir 306. Pressure-driven flow can also be implemented in other ways along with or as an alternative to the gas-to-reservoir supply. For example, in some embodiments, a displacement mechanism such as a diaphragm or piston can be used to control the gas pressure in reservoir 306.

[0163] Figure 14This is a schematic diagram showing a portion of a jet chip 300 having a reservoir 306 containing a background electrolyte solution 314. A top space 350 is located above the background electrolyte solution 314. To control the pressure within the reservoir 306 (and thus the pressure applied to the background electrolyte solution 314), a piston 1480 forms part of the top wall of the reservoir 306. The piston 1480 is coupled to an electronic processor 390 via a control line 1482.

[0164] The processor 390 can adjust the pressure within the headspace 350 by advancing the piston 1480 into the reservoir 306 (to increase pressure) or withdrawing the piston 1480 from the reservoir 306 (to decrease pressure). In this way, the processor 390 can eject sample components from the emitter 310, as discussed above, and can adjust the operation of the chip 300 based on the measured parameters of the plume 340.

[0165] Pressure control within the reservoir 306 can also be achieved by adjusting the temperature within the top space. Temperature regulation can be implemented in various ways. For example, in Figure 14 In this configuration, heating element 1484 is positioned along the wall of reservoir 306 and connected to processor 390 via control line 1486. ​​By heating reservoir 306 (and background electrolyte solution 314) via heating element 1484, electronic processor 390 can increase the gas pressure within reservoir 306. Conversely, by (e.g., via...) Figure 14 The electronic processor 390 can reduce the gas pressure within the reservoir 306 by cooling elements (not shown) or by reducing the temperature of the heating element 1484.

[0166] Because a pressure gradient exists between the solution in reservoir 306 and the solution at or near emitter 310, pressure-driven flow of the background electrolyte solution 314 occurs. Therefore, in some embodiments, pressure-driven flow of the background electrolyte solution 314 can be achieved by reducing the pressure applied to the background solution 314 near emitter 310. Figure 15 A schematic diagram of a portion of the jet chip 300 is shown. For clarity, many features of the chip 300 have been omitted, but it should be understood that the chip 300 may generally include any features discussed herein regarding various jet chips.

[0167] exist Figure 15In this configuration, a vacuum source 1594 is positioned adjacent to the transmitter 310. The vacuum source 1594 is connected to an electronic processor 390 via a control line 1596. During operation, the processor 390 can adjust the pressure applied to the background electrolyte solution 314 at or near the transmitter 310 by selectively connecting the vacuum source 1594 to the pumping channel 308 and disconnecting the vacuum source 1594 from fluid communication with the pumping channel 308, for example, by opening or closing a valve within the vacuum source 1594. Typically, a wide variety of vacuum sources can be used, including but not limited to vacuum pumps, evacuated volumes, and displacement mechanisms such as pistons and diaphragms.

[0168] Typically, any of the foregoing methods can be used alone or in combination in the jet chip disclosed herein to allow the electronic processor 390 to control the pressure gradient across the background electrolyte solution 314 in the pumping channel 308 by adjusting the pressure within the reservoir 306, by adjusting the pressure at or near the transmitter 310, or both.

[0169] III. Connect to the mass spectrometry detection system via interface.

[0170] As discussed above, in some embodiments, the discharge from emitter 310 may be directly coupled into the inlet of the mass spectrometry system to facilitate the analysis of the separated sample components as they are discharged. In some embodiments, the discharge from emitter 310 may be located in the spatial region where the inlet of the mass spectrometry system is situated, and the system may sample the discharge through the inlet, allowing a portion of the discharge to enter for analysis. For example, additional aspects of suitable mass spectrometry systems that can be used with the jet chips disclosed herein are disclosed in, for example, U.S. Patent Application Publication No. 2015 / 0200083, U.S. Patent No. 9,502,226, and U.S. Patent Application Publication No. 2016 / 0099137, the entire contents of each of which are incorporated herein by reference.

[0171] Although the coupling between the emitter 310 of the chip 300 and the inlet of the mass spectrometry detection system can typically be achieved in a variety of ways, one such coupling method involves generating an ionized electrospray from the emitter 310, which is then guided into the inlet. Figure 13 This is a schematic diagram showing the jet chip 300 coupled to the mass spectrometry detection system 1310. Figure 13 For clarity, many features of chip 300 have been omitted. However, it should be understood that... Figure 13 The chip 300 in this document may typically include any features disclosed herein regarding various jet chips.

[0172] exist Figure 13In this process, sample components are emitted from emitter 310 via electrodes 1302 and 1304, which are connected to electronic processor 390 via control lines 1305 and 1307. During operation, processor 390 applies a potential to electrodes 1302 and 1304 to establish a potential difference between the electrodes, referred to above as the electrospray ionization voltage. As discussed earlier, processor 390 can adjust this voltage based on measurements from detector 704 and / or mass spectrometry detection system 1310.

[0173] Electrode 1304 forms part of inlet 1318 of detection system 1310, which also includes optional ion source 1312, ion trap 1314, and ion detector 1316. Components of detection system 1310 are connected to processor 390 via control line 1309. It should be noted that processor 390 can be configured as follows: Figure 13 The processor 390 is shown to be separate from the detection system 1310, or alternatively, it may be integrated within the detection system 1310. The processor 390 can typically control various components of one or both of the chip 300 and the detection system 1310 and perform functions associated with one or both of the chip 300 and the detection system 1310.

[0174] When processor 390 applies an electrospray ionization voltage between electrodes 1302 and 1304, an electrospray plume 340 is generated and enters inlet 1318. As sample components separate within separation channel 304, they are ejected through emitter 310, ionized between electrodes 1302 and 1304, and coupled into inlet 1318. Once inside ion trap 1314, the ionized sample components can optionally undergo further ionization in ion source 1312 before being captured and selectively ejected from ion trap 1314 for detection by detector 1316. Mass spectrometric information, including the mass-to-charge ratio of the detected ions, can be transmitted to processor 390 via control line 1309.

[0175] IV. Additional System Hardware and Software Components

[0176] Any method steps, features, and / or attributes disclosed herein can be executed by an electronic processor 390 and / or one or more additional electronic processors (e.g., a computer or a pre-programmed integrated circuit) that executes a program based on standard programming techniques. Such programs are designed to execute on a programmable computing device or a specially designed integrated circuit, each of which includes a processor, a data storage system (including memory and / or storage elements), at least one input device, and at least one output device, such as a display or printer. Program code is applied to input data to perform functions and generate output information applied to one or more output devices. Each such computer program can be implemented in a high-level procedural or object-oriented programming language, or assembly or machine language. Furthermore, the language can be a compiled or interpreted language. Each such computer program can be stored on a computer-readable storage medium (e.g., an optical storage medium, magnetic storage medium, and / or persistent solid-state storage medium such as a CD-ROM or DVD), which, when read by a computer, processor, or electronic circuit, enables the computer, processor, or electronic circuit to perform the analysis and control functions described herein.

[0177] Other embodiments

[0178] Many embodiments have been described. However, it should be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Therefore, other embodiments are within the scope of the appended claims.

Claims

1. A jet analysis system, comprising: A jet chip, the jet chip being formed on a planar substrate, and comprising: A sample reservoir connected to the first end of the electrophoresis separation channel; A background electrolyte reservoir is connected to the electrophoretic separation channel via a pumping channel; A hole extending from the second end of the electrophoretic separation channel to the outer surface of the jet chip; A first electrode extends from the outer surface of the jet chip into the sample reservoir; and A second electrode extends from the outer surface of the jet chip into the background electrolyte reservoir; A pressurization mechanism, which is connected to the background electrolyte reservoir; A detector configured to acquire information about an electrospray plume generated by the discharge of a background electrolyte solution through the orifice; and An electronic processor, connected to the detector and configured to: The pressurization mechanism is activated to adjust the gas pressure in the background electrolyte reservoir so that the gas pressure in the background electrolyte reservoir is greater than the gas pressure outside the orifice, thereby generating the flow of the background electrolyte solution from the background electrolyte reservoir through the pumping channel and out of the orifice; Receive the information about the electrospray plume from the detector; and The characteristics of the electrospray plume are adjusted based on the received information.

2. The system according to claim 1, wherein, The detector is configured to acquire an image of the electrospray plume.

3. The system according to claim 2, wherein, The information regarding the electrospray plume includes image information of the electrospray plume.

4. The system according to claim 2, wherein, The information regarding the electrospray plume includes the spatial distribution of the electrospray plume.

5. The system according to claim 2, wherein, The information regarding the electrospray plume includes the shape of the electrospray plume.

6. The system according to claim 1, wherein, The detector is configured to obtain spectral information of the electrospray plume.

7. The system according to claim 6, wherein, The spectral information includes the color of the electrospray plume.

8. The system according to claim 1, wherein, The detector is configured to measure the light transmitted through the electrospray plume.

9. The system according to claim 1, wherein, The detector is configured to measure the light reflected from the electrospray plume.

10. The system according to claim 1, wherein, The detector is configured to measure the light scattered from the electrospray plume.

11. The system according to claim 1, wherein, The detector is configured to measure light absorbed by the electrospray plume as a function of wavelength.

12. The system according to claim 1, wherein, The detector is configured to measure at least one member selected from the group consisting of: light refracted by the electrospray plume and light diffracted by the electrospray plume.

13. The system according to claim 1, wherein, The detector is configured to obtain electrical information about the electrospray plume.

14. The system according to claim 13, wherein, The electrical information includes the voltage associated with the electrospray plume.

15. The system according to claim 13, wherein, The electrical information includes the current associated with the electrospray plume.

16. The system according to claim 1, wherein, The electronic processor is configured to stabilize the electrospray plume by adjusting the characteristics of the electrospray plume.

17. The system according to claim 2, wherein, The electronic processor is configured to analyze the image and, based on the analysis, determine adjustments to the characteristics of the electrospray plume.

18. The system according to claim 17, wherein, The electronic processor is configured to analyze the image by detecting droplets in the image.

19. The system according to claim 18, wherein, The electronic processor is configured to determine the cross-sectional dimensions of one or more droplets in the image.

20. The system of claim 19, further comprising an electrode connected to the electronic processor and positioned along a flow path of the background electrolyte solution, wherein the electronic processor is configured to adjust a voltage applied to the electrode to ionize the background electrolyte solution in response to a determined cross-sectional size of the one or more droplets.

21. The system of claim 19, further comprising a valve connected to the electronic processor and positioned along a flow path of the background electrolyte solution, wherein the electronic processor is configured to adjust the valve to control the flow rate of the background electrolyte solution in response to a determined cross-sectional size of the one or more droplets.

22. The system according to claim 18, wherein, The electronic processor is configured to identify the absence of droplets in the image.

23. The system of claim 22, further comprising an electrode connected to the electronic processor and positioned along a flow path of the background electrolyte solution, wherein the electronic processor is configured to increase a voltage applied to the electrode to ionize the background electrolyte solution in response to identifying a lack of droplets in the image.

24. The system according to claim 23, wherein, The electronic processor is configured to maintain a constant flow rate of the background electrolyte solution through the electrophoretic separation channel after the applied voltage is increased.

25. The system according to claim 18, wherein, The electronic processor is configured to identify droplets in the image that are in contact with the edge of the jet chip.

26. The system of claim 25, further comprising an electrode connected to the electronic processor and positioned along a flow path of the background electrolyte solution, wherein the electronic processor is configured to increase a voltage applied to the electrode to ionize the background electrolyte solution in response to identifying a droplet in the image that contacts the edge of the jet chip.

27. The system according to claim 4, wherein: The detector is configured to acquire multiple images of the electrospray plume changing over time; as well as The electronic processor is configured to analyze the plurality of images to identify temporal variations in the spatial distribution of the electrospray plume.

28. The system according to claim 27, wherein, The electronic processor is configured to identify the temporal variation of the starting point of the electrospray plume in the plurality of images.

29. The system of claim 28, further comprising an electrode connected to the electronic processor and positioned along a flow path of the background electrolyte solution, wherein the electronic processor is configured to increase a voltage applied to the electrode to ionize the background electrolyte solution in response to a time variation in the initiation point of the electrospray plume in the plurality of images.

30. The system according to claim 27, wherein, The electronic processor is configured to identify changes in the boundary region of the electrospray plume in the plurality of images.

31. The system of claim 30, further comprising an electrode connected to the electronic processor and positioned along a flow path of the background electrolyte solution, wherein the electronic processor is configured to increase a voltage applied to the electrode to ionize the background electrolyte solution in response to a change in the boundary region of the electrospray plume in the plurality of images.

32. The system according to claim 31, wherein, The detector is configured to measure the mass spectrometry information of the electrospray plume.

33. The system according to claim 32, wherein, The electronic processor is configured to determine the flow rate of the electrospray plume based on the ion signals in the mass spectrometry information.

34. The system of claim 33, further comprising a valve connected to the electronic processor and positioned along a flow path of the background electrolyte solution, wherein the electronic processor is configured to adjust the valve based on a determined flow rate to control the flow rate of the background electrolyte solution.

35. The system according to claim 15, wherein, The electronic processor is configured to determine the time variation of the flow rate of the electrospray plume based on the current.

36. The system of claim 35, further comprising an electrode connected to the electronic processor and positioned along a flow path of the background electrolyte solution, wherein the electronic processor is configured to adjust a voltage applied to the electrode to ionize the background electrolyte solution based on the time variation of the flow rate of the electrospray plume.

37. The system according to claim 1, wherein, The detector is configured to obtain at least two of the following: Image information of the electrospray plume; The electrical information of the electrospray plume; and Mass spectrometry information of the electrospray plume.

38. The system according to claim 37, wherein, The electronic processor is configured to adjust multiple operating characteristics of the jet chip based on information received from the detector.

39. The system of claim 1 further includes a light source configured to guide illumination radiation incident on the electrospray plume.

40. A jet analysis method, comprising: Obtain information about an electrospray plume comprising a background electrolyte solution discharged from an orifice of a jet chip in the jet analysis system according to claim 1; and The electrospray plume of the jet chip is adjusted based on the received information.

41. The method according to claim 40, wherein, The information includes at least one member selected from the group consisting of: image information of the electrospray plume, spectral information of the electrospray plume, electrical information of the electrospray plume, and mass spectrometry information of the electrospray plume.

42. The method according to claim 41, wherein, This includes determining at least one member selected from the group consisting of: the spatial distribution of the electrospray plume, the shape of the electrospray plume, and the color of the electrospray plume.

43. The method of claim 40, further comprising obtaining the information by measuring at least one member of the group consisting of: light transmitted through the electrospray plume, light reflected from the electrospray plume, light scattered from the electrospray plume, light absorbed by the electrospray plume as a function of wavelength, light refracted by the electrospray plume, and light diffracted by the electrospray plume.

44. The method according to claim 41, wherein, The electrical information includes at least one member selected from the group consisting of: voltage associated with the electrospray plume and current associated with the electrospray plume.

45. The method of claim 41, further comprising analyzing the image information to detect droplets in the image information.

46. ​​The method of claim 45, further comprising determining the cross-sectional dimensions of one or more droplets in the image information, and adjusting the voltage applied to ionize the background electrolyte solution based on the determined cross-sectional dimensions of the one or more droplets.

47. The method of claim 45, further comprising determining the cross-sectional dimensions of one or more droplets in the image information, and adjusting the flow rate of the background electrolyte solution based on the determined cross-sectional dimensions of the one or more droplets.

48. The method of claim 45, further comprising identifying a lack of droplets in the image information, and in response to identifying a lack of droplets in the image, increasing the voltage applied to ionize the background electrolyte solution.

49. The method of claim 45, further comprising identifying droplets in the image information that are in contact with the edge of the jet chip, and in response to identifying droplets in the image information that are in contact with the edge of the jet chip, increasing the voltage applied to ionize the background electrolyte solution.

50. The method of claim 42, further comprising identifying temporal variations in the spatial distribution of the electrospray plume from the image information.

51. The method according to claim 50, wherein, The temporal variation of the spatial distribution includes at least one member selected from the group consisting of: the temporal variation of the starting point of the electrospray plume and the variation of the boundary region of the electrospray plume.

52. The method of claim 51, further comprising increasing the voltage applied to ionize the background electrolyte solution in response to at least one of a time variation in the initiation point of the electrospray plume and a variation in the boundary region of the electrospray plume.

53. The method of claim 41, further comprising determining the flow rate of the electrospray plume based on ion signals in the mass spectrometry information, and adjusting the flow rate of the background electrolyte solution based on the flow rate of the electrospray plume.

54. The method of claim 44, further comprising determining a time variation in the flow rate of the electrospray plume based on the current, and adjusting a voltage applied to ionize the background electrolyte solution based on the time variation in the flow rate of the electrospray plume.

55. The method of claim 41, further comprising adjusting a plurality of operating characteristics of the jet chip based on the received information.

56. A jet analysis system, comprising: The jet chip includes: Sample reservoir connected to the electrophoresis separation channel; A background reservoir connected to the electrophoretic separation channel via a pumping channel; A hole near one end of the electrophoretic separation channel; The first electrode extends into the sample reservoir; A second electrode extending into the background reservoir; A pressurization mechanism connected to the background reservoir; and An electronic processor, connected to the electrodes and the pressurization mechanism, and configured to: The pressurization mechanism is activated to adjust the gas pressure in the background reservoir so that the gas pressure in the background reservoir is greater than the gas pressure outside the orifice, thereby generating a flow of background electrolyte solution from the background reservoir through the pumping channel and out of the orifice; The flow rate of the background electrolyte solution through the electrophoretic separation channel is controlled by adjusting the pressurization mechanism; and The relative separation of components in the sample in the electrophoretic separation channel is controlled by adjusting the electrical signal applied to the electrode.

57. A method for analyzing a sample using the jet chip in the jet analysis system according to claim 1, comprising: An electrical signal is applied to the electrodes of the jet chip to control the relative separation of sample components along the electrophoretic separation channels of the jet chip; as well as The pressure of the background electrolyte solution in the jet chip is adjusted to control the discharge rate of the background electrolyte solution from the orifice of the jet chip.

58. A jet chip in a jet analysis system according to claim 1, comprising: Electrolyte reservoir; as well as A piston, the piston including a surface that forms part of the wall of the electrolyte reservoir.

59. A method for analyzing a sample using the jet chip in the jet analysis system according to claim 1, comprising: The coupling agent in the sample and the background electrolyte solution is combined on the jet chip; The components of the sample are discharged from the jet chip; as well as Determine the mass spectrometry information of the components of the discharged sample.

60. A mass spectrometry system, comprising: Ion source; Ion trap; as well as Ion detector, The mass spectrometry system is configured to receive sample components from the jet chip in the jet analysis system according to claim 1.

61. A system for jet delivery of a sample, comprising: The jet chip in the jet analysis system according to claim 1, the jet chip includes a channel for delivering a sample; as well as A fluid pressure regulating mechanism, the fluid pressure regulating mechanism including a diaphragm to regulate the fluid flowing through the channel.

Images