Microfluidic analysis of biological samples

Abstract

To provide mass analysis and a method for measuring mass spectrum information by using a microfluidic analysis sample handling system.SOLUTION: A method and a system for processing a sample includes the steps of: introducing a sample characteristically having one or more components in a first electrolyte solution into a separation channel of a fluid device; applying an electrical potential difference across the sample in order to move at least one sample component toward an end of the separation channel; adjusting at least one of gas pressure in a tank and gas pressure outside an aperture such that the gas pressure in the tank including a second electrolyte solution becomes greater than the gas pressure outside the aperture arranged at the end of the separation channel; and directing the flow of the second electrolyte solution to the outside of the aperture through a pumping channel in response to the gas pressure in order to discharge at least one sample component through the aperture.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 478,689, filed March 30, 2017, which is incorporated herein by reference in its entirety.

[0002] Technical Field The present disclosure provides mass spectrometry and methods for measuring mass spectral information using a microfluidic sample handling system. [Background technology]

[0003] Mass spectrometers are widely used to detect chemical compounds. In a typical mass spectrometer, molecules or particles are excited or ionized, and these excited species often break down to form ions of lower mass or react with other species to form other characteristic ions. This ion formation pattern can be interpreted by the system operator to infer the identity of the compound.

[0004] Biomolecules can sometimes be difficult to analyze using mass spectrometry. In particular, the transport and ionization of certain large biomolecules prior to mass spectrometric analysis can be a significant obstacle. While fluidic transport techniques can be used to handle such samples, specialized ionization methods, such as electrospray ionization, have been developed to generate ions that can then be analyzed. Summary of the Invention

[0005] Transporting biological samples on specially designed fluidic chips provides a convenient and reproducible method for handling such samples. Such chips can include one or more built-in electrospray emitters to efficiently eject such samples and form a homogeneous, finely dispersed sample vapor that can be injected into a mass spectrometer. Conventional techniques for flowing such samples through fluidic channels include, for example, electroosmotic pumping, in which an electric potential is applied to the ends of a flow channel to move a fluid within the channel.

[0006] In conventional chips that implement electroosmotic pumping, the chemical environment of the flow channel walls is carefully controlled to ensure proper sample flow within the channel. Control over the chemical environment of the channel walls, along with the potential difference applied between the ends of the channel, is used to modulate the electroosmotic and electrophoretic mobilities of analytes and sample matrix components within the channel.

[0007] The present disclosure features systems and methods that use differential pressure, rather than electroosmotic pumping, to generate sample flow and achieve separation of sample components within microfluidic chips and other fluid transport systems. Differential pressure allows transport of sample components through a fluid channel even under applied potential differences that would otherwise be too small to transport the components. The methods and systems disclosed herein can transport samples even when no potential difference is applied across the flow channel.

[0008] By decoupling the applied potential difference from the flow rate of sample components through the fluidic chip or system, the flow rate within the flow channel can be controlled independently of the potential difference applied across the channel. This allows separation of sample components to be performed within the flow channel at a desired flow rate range. In effect, the flow rate and degree of separation of sample components within the flow channel can be independently varied, allowing for significantly greater control of sample separation and analysis within a mass spectrometry system.

[0009] In general, in a first aspect, the disclosure provides a method for processing a sample, the method comprising: introducing a sample, the sample characterized by one or more components in a first electrolyte solution, into a separation channel of a fluidic device; applying a potential difference across the sample to migrate at least one sample component toward an end of the separation channel; adjusting at least one of the gas pressure in a tank containing a second electrolyte solution and the gas pressure outside an opening disposed at the end of the separation channel such that the gas pressure in the tank is greater than the gas pressure outside the opening, the tank being connected to a pumping channel, the pumping channel being connected to the separation channel near the end of the separation channel; and directing a flow of the second electrolyte solution through the pumping channel and out of the opening in response to the gas pressure to expel the at least one sample component through the opening.

[0010] Embodiments of the method can include one or more of the following features.

[0011] Regulating the gas pressure within the tank can include one or more of introducing a gas into the tank, compressing the gas within the tank, and heating the tank. Regulating the gas pressure outside the opening can include operating a vacuum source proximate to the opening. The gas pressure within the tank can be at least 0.5 psi (e.g., 2.0 psi) higher than the gas pressure outside the opening.

[0012] The flow rate of the electrolyte in the pumping channel can be 50 nL / min or more (e.g., 200 nL / min or more). The first and second electrolytes can have common components. Alternatively, the composition of the second electrolyte can be different from the composition of the first electrolyte. The concentration of one or more organic modifiers in the second electrolyte can be higher than the concentration of the one or more organic modifiers in the first electrolyte.

[0013] The magnitude of the potential difference can be between 0 V and 20 kV (e.g., between 0 V and 10 kV). The at least one sample component can travel through a dead volume of 500 pL or less (e.g., 100 pL or less) before being expelled from the opening.

[0014] The method can include acquiring information about an exhaust plume generated by discharging a fluid including the second electrolyte through the opening. To acquire the information, the method can include measuring one or more of 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. To adjust the exhaust plume, the method can include diverting a portion of the flow of the second electrolyte into an exhaust channel based on the acquired information.

[0015] The method can include adjusting a gas pressure in an auxiliary tank containing a third liquid so that the gas pressure in the auxiliary tank is greater than the gas pressure outside the opening, the auxiliary tank being connected to an auxiliary channel, the auxiliary channel being connected to the separation channel near the end of the separation channel, and directing a flow of the third liquid through the auxiliary channel into the separation channel in response to the gas pressure in the auxiliary tank. The third liquid can include a mass spectrometry calibration compound, and the calibration compound is discharged through the opening. The third liquid can include a mass spectrometry coupling agent, and the coupling agent is discharged through the opening.

[0016] The method can include obtaining information regarding an actual or expected migration time of the at least one sample component within the separation channel; and adjusting 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, the first potential difference and the second potential difference having different magnitudes. The first potential difference can be between 10 kV and 20 kV. The second potential difference can be 10 kV or less (e.g., 5.0 kV or less). The second potential difference can be 0 V.

[0017] The method can include obtaining the information regarding the actual migration time of the at least one sample component by detecting a portion of the at least one sample component released through the opening. The method can include detecting the portion of the at least one sample component using a mass spectrometry detection system.

[0018] The method can include obtaining the information regarding the expected migration time of the at least one sample component from a database of reference information for the at least one sample component. The at least one sample component can include multiple sample components, and the method can include adjusting the applied potential difference for each sample component or group of components such that different potential differences are applied during different portions of the migration of that component or group of components. The applied potential difference can alternate between successively larger and smaller values ​​during migration of the multiple sample components.

[0019] Embodiments of the method may include any other features disclosed herein, including features disclosed in the context of different embodiments, in any combination, unless expressly stated otherwise.

[0020] In another aspect, the disclosure provides a fluidic analytical system including: a fluidic chip formed on a planar substrate, the fluidic chip including: a sample reservoir connected to a separation channel at a first end thereof; a background electrolyte reservoir connected to a pumping channel, the pumping channel connected to the separation channel near a second end thereof opposite the first end; an opening 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 pressure mechanism connected to the background electrolyte reservoir; and an electronic processor connected to the first and second electrodes and to the pressure mechanism. and an electronic processor configured, during operation of the system, to: apply a potential difference between the first electrode and the second electrode to move at least one component of a sample contained in the separation channel toward the second end; operate the pressurizing mechanism to adjust the gas pressure in the background electrolyte tank so that the gas pressure in the background electrolyte tank is greater than the gas pressure outside the opening; and generate a flow of background electrolyte from the background electrolyte tank through the pumping channel and out of the opening; wherein when the at least one component of the sample reaches the second end, the at least one component is released through the opening by the flow of background electrolyte.

[0021] Embodiments of the system may include one or more of the following features.

[0022] The pressurizing mechanism can include at least one of a piston, a diaphragm, and a heating device. The gas pressure in the background electrolyte tank can be at least 0.5 psi higher than the gas pressure outside the opening. The flow rate of the background electrolyte in the pumping channel can be 50 nL / min or greater. The magnitude of the potential difference can be between 0 V and 20 kV.

[0023] The system may include a junction between the separation channel and the pumping channel near the second end that defines a dead volume of the separation channel, and the dead volume may be 500 pL or less (e.g., 100 pL or less).

[0024] The system can include at least one detector coupled to the electronic processor to obtain information about an exhaust plume generated by exhausting the background electrolyte-containing fluid through the opening, wherein the at least one detector can be configured to measure one or more of 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 to obtain the information.

[0025] The system can include a drain channel connected to the pumping channel through a drain valve, the electronic processor connected to the drain valve and configured to receive the information from the at least one detector and, based on the received information, selectively actuate the drain valve to divert a portion of the flow of the background electrolyte into the drain channel.

[0026] The fluidic chip may include an auxiliary tank connected to an auxiliary pressurizing mechanism and an auxiliary channel connected to the auxiliary tank and near the second end of the separation channel, and the electronic processor may be connected to the auxiliary pressurizing mechanism and configured to operate the auxiliary pressurizing mechanism to adjust the gas pressure in the auxiliary tank so that the gas pressure in the auxiliary tank is greater than the gas pressure outside the tank, thereby generating a flow of auxiliary liquid from the auxiliary tank through the auxiliary channel and into the separation channel. The auxiliary liquid may include a mass spectrometry calibration compound, and the calibration compound is discharged through the opening. The auxiliary liquid may include a mass spectrometry coupling agent, and the coupling agent is discharged through the opening.

[0027] The electronic processor can be further configured to obtain information regarding an actual or expected migration time of the at least one sample component within the 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, the first potential difference and the second potential difference having different magnitudes. The first potential difference can be between 10 kV and 20 kV. The second potential difference can be 10 kV or less (e.g., 5.0 kV or less). The second potential difference can be 0 V.

[0028] The electronic processor can be configured to obtain the information regarding the actual migration time of the at least one sample component from a detection system configured to detect a portion of the at least one sample component released through the opening. 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 be configured to obtain the information regarding the expected migration time of the at least one sample component from a database of reference information for the at least one sample component.

[0029] The at least one sample component can include multiple sample components, and for each sample component or group of components, the electronic processor can be configured to adjust the applied potential difference such that different potential differences are applied during different portions of the movement of the component or group of components. The electronic processor can be configured to adjust the applied potential difference such that the applied potential difference alternates between successively larger and smaller values ​​during movement of the multiple sample components.

[0030] Embodiments of the system may include any other features disclosed herein, including features disclosed in connection with different embodiments, in any combination unless expressly stated otherwise.

[0031] Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter herein; suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, these materials, methods, and examples are illustrative and not intended to be limiting.

[0032] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will become apparent from the detailed description, the drawings, and the claims. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram of a capillary electrophoresis system. [Figure 2] FIG. 1 is a schematic diagram of a capillary electrophoresis system using pressure-based transport of background electrolyte. [Figure 3] FIG. 1 is a schematic diagram of a fluidic chip. [Figure 4] FIG. 1 is a schematic diagram showing a portion of a separation channel. [Figure 5] FIG. 4 is a schematic diagram showing a portion of the fluidic chip of FIG. 3. [Figure 6] FIG. 4 is a schematic diagram showing another portion of the fluidic chip of FIG. 3. [Figure 7] FIG. 1 is a schematic diagram of a fluidic chip connected to an electronic processor. [Figure 8] FIG. 1 is a schematic diagram of another fluidic chip. [Figure 9] FIG. 1 is a schematic diagram of a further fluidic chip. [Figure 10] 1 is a flow chart showing a series of steps for separating sample components in a fluidic chip. [Figure 11] FIG. 1 is a schematic diagram of ejection from a fluidic chip. [Figure 12] FIG. 1 is a schematic diagram of a plume emitted from a fluidic chip. [Figure 13] FIG. 1 is a schematic diagram of a fluidic chip coupled to a mass spectrometry detection system. [Figure 14] FIG. 1 is a schematic diagram of a fluidic chip including a displacement mechanism connected to a fluid reservoir. [Figure 15] FIG. 1 is a schematic diagram of a fluidic chip including a vacuum source near an emitter.

[0034] Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION OF THE INVENTION

[0035] The analysis of biological molecules and sample components can be difficult due to the complex nature of such molecules and components, their relative fragility in some cases, and the environment they and the sample reside in. Often, biological analytes of interest, such as proteins, nucleic acids, lipids, and carbohydrates, are present in a sample along with a variety of other species not of interest (collectively referred to as "matrix" or "matrix components").

[0036] The accuracy and reproducibility of these molecules and components can be significantly improved if they are at least partially separated from the matrix before being subjected to quantitative analytical techniques, thereby ensuring that confounding effects on the analytical signal from the matrix, which tend to obscure the signal from the analyte of interest, are reduced or eliminated.

[0037] Several techniques can be used to separate a single biological sample into analytes of interest and matrix components not intended for analysis. Among these techniques, capillary electrophoresis is particularly useful due to its general applicability to a wide range of samples and its ability to achieve high-quality separation of analytes of interest even with relatively small total sample volumes. Capillary electrophoresis is also a relatively mild technique well suited to preserving the biochemical and stereochemical properties of analytes.

[0038] The following sections describe some general aspects of fluid handling and sample separation via electrophoretic techniques, followed by a description of methods and systems for performing sample transport and electrospray ionization via pressure-driven flow.

[0039] I. Handling and Transport of Fluid Samples Capillary electrophoresis is a technique in which components of a sample are separated from one another along the length of a separation column or channel based on their differential migration in the presence of an electric field. EP is its electrophoretic velocity μ in the electric field E EP is defined as follows:

[0040]

number

[0041] The total velocity of the components moving is the electroosmotic mobility μ EO Electroosmotic flow V depends on EO In capillary electrophoresis systems, it depends on:

[0042]

number

[0043] The electroosmotic mobility of components depends on their interaction with the channel through which they move (via their zeta potential) and the relative dielectric constant of the buffer. The overall rate of movement of the components within the channel also depends on the electroosmotic flow of the buffer within the channel.

[0044] In an electrophoresis system in which the electroosmotic flow of buffer solution is directed toward a negatively charged cathode, positively charged sample components migrate toward the cathode, moving in the same direction and with the electroosmotic flow. In contrast, negatively charged sample components tend to migrate in the opposite direction to the electroosmotic flow, toward the positively charged anode. When the magnitude of the electroosmotic flow velocity is greater than the magnitude of the electrophoretic velocity, all sample components migrate in the same direction as the electroosmotic flow.

[0045] In conventional capillary electrophoresis systems, separation of sample components occurs by migration of the components within a separation channel. An electric field applied to the channel controls the electrophoretic velocity of the components within the channel and the rate of electroosmotic flow. Furthermore, the surface chemistry of the channel walls can be controlled to adjust the electroosmotic mobility of each component, and the direction of electroosmotic flow can be controlled by the choice of buffer used. By appropriately selecting each of these factors, sample components of interest (i.e., analytes) can be separated from matrix components, and analytes can be separated from each other for analysis.

[0046] 1 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 realized as a small orifice near the intersection of the separation channel 104 and the pumping channel 108, and functions as an electrospray emitter for generating vapor containing sample components.

[0047] During operation of system 100, a potential V1 is applied via electrode 116 to background electrolyte 112 in sample tank 102, which also contains the sample of interest. A potential V2 is applied via electrode 118 to background electrolyte 114 in background electrolyte tank 106. The potential difference ΔV between background electrolyte 112 in tank 102 and background electrolyte 114 in tank 106 is sep = V1-V2 generates an electric field between tanks 102 and 106. This electric field extends through junction 120 where separation channel 104 and pumping channel 108 intersect.

[0048] As discussed above in connection with Equations 1 and 2, ions and dipolar sample components move at different velocities in response to the electric field generated in the separation channel 104. Thus, different sample components arrive at the junction 120 at different times. Once separated in this manner, these different sample components can be analyzed using techniques such as mass spectrometry. To begin mass analysis of the sample components, the components are first ejected from the separation channel 104 via the ejector 110 and then introduced into a mass spectrometry system.

[0049] Electrospray ejection via the ejector 110 is a technique particularly suited to biological sample components that may otherwise be difficult to introduce into a mass spectrometry system. Many biological sample components include large molecules, such as proteins, peptides, and nucleic acids, that are poorly suited to vaporization into the gas phase. Conversely, the amount of heat required to vaporize such components can result in undesirable side effects, such as protein denaturation, rendering analysis of such components inaccurate and ineffective. Electrospray ejection converts many biological sample components into the gas phase under relatively mild conditions, thereby avoiding undesirable denaturation of the structure of these components.

[0050] 1 , electrospraying of separated sample components through emitter 110 is achieved by electroosmotic pumping of background electrolyte 114 from background electrolyte tank 106 through emitter 110. When voltages V1 and V2 are applied via electrodes 116 and 118 as described above, electroosmotic flow of background electrolyte 114 occurs from background electrolyte tank 106 through pumping channel 108 and junction 120. To assist the electroosmotic flow of background electrolyte 114, the interior surface of pumping channel 108 can include a surface coating that interacts with background electrolyte 114 to drive electroosmotic flow.

[0051] As background electrolyte 114 passes through junction 120, sample components that separate from one another and propagate through separation channel 104 are swept away by the electroosmotic flow of background electrolyte 114 and ejected through ejector 110. In this way, a detailed gas-phase spatial distribution of each component is generated for subsequent analysis, for example, using mass spectrometry.

[0052] The generation and velocity of electroosmotic flow within pumping channel 108 and subsequent electrospray through emitter 110 depend on the potential difference applied between electrodes 116 and 118 and on the interior surface chemistry of pumping channel 108. Generally, the interior surface of pumping channel 108 is charged and has a potential difference ΔV sep When a potential difference ΔV is applied, making electrode 116 an anode (i.e., positively charged) and electrode 118 a cathode (i.e., negatively charged), the cationic moieties in background electrolyte 114 tend to migrate toward electrode 118, while the anionic moieties tend to migrate toward electrode 116. If an anionic charge accumulates on the inner channel wall, the positively charged portion of the background electrolyte forms two cationic layers ("diffuse double layer" or "electrical double layer") on the inner channel wall. The first layer, closest to the channel wall, is more tightly bound than the second layer, which we call the "mobile layer." The potential difference ΔV sepWhen a voltage is applied, it is the movable layer that is attracted toward the anionic cathode (i.e., electrode 118). The cationic moieties that make up the movable layer are solvated in the background electrolyte, and bulk transport of the background electrolyte occurs in the direction away from electrode 118. That is, both the cationic and anionic moieties in background electrolyte 114 are transported toward electrode 118 via the process of electroosmotic flow.

[0053] Thus, the discharge rate and other physical attributes of the electrospray through emitter 110 are strongly influenced by the electroosmotic flow rate through pumping channel 108 and junction 120. The electroosmotic flow rate then varies with the applied potential difference ΔV sep and can be controlled by adjusting the surface chemistry of the inner wall of the pumping channel 108. Generally, the applied potential difference ΔV sep As the value of is increased, the electroosmotic flow rate increases.

[0054] Engineering the surface chemistry of the interior walls of channel 108 is more complex. In the example above, anionic charges can be generated on the channel walls by selecting an appropriate material for forming the walls of channel 108 (or a material used to functionalize the walls of channel 108). The number of available anionic charges can, in some embodiments, be adjusted by flowing a solution of appropriate pH through channel 108 (e.g., to generate anionic functional groups on the channel walls via deprotonation).

[0055] Thus, a method for controlling the rate of electroosmotic flow within the pumping channel 108 and junction 120 involves adjusting the surface chemistry of the inner walls of the pumping channel 108. In the example described above, strong anionic groups functionalize the walls of the channel 108, allowing for relatively strong electroosmotic flow, leading to a relatively high rate of bulk transport of background electrolyte 114 through the pumping channel 108 and junction 120 and a relatively high rate of ejection through the emitter 110. Conversely, to reduce electroosmotic flow within the channel 108, junction 120, and emitter 110, the inner walls of the channel 108 can be coated with materials such as various polymers and / or surfactants, which prevent the formation of charged functional groups on the wall surfaces and thus reduce the rate of electroosmotic flow. In such a situation, the rate of bulk transport of background electrolyte 114 slows sufficiently to allow the anionic portion of the electrolyte 114 to resume natural migration toward the junction 120 (i.e., toward the cathode electrode 116).

[0056] The flow rate of background electrolyte 114 through pumping channel 108 and junction 120 and out of emitter 110 affects not only the electrospray fluid ejection rate, but also the rate at which sample components move through separation channel 104. As background electrolyte 114 passes through junction 120, it mixes with background electrolyte 112 from separation channel 104. This mixture of fluids is ejected from emitter 110. Thus, as the electroosmotic flow rate of background electrolyte 114 increases, the rate at which background electrolyte 112 is displaced through separation channel 104 also increases. This causes sample components to move through separation channel 104 at a faster rate.

[0057] In summary, electroosmotic pumping, by achieving electroosmotic flow of background electrolyte 114 in pumping channel 108, allows for controlled electrospray of sample components separated from one another in separation channel 104 via emitter 110. By controlling the flow rate of the electroosmotic flow in pumping channel 108, the fluid volume of the electrospray emission and the migration time of individual sample components through channel 104 can be adjusted.

[0058] The potentials V1 and V2 applied to electrodes 116 and 118, respectively, also affect the rate of electroosmotic flow in separation channel 104. The background electrolyte 112 present in sample reservoir 102 and separation channel 104 is proportional to the applied potential difference ΔV sep = V1-V2. The rate of electroosmotic flow of background electrolyte 112 within separation channel 104 is also strongly influenced by the surface chemistry of the inner walls of separation channel 104. Depending on the nature of the inner wall functionalization, electroosmotic flow of background electrolyte 112 within separation channel 104 can occur in a direction toward sample reservoir 102 or junction 120.

[0059] From the above description, it is clear that regulating the electroosmotic flow rate in the pumping channel 108 and the separation channel 104 involves carefully balancing several factors. For example, the electroosmotic flow rate in the pumping channel 108 and the separation channel 104 is affected by the potential difference ΔV sep , which in turn depends on the potentials V1 and V2 applied to electrodes 116 and 118. However, the electrophoretic mobilities of individual sample components within separation channel 104 also depend on the potential difference ΔV sep Thus, modulation of the electroosmotic flow rate in the separation channel 104 and pumping channel 108 is coupled, at least in part, to modulation of the electrophoretic mobility of sample components in the separation channel 104.

[0060] The electroosmotic flow rates in channels 104 and 108 also depend on the surface chemistry of the inner walls of these channels, and in principle, these flow rates can be adjusted by manipulating the surface chemistry within the channels. However, in practice, achieving uniform coatings in small-diameter channels presents manufacturing challenges. Furthermore, changing the surface chemistry in situ by flowing a solution through channels 104 and / or 108 may not be easily achieved depending on the surface chemistry of the channel walls. Thus, depending on the nature of the background electrolytes 112 and 114, it may not always be possible to adjust the surface chemistry of channels 104 and 108 as controllably as desired.

[0061] II. Sample transport via pressure-driven flow and electrospray ejection Based on the above description, it is clear that greater control over the rate of sample component migration within the separation channel 104 and the amount of fluid released through the releaser 110 can be achieved by using an alternative method of fluid transport in the pumping channel 108 that is isolated from the electroosmotic flow of background electrolyte and electrophoretic migration of sample components occurring within the separation channel 104.

[0062] As an alternative to the electroosmotic flow-based pumping system described above, Figure 2 shows a schematic diagram of an electrophoresis-based separation system 200 that uses pressure-based transport of a background electrolyte in a pumping channel. System 200 includes a sample reservoir 202, a separation channel 204, a background electrolyte reservoir 206, and a pumping channel 208. A mixture of background electrolyte 212 and sample is contained in sample reservoir 202, and background electrolyte 214 is contained in background electrolyte reservoir 206. Electrodes 216 and 218 extend into sample reservoir 202 and electrolyte reservoir 206, respectively.

[0063] In operation, as described above, potentials V1 and V2 are applied to electrodes 216 and 218, respectively, creating a potential difference ΔV sep= V1 - V2. This electric field imparts electrophoretic velocities to the components of the sample, which move at different speeds along separation channel 204 from reservoir 202 to junction 220. At the same time, the surface chemistry of the pumping channel 208 interior walls ensures that the electroosmotic mobility of background electrolyte 214 within pumping channel 208 is relatively small or zero. That is, in contrast to the pumping scheme shown in FIG. 1 , electroosmotic flow of background electrolyte 214 within pumping channel 208 does not occur as a result of the potential difference between electrodes 216 and 218.

[0064] 2 , background electrolyte tank 206, in addition to being connected to pumping channel 208, is also connected to gas source 230 via conduit 232. Gas source 230 supplies gas 234 to the headspace above background electrolyte 214 in tank 206. In this manner, gas source 230 is configured to pressurize the volume contained within tank 206. By applying an appropriate pressure to the headspace (i.e., gas-filled portion) within tank 206, gas source 230 drives the transport of background electrolyte 214 from tank 206 through pumping channel 208 and junction 220. Background electrolyte 214 mixes with background electrolyte 212 and sample components from separation channel 204, and this mixture is expelled through emitter 210.

[0065] As discussed above in connection with FIG. 1 , the flow rate of background electrolyte 214 through junction 220 affects the fluid output through emitter 210 and, by mixing with fluid from the separation channel 204, the migration rate of sample components from reservoir 206 through the separation channel 204. As the flow rate of background electrolyte 214 increases, the migration rate of sample components in separation channel 204 also increases, resulting in a decrease in the migration time of each sample component through the separation channel 204. The flow rate of background electrolyte 214 can be controllably adjusted by varying the gas pressure in the headspace of reservoir 206. Generally, increasing the gas pressure in reservoir 206 increases the pressure gradient between reservoir 206 and emitter 210, increasing the flow rate of background electrolyte 214 through pumping channel 208, junction 220, and emitter 210. As discussed above, increasing the flow rate of background electrolyte 214 also increases the migration rate of sample components through separation channel 204.

[0066] In addition to the effect of the flow rate of background electrolyte 214, the migration rate of sample components through separation channel 204 is also determined by the electrophoretic velocity imparted to each sample component by the electric field generated by the voltage applied at electrodes 216 and 218. Thus, by utilizing pressure-based fluid transport within pumping channel 208, two aspects that contribute to the overall migration rate of sample components through separation channel 204 can be independently controlled: the electrophoretic mobility (potential difference ΔV) of the sample components, and the electrophoretic velocity (potential difference ΔV) of the sample components. sep 2) and the component of the velocity of each sample component due to the amount of fluid ejected through the ejector 210 (via adjustment of the flow rate of the background electrolyte 214). These aspects can be adjusted independently, allowing a greater degree of control over the movement of sample components within the separation channel 204 and over the characteristics of the electrospray plume ejected from the ejector 210 than is possible using the electroosmotic pumping scheme shown in FIG.

[0067] An important advantage of using pressure-based control over the flow rate of background electrolyte 214 is that it is not necessary to adjust the surface chemistry of the interior walls of pumping channel 208 to generate electroosmotic flow. As discussed above in connection with FIG. 1 , in some embodiments, electroosmotic flow is controlled or influenced by adjusting the surface chemistry of the channel walls through which the flow occurs. This surface chemistry is typically tailored to the specific background electrolyte and / or sample components to achieve appropriate migration times for the sample components. To adapt an electrophoresis system to handle different samples, it may be necessary to modify the surface chemistry of the channel walls, for example, by introducing a strongly basic or strongly acidic solution into the channel to alter the ionic state of the surface functional groups attached to the walls. During channel fabrication, it can be challenging to provide a thin, uniform coating on the surface of the channel walls, especially when the channel diameter is only a few microns.

[0068] Pressure-based control of the flow rate of background electrolyte 214 eliminates the fabrication difficulties discussed above. Because fluid flow occurs based on a pressure gradient rather than electroosmosis, adjustments to the surface chemistry of the channel walls are generally not required during fabrication or sample change. Because background electrolytes with specific properties do not "match" the surface chemistry of the pumping channel 208, a wide variety of different background electrolytes can be used for pumping. As a result, electrophoretic separation systems are easier to fabricate and can be readily adapted to different analytical conditions and samples of interest.

[0069] The electrophoresis sample analysis systems disclosed herein are typically implemented, at least in part, on a fluidic chip. Figure 3 is a schematic diagram illustrating a fluidic chip 300 including a sample reservoir 302, a separation channel 304, a background electrolyte 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. An emitter 310 is located near the junction 320. The above-described components of the fluidic chip operate generally similarly to the corresponding components in Figure 2. A gas-phase electrospray plume 340 containing background electrolyte from the separation channel 304 and the pumping channel 308, and optionally one or more sample components, is emitted from the emitter 310 during operation.

[0070] Tank 306 contains background electrolyte 314 and a gas-filled sealed headspace region 350. Tank 306 is coupled via conduit 332 and gas-tight interface 336 to a gas source 330 that supplies gas to the sealed headspace of tank 306.

[0071] Tank 302 contains a sample 360 ​​of interest that is to be separated into components on chip 300. For electrophoretic separation of the components of sample 360 ​​in separation channel 304, tank 302 may also contain a background electrolyte 312.

[0072] Electrodes 316 and 318 and gas source 330 can be connected to electronic processor 390 via control lines 391, 392, and 393. In operation, electronic processor 390, if present, applies appropriate voltages to electrodes 316 and 318 to initiate electrophoretic movement of sample components from reservoir 302 through separation channel 304 toward junction 320, and activates gas source 330 to pump gas into the headspace of reservoir 306, transporting background electrolyte 314 from reservoir 306 through pumping channel 308 and junction 320, and expelling background electrolyte 314 through emitter 310.

[0073] In some embodiments, chip 300 may be configured to electrochemically inject sample into separation channel 304. Chip 300 may optionally include a background electrolyte tank 370 and a waste tank 372 positioned on opposite sides of separation channel 304 to form an "injection crossover" 376. Optional electrode 374 is coupled to electronic processor 390 via control line 390, allowing electronic processor 390 to apply an electrical potential to background electrolyte 312 in tank 370. By alternately pumping multiple volumes of fluid from tanks 302 and 370 into separation channel 304 (and directing waste into tank 372), sample "plugs" can be introduced into separation channel 304 for subsequent electrophoretic separation in separation channel 304. Additional aspects of electrokinetic sample injection are disclosed, for example, in U.S. Published Patent Application No. 2013 / 0327936, PCT Published Patent Application No. WO 2013 / 191908, and U.S. Published Patent Application No. 15 / 079,541, the entire contents of which are incorporated herein by reference. [Patent Document 1] U.S. Patent Publication No. 2013 / 0327936 [Patent Document 2] PCT Patent Publication No. WO 2013 / 191908 [Patent Document 3] U.S. Patent Application Serial No. 15 / 079,541

[0074] A schematic diagram of a portion of the separation channel 304 is shown in FIG. 4. The fluidic chip 300 is implemented as a planar structure extending in the x and y dimensions and having a thickness in the z dimension (i.e., perpendicular to the plane of FIG. 4). The separation channel 304 extends axially in the xy plane and is defined by a central axis 402 that defines the axial direction. The axis 402 may be linear or may follow a nonlinear (e.g., curved) path. The width w of the separation channel 304 is measured in a direction perpendicular to the axis 402 in the xy plane. The width w is generally selected based on factors such as the desired flow rate of the sample and background electrolyte through the separation channel 304 and the desired interaction between the sample 360 ​​and the walls of the separation channel 304 during sample migration. Typically, w is about 1 micron or greater (e.g., 5 microns or greater, 10 microns or greater, 20 microns or greater, 30 microns or greater, 40 microns or greater, 50 microns or greater, 60 microns or greater, 70 microns or greater, 80 microns or greater, 90 microns or greater, 100 microns or greater, 120 microns or greater, 150 microns or greater, 200 microns or greater, 300 microns or greater, 500 microns or greater, 750 microns or greater, 1 mm or greater).

[0075] The depth of the separation channel 304, measured in the z-direction perpendicular to the xy-plane, may be selected as desired based on the thickness of the chip 300 to control the amount of sample and background electrolyte that the separation channel 304 can handle during sample separation and analysis. The depth of the separation channel 304 may be selected to control Joule heating within the channel, adjust the hydraulic flow resistance of the channel, and control the overall flow rate of fluid within the channel. Typically, the separation channel 304 has a depth of about 10 microns. More commonly, the depth of the separation channel 304 may be 1 micron or greater (e.g., 5 microns or greater, 10 microns or greater, 20 microns or greater, 30 microns or greater, 40 microns or greater, 50 microns or greater, 75 microns or greater, 100 microns or greater).

[0076] 3, the overall length of the separation channel 304 may be selected as desired to provide a sufficient flow path for the different components of the sample to separate before arriving at the ejector 310. By selecting the appropriate length of 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., as the sample components exit) can be controlled. The overall length corresponds to the path length defined by the axis 402 of the separation channel 304 between the reservoir 302 and the junction 310 and may 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).

[0077] Generally, the transverse dimensions of the pumping channel 308 are similar to the transverse dimensions of the separation 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 may be the same as any of the widths and depths described above for the separation channel 304. In some embodiments, the width and / or depth of the pumping channel 308 may be the same as the separation channel 304. However, more generally, the pumping channel 308 may have a width and / or depth that differs from the width and / or depth of the separation channel 304.

[0078] A central axis extends along the length of the pumping channel 308, similar to 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. Generally, the length of the pumping channel 308 may be selected as desired to accommodate a desired amount of background electrolyte 314. In some embodiments, it may be advantageous to use a pumping channel length significantly shorter than the length of the separation channel 304. Because separation of sample components does not occur within the pumping channel 308, this length does not need to be a consideration for sample component separation. Furthermore, shortening the length of the pumping channel 308 can increase the spatial pressure gradient within the pumping channel 308, which increases the driving force applied to each volume unit of background electrolyte within the pumping channel. In some embodiments, the pumping channel has a length of 0.5 cm or more (eg, 1.0 cm or more, 1.5 cm or more, 2.0 cm or more, 2.5 cm or more, 3.0 cm or more, 4.0 cm or more, 5.0 cm or more, 7.0 cm or more).

[0079] With no pressure applied to the headspace of the tank 306, the background electrolyte 314 is not transported through the pumping channel 308 and the ejector 310. Because the flow of background electrolyte 314 transports the separated sample components from the separation channel 304 through the junction 310 after the components have passed through the ejector 310, the lack of applied gas pressure in the headspace of the tank 306 ensures that the sample components are not expelled through the ejector 310.

[0080] The term "pressure," when used herein to describe the gas pressure in the headspace of the tank 306, is relative to the atmospheric or ambient pressure at which the chip 300 operates, i.e., the pressure at the emitter 310. Even in the absence of gas pressure applied to the tank 306, the background electrolyte 314 is subjected to atmospheric or ambient gas pressure. However, there is no pressure gradient between the tank 306 and the emitter 310.

[0081] The application of gas pressure to the headspace of the tank 306 creates a pressure gradient between the tank 306 and the emitter 310 that enables fluid transport through the pumping channel 308. Thus, the "gas pressure" in the headspace of the tank 306 refers to the pressure difference between the absolute pressure in the tank 306 and the ambient pressure at the emitter 310.

[0082] Generally, to initiate and maintain transport of background electrolyte 314 between tank 306 and emitter 310, the applied gas pressure is selected based on the desired flow rate of the background electrolyte. 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).

[0083] In some embodiments, the gas pressure outside and adjacent to the emitter 310 can be different from atmospheric pressure. To initiate and maintain the transfer of background electrolyte 314 between the tank 306 and the emitter, the pressure difference between the gas pressure in the tank 306 and the gas pressure outside the emitter 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).

[0084] In operation, the flow rate of the background electrolyte 314 in the pumping channel 308 is selected to control the rate of fluid emission from the emitter 310. A variety of different flow rates may be selected depending on the desired volume of fluid per unit time in the electrospray plume 340. In some embodiments, the flow rate of the background electrolyte 314 in the pumping channel 308 is, for example, 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).

[0085] In some embodiments, it has been experimentally observed that improved ionization of sample components can be achieved under certain conditions at relatively low flow rates of background electrolyte 314. Thus, for example, in certain embodiments, more efficient ionization of components can occur when the flow rate of background electrolyte 314 is between 50 nL / min and 500 nL / min (e.g., between 100 nL / min and 250 nL / min).

[0086] A variety of different background electrolytes can be used in chip 300. In some embodiments, background electrolyte 312 and background electrolyte 314 have the same composition. More generally, background electrolytes 312 and 314 may have different compositions. The background electrolytes used in chip 300 are generally aqueous and include one or more additional components (i.e., in addition to water) that aid in the separation of sample components and / or the generation of the electrospray plume. For example, the background electrolyte can include one or more compounds that act as organic modifiers, such as, but not limited to, methanol and acetonitrile. The background electrolyte can include one or more weak acids, such as, but not limited to, formic acid.

[0087] It has been experimentally observed that in some embodiments, it can be advantageous to ensure that the background electrolyte contains a higher concentration of one or more organic modifiers and / or a lower concentration of salts than the background electrolyte 312. In general, using a background electrolyte 314 with a relatively low ionic strength, for example, 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), can aid in the efficient generation of an electrospray plume. Furthermore, using a background electrolyte 314 with an increased concentration of one or more organic modifiers relative to the background electrolyte 312 can aid in the efficient generation of an electrospray plume, because the one or more organic modifiers reduce the surface tension of the background electrolyte 314 and promote droplet formation.

[0088] Additionally, in some embodiments, the background electrolyte 312 can have a composition adapted to promote favorable surface chemistry on the walls of the separation channel 304 to improve separation of individual sample components as they migrate through the channel. The composition of the background electrolyte 312 can be changed as different samples are introduced into the chip 300 for analysis.

[0089] As noted above, background electrolyte 314 can have the same or a different composition as background electrolyte 312. Due to the mixing and dilution that occurs in the region of junction 320, a background electrolyte designed to promote high-quality separation in channel 304 is not necessarily optimized to promote high-quality electrospray ejection. By altering the composition of background electrolyte 314 relative to the composition of background electrolyte 312 (e.g., by adjusting the concentrations of one or more organic modifiers and / or one or more acids), both high-quality sample component separation and high-quality electrospray ejection of the separated sample components can be achieved.

[0090] During operation of the chip 300, the potential V2 applied to the electrode 318 may generally be selected as desired. In some embodiments, V2 is relatively large compared to the external ground voltage (which is nominally zero, i.e., represents ground). By maintaining a relatively large voltage at the electrode 318, ionization of the separated sample components occurs as they exit the separation channel 304 and are ejected from the emitter 310. In this manner, the electrospray plume 340 contains a population of ionized molecules of each sample component isolated within the separation channel 304. These ionized molecules can then be directly coupled to a mass analyzer for analysis.

[0091] Typically, potential V2 has a positive sign relative to external ground potential. However, more commonly, potential V2 can be negative relative to external ground potential, depending on the nature of the sample components. Potential V2 is typically 3.5 kV, but can vary depending on the sample components 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).

[0092] Like V2, potential V1 can have a positive or negative sign relative to an external ground potential, depending on the nature of the sample components being analyzed. The magnitude of potential V1 can also vary depending on the sample being analyzed. In certain 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).

[0093] Potential difference ΔV sep= V1-V2 may generally be adjusted to any value to control the electrophoretic mobility of the sample components in the separation channel 304. sep The larger ΔV, the greater the electrophoretic mobility of each sample component and the faster each component moves through separation channel 304. sep = 0, the sample components are not subjected to an electrophoretic driving force, but some movement still occurs due to bulk fluid transport within the separation channel 304. sep If is non-zero, ΔV can be adjusted as desired based on the nature of the sample being analyzed. sep The sign of can be positive (i.e. V1-V2 > 0) or ΔV sep The sign of may be negative (i.e. V1-V2 < 0).

[0094] In general, ΔV sep The magnitude of ΔV may be 0 V or greater (e.g., 100 V or greater, 200 V or greater, 300 V or greater, 500 V or greater, 750 V 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, 18.0 kV or greater). As noted above, ΔV sep If σ is greater than zero, the electrophoretic velocity of each charged or bipolar sample component within separation channel 304 is greater than zero, and the sample components tend to migrate along the length of separation channel 304 .

[0095] FIG. 5 shows a schematic diagram of a portion of the fluidic chip 300 in the region of the emitter 310. In particular, FIG. 5 shows the intersection of the separation channel 304 and the pumping channel 308 at junction 320. While the junction between the separation channel and the pumping channel can be realized using a wide variety of geometries, FIG. 5 shows one possible geometry for illustrative purposes. Channels 304 and 308 intersect at an internal point 386. Extending from internal point 386 are two imaginary lines 382 and 384. Line 382 extends perpendicular to the central axis of channel 304 in the xy plane and indicates the end of channel 304. Similarly, line 384 extends perpendicular to the central axis of channel 308 in the xy plane and indicates the end of channel 308. Region 380 in Figure 5 is bounded by lines 382 and 384, by emitter 310, and by portions of the walls of channels 304 and 308, and corresponds to the "dead volume" of fluidic chip 300, which is the volume region through which separated sample components are transported after exiting separation channel 304 and before being expelled from emitter 310.

[0096] An important advantage of the pressure-based fluid transport methods described herein is that the dead volume of the chip 300 is relatively small. Maintaining a small dead volume reduces diffusion (i.e., spatial spread) of separated sample components and ensures that the concentration of each component in the electrospray plume 340 is not significantly diluted by the background electrolyte 314. In certain embodiments, for example, the dead volume 380 can 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).

[0097] 6 shows a schematic diagram of a portion of a fluidic chip 300, including a separation channel 304, a pumping channel 308, and a junction 310. A reservoir 306 is located at the end of the pumping channel 308 opposite the emitter 310 and is connected to a conduit 332 via an interface 336. In general, the interface 336 is an airtight interface that can be implemented in a variety of ways. For example, in some embodiments, the interface 336 includes one or more sealing members, such as an O-ring, that connect the conduit 332 to the reservoir 306.

[0098] Conduit 332 is connected to gas source 330 via valve 335, which is connected to electronic processor 390 via communication line 395. During operation of chip 300, electronic processor 390 can adjust the gas pressure within reservoir 306, and therefore the flow rate of background electrolyte 314 through pumping channel 308, by opening or closing valve 335. In some embodiments, pressure detector 337 is disposed within or coupled to reservoir 306 and is further connected to electronic processor 390 via communication line 396. Pressure detector 337 can send a measurement signal to processor 390 that includes information regarding the gas pressure within the sealed headspace of reservoir 306. Processor 390 can then adjust the gas pressure within reservoir 306 by opening or closing valve 335 based on this measurement information.

[0099] In some embodiments, chip 300 includes another valve 341 connected to the sealed headspace of reservoir 306 and also connected to electronic processor 390 via communication line 397. Processor 390 can decrease the flow rate of background electrolyte 314 in pumping channel 308 by decreasing the gas pressure in reservoir 306. Reducing the gas pressure can be accomplished by opening valve 341 to allow excess gas to escape 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 admit more gas from gas source 330 (e.g., increasing the gas pressure in reservoir 306) or by opening valve 341 to vent excess gas pressure from the reservoir (e.g., decreasing the gas pressure in the reservoir). Thus, electronic processor 390 can exercise complete control over the flow rate of background electrolyte 314 over chip 300. Because the flow of background electrolyte 314 largely determines the spatial extent and fluid volume of the electrospray plume, electronic processor 390 can adjust the plume characteristics through the operation of valves 335 and 341 .

[0100] In some embodiments, to further control the electrospray plume, chip 300 can include a valve 343 for isolating pumping channel 308 from fluid exhaust channel 345. Valve 343 is connected to electronic processor 390 via communication line 398. In certain applications where a high flow rate of background electrolyte 314 is desired, but a relatively small overall volume of fluid flow generating the electrospray plume, electronic processor 390 can open valve 343 to divert a certain amount of background electrolyte 314 from pumping channel 308 into fluid exhaust channel 345. In this manner, the amount of background electrolyte reaching emitter 310 per unit time is reduced, while the flow rate of background electrolyte 314 in pumping channel 308 (through emitter 310) is maintained.

[0101] Figure 7 shows a schematic diagram of fluidic chip 300 including many of the features described above. Figure 7 also shows detector 704 connected to electronic processor 390 via communication line 705. In operation, detector 704 can be used to measure one or more characteristics of electrospray plume 340 and send a measurement signal containing information about the electrospray plume to electronic processor 390. Based on this information, electronic processor 390 can adjust the flow rate and / or volume of fluid in pumping channel 308 and / or the voltages V1 and / or V2 applied to electrodes 316 and 318, respectively, as described above.

[0102] 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 regarding the spatial distribution of the plume, the shape of the plume, and the 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 photometric detector (e.g., a photodiode) and / or a spectral detector (e.g., a grating or other dispersion-based spectrometer). Measurement information, including one or more of light transmitted through the plume, light reflected from the plume, light scattering by the plume, wavelength-differential absorption by the plume, and / or light refraction / diffraction by the plume, can be used by electronic processor 390 to adjust the operation of chip 300.

[0103] In some embodiments, detector 704 may include a detection device configured to measure the voltage, current, and / or another electrical property of the electrospray plume. Information obtained from measuring the plume's electrical properties may be used by electronic processor 390 to adjust the operation of chip 300, for example, to achieve a stable plume and eject sample components into a mass spectrometry detection system.

[0104] Thus, both optical and non-optical measurements can be used to adjust the operation of the tip 300. As an example, in some embodiments, the detector 704 can be used to acquire images of the tip 300, particularly images of the emitter 310 and the electrospray plume 340 emitted from the emitter. The electronic processor 390 can then analyze the images to determine information about the electrospray plume and adjust the operation of the tip 300 based on this information.

[0105] 11 is a schematic diagram showing a representative image 1100 of the tip 300 acquired by the detector 704. In this image, multiple liquid particles 1104 forming the electrospray plume 340 are visible. Liquid particles 1102 formed on the emitter 310 are also visible. The electronic processor 390 is configured to analyze the image 1110 to determine the maximum dimension (i.e., maximum cross-section or diameter) of the liquid particles 1104. The electronic processor 390 can then use the maximum dimension information to adjust the operation of the tip 300.

[0106] For example, the electronic processor 390 can determine the average maximum dimension of the droplets 1104 and compare this average maximum dimension to a threshold average maximum dimension. It has been observed that when the droplets 1104 become too large (i.e., the average maximum dimension exceeds the threshold), the voltage applied by the processor 390 to ionize the electrospray plume may be too low. Alternatively or additionally, when the droplets 1104 become too large, the flow rate of ejected sample components and background electrolyte through the emitter 310 may be too high.

[0107] Thus, in some embodiments, electronic processor 390 is configured to increase the magnitude of the voltage applied to ionize the electrospray plume. Alternatively or additionally, in some embodiments, electronic processor 390 is configured to reduce the flow rate of background electrolyte and sample components through emitter 310 by adjusting the pressure applied to the headspace of reservoir 306 such that the pressure gradient between reservoir 306 and the ambient pressure outside chip 300 is reduced, i.e., by reducing the pressure applied to the headspace.

[0108] Electronic processor 390 can also be configured to acquire one or more additional images of tip 300 and electrospray plume 340 after adjusting the operation of tip 300 as described above. Processor 390 analyzes the one or more additional images to identify plume particles in those images, iterates to calculate the maximum dimension of each identified plume particle, calculates a new average maximum dimension for those particles, and then compares the new average maximum dimension to a threshold value to determine whether the electrospray ionization voltage and / or the flow rate through emitter 310 should be further adjusted. This process is repeated until processor 390 determines that the maximum dimension of the particles forming electrospray plume 340 has fallen below the threshold value.

[0109] If the electrospray ionization voltage is too low, the electrospray plume 340 may disappear entirely from the image 1100 (i.e., the liquid particles 1104 are not visible in the image 1100 and cannot be identified by the processor 390). If the electronic processor 390 determines that the liquid particles are not visible in the image 1110 (or more generally, if the number of identified liquid particles is a threshold percentage less than the expected number of liquid particles, e.g., 50% less, 60% less, 70% less, 80% less, 90% less, or even less), the electronic processor 390 can be configured to increase the applied electrospray ionization voltage. Because the electronic processor 390 recognizes that the low electrospray ionization voltage is the reason for the absence of the plume 340, this change can be made while maintaining a substantially constant flow rate through the emitter 310.

[0110] A very low electrospray ionization voltage can cause liquid particles 1102 to appear at the end of the tip 300, as shown in Figure 11. When the electronic processor 390 analyzes the image 1110 and detects the presence of liquid particles 1102 overlying the portion of the tip 300 near the emitter 310, the electronic processor 390 can be configured to increase the magnitude of the applied electrospray ionization voltage. Because the electronic processor 390 recognizes that a low electrospray ionization voltage is the reason for the presence of the liquid particles 1102 at the end of the tip 300, this change can be made while maintaining a substantially constant flow rate through the emitter 310.

[0111] It has been observed that if the electrospray ionization voltage is too high, the electrospray plume 340 may "twitch" or appear at different points within the emitter 310 as a function of time. In some embodiments, the electronic processor 390 can determine a shift in the spatial location of the electrospray plume 340 and adjust the operation of the tip 300 based on this shift.

[0112] 12 is a schematic diagram illustrating an exemplary image 1200 acquired by detector 704 showing an electrospray plume 340 emerging from emitter 310 of chip 300. Electronic processor 390 is configured to analyze image 1200 to determine whether plume 340 emerges from different spatial locations as a function of time. For example, processor 390 can acquire a first image of plume 340 using detector 704 at a first time t1 and analyze the first image to identify a boundary region 1202 surrounding plume 340 and an origin 1206 of plume 340 within the image. Processor 390 can acquire a second image of plume 340 via detector 704 at a later second time t2 and analyze the second image to identify a second boundary region 1204 surrounding plume 340 and a second origin 1208 of plume 340 within the image.

[0113] Based on boundary regions 1202 and 1204 and / or origins 1206 and 1208, processor 390 can then determine whether plume 340 has "twitched" as a function of time. For example, if boundary regions 1202 and 1204 are displaced from one another in the two (overlaid) images by an amount greater than a threshold, processor 390 can determine that plume 340 is twitching as a function of time, as shown generally in FIG. 12. Alternatively or additionally, if origins 1206 and 1208 are displaced from one another in the two images by an amount greater than a threshold, processor 390 can determine that plume 340 is twitching as a function of time, as shown generally in FIG. 12. If processor 390 determines that plume 340 has twitched, processor 390 can be configured to adjust operation by reducing the electrospray voltage applied to eject sample components from emitter 310 into the mass spectrometry detection system, as described in more detail below.

[0114] In some embodiments, a mass spectrometry detection system oriented to receive the sample components ejected from the emitter 310 can measure one or more characteristics of the plume 340, and the processor 390 can use these measured characteristics to adjust the operation of the chip 300. For example, the mass spectrometry detection system can be used to determine the flow rate of particles in the plume 340 by detecting the particles as they are introduced into the mass spectrometry detection system. If the flow rate is less than a threshold, the processor 390 can be configured to increase the flow rate through the emitter 310. For example, the processor 390 can apply increased pressure via the gas source 330 to the headspace 350 of the tank 306, which increases the rate at which fluid is ejected through the emitter 310.

[0115] In some embodiments, detector 704 may be an electrical detector that measures one or more electrical properties of plume 340 in order to adjust operation of chip 300. For example, detector 704 may measure the current of plume 340, and processor 390 may use the current measurement to estimate the flow rate of plume 340. Processor 390 may further calculate the rate of change of current and / or the variation of current as a function of time. If the rate of change or variation of current is too large, processor 390 may adjust operation by increasing the electrospray voltage (if plume 340 sputters) or decreasing the electrospray voltage (if plume 340 twitches).

[0116] As is apparent from the above, electronic processor 390 can use a number of measurements to adjust the operation of chip 300. For example, detector 704 can include imaging detectors, such as cameras and electrical detectors, each of which can perform any of the measurements described above. Additionally, plume 340 can be directed to the inlet of a mass spectrometry detection system in communication with electronic processor 390. Electronic processor 390 can then adjust various operating parameters of chip 300 based on any combination of the measurements described above.

[0117] Optionally, a light source 702 may be provided and connected to the electronic processor 390 via communication line 703. In operation, the light source 702 may provide illumination radiation that interacts with the electrospray plume 340. Light transmitted, reflected, scattered, or otherwise emitted from any of the various processes from the electrospray plume 340 is detected by a detector 704, which generates a measurement signal. The light source 702 may generally include any of a variety of different sources, including laser-based sources, diode sources, incandescent sources, and other light-emitting elements. If the light source 702 is not provided, 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.

[0118] In some embodiments, at least some components of the separation system 200 described in connection with FIG. 2 can be implemented within a modular housing that interfaces with an existing mass spectrometry detection system. In FIG. 7, one or more of the electronic processor 390, gas source 330, valve 335, light source 702, and detector 704 can be disposed within a modular housing 710. The housing 710 can also include a support structure (not shown in FIG. 7) for securing the chip 300 so that various electrical and fluid connections can be made between the chip 300 and the components within the housing. Additionally, the housing 710 can include a communications interface 712 connected to the electronic processor 390 via communications line 707, which connects and interfaces with the mass spectrometry detection system. In 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.

[0119] 8 shows a schematic diagram of a fluidic chip 300 that includes many of the features described above. Generally, chip 300 can also include various other sample and fluid handling and transport elements. For example, chip 300 can include one or more valves, conduits, gates, liquid reservoirs, gas reservoirs, electrodes, and other components, whether implemented within or connected to reservoir 302, separation channel 304, reservoir 306, and / or pumping channel 308. Actuable elements such as valves, gates, and electrodes can be connected to electronic processor 390 via communication lines. A gas source, optionally also connected to electronic processor 390, can be connected to the gas reservoir and transports fluid by pressurizing the sealed headspace within the gas reservoir in the manner described above.

[0120] In some embodiments, as shown in Figure 8, additional channels and reservoirs can be coupled to pumping channel 308 to deliver additional compounds to the fluid mixture (e.g., one or more background electrolytes and sample components) that are ejected via emitter 310. In Figure 8, auxiliary reservoir 802 is connected to pumping channel 308 via channel 804, and auxiliary reservoir 806 is connected to pumping channel 304 via channel 808. In general, any number of auxiliary reservoirs and channels may be coupled to pumping channel 308.

[0121] Fluid transport from reservoirs 802 and / or 806 through channels 804 and 808, respectively, can be initiated using a variety of methods. In some embodiments, for example, reservoirs 802 and / or 806 and channels 804 and / or 808 can include electrodes connected to electronic processor 390. Processor 390 applies a voltage to the electrodes, and an electric field generated within channels 804 and / or 808 causes fluid transport within the channels. In some embodiments, reservoirs 802 and / or 806 can be connected to a gas source, and electronic processor 390 regulates fluid transport within the channels by opening or closing valves coupled to the reservoirs and / or channels, as described above.

[0122] Auxiliary reservoirs and channels can be used to deliver various agents into the background electrolyte 314 in the pumping channel 308. Because the pumping channel 308 intersects the separation channel 304 close to the emitter 310, the separated sample components interact with these agents just before the components are ejected into the electrospray plume, ensuring that the added agents do not alter the sample components in a way that would adversely affect the components' subsequent analysis in the mass spectrometry system. Furthermore, because the agents are introduced just before ejection via the emitter 310, they do not travel upward in the separation channel 304, away from the emitter 310.

[0123] A variety of different agents can be introduced via auxiliary reservoirs and channels coupled to pumping channel 308. In some embodiments, for example, one or more coupling agents can be added to background electrolyte 314. These coupling agents can improve the accuracy of subsequent mass spectrometric characterization. Suitable coupling agents are disclosed, for example, in the following publications: Remsburg et al., J. Am. Soc. Mass Spectrom 19: 261 (2008); and Soukup-Hein et al., Anal. Chem. 80: 2612 (2008), the entire contents of each of which are incorporated herein by reference. [Non-Patent Document 1] Remsburg et al., J. Am. Soc. Mass Spectrom 19: 261 (2008); and Soukup-Hein et al., Anal. Chem. 80: 2612 (2008)

[0124] In some embodiments, one or more calibration compounds can be introduced through auxiliary reservoirs and channels. These calibration compounds can be added to the background electrolyte 314, released into the electrospray plume, and introduced into the mass spectrometry system. The calibration compounds provide standard markers for calibrating the mass analysis of sample components. A wide variety of calibration compounds can be used. One such example is glu-fibrinopeptide, which is useful for calibrating peptide analysis.

[0125] In some embodiments, a fluidic chip can include multiple emitters through which sample components are ejected. Figure 9 is a schematic diagram showing a chip 300 including three sample reservoirs 302a-c connected to three separation channels 304a-c, each terminating in an emitter 310a-c. The chip 300 also includes three background electrolyte reservoirs 306a-c connected to three pumping channels 308a-c, each terminating in one of the emitters 310a-c. In operation, a voltage difference ΔV sep can be applied to one or more separation channels 304a-c. Generally, the voltage separation applied to each channel can be the same or different. By selectively driving a flow of background electrolyte from one of the reservoirs 306a-c through one of the corresponding channels 308a-c and ejecting it from one of the emitters 310a, 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 ejected from the chip 300.

[0126] The multiple emitter configuration shown in FIG. 9 may be implemented with, for example, an isolation voltage ΔV sepThis can be advantageous because a voltage applied to multiple separation channels can be applied simultaneously. Thus, for example, a sample can be analyzed with a separation voltage applied at all times. For a common sample in each sample reservoir and separation channel, different processing conditions (e.g., separation voltage, background electrolyte) can be used in each combination of sample reservoir and separation channel, allowing a single sample to be analyzed under different combinations of conditions. If different samples are contained in at least some of these sample reservoirs, the multi-channel configuration of FIG. 9 can be used to multiplex the analysis of these different samples, which may have significantly different separation conditions and / or times. For example, sample components that separate and move relatively quickly within the corresponding separation channel can be ejected first from their respective ejectors, while sample components that separate and move relatively slowly can be ejected later upon reaching the end of the corresponding separation channel. In this manner, the total time required to analyze multiple samples can be reduced compared to sequential analysis of these samples in a chip with a single separation channel.

[0127] The chip 300 shown in FIG. 9 includes three sample reservoirs, three separation channels, three background electrolyte reservoirs, and three pumping channels, but more generally, the chip 300 may include two or more of these components (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, or more).

[0128] 10 is a flowchart 1000 showing a series of steps for performing pressure-based electrospray and / or injection of separated sample components. In a first step 1002, a sample with components to be separated and / or analyzed is introduced into the sample reservoir 302. Introduction can be via a port coupled to the reservoir, via a fluid channel communicating with the reservoir, or by injection via any of a variety of other methods. Typically, the sample is introduced in background electrolyte, i.e., in a partially or fully solvated form.

[0129] Next, in step 1004, electronic processor 390 applies a potential difference between sample reservoir 302 and the end of separation channel 304 via electrodes 316 and 318. Typically, for example, electronic processor 390 activates one or more power sources (not shown in the drawings) to apply the potential difference. Application of the potential difference causes components of the sample to electrophoretically migrate from sample reservoir 302 through separation channel 304 toward emitter 310, with the migration rates of individual components varying depending on their individual electrophoretic mobilities and, in some embodiments, interactions with the walls of separation channel 304.

[0130] In step 1006, electronic processor 390 pressurizes the headspace region of background electrolyte tank 306, causing gas to flow from tank 306 through pumping channel 308 and out of emitter 310. Typically, for example, electronic processor 335 controls the gas pressure in the headspace region by opening and closing valve 335, with gas being supplied from gas source 330.

[0131] Pressure-driven flow of background electrolyte from reservoir 306, through pumping channel 308, and out of ejector 310 causes the sample components to be individually ejected from ejector 310 in step 1008 after the components have separated from one another during electrophoretic migration through separation channel 304. The different electrophoretic mobilities of the sample components cause the individual components to be ejected from ejector 310 at different times. Thereafter, in step 1010, when the components are injected or introduced into a mass spectrometry system (or into another type of analytical system), they are introduced at different times, simplifying component handling and analysis. The process ends in step 1012.

[0132] In some embodiments, it may be advantageous to ensure that the flow of background electrolyte through the pumping channel 308 and out of the emitter 310 in steps 1004 and 1006 is initiated simultaneously or nearly simultaneously with the application of the potential difference between the end of the sample reservoir 302 and the separation channel 304. It has been found experimentally that if the initiation of background electrolyte flow and the application of the potential difference do not occur within a short time of each other, it can be difficult to generate a high-quality electrospray plume from the emitter 310. Thus, in some embodiments, the time difference between the initiation of background electrolyte flow and the application of the potential difference (whichever occurs 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 ms or less, 200 ms or less, 100 ms or less).

[0133] As described above, by using a pressure-driven flow of background electrolyte to eject sample components from the emitter 310, the process of generating an electrospray plume can be decoupled from the process of separating sample components within the separation channel. The generation of the appropriate electrospray plume is controlled by the pressure-driven flow of background electrolyte. Ion generation from the separated sample components is controlled by the difference between the potential at the emitter 310 and a ground or reference potential external to the chip 300.

[0134] Thus, the potential difference between electrodes 316 and 318 is used to control only the electrophoretic separation of sample components within separation channel 304. This "separation" can also occur with a zero volt potential difference between electrodes 316 and 318. Sample components do not significantly migrate under these conditions, but emission from emitter 310 does occur (in the form of background electrolyte), which, as described above, allows the characteristics of the electrospray plume to be measured, confirmed, and adjusted through adjustment of one or more operating parameters.

[0135] More generally, electronic processor 390 is configured to implement various adaptive processing techniques, including adjustment of the applied potential difference, to improve sample component separation and analytical performance. For example, in some embodiments, electronic processor 390 is configured to adjust the migration rate of sample components through separation channel 304 by modifying the potential difference applied between electrodes 316 and 318. Because ejection from ejector 310 is achieved via pressure-driven flow of background electrolyte, the applied potential difference can generally be adjusted as desired to adjust component migration times without adversely affecting component ejection.

[0136] For example, electronic processor 390 can apply a relatively high first potential difference between electrodes 316 and 318 before the separated sample components first reach the end of separation channel 304. The exhaust from emitter 310 is coupled or introduced into a mass spectrometry system, which analyzes the exhaust to determine whether components of interest are present in the exhaust. Information regarding the presence or absence of the components of interest is identified or received by processor 390. While the components of interest are not detected, electronic processor 390 maintains the relatively high first potential difference between electrodes 316 and 318 to shorten the migration time of the sample components through separation channel 304.

[0137] 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 second, lower potential difference, effectively reducing the migration rate of the sample components through the separation channel 304. Individual components can then be analyzed individually and in temporal order as they arrive at the end of the channel 304 and exit the emitter 310. Varying the applied potential difference in this manner effectively changes the temporal order between the sample components arriving at the mass spectrometry system for analysis.

[0138] For complex samples with many components of interest, electronic processor 390 can be configured to cycle between high and low applied potential differences multiple times to regulate the flow rate of the components within separation channel 304. Processor 390 is not limited to alternating between only two applied potential differences; that is, more generally, at any given time, processor 390 can select any potential difference to apply between electrodes 316 and 318 to control the migration time of sample components within separation channel 304.

[0139] In some embodiments, the electronic processor 390 can be configured to execute an analysis program configured to identify one or more specific components within the sample. That is, the processor is configured to specifically look for the presence or absence of the specific component(s) regardless of the presence or absence of other components within the sample. The electronic processor 390 can receive configuration information regarding the migration time of the specific component(s) (e.g., as part of the program or from another source, such as a database or a system user). To shorten the overall analysis time for a particular sample, the electronic processor 390 can apply a relatively high potential difference between the electrodes 316 and 318 until just before the first of the specific components is expected to arrive at the end of the 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 ejected from the emitter 310, it will be accurately detected.

[0140] The electronic processor 390 then increases the applied potential difference until just before the next of the specific components is expected to arrive at the end of the separation channel 304 based on the configuration information, after which the applied potential difference is again decreased by the processor 390 to facilitate detection of the second specific component, if present in the sample. Alternatively, the processor 390 may increase or decrease the applied potential difference in this manner to decrease the overall analysis time of the sample while at the same time ensuring that the specific components of interest are sufficiently separated in time for detection.

[0141] Adjustment of the applied potential difference within the separation channel 304 can also be advantageous in situations where there is a limited time frame for analyzing a particular sample. For example, in proton exchange mass spectrometry, the sample can be treated to exchange certain hydrogen atoms for deuterium atoms for labeling purposes. Following sample digestion, the deuterium-labeled proteins are analyzed to locate the deuterium-labeled proteins within the structure.

[0142] However, deuterium atoms remain relatively unstable within sample proteins and tend to exchange again with normal hydrogen atoms. Thus, analysis of deuterium-labeled proteins can be performed relatively quickly, before this exchange occurs to a greater extent. To analyze sample components with a time-limited lifetime or analysis window, such as deuterium-labeled proteins, electronic processor 390 can adjust the potential difference applied between electrodes 316 and 318 to reduce the overall analysis time of the sample, as described above. Specifically, processor 390 can maintain a relatively large potential difference while the components of interest flow through channel 304, reducing the time it takes for them to travel the entire length of the channel. When components of interest are expected to arrive at the end of channel 304 (or begin to be detected as they exit emitter 310), electronic processor 390 reduces the potential difference between electrodes 316 and 318 as each sample component is analyzed. After a particular component is ejected, processor 390 again increases the potential difference until the next component of interest arrives at the end of channel 304 or is detected in the emission from emitter 310. In this manner, electronic processor 390 can implement various programs / sequences suitable for the analysis of time-limited samples, such as proton exchange mass spectrometry.

[0143] In the embodiments described above, pressure-driven flow of background electrolyte occurs under the control of processor 390, which directs gas source 330 to supply gas to headspace 350 of tank 306. Pressure-driven flow may be accomplished in other ways in addition to or instead of supplying gas to the tank. For example, in some embodiments, control of gas pressure within tank 306 may be achieved using a displacement mechanism such as a diaphragm or piston.

[0144] 14 is a schematic diagram of a portion of a fluidic chip 300 including a reservoir 306 containing a background electrolyte 314. A headspace 350 is located above the background electrolyte 314. A piston 380 forms part of the top wall of the reservoir 306 to control the pressure within the reservoir 306 (and therefore the pressure applied to the background electrolyte 314). The piston 380 is coupled to an electronic processor 390 via a control line 382.

[0145] Processor 390 can adjust the pressure in headspace 350 by advancing piston 380 into (to increase pressure) or retracting piston 380 from (to decrease pressure) reservoir 306. In this manner, processor 390 can expel sample components from emitter 310 and, as described above, adjust the operation of tip 300 based on measured parameters of plume 340.

[0146] Pressure regulation within tank 306 can also be controlled by regulating the temperature within the headspace. Temperature regulation can be accomplished in a variety of ways. For example, in FIG. 14 , heating element 384 is disposed along the wall of tank 306 and is connected to processor 390 via control lines 386. By heating tank 306 (and background electrolyte 314) via heating element 384, electronic processor 390 increases the gas pressure within tank 306. Conversely, by cooling tank 306 (e.g., via a cooling element not shown in FIG. 14 or by reducing the temperature of heating element 384), electronic processor 390 can decrease the gas pressure within tank 306.

[0147] Pressure-driven flow of background electrolyte 314 occurs because a pressure gradient exists between the electrolyte in tank 306 and the fluid at or adjacent to emitter 310. Thus, in some embodiments, pressure-driven flow of background electrolyte 314 can be induced by reducing the pressure applied to background electrolyte 314 adjacent to emitter 310. Figure 15 shows a schematic diagram of a portion of housing 300. Some features of chip 300 have been omitted for clarity, but generally, chip 300 can include any of the features described in connection with the various fluidic chips.

[0148] 15 , vacuum source 394 is positioned proximate to emitter 310. Vacuum source 394 is connected to electronic processor 390 via communication line 396. In operation, by selectively connecting and disconnecting vacuum source 394 from fluid communication with pumping channel 308, for example, by opening or closing a valve in vacuum source 394, processor 390 can adjust the pressure applied to background electrolyte 314 at or proximate to emitter 310. Generally, 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.

[0149] Generally, any of the above methods can be used alone or in combination with the above-described fluidic chip, and the electronic processor 390 can control the pressure gradient in the background electrolyte 314 in the pumping channel 308 by adjusting the pressure in the tank 306, by adjusting the pressure at or adjacent to the emitter 310, or both.

[0150] III. Connection to a mass spectrometry detection system As noted above, in some embodiments, the output from the emitter 310 can be directly connected to the inlet of a mass spectrometry system to facilitate analysis of the separated sample components as they are ejected. In some embodiments, the output from the emitter 310 can be in a spatial region where the inlet of a mass spectrometry system is located, which can sample the output via the inlet and incorporate a portion for analysis. Additional features of suitable mass spectrometry systems that can be used with the fluidic chips disclosed herein are disclosed in U.S. Patent Publication No. 2015 / 0200083, published as U.S. Patent No. 9,502,226, and U.S. Patent Publication No. 2016 / 0099137, the entire contents of each of which are incorporated herein by reference. [Patent Document 4] U.S. Patent Publication No. 2015 / 0200083 [Patent Document 5] U.S. Patent Publication No. 2016 / 0099137

[0151] Generally, the emitter 310 of the chip 300 and the inlet of the mass spectrometry detection system can be coupled in a variety of ways, one such method including generating an ionized electrospray from the emitter 310, with the emitter directed toward the inlet. Figure 13 is a schematic diagram showing the fluidic chip 300 coupled to a mass spectrometry detection system 1310. In Figure 13, some features of the chip 300 have been omitted for clarity. However, the chip 300 of Figure 13 can generally include any of the features disclosed herein in connection with the various fluidic chips.

[0152] 13, sample components are ejected from emitter 310 via electrodes 1302 and 1304, which are coupled to electronic processor 390 via control lines 1305 and 1307. In operation, processor 390 applies a potential to electrodes 1302 and 1304, creating a potential difference between them, referred to as the electrospray ionization voltage, as described above. As described above, processor 390 can adjust this voltage based on measurements from detector 704 and / or mass spectrometry detection system 1310.

[0153] Electrode 1304 forms part of an inlet 1318 to a detection system 1310, which also includes an optional ion source 1312, ion trap 1314, and ion detector 1316. The components of the detection system 1310 are connected to a processor 390 via control lines 1309. Processor 390 may be separate from detection system 1310, as shown in FIG. 13, or alternatively, may be incorporated within detection system 1310. Processor 390 generally controls and can perform functions in conjunction with various components of chip 300 and / or detection system 1310.

[0154] When processor 390 applies an electrospray ionization voltage between electrodes 1302 and 1304, an electrospray plume 340 is generated and enters inlet 1318. Once sample components are separated in separation channel 304, they are ejected from emitter 310, ionized between electrodes 1302 and 1304, and coupled into inlet 1318. Once in ion trap 1314, the ionized sample components are optionally further ionized in ion source 1312 and then trapped and selectively ejected from ion trap 1314 for detection by detector 1314. Mass spectral information, including the mass-to-charge ratios of the detected ions, is communicated to processor 390 via control line 1309.

[0155] IV. ADDITIONAL SYSTEM HARDWARE AND SOFTWARE COMPONENTS Any method step, feature, and / or attribute disclosed herein can be performed by electronic processor 390 and / or one or more additional electronic processors (such as a computer or a programmed integrated circuit) executing a program according to standard programming techniques. Such programs are designed to run on programmable computing devices or specially designed integrated circuits, 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. The program code is applied to input data to perform a function and to generate output information that is 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 in an assembly or machine language. Moreover, such a language may be a compiled or interpreted language. Each such computer program can be stored on a computer-readable storage medium (e.g., optical storage medium such as a CD-ROM or DVD, magnetic storage medium, and / or non-transitory solid-state storage medium) that, when read by a computer, processor, or electronic circuit, causes the computer, processor, or electronic circuit to perform the analysis and control functions described herein.

[0156] Other embodiments Although several embodiments have been described, it will be understood that many modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A fluid analysis system comprising: Fluidic chips fabricated on planar substrates: a sample reservoir connected to the electrophoretic separation channel at a first end of the electrophoretic separation channel; a background electrolyte tank connected to the electrophoretic separation channel via a pumping channel; a fluidic chip including an opening extending from the second end of the electrophoretic separation channel to an exterior surface of the fluidic chip; a detector configured to obtain information regarding an exhaust plume generated by the exhaust of fluid through the opening; an electronic processor connected to the detector that, during operation of the fluid analysis system, receiving the information regarding the exhaust plume from the detector; and an electronic processor configured to adjust the exhaust plume based on the received information.

2. The system of claim 1 , wherein the detector is an optical detector configured to measure one or more optical properties of the exhaust plume.

3. 3. The system of claim 2, wherein the one or more optical properties include at least one member of the group consisting of 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.

4. The system of claim 1 , wherein the detector is an imaging detector configured to measure image information of the exhaust plume.

5. The system of claim 4 , wherein the image information includes at least one member of the group consisting of spatial distribution information of the exhaust plume, a shape of the exhaust plume, and a color of the exhaust plume.

6. The system of claim 1 , wherein the detector is an electrical detector configured to measure one or more electrical properties of the exhaust plume.

7. The system of claim 6 , wherein the one or more electrical properties include at least one component selected from the group consisting of voltage and current.

8. The background electrolyte tank includes a gas headspace, and the system: a pressurization mechanism connected to the gas headspace of the background electrolyte tank via a first valve; a second valve connected to the gas headspace of the background electrolyte tank; the first and second valves are connected to the electronic processor; 2. The system of claim 1, wherein the electronic processor is configured to regulate the exhaust plume by actuating at least one of the first and second valves to regulate gas pressure in the gas headspace of the background electrolyte tank.

9. 9. The system of claim 8, wherein the electronic processor is configured to open the first valve to increase gas pressure in the gas headspace of the background electrolyte tank, thereby increasing the flow rate of background electrolyte through the electrophoresis separation channel to the opening.

10. 9. The system of claim 8, wherein the electronic processor is configured to open the second valve to reduce gas pressure in the gas headspace of the background electrolyte tank, thereby reducing the flow rate of background electrolyte through the electrophoresis separation channel to the opening.

11. 2. The system of claim 1, wherein the fluid chip includes a fluid exhaust channel connected to the pumping channel through a third valve connected to the electronic processor, the electronic processor configured to regulate the exhaust plume by actuating the third valve.

12. 12. The system of claim 11, wherein the electronic processor is configured to open the third valve to divert a portion of the background electrolyte through the pumping channel and into the fluid exhaust channel, thereby reducing the amount of background electrolyte exhausted from the opening.

13. further comprising one or more electrodes extending into at least one channel or reservoir of the fluidic chip; the one or more electrodes are connected to the electronic processor; The system of claim 1 , wherein the electronic processor is configured to regulate the exhaust plume by controlling an electrical signal applied to the one or more electrodes.

14. The system of claim 4 , wherein the image information includes an image of one or more liquid particles in the exhaust plume.

15. 15. The system of claim 14, wherein the electronic processor is configured to calculate an average size of the one or more liquid particles based on the image of the one or more liquid particles and adjust the exhaust plume based on the calculated average size of the liquid particles.

16. 16. The system of claim 15, wherein the electronic processor is configured to adjust the exhaust plume by adjusting an ionization voltage applied to background electrolyte exhausted from the opening.

17. 16. The system of claim 15, wherein the electronic processor is configured to adjust the exhaust plume by adjusting a flow rate of background electrolyte through the opening.

18. the detector is configured to measure image information of the exhaust plume at a plurality of successive time points, the image information including images of one or more liquid particles of the exhaust plume; 16. The system of claim 15, wherein the electronic processor is configured to calculate, for each successive point in time, an average size of the liquid particles in the exhaust plume at that point in time, and adjust the exhaust plume based on the calculated average size of the liquid particles at that point in time.

19. 20. The system of claim 18, wherein the electronic processor is configured to compare the calculated average size of the droplets at a time to the calculated average size of the droplets at a later time to determine appropriate adjustments to the exhaust plume.

20. 5. The system of claim 4, wherein the electronic processor is configured to adjust an ionization voltage applied to the background electrolyte discharged from the opening when the number of liquid particles in the image information is below a threshold value.

21. 5. The system of claim 4, wherein if the image information includes an image of a liquid particle overlapping a portion of the fluidic tip proximate the opening, the electronic processor is configured to adjust an ionization voltage applied to background electrolyte ejected from the opening.

22. the detector is configured to measure image information of the exhaust plume at a plurality of consecutive time points; The system of claim 4 , wherein the electronic processor is configured to determine whether a spatial distribution of the exhaust plume changes at the successive time points based on the image information at the successive time points.

23. 23. The system of claim 22, wherein the electronic processor is configured to identify an origin and spatial boundaries of the exhaust plume at the successive times and determine whether the spatial location of the exhaust plume changes based on the origin and spatial boundaries of the exhaust plume at the successive times.

24. 24. The system of claim 23, wherein the electronic processor is configured to determine whether the spatial location of the exhaust plume changes based on displacement of a spatial boundary of the exhaust plume at different times.

25. 24. The system of claim 23, wherein the electronic processor is configured to determine whether the spatial location of the exhaust plume changes based on the point of origin of the exhaust plume at different times.

26. 23. The system of claim 22, wherein the electronic processor is configured to adjust a voltage applied to the background electrolyte exhausted from the opening if the spatial distribution of the exhaust plume is determined to change at successive times.

27. The system of claim 1 , wherein the detector is a mass spectrometry detector configured to detect one or more components of the exhaust plume.

28. 28. The system of claim 27, wherein the electronic processor is configured to receive a mass spectrometry signal from the mass spectrometry detector and determine a flow rate of background electrolyte discharged from the opening based on the mass spectrometry signal.

29. 30. The system of claim 28, wherein the electronic processor is configured to regulate the flow rate of background electrolyte discharged from the opening.

30. 1. A method for processing a sample, comprising: introducing a sample containing one or more components into an electrophoretic separation channel of a fluidic chip; migrating at least one sample component toward an end of the electrophoretic separation channel; directing a flow of background electrolyte through a pumping channel of the fluidic chip into the separation channel and out of the opening to expel the at least one sample component through an opening of the fluidic chip; obtaining information about an exhaust plume emerging from the opening; adjusting the exhaust plume based on the information; The method, wherein the information includes at least one component selected from the group consisting of image information of the exhaust plume, optical information of the exhaust plume, and electrical information of the exhaust plume.

31. 31. The method of claim 30, wherein the optical information includes at least one member of the group consisting of 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.

32. 31. The method of claim 30, wherein the electrical information of the exhaust plume includes at least one component selected from the group consisting of voltage and current.

33. 31. The method of claim 30, wherein the image information of the exhaust plume includes at least one member of the group consisting of spatial distribution information of the exhaust plume, a shape of the exhaust plume, and a color of the exhaust plume.

34. 31. The method of claim 30, comprising adjusting the exhaust plume by adjusting gas pressure in a gas headspace of a background electrolyte tank of the fluidic chip.

35. 31. The method of claim 30, comprising adjusting the exhaust plume by controlling an electrical signal applied to one or more electrodes of the fluidic chip.

36. 31. The method of claim 30, wherein the image information of the exhaust plume includes an image of one or more liquid particles in the exhaust plume, the method further comprising calculating an average size of the liquid particles.

37. 37. The method of claim 36, including adjusting the exhaust plume based on the calculated average size of the liquid particles.

38. 38. The method of claim 37, comprising adjusting the exhaust plume by adjusting an ionizing voltage applied to background electrolyte exhausted from the opening.

39. 38. The method of claim 37, comprising adjusting the exhaust plume by adjusting the flow rate of background electrolyte through the opening.

40. measuring image information of the discharge plume at a plurality of successive time points, the image information including images of one or more liquid particles of the discharge plume; calculating, for each successive point in time, an average size of liquid particles in the exhaust plume at that point in time; 31. The method of claim 30, including adjusting the exhaust plume based on the calculated average size of the liquid particles at that time.

41. 41. The method of claim 40, comprising comparing the calculated average size of the liquid particles at a time to the calculated average size of the liquid particles at a later time to determine appropriate adjustments to the exhaust plume.

42. 37. The method of claim 36, including adjusting an ionizing voltage applied to background electrolyte discharged from the opening when the number of liquid particles in the image information is below a threshold value.

43. measuring image information of the exhaust plume at a plurality of successive time points; 31. The method of claim 30, comprising determining whether the spatial distribution of the exhaust plume changes at the successive time points based on the image information at the successive time points.

44. 44. The method of claim 43, comprising identifying at least one of an origin and a spatial boundary of the exhaust plume at said successive times to determine whether the spatial location of the exhaust plume changes at said successive times.

45. 45. The method of claim 44, comprising adjusting a voltage applied to the background electrolyte exhausted from the opening if the spatial distribution of the exhaust plume is determined to change at successive times.

Images