Patent Application
20240087865
This invention relates to systems and methods for analysis utilizing segmented flows.
Segmented flow analysis is a common microfluidic approach to control sample dispersion for analytical techniques such as mass spectrometry and high-performance liquid chromatography. During segmented flow analysis, a sample, typically mixed with a carrier liquid, is ejected/dispensed into a transport capillary carrying a continuous flow of segmenting liquid. The segmenting liquid, which is immiscible with the capture liquid, effectively prevents dispersion and dilution of the sample droplets traveling through the capillary.
Segmented flow loading is particularly challenging, and requires a large volume of segmenting liquid. The oily nature of most immiscible segmenting liquids, typically fluorinated hydrocarbons, can cause contamination of a downstream ionization source and mass spectrometer, particularly when above nL/min flow rates are used. Further, under certain conditions, sample dispersion and dilution can benefit downstream analysis by preventing or reducing sample matrix effects for mass spectrometry, HPLC, and other analytical techniques.
Described herein are novel systems and methods for segmented flow analysis that reduce segmenting liquid volume, and allow for controlled dispersion and dilution of ejected/dispensed sample.
The disclosure generally provides systems and methods of segmented flow analysis. In various aspects and embodiments, the systems and methods of segmented flow provided by the disclosure may be used in combination with any variety of analytical instruments and techniques that are commonly used to detect, characterize, identify, separate, and/or purify one or more molecules in a sample.
In one aspect, a system for segmented flow analysis of a sample can include (a) a transport capillary for receiving a sample, and a segmenting liquid; (b) a droplet dispenser for providing the sample and the segmenting liquid to the transport capillary, wherein the sample and the segmenting liquid are dispensed into the transport capillary as discrete droplets; and (c) a conduit fluidly connected to the transport capillary that provides a capture liquid to the transport capillary.
In some embodiments, a system is provided for loading sample into a segmented flow. The system may include: a transport capillary for receiving at least one liquid sample and a flow of segmenting liquid; a droplet dispenser for dispensing the at least one liquid sample into the segmenting liquid at an open end of the transport capillary; and a conduit fluidly connected to the transport capillary that provides the segmenting liquid to the open end.
In embodiments, the segmenting liquid is immiscible with the sample and the capture liquid (e.g., selection can be based on immiscibility arising from difference(s) in polarity, hydrophobicity, hydrophilicity, aqueous, organic, etc.). Exemplary segmenting liquids may include, for instance, hydrocarbons (e.g., alkanes and cycloalkanes such as, e.g., heptane, octane) and halogenated solvents (e.g., perfluorodecalin, Novec HFE 7500, Fluorinet 40 (FC-40), and perfluorooctanol (FC-72)). In some aspects, the capture liquid is methanol.
In some aspects, the droplet dispenser comprises an acoustic droplet dispenser, a gravity dispenser, an electrostatic dispenser, a piezoelectric dispenser, a mechanical drive, or a pneumatic dispenser.
In some embodiments, the system can further include an ionization source that ionizes the sample and capture liquid when expelled from the transport capillary and/or a mass spectrometer operative to conduct mass analysis on the resulting sample ions. In certain aspects, the transport capillary has a typical geometry and dimensions, and in some embodiments may comprise an internal diameter of about 250 μm.
In embodiments the volume of an ejected sample droplet may be about 1-10 nanoliters, tens of nanoliters, or hundreds of nanoliters. In some embodiments the volume of an ejected sample droplet may be in the range of picoliters. In some embodiments the volume of a sample droplet may be about 2.5 nL.
The present disclosure further provides a method for segmented flow analysis of a sample, wherein the method can include (a) ejecting a sample into a open end of a transport capillary; (b) ejecting a segmenting liquid into the open end transport capillary, wherein the sample and segmenting liquid are alternately ejected into the transport capillary; and (c) running a capture liquid through the transport capillary to carry the sample and the segmenting liquid toward a sample outlet of the transport capillary.
In some embodiments, a method is provided for segmented flow analysis of a sample. The method may include: supplying a segmenting liquid to an open end of a transport capillary; dispensing a first liquid sample into the segmenting liquid at the open end; and drawing the segmenting liquid and first liquid sample through the transport capillary for delivery to an analytical device.
In embodiments of the methods, the segmenting liquid is immiscible with the sample and capture liquid. In embodiments of the method, some exemplary segmenting liquids include perfluorodecalin, Novec HFE 7500, Fluorinet 40 (FC-40), and perfluorooctanol (FC-72). In some aspects of the method, the capture liquid is methanol.
In some embodiments, the dispensing may be performed by non-contact droplet loading using an acoustic droplet ejector (ADE). In some embodiments, the sample and segmenting liquid are each separately ejected as one or more discrete droplets.
In some embodiments, the methods may further comprise ejecting a plurality of different samples into the open end of the transport capillary and alternately ejecting segmenting liquid between each of the samples.
In some aspects, the methods further include ionizing the sample carried in the transport capillary, wherein the ionization method can be Electron Impact Ionization (EI), Fast Atom Bombardment (FAB), Electrospray Ionization (ESI), Atmosphere Pressure Chemical Ionization (APCI) or Matrix Assisted Laser Desorption Ionization (MALDI). In some further embodiments the method comprises ESI.
In some aspects, the methods further include performing mass spectrometry on the sample.
In some aspects, the methods further comprise contacting the interior of the transport capillary with a dynamic or a permanent surface coating comprising a wetting agent that increases the contact interaction between the capillary surface and the segmenting liquid. In embodiments comprising a fluorinated segmenting liquid, the surface coating may comprise a derivatizing agent such as, for example, trichloro(1H,1H,2H,2H-perfluoroocytyl)silane.
In some aspects, the methods further include adjusting the volume of the capture liquid to control dispersion and dilution of the sample traveling through the transport capillary.
Described herein are systems and methods of loading a sample and a liquid or solvent into an apparatus for Segmented Flow Analysis. “Segmented Flow Analysis”, “segmented flow delivery”, or “SFA” as used herein refers to the introduction of a sample into the transport tubing, capillary, or coil(s) of a first apparatus having a first function, wherein the sample is transported as discrete or disconnected segments and delivered to a second apparatus having a second function. The methods described herein can involve independent droplet ejection of a plurality of different liquid samples and a segmenting liquid, immiscible with the capture liquid of the liquid samples, into the continuous flow of a transport or capture liquid such that each liquid sample is separated by a droplet, or volume, of segmenting liquid. Each of the liquid samples may be ejected as one or more discrete droplets to make up a volume of liquid sample ejected into the capture liquid and separated by a volume of segmenting liquid from other liquid samples.
As discussed herein, a suitable segmenting liquid can be selected based on its immiscibility with the liquid sample and capture liquid. In some embodiments segmenting liquid selection can be based on immiscibility arising from difference(s) in polarity, hydrophobicity, hydrophilicity, aqueous component, organic component, etc. between the segmenting liquid and/or the liquid sample, and capture liquid. In embodiments comprising polar (i.e., aqueous) liquid sample and a capture liquid that is miscible with the liquid sample, exemplary segmenting liquids can include hydrocarbons (e.g., alkanes and cycloalkanes such as, e.g., heptane, octane) and halogenated solvents (e.g., perfluorodecalin, Novec HFE 7500, Fluorinet 40 (FC-40), and perfluorooctanol (FC-72)). In some aspects, the capture liquid may comprise lower alcohols (e.g., C1-C3 alcohols such as methanol or ethanol).
In some further embodiments the segmenting liquid may be selected based on its compatibility with any analytical device(s) that are coupled with the segmented flow systems disclosed herein. For example, in such embodiments the selected analytical device/technique may be “blind” to one or more available segmenting liquid(s), and the segmenting liquid may be selected based on its ability to generate little to no signal when delivered to an analytical instrument for analysis (e.g., the segmenting liquid is selected to have little to no absorbance within a given wavelength, does not ionize/ions are not detectable, etc.).
Any variety of transport tubing, capillaries, or coils may be used in accordance with aspects and embodiments of the disclosure. In some embodiments, the tubing, capillary, or coil may be selected based on the scale, application(s), and/or techniques that are incorporated or combined with the methods and systems disclosed herein. Some embodiments comprise a capillary (i.e., “transport capillary”) comprising dimensions and materials that are generally known and available to one of skill in the art. In some embodiments a capillary comprises an internal diameter of from about 50 μm to about 500 μm (e.g., about 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or about 500 μm), or from about 50-250 μm, or from about 100-250 μm.
In some embodiments the capillary material comprises stainless steel, glass, fused silica, or a polymer (e.g., plastics, PEEK, etc.) as known in the art. In accordance with various aspects and embodiments of the disclosure, adequate contact and interaction between the segmenting liquid and the inner wall of the transport capillary (e.g., wettability) can help ensure that adequate segmented flow is achieved. In some embodiments, the systems and methods can comprise contacting the interior of the transport capillary with a dynamic or a permanent surface coating comprising a wetting agent that increases the contact interaction between the capillary surface and the segmenting liquid. Any suitable wetting agent or derivatizing agent may be used in accordance with such embodiments, and may be selected based on the capillary, tubing, or coil material and any existing surface modification it may comprise, and/or the segmenting liquid (e.g., a fluorinated segmenting liquid). In some embodiments that comprise a fluorinated segmenting liquid, the surface coating may comprise a derivatizing agent that enhances wettability between the interior surface and the fluorinated segmenting liquid such as, for example, trichloro(1H,1H,2H,2H-perfluoroocytyl)silane.
Methods, equipment, and means for droplet ejection or delivery (i.e., via a droplet dispenser) of a sample and/or segmenting liquid to a transport capillary can include a variety of droplet dispensers including, for instance, acoustic droplet ejectors (i.e. ADE) or dispensers, gravity dispensers, electrostatic dispensers, piezoelectric dispensers, mechanical drive dispensers, and pneumatic dispensers etc. In some embodiments, gravity dispensers can be used to dispense larger sample volumes (e.g. ≥1 μL). In other embodiments, electrostatic, piezoelectric, mechanical drive, or pneumatic dispensers can be used to dispense smaller sample volumes (e.g. ≥1 nL, 1.0 nL-100 nL, etc.). Exemplary pneumatic dispensers include, for instance, Immediate Drop on Demand Technology (I-DOT; Dispendix).
In accordance with the aspects and embodiments described herein, the droplet dispenser and the transport capillary may comprise any suitable arrangement and orientation that allows for the delivery of the liquid sample droplet and/or the segmenting liquid to the transport capillary. In some embodiments, the droplet dispenser may be oriented opposite from the transport capillary (e.g., an open end of the transport capillary), at an approximate 180 degree angle (i.e., above or below, left or right, etc.). In some embodiments, the droplet dispenser and the transport capillary may be arranged in an orientation that is generally adjacent to each other (e.g., at an angle ranging from about 20-160 degrees). In yet other embodiments, the droplet dispenser and the transport capillary may be in direct fluid communication via, for example and integrated connection from the dispenser to the transport capillary.
In accordance with some aspects and embodiments, the systems and methods can comprise an acoustic droplet dispenser that dispenses liquid sample from a sample source, such as a microtiter plate, and delivers the liquid sample for SFA. In such embodiments, and other embodiments that provide a non-contact droplet delivery of liquid sample, the methods and systems are operative using low sample volumes, and eliminate the need for high-pressure pumps and ejector valves. The embodiments further reduce the potential for liquid sample carryover and contamination between samples, and can separate the dispensing and delivery from any subsequent and analytical processes. As used herein, “non-contact droplet loading” or “non-contact sample delivery” refers to direct delivery of a liquid sample from a sample source to a transport capillary without use of an intermediate device that contacts the sample, such as a pipette, tubing, or ejector pump. Exemplary droplet generators include the piezoelectric PolyPico dispensing head (PolyPico Technologies Ltd.), which dispenses low sample volumes (20-120 pL) and the Labcyte Echo® which can acoustically dispense droplets in volumes on the order of nL. In some particular embodiments, the methods and systems can comprise the Sciex Echo® MS system, which is operative to dispense droplets in the range of nL directly into an open port interface (OPI) and capture liquid flowing from 100-1000 μL/min. The OPI and transport capillary can deliver a segmented flow comprising a liquid sample and segmenting liquid to a conventional electrospray ion source for mass analysis.
In another aspect, the disclosure relates to segmented flow delivery of a liquid sample to other types of secondary devices and systems for chemical analysis, such as liquid chromatographic systems (e.g., high performance liquid chromatography (HPLC)), electrophoresis, GC-MS, ICP-MS, UV-Vis or other analytical systems and techniques.
SFA can be used to prevent or reduce the spreading or dispersion and dilution of an eject sample as it travels through a capillary. As illustrated in
However, under certain conditions, sample dispersion and dilution (
The systems and methods disclosed herein can be used to control, modulate, or manipulate the dispersion and dilution of captured sample. An advantage of controlled dispersion and dilution is eliminating or reducing sample matrix effects. In an alternate SFA embodiment, as illustrated in
In addition to controlled dispersion and dilution, the segmented flow of
In some embodiments, the methods can be adapted to provide droplet sequences or patterns, e.g., sequences or patterns that comprise segmenting liquid droplets, liquid sample droplets, and/or capture fluid that can be detected. In such embodiments a sequence or pattern can be used to identify one or more features of the SFA being detected such as, for example, transitions between liquid sample droplets or mixtures of two or more liquid sample droplets, timing for droplet delivery to the capture liquid, adjustment for capture liquid flow rate, adjustment for liquid sample and/or segmenting fluid droplet size and/or pattern, and the like. The sequences or patterns can be identified by a detectable signal or the absence of a detectable signal, either of which would have a characteristic ‘signature’ pattern that allows for its specific identification. Such embodiments relating to this type of “bar coding” pattern recognition can allow for variations in experimental settings without the need to recalibrate the settings based on consumption of sample.
The volume of the segmenting liquid introduced between samples should be sufficient to span the internal diameter of a transfer capillary and maintain a contiguous boundary during transport to the ionization source. For example, a transfer capillary having an internal diameter of 250 μm requires a minimum segmenting liquid volume of about 8-10 nL to provide consistent segmenting between samples. As described herein, the segmenting liquid is delivered to the transport capillary (or tube, coil, etc.) in an amount effective to provide a complete separation between sample droplets and/or capture fluid across the entire diameter of the particular capillary. The interaction between the segmenting liquid and the interior surface of the capillary (or tube, coil, etc.) can be enhanced using the surface modification techniques disclosed herein and as may be known in the art.
To control or modulate the dispersion and dilution of an ejected sample, the volume of capture liquid between the segmenting droplets can be varied and manipulated. In general, where the capture liquid is a solvent, dispersion and dilution of sample may vary with type of solvent, velocity gradients or turbulence within the transfer capillary, distance of transfer, etc. In some embodiments, sample dispersion and dilution may be moderated by providing a less turbulent flow and reducing the transfer distance.
When samples and segmenting liquid are ejected from a microtiter plate, the spacing between sample droplet dilutions and delimiting segmenting liquid droplets can be defined by the rate at which the ejector or dispenser can travel between sample and segmenting liquid wells. In some embodiments, the time to dispense a sample droplet and a segmenting droplet from their respective wells can be on the order of microseconds or seconds, e.g., from about 1-1,000 or more ms. For purposes of illustration
As discussed herein, the liquid sample droplet size can vary similarly to the segmenting liquid droplet size. A plurality of sample droplets or segmenting liquid droplets may be rapidly ejected to make up a larger volume captured in the capture liquid. In some example embodiments, the sample droplet volume is in the range of picoliters. In some example embodiments, the sample droplet volume is from about 1.0 nL to about 10.0 nL, and may vary depending on transport capillary size. In some embodiments the sample droplet may be about 2.5 nL with a resulting radius of about 168 μm in a transfer capillary having an internal diameter of 250 μm. In some embodiments liquid sample droplet dispersion may be controlled. For example, liquid sample droplet dispersion may increase as segmenting time increases, which may result in and be observed as sample dispersion between different regions of segmenting liquid.
In another aspect, the systems and methods described herein can be used to eject more than one sample volume between segmenting liquid volumes. As illustrated in
In another aspect, the present disclosure relates to systems for delivering segmented flow of a sample to a secondary apparatus for chemical analysis, such as high performance liquid chromatography (HPLC), optical detectors, or mass spectrometry. In a particular aspect, the systems disclosed herein deliver a segmented sample to an ionization source for subsequent mass spectrometry. Such systems can be coupled with or integrated into the open port sample interface (OPSI, or OPI) as disclosed in U.S. Pat. No. 9,632,066, which is incorporated by reference herein in its entirety. In some embodiments, the OPI is a continuous flow, open interface for transferring samples into a capillary solvent stream for subsequent dilution and transfer to an ion source for ionization and subsequent mass spectrometric analysis. In some embodiments, the systems can be coupled to an Echo® MS (SCIEX) for Acoustic Ejection Mass Spectrometry. The Echo® MS uses an OPSI/OPI to capture low (e.g. nL) volumes of acoustically generated sample droplets from individual wells of a microtiter plate and transfers them to an ionization source for mass spectrometry. An exemplary AEMS system is described, for instance, in WO2019/104235, which is incorporated herein in its entirety.
In accordance with the disclosure, a representative system that may be utilized in aspects and example embodiments of the disclosed methods and compositions is illustrated in
The acoustic droplet ejection device 11 includes at least one reservoir, with a first reservoir shown at 13 and an optional second reservoir 31. In some embodiments a further plurality of reservoirs may be provided. Each reservoir is configured to house a fluid sample having a fluid surface, e.g., a first fluid sample 14 and a second fluid sample 16 having fluid surfaces respectively indicated at 17 and 19. When more than one reservoir is used, as illustrated in
The ADE comprises acoustic ejector 33, which includes acoustic radiation generator 35 and focusing element 37 for focusing the acoustic radiation generated at a focal point 47 within the fluid sample, near the fluid surface. As shown in
The acoustic droplet ejector 33 may be in either direct contact or indirect contact with the external surface of each reservoir. With direct contact, in order to acoustically couple the ejector to a reservoir, it is preferred that the direct contact be wholly conformal to ensure efficient acoustic energy transfer. That is, the ejector and the reservoir should have corresponding surfaces adapted for mating contact. Thus, if acoustic coupling is achieved between the ejector and reservoir through the focusing means, it is desirable for the reservoir to have an outside surface that corresponds to the surface profile of the focusing means. Without conformal contact, efficiency and accuracy of acoustic energy transfer may be compromised. In addition, since many focusing means have a curved surface, the direct contact approach may necessitate the use of reservoirs that have a specially formed inverse surface.
Optimally, acoustic coupling is achieved between the ejector and each of the reservoirs through indirect contact, as illustrated in
In operation, reservoir 13 and optional reservoir 15 of the device are filled with first and second fluid samples 14 and 16, respectively, as shown in
The structure of OPI 51 is also shown in
Fluid flow within the OPI 51 carries the sample, or analyte-solvent dilution, through a sample transport capillary 61 provided by inner capillary tube 73 toward sample outlet 63 the transport capillary 61 for subsequent transfer to an analytical instrument. A sampling pump (not shown) can be provided that is operably connected to and in fluid communication with the sample transport capillary 61, to control the output rate from outlet 63. In a preferred embodiment, a positive displacement pump is used as the capture liquid pump, e.g., a peristaltic pump, and, instead of a sampling pump, an aspirating nebulization system is used so that the sample or analyte-solvent dilution is drawn out of the sample outlet 63 by the Venturi effect caused by the flow of the nebulizing gas introduced from a nebulizing gas source 65 via gas inlet 67 (shown in simplified form in
The capture liquid transport capillary 59 and sample transport capillary 61 are provided by outer capillary tube 71 and inner capillary tube 73 substantially co-axially disposed therein, where the inner capillary tube 73 defines the sample transport capillary, and the annular space between the inner capillary tube 73 and outer capillary tube 71 defines the capture liquid transport capillary 59.
The system can also include an adjuster 75 coupled to the outer capillary tube 71 and the inner capillary tube 73. The adjuster 75 can be adapted for moving the outer capillary tube tip 77 and the inner capillary tube tip 79 longitudinally relative to one another. The adjuster 75 can be any device capable of moving the outer capillary tube 71 relative to the inner capillary tube 73. Exemplary adjusters 75 can be motors including, but are not limited to, electric motors (e.g., AC motors, DC motors, electrostatic motors, servo motors, etc.), hydraulic motors, pneumatic motors, translational stages, and combinations thereof. As used herein, “longitudinally” refers to an axis that runs the length of the probe 51, and the inner and outer capillary tubes 73, 71 can be arranged coaxially around a longitudinal axis of the probe 51, as shown in
Additionally, as illustrated in
As shown in
In the depicted embodiment, the ionization chamber 112 can be maintained at an atmospheric pressure, though in some embodiments, the ionization chamber 112 can be evacuated to a pressure lower than atmospheric pressure. The ionization chamber 112, within which the analyte can be ionized as the analyte-solvent dilution is discharged from the electrospray electrode 164, is separated from a gas curtain chamber 114 by a plate 114a having a curtain plate aperture 114b. As shown, a vacuum chamber 116, which houses the mass analyzer 170, is separated from the curtain chamber 114 by a plate 116a having a vacuum chamber sampling orifice 116b. The curtain chamber 114 and vacuum chamber 116 can be maintained at a selected pressure(s) (e.g., the same or different sub-atmospheric pressures, a pressure lower than the ionization chamber) by evacuation through one or more vacuum pump ports 118.
It will also be appreciated by a person skilled in the art, and in light of the guidance herein, that the mass analyzer 170 can have a variety of configurations. Generally, the mass analyzer 170 is configured to process (e.g., filter, sort, dissociate, detect, etc.) sample ions generated by the ion source 160. By way of non-limiting example, the mass analyzer 170 can be a triple quadrupole mass spectrometer, or any other mass analyzer known in the art and modified in accordance with the teachings herein. Other non-limiting, exemplary mass spectrometer systems that can be modified in accordance various aspects of the systems, devices, and methods disclosed herein can be found, for example, in an article entitled “Product ion scanning using a Q-q-Qlinear ion trap (Q TRAP®) mass spectrometer,” authored by James W. Hager and J. C. Yves Le Blanc and published in Rapid Communications in Mass Spectrometry (2003; 17: 1056-1064), and U.S. Pat. No. 7,923,681, entitled “Collision Cell for Mass Spectrometer,” which are hereby incorporated by reference in their entireties. Other configurations, including but not limited to those described herein and others known to those skilled in the art, can also be utilized in conjunction with the systems, devices, and methods disclosed herein. For instance other suitable mass spectrometers include single quadrupole, triple quadrupole, ToF, trap, and hybrid analyzers. It will further be appreciated that any number of additional elements can be included in the system 110 including, for example, an ion mobility spectrometer (e.g., a differential mobility spectrometer) that is disposed between the ionization chamber 112 and the mass analyzer 170 and is configured to separate ions based on their mobility through a drift gas in high- and low-fields rather than their mass-to-charge ratio). Additionally, it will be appreciated that the mass analyzer 170 can comprise a detector that can detect the ions which pass through the analyzer 170 and can, for example, supply a signal indicative of the number of ions per second that are detected.
Table 1 compares the flow rate and elution time of 2.5 nL sample droplets ejected into capillaries having an internal diameter of 250 μm or 127 μm. Samples were ejected into a continuous (i.e. non-discrete) flow of segmenting liquid.
Table 2 compares the dilution factor of various sample volumes ejected into a capillary having a 250 μm internal diameter. Values are based on a methanol flow rate of 250 μL/min, and a 600 ms spacing between segmenting droplets.
The presently described technology is now described in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, to practice the same. It is to be understood that the foregoing describes preferred aspects of the technology and that modifications may be made therein without departing from the spirit or scope of the invention as set forth in the appended claims.
This application claims the benefit of priority from U.S. provisional application No. 63/119,279, filed on Nov. 30, 2020, the entire contents of which is hereby incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/061142 | 11/30/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63119279 | Nov 2020 | US |
