System and method for identifying, selecting and purifying particles

Abstract

A method for purifying particles generates charged particles from a sample, measures at least one of mass, charge magnitude, and mobility of the generated charged particles, and selectively passes each of the measured charged particles having at least one of: (a) a measured mass equal to a selected mass or within a selected range of particle masses, (b) a measured charge magnitude equal to a selected charge magnitude or within a selected range of charge magnitudes, (c) a mass-to-charge ratio equal to a selected mass-to-charge ratio or within a selected range of mass-to-charge ratios, and (d) a measured mobility equal to a selected mobility or within a selected range of mobilities, to a particle collection target. In some embodiments, the collected particles can be harvested and expanded.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims the benefit and priority of U.S. Provisional Patent Application Serial No. 62 / 913,460, filed October 10, 2019; U.S. Provisional Patent Application Serial No. 62 / 949,559, filed December 18, 2019; and U.S. Provisional Patent Application Serial No. 62 / 972,403, filed February 10, 2020, the entire disclosure of which is expressly incorporated herein by reference.

[0003] Government rights

[0004] This invention was completed with government support under GM131100 granted by the National Institutes of Health. The U.S. government owns certain rights to this invention. Technical Field

[0005] This disclosure generally relates to instruments and methods for identifying, selecting and purifying particles, and more specifically, to instruments and methods for identifying, selecting and purifying particles based on one or more molecular properties. Background Technology

[0006] Spectroscopic instruments provide identification of a substance's chemical composition by measuring one or more molecular properties. Some such instruments are configured to analyze substances in solution, while others are configured to analyze charged particles of substances in the gas phase. The molecular information generated by many such charged particle measuring instruments is limited in the number and types of measurable molecular properties. Consequently, the purification of particles using such instruments is also limited. Summary of the Invention

[0007] This disclosure may include one or more features listed in the appended claims, and / or one or more of the following features and combinations thereof. In one aspect, a particle purification apparatus may include: an ion generator configured to generate charged particles from a sample; an ion processing region configured to receive the charged particles generated by the ion generator and measure at least one of the mass and charge magnitude of the generated charged particles; a particle collection target; means for selectively transferring charged particles leaving the ion processing region to the particle collection target; a processor; and a memory storing instructions executable by the processor to control the means for selectively transferring charged particles to transfer each of the measured charged particles having at least one of the following characteristics to the particle collection target: (a) a measured mass equal to a selected mass or within a selected particle mass range, (b) a measured charge magnitude equal to a selected charge magnitude or within a selected charge magnitude range, and (c) a mass-to-charge ratio equal to a selected mass-to-charge ratio or within a selected mass-to-charge ratio range.

[0008] In another aspect, a method for purifying particles may include generating charged particles from a sample, measuring at least one of the mass and charge size of the generated charged particles, and selectively transferring each of the measured charged particles having at least one of the following to a particle collection target: (a) a measured mass equal to a selected mass or within a selected particle mass range, (b) a measured charge size equal to a selected charge size or within a selected charge size range, and (c) a mass-to-charge ratio equal to a selected mass-to-charge ratio or within a selected mass-to-charge ratio range.

[0009] In another aspect, a method for purifying particles may include generating charged particles from a sample, measuring the charge size of the generated charged particles, and selectively delivering each measured charged particle with a charge size equal to or within a selected charge size range to a particle collection target.

[0010] In another aspect, a method for purifying particles may include generating charged particles from a sample, measuring the mass of the generated charged particles, and selectively transferring each measured charged particle with a mass equal to or within a selected mass range to a particle collection target.

[0011] In another aspect, a method for purifying particles may include generating charged particles from a sample, measuring the mass and charge size of the generated charged particles, calculating the mass-to-charge ratio of the measured charged particles based on the measured mass and charge size, and selectively transferring each measured charged particle with a calculated mass-to-charge ratio equal to or within a selected mass-to-charge ratio range to a particle collection target.

[0012] In another aspect, a method for purifying particles may include generating charged particles from a sample, measuring at least one of the mass, charge size, and mobility of the generated charged particles, and selectively transferring each of the measured charged particles having at least one of the following to a particle collection target: (a) a measured mass equal to a selected mass or within a selected particle mass range, (b) a measured charge size equal to a selected charge size or within a selected charge size range, (c) a mass-to-charge ratio equal to a selected mass-to-charge ratio or within a selected mass-to-charge ratio range, and (d) a measured mobility equal to a selected mobility or within a selected mobility range.

[0013] In another aspect, a method for purifying particles may include generating charged particles from a sample, measuring the mobility of the generated charged particles, and selectively delivering each measured charged particle with a measured mobility equal to or within a selected mobility range to a particle collection target.

[0014] In another aspect, a method for measuring particles in an extracellular vesicle preparation may include generating ions from the extracellular vesicle preparation and measuring the mass and charge of at least a subset of the generated ions using a charge detection mass spectrometer.

[0015] On the other hand, a method for measuring exosomes in a sample preparation may include generating ions from the sample preparation, measuring the mass and charge of at least some of the generated ions using a charge detection mass spectrometer, and identifying a subset of the measured ions as exosome ions from the measured mass of at least some of the generated ions. Attached Figure Description

[0016] Figure 1 This is a simplified diagram of the instruments and methods used to purify particles.

[0017] Figure 2 It is used for control Figure 1 A simplified flowchart of an embodiment of the process of using an instrument to generate and measure charged particles and produce the resulting spectrum from which a subgroup of charged particles can be identified or selected for purification.

[0018] Figure 3 It is used for control Figure 1 A simplified flowchart of an embodiment of an instrument for purifying particles by generating, measuring, and filtering charged particles, and of a process for collecting the purified particles.

[0019] Figure 4A It is by Figure 1 An embodiment of the instrument is a scatter plot of particle charge versus mass generated from a urine exome sample, wherein the ion processing region is implemented in the form of a charge detection mass spectrometer.

[0020] Figure 4B yes Figure 4A The image, overlaid with... Figure 1 The instrument provides an example selection of particle subpopulations for purification, where the selected subpopulations are limited by a range of particle mass values.

[0021] Figure 4C yes Figure 4A The figure shows an example selection of particle subgroups for purification, overlaid with another example selection, where the selected subgroups are limited by a range of specified particle charge values.

[0022] Figure 4D yes Figure 4A The figure shows another example selection of particle subgroups for purification, overlaid with a specified range of particle charge values ​​and a specified range of particle mass values.

[0023] Figure 4E yes Figure 4AThe figure shows another example selection of particle subgroups for purification, overlaid with a specified range of particle mass-to-charge ratio values.

[0024] Figure 4F yes Figure 4A The figure shows an example selection of particle subgroups for purification, overlaid with a specified range of particle mass-to-charge ratio values ​​and a specified range of particle mass values.

[0025] Figure 4G yes Figure 4A The figure shows another example selection of particle subgroups for purification, overlaid with a specified range of particle mass-to-charge ratio values ​​and a specified range of particle charge values.

[0026] Figure 4H yes Figure 4A The figure shows another example selection of particle subgroups for purification, overlaid with a specified range of particle mass-to-charge ratio values, a specified range of particle mass values, and a specified range of particle charge values.

[0027] Figure 5 It is used for control Figure 1 A simplified flowchart of an embodiment of an instrument for identifying, collecting, and / or purifying groups and / or subgroups of charged particles of a specified type.

[0028] Figure 6A Through Figure 1 Examples of instruments and their use Figure 5 The process shown is a scatter plot of particle charge versus mass generated from an exosome-rich milk sample, where the ion processing region of the instrument is implemented in the form of a charge detection mass spectrometer.

[0029] Figure 6B yes Figure 6A The scatter plot, which is covered by multiple boundaries, shows the process of processing the plotted data into various subgroups of charged particles. Detailed Implementation

[0030] To facilitate an understanding of the principles of this disclosure, reference will now be made to several illustrative embodiments shown in the accompanying drawings, and these embodiments will be described using specific language.

[0031] This disclosure relates to apparatus and techniques for identifying and / or purifying particles based on one or more molecular properties, examples of which may include, but are not limited to, mass, charge, mass-to-charge ratio, mobility, etc. For the purposes of this document, the terms "charged particle" and "ion" are used interchangeably, and both terms are intended to refer to any particle having a net positive or negative charge. The terms "purify" and "purification" refer to the identification, extraction, i.e., separation, of a subgroup of charged particles generated from a sample based on one or more molecular properties.

[0032] Now for reference Figure 1 A schematic diagram of the instrument 10 used for purifying particles is shown. Figure 1 An example process 12 for collecting and, in some embodiments, processing the collected purified particles is also depicted. In the illustrated embodiment, instrument 10 illustratively includes an ion source region 14 having an outlet coupled to an inlet of a charged particle processing region 16. The outlet of the charged particle processing region 16 is coupled to an inlet of a charged particle deflector (CPD) or directional device (CPSD) 18. In some embodiments, instrument 10 may further optionally include a conventional ion trap (IT) 20 having an inlet coupled to an outlet of the charged particle deflector or directional device 18, and an outlet opposite the inlet, such as... Figure 1 The dashed line indicates this. In such an embodiment, the outlet of the ion trap 20 defines the charged particle outlet of the instrument 10. In other embodiments where the ion trap 20 is omitted, the outlet of the instrument 10 is the outlet of the charged particle deflector or steering device 18.

[0033] Ion source region 14 illustratively includes an ion generator 22 configured to generate ions, i.e., charged particles, from sample 24. Ion generator 22 is illustratively implemented in the form of any conventional device or apparatus for generating ions from a sample. As an illustrative example, ion generator 22 may be or include a conventional electrospray ionization (ESI) source, a matrix-assisted laser desorption / ionization (MALDI) source, or other conventional ion generator configured to generate ions from sample 24; this example should not be considered as limiting in any way. Sample 24 from which ions are generated may be any biological or other material. In some embodiments, sample 24 may be dissolved, dispersed, or otherwise supported in a solution, although in other embodiments the sample may not be in solution or not part of a solution.

[0034] In the illustrated embodiment, voltage source VS1 is electrically connected to processor 26 via F signal paths, where F can be any positive integer, and further electrically connected to ion source region 14 via G signal paths, where G can also be any positive integer. In some embodiments, voltage source VS1 may be implemented as a single voltage source, and in other embodiments, voltage source VS1 may include any number of independent voltage sources. In some embodiments, voltage source VS1 may be configured or controlled to generate and supply one or more time-invariant (i.e., DC) voltages of selectable amplitude. Alternatively or additionally, voltage source VS1 may be configured or controlled to generate and supply one or more switchable time-invariant voltages, i.e., one or more switchable DC voltages. Alternatively or additionally, voltage source VS1 may be configured or controllable to generate and supply one or more time-varying signals of selectable shape, duty cycle, peak amplitude, and / or frequency.

[0035] Processor 26 is illustratively conventional and may include a single processing circuit or multiple processing circuits. Processor 26 illustratively includes or is coupled to a memory 28 in which instructions are stored, which, when executed by processor 26, cause processor 26 to control voltage source VS1 to generate one or more output voltages for selectively controlling the operation of ion generator 22. In some embodiments, processor 26 may be implemented as one or more conventional microprocessors or controllers, and in such embodiments, memory 28 may be implemented as one or more conventional memory cells storing instructions in the form of one or more microprocessor-executable instructions or instruction sets. In other embodiments, processor 26 may alternatively or additionally be implemented as a field-programmable gate array (FPGA) or similar circuitry, and in such embodiments, memory 28 may be implemented as a programmable logic block contained within and / or outside the FPGA, where instructions may be programmed and stored within the FPGA. In other embodiments, processor 26 and / or memory 28 may be implemented as one or more application-specific integrated circuits (ASICs). Those skilled in the art will recognize that other forms of the processor 26 and / or memory 28 can be implemented therein, and will understand that any such other implementations are contemplated by this disclosure and are intended to fall within the scope of this disclosure. In some alternative embodiments, the voltage source VS1 itself may be programmable to selectively generate one or more constant and / or time-varying output voltages.

[0036] In the illustrated embodiment, voltage source VS1 is illustratively configured to generate one or more voltages in response to a control signal generated by processor 26, thereby causing ion generator 22 to generate ions from sample 24. In some embodiments, sample 24 is positioned within ion source region 14, such as... Figure 1As shown, and in other embodiments, sample 24 may be located outside ion source region 14. In an example embodiment, which should not be considered as limiting in any way, sample 24 is provided in solution form, and ion generator 22 is a conventional electrospray ionization (ESI) source configured to generate ions from sample 24 in the form of a fine mist of charged droplets in response to one or more voltages supplied by VS1. It should be understood that, as stated above, ESI and MALDI are merely two examples of numerous conventional ion generators, and ion generator 22 may be or include any such conventional means or apparatus for generating ions from a sample, whether or not the sample is in solution.

[0037] Ion processing area 16 illustratively includes M ion processing stages or devices 161-16 M Where M can be any positive integer. One or more ion processing devices 161-16 M The method is illustratively operable to process charged particles generated in ion source region 14 and transferred to ion processing region 16 in a manner that measures one or more molecular properties of the charged particles, filters charged particles based on one or more molecular properties to provide a subgroup or subset of charged particles having at least one specified molecular property, and / or dissociates, for example, fragments, charged particles.

[0038] In the illustrated embodiment, voltage source VS2 is electrically connected to processor 26 via H signal paths, where H can be any positive integer, and is also electrically connected to ion processing region 16 via J signal paths, where J can also be any positive integer. In some embodiments, voltage source VS2 may be implemented as a single voltage source, and in other embodiments, voltage source VS2 may include any number of independent voltage sources. In some embodiments, voltage source VS2 may be configured or controlled to generate and supply one or more time-invariant (i.e., DC) voltages of selectable amplitude. Alternatively or additionally, voltage source VS2 may be configured or controlled to generate and supply one or more switchable time-invariant voltages, i.e., one or more switchable DC voltages. Alternatively or additionally, voltage source VS2 may be configured or controllable to generate and supply one or more time-varying signals of selectable shape, duty cycle, peak amplitude, and / or frequency. Typically, one or more outputs of voltage source VS2 are illustratively coupled to one or more ion processing devices 161-16 in ion processing region 16. M Each of these, and it should be understood, will have its quantity of output and / or the type of voltage generated therein depend on the composition of one or more ion processing devices 161-16 MThe number and / or type of ion processing devices. In any case, memory 28 illustratively contains instructions stored therein that, when executed by processor 26, cause processor 26 to control voltage source VS2 to generate one or more output voltages for selectively controlling one or more ion processing devices 161-16 in ion processing region 16. M The operation.

[0039] Ion treatment device 161-16 M Examples may include, but are not limited to, one or more devices and / or instruments in any order and / or combination for separating, collecting, and / or filtering charged particles based on one or more molecular properties, and / or one or more devices and / or instruments for dissociating (e.g., fragmenting) charged particles. Examples of one or more devices and / or instruments for separating charged particles based on one or more molecular properties may include, but are not limited to, one or more mass spectrometers or mass analyzers, one or more ion mobility spectrometers, one or more gas chromatographs, etc. Including ion processing devices 161-16 M One or more of the ion processing devices 161-16 M Examples of mass spectrometers in this embodiment include, but are not limited to, any mass spectrometer operable to measure at least the ion mass-to-charge ratio and to transfer the measured ions from the mass spectrometer to the charged particle deflector or steering device 18. In embodiments where the mass spectrometer is operable to measure only the ion mass-to-charge ratio, the mass spectrometer may be conventional. In other such embodiments, the mass spectrometer may be provided illustratively as a mass spectrometer configured to measure the mass and charge magnitude of charged particles generated in the ion source region 14 and transferred to the ion processing region 16. In one example of this embodiment, which should not be considered as limiting in any way, the mass spectrometer may be implemented illustratively as a charge detection mass spectrometer (CDMS), wherein the ion processing devices 161-16 MThis includes a conventional through-pass ion mass spectrometer or mass analyzer and one or more corresponding CDMS charge detectors. In some embodiments, the one or more CDMS charge detectors may be provided in the form of one or more electrostatic linear ion traps (ELITs), and in other embodiments, the one or more CDMS charge detectors may be provided in the form of at least one orbital trap. In some embodiments, the CDMS detector may include at least one ELIT and at least one orbital trap. Illustratively, CDMS is a single-particle technique that is generally operable to measure the mass and charge magnitude values ​​of a single ion, although some CDMS detectors have been designed and / or operated to measure the mass and charge of more than one charged particle at a time. Examples of CDMS instruments and / or techniques, as well as CDMS charge detectors and / or techniques, which can be implemented in a mass spectrometer, are disclosed in co-pending international applications PCT / US2019 / 013251, PCT / US2019 / 013274, PCT / US2019 / 013277, PCT / US2019 / 013278, PCT / US2019 / 013280, PCT / US2019 / 013283, PCT / US2019 / 013284, and PCT / US2019 / 013285 (all filed January 11, 2019). Figure 1 Ion processing device 161-16 M Or a portion thereof, and all of its publications are incorporated herein by reference in their entirety.

[0040] In other embodiments of a mass spectrometer, including one configured to measure both the mass and charge magnitude of charged particles generated in ion source region 14 and transferred to ion processing region 16, such a mass spectrometer may be provided in the form of a conventional mass analyzer (e.g., a quadrupole mass analyzer, etc.) configured to selectively pass ions of a specified mass-to-charge ratio or ions within a specified mass-to-charge ratio range through it, or in the form of a through-pass ion mass spectrometer with the same configuration, either followed by a field-drift-free region comprising a charge detector array (CDA) configured to measure the charge magnitude or charge state of charged particles leaving the mass analyzer or mass spectrometer. Some example configurations of such mass spectrometers are disclosed in co-pending U.S. Patent Application Serial No. 62 / 949,555 and / or co-pending U.S. Patent Application Serial No. 62 / 949,554 (both filed December 18, 2019), which can be implemented as follows: Figure 1 Ion processing device 161-16 M Or a portion thereof, the publicly available content of which is incorporated herein by reference in its entirety.

[0041] In some embodiments, ion processing devices 161-16 M This may include a mass spectrometer configured as described above to measure both the mass and charge of charged particles supplied by ion source region 14, with an associated charge detector or array electrically connected to the input of each of N charge detection amplifiers CA, and the outputs of the N charge detection amplifiers CA electrically connected to processor 26, as shown. Figure 1 As shown, N can be any positive integer. Each charge amplifier CA is illustratively a conventional amplifier and, in response to a charge induced by a charged particle on one or more corresponding charge detectors, generates a corresponding charge detection signal at its output and supplies the charge detection signal to the processor 26.

[0042] In any embodiment that includes one or more conventional mass spectrometers, such mass spectrometers may be provided in the form of one or any combination of time-of-flight (TOF) mass spectrometers, reflectance mass spectrometers, Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, quadrupole mass spectrometers, triple quadrupole mass spectrometers, magnetic fan mass spectrometers, and orbital trap mass spectrometers.

[0043] Including ion processing devices 161-16 M One or more of the ion processing devices 161-16 M In the embodiments, examples of ion mobility spectrometers include, but are not limited to, single-tube linear ion mobility spectrometers, multi-tube linear ion mobility spectrometers, and circular tube ion mobility spectrometers. In the ion processing apparatus 161-16 M One or more of the ion processing devices 161-16 M In embodiments, examples of one or more devices and / or instruments for collecting charged particles include, but are not limited to, quadrupole ion traps, hexapole ion traps, ion funnels, etc. In the inclusion of ion processing devices 161-16 M One or more of the ion processing devices 161-16 M In the embodiments, examples of one or more devices and / or instruments for filtering charged particles include, but are not limited to, one or more devices or instruments for filtering charged particles according to mass-to-charge ratio, one or more devices or instruments for filtering charged particles according to particle mobility, etc. In the ion processing apparatus 161-16 M One or more of the ion processing devices 161-16 M In the embodiments, examples of one or more devices and / or instruments for dissociating charged particles include, but are not limited to, one or more devices or instruments for dissociating charged particles by collision-induced dissociation (CID), surface-induced dissociation (SID), electron capture dissociation (ECD), and / or photo-induced dissociation (PID).

[0044] It should be understood that the ion processing device 161-16 M Any of the instruments, devices, or stages described above may be included in any order, or any combination thereof, and some embodiments may include multiple such instruments, devices, or stages that are adjacent or spaced apart. Figure 1 A non-limiting example embodiment of the instrument 10 illustrated in the figure, ion processing apparatus 161-16 M It may include a single CDMS, which is configured as described above to measure the mass and charge of charged particles and sequentially supply the measured charged particles to the charged particle deflector or charged particle steering device 18. Figure 1 Other non-limiting example embodiments of the instrument 10 illustrated in the figure, ion processing devices 161-16 M It may include a single mass spectrometer comprising a charge detector array configured, as briefly described above, to measure the mass and charge of charged particles and to supply the measured charged particles to a charged particle deflector or charged particle steering device 18. In any of these examples, the processor 26 is illustratively programmed to control a voltage source VS2 to enable the mass spectrometer instrument to measure the mass and charge of charged particles. Figure 1 Another non-limiting example embodiment of the instrument 10 shown is the ion processing apparatus 161-16. M This may include a mass-to-charge ratio filter, for example, in the form of a quadrupole mass analyzer. In this example, processor 26 is illustratively programmed to control voltage source VS2 such that the mass-to-charge ratio filter selectively passes only ions with a specified mass-to-charge ratio, or only ions with mass-to-charge ratios within a specified range, through it to the charged particle deflector or charged particle redirection device 18. In some such embodiments, ion processing devices 161-16 M It may further include a mass spectrometer disposed between the mass-to-charge ratio filter and the charged particle deflector or charged particle steering device 18, and configured to measure the mass and charge of charged particles exiting the mass-to-charge ratio filter. In some such embodiments, ion processing devices 161-16 M It may also include a particle dissociation stage or device disposed between the mass-to-charge ratio filter and the mass spectrometer, and configured to dissociate charged particles leaving the mass-to-charge ratio filter. In such an example, processor 26 is illustratively programmed to control voltage source VS2 to operate the example device and / or stage in a conventional manner. Those skilled in the art will contemplate ion processing devices 161-16. MOther examples and combinations thereof, and it will be understood that all such examples and combinations thereof are intended to fall within the scope of this disclosure. In any case, processor 26 is configured (e.g., programmed) to control voltage source VS2 to generate one or more voltages for controlling ion processing devices 161-16 M Operate in the usual manner and / or as described herein.

[0045] In embodiments including a charged particle deflector or charged particle redirection device 18, the charged particle deflector or charged particle redirection device 18 is illustratively configured to selectively pass only charged particles having one or more specified molecular properties or one or more molecular properties within a range of molecular properties through its outlet. The remaining charged particles are directed away from the outlet from which they are collected (e.g., through another channel or outlet from which they do not collect or store charged particles) if the charged particle deflector is blocked (e.g., by guiding such charged particles into a conductive structure, or in the case of the charged particle redirection device).

[0046] In one example embodiment, the charged particle deflector or steering device 18 may be implemented as a conventional single-inlet, single-outlet charge deflector configured and controllable to selectively allow ions to pass through it or block ions from passing through it. In another example embodiment, the charged particle deflector or steering device 18 may be implemented as a conventional single-inlet, multi-outlet charge steering device configured and controllable to selectively deflect ions entering a single inlet through one of a plurality of different ion outlets, from which purified charged particles are collected. In either case, another voltage source VS3 is electrically connected to the processor 26 via K signal paths, where K can be any positive integer, and is also electrically connected to the charged particle deflector or steering device 18 via L signal paths, where L can also be any positive integer. In some embodiments, the voltage source VS3 may be implemented as a single voltage source, and in other embodiments, the voltage source VS3 may include any number of independent voltage sources. In some embodiments, the voltage source VS3 may be configured or controlled to generate and supply one or more time-invariant (i.e., DC) voltages of selectable amplitudes. Alternatively or additionally, the voltage source VS3 may be configured or controlled to generate and supply one or more switchable time-invariant voltages, i.e., one or more switchable DC voltages. Alternatively or additionally, the voltage source VS3 may be configured or controllable to generate and supply one or more time-varying signals of selectable shape, duty cycle, peak size, and / or frequency. Typically, one or more outputs of the voltage source VS3 are illustratively coupled to the charged particle deflector or steering device 18, and it will be understood that the number of such outputs and / or the type of voltage generated therein will depend on the type of charged particle deflector or steering device 18 implemented. In any case, the memory 28 illustratively has instructions stored therein that, when executed by the processor 26, cause the processor 26 to control the voltage source VS3 to generate one or more output voltages for selectively controlling the operation of the charged particle deflector or steering device 18.

[0047] In some embodiments where the charged particle deflector or steering device 18 is implemented as a single-inlet, single-outlet charge deflector, the processor 26 is illustratively operable to deflect and accelerate charged particles P entering its inlet into a conductive structure, such as a conductive plate, tube, or rod, by controlling the voltage source VS3 to generate an electric field E of sufficient magnitude. In such embodiments, the processor 26 is illustratively operable to allow charged particles entering the inlet to pass through its outlet by controlling the voltage source VS3 to generate conditions (e.g., a small electric field or no electric field) within the deflector that allow charged particles to pass through it. In some embodiments where the charged particle deflector or steering device 18 is implemented as a single-inlet, multi-outlet charge deflector, the processor 26 is illustratively operable to deflect charged particles entering its inlet through an outlet such that charged particles P enter the channel and / or through an outlet from which they do not collect purified charged particles. In such an embodiment, processor 26 is illustratively operable to pass charged particles entering the inlet through its outlet (from which purified charged particles are collected) by controlling voltage source VS3 to generate conditions within the charge redirection device that allow charged particles to pass through a corresponding outlet. Several alternative embodiments of the charged particle deflector or redirection device 18 are illustrated and described in U.S. Patent Application No. 62 / 52 / 949,555, filed December 18, 2019, and that patent application is incorporated herein by reference, but it will be understood that these embodiments are provided by way of example only. Other charged particle deflection and / or redirection instruments or devices will occur to those skilled in the art, and it will be understood that any other such charged particle deflection and / or redirection instruments or devices are intended to fall within the scope of this disclosure.

[0048] In some embodiments, as briefly described above and Figure 1As shown by the dashed lines, the ion trap 20 can be coupled to a charged particle deflector or steering device 18. In such an embodiment, another voltage source VS4 is electrically connected to the processor 26 via P signal paths, where P can be any positive integer, and is also electrically connected to the ion trap 20 via Q signal paths, where Q can also be any positive integer. In some embodiments, the voltage source VS4 can be implemented as a single voltage source, and in other embodiments, the voltage source VS4 can include any number of independent voltage sources. In some embodiments, the voltage source VS4 can be configured or controlled to generate and supply one or more time-invariant (i.e., DC) voltages of selectable amplitude. Alternatively or additionally, the voltage source VS4 can be configured or controlled to generate and supply one or more switchable time-invariant voltages, i.e., one or more switchable DC voltages. Alternatively or additionally, the voltage source VS4 can be configured or controllable to generate and supply one or more time-varying signals of selectable shape, duty cycle, peak amplitude, and / or frequency. One or more outputs of voltage source VS4 are illustratively coupled to ion trap 20, and memory 28 illustratively has instructions stored therein that, when executed by processor 26, cause processor 26 to control voltage source VS4 to generate one or more output voltages for controlling ion trap 20 to selectively capture and store charged particles therein, and to generate one or more output voltages for controlling ion trap 20 to selectively release and accelerate captured particles therefrom.

[0049] Processor 26 is also illustratively coupled to one or more peripheral devices 30 (PDs) via R signal paths, where R can be any positive integer. The one or more peripheral devices 30 may include one or more means for providing signal input to processor 26 and / or one or more means for processor 26 to provide signal output thereto. In some embodiments, peripheral devices 30 include at least one of conventional display monitors, printers, and / or other output devices, and in such embodiments, memory 28 has instructions stored therein that, when executed by processor 26, cause processor 26 to control one or more such output peripheral devices 30 to display and / or record analysis of the operation of instrument 10, including, for example, but not limited to, particle spectrum information measured by instrument 10.

[0050] As briefly described above, instrument 10 is illustratively operable, under the control of processor 26, via control voltage sources VS1, VS2, VS3, and in some embodiments via control voltage source VS4, to purify charged particles generated by ion generator 22 by selectively passing through only a subset of charged particles having one or more molecular properties or one or more molecular properties within a range of one or more molecular properties. For example, in some embodiments, the subset may illustratively include only charged particles of a specified mass or charged particles with a mass within a specified mass range. In other embodiments, the subset may illustratively include only charged particles of a specified charge or charged particles with a mass within a specified charge size or charge state range. In other embodiments, the subset may illustratively include only charged particles of a specified mass along with a specified charge or charge size or charge state range, or particles with a mass value within a specified mass range along with a specified charge or charge size or charge state range. In other embodiments, a subgroup or subset may illustratively include only charged particles with a specified mass-to-charge ratio or charged particles with a mass-to-charge ratio value within a specific range of mass-to-charge ratio values. In some such embodiments, a subgroup or subset may further include only charged particles that also have a specified mass value or a mass value within a specified range of mass values, and / or only include charged particles that also have a specified charge magnitude or charge state value or a charge magnitude or charge state value within a specified range of charge magnitude values ​​or charge state values. In even further embodiments, a subgroup or subset may illustratively include only charged particles with a specified mobility, or only include charged particles with a mobility within a specified mobility value range. In some such embodiments, a subgroup or subset may be further limited to a specified charged particle mass value or mass value range, a specified charge magnitude or charge state or a specified range thereof, a specified mass-to-charge ratio or a range thereof, or any combination just described. Those skilled in the art will recognize that the charged particle instruments 161-16 implemented in any particular embodiment of instrument 10 M The quantity and type will depend on the specific subgroup or subset of charged particles for which purification is sought, and the aforementioned charged particle instruments 161-16 M Various types and combinations of molecular properties can be used to collect desired subgroups or subsets. Furthermore, those skilled in the art will recognize that other molecular property subgroups or subgroups and / or combinations thereof may be sought for purification, and will understand that such other molecular property subgroups or subgroups and / or combinations thereof, as well as the various instruments and instrument combinations used to collect them, are intended to fall within the scope of this disclosure.

[0051] Figure 1The document also describes a simplified process 12 for collecting and, in some embodiments, processing the collected purified particles. In some embodiments, for example, a subgroup or subset of charged particles exiting instrument 10 is collected on surface 40A of particle collection target 40 via particle deposition (e.g., via low-energy deposition) or other conventional particle collection techniques. Particle collection target 40, or at least its surface 40A, is illustratively a non-reactive or inert material so as not to bind to or otherwise react with the purified charged particles exiting instrument 10. In some embodiments, particle collection surface 40A of particle collection target 40 may be viscous or oily, or otherwise configured or constructed such that purified charged particles exiting instrument 10 can be effectively collected thereon over a period of time. In other embodiments including ion trap 20, purified charged particles exiting instrument 10 may be trapped and collected within ion trap 20 over a period of time, and then released in large quantities from ion trap 20 onto surface 40A of particle collection target 40. In any case, the particle collecting surface 40A of the particle collecting target 40 is illustratively configured to not only collect purified charged particles exiting the instrument 10, but also to provide harvesting of the purified particles collected therefrom. In some embodiments, for example, the purified particles collected on the surface 40A of the particle collecting target 40 can be harvested by rinsing the surface 40A with a liquid solution 45 dispensed from the solution source 42 and guiding the resulting combination 46 of the solution 45 carrying the purified particles into a suitable container 44. Those skilled in the art will recognize other techniques, instruments, apparatuses, etc., for harvesting the purified particles collected on the collecting surface 40A of the particle collecting target 40, and will understand that any such other techniques, instruments, apparatuses, etc., are intended to fall within the scope of this disclosure.

[0052] In some embodiments, the harvested purified particle set may be amplified, i.e., replicated or otherwise multiplied, in a conventional particle amplifier or particle amplification process 48. In embodiments where the purified particles are or include DNA, for example, the particle amplifier or amplification process 48 may take the form of a conventional polymerase chain reaction (PCR) instrument or process to amplify or replicate particles on several orders of magnitude, such as thousands or millions of copies. Those skilled in the art will recognize other instruments and / or methods for amplifying the harvested, purified particles, whether they are or include DNA and / or other molecular components, and will understand that any such other particle amplification instruments and / or processes are intended to fall within the scope of this disclosure.

[0053] In some cases, it may be desirable to observe all or at least part of the molecular characterization spectra of sample 24 in order to identify, or facilitate the identification of, its subgroups or subsets for purification. In this respect, Figure 2 A simplified flowchart is shown, which depicts the operation. Figure 1The instrument 10 is used to measure one or more molecular properties of charged particles generated from sample 24 and to process such measurements to generate a multidimensional (e.g., two or more) molecular property spectrum 100. At least some steps of process 100 are stored in memory 28 in the form of instructions executable by processor 26 to perform the measurement, analysis, and visualization of the spectrum. Process 100 begins at step 102, where processor 26 is illustratively operable to control voltage source VS1 to cause ion generator 22 to generate charged particles from sample 24 and supply the generated charged particles to ion processing region 16. Subsequently, at step 104, processor 26 is operable to control voltage source VS2 to cause one or more instruments or devices in ion processing region 16 to measure two or more molecular properties.

[0054] In some embodiments, as described above regarding Figure 1 The ion processing region 16 may include or be implemented in the form of a mass spectrometer configured to measure particle mass and particle charge. In some such embodiments, for example, such a mass spectrometer may be implemented in the form of a charge detection mass spectrometer (CDMS), and in other embodiments, such a mass spectrometer may be implemented in the form of a mass analyzer, a mass-to-charge ratio filter, or other instrument configured to measure mass-to-charge ratio (conventional MS) followed by a charge detector array (CDA), some examples of which are described and published in U.S. Patent Application Serial No. 62 / 949,555, filed December 18, 2019, the disclosure of which is incorporated herein by reference. In other embodiments, the ion processing region 16 may include or be implemented in the form of an ion mobility spectrometer (IMS) followed by such a charge detector array. In other embodiments, the ion processing region 16 may include a mass spectrometer, an ion mobility spectrometer, and a charged particle charge measurement instrument or apparatus, or a combination thereof. In some such embodiments, for example, the ion processing region 16 may include an IMS followed by a CDMS, or an IMS followed by a conventional MS followed by a CDA. In other such embodiments, as further examples, the ion processing region 16 may include conventional MS followed by IMS followed by CDA, conventional MS followed by CDA followed by IMS, or CDMS followed by IMS. In these example embodiments of the ion processing region 16, the processor 26 is illustratively operable in step 104 to control the voltage source VS2, thereby enabling the spectrometer instrument to measure the charge magnitude or charge state of the generated charged particles and the mass and / or mobility values ​​of the generated charged particles, such as... Figure 2 As shown.

[0055] Following step 104, processor 26 is operable to process the measurements performed in step 104 and thereby generate a charged particle spectrum. As an illustrative example, where sample 24 is a liquid solution of urinary exons, and ion processing region 16 is implemented in the form of CDMS or conventional MS followed by CDA, processor 26 is illustratively operable in step 106 to generate a scatter plot of charged particle charge magnitude (in elementary charge e) versus charged particle mass (in megadaltons MDa), as shown below. Figure 4A As shown.

[0056] Following step 106, process 100 proceeds to step 108, whereby the spectrum generated in step 106 is analyzed, for example by processor 26, visually or automatically, to determine a suitable subgroup or subset of particles to be purified. Illustratively, a subgroup or subset of particles may be selected based on one or any combination of particle mass, mass-to-charge ratio, charge (size or charge state), or mobility value or range.

[0057] Now for reference Figure 3 This shows the use of Figure 1 A simplified flowchart of a process 200 for purifying particles from sample 24 using any of the various embodiments of the instrument 10 shown. In some embodiments, for performing... Figure 2 The embodiment of instrument 10 in process 100 shown can also be used after process 100 to perform... Figure 3 The process 200 is shown. In other embodiments, for example, the molecular characteristic values ​​and / or ranges used for purification are known in advance. Figure 2 The process 100 shown may not be performed, and the configuration of instrument 10 can be specifically selected to achieve or facilitate the desired purification. In any case, at least some steps of process 200 are illustratively stored in memory 28 in the form of instructions executable by processor 26, for processing from... Figure 1A selected subgroup or subset of charged particles generated in sample 24 is purified. Process 200 begins at step 202, where processor 26 is illustratively operable to control voltage source VS1 to cause ion generator 22 to generate charged particles from sample 24 and supply the generated charged particles to ion processing region 16. Subsequently, at step 204, processor 26 is operable to control voltage source VS2 to cause one or more instruments or devices in ion processing region 16 to measure two or more molecular properties. Various combinations of instruments or devices can be implemented in ion processing region 16 to measure any two or more molecular properties, and several examples of such instruments or devices and such one or more molecular properties are given in the description of process 100 above. In these example embodiments of ion processing region 16, processor 26 is illustratively operable at step 204 to control voltage source VS2, thereby causing spectrometer instruments to measure the charge magnitude or charge state of the generated charged particles and the mass and / or mobility values ​​of the generated charged particles, such as... Figure 3 As illustrated in the example, although it should be understood that in step 204, the ion treatment region 16 may alternatively be implemented in different forms (i.e., using different instruments), and / or one or more molecular properties may be measurable molecular properties other than or additional to particle mass, mass-to-charge ratio, mobility and charge (size or charge state).

[0058] Following step 204, process 200 proceeds to step 206, where processor 26 is operable to control voltage source VS3 so that the charged particle deflector or charged particle redirection device 18 delivers only charged particles from a selected subgroup or subset of charged particles generated by ion generator 22 through its charged particle outlet, or through a designated one of its multiple charged particle outlets. As described above, the subgroup or subset of charged particles generated by ion generator 22 can be selected based on one or any combination of measurements of particle mass, mass-to-charge ratio, charge (size or charge state), or mobility value or range. When the corresponding charged particles leave ion processing region 16, processor 26, using such measurements known to processor 26, is operable to control the charged particle deflector or charged particle redirection device 18, for example, via control voltage source VS3, to selectively deliver them through it, thereby collecting only those charged particles having one or a combination of measured molecular characteristic values ​​defined by the selected subgroup or subset of charged particles generated by ion generator 22. To demonstrate steps 204 and 206 of process 200, the following will refer to... Figure 4B-4H describe Figure 4A The illustration shows some example subgroups or subsets of the urine exon spectrum, as well as some example configurations and implementations of the instrument 10 used to purify these subgroups.

[0059] Following step 206, process 200 proceeds to step 208, in which charged particles exiting the charged particle deflector or charged particle deflector 18 through a single outlet or a selected one of its multiple outlets are collected. In an embodiment of the instrument 10 including ion trap 20, step 208 illustratively includes being controlled by processor 26 of voltage source VS4 to supply one or more voltages to ion trap 20, thereby causing ion trap 20 to collect and store therein charged particles exiting the charged particle deflector or charged particle deflector 18 through a single outlet or a selected one of its multiple outlets. After the collection period in which the ion trap 20 is operable to collect and store departing charged particles has elapsed, the processor 26 is further operable in step 208 to control the voltage source VS4 to supply one or more voltages to the ion trap 20, causing the ion trap 20 to release and guide the stored ions toward and onto the collection surface 40A of the collection target 40. In an embodiment of the instrument 10 excluding the ion trap 20, step 208 illustratively includes collecting charged particles on the collection surface 40A of the collection target 40 as they depart from the charged particle deflector or charged particle steering device 18. Subsequently, in step 210, the purified particles collected on the collection surface 40A of the particle collection target 40 are harvested, for example, as described above regarding Figure 1 In some embodiments, process 200 includes another step 212 following step 210, in which the harvested particles are amplified, i.e., replicated or multiplied, in a conventional manner as described above.

[0060] Now for reference Figure 5 A simplified flowchart of process 500 is shown, which is used to control... Figure 1Any of the various embodiments of instrument 10 is used to identify, collect, and / or purify groups and / or subgroups of charged particles of a specified type from sample 24. Process 500 begins at step 502, in which a sample is provided in which particles of a specified type are present. The specified particles can be any particles, such as molecules or collections thereof, which are present in or part of a cell, and / or transported between cells, and have a mass greater than or equal to the megadalton range. Examples of particle types present in the sample and the sample selected and provided for them can include, but are not limited to, exomes, endosomes, typically microvesicles, exosomes, apoptotic bodies, retroviruses, exosomes, chylomicrons, DNA, RNA, proteins, fats, acids, carbohydrates, enzymes, viruses, bacteria, etc. Examples of other samples and / or particles of interest present in the samples (all of which are intended to fall within the scope of this disclosure) include, but are not limited to, any cell that emits exosomes or extracellular vesicles, any molecules or aggregates thereof enclosed in a biolayer (e.g., viruses), any organelles aggregated but not bound to the biolayer or any non-compartmentalized organelles in the biolayer, any extracellular vesicles that have been altered in a manner that results in a detectable mass shift (e.g., by adding one or more small molecules to them, by adding drugs such as anticancer drugs, etc.), any biological tissue, fluid, cell and / or other biological material thereof, or part thereof.

[0061] In some embodiments, the sample provided in step 502 (in which a specific type of particle is present) may be Figure 1 The sample 24 depicted in the diagram generates charged particles for analysis by instrument 10. In some alternative embodiments, process 500 may include step 504, as shown in the dashed configuration, in which the sample provided in step 502 is enriched for a specified particle type. After step 504, in embodiments including step 504, in one embodiment, process 500 illustratively proceeds to step 506, in which the enriched sample 24, i.e., for the specified particle type, provided in step 502 and enriched in step 504, is used to perform... Figure 2 The process 100 is shown. In an embodiment excluding step 504, step 506 is performed after step 502, such that the sample 24 in which the specified type of particles is present is used for execution. Figure 2 The process 100 is shown below. In some embodiments, step 108 of process 100 (wherein subgroups of the particle spectrum are identified and / or selected for purification) may include performing one or more conventional statistical and / or modeling procedures on the particle dataset for the purpose of identifying and / or selecting one or more subgroups of particles. An example of such a statistical procedure will be described below with reference to Example 8.

[0062] In some embodiments, process 500 ends after performing step 506. In some alternative embodiments, process 500 proceeds from step 506 to step 508, in which enrichment sample 24 is used for execution. Figure 3 The illustrated process 200 is used to purify a specific type of particle or one or more subgroups thereof identified in step 506. In some alternative embodiments, process 500 may proceed directly from step 504 to step 508 in embodiments that include step 504, or directly from step 502 in embodiments that do not include step 504, as described above regarding... Figure 3 As stated above.

[0063] In some embodiments including step 504, the process for enriching a sample of a specified particle type may depend on the sample type and / or the specified particle type, and will be known to those skilled in the art in any case. In such embodiments, the enriched sample obtained from step 504 will be Figure 1 The sample 24 depicted in the image generates charged particles for analysis by instrument 10. An example of this method for enriching exosomes from a milk sample is described in Example 8 below, and this example should not be considered as limiting in any way. In other embodiments, including or excluding step 504, various configurations and / or implementations of the ion processing region 16 of instrument 10 may be used to enrich and / or assist in enriching samples of a specified type of particles. For example, in some embodiments, the sample may include one or more unwanted particles known to be present in a range of mass, mass-to-charge ratio, and / or mobility that differs from the range of particles in a specified type of sample, and in such embodiments, the ion processing region 16 may be configured differently, as described above, to filter out some or all of such unwanted particles prior to performing steps 506 and / or 508.

[0064] Example

[0065] Example 1

[0066] Now for reference Figure 4B It reproduces Figure 4A A urine exon map, overlaid with data based on... Figure 3 Steps 204 and 206 of process 200 shown are passed through Figure 1The instrument selects an example of a subpopulation or subset 300 of particles for purification. In this example, the selected subpopulation 300 is limited only by a specified particle mass value range between 20 and 30 MDa. To enable the processor 26 to control the voltage source VS3 in step 206 so that the charged particle deflector or charged particle steering device 18 delivers only charged particles with a mass within the specified particle mass range of 20-30 MDa through it to the particle target 40, particle measurement information generated by one or more instruments or devices in the ion processing region 16 must include particle mass information or particle measurement information, which the processor 26 can use to determine the particle mass prior to step 206. In this example, as described above regarding... Figure 4A The ion processing region 16 is illustratively implemented in the form of CDMS or conventional MS followed by CDA, either of which is configured to directly measure particle mass in step 204 or to determine particle mass based on charged particle measurements performed by the instrument. However, it should be understood that the ion processing region 16 may alternatively be or include any instrument or device or combination thereof configured to measure particle mass or to measure one or more characteristics or properties of particles whose mass can be determined or estimated by it. In any case, using the particle mass information determined in step 204, the processor 26 is operable in step 206 to control the voltage source VS3, thereby causing the charged particle deflector or charged particle steering device 18 to deliver the charged particle to the particle target 40 only if the mass of the charged particle leaving the ion processing region 16 is within a specified particle mass range of 20-30 MDa, or otherwise controlling the voltage source VS3 to prevent the charged particle deflector or charged particle steering device 18 from reaching the target 40 or to deflect the charged particle away from the target 40.

[0067] Example 2

[0068] Now for reference Figure 4C It has reappeared once again. Figure 4A An image of urine exons, overlaid with images based on... Figure 3 Steps 204 and 206 of process 200 shown are passed through Figure 1The instrument selects another example of a subgroup or subset 302 of particles for purification. In this example, the selected subgroup 302 is limited only by a range of particle charge magnitude values ​​between 750 and 900 e. For the processor 26 to control the voltage source VS3 in step 206 so that the charged particle deflector or charged particle steering device 18 only passes charged particles with particle charge values ​​within the specified particle charge range of 750-900 e through it to the particle target 40, particle measurement information generated by one or more instruments or devices in the ion processing region 16 must include particle charge information or particle measurement information, which the processor 26 can determine based on prior to step 206. In this example, as described above regarding... Figure 4A The ion processing region 16 is illustratively implemented in the form of CDMS or conventional MS followed by CDA, either of which is configured to directly measure particle charge in step 204 or to determine particle charge based on charged particle measurements performed by the instrument. However, it should be understood that the ion processing region 16 may alternatively be or include any instrument or device or combination thereof configured to measure particle charge or to measure one or more characteristics or properties of the particles from which the particle charge can be determined or estimated. In any case, using the particle charge information determined in step 204, the processor 26 is operable in step 206 to control the voltage source VS3, such that the charged particle deflector or charged particle deflector 18 delivers the charged particle to the particle target 40 only when the charge size of the charged particle leaving the ion processing region 16 is within a specified particle charge size range of 750-900e, or otherwise controls the voltage source VS3 to prevent the charged particle deflector or charged particle deflector 18 from reaching the target 40 or to deflect the charged particle away from the target 40.

[0069] Example 3

[0070] Now for reference Figure 4D It has reappeared once again. Figure 4A A urine exon map, overlaid with data based on... Figure 3 Steps 204 and 206 of process 200 shown are by Figure 1This is yet another example selection of the instrument used to purify another subgroup or subset 304 of particles. In this example, the selected subgroup 304 is defined by a range of particle mass values ​​specified between 10 and 15 MDa, and also by a range of charge magnitude values ​​between 600 and 700 e. In order for the processor 26 to control the voltage source VS3 in step 206 so that the charged particle deflector or charged particle steering device 18 only transmits charged particles with particle mass values ​​within the specified particle mass range of 10–15 MDa and charge values ​​within the specified particle charge range of 600–750 e through it to the particle target 40, particle measurement information generated by one or more instruments or devices in the ion processing region 16 must include particle mass and charge information or particle measurement information that the processor 26 can determine based on before step 206. In this example, as mentioned above regarding… Figure 4A The ion processing region 16 is illustratively implemented in the form of CDMS or conventional MS followed by CDA, either of which is configured to directly measure particle mass and charge in step 204, or to determine particle mass and charge based on charged particle measurements performed by the instrument. However, it should be understood that the ion processing region 16 may alternatively be or include any instrument or apparatus or combination thereof configured to measure particle mass and charge, or configured to measure one or more characteristics or properties of the particles by which both particle mass and charge can be determined or estimated. In any case, using the particle mass and charge information determined in step 204, processor 26 is operable in step 206 to control voltage source VS3 so that charged particle deflector or charged particle deflector 18 delivers the charged particle to particle target 40 only when the mass of the charged particle leaving ion processing region 16 is within a specified particle mass range of 10-15 MDa and the charge size of the particle is within a specified particle charge size range of 600-750e, or otherwise controls voltage source VS3 to block the charged particle deflector or charged particle deflector 18 from passing through to target 40 or to deflect the charged particle away from target 40.

[0071] Example 4

[0072] Now for reference Figure 4E It has reappeared once again. Figure 4A A urine exon map, overlaid with data based on... Figure 3 Steps 204 and 206 of process 200 shown are by Figure 1 The instrument is selected for yet another example of 400 samples for purifying another subgroup or subset of particles. From Figure 4AAs is evident in the diagram, the total population of urinary exons appears to fall into multiple distinct diagonal or slanted subgroups, subsets, or families, each grouped approximately or along different values ​​or ranges of a constant mass-to-charge ratio. In this example, the selected subgroup 400 is defined by a specified value or range of such particle mass-to-charge ratios. For the processor 26 to control the voltage source VS3 in step 206 so that the charged particle deflector or charged particle steering device 18 only passes charged particles with a specified mass-to-charge ratio value or a mass-to-charge ratio within a specified range through it to the particle target 40, particle measurement information generated by one or more instruments or devices in the ion processing region 16 must include particle mass-to-charge ratio information or particle measurement information, which the processor 26 can determine based on this information prior to step 206. In this example, as described above regarding... Figure 4A The ion processing region 16 is illustratively implemented in the form of CDMS or conventional MS followed by CDA, either of which is configured to directly measure particle mass and charge in step 204, or to determine particle mass and charge based on charged particle measurements performed by the instrument. However, it should be understood that the ion processing region 16 may alternatively be or include any instrument or apparatus or combination thereof configured to measure particle mass and charge, or configured to measure one or more characteristics or properties of the particles by which the particle mass and charge can be determined or estimated. In any case, the processor 26 in this embodiment is operable to calculate the particle mass-to-charge ratio based on the measured particle mass and charge.

[0073] Using the particle mass-to-charge ratio information determined in step 204, processor 26 can operate in step 206 to control voltage source VS3 so that charged particle deflector or charged particle deflector 18 delivers the charged particle to particle target 40 only when the charged particle leaving ion processing region 16 has a specified mass-to-charge ratio or the mass-to-charge ratio is within a specified particle mass-to-charge ratio range, or otherwise controls voltage source VS3 to block particles from reaching target 40 or deflect charged particles away from target 40.

[0074] In an alternative embodiment of instrument 10, the charged particle deflector or charged particle steering device 18 can be omitted, and the ion processing region 16 can be implemented in the form of a conventional mass analyzer or mass-to-charge ratio filter, such as a quadrupole mass-to-charge ratio filter. In this embodiment, it is not necessary to measure the particle charge through the ion processing region 16, and therefore one or more charge amplifiers CA can also be omitted. In this embodiment of instrument 10, step 206 of process 200 can be omitted, and the processor 26 can be operable in step 204 to control the voltage source VS2 so that the mass analyzer or mass-to-charge ratio filter only passes charged particles with mass-to-charge ratio values ​​within a selected mass-to-charge ratio range 400 through it to the particle collection target 40.

[0075] Example 5

[0076] Now for reference Figure 4F It has reappeared once again. Figure 4A A urine exon map, overlaid with data based on... Figure 3 Steps 204 and 206 of process 200 shown are passed through Figure 1 The instrument selects another example of a subgroup or subset 402 of particles for purification. In this example, similar to Example 4, the selected subgroup 402 is defined by a specified one of a plurality of different families of constant mass-to-charge ratios or mass-to-charge ratio ranges, and further defined by a specified mass value range between 10 and 20 MDa. In order for the processor 26 to control the voltage source VS3 in step 206 so that the charged particle deflector or charged particle steering device 18 delivers only charged particles having a specified mass-to-charge ratio or a mass-to-charge ratio within a specified mass-to-charge ratio range and a mass value within a specified mass value range through it to the particle target 40, the particle measurement information generated by one or more instruments or devices in the ion processing region 16 must include particle mass and mass-to-charge ratio information or particle measurement information, which the processor 26 can determine based on the particle mass and mass-to-charge ratio before step 206. In this example, as mentioned above regarding Figure 4A The ion processing region 16 is illustratively implemented in the form of CDMS or conventional MS followed by CDA, either of which is configured to directly measure particle mass and charge in step 204, or to determine particle mass and charge based on charged particle measurements performed by the instrument. However, it should be understood that the ion processing region 16 may alternatively be or include any instrument or apparatus or combination thereof configured to measure particle mass and charge, or configured to measure one or more characteristics or properties of the particles by which the particle mass and charge can be determined or estimated. In any case, the processor 26 is operable to calculate the particle mass-to-charge ratio based on the measured particle mass and charge.

[0077] Using the particle mass and mass-to-charge ratio information determined in step 204, processor 26 can operate in step 206 to control voltage source VS3, thereby causing charged particle deflectors or charged particle steering devices 18 to deliver charged particles to particle target 40 only when the mass-to-charge ratio and mass of charged particles leaving ion processing region 16 are within the specified range 402 of particle mass and mass-to-charge ratio, or otherwise controlling voltage source VS3 to cause charged particle deflectors or charged particle steering devices 18 to block particles from reaching target 40 or to deflect charged particles away from target 40.

[0078] Example 6

[0079] Now for reference Figure 4GIt has reappeared once again. Figure 4A A urine exon map, overlaid with data based on... Figure 3 Steps 204 and 206 of process 200 shown are by Figure 1 This is yet another example of instrument selection for another subgroup or subset 404 of particles used for purification. In this example, as in Examples 4 and 5, the selected subgroup 404 is defined by specifying one of a plurality of different families of constant mass-to-charge ratios or mass-to-charge ratio ranges, and further defined by a specified range of charge magnitude values ​​between 300 and 450 e. In order for processor 26 to control voltage source VS3 in step 206 so that charged particle deflectors or charged particle steering devices 18 pass only charged particles having a specified mass-to-charge ratio or mass-to-charge ratio within a specified mass-to-charge ratio range and also a charge magnitude value within a specified charge magnitude value range through it to particle target 40, particle measurement information generated by one or more instruments or devices in ion processing region 16 must include particle mass-to-charge ratio and charge magnitude information or particle measurement information that processor 26 can determine based on the particle mass-to-charge ratio and charge magnitude prior to step 206. In this example, as described above regarding... Figure 4A The ion processing region 16 is illustratively implemented in the form of CDMS or conventional MS followed by CDA, either of which is configured to directly measure particle mass and charge in step 204, or to determine particle mass and charge based on charged particle measurements performed by the instrument. However, it should be understood that the ion processing region 16 may alternatively be or include any instrument or apparatus or combination thereof configured to measure particle mass and charge, or configured to measure one or more characteristics or properties of the particles by which the particle mass and charge can be determined or estimated. In any case, the processor 26 is operable to calculate the particle mass-to-charge ratio based on the measured particle mass and charge.

[0080] Using the particle mass-to-charge ratio and charge size information determined in step 204, processor 26 can operate in step 206 to control voltage source VS3, thereby causing charged particle deflector or charged particle steering device 18 to deliver the charged particle to particle target 40 only when the mass-to-charge ratio and charge size of the charged particle leaving ion processing region 16 are within the range 404 of particle mass-to-charge ratio and specified charge size values, or otherwise controlling voltage source VS3 to cause charged particle deflector or charged particle steering device 18 to block the particle from reaching target 40 or to deflect the charged particle away from target 40.

[0081] Example 7

[0082] Now for reference Figure 4H It has reappeared once again. Figure 4A A urine exon map, overlaid with data based on... Figure 3Steps 204 and 206 of process 200 shown are by Figure 1 This is yet another example of instrument selection for another subgroup or subset 406 of particles used for purification. In this example, like those in Examples 4, 5, and 6, the selected subgroup 406 is defined by specifying one of a plurality of different families of constant mass-to-charge ratios or mass-to-charge ratio ranges, and further defined by a specified mass value range between 15 and 25 MDa and a specified charge magnitude value range between 300 and 450 e. In order for processor 26 to control voltage source VS3 in step 206 so that charged particle deflectors or charged particle steering devices 18 pass only charged particles having a specified mass-to-charge ratio or mass-to-charge ratio within a specified mass-to-charge ratio range and a charge magnitude value within a specified charge magnitude value range and a mass value within a specified mass value range through it to particle target 40, particle measurement information generated by one or more instruments or devices in ion processing region 16 must include mass and charge magnitude information, mass-to-charge ratio and charge magnitude information, or particle measurement information, which processor 26 can determine based on the particle mass, mass-to-charge ratio, and charge magnitude values ​​prior to step 206. In this example, as described above regarding... Figure 4A The ion processing region 16 is illustratively implemented in the form of CDMS or conventional MS followed by CDA, either of which is configured to directly measure particle mass and charge in step 204, or to determine particle mass and charge based on charged particle measurements performed by the instrument. However, it should be understood that the ion processing region 16 may alternatively be or include any instrument or apparatus or combination thereof configured to measure particle mass and charge, or configured to measure one or more characteristics or properties of the particles, thereby determining or estimating both particle mass and charge. In any case, the processor 26 is operable to calculate the particle mass-to-charge ratio based on the measured particle mass and charge.

[0083] Using the particle mass-to-charge ratio and charge magnitude information determined in step 204, processor 26 can operate in step 206 to control voltage source VS3, thereby causing charged particle deflector or charged particle steering device 18 to deliver the charged particle to particle target 40 only if the mass-to-charge ratio, charge magnitude, and mass of the charged particle leaving ion processing region 16 are within the specified range 406 of particle mass-to-charge ratio, charge magnitude, and mass values, or otherwise controlling voltage source VS3 to cause charged particle deflector or charged particle steering device 18 to block the particle from reaching target 40 or to deflect the charged particle away from target 40.

[0084] It should be understood that, although in Figures 4A-4HThe example shown uses urinary exons as sample 24; however, in other applications, sample 24 can be any material, whether biological in nature or not, and whether in solution or otherwise. Other examples of biological substances or materials that can be used as sample 24 include, but are not limited to, other exons, endosomes, typically microvesicles, exosomes, apoptotic bodies, retroviruses, exosomes, chylomicrons, DNA, RNA, proteins, fats, acids, carbohydrates, enzymes, viruses, bacteria, etc. In some embodiments, purified (and in some cases amplified) particles can be used for research, assembly, and / or manufacturing of gene therapy products and / or other products. It will also be understood that, although Figures 4A-4H The examples shown illustrate a subgroup or subset of charged particles generated from sample 24, defined by various values ​​or ranges of particle mass, charge, and / or mass-to-charge ratio. Figure 1 The instrument 10 shown and the processes 100, 200 for operating the instrument 10 are not limited thereto. In particular, it should be understood that the instrument 10 can be configured, and processes 100 and / or 200 can be modified to collect subgroups of charged particles that are alternatively or additionally defined by values ​​or ranges of particle mobility and / or other molecular properties. As a specific example, and not to be considered as limiting in any way, the ion processing region 16 can be configured to include instruments for measuring or otherwise determining particle mass, charge, and mobility, and various purified multidimensional subgroups of charged particles can be defined by values ​​and / or ranges of particle mass, mass-to-charge ratio, charge size or charge state, and mobility.

[0085] Example 8

[0086] Refer again Figure 5Method 500 was performed using milk samples, where the specified particle type was exosomes. In step 502, concentrated unprocessed (raw) milk (from approximately 20 animals) was provided. Subsequently, in step 504, the raw milk samples were enriched for exosomes as follows: Within approximately 200 minutes of collecting the milk samples, 50 mL aliquots of raw milk were defatted and then further centrifuged to reduce the amount of apoptotic bodies in the samples. For example, this was achieved by centrifuging the raw milk at 2000 × g for 10 minutes at 4°C to remove the concentrated milk fat layer. The remaining suspension was separated and centrifuged at 12,000 × g for 20 minutes at 4°C to remove cells and other debris. Acetic acid (1% by volume) was then added to the supernatant and mixed for 5 minutes to induce precipitation of non-EV (non-extracellular vesicle) proteins, particularly casein (with an isoelectric point of 4.6). The precipitate was separated by centrifugation at 10,000 × g for 10 minutes at 4°C, following standard procedures. The resulting supernatant, a mixture of proteins, lipids, and other substances (including EVs (named whey)), was ultracentrifuged at 210,000 × g for 70 minutes at 4 °C. The resulting precipitate was redissolved in 500 μL of 100 mM ammonium acetate and centrifuged at 10,000 × g for 5 minutes at 4 °C to remove residual precipitate. The resulting EV supernatant containing exosomes was then diluted 100-fold in 100 mM ammonium acetate solution to prepare Sample 24, which was used... Figure 1 The instrument 10 shown generates ions from the sample.

[0087] The instrument 10 is configured as follows, although it should be understood that the following configuration of the instrument 10 is merely as described. Figure 1This is one of several different possible configurations of the instrument 10 shown and described above. For this particular example, the ion generator 22 is an electrospray ionization (ESI) unit with a ~5 µm diameter borosilicate capillary emitter and generates ions from the enriched sample 24 using an emitter potential of ~1.4 kV. The electrosprayed ions are transported to the source region of the instrument 10 via a capillary interface, which is illustratively configured as a CDMS instrument as described above. In this particular example, the ion processing region 16 includes a mixing ion funnel-ion blanket interface, for example, illustrated and described in co-pending international application PCT / US2019 / 0132274, which is incorporated herein by reference, through which ions from the source capillary are transported. Following this interface, the ions are transported through a RF-only hexapole, where they undergo collisions, thermalizing the ion kinetic energy distribution. As ions exit the hexapole, they enter an RF-only quadpole, which acts as a low-pass filter tuned to transport large ions with a mass-to-charge ratio (m / z) higher than ~12,000. Eliminating low m / z species ensures optimized measurement time for high m / z ions. A 100 V DC offset voltage across the hexapole is used to set the nominal ion energy per charge. The ions are then focused into the inlet of a dual-hemispherical deflection energy analyzer, and the energy-selected ions exiting the energy analyzer are then introduced into an electrostatic linear ion trap (ELIT) containing a charge-detection cylinder.

[0088] When each ion enters the ELIT, it induces a charge on the charge detection cylinder. At the start of each capture event, both end caps of the ELIT are in transport mode, allowing ions to travel through the trap. The capture event is initiated by switching the rear end cap from transport mode to capture mode, thus reflecting the ions back through the charge detection cylinder and to the ELIT inlet. After a brief delay of 0.3 ms, the front end cap switches to capture mode, and the captured ions oscillate back and forth within the ELIT. After 100 ms, the capture event terminates, and both end caps of the ELIT switch back to transport mode. This process is repeated for each ion after a 1 ms delay. During the 100 ms measurement period, each ion oscillates through the charge detection cylinder of the ELIT, inducing a periodic signal, which is amplified and digitized by a charge-sensitive preamplifier (CA) and then analyzed using a fast Fourier transform. The mass-to-charge ratio of the ion is derived from the measured fundamental frequency, and the charge is derived from the amplitude of the fundamental frequency. Mass distribution is generated by multiplying the mass-to-charge ratio by the measured charge of each ion and grouping the resulting masses.

[0089] In step 506, the procedure is performed as described above. Figure 2In process 100, and in step 106 of process 100, a scatter plot of the charged particle charge magnitude (in terms of elementary charge e) versus the charged particle mass (in terms of megadaltons) is generated, as shown below. Figure 6A As shown. Subsequently, in step 108, the scatter plot is processed to determine a subset of the graph data that can be identified as exosomes. In one embodiment of process 500, all particles with a mass greater than 9.8 megadaltons (generally understood as the minimum mass of an exosome) are considered exosomes, and... Figure 6A The exosome quality threshold line EM is identified as a vertical, dashed line in the middle. TH All charged particles on the right. In some embodiments, process 200 may be performed illustratively as described above to collect and / or purify the exosomes identified in step 108.

[0090] In an alternative embodiment of process 500, the process is performed in step 108. Figure 6A The data in the scatter plot is used to determine one or more subgroups of charged particles that can be identified as exosomes, and / or to determine whether there are multiple subgroups of charged particles that are distinguishable from each other, for example, whether there are subgroups that can be resolved into families of particles from CDMS data. At this point, processor 26 is programmed in step 108 to perform a conventional two-dimensional Gaussian mixture model (GMM) to fit the data. Figure 6A The two-dimensional mass-to-charge data assumes that subgroups of particles fall into families of related masses and charges, and that these families are normally distributed. Under this assumption, processor 26 is programmed to perform conventional clustering analysis on the charged particle data, resulting in multiple distributions of two-dimensional mass-to-charge subgroups. When combined, the sum of these subgroups captures the main features of the two-dimensional CDMS data. For simplicity, the number of possible subgroups is limited to between 1 and 10 two-dimensional Gaussian distributions. Apart from this limitation, the analysis is unsupervised, and the algorithm determines the number of subgroups, as well as the location and width associated with each subgroup, to best fit the two-dimensional CDMS dataset when summed. Figure 6A The CDMS dataset shown in the analysis converges on the best-fit model, which consists of six independent subgroups S1-S6, as follows: Figure 6B As shown in the example. It should be understood that, because... Figure 6B Each of the six subgroups of charged particles shown is Gaussian distributed, so the boundaries of each of S1-S6 are only approximations, and include... Figure 6B The above is merely to illustrate the position and approximate size of subgroups S1-S6 relative to each other. In alternative embodiments, one or more other conventional statistical models can be used to analyze the particle mass and charge dataset generated by CDMS 10.

[0091] Figure 6BThe data show that the lowest-mass subgroup S1 observed in Sample 24 corresponds to a relatively narrow distribution, concentrated at mass (m) = 5.7 ± 1.6 MDa and charge (z) = 145 ± 38 e. This subgroup S1 comprises approximately 27% of the total number of charged particles in the dataset (975 out of 3586). The highest-mass subgroup S6 observed in Sample 24 corresponds to a wide distribution, concentrated at m = 27.7 ± 5.4 MDa and z = 594 ± 76 e. This subgroup S6 comprises approximately 22% of the total number of charged particles in the dataset (772 out of 3586). The S2 subgroup (or family) concentrated at m = 10.2 ± 1.9 MDa and z = 189 ± 44 e accounts for only 3% of the total number of charged particles in the dataset, making it the least abundant subgroup. The S3 (m=12.5±2.9 MDa, z=296±31 e) subgroup, comprising approximately 4% of the total number of charged particles, and the S4 (m=17.6±2.6 MDa, z=488±76 e) subgroup, comprising approximately 18% of the total number of charged particles, are more easily distinguished by charge than by mass. This suggests that these subgroups or families consist of particles of similar size that are substantially different at the molecular level, and therefore the charge of each particle has a greater influence than its mass. The S5 (m=23.4±3.4 MDa, z=550±113 e) subgroup (comprising approximately 26% of the total number of charged particles) appears to be more easily distinguished by mass than by charge, indicating that they are more similar in charge properties than in size.

[0092] Based on the Gaussian clustering analysis performed in step 108 of method 100 just described, the proportion of particles in the dataset that are exosomes can be estimated. Regarding the average mass of the various subgroups, only the S1 subgroup (m = 5.7 ± 1.6 MDa) is too small to be an exosome (based on the minimum exosome mass approximation of 9.8 MDa). Since S1 represents 27% of the total number of charged particles in the dataset, the remaining 73% of the charged particles in the dataset are within the expected mass range for exosomes. In some embodiments, process 200 may be performed illustratively as described above to collect and / or purify the exosomes identified in this embodiment in step 108.

[0093] Although this disclosure has been illustrated and described in detail in the foregoing figures and description, it should be considered illustrative in nature and not restrictive. It should be understood that only illustrative embodiments are shown and described, and all variations and modifications within the spirit and scope of this disclosure are intended to be protected. For example, in some embodiments in which the ion processing region 16 is implemented in the form of a CDMS, the charge detector of the CDMS can be illustratively controlled to selectively release charged particles or block particles from being released therefrom, for example, by selective control of the voltage source VS2. In embodiments of the CDMS where the charge detector is an electrostatic linear ion trap (ELIT), for example, the voltage applied by the voltage source VS2 to one or both of its end caps can be illustratively controlled to allow ions trapped and oscillating therein to leave the ELIT in the direction of the particle collection target 40, or to cause the ion oscillations within the ELIT to become unstable and contact structures therein, such that ions are not released from the ELIT. In such embodiments, this control of the ELIT can render the charged particle deflector or steering device 18 unnecessary, allowing it to be omitted. In embodiments where the charge detector is a CDMS with an orbital trap, the voltage source VS2 can be similarly controlled with the same effect.

Claims

1. A particle purification device, comprising: An ion generator, configured to produce charged particles from a sample. An ion processing region is configured to receive charged particles generated by the ion generator and to measure at least one of the mass and charge magnitude of the generated charged particles. Particle collection target, A device for selectively transferring charged particles leaving the ion processing region. An ion trap is disposed between the particle collection target and the device for selectively transferring charged particles leaving the ion processing region. processor, and A memory having instructions stored therein, the instructions being executable by the processor to (i) cause the processor to control means for selectively delivering charged particles to deliver each of the measured charged particles having at least one of the following: (a) a measured mass equal to a selected mass or within a selected particle mass range, (b) a measured charge equal to a selected charge size or within a selected charge size range, and (c) a mass-to-charge ratio equal to a selected mass-to-charge ratio or within a selected mass-to-charge ratio range, and (ii) The processor controls the ion trap to capture charged particles leaving the means for selectively transferring charged particles leaving the ion processing region, and controls the ion trap to release the captured charged particles and accelerate the charged particles toward the particle collection target.

2. The particle purification apparatus according to claim 1, wherein, The instructions stored in the memory also include instructions that can be executed by the processor to cause the processor to control means for selectively delivering charged particles, wherein the delivery of the measured charged particles is blocked when the instructions are not executed.

3. The particle purification apparatus according to claim 1 or 2, wherein, The ion processing area includes a charge detection mass spectrometer configured to receive charged particles generated by the ion generator and to measure the mass and charge of the generated charged particles.

4. The particle purification apparatus according to claim 3, wherein, The apparatus for selectively transferring charged particles leaving the ion processing region includes a charged particle deflection or redirection device, which can be controlled by the processor to selectively transfer charged particles through the charged particle deflection or redirection device.

5. The particle purification apparatus according to claim 3, wherein, The apparatus for selectively transferring charged particles leaving the ion processing region includes a charge detector of a charge detection mass spectrometer, the charge detector being controllable by the processor to selectively transfer charged particles through the charge detector.

6. The particle purification apparatus according to claim 1 or claim 2, wherein, The ion processing region includes one of a mass analyzer, a mass spectrometer, and a mass-to-charge ratio filter, wherein the mass analyzer, mass spectrometer, and mass-to-charge ratio filter are configured to pass charged particles with a selected mass-to-charge ratio or charged particles with a mass-to-charge ratio within a selected mass-to-charge ratio range through the mass analyzer, mass spectrometer, and mass-to-charge ratio filter, followed by a charge detector array configured to measure the charge magnitude of the charged particles leaving the mass analyzer, mass spectrometer, and mass-to-charge ratio filter.

7. The particle purification apparatus according to claim 6, wherein, The apparatus for selectively transferring charged particles leaving the ion processing region includes a charged particle deflection or redirection device, which can be controlled by the processor to selectively transfer charged particles through the charged particle deflection or redirection device.

8. A method for purifying particles, comprising: Generate charged particles from the sample. Measure at least one of the mass, charge magnitude, and mobility of the generated charged particles. Each charged particle of the measured charged particles having at least one of the following characteristics is selectively transferred to an ion trap for capture therein: (a) a measured mass equal to a selected mass or within a selected particle mass range, (b) a measured charge size equal to a selected charge size or within a selected charge size range, (c) a measured charge ratio equal to a selected mass-to-charge ratio or within a selected mass-to-charge ratio range, and (d) a measured mobility equal to a selected mobility or within a selected mobility range. The ion trap is controlled to release the charged particles captured therein and to accelerate the charged particles toward the particle collection target.

9. The method of claim 8, further comprising collecting the measured charged particles accelerated thereto on the collection surface of the particle collection target.

10. The method of claim 9, further comprising harvesting charged particles collected on the collection surface of the particle collection target.

11. The method of claim 10, further comprising amplifying the harvested charged particles.

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