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WO2025188838A1 - Cytométrie de flux magnétique à multiplicité améliorée - Google Patents

Cytométrie de flux magnétique à multiplicité améliorée

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Publication number
WO2025188838A1
WO2025188838A1 PCT/US2025/018481 US2025018481W WO2025188838A1 WO 2025188838 A1 WO2025188838 A1 WO 2025188838A1 US 2025018481 W US2025018481 W US 2025018481W WO 2025188838 A1 WO2025188838 A1 WO 2025188838A1
Authority
WO
WIPO (PCT)
Prior art keywords
magnetic
flow cytometry
label
particle
detector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/018481
Other languages
English (en)
Other versions
WO2025188838A8 (fr
Inventor
Kai Wu
Jian-Ping Wang
Vinit Kumar CHUGH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Minnesota Twin Cities
Texas Tech University TTU
Texas Tech University System
University of Minnesota System
Original Assignee
University of Minnesota Twin Cities
Texas Tech University TTU
Texas Tech University System
University of Minnesota System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Minnesota Twin Cities, Texas Tech University TTU, Texas Tech University System, University of Minnesota System filed Critical University of Minnesota Twin Cities
Publication of WO2025188838A1 publication Critical patent/WO2025188838A1/fr
Publication of WO2025188838A8 publication Critical patent/WO2025188838A8/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1023—Microstructural devices for non-optical measurement
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0038—Investigating nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1024—Counting particles by non-optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1028—Sorting particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/0017—Means for compensating offset magnetic fields or the magnetic flux to be measured; Means for generating calibration magnetic fields
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/12—Measuring magnetic properties of articles or specimens of solids or fluids
    • G01R33/1269—Measuring magnetic properties of articles or specimens of solids or fluids of molecules labeled with magnetic beads

Definitions

  • Embodiments are further related to cytometry. Embodiments are also related to flow cytometry. Embodiments are further related to magnetic flow cytometry with enhanced multiplicity. BACKGROUND [0003] Cytometry refers generally to measurements relating to cells. Cytometry is used in numerous applications. For example, it can be used for cell counting, cell sorting, determining cell characteristics and function, detecting microorganisms, protein molecules and biomarker detection, as well as diagnosing diseases, such as circulating tumor cells (CTCs). [0004] One common method of cytometry is known as flow cytometry. Flow cytometry makes use of cells or biological molecules which are labeled by fluorescent reporters (fluorophores).
  • the reporters emit light at varying wavelengths when excited, usually by a laser.
  • the fluorescent light is filtered and channeled to various photodetectors.
  • Optical flow cytometry has been a critically important improvement in cell analysis.
  • interference is a common problem with optical flow cytometry, because optical background noise is often generated by biological samples.
  • extensive sample preparation is required before Attorney Docket No. TTU-1079PCT PATENT APPLICATION analysis.
  • fluorescent labels suffer from poor stability and short lifetime. For example, photobleaching (loss of fluorescence upon continuous light excitation) limits the stable lifetime for detection using optical flow cytometry.
  • optical flow cytometry has limited multiplicity.
  • a flow cytometry system comprises a fluidic transport system comprising a magnetic separator stage and a detector region, a magnetic detector configured about the detector region of the fluidic transport system, an electronic conditioning system configured to condition an input analog signal from the magnetic detector and output a digital signal, and a computer system further comprising: at least one processor, and a computer-usable medium embodying computer program code, the computer-usable medium capable of communicating with the at least one processor, the computer program code comprising instructions executable by the at least one processor and configured for: identifying higher harmonics in the digital signal, and identifying at least one biological molecule according to the identified higher harmonics.
  • the flow cytometry system further comprises an incubation well for mixing at least one magnetic label to at least one of the biological molecules.
  • the at least one magnetic label comprises at least one antibody and at least one superparamagnetic nanoparticle.
  • the magnetic separator stage further comprises: a magnet configured to apply a magnetophoretic force and a separator channel, wherein the magnetophoretic force drives unbound magnetic labels into the separator channel.
  • the magnetic detector further comprises: at least one excitation coil configured to generate a magnetic field and at least one pick-up coil configured to convert a magnetic response into a voltage.
  • the magnetic field generated by the at least one excitation coil comprises one of a sinusoid wave, a sinusoidal wave with constant shift, two sinusoidal waves, or three or more sinusoidal waves.
  • the magnetic detector further comprises at least one compensation coil configured to cancel a feed through signal via inductive dissipation.
  • identifying the at least one biological molecule according to the identified higher harmonics further comprises correlating the identified higher harmonics with the magnetic label bound to the biological molecule.
  • the electronic conditioning system further comprises a differential amplifier for amplifying an incoming signal, a first signal filter, a lock-in stage to lock the signal to a harmonic component, a second signal filter, and an analog to digital converter.
  • a magnetic label comprises at least one magnetic particle and at least one antibody bound to a surface of the magnetic particle.
  • the at least one antibody is bound to the surface of the magnetic particle using a covalent bond between the antibody’s functional group and the particle surface.
  • the at least one antibody is bound to the surface of the magnetic particle using an affinity reaction between the antibody’s Fc region and protein molecules on the particle’s surface.
  • the at least one antibody is bound to the surface of the magnetic particle according to a biotin-streptavidin affinity reaction.
  • the at least one magnetic particle comprises a plurality of superparamagnetic nanoparticles.
  • each of the plurality of superparamagnetic nanoparticles has a unique harmonic spectra when subjected to a magnetic driving field.
  • the at least one magnetic particle comprises a plurality of superparamagnetic nanoparticles and a polymer shell.
  • a flow cytometry method comprises binding at least one magnetic label to at least one biological molecule in a sample, removing unbound magnetic Attorney Docket No.
  • TTU-1079PCT PATENT APPLICATION labels in a magnetic separator stage of a fluidic transport system, generating an excitation magnetic field with an excitation coil, collecting a voltage indicative of a magnetic response from the magnetic label with a magnetic detector, conditioning the voltage from the magnetic detector and outputting a digital signal with an electronic conditioning system, identifying higher harmonics in the digital signal, and identifying the at least one biological molecule according to the identified higher harmonics.
  • the magnetic label comprises at least one antibody and at least one superparamagnetic nanoparticle.
  • removing unbound magnetic labels in a magnetic separator stage of the fluidic transport system further comprises generating a magnetophoretic force with a magnet and driving the unbound magnetic labels into a separator channel with the magnetophoretic force.
  • identifying at least one biological molecule according to the identified higher harmonics further comprises correlating the identified high harmonics with the magnetic label bound to the biological molecule.
  • FIG.1A depicts a block diagram of a flow cytometry system, in accordance with the disclosed embodiments; [0019] FIG.
  • FIG. 1B depicts aspects of a method for flow cytometry, in accordance with the disclosed embodiments;
  • FIG.2 depicts an incubation well for binding a magnetic label to a biological particle, in accordance with the disclosed embodiments;
  • FIG.3 depicts a magnetic separator stage associated with a flow cytometry system, in accordance with the disclosed embodiments;
  • FIG. 4A depicts a detector region of a fluidic transport system and associated magnetic detector, in accordance with the disclosed embodiments; [0023] FIG.4B depicts a chart of harmonic responses in a single AC field, in accordance with the disclosed embodiments; [0024] FIG.4C depicts a chart of harmonic responses in a one DC field and one AC field, in accordance with the disclosed embodiments; [0025] FIG.4D depicts a chart of harmonic responses in two AC fields, in accordance with the disclosed embodiments; [0026] FIG. 5A depicts a configuration of a detector, in accordance with the disclosed embodiments; [0027] FIG. 5B depicts another configuration of a detector, in accordance with the Attorney Docket No.
  • FIG. 5C depicts another configuration of a detector, in accordance with the disclosed embodiments; [0029] FIG. 5D depicts another configuration of a detector, in accordance with the disclosed embodiments; [0030] FIG.6A illustrates an exemplary magnetic label, in accordance with the disclosed embodiments; [0031] FIG. 6B depicts a chart of harmonic responses in a time domain and frequency domain, in accordance with the disclosed embodiments; [0032] FIG. 6C depicts exemplary harmonic responses in a frequency domain, in accordance with the disclosed embodiments; [0033] FIG.
  • FIG. 7 depicts steps in a method for cell counting/sorting, in accordance with the disclosed embodiments;
  • FIG. 8 illustrates another exemplary magnetic label, in accordance with the disclosed embodiments;
  • FIG. 9 illustrates exemplary magnetic labels bound to biological particles, in accordance with the disclosed embodiments;
  • FIG. 10A illustrates an electronic conditioning system, in accordance with the disclosed embodiments; Attorney Docket No. TTU-1079PCT PATENT APPLICATION [0037]
  • FIG.10B illustrates another electronic conditioning system, in accordance with the disclosed embodiments; [0038] FIG.
  • FIG. 11 depicts a block diagram of a computer system which is implemented in accordance with the disclosed embodiments; [0039] FIG.12 depicts a graphical representation of a network of data-processing devices in which aspects of the present embodiments may be implemented; and [0040] FIG.13 depicts a computer software system for directing the operation of the data- processing system depicted in FIG.11, in accordance with an example embodiment.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • BB BB
  • AAA AAA
  • AB BBC
  • AAABCCCCCC CBBAAA
  • CABABB CABABB
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Embodiments disclosed herein are generally related to methods and systems for counting or sorting biological samples (such as cells) using magnetic labeling and detection in a process called “flow cytometry” or “magnetic flow cytometry”.
  • Cells in a sample can first be bound to a magnetic particle.
  • Various magnetic particles can be used for this purpose, but in an exemplary embodiment, supraparticle magnetic labels can be bound to surface proteins on the cells.
  • any unbound labels can be separated by applying a magnetophoretic force in a magnetic separation channel. The cells with magnetic labels can then be passed through a detector for multiplexed magnetic detection.
  • FIG. 1A illustrates a high-level block diagram of a system 100 for magnetic flow cytometry.
  • An incubation well 102 is used to mix sample cells 128 with magnetic labels 112 forming a mixture 104, aspects of which are further detailed herein.
  • the mixture 104 is provided to a fluidic transport system 106.
  • the incubation well 102 allows the magnetic labels 112 to bind with sample cells 128, as further detailed herein, forming magnetic labeled cells 118.
  • the fluidic transport system 106 can comprise a tube or channel, configured in some cases, to flow the sample mixture 104 at pressure.
  • the fluidic transport system 106 can include a magnetic separator stage 108.
  • a magnet 110 e.g., an electromagnet, or other such magnet
  • the unbound magnetic labels 112 can be drawn to a separator channel 114 and discarded or reused, as illustrated by arrow 116.
  • the remaining magnetic labeled cells 118 in the sample proceed through the fluidic transport system 106 to a detector region 120, aspects of which are provided herein.
  • an excitation coil is used to generate a magnetic field
  • a pickup coil is used to collect a signal from the magnetic labeled cells 118 resulting from the magnetic field.
  • the analog signal (embodied as a voltage) is provided to an electronic conditioning system 122 for conditioning the analog signal, and then converting the analog signal to a digital signal.
  • the digital signal is then provided to a computing device 124 for read out. After the cells have cleared the detector region 120, they are output as waste, as illustrated by arrow 126.
  • FIG.1B illustrates a high-level flow chart of steps associated with a method 150 for magnetic flow cytometry in accordance with the disclosed embodiments. It should be appreciated that the steps in method 150 can be accomplished using, for example, the magnetic flow cytometry system 100, and associated components.
  • Attorney Docket No. TTU-1079PCT PATENT APPLICATION [0059]
  • the constituents in a biological sample are labeled in an incubation well.
  • the sample can comprise a plurality of cells. The unique surface proteins from each cell in the sample can be identified. Then the antibodies that specifically bind to each of those types of proteins are selected, for use as a part of the magnetic labels.
  • FIG.2A illustrates, an exemplary sample 205 comprising three cell types 210, 212, and 214 distributed in a reaction or incubation well 230, along with 3 supraparticle magnetic labels 215, 217, and 219. Each type of label is surface functionalized with one specific type of antibody 220, 222, and 224 respectively.
  • Surface functionalization can be achieved in various ways. One option is to make use of covalent chemistry. In this case, the antibodies’ native functional groups bind to the particles’ surface through bifunctional linkers (e.g., glutaraldehyde, EDC/NHS, sulfo-EMCS, sulfo-SPDP).
  • bifunctional linkers e.g., glutaraldehyde, EDC/NHS, sulfo-EMCS, sulfo-SPDP.
  • FIG.3 illustrates a diagram 300 showing the mechanism for unbound antibody removal, in accordance with the disclosed embodiments.
  • a magnetophoretic force (herein referred to as “Fmag” 305) can be exerted on each magnetic label flowing through a fluidic channel 310, by an external gradient magnetic field (or B-field) 315.
  • a drag force (herein referred to as “Fdrag” 320) is exerted on each magnetic label or magnetically labeled cell.
  • the drag force 320 is proportional to the cross-sectional area of the Attorney Docket No. TTU-1079PCT PATENT APPLICATION associated object.
  • a gravitational force (herein referred to as “Fg” 330) may or may not be relevant depending on the orientation of the fluidic channel 310.
  • the unbound magnetic labels 340 can be drawn away from the bound labels 335.
  • the magnetophoretic force 305 will act equally on both the bound labels/cells 335 and unbound labels 340.
  • the additional drag force 320 and gravitational force 330 exerted on the bound labels/cells 335, which are bound to the associated cells will cause them to separate from the unbound magnetic labels 340, which experience a smaller drag force 320.
  • the separated unbound magnetic labels 340 can then be removed from the fluidic channel 310.
  • the gradient B field 315, flow velocity ⁇ 345, and fluidic channel 310 width can be selected in order to optimize the separation result.
  • Step 165 can be achieved using an apparatus including an excitation coil and pickup coil.
  • FIG. 4A illustrates aspects of a detector system 400 in accordance with the disclosed embodiments.
  • the excitation coil 405 can generally be configured external to a detection portion 410 of the fluidic channel.
  • the excitation coil 405 is used to generate a magnetic field to periodically saturate the magnetic labels 415, which causes a nonlinear magnetic response from the labels as further detailed herein.
  • the waveform of the drive field generated by the excitation coil 405 can be a sinusoidal wave (e.g., one AC field), as given by equation (1).
  • FIG.4B illustrates a chart 490 of exemplary harmonics associated with one AC field.
  • the waveform of the drive field generated by the excitation coil can be a sinusoidal wave with a constant shift (e.g., one DC field + one AC field), as given by equation (2).
  • FIG. 4C illustrates a chart 492 of exemplary harmonics associated with one DC field and one AC field.
  • ⁇ ⁇ + ⁇ ⁇ 2 ⁇ (2)
  • the waveform of the drive field generated by the excitation coil can be two sinusoidal waves (e.g., two AC fields), as given by equation (3).
  • FIG.4D illustrates a chart 494 of exemplary harmonics associated with two AC fields.
  • the waveform of the drive field generated by the excitation coil can be three or more sinusoidal waves with or without a constant shift field, as given by equation (4).
  • ⁇ ⁇ + ⁇ ⁇ ⁇ 2 ⁇ (4)
  • the pickup coil 420 coverts the dynamic magnetic response of the magnetic labels 415 into a signal (e.g., a voltage). The voltage can then be analyzed to determine which magnetic label caused the voltage and in turn which cell passed through the detector region.
  • FIGs. 5A-5C illustrate various configurations of the pickup coil and excitation coil, which can be used to cancel voltage response.
  • the pickup and/or excitation coil can comprise planar coils arranged proximate to the channel 410 in the detection region 420.
  • the pickup coil and/or excitation coil can Attorney Docket No. TTU-1079PCT PATENT APPLICATION comprise coils wound around the channel 410 in the detection region 120.
  • both planar coils and coils wound around the channel 410 in the detection region can be used.
  • one or more turns 528 of the planar pickup coil 502 can be configured proximate to the channel 410, with separate turns 504 not proximate to the channel 410.
  • the planar excitation coil 530 can surround both the planar pickup coil 502 and the channel 410. This arrangement is configured to cancel the feed through signal with the associated gradiometer geometry.
  • FIG.5B illustrates another embodiment, where turns 510 of the planar pickup coil 502 are configured proximate to the channel 410.
  • the excitation coil 532 is wound around the channel 410. This arrangement is configured to cancel the feed through signal via spatial arrangement of the respective coils.
  • FIG.5C illustrates another embodiment, which includes a primary planar excitation coil 520 and secondary planar excitation coil 522.
  • the primary planar excitation coil 520 surrounds the planar pickup coil 502 with turns 526 arranged proximate to the channel 410.
  • a planar compensation coil 524 is surrounded by the planar secondary excitation coil 522. This arrangement is configured to cancel the feed through signal via inductive dissipation with the planar compensation coil 524.
  • FIG.5D illustrates a cross sectional view of another embodiment which includes a primary excitation coil 534 wound around the primary pickup coil 536.
  • the primary pickup coil 536 is wound around the channel 410.
  • the primary excitation coil 534 surrounds the primary pickup coil 536.
  • a compensation coil 538 is surrounded by a planar secondary coil 540. This arrangement is configured to cancel the feed through signal via inductive dissipation with the compensation coil 538.
  • Cell counting and sorting can be completed at step 170, with an electronic (e.g., Attorney Docket No. TTU-1079PCT PATENT APPLICATION computer) system as further detailed herein.
  • an electronic e.g., Attorney Docket No. TTU-1079PCT PATENT APPLICATION computer
  • Each supraparticle label can be a micron-sized bead that comprises plural types of superparamagnetic nanoparticles.
  • FIG.6A illustrates an exemplary supraparticle label 600, comprising three types of superparamagnetic nanoparticles: Particle A 602, Particle B 604, and Particle C 606. It should be appreciated that this is exemplary, and in other embodiments, other numbers, and types of superparamagnetic nanoparticles can be a part of the supraparticle label 600.
  • the magnetization responses (MH curves) of the superparamagnetic nanoparticles are linear in a small field and nonlinear in larger field.
  • FIG. 6B illustrates this principle. As illustrated in FIG.
  • a sinusoidal driving field illustrated in chart 650 will generate a magnetic response as illustrated in chart 652.
  • chart 654 illustrates the driving field in the frequency domain.
  • the voltage response will result in characteristic high harmonics in the frequency domain.
  • the nonlinear responses of superparamagnetic nanoparticles will cause higher harmonics that are unique for each type of nanoparticle.
  • the nonlinear magnetic response of superparamagnetic nanoparticles causes higher odd harmonics at 3 ⁇ , 5 ⁇ , 7 ⁇ , etc. as illustrated in chart 656.
  • FIG.6C illustrates this principle for exemplary supraparticle 600.
  • the harmonics 675 for Particle A 602 are unique to Particle A 602.
  • the harmonics 677 for Particle B 604 are unique to Particle B 604.
  • the harmonics 679 for Particle C 606 are unique to Particle C 606.
  • Each type of superparamagnetic nanoparticle 600 has its unique harmonic spectra when subjected to magnetic driving fields.
  • each type of nanoparticle has its unique harmonic spectra
  • a plural number of supraparticle labels can be included, creating the desired multiplicity.
  • the available n multiplicity in the embodiments Attorney Docket No. TTU-1079PCT PATENT APPLICATION provided here is dependent on 1) the minimum weight of A, B, C, ..., nanoparticles the detector can recognize; 2) the difference of harmonic spectra between each type of nanoparticles used (the larger differences, the easier to distinguish); and/or 3) the size of the desired supraparticle.
  • Each type of supraparticle 600 has its own unique harmonic spectra. This is due to different compositions of, for example, A, B, and C nanoparticles, in the supraparticle 600.
  • a type X supraparticle composed of ⁇ ⁇ parts of Particle A, ⁇ ⁇ parts of Particle B, ⁇ ⁇ parts of Particle C, measured under one AC drive field, higher harmonics are found at 3f, 5f, 7f, 9f, .... etc.
  • the higher harmonics can be identified as a weighted sum of higher harmonics from A, B, and C based on the composition (e.g., A3 is the harmonic of A at 3f, etc.).
  • Equation (5) This is illustrated by equation (5) as: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ (5) and sorting 170, in accordance with the disclosed embodiments.
  • the harmonic spectra of a supraparticle can be collected and recorded. This step can be achieved with a detector as illustrated in FIGs.4 and 5.
  • the composition of nanoparticle e.g., Particle A 602, Particle B 604, and Particle C 606
  • the type of supraparticle and associated antibody that was originally functionalized on it can be confirmed. This allows for the identification of the cell, which can be counted or sorted.
  • FIG. 8 illustrates an exemplary multi-core magnetic bead 800 in accordance with aspects of the disclosed embodiments.
  • Multi-core magnetic beads 800 are composed of smaller superparamagnetic nanoparticles, for example, superparamagnetic nanoparticle A 802, superparamagnetic nanoparticle B 804, and superparamagnetic nanoparticle C 806, embedded in a biocompatible polymer matrix 808.
  • the superparamagnetic nanoparticles illustrated are exemplary and different numbers of superparamagnetic nanoparticle can be included in other embodiments.
  • different magnetic beads can be configured, each of which will exhibit unique harmonics, for labeling purposes.
  • the multi-core magnetic beads are not necessarily spherical. They can be cube-shaped, rod-shaped, ellipsoid-shaped, etc.
  • Superparamagnetic nanoparticles e.g., nanoparticle A 950, nanoparticle B 955, nanoparticle C 960, nanoparticle D 965, nanoparticle E 970, etc. illustrated in FIG. 9
  • Each superparamagnetic nanoparticle has unique higher harmonics.
  • Each cell in a sample generally has more than one surface protein (say, proteins PA, PB, PC, PD, PE, etc.).
  • FIG. 9 Cell 1905 is labeled by nanoparticle A 950 and nanoparticle B 955.
  • Cell 2910 is labeled by nanoparticle B 955, nanoparticle C 960, and nanoparticle D 965.
  • Cell 3915 is labeled by nanoparticle C 960 and nanoparticle E 970.
  • Each of nanoparticle A 950, nanoparticle B 955, nanoparticle C 960, nanoparticle D 965, and nanoparticle E 970 has its own unique harmonics.
  • An algorithm (as detailed previously) can be used to separate the harmonics.
  • an electronic system can be used for signal conditioning, amplification, and/or conversion from an analog to digital signal.
  • FIG.10A illustrates aspects of the electronic conditioning system 1000 associated with the magnetic flow cytometry systems and methods disclosed herein.
  • the signal from the pickup coils 420 can be provided to a single frequency electronic conditioning system 1000.
  • the input signal is amplified with a differential amplifier 1010, and filtered with a filter implementation 1015.
  • a lock-in stage 1020 is provided to multiplex and shift the signal to a lower frequency regime so that the F frequency is shifted to zero.
  • the signal is then filtered again with the second filter implementation 1025.
  • FIG.10B illustrates aspects of the detection system 1050.
  • the signal from the pickup coils 420 can be provided to a dual-frequency electronic system 1055.
  • the input signal is amplified with a differential amplifier 1060, and filtered with a filter implementation 1065.
  • a lock-in stage 1070 is provided to multiplex and shift the signal to a lower frequency regime so that the F2 frequency (the secondary harmonic component) is shifted to zero.
  • the signal is then filtered again with the second filter implementation 1075.
  • the analog signal is then converted to a digital signal with ADC 1080, and provided to a processing unit 1100, as further detailed herein.
  • FIGs.11-13 are provided as exemplary diagrams of data-processing environments in which embodiments may be implemented. It should be appreciated that FIGs.11-13 are only exemplary and are not intended to assert or imply any limitation with regard to the environments in which aspects or embodiments of the disclosed embodiments may be implemented. Many modifications to the depicted environments may be made without departing from the spirit and scope of the disclosed embodiments.
  • FIG.11 A block diagram of a computer system 1100 that executes programming for implementing parts of the methods and systems disclosed herein is provided in FIG.11.
  • TTU-1079PCT PATENT APPLICATION peripheral devices may include one or more processing units 1102, memory 1104, removable storage 1112, and non-removable storage 1114.
  • Memory 1104 may include volatile memory 1106 and non-volatile memory 1108.
  • Computer 1110 may include or have access to a computing environment that includes a variety of transitory and non-transitory computer-readable media such as volatile memory 1106 and non-volatile memory 1108, removable storage 1112 and non-removable storage 1114.
  • Computer storage includes, for example, random access memory (RAM), read only memory (ROM), erasable programmable read-only memory (EPROM) and electrically erasable programmable read-only memory (EEPROM), flash memory or other memory technologies, compact disc read-only memory (CD ROM), Digital Versatile Disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage, or other magnetic storage devices, or any other medium capable of storing computer-readable instructions, as well as data including image data.
  • Computer 1110 may include, or have access to, a computing environment that includes input 1116, output 1118, and a communication connection 1120.
  • the computer may operate in a networked environment using a communication connection 1120 to connect to one or more remote computers, remote sensors and/or controllers, detection devices, hand- held devices, multi-function devices (MFDs), speakers, mobile devices, tablet devices, mobile phones, Smartphone, or other such devices.
  • the remote computer may also include a personal computer (PC), server, router, network PC, RFID enabled device, a peer device or other common network node, or the like.
  • the communication connection may include a Local Area Network (LAN), a Wide Area Network (WAN), Bluetooth connection, or other networks. This functionality is described more fully in the description associated with FIG.12 below.
  • Output 1118 is most commonly provided as a computer monitor, but may include any output device.
  • Output 1118 and/or input 1116 may include a data collection apparatus associated with computer system 1100.
  • input 1116 which commonly includes a computer keyboard and/or pointing device such as a computer mouse, computer track pad, or the like, allows a user to input instructions to computer system 1100.
  • a user interface can be provided using output 1118 and input 1116.
  • Output 1118 may function as a display for Attorney Docket No. TTU-1079PCT PATENT APPLICATION displaying data and information for a user, and for interactively displaying a graphical user interface (GUI) 1130.
  • GUI graphical user interface
  • GUI generally refers to a type of environment that represents programs, files, options, and so forth by means of graphically displayed icons, menus, and dialog boxes on a computer monitor screen.
  • a user can interact with the GUI to select and activate such options by directly touching the screen and/or pointing and clicking with a user input device 1116 such as, for example, a pointing device such as a mouse, and/or with a keyboard.
  • a user input device 1116 such as, for example, a pointing device such as a mouse, and/or with a keyboard.
  • a particular item can function in the same manner to the user in all applications because the GUI provides standard software routines (e.g., module 1125) to handle these elements and report the user’s actions.
  • the GUI can further be used to display the electronic service image frames as discussed below.
  • Computer-readable instructions for example, program module or node 1125, which can be representative of other modules or nodes described herein, are stored on a computer- readable medium and are executable by the processing unit 1102 of computer 1110.
  • Program module or node 1125 may include a computer application.
  • a hard drive, CD-ROM, RAM, Flash Memory, and a USB drive are just some examples of articles including a computer-readable medium.
  • FIG.12 depicts a graphical representation of a network of data-processing systems 1200 in which aspects of the present invention may be implemented.
  • Network data- processing system 1200 can be a network of computers or other such devices, such as mobile phones, smart phones, sensors, controllers, actuators, speakers, “internet of things” devices, and the like, in which embodiments of the present invention may be implemented.
  • the system 1200 can be implemented in the context of a software module such as program module 1125.
  • the system 1200 includes a network 1202 in communication with one or more clients 1210, 1212, and 1214.
  • Network 1202 may also be in communication with one or more devices 1204, servers 1206, and storage 1208.
  • Network 1202 is a medium that can be used to provide communications links between various devices and computers connected together within a networked data processing system such as computer system 1100.
  • Network 1202 may include connections such as wired communication links, wireless Attorney Docket No.
  • Network 1202 can communicate with one or more servers 1206, one or more external devices such as device 1204, and a memory storage unit such as, for example, memory or database 1208.
  • device 1204 may be embodied as a detector device, magnetic detector, electronic conditioning system, controller, receiver, transmitter, transceiver, transducer, driver, signal generator, testing apparatus, or other such device.
  • device 1204, server 1206, and clients 1210, 1212, and 1214 connect to network 1202 along with storage unit 1208.
  • Clients 1210, 1212, and 1214 may be, for example, personal computers or network computers, handheld devices, mobile devices, tablet devices, smart phones, personal digital assistants, controllers, recording devices, speakers, MFDs, etc.
  • Computer system 1100 depicted in FIG. 11 can be, for example, a client such as client 1210 and/or 1212 and/or 1214.
  • Computer system 1100 can also be implemented as a server such as server 1206, depending upon design considerations.
  • server 1206 provides data such as boot files, operating system images, applications, and application updates to clients 1210, 1212, and/or 1214.
  • Clients 1210, 1212, and 1214 and device 1204 are clients to server 1206 in this example.
  • Network data-processing system 1200 may include additional servers, clients, and other devices not shown. Specifically, clients may connect to any member of a network of servers, which provide equivalent content.
  • network data-processing system 1200 is the Internet, with network 1202 representing a worldwide collection of networks and gateways that use the Transmission Control Protocol/Internet Protocol (TCP/IP) suite of protocols to communicate with one another.
  • TCP/IP Transmission Control Protocol/Internet Protocol
  • At the heart of the Internet is a backbone of high-speed data communication lines between major nodes or host computers consisting of thousands of commercial, government, educational, and other computer systems that route data and messages.
  • network data-processing system 1200 may also be implemented as a number of different types of networks such as, for example, an intranet, a local area network (LAN), or a wide area network (WAN).
  • FIGs.11 and 12 are intended as examples and not as architectural limitations for different embodiments of the present invention.
  • Attorney Docket No. TTU-1079PCT PATENT APPLICATION [00107]
  • FIG. 13 illustrates a software system 1300, which may be employed for directing the operation of the data-processing systems such as computer system 1100 depicted in FIG.11.
  • Software application 1305, may be stored in memory 1104, on removable storage 1112, or on non-removable storage 1114 shown in FIG.11, and generally includes and/or is associated with a kernel or operating system 1310 and a shell or interface 1315.
  • One or more application programs, such as module(s) or node(s) 1125, may be "loaded” (i.e., transferred from removable storage 1112 into the memory 1104) for execution by the data-processing system 1100.
  • the data-processing system 1100 can receive user commands and data through user interface 1315, which can include input 1116 and output 1118, accessible by a user 1320.
  • program modules can include, but are not limited to, routines, subroutines, software applications, programs, objects, components, data structures, etc., that perform particular tasks or implement particular abstract data types and instructions.
  • module or “node” as utilized herein may refer to a collection of routines and data structures that perform a particular task or implements a particular abstract data type.
  • Modules may be composed of two parts: an interface, which lists the constants, data types, variables, and routines that can be accessed by other modules or routines; and an implementation, which is typically private (accessible only to that module), and which includes source code that actually implements the routines in the module.
  • the term module may also simply refer to an application such as a computer program designed to assist in the Attorney Docket No. TTU-1079PCT PATENT APPLICATION performance of a specific task such as word processing, accounting, inventory management, etc., or a hardware component designed to equivalently assist in the performance of a task.
  • the interface 1315 (e.g., a graphical user interface 1130) can serve to display results, whereupon a user 1320 may supply additional inputs or terminate a particular session.
  • operating system 1310 and GUI 1130 can be implemented in the context of a “windows” system. It can be appreciated, of course, that other types of systems are possible. For example, rather than a traditional “windows” system, other operation systems such as, for example, a real-time operating system (RTOS) more commonly employed in wireless systems may also be employed with respect to operating system 1310 and interface 1315.
  • the software application 1305 can include, for example, module(s) 1125, which can include instructions for carrying out steps or logical operations such as those shown and described herein.
  • a flow cytometry system comprises a fluidic transport system comprising a magnetic separator stage and a detector region, a magnetic detector configured about the detector region of the fluidic transport system, an electronic conditioning system configured to condition an input analog signal from the magnetic detector and output a digital signal, and a computer system Attorney Docket No.
  • TTU-1079PCT PATENT APPLICATION further comprising: at least one processor, and a computer-usable medium embodying computer program code, the computer-usable medium capable of communicating with the at least one processor, the computer program code comprising instructions executable by the at least one processor and configured for: identifying higher harmonics in the digital signal, and identifying at least one biological molecule according to the identified higher harmonics.
  • the flow cytometry system further comprises an incubation well for mixing at least one magnetic label to at least one of the biological molecules.
  • the at least one magnetic label comprises at least one antibody and at least one superparamagnetic nanoparticle.
  • the magnetic separator stage further comprises: a magnet configured to apply a magnetophoretic force and a separator channel, wherein the magnetophoretic force drives unbound magnetic labels into the separator channel.
  • the magnetic detector further comprises: at least one excitation coil configured to generate a magnetic field and at least one pick-up coil configured to convert a magnetic response into a voltage.
  • the magnetic field generated by the at least one excitation coil comprises one of a sinusoid wave, a sinusoidal wave with constant shift, two sinusoidal waves, or three or more sinusoidal waves.
  • the magnetic detector further comprises at least one compensation coil configured to cancel a feed through signal via inductive dissipation.
  • identifying the at least one biological molecule according to the identified higher harmonics further comprises correlating the identified higher harmonics with the magnetic label bound to the biological molecule.
  • the electronic conditioning system further comprises a differential amplifier for amplifying an incoming signal, a first signal filter, a lock-in stage to lock the signal to a harmonic component, a second signal filter, and an analog to digital converter.
  • a magnetic label comprises at least one magnetic particle and at least one antibody bound to a surface of the magnetic particle.
  • the at least one antibody is bound to the surface of the magnetic particle using a covalent bond between the antibody’s functional group and the particle surface.
  • the at least one antibody is bound to the surface of the magnetic particle using an affinity reaction between the antibody’s Fc region and protein molecules on the particle’s surface. In an embodiment, the at least one antibody is bound to the surface of the magnetic particle Attorney Docket No. TTU-1079PCT PATENT APPLICATION according to a biotin-streptavidin affinity reaction.
  • the at least one magnetic particle comprises a plurality of superparamagnetic nanoparticles. In an embodiment, each of the plurality of superparamagnetic nanoparticles has a unique harmonic spectra when subjected to a magnetic driving field. In an embodiment, the at least one magnetic particle comprises a plurality of superparamagnetic nanoparticles and a polymer shell.
  • a flow cytometry method comprises binding at least one magnetic label to at least one biological molecule in a sample, removing unbound magnetic labels in a magnetic separator stage of a fluidic transport system, generating an excitation magnetic field with an excitation coil, collecting a voltage indicative of a magnetic response from the magnetic label with a magnetic detector, conditioning the voltage from the magnetic detector and outputting a digital signal with an electronic conditioning system, identifying higher harmonics in the digital signal, and identifying the at least one biological molecule according to the identified higher harmonics.
  • the magnetic label comprises at least one antibody and at least one superparamagnetic nanoparticle.
  • removing unbound magnetic labels in a magnetic separator stage of the fluidic transport system further comprises generating a magnetophoretic force with a magnet and driving the unbound magnetic labels into a separator channel with the magnetophoretic force.
  • identifying at least one biological molecule according to the identified higher harmonics further comprises correlating the identified higher harmonics with the magnetic label bound to the biological molecule.

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Abstract

L'invention concerne un système et un procédé de cytométrie de flux comprenant un système de transport fluidique avec un étage de séparation magnétique et une région de détection avec un détecteur magnétique conçu à proximité de la région de détection du système de transport fluidique ; un système de conditionnement électronique conçu pour conditionner un signal analogique d'entrée provenant du détecteur magnétique et fournir un signal numérique ; et un système informatique avec un logiciel associé pour identifier des harmoniques supérieures dans le signal numérique, et identifier au moins une molécule biologique en fonction des harmoniques supérieures identifiées.
PCT/US2025/018481 2024-03-07 2025-03-05 Cytométrie de flux magnétique à multiplicité améliorée Pending WO2025188838A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9778225B2 (en) * 2010-11-15 2017-10-03 Regents Of The University Of Minnesota Magnetic search coil for measuring real-time brownian relaxation of magnetic nanoparticles
US20180095067A1 (en) * 2015-04-03 2018-04-05 Abbott Laboratories Devices and methods for sample analysis
US20220120833A1 (en) * 2020-10-19 2022-04-21 Esaote S.P.A. Method for correcting inhomogeneity of the static magnetic field particularly of the static magnetic field generated by the magnetic structure of a machine for acquiring nuclear magnetic resonance images and mri system for carrying out such method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9778225B2 (en) * 2010-11-15 2017-10-03 Regents Of The University Of Minnesota Magnetic search coil for measuring real-time brownian relaxation of magnetic nanoparticles
US20180095067A1 (en) * 2015-04-03 2018-04-05 Abbott Laboratories Devices and methods for sample analysis
US20220120833A1 (en) * 2020-10-19 2022-04-21 Esaote S.P.A. Method for correcting inhomogeneity of the static magnetic field particularly of the static magnetic field generated by the magnetic structure of a machine for acquiring nuclear magnetic resonance images and mri system for carrying out such method

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