Magnetic flow cytometry with enhanced multiplicity

Abstract

A flow cytometry system and method including a fluidic transport system with a magnetic separator stage and a detector region with a magnetic detector configured proximate to 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 with associated software for identifying higher harmonics in the digital signal, and identifying at least one biological molecule according to the identified higher harmonics.

Description

Attorney Docket No. TTU-1079PCT PATENT APPLICATION MAGNETIC FLOW CYTOMETRY WITH ENHANCED MULTIPLICITY CROSS REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the priority and benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No. 63 / 562,374 filed March 7, 2024, entitled “MAGNETIC FLOW CYTOMETRY WITH ENHANCED MULTIPLICITY.” U.S. Provisional Patent Application Serial Number 63 / 562,374 is herein incorporated by reference in its entirety. TECHNICAL FIELD

[0002] Embodiments are generally related to the field of detection. 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.

[0005] Optical flow cytometry has been a critically important improvement in cell analysis. However, there are significant limitations to optical flow cytometry. For example, interference is a common problem with optical flow cytometry, because optical background noise is often generated by biological samples. As such, extensive sample preparation is required beforeAttorney Docket No. TTU-1079PCT PATENT APPLICATION analysis.

[0006] Furthermore, 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.

[0007] In addition, optical flow cytometry has limited multiplicity. Since the emission bands are often too broad, using multiple dyes can cause spectral overlap. Deconvolution of overlapping spectra by sophisticated correction algorithms is therefore required.

[0008] Given the critical need for flow cytometry, and in light of the limitations outlined above with respect to classic optical flow cytometry, there is a need for improved systems and methods for magnetic flow cytometry with enhanced multiplicity as disclosed herein.Attorney Docket No. TTU-1079PCT PATENT APPLICATION SUMMARY

[0009] The following summary is provided to facilitate an understanding of some of the innovative features unique to the embodiments disclosed and is not intended to be a full description. A full appreciation of the various aspects of the embodiments can be gained by taking the entire specification, claims, drawings, and abstract as a whole.

[0010] It is, therefore, one aspect of the disclosed embodiments to provide cytometry.

[0011] It is another aspect of the disclosed embodiments to provide methods and systems for flow cytometry.

[0012] It is another aspect of the disclosed embodiments to provide methods and systems for magnetic flow cytometry.

[0013] It is another aspect of the disclosed embodiments to provide methods, systems, and apparatuses for magnetic flow cytometry with enhanced multiplicity.

[0014] It will be appreciated that the methods and systems can be achieved according to the embodiments disclosed herein. In an embodiment, 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. In an embodiment, the flow cytometry system further comprises an incubation well for mixing at least one magnetic label to at least one of the biological molecules. In an embodiment, the at least one magnetic label comprises at least one antibody and at least one superparamagnetic nanoparticle. InAttorney Docket No. TTU-1079PCT PATENT APPLICATION an embodiment, 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. In an embodiment, 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. In an embodiment, 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. In an embodiment, the magnetic detector further comprises at least one compensation coil configured to cancel a feed through signal via inductive dissipation. In an embodiment, 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. In an embodiment, 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.

[0015] In an embodiment, a magnetic label comprises at least one magnetic particle and at least one antibody bound to a surface of the magnetic particle. In an embodiment, 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. In an embodiment, 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 according to a biotin-streptavidin affinity reaction. In an embodiment, 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.

[0016] In an embodiment, a flow cytometry method comprises binding at least one magnetic label to at least one biological molecule in a sample, removing unbound magneticAttorney 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. In an embodiment, the magnetic label comprises at least one antibody and at least one superparamagnetic nanoparticle. In an embodiment, 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. In an embodiment, 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.Attorney Docket No. TTU-1079PCT PATENT APPLICATION BRIEF DESCRIPTION OF THE FIGURES

[0017] The accompanying figures, in which like reference numerals refer to identical or functionally similar elements throughout the separate views and which are incorporated in and form a part of the specification, further illustrate the embodiments and, together with the detailed description, serve to explain the embodiments disclosed herein.

[0018] FIG.1A depicts a block diagram of a flow cytometry system, in accordance with the disclosed embodiments;

[0019] FIG. 1B depicts aspects of a method for flow cytometry, in accordance with the disclosed embodiments;

[0020] FIG.2 depicts an incubation well for binding a magnetic label to a biological particle, in accordance with the disclosed embodiments;

[0021] FIG.3 depicts a magnetic separator stage associated with a flow cytometry system, in accordance with the disclosed embodiments;

[0022] 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 theAttorney Docket No. TTU-1079PCT PATENT APPLICATION disclosed embodiments;

[0028] 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. 7 depicts steps in a method for cell counting / sorting, in accordance with the disclosed embodiments;

[0034] FIG. 8 illustrates another exemplary magnetic label, in accordance with the disclosed embodiments;

[0035] FIG. 9 illustrates exemplary magnetic labels bound to biological particles, in accordance with the disclosed embodiments;

[0036] 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. 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.Attorney Docket No. TTU-1079PCT PATENT APPLICATION DETAILED DESCRIPTION

[0041] Embodiments and aspects of the disclosed technology are presented herein. The particular embodiments and configurations discussed in the following non-limiting examples can be varied, and are provided to illustrate one or more embodiments, and are not intended to limit the scope thereof.

[0042] Reference to the accompanying drawings, in which illustrative embodiments are shown are provided herein. The embodiments disclosed can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art. Like numbers refer to like elements throughout.

[0043] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0044] Throughout the specification and claims, terms may have nuanced meanings suggested or implied in context beyond an explicitly stated meaning. Likewise, the phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment and the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment. It is intended, for example, that claimed subject matter include combinations of example embodiments in whole or in part.

[0045] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. ItAttorney Docket No. TTU-1079PCT PATENT APPLICATION will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0046] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0047] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0048] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0049] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.Attorney Docket No. TTU-1079PCT PATENT APPLICATION

[0050] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “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. Continuing with this example, 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. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0051] All of the 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.

[0052] 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.

[0053] Once the magnetic labels are attached to 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. An electronic system is then used for cell counting, cell sorting, to determine cell characteristics and function, to detect microorganisms, to identify protein molecules, for biomarker detection, for diagnosis of diseases such as circulating tumor cells (CTCs), or other such applications.Attorney Docket No. TTU-1079PCT PATENT APPLICATION

[0054] 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.

[0055] 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. In the magnetic separator stage 108, a magnet 110 (e.g., an electromagnet, or other such magnet) is used to create a magnetophoretic field which is used to separate unbound magnetic labels 112 from the magnetic labeled cells 118. The unbound magnetic labels 112 can be drawn to a separator channel 114 and discarded or reused, as illustrated by arrow 116.

[0056] 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. In the detector region 120, an excitation coil is used to generate a magnetic field, and a pickup coil is used to collect a signal from the magnetic labeled cells 118 resulting from the magnetic field.

[0057] 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.

[0058] 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] At step 155, the constituents in a biological sample are labeled in an incubation well. In an exemplary embodiment, 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.

[0060] 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.

[0061] 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). Another option is to leverage affinity reactions between an antibodies’ Fc region and protein A or G molecules on the particles’ surface. Another option is the use of biotinylation of the antibody for binding via the biotin–streptavidin affinity reaction.

[0062] Next, the surface functionalized magnetic labels are mixed with the biological sample, which can comprise a biofluidic sample, that contains target cells / biomolecules. Varying incubation times and incubation conditions may be necessary for the specific binding and labeling application. The antibodies on the magnetic labels will attach to cell proteins, such that the cells of interest are bound to magnetic labels.

[0063] At step 160, unbound magnetic labels can be removed from the sample via magnetic separation. FIG.3 illustrates a diagram 300 showing the mechanism for unbound antibody removal, in accordance with the disclosed embodiments.

[0064] As illustrated in FIG.3, 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. When the object is moving relative to the fluid 325, the drag force 320 is proportional to the cross-sectional area of theAttorney Docket No. TTU-1079PCT PATENT APPLICATION associated object. Likewise, a gravitational force (herein referred to as “Fg” 330) may or may not be relevant depending on the orientation of the fluidic channel 310.

[0065] Using magnetic forces exerted on unbound magnetic labels 340 and bound labels / cells 335, 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. However, 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.

[0066] The gradient B field 315, flow velocity ^ 345, and fluidic channel 310 width can be selected in order to optimize the separation result. Removing the unbound magnetic labels 340 can benefit later detection steps. Removing the unbound magnetic labels 340 reduces or eliminates magnetic signals that might result in invalid sorting / counting of cells.

[0067] Once the constituent particles in the sample have been labeled and the unbound magnetic labels 340 have been removed, the method 150 proceeds to step 165 where magnetic detection is used to identify the labeled cells 335.

[0068] 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.

[0069] 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.

[0070] In an exemplary embodiment, 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.Attorney Docket No. TTU-1079PCT PATENT APPLICATION^^^^ = ^^^^ ^2^^^^ (1)

[0071] In another exemplary embodiment, 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)

[0072] In another exemplary embodiment, 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.^^^^ = ^^^^^ ^2^^^^^ + ^^^^^ ^2^^^^^ (3)

[0073] In another exemplary embodiment, 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)

[0074] Returning to FIG. 4A, the pickup coil 420 is similarly configured external to the detection portion 410 of the fluidic channel. 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. It should be appreciated that the geometry of this arrangement can be selected to cancel any voltage response generated as a result of the excitation signal.

[0075] FIGs. 5A-5C illustrate various configurations of the pickup coil and excitation coil, which can be used to cancel voltage response. In some embodiments, the pickup and / or excitation coil can comprise planar coils arranged proximate to the channel 410 in the detection region 420. In other embodiments, the pickup coil and / or excitation coil canAttorney Docket No. TTU-1079PCT PATENT APPLICATION comprise coils wound around the channel 410 in the detection region 120. In still other embodiments, both planar coils and coils wound around the channel 410 in the detection region can be used.

[0076] For example, in FIG.5A, 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.

[0077] 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. In this embodiment, the planar pickup coil 502 is parallel to the magnetic flux produced by the excitation coil 532 wound around the channel 410, so it is not recording the signal due to the time varying excitation field. Thus, the planar pickup coil 502 only records the magnetizations of nanoparticles passing by.

[0078] 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.

[0079] 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. In this way, 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.

[0080] 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. 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.

[0081] 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. 6B, a sinusoidal driving field illustrated in chart 650, will generate a magnetic response as illustrated in chart 652. However, chart 654 illustrates the driving field in the frequency domain. As illustrated in chart 656, the voltage response will result in characteristic high harmonics in the frequency domain.

[0082] Thus, as one or more sinusoidal AC magnetic fields is applied (for example, one AC field), the nonlinear responses of superparamagnetic nanoparticles will cause higher harmonics that are unique for each type of nanoparticle.

[0083] For example, take the AC magnetic field represented by equation (1) as an illustrative case. The nonlinear magnetic response of superparamagnetic nanoparticles causes higher odd harmonics at 3^, 5^, 7^, etc. as illustrated in chart 656.

[0084] These harmonics are unique for each type of nanoparticle, making multiplicity possible. FIG.6C illustrates this principle for exemplary supraparticle 600. As illustrated, the harmonics 675 for Particle A 602 are unique to Particle A 602. Likewise, 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. Furthermore, because each type of nanoparticle has its unique harmonic spectra, by mixing different ratios of, for example, Particle A 602, Particle B 604, and Particle C 606 in the supraparticle 600, a plural number of supraparticle labels can be included, creating the desired multiplicity.

[0085] It should be appreciated that the available n multiplicity in the embodimentsAttorney 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.

[0086] 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. For example, in 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.). This is illustrated by equation (5) as: ^^^^ ^^^^^ ^^^^^ ^ ^ ^ ^ ^ ^(5)and sorting 170, in accordance with the disclosed embodiments. At step 705 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. Next, at step 710 the composition of nanoparticle (e.g., Particle A 602, Particle B 604, and Particle C 606) can be calculated. With this information, at step 715 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.

[0088] It should be appreciated that various controllable aspects of the methods and systems disclosed herein can be advantageously selected to change the multiplicity of the system. For example, the algorithm used to solve the composition matrix given by equation (5) can be selected. Likewise, using uniformly sized superparamagnetic nanoparticles to assemble the beads can change the multiplicity. Another option to change multiplicity is to select each type of superparamagnetic nanoparticles (for example, particles A, B, C) to have intrinsically different harmonic spectra.Attorney Docket No. TTU-1079PCT PATENT APPLICATION

[0089] Supraparticles have been disclosed in association with various embodiments herein. In other embodiments other magnetic particles can be used for similar purposes. For example, in certain embodiments, multi-core magnetic beads can be used.

[0090] 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. It should be appreciated that the superparamagnetic nanoparticles illustrated are exemplary and different numbers of superparamagnetic nanoparticle can be included in other embodiments. By varying the superparamagnetic nanoparticle cores, different magnetic beads can be configured, each of which will exhibit unique harmonics, for labeling purposes. Note, the drawings in FIG.8 are simplified for purposes of illustration. The multi-core magnetic beads are not necessarily spherical. They can be cube-shaped, rod-shaped, ellipsoid-shaped, etc.

[0091] Superparamagnetic nanoparticles (e.g., nanoparticle A 950, nanoparticle B 955, nanoparticle C 960, nanoparticle D 965, nanoparticle E 970, etc. illustrated in FIG. 9) can also be used in accordance with the disclosed embodiments. Each superparamagnetic nanoparticle has unique higher harmonics.

[0092] Each cell in a sample generally has more than one surface protein (say, proteins PA, PB, PC, PD, PE, etc.). Using this property, different superparamagnetic nanoparticles can be designed, each functionalized with one type of antibody, to allow them to specifically bind to target proteins.

[0093] For example, as illustrated in 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.Attorney Docket No. TTU-1079PCT PATENT APPLICATION

[0094] In certain embodiments 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.

[0095] 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. The analog signal is then converted to a digital signal with ADC 1030, and provided to a processing unit 1100, as further detailed herein.

[0096] Similarly, FIG.10B illustrates aspects of the detection system 1050. Here 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.

[0097] 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.

[0098] 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. A computing device in the form of a computer 1110 configured to interface with controllers,Attorney Docket No. TTU-1079PCT PATENT APPLICATION peripheral devices, and other elements disclosed herein 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.

[0099] 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.

[0100] 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. In addition, 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 forAttorney Docket No. TTU-1079PCT PATENT APPLICATION displaying data and information for a user, and for interactively displaying a graphical user interface (GUI) 1130.

[0101] Note that the term “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 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.

[0102] 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.

[0103] 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. Note that 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, wirelessAttorney Docket No. TTU-1079PCT PATENT APPLICATION communication links of various types, and fiber optic cables. 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. It should be understood that 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.

[0104] In the depicted example, 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.

[0105] Computer system 1100 can also be implemented as a server such as server 1206, depending upon design considerations. In the depicted example, 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.

[0106] In the depicted example, 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. 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. Of course, 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

[0107] 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. These inputs may then be acted upon by the computer system 1100 in accordance with instructions from operating system 1310 and / or software application 1305 and any software module(s) 1125 thereof.

[0108] Generally, program modules (e.g., module 1125) 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. Moreover, those skilled in the art will appreciate that elements of the disclosed methods and systems may be practiced with other computer system configurations such as, for example, hand-held devices, mobile phones, smart phones, tablet devices multi- processor systems, microcontrollers, printers, copiers, fax machines, multi-function devices, data networks, microprocessor-based or programmable consumer electronics, networked personal computers, minicomputers, mainframe computers, servers, medical equipment, medical devices, and the like.

[0109] Note that the term “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 theAttorney 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.

[0110] 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. In some embodiments, 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.

[0111] The following description is presented with respect to embodiments of the present invention, which can be embodied in the context of, or require the use of, a data-processing system such as computer system 1100, in conjunction with program module 1125, and data- processing system 1200 and network 1202 depicted in FIGs.11-13. The present invention, however, is not limited to any particular application or any particular environment. Instead, those skilled in the art will find that the system and method of the present invention may be advantageously applied to a variety of system and application software including database management systems, word processors, and the like. Moreover, the present invention may be embodied on a variety of different platforms including Windows, Macintosh, UNIX, LINUX, Android, Arduino, LabView and the like. Therefore, the descriptions of the exemplary embodiments, which follow, are for purposes of illustration and not considered a limitation.

[0112] Based on the foregoing, it can be appreciated that a number of embodiments, preferred and alternative, are disclosed herein. For example, in an embodiment, 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 systemAttorney 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. In an embodiment, the flow cytometry system further comprises an incubation well for mixing at least one magnetic label to at least one of the biological molecules. In an embodiment, the at least one magnetic label comprises at least one antibody and at least one superparamagnetic nanoparticle. In an embodiment, 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. In an embodiment, 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. In an embodiment, 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. In an embodiment, the magnetic detector further comprises at least one compensation coil configured to cancel a feed through signal via inductive dissipation. In an embodiment, 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. In an embodiment, 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.

[0113] In an embodiment, a magnetic label comprises at least one magnetic particle and at least one antibody bound to a surface of the magnetic particle. In an embodiment, 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. In an embodiment, 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 particleAttorney Docket No. TTU-1079PCT PATENT APPLICATION according to a biotin-streptavidin affinity reaction. In an embodiment, 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.

[0114] In an embodiment, 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. In an embodiment, the magnetic label comprises at least one antibody and at least one superparamagnetic nanoparticle. In an embodiment, 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. In an embodiment, 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.

[0115] It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, it should be appreciated that various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims

Attorney Docket No. TTU-1079PCT PATENT APPLICATION CLAIMS What is claimed is:

1. A flow cytometry system comprising: 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.

2. The flow cytometry system of claim 1 further comprising: an incubation well for mixing at least one magnetic label to at least one of the biological molecules.

3. The flow cytometry system of claim 2 wherein the at least one magnetic label comprises: at least one antibody; and at least one superparamagnetic nanoparticle.

4. The flow cytometry system of claim 1 wherein the magnetic separator stage further comprises:Attorney Docket No. TTU-1079PCT PATENT APPLICATION a magnet configured to apply a magnetophoretic force; and a separator channel, wherein the magnetophoretic force drives unbound magnetic labels into the separator channel.

5. The flow cytometry system of claim 1 wherein 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.

6. The flow cytometry system of claim 5 wherein 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.

7. The flow cytometry system of claim 1 wherein the magnetic detector further comprises: at least one compensation coil configured to cancel a feed through signal via inductive dissipation.

8. The flow cytometry system of claim 1 wherein 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 at least one biological molecule.

9. The flow cytometry system of claim 1 wherein 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.Attorney Docket No. TTU-1079PCT PATENT APPLICATION 10. A magnetic label comprising: at least one magnetic particle; and at least one antibody bound to a surface of the at least one magnetic particle.

11. The magnetic label of claim 10 wherein the at least one antibody is bound to the surface of the at least one magnetic particle using a covalent bond between the antibody’s functional group and the particle surface.

12. The magnetic label of claim 10 wherein 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.

13. The magnetic label of claim 10 wherein the at least one antibody is bound to the surface of the magnetic particle according to a biotin-streptavidin affinity reaction.

14. The magnetic label of claim 10 wherein the at least one magnetic particle comprises: a plurality of superparamagnetic nanoparticles.

15. The magnetic label of claim 14 wherein each of the plurality of superparamagnetic nanoparticles has a unique harmonic spectra when subjected to a magnetic driving field.

16. The magnetic label of claim 10 wherein the at least one magnetic particle comprises: a plurality of superparamagnetic nanoparticles; and a polymer shell.

17. A flow cytometry method comprising: 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;Attorney Docket No. TTU-1079PCT PATENT APPLICATION 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.

18. The flow cytometry method of claim 17 wherein the magnetic label comprises: at least one antibody; and at least one superparamagnetic nanoparticle.

19. The flow cytometry method of claim 17 wherein 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.

20. The flow cytometry method of claim 17 wherein 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.

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