Use of magnetic nanoparticles for the detection and quantification of analytes

Abstract

Described are methods and devices for detecting an analyte in a sample, the methods and devices including contacting a sample containing a target analyte with magnetizable particles, the particles being coated with binding molecules complementary to the target analyte, resulting in bound and unbound binding agent complexes; placing the magnetizable particles containing both bound and unbound binding agent complexes in proximity to a magnetic field sensor; changing the magnetic field sufficient to release at least a portion of the magnetizable particles containing both bound and unbound binding agent complexes from their proximity to the magnetic field sensor; and measuring a change in a magnetic signal detected from the net movement, which is either translational or rotational movement, of the magnetizable particles relative to the magnetic sensor.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting an analyte in a sample, and more particularly to the use of magnetizable nanoparticles and a magnetic sensor system. The present invention also relates to a device for detecting an analyte based on the use of magnetizable nanoparticles. [Background technology]

[0002] Many methods are known for detecting and quantifying an analyte in a sample. Such systems require an indirect method of quantifying the analyte by detecting and measuring the complex bound to the analyte. Typically, such methods rely on binding or recognition systems in which a visualization aid is coated or linked to a binding molecule that binds to the analyte in the sample.

[0003] Binding molecules may include antibodies, enzymes, or pharmacological agents specifically selected based on their affinity for the target analyte. Molecules that directly bind to the analyte may themselves be labeled with an enzyme or fluorophore (in the case of fluorescent labels).

[0004] Alternatively, the molecule that directly binds to the analyte may itself be unlabeled, but instead be conjugated to another binding agent that is itself labeled with an enzyme or fluorophore. This additional labeling step can amplify the signal and reduce background staining. Well-known conjugates are the avidin-biotin complex and the peroxidase-antiperoxidase approach.

[0005] Technologies for detecting and quantifying analytes in samples need to be rapid, sensitive, qualitative, and / or miniaturizable to meet the needs of in vitro diagnostics. Miniaturization of devices can result in slow and inefficient mixing of fluids due to increased viscous forces.

[0006] Point-of-care instantaneous testing would reduce turnaround time for diagnostic testing, providing improved workflow and thus potentially contributing to improved patient care. Such systems must include sensing technologies to detect biomarkers (e.g., protein or nucleic acid markers). Magnetizable particles have been used to detect analytes in everything from manual assays for basic research to high-throughput testing.

[0007] Many existing devices for detecting analytes attached to magnetizable particles require complex configurations that are not suitable or easily adapted for miniaturization in point-of-care rapid testing applications.

[0008] The use of magnetizable particles relies on the functionalization of the particles with binding molecules (e.g., antibodies with high affinity for the target analyte) that enable binding to the target analyte, followed by a fluid exchange step to achieve separation and purification. It has been reported that the analyte capture rate is proportional to the total surface area of ​​the suspended particles and therefore to the particle concentration. However, high particle concentrations generally increase nonspecific particle-particle and particle-surface interactions, promote electric field-induced particle aggregation, cause steric hindrance during the particle concentration step, interfere with particle chemical reactions, and sterically inhibit reactions between the particles and the biosensing surface. Therefore, the use of very high particle concentrations is disadvantageous for downstream processes in integrated multi-step lab-on-a-chip assays.

[0009] The target analyte may be present at low concentrations in a sample containing high concentrations of background material, such as blood or saliva. In such complex matrices, nonspecific attachment of non-target molecules to magnetizable particles can reduce the effectiveness of the assay.

[0010] The process of magnetic particle-based capture of target analytes consists of an encounter between two components (the target analyte and the magnetic particle) and may depend on the two components aligning their outer surfaces in a highly specific manner relative to each other. Thus, the binding rate of the two components may be limited by diffusion and the geometric constraints of the binding sites of the two components, and may also be reduced by the eventual chemical reaction.

[0011] Analytes can be captured in flowing or static fluids. Without fluidity, methods relying on surface-immobilized antibodies can be diffusion-limited, resulting in poor binding rates.

[0012] After the capture of the target analyte by the magnetic particles, additional processing is required for detection. When used only as a carrier, the magnetizable particles are typically bound to an identification molecule such as a luminescent label or a fluorescent molecule. For accurate detection, it is important that only the bound analyte is labeled and only the bound label is detected. This requires several washing or separation steps.

[0013] Magnetizable particles can also be used as labels to indicate the binding of target analytes to the sensing surface. Agglutination assays utilize the process by which particle aggregates form when a specific analyte is present in a sample fluid. The degree of agglutination is a measure of the concentration of the analyte in the fluid. Agglutination assays are performed in one step without separation or rigor, which makes them reagent-intensive.

[0014] In magnetic agglutination assays, particle cluster formation is accelerated by collecting particles under the influence of a magnetic field. A problem with this methodology is that when the analyte concentration is much smaller than the magnetizable particle concentration, a small number of particle aggregates, governed by Poisson statistics, form. The application of a magnetic field can be enhanced by applying a magnetic field during incubation. However, the magnetic field can also increase nonspecific binding between particles. Nonspecific binding (i.e., binding not mediated by the target analyte) results in false-positive signals. Nonspecific binding can be attributed to several types of interactions, such as van der Waals interactions, electrostatic interactions, and hydrophobic interactions, which cause background levels and statistical fluctuations in results, thus affecting the quantitation limit and precision of the method.

[0015] The use of magnetisable particles means that additional forces can be applied to the particles, for example to separate bound particles from unbound particles.

[0016] The assessment of analytical performance of a detection methodology is based on the limit of quantification (LoQ), i.e., the lowest biomarker concentration that can be quantified with a given required precision.

[0017] Optimizing magnetizable particles for specific applications and selecting appropriate detection methods remains challenging for the magnetic nanotechnology community due to increasing demands for detection sensitivity, molecular specificity, and application complexity.

[0018] The use of GMR in immunoassays has been used in sandwich-type approaches (such as ELISA) in which molecular targets are immobilized on a sensor surface to which tagged magnetic probes have been added (Non-Patent Document 1 and Non-Patent Document 2).

[0019] Some techniques use superconducting quantum interference devices (SQUIDs) to detect and measure Néel relaxation (magnetic dipole misalignment) in magnetically labeled bacteria. In such techniques, a magnetic field is pulsed to induce magnetic dipole alignment, and the subsequent dipole misalignment is detected. [Prior art documents] [Non-patent literature]

[0020] [Non-Patent Document 1] Koh and Josephson, "Magnetic nanoparticle sensors," Sensors 2009:9;8130-45 [Non-patent document 2] Yao and Xu “Detection of magnetic nanomaterials in molecular imaging and diagnosis applications” Nanotechnol.Rev 2014:3;247-268 Summary of the Invention [Problem to be solved by the invention]

[0021] It is an object of the present invention to address one or more of the problems discussed above and / or to provide a method for detecting an analyte in a sample and / or at least to provide the public with a useful choice. [Means for solving the problem]

[0022] In a first aspect, there is provided a method for detecting an analyte in a sample, comprising: contacting the sample containing the target analyte with magnetizable particles, the particles being coated with binding molecules complementary to the target analyte, resulting in bound and unbound binding agent complexes; placing the magnetizable particles, including both the bound and the unbound binding agent complexes, in proximity to a magnetic field sensor; varying the magnetic field sufficient to release at least a portion of the magnetizable particles, including both the bound and the unbound binding agent complexes, from their proximity to the magnetic field sensor; measuring a change in a magnetic signal detected from the magnetizable particles as a result of net movement (translation or rotation) of the magnetizable particles relative to the magnetic sensor.

[0023] In a further aspect, there is provided a method for detecting an analyte in a sample, comprising: a sample well or sample reservoir; one or more magnets for generating a magnetic field in the sample well or the sample reservoir; a magnetic field sensor for measuring changes in the magnetic field over time in the sample well or sample reservoir; contacting the sample containing the target analyte with magnetizable particles in the sample well, the particles being coated with binding molecules complementary to the target analyte; placing said magnetizable particles in proximity to a magnetic sensor; Varying the magnetic field sufficiently to allow the magnetizable particles to move (translationally or rotationally) relative to the magnetic sensor.

[0024] In a further aspect, there is provided a method for detecting an analyte, the method comprising: a) generating within 10 seconds a magnetic signal sufficient to detect and / or measure the amount of target analyte in the sample; or b) has a limit of detection (LOD) of at least about 0.05 pg / mL; or c) has a limit of quantitation (LOQ) of at least about 0.1 pg / mL; or d) A method is described, wherein the method is one or more of (a) through (c).

[0025] In a further aspect, there is provided a device for detecting an analyte in a sample, comprising: a sample well or sample reservoir; one or more magnets for generating a magnetic field in the sample well; a magnetic field sensor for measuring changes in the magnetic field over time in the sample well; A device is described in which the one or more magnets and magnetic sensors are adapted for use such that the magnetic sensor can detect changes in the magnetic field based on the net movement (translational or rotational) of magnetizable particles relative to the magnetic sensor.

[0026] In a further aspect, there is provided a diagnostic system for detecting an analyte in a sample, the system comprising: contacting the sample containing the target analyte with magnetizable particles, the particles being coated with binding molecules complementary to the target analyte; placing magnetizable particles in proximity to a magnetic field sensor; varying the magnetic field sufficiently to release at least a portion of the magnetizable particles from their proximity to the magnetic field sensor; measuring a change in a magnetic signal detected from the magnetizable particles as the magnetizable particles move (translationally or rotationally) relative to the magnetic sensor; The diagnostic system a) obtaining within 20 seconds a magnetic signal sufficient to detect and / or measure the amount of said target analyte in said sample; or b) has a limit of detection (LOD) of at least about 0.05 pg / mL; or c) has a limit of quantitation (LOQ) of at least about 0.1 pg / mL; or d) A system is described, wherein the system is one or more of (a) through (c).

[0027] Any one or more of the following embodiments may relate to any of the above aspects.

[0028] In one configuration, the device or diagnostic system acquires a magnetic signal sufficient to detect and / or measure the amount of target analyte in the sample within 5, 10, 15, or 20 seconds, and a suitable range can be selected between any of these values.

[0029] In one configuration, a magnetic field is applied to position the magnetizable particles in proximity to a magnetic field sensor.

[0030] In one configuration, the magnetic field mixes the sample.

[0031] In one configuration, the detection and quantification of an analyte in a sample relies on the amount of magnetizable particles detected via a magnetic field sensor.

[0032] In one configuration, the magnetizable particles are positioned using centrifugal force, acoustics or piezoelectricity.

[0033] In one configuration, magnetizable particles are functionalized with molecules that specifically bind to the analyte.

[0034] In one configuration, the sample and magnetizable particles are processed by a microfluidic device, preferably the microfluidic device promotes binding between the magnetizable particles and the analyte.

[0035] In one configuration, the magnetic field enhances or strengthens the binding between the magnetizable particles and the target analyte.

[0036] In one configuration, the magnetizable particles are magnetic particles.

[0037] In one configuration, the magnetizable particles are paramagnetic.

[0038] In one configuration, the magnetizable particles are ferromagnetic.

[0039] In one configuration, detection is provided by a lab-on-a-chip device, preferably comprising a microfluidic device.

[0040] In one configuration, the chip device has a multi-chipset design.

[0041] In one configuration, the magnetizable particles have an average particle size of about 5 to about 500 nm, and a suitable range can be selected between any of these values.

[0042] In one configuration, the magnetizable particles have an average particle size of about 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 nm, and a suitable range may be selected between any of these values.

[0043] In one configuration, the magnetizable particles have an average particle size of about 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nm, and a suitable range may be selected between any of these values.

[0044] In one configuration, the magnetizable particles have an average particle size of about 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 nm, and a suitable range may be selected between any of these values.

[0045] In one configuration, the microfluidic device places the magnetizable particles and the analyte in close proximity to a magnetic sensor.

[0046] In one configuration, the magnetizable particles and analyte are brought within 1, 10, 100, 500, 1000, 2000, 3000, 4000 or 5,000 μm of the sensing element of the magnetic sensor, and a useful range can be selected between any of these values.

[0047] In one configuration, one or more magnets (or electromagnets) align the magnetizable particles.

[0048] In one configuration, one or more magnets generate a time-varying magnetic field.

[0049] In one configuration, the magnetic field generator is capable of generating a continuum of magnitudes.

[0050] In one configuration, the magnetic field generator can alternately switch the magnetic field between on and off.

[0051] In one configuration, the magnetic field is generated and positioned to maximize its effect on the magnetizable particles but minimize its effect on the magnetic sensor.

[0052] In one configuration, the magnetic field sensor is adapted to maximize sensing of magnetizable particles and minimize sensing from magnets.

[0053] In one configuration, when the microfluidic device processes magnetizable particles and is placed in close proximity to the magnetic sensor, data acquisition by the sensor is synchronized with the microfluidic device such that the magnetic field signal from the sensor is distinguishable as data from the sample.

[0054] In one arrangement, the data is obtained continuously from the sensor, preferably by processing signals from the magnetic sensor.

[0055] In one configuration, the acquired data is flagged as either 1) environmental and / or ambient, or (2) inspection data. Preferably, the classification of the data into (1) environmental and / or ambient, or (2) inspection data depends on synchronization of the data acquisition with the operation of the microfluidic device.

[0056] In one configuration, the method is calibrated based on synchronization of signal acquisition with operation of the microfluidic device.

[0057] In one configuration, data is acquired over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 90 or 120 seconds, and a useful range can be selected between any of these values.

[0058] In one configuration, the signal output from the magnetic sensor is increased by a signal amplifier.

[0059] In one configuration, the signal output from the sensor is a voltage reading proportional to the magnetic field strength it senses.

[0060] In one configuration, the voltage from the sensor is increased in magnitude to a higher voltage while keeping all changes proportional to the original signal, within a range compatible with the data processing and acquisition electronics.

[0061] In one configuration, the amplified signal is converted from a voltage reading to a digital bitstream and recorded by a computer.

[0062] In one configuration, the conversion is performed by an analog-to-digital converter (ADC).

[0063] In one configuration, the conversion or sampling rate may be between 50 and 500,000 hertz.

[0064] In one configuration, the conversion or sampling resolution can be 16 to 32 bits.

[0065] In one configuration, the signal output is digitally processed with mathematical operations to generate a readout that can be used for interpretation and analysis.

[0066] In one configuration, the device or diagnostic system use has an LOD of at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, or 0.20 pg / mL, and a useful range can be selected between any of these values.

[0067] In one configuration, the device or diagnostic system used has an LOD of at least 0.1 pg / mL.

[0068] In one configuration, the device or diagnostic system used has an LOQ of at least 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20 pg / mL, and a useful range can be selected between any of these values.

[0069] In one configuration, the device or diagnostic system used has an LOQ of at least 0.1 pg / mL.

[0070] As used herein, the term "comprising" means "consisting at least in part of." When interpreting descriptions herein that include the term, all features preceded by the term in each description must be present, although other features may also be present. Related terms such as "comprise" and "comprised" should be interpreted similarly.

[0071] Reference to a range of numbers disclosed herein (e.g., 1 to 10) also incorporates reference to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10), and is intended to incorporate any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7).

[0072] The present invention also relates to the components, elements and features individually or collectively referred to or indicated in the specification of this application, and any or all combinations of any two or more of said components, elements or features, and where a particular integer having known equivalents in the art to which the invention pertains is referred to herein, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0073] Numerous modifications in the structure of the present invention and widely different embodiments and applications will suggest themselves to those skilled in the art to which this invention pertains without departing from the scope of the invention as defined in the appended claims. The disclosures and descriptions herein are purely illustrative and are not intended to be in any sense limiting. [Brief explanation of the drawings]

[0074] The invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0075] [Figure 1] FIG. 1 is a flow diagram showing the set-up of the method as described. [Figure 2] FIG. 2 is a schematic diagram of a microfluidic device. [Figure 3] FIG. 3 is a graph showing a plot of signal versus sensitivity showing an LoQ of approximately 0.5 pg. [Figure 4] FIG. 4 is a graph showing signal acquisition over time for a control, 50 pg of particles, and 500,000 pg of particles. DETAILED DESCRIPTION OF THE INVENTION

[0076] Described is a method for detecting an analyte in a sample, comprising: contacting the sample containing the target analyte with magnetizable particles, the particles being coated with binding molecules complementary to the target analyte, resulting in bound and unbound binding agent complexes; applying a magnetic field to place the magnetizable particles, including both the bound and the unbound binding agent complexes, in proximity to a magnetic field sensor (a "capture" step); changing the magnetic field sufficient to release at least a portion of the magnetizable particles, including both the bound and the unbound binding agent complexes, from their proximity to the magnetic field sensor (a "releasing" step); and measuring a change in a magnetic signal detected from the magnetizable particles as a result of net movement of the magnetizable particles relative to the magnetic sensor, the movement being either translational or rotational.

[0077] The described method is based on the concept of bringing magnetizable particles and analyte complexes into close proximity with a magnetic field sensor. The magnetic field strength is adjusted (i.e., by translational or rotational movement) to allow the magnetizable particles and analyte complexes to diffuse away from the magnetic field sensor. The magnetic field sensor then measures the change in magnetic field strength over time that the magnetizable particles generate due to Brownian rotation or diffusion, quantifying the amount of magnetizable particle-analyte complexes and thereby determining the amount of analyte in the sample. That is, bound and unbound binder complexes are distinguished based on their diffusion properties. The magnetizable beads (i.e., both bound and unbound complexes) physically move relative to the magnetic field sensor, allowing the distinction between bound and unbound complexes (assuming they move to different degrees due to different diffusion properties).

[0078] Broadly speaking, the sample analysis method has three stages. The first stage can be a pre-sample baseline sensing stage. This stage is performed in the absence of sample to obtain a baseline reading. The baseline reading provides a base comparison for subsequent sample readings. The pre-sample baseline sensing stage can take 1, 2, 3, 4, or 5 seconds, and an appropriate range can be selected from any of these values ​​(e.g., about 1 to about 5, about 1 to about 4, about 2 to about 5, about 2 to about 3, or about 3 to about 5 seconds).

[0079] The second step can be loading the sample into the device. This step can involve sample mixing and analyte-binding complexation (i.e., functionalized magnetizable particles binding to the analyte). This step can take about 3, 4, 5, 6, 7, or 8 minutes, and a suitable range can be selected from any of these values ​​(e.g., about 3 to about 8, about 3 to about 7, about 3 to about 5, about 4 to about 8, about 4 to about 6, or about 5 to about 8 minutes).

[0080] The third step can be a sample read step. That is, the magnetizable particles are placed in proximity to a magnetic field sensor, the magnetic field is changed to release at least a portion of the bound and unbound binding agent complexes, and the magnetic sensor measures the change in the magnetic signal detected from the magnetizable particles as a result of their net movement relative to the magnetic sensor. This step can take about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 seconds, and a suitable range can be selected from any of these values ​​(e.g., about 10 to about 20, about 10 to about 18, about 10 to about 15, about 11 to about 20, about 11 to about 19, about 11 to about 16, about 11 to about 15, about 12 to about 20, about 12 to about 18, about 12 to about 15, about 13 to about 20, about 13 to about 19, about 13 to about 17, or about 13 to about 15 seconds).

[0081] As described above, the amount of analyte in a sample is determined based on a change in the magnetic signal detected by a magnetic sensor. The magnetic sensor detects the change based on the net movement of the magnetizable particles. Upon release from their proximity to the magnetic field sensor, the magnetizable particles, including both bound and unbound binding agent complexes, move away from the magnetic field sensor. This movement is random based on Brownian diffusion.

[0082] Typically, the magnetic field sensor is positioned close to or adjacent (non-sample side) to the surface of the sample well or sample reservoir. When the bound and unbound magnetizable particles are positioned close to the magnetic field sensor, they may be positioned at or close to the surface of the wall of the sample well or sample reservoir until they are released. When released from their proximity to the magnetic field sensor, the magnetizable particles may move translationally or rotationally. Given their proximity to the surface of the sample well or sample reservoir, the bound and unbound magnetizable particles may typically move with a 180° degree of freedom of movement relative to the surface of the sample well or sample reservoir. Brownian diffusion means that the magnetizable particles can move in any direction, including toward the magnetic field sensor. The magnetic signal detected by the magnetic field sensor is based on the net movement of the bound and unbound magnetizable particles.

[0083] Advantages of the present invention may include rapid detection (see, eg, Example 2) and highly sensitive detection methodologies (see, eg, Examples 1 and 3).

[0084] Considering the encounter between a free analyte in solution and a magnetizable particle, the diffusive encounter step can be divided into (1) the process of diffusive transport through the fluid volume and (2) the process of near-surface alignment. While volume transport generates the initial encounter between the particle and the target analyte, the subsequent near-surface alignment process deals with the alignment rate of the reactant's binding sites. While volume transport is essentially a translational process, alignment is determined by both the translational and rotational mobility of the reactants.

[0085] When free components react in solution, alignment processes (i.e., rotational diffusion) become a significant limitation due to very specific alignment constraints, but volume transport (i.e., translational diffusion) is not. In cases where one of the components is attached to a surface, volume transport can become limiting.

[0086] The magnetic properties of nano- and micron-sized magnetic materials differ from those of the corresponding bulk magnetic materials. Typically, magnetizable particles are classified as paramagnetic, ferromagnetic, ferrimagnetic, antiferromagnetic, or superparamagnetic based on their magnetic behavior in the presence and absence of an applied magnetic field.

[0087] Diamagnetic materials exhibit no dipole moment in the absence of a magnetic field, but align against the direction of the magnetic field in the presence of a magnetic field.

[0088] Paramagnetic particles exhibit random dipole moments in the absence of a magnetic field, and align in the direction of the magnetic field when a magnetic field is present.

[0089] Ferromagnetic materials exhibit aligned dipole moments.

[0090] Ferrimagnetic and antiferromagnetic materials exhibit alternatingly aligned dipole moments.

[0091] In one embodiment, the magnetizable particles are paramagnetic particles. Such particles become magnetic when exposed to a magnetic field. When the magnetic field is removed, the particles begin to lose their magnetic properties.

[0092] In another embodiment, the magnetizable particles are ferromagnetic particles, i.e., they always exhibit magnetic properties, regardless of the presence or absence of a magnetic field.

[0093] Commercially available magnetizable particles include Dynaparticles M-270, Dynaparticles M-280, Dynaparticles MyOne T1, and Dynaparticles MyOne C1 from Thermo Fisher Scientific, μMACS MicroParticles from Miltenyi Biotec, SPHERO™ Superparamagnetic Particles, SPHERO™ Paramagnetic Particles, and SPHERO™ Ferromagnetic Particles from Spherotech.

[0094] Magnetizable particles can be formed by ferrites (such as magnetite and maghemite), which are themselves formed from iron oxide. Various methods are known for synthesizing iron oxide and metal-substituted ferrite magnetizable particles, including co-precipitation, pyrolysis, and hydrothermal synthesis. The co-precipitation process uses stoichiometric amounts of ferrous and ferric salts in alkaline solution in combination with a water-soluble surface coating material, such as polyethylene glycol (PEG), and the coating provides colloidal stability and biocompatibility. The size and characteristics of the magnetizable particles can be controlled by a variety of factors, including reducing agent concentration, pH, ionic strength, temperature, iron salt source, or Fe. 2+ and Fe 3+ can be controlled by adjusting the ratio of

[0095] The size and shape of the magnetizable particles can be tuned by varying the reaction conditions, such as the type of organic solvent, heating rate, surfactant, and reaction time. This method results in a narrow size distribution of magnetizable particles in the size range of 10-100 nm. 2+ can be substituted with other metals to increase the saturation magnetization.

[0096] The magnetizable particles may be coated with a hydrophobic coating during the synthesis process, and if so, the method of producing the magnetizable particles may include an additional step of ligand exchange so that the magnetizable particles can be dispersed in water for further use.

[0097] Magnetizable particles can be produced by polyol hydrothermal reduction, which produces water-dispersible magnetizable particles in the size range of tens to hundreds of nanometers. The size and surface functionalization of iron oxide magnetizable particles can be optimized by adjusting the solvent system, reducing agent, and type of surfactant used. This process can be used to synthesize FePt magnetizable particles.

[0098] Magnetizable particles can be produced by the inverse water-in-oil micelle method, which involves forming a microemulsion of aqueous nanodroplets of an iron precursor stabilized by a surfactant in an oil phase with magnetic nanoparticles obtained by precipitation. Iron oxide nanocrystals can be assembled by combining the microemulsion with silica sol-gel, which can be co-precipitated into magnetizable particles with diameters exceeding 100 nm.

[0099] Metallic magnetizable particles can be either monometallic (e.g., Fe, Co, or Ni) or bimetallic (e.g., FePt and FeCo). Alloy magnetizable particles can be synthesized by physical methods, including vacuum deposition and vapor phase evaporation. These methods can produce FeCo magnetizable particles with high saturation magnetization (about 207 emu / g), and Fe 3+ and Co 2+ It can be synthesized via reduction of a salt.

[0100] The magnetizable particles may comprise a single metal or metal oxide core. The magnetizable particles may comprise multiple cores, multiple layers of magnetic and non-magnetic materials. The magnetizable particles may comprise a coating of a silica or polymer core with a magnetic shell. The non-magnetic core particles may comprise silica or other polymers.

[0101] The magnetizable particles may comprise a dielectric silica core coated with a magnetic shell. The magnetic shell may be formed from Co, FePt, or Fe3O4. The shell may also comprise a stabilizer, such as a silica shell or a polyelectrolyte layer. The magnetizable particles may be mesoporous magnetizable particles.

[0102] Coatings on magnetizable particles can define the interaction between the magnetizable particles and biological molecules (such as analytes) and their biocompatibility. Coatings can be used to define the surface charge, which, in turn, can change the hydrodynamic size of the magnetic particles. The hydrodynamic size of the magnetizable particles can change the functionality of the magnetic particles.

[0103] The magnetizable particles can be coated with specific coatings that provide electrostatic and steric repulsion, which can help stabilize the magnetizable particles, preventing them from agglomerating or settling.

[0104] The magnetizable particles may have a coating formed from an inorganic material. Such magnetizable particles may be formed with a core-shell structure. For example, biocompatible silica or gold-coated magnetizable particles (e.g., silica-coated alloy magnetic nanoparticles, FeCo and CoPt). The shell may provide a platform for modifying the magnetizable particles with ligands (e.g., thiols). Other inorganic coating materials may include titanates or silver. For example, silver-coated iron oxide magnetizable particles may be synthesized and integrated with carbon paste.

[0105] The shell can be formed from silica. The advantage of coating with silica is that silica-coated magnetizable particles can be covalently bonded to versatile functional molecules and surface reactive groups. Silica shells can be produced, for example, by the Stöber method, which uses the sol-gel principle, or the Philips method, or a combination thereof. The core of the magnetizable particle can be coated with tetraethoxysilane (TEOS), for example, by hydrolysis of TEOS under basic conditions, which condenses and polymerizes TEOS into a silica shell on the surface of the magnetic core. Cobalt magnetizable particles can be coated using a modified Stöber method combining 3-aminopropyltrimethoxysilane and TEOS.

[0106] The Philipse method forms a silica shell of sodium silicate on the magnetic core. A second layer of silica can be deposited by the Stober method. The reverse microemulsion method can be used to coat with silica. This method can be used with a surfactant. The surfactant can be selected from Igepal CO-520 to provide a silica shell about 5 to about 20 nm thick. Preferably, the reagent for producing the silica shell is selected from an amino-terminated silane or an alkene-terminated silane. Preferably, the amino-terminated silane is (3-aminopropyl)trimethoxysilane (APTMS). Preferably, the alkene-terminated silane is (3-methacryloxypropyl)trimethoxysilane.

[0107] The magnetizable particles may be coated with gold. Gold-coated iron oxide nanoparticles may be synthesized by any of chemical, reverse microemulsion, and laser enhancement methods. Gold-coated magnetizable particles may be synthesized by directly coating gold onto the magnetizable particle core. Alternatively, gold-coated magnetizable particles may be synthesized by using silica as an intermediate layer for the gold coating. Preferably, reduction is used as a method for depositing a gold shell on the magnetizable particles.

[0108] A metal oxide or silica-coated magnetic core can be first functionalized with (3-aminopropyl)trimethoxysilane, followed by electrostatic deposition of approximately 2 to approximately 3 nm gold nanocrystal seeds (from chloroauric acid) on the surface, followed by the addition of a reducing agent to form a gold shell. Preferably, the reducing agent is a mild reducing agent selected from sodium citrate or tetrakis(hydroxymethyl)phosphonium chloride. In some embodiments, the gold shell is formed from the reduction of gold(III) acetate (Au(OOCCH3)3). In some embodiments, the gold shell is formed on a metal magnetic core (e.g., nickel and iron) by reverse micellization.

[0109] Magnetizable particles can be functionalized with organic ligands. This can be done in situ (i.e., functional ligands provided on the magnetizable particles during the synthesis process) or post-synthesis. Magnetizable particles can be functionalized with terminal hydroxyl (-OH), amino (-NH), and carboxyl (-COOH) groups. This can be achieved by varying the surfactant (e.g., dextran, chitosan, or poly(acrylic acid)) used in the hydrothermal synthesis.

[0110] Post-synthesis functionalization of magnetizable particles can allow for customized ligand functionalization on any magnetizable particle surface. Post-synthesis functionalization can be performed by ligand addition and ligand exchange. Ligand addition involves the adsorption of amphiphilic molecules (containing both hydrophobic segments and hydrophilic components) to form a bilayer structure. Ligand exchange replaces the original surfactant (or ligand) with a new functional ligand. Preferably, the new ligand contains a functional group that can bind to the magnetizable particle surface via either strong chemical bonds or electrostatic attraction. In some embodiments, the magnetizable particles also contain functional groups for stabilization in water and / or biofunctionalization.

[0111] Magnetizable particles can be coated with ligands that enhance ionic stability. Functional groups can be selected from carboxylate, phosphate, and catechol (e.g., dopamine). The ligand can be a siloxane group for coating hydroxyl-rich surfaces (e.g., metal oxide magnetic particles or silica-coated magnetic particles). The ligand can be a small silane ligand linking the magnetizable particle and various functional ligands (e.g., amine, carboxylate, thiol, and epoxide). The silane ligand can be selected from N-(trimethoxysilylpropyl)ethylenediaminetriacetic acid and (triethoxysilylpropyl)succinic anhydride to provide carboxylate-terminated magnetic particles. The functional group can be selected from phosphonic acid and catechol (to provide a hydrophilic tail group). The functional group can be selected from amino-terminated phosphonic acid. The functional group can be selected from 3-(trihydroxysilyl)propyl methylphosphonate for dispersion in aqueous solutions. The ligand may be selected from dihydroxyhydrocinnamic acid, citric acid, or thiomalic acid for the magnetizable particles to be dispersed in water.

[0112] In some embodiments, the magnetizable particles are functionalized with polymeric ligands, which may be selected from natural polymers (e.g., starch, dextran, or chitosan), PEG, polyacrylic acid (PAA), poly(methacrylic acid) (PMAA), poly(N,N-methylene-bisacrylamide) (PMBBAm), and poly(N,N / methylenebisacrylamide-coglycidyl methacrylate) (PMG).

[0113] The functional groups on the surface of the magnetizable particles function as linkers for binding with complementary biomolecules. The biomolecules can be small biomolecules. The small biomolecules can be selected from vitamins, peptides, and aptamers. The biomolecules can be larger biomolecules. The larger biomolecules can be selected from DNA, RNA, and proteins.

[0114] Regarding nucleic acid binding, nucleic acids can be bound by non-chemical methods (e.g., electrostatic interactions) or chemical methods (e.g., covalent bonds). The nucleic acid chain can be modified with functional groups. The functional groups can be selected from thiols or amines, or any combination thereof.

[0115] Binding of larger biomolecules can rely on specific binding interactions with a wide range of subtractive and synthetic analogs, such as specific receptor-substrate recognition (i.e., antigen-antibody and biotin-avidin interactions).

[0116] Specific protein pairs can be used to immobilize species on magnetic particles. Physical interactions include electrostatic, hydrophilic-hydrophobic, and affinity interactions.

[0117] In some embodiments, the biomolecule has a charge opposite to that of the magnetic polymer coating (e.g., polyethyleneimine or polyethylenimine), for example, a positively charged magnetizable particle that binds to negatively charged DNA.

[0118] Magnetizable particles can utilize biotin-avidin interactions, where biotin molecules and tetrameric streptavidin have site-specific attractive forces with minimal non-specific binding to control the orientation of interacting biomolecules, such as the exposure of the Fab region of an antibody to an antigen.

[0119] Magnetizable particles can be attached to biomolecules using covalent bonds selected from homobifunctional / heterobifunctional crosslinkers (amino groups), carbodiimide coupling (carboxyl groups), maleimide coupling (amino groups), direct reaction (epoxide groups), maleimide coupling (thiol groups), Schiff base condensation (aldehyde groups), and click reaction (alkyne / azide groups).

[0120] The magnetizable particles may have an average particle size of about 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm, and a suitable range may be selected from between any of these values ​​(e.g., about 5 to about 500, about 5 to about 400, about 5 to about 250, about 5 to about 100, about 5 to about 50, about 10 to about 500, about 10 to about 450, about 10 to about 300, about 10 to about 150, about 10 to about 50, about 50 to about 500, about 50 to about 350, about 50 to about 250, about 50 to about 150, about 100 to about 500, about 100 to about 300, about 150 to about 500, about 150 to about 450, or about 200 to about 500 nm).

[0121] The magnetizable particles may have an average particle size of about 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nm, and a suitable range may be selected from between any of these values ​​(e.g., about 500 to about 1000, about 500 to about 850, about 500 to about 700, about 550 to about 1000, about 550 to about 800, about 600 to about 1000, about 600 to about 900, about 650 to about 1000, about 650 to about 950, about 650 to about 800, or about 700 to about 1000 nm).

[0122] The magnetizable particles may have an average particle size of about 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 nm, and a suitable range may be selected between any of these values ​​(e.g., about 1000 to about 5000, about 1000 to about 4000, about 1500 to about 5000, about 1500 to about 4500, about 1500 to about 3500, about 2000 to about 5000, about 2000 to about 4000, about 2500 to about 5000, about 2500 to about 3500, about 3000 to about 5000 nm).

[0123] The variation in particle size of the magnetizable beads may be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%, and a suitable range may be selected between any of these values.

[0124] Microfluidics allows for faster analysis and shorter response times. Microfluidic systems also offer the ability to automate sample preparation, thereby reducing the risk of contamination and false positives due to human error. Additionally, microfluidic systems require small sample volumes. Microfluidics can reduce diffusion distances by increasing the surface area-to-volume ratio, reducing reagent consumption through micro- and nano-fabricated channels and chambers, and / or automating all steps of the process.

[0125] Microfluidics allows for miniaturization that enables lab-on-a-chip applications. Microfluidics can be used, for example, as part of a biosensor that includes channels for acquiring biological samples (e.g., saliva and / or gingival crevicular fluid), processing the fluid (e.g., combining it with one or more reagents and / or detecting interactions with biomolecules, etc.).

[0126] Microfluidics may require some degree of sample preparation. Sample preparation may include cell lysis, washing, centrifugation, separation, filtration, and elution. In some embodiments, sample preparation is performed off-chip. Alternatively, sample preparation is performed on-chip.

[0127] In some configurations, the microfluidic system may include rigid or flexible materials and may include electronics that may be integrated into the device. The electronics may include wireless communication electronics.

[0128] The microfluidic system can be a flow-through or a stationary system. For example, the microfluidic system can include a magnetic field sensor that is stationary relative to the microfluidic system.

[0129] Microfluidic systems can operate passively, for example, they can operate under passive diffusion, i.e., they do not require actively generated flow to perform effectively.

[0130] A microfluidic system can include a network of reservoirs, connected by microfluidic channels, which can be configured for active or passive metering, which can allow sample fluid to be drawn into the microfluidic channels and passed through to a sample chamber.

[0131] A microfluidic system may include microfluidic channels configured to allow access to various sample and / or detection regions on the device at different times. For example, a microfluidic device integrated within or on an aligner may be configured to provide timing through temporal sampling of fluids. For example, a microfluidic system can be designed to allow sampling in a chronological order and with controlled timing. In some variations, the timing of fluids within a microchannel may be actively timed, for example, by opening the channel via the release of a valve (e.g., electromechanical valve, solenoid valve, pressure valve). Examples of valves that control fluids in microfluidic networks include piezoelectric, electrokinetic, and chemical approaches.

[0132] The microfluidic device may include multiple microfluidic channels arranged in series. Fluid may be drawn into the microfluidic device at a metered rate. The timing of sample access to the channels may be staggered.

[0133] The device may perform signal multiplexing. That is, the device may be used to sample and / or measure multiple biomarkers at controlled intervals. For example, the device may be used to provide access to one or more sample chambers. The device may include one or more valves controlled by a control circuit in the device. The one or more valves may be connected to each other. Thus, the device may be adapted to perform simultaneous detection of multiple analytes in a common sample body. Additionally or alternatively, the device may be configured to perform simultaneous multiplex detection of multiple samples of the same target.

[0134] The microfluidic channels are approximately 0.001 to 0.01 mm 2 , 0.01~0.1mm 2 , 0.1~0.25mm 2 , 0.25~0.5mm 2 , 0.1~1mm 2 , 0.5~1mm, 2 , 1~2mm 2 , or 2 to 10 mm 2 and a useful range can be selected between any of these values.

[0135] In some embodiments, the microfluidic device accepts a predetermined sample volume in the range of about 0.1-1 μL, 1-5 μL, 5-10 μL, 10-20 μL, or 20-50 μL or more, and a useful range can be selected between any of these values.

[0136] 2 is an example of a microfluidic device 1. The microfluidic device 1 may include a plurality of channels 2 arranged to direct the flow of liquids and particles from a sample insertion region 4 towards a sensor 3.

[0137] The channels have cross-sectional dimensions as described above, more preferably about 0.1 mm 2The channels may have a diameter of 0.1 mm x 1.0 mm. The channels may be of variable length. For example, the channels may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, or 300 mm long, and a useful range may be selected between any of these values ​​(e.g., lengths of about 1-10, 1-20, 1-50, 1-100, 1-200, 1-300, 1-20, 10-40, 10-60, 10-80, 10-100, 50-100, 50-150, 50-200, 50-250, 50-300, 100-200, or 100-300 mm).

[0138] The above dimensions of the channel promote passive capillary flow.

[0139] In use, a sample is introduced into the microfluidic device 1 via the sample insertion region 4 .

[0140] In some embodiments, a filter membrane may be present in the insert region 4 to separate and pass desired components of the sample. For example, it may allow plasma from blood to pass through the microfluidic device 1, but not cells. The presence of a filter membrane depends on the nature of the sample and whether it contains components that are desired not to pass through the microfluidic device 1.

[0141] Once introduced into the insertion region 4, the sample then comes into contact with the microfluidic channel 2 and flows through the remainder of the channel circuit.

[0142] The microfluidic device 1 may include one or more magnetic sensors 3 in close proximity to the channels 2. For example, the microfluidic device 1 may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 magnetic sensors arranged around the microfluidic device 1. As shown in Figure 5, the microfluidic device 1 includes six magnetic sensors (6) located at the junctions of the channels 2.

[0143] In one embodiment, microfluidic device 1 includes two or more magnets, e.g., permanent magnets or electromagnets, arranged in close proximity to the channel 2 that can attract and activate magnetizable particles through the liquid in channel 2 to facilitate mixing. Mixing can be performed for, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, and an appropriate range can be selected between any of these values. The timing of mixing can depend on assay requirements such as sample volume, viscosity, composition, and detection range of the target analyte.

[0144] To effect mixing, magnets (e.g., electromagnets) can be placed at substantially opposing ends of the channel or of the microfluidic device 1. For example, the magnets can be controlled or switched to attract the magnetizable particles toward one end of the channel or microfluidic device 1 and then reversed to attract the magnetizable particles toward the other end of the channel or microfluidic device 1. This cycle can be repeated multiple times until the desired level of mixing is achieved.

[0145] The magnet can be an electromagnet that can exert a field strength of about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 Gauss, and a suitable range can be selected between any of these values.

[0146] When the sample is prepared for analysis, the magnet can then be controlled or switched to place the magnetizable particles in close proximity to the magnetic sensor. The magnet can exert a magnetic field strength of approximately 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 50, or 100 Gauss, and an appropriate range can be selected between any of these values. Sample data can then be acquired as described.

[0147] The magnetizable particles are sensed by a magnetic sensor.

[0148] The magnetic sensor may be selected from a spintronic sensor, an atomic magnetometer (AM), a nuclear magnetic resonance (NMR) system, a fluxgate sensor, a Faraday induction coil sensor, a diamond magnetometer, and a domain wall-based sensor.

[0149] Volume-based sensors, such as planar Hall effect (PHE) sensors, offer simple and rapid sample preparation and detection. Surface-based sensors, such as giant magnetoresistance (GMR), offer low detection limits (single particle) due to the short distance between the magnetizable particle and the sensor. However, these techniques typically require laborious sample and / or substrate preparation. Optimizing magnetizable particles for specific applications and selecting appropriate detection methods remains challenging for the magnetic nanotechnology community due to increasing demands for detection sensitivity, molecular specificity, and application complexity. Spintronic sensors can be selected from giant magnetoresistance (GMR), tunneling magnetoresistance (TMR), anisotropic magnetoresistance (AMR), and planar Hall effect (PHE) sensors.

[0150] The GMR effect was discovered in the 1980s and has traditionally been used in data recording. Spin valves offer higher sensitivity in micron-sized designs. Spin valve GMR sensors consist of an artificial magnetic structure with alternating ferromagnetic and non-magnetic layers. The magnetoresistance effect is caused by spin-orbit coupling between conduction electrons across different layers. Changes in magnetoresistance provide quantitative analysis with this spin-dependent sensor. GMR sensors can be used to detect DNA-DNA or protein (antibody)-DNA interactions. The dimensions of the sensor array can be tailored for the detection of individual magnetizable particles. GMR sensors can be used in combination with antiferromagnetic particles.

[0151] The planar Hall effect is an exchange-biased Permalloy planar sensor based on the anisotropic magnetoresistance effect of ferromagnetic materials. The PHE sensor can be a spin valve PHE or a PHE bridge sensor. The PHE sensor can perform single particle sensing.

[0152] Described is a method for detecting an analyte in a sample, comprising: contacting the sample containing the target analyte with magnetizable particles, the particles being coated with binding molecules complementary to the target analyte, resulting in bound and unbound binding agent complexes; placing the magnetizable particles, including both the bound and the unbound binding agent complexes, in proximity to a magnetic field sensor; varying the magnetic field sufficient to release at least a portion of the magnetizable particles, including both the bound and the unbound binding agent complexes, from their proximity to the magnetic field sensor; measuring a change in a magnetic signal detected from the net movement (i.e., translational or rotational movement) of said magnetizable particle relative to said magnetic sensor.

[0153] As shown in FIG. 1, a setup according to one embodiment of this method may broadly comprise a microfluidic device, a sensor, a magnet, a signal amplifier, an analog-to-digital converter, and a computer.

[0154] The target analyte can be any substance or molecule that is complementary to and capable of being bound by the binding molecule provided on the magnetizable particle. For example, the target analyte can be selected from the group consisting of proteins, peptides, nucleic acids, lipids, or carbohydrates.

[0155] The target analyte may be a protein or fragment thereof selected from the group consisting of an antibody, an enzyme, a signaling molecule, or a hormone.

[0156] The target analyte can be a nucleic acid selected from the group consisting of DNA, RNA, cDNA, mRNA, or rRNA.

[0157] The method can detect multiple target analytes in a single sample, for example, the method can detect two or more, three or more, four or more, five or more, ten or more, fifteen or more, twenty or more target analytes in a single sample.

[0158] The sample to be analyzed can be any sample that can contain one or more target analytes. For example, the sample can be a clinical, veterinary, environmental, food, forensic, or other suitable biological sample.

[0159] The clinical sample may be selected from a body fluid, for example, the body fluid may be selected from blood, sweat, saliva, urine, sputum, semen, mucus, tears, cerebrospinal fluid, amniotic fluid, gastric juice, gingival crevicular fluid, or interstitial fluid.

[0160] The environmental sample may be selected from the group consisting of water, soil, or aerosol.

[0161] An advantage of the present invention can be that sample preparation is not labor intensive or difficult to prepare, utilizing established biochemistry for the functionalization and attachment of molecules to either microfluidic or magnetizable particle surfaces.

[0162] The sample to be analyzed can be added directly to the sample well or microfluidic device without further processing.

[0163] The sample may be subjected to one or more sample processing steps. It will be understood that the appropriate sample processing step may depend on the type and / or nature of the sample being analyzed. In some embodiments, the sample processing step may be selected from the group including dilution, filtration, or extraction (e.g., liquid-liquid, solid-phase). For example, a whole blood sample may be filtered using a cellulose-based filter to separate the plasma that is analyzed.

[0164] The first step of the method may involve combining the sample to be analyzed with a preparation containing freely diffusible magnetizable particles coated with binding molecules (binder complexes) complementary to the target analyte in a sample well or sample reservoir. Where appropriate, the term "binder complexes" may be used interchangeably to refer to magnetizable particles that are coated with binding molecules.

[0165] In some embodiments, the magnetizable particles may have limited diffusivity. This may occur when the magnetizable particles are crosslinked or derivatized with a macromolecule. The macromolecule may be a hydrogel or a PEG linker. This may occur when the device is used in a multiplexed assay to detect multiple targets or samples in one sample.

[0166] The present method can improve the binding rate of binding molecules to target analytes by providing a binding agent complex that is mobile and freely diffusible in solution. When a sample and a preparation of the binding agent complex are combined, the binding agent complex is free to diffuse, and the binding molecule can interact with the target analyte throughout the sample volume. Because both the binding agent complex and the target analyte are freely diffusible and suspended within the sample volume, the average physical distance between the target analyte and the binding agent complex is likely to be small. Therefore, the binding rate is improved, and binding equilibrium can be achieved significantly faster.

[0167] In detection assays such as ELISA, binding molecules such as antibodies are immobilized on macroscale objects, such as the surface of a test well. In such methods, the physical distance between the target analyte and the antibody can vary significantly depending on the analyte's location in the sample volume. For example, a target analyte located near the top of the sample volume may be significantly farther away from the immobilized antibody and less likely to be captured and bound. Thus, the binding rate may be limited by the rate at which the target analyte diffuses within the sample volume toward the immobilized antibody.

[0168] The sample and the binding agent complex can be combined for an appropriate time to allow the binding molecules to reach binding equilibrium. In some embodiments, an appropriate time to allow binding to reach equilibrium can be about 1, 2, 3, 4, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, or 360 seconds, and a useful range can be selected between any of these values ​​(e.g., about 1 to 30, 1 to 60, 1 to 120, 10 to 30, 10 to 60, 10 to 90, 30 to 60, 30 to 90, 30 to 120, 60 to 90, 60 to 120, 60 to 180, 90 to 120, 90 to 180, 90 to 240, 180 to 240, 180 to 300, or 180 to 360 seconds).

[0169] A magnetic field generator can be used to induce magnetohydrodynamic mixing of the sample and improve the rate at which binding equilibrium is reached. In such embodiments, the magnetic field generator is used to induce movement of binding agent complexes within the sample volume.

[0170] A signal that allows for quantification of the analyte in the sample is generated by measuring the change in the magnetic field as the bound analyte migrates away from the magnetic field sensor.

[0171] The magnetic field sensor may be an on-chip magnetometer. The magnetic field sensor may have a sensitivity of at least 1 mV / V / Gauss. In some embodiments, the magnetic field sensor may detect and / or measure a magnetic field of at least about 10 mGauss, 1 mGauss, 100 μGauss, or 10 μGauss.

[0172] The magnetic field sensor may have multiple axes, for example, one, two, or three axes.

[0173] The magnetic field sensor may be a Honeywell HMC 1021S magnetometer. In another embodiment, the magnetic field sensor may be a Honeywell HMC1041Z magnetometer. In other embodiments, the magnetic field sensor may be selected from the group including a Honeywell HMC 1001, HMC 1002, HMC 1022, HMC 1051, HMC 1052, HMC 1053, or HMC 2003 magnetometer.

[0174] The magnetic field sensor may comprise a custom magnetic field sensor having custom components.

[0175] Multiple magnetic field sensors can be used simultaneously to measure changes in the magnetic field, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 magnetic field sensors for small, portable applications.

[0176] The magnetic field sensors can be provided in a relatively small area within the device. For example, 24 magnetic field sensors can be provided in an area of ​​approximately 13 mm x 19 mm. Such a configuration allows for faster sample-to-data times due to the shorter microfluidic channels used in this magnetic field sensor configuration, as discussed above in paragraphs

[0081] -

[0082] . This configuration allows for a more compact and portable device.

[0177] The device is mounted on a 1cm 2 The system may have approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 magnetic field sensors per square centimeter, and a useful range may be selected between any of these values ​​(e.g., 1 cm of a printed circuit board). 2 about 5 to about 15, about 5 to about 13, about 5 to about 10, about 6 to about 15, about 6 to about 12, about 6 to about 9, about 7 to about 15, about 7 to about 14, about 7 to about 13, about 7 to about 10, about 8 to about 15, about 8 to about 14, about 8 to about 11, about 9 to about 15, about 9 to about 13, or about 10 to about 15 sensors per

[0178] In some embodiments, multiple magnetic field sensors can be used simultaneously to measure changes in the magnetic field, for example, 50, 60, 70, 80, 90, 100, 110, or 120 magnetic field sensors for small portable applications and in situ laboratory or clinical applications, and a useful range can be selected between any of these values ​​(e.g., about 50 to about 120, about 50 to about 100, about 50 to about 90, about 50 to about 80, about 60 to about 120, about 60 to about 110, about 60 to about 90, about 70 to about 110, about 70 to about 90, about 80 to about 120, or about 80 to about 110 magnetic field sensors).

[0179] In some embodiments, multiple magnetic field sensors can be used simultaneously to measure changes in a magnetic field, for example, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, or 3000 magnetic field sensors for laboratory or clinical, research, or industrial applications.

[0180] A further step of the method may include applying a magnetic field to the sample to place the binding agent complexes in proximity to a magnetic field sensor. The magnetic field generator described in paragraph

[0171] may be used to generate a magnetic field to manipulate the bound and unbound binding agent complexes to a position where the magnetic field sensor can effectively measure changes in the magnetic field generated by the magnetizable particles.

[0181] In some embodiments, the binding agent complex may be placed in proximity to the magnetic field sensor using microfluidic, acoustic, piezoelectric, or other suitable means, hi other embodiments, the binding agent complex may be placed by centrifugation.

[0182] In some embodiments, the magnetic field can be generated in a direction that moves the magnetizable particles in the sample volume toward the magnetic field sensor. The magnetic field sensor can be provided at any location relative to the test well or the microfluidic device. For example, if the magnetic field sensor is located below the test well or sample reservoir, the magnetic field moves the magnetizable particles toward the bottom of the test well or sample reservoir. In another example, if the magnetic field sensor is located above the test well or sample reservoir, the magnetic field moves the magnetizable particles toward the top of the test well or sample reservoir.

[0183] In some embodiments, the magnetic field generated can be static or dynamic.

[0184] In some embodiments, the strength of the generated magnetic field can be adjusted.

[0185] Without wishing to be bound by theory, the adjustment of this magnetic field (i.e., the bias field) has the primary function of aligning the magnetizable particles to the sensor during detection to achieve the highest sensitivity of detection. In the case of ferromagnetic particles, if they have their own permanent magnetic field, the bias field will be turned off, resulting in misalignment of the magnetic particles. In the case of paramagnetic (or superparamagnetic) particles, their magnetic field needs to be induced by an external magnetic field, so the bias field serves the additional function of inducing such a magnetic field.

[0186] The bias field can be adjusted to support different magnetizable particles, as different particles (whether due to chemical composition or physical size) may require different bias field strengths and configurations.

[0187] In some embodiments, the magnetic field can be generated and positioned to maximize its effect on the magnetizable particles but minimize its effect on the magnetic sensor. The magnetic field generator can be generated and / or positioned in close proximity to the magnetic field sensor. In some embodiments, the magnetic field generator is positioned above, below, or to the side of the magnetic field sensor. In some embodiments, the magnetic field generator can be positioned in the same vertical or horizontal plane as the magnetic field sensor.

[0188] A further step of the method may include varying the magnetic field sufficiently to release at least a portion of the binding agent complexes from their proximity to the magnetic field sensor when the bound and unbound binding agent complexes are placed in proximity to the magnetic field sensor.

[0189] In some embodiments, the magnetic field may be gradually decreased.

[0190] In some embodiments, the magnetic field may be immediately removed.

[0191] In some embodiments, the magnetic field can be variable in shape.

[0192] As the magnetic field applied to the sample is reduced and / or removed, the bound and unbound binding agent complexes are released from the magnetic field and may freely diffuse (translational movement) from their proximity to the magnetic field sensor. The binding agent complexes may also rotate (rotational movement) relative to the magnetic field sensor as the magnetic field applied to the sample is reduced and / or removed.

[0193] According to this method, bound and unbound binder complexes can be distinguished based on changes in molecular diffusion properties according to Graham's law of molecular diffusion, which states that the diffusion rate is inversely proportional to the square root of its molecular weight. The diffusion rate can be calculated using the following equation:

number

[0194] The binding agent complex bound to the target analyte has a larger molecular weight compared to the unbound binding agent complex, which will have a faster diffusion rate according to Graham's law. Therefore, the bound and unbound binding agent complexes can be distinguished based on their kinetic profiles.

[0195] A further step of the method may include measuring changes in the magnetic signal detected from the magnetizable particles as they move (via translational or rotational motion) relative to a magnetic field sensor. The magnetic field sensor measures changes in the magnetic field strength generated by the magnetizable particles over time, as described in detail in the previous paragraph. The method uses magnetic field changes over time, requiring only one binding molecule for binding of the target analyte.

[0196] In some embodiments, the magnetic field change over time can be determined by measuring the magnetoresistance effect and the signal decay over time.

[0197] The magnetic field signal generated by a magnetizable particle with respect to a magnetic field sensor follows the magnetic dipole field equation.

number

number

[0198] Based on the magnetic dipole field equation, the detected signal falls off as the cube of the distance from the magnetic field sensor. This phenomenon, combined with the diffusion kinetics described above, can be used for the signal generation described in the previous paragraph.

[0199] Due to the high diffusion rate of unbound binding agent complexes, they can move away from the sensor at a faster rate than binding agent complexes bound to the target analyte. The difference in diffusion rate generates a magnetic field decay signal over time. The decay rate depends on the molecular weight of the bound and unbound binding agent complexes, with unbound binding agent complexes having a faster decay rate than bound binding agent complexes.

[0200] The decay rate can be modeled with a decay curve, which can be used to distinguish between bound and unbound binding agent conjugates. For example, an accelerated decay curve can indicate an unbound binding agent conjugate, and a slowed decay curve can indicate a bound binding agent conjugate.

[0201] The method may involve multiple repetitions of the following steps to generate a signal curve over time that distinguishes between bound and unbound binding agent complexes to quantify the target analyte. Applying a magnetic field to place magnetizable particles in proximity to a magnetic field sensor. · Varying the magnetic field sufficiently to release at least a portion of the magnetizable particles from their proximity to the magnetic field sensor. Measuring the change in the magnetic signal detected from the magnetic particle as it moves away from the magnetic sensor.

[0202] The method may include a reference calibration step by measuring the total magnetic field strength generated by the bound or unbound binding agent complex.

[0203] The magnetic field signal generated by the magnetizable particles may be due to intrinsic properties of the magnetizable particles or may be induced by an external magnetic field.

[0204] The magnetic field sensor is positioned to maximize its sensing of the magnetizable particles but minimize its sensing of the magnetic field generator.

[0205] The magnetic field or signal from a magnetizable particle can be intrinsic to its atomic structure or can be induced by an external magnetic field.

[0206] Data acquisition by the sensor can be synchronized with the microfluidic device. This can allow detected data from the sensor to be characterized as either sample data or environmental or ambient data. For example, detection of a signal by a magnetic sensor without sample injection into the microfluidic device characterizes the data as environmental or ambient data. Characterizing the data as environmental or ambient data can be useful for establishing background and can also be useful for preparing calibration data.

[0207] If the magnetic sensor detects a signal following injection of a sample into a microfluidic device that is consistent with the placement of magnetizable particles in close proximity to the magnetic sensor, such data can be characterized as sample data.

[0208] Data acquisition from the sensor can be continuous, i.e., the magnetic sensor transmits a signal continuously, and the data is characterized as sample data or background data based on the synchronization of data collection with the injection of the sample into the microfluidic device.

[0209] Sensor data can be acquired over a period of time to measure changes in the magnetic signal from the magnetizable particles. Actions or events can be inferred from changes in the sensed magnetic signal. Actions or events can include movement of the magnetizable particles from fluid flow, from external magnetic forces, or from diffusion.

[0210] The method may include processing raw data output from the magnetic field sensor to quantify the amount of target analyte in the sample. The raw data processing may be performed using a combination of hardware and software implementations as detailed in the previous paragraph.

[0211] Processing the output raw data may include amplifying the signal output from the magnetic field sensor using a signal amplifier. The signal output from the magnetic field sensor may be a voltage reading proportional to the sensed magnetic field. In some embodiments, the signal amplifier is a Texas Instruments INA819 amplifier.

[0212] The voltage reading from the magnetic field sensor can be amplified in magnitude to a higher voltage (proportional to the original voltage reading) that is compatible with the data processing and acquisition electronics.

[0213] Processing the output raw data may further include converting the analog data output from the magnetic field sensor to output digital data, for example, voltage readings may be converted to a digital bit stream that can be recorded by a computer.

[0214] The analog to digital conversion may be performed using an analog-to-digital converter (ADC).

[0215] The conversion or sampling resolution can be 16, 24, 32, 64, 128, 256, or 512 bits.

[0216] The analytical performance of a detection method is often evaluated by measuring a dose-response curve from which the limit of detection (LoD) can be derived. The LoD is the lowest amount of a substance, such as a biomarker, that can be detected with a selected confidence level. The selected assay (biomarker, biological material, sample matrix, incubation time, etc.) can have a strong influence on the LoD. Alternatively, the limit of quantification (LoQ), which is the lowest biomarker concentration that can be quantified with a specific required accuracy, is used. The LoQ approaches the LoD when the dose-response curve has good sensitivity, i.e., when the signal changes significantly as a function of target concentration.

[0217] The method may provide an LoQ of about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 pg / mL, and a suitable range may be selected between any of these values.

[0218] The method may provide an LoD of about 0.1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pg / mL, and a suitable range may be selected between any of these values.

[0219] The present invention describes methods, reagents and systems for detecting and quantifying an analyte in a sample.

[0220] High-throughput screening (HTS) systems allow for a large number of assays to be performed in a relatively short period of time. HTS systems may include microplates, microplate readers, robotic liquid handling, and microplate handling platforms. In some embodiments, one or more steps of the presently described methods may be performed using an HTS system.

[0221] In some embodiments, the microplate may include, for example, 6, 12, 24, 48, 96, 384, or 1536 sample wells.

[0222] In some embodiments, the magnetic sensors may be provided in a microplate.

[0223] In some embodiments, a robotic liquid handling device may be used to dispense samples and / or reagents onto the microplates.

[0224] In some embodiments, the methods can be partially or fully automated using an HTS system.

[0225] The device for detecting an analyte may be operable in any orientation. The operation of the device or the performance of the method does not depend on gravity to function effectively. That is, the device can perform the method regardless of how the device is oriented. For example, the device may be operable in an inverted configuration in which the magnetic field sensor is oriented above the sample reservoir or microfluidic device.

[0226] It will be appreciated that the present method may be broadly used in any application requiring the detection and / or quantification of a target analyte. In particular, the present method is useful for detecting the presence of a target analyte in a sample. i) Rapid decision; or ii) Highly sensitive determination, or iii) a quantitative determination; or iv) Any combination of (i) to (iii) It can be used in applications requiring

[0227] For example, suitable applications may include clinical, veterinary, environmental, food safety, or forensic applications.

[0228] In some embodiments, clinical applications may include the diagnostic detection of biomarkers in samples that may indicate a clinical condition. In one example, the method may be used for the rapid, sensitive, and quantitative diagnostic detection of specific antibodies in blood samples that may indicate possible infection by a pathogen. In a further example, the method may be used for the diagnostic detection of specific protein biomarkers that are overexpressed in cancer. Diagnostic detection may be performed on samples across different species.

[0229] The clinical condition may be selected from infectious diseases such as infections from bacteria, fungi, viruses (e.g., hepatitis and HIV) (e.g., biomarkers such as hepatitis and HIV antibodies), parasites (e.g., microbial parasites [e.g., malaria], nematodes, insect parasites).

[0230] The clinical condition may be selected from diseases such as cardiac disease (biomarkers such as BNP), cancer (e.g., solid organ cancer, blood cancer, other cancers), (e.g., biomarkers such as Ca-125 and other tumor markers), neurological diseases (e.g., multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease) (e.g., biomarkers such as CNS immunoglobulins), respiratory diseases (e.g., biomarkers such as serum ACE), liver diseases (e.g., biomarkers such as liver function tests and albumin), kidney diseases (e.g., biomarkers such as creatinine and protein).

[0231] The clinical condition may be selected from organ injury or failure such as brain injury (e.g., a biomarker such as glial fibrillary acidic protein or GFAP), kidney injury (e.g., a biomarker such as serum creatine), heart injury (e.g., a biomarker such as creatine kinase-muscle), lung injury (e.g., a biomarker such as intercellular adhesion molecule-1 or ICAM1), or liver injury (e.g., a biomarker such as alkaline phosphatase).

[0232] The clinical condition may be selected from endocrine diseases such as diabetes (e.g., biomarkers such as insulin, hypertension, HbA1C, thyroid dysfunction, thyroid hormones), pituitary disorders (e.g., biomarkers such as ACTH, prolactin, gonadotrophins, thyroid-stimulating hormone, growth hormone, antidiuretic hormone), parathyroid disorders (e.g., biomarkers such as parathyroid hormone), adrenal disorders (e.g., biomarkers such as cortisol, aldosterone, adrenaline, DHEAS), sex hormone imbalance (e.g., biomarkers such as androgens and estrogens), carcinoid tumors (e.g., biomarkers such as 5-HIAA, VIPoma, serum VIP), and elevated bone turnover (e.g., biomarkers such as P1NP).

[0233] The clinical condition may be selected from lipid disorders (eg, biomarkers such as cholesterol and triglycerides).

[0234] The clinical condition may be selected from a nutritional disorder (eg, vitamin deficiency, malabsorption syndrome, malnutrition, vitamin metabolism disorder), (eg, biomarkers of vitamin levels, iron levels, mineral levels, etc.).

[0235] The clinical condition may be selected from inflammation or inflammatory disorders (eg, biomarkers such as ESR, Crp and other acute phase proteins).

[0236] The clinical condition may be selected from an autoimmune disease (eg, a biomarker such as a specific antibody marker).

[0237] The clinical condition may be selected from allergic diseases (eg, biomarkers such as tryptase).

[0238] The clinical condition may be selected from physical trauma such as electrocution (eg, a biomarker such as creatine kinase).

[0239] The clinical condition can be selected from an immunodeficiency disorder (eg, common variable immunodeficiency) (eg, biomarkers such as complement, leukocytes, and immunoglobulins).

[0240] The clinical condition may be selected from a coagulation disorder (eg, thrombosis) (eg, biomarkers such as biomarkers for clotting factors and other markers).

[0241] The clinical condition may be selected from inherited or acquired enzyme disorders, deficiencies or excesses, and other congenital or acquired metabolic disorders (e.g., Bartter's syndrome, congenital adrenal hyperplasia) (e.g., biomarkers such as electrolytes, enzyme levels, metabolic products of enzymes, etc.).

[0242] The clinical condition may be selected from electrolyte disorders (eg, biomarkers such as electrolytes) such as hyperkalemia and hypernatremia.

[0243] The clinical condition may be selected from drug side effects or addiction (eg, biomarkers such as drug levels and drug metabolite levels).

[0244] Specifically for veterinary medicine, the clinical condition may be selected from renal failure, FIV / AIDS (feline), cancer, and any biomarker for organ function / failure.

[0245] In some embodiments, the clinical condition can be a condition in a veterinary subject, such as a feline, canine, bovine, ovine, equine, porcine, or murine.

[0246] In some embodiments, environmental applications may include the detection of contaminants in environmental samples. The environmental contaminants may be selected from contaminants such as lead, particulate matter, microplastics, and hormones.

[0247] For example, the method can be used to monitor and quantify heavy metals in water samples.

[0248] In some embodiments, food safety applications may include the detection of pathogens in food samples. For example, the method may be used to rapidly and sensitively detect post-pasteurization contamination in milk with bacterial pathogens. [Example]

[0249] Example 1: Sensitivity and Limit of Detection The aim of this study was to examine the sensitivity of detection.

[0250] A specific amount of magnetizable particles was added to the microfluidic system for detection. The system setup is summarized below. Magnetic sensor: Honeywell HMC 1021S magnetometer Magnetic particles: Thermo Fisher Dynaparticles T1 (1 μm) streptavidin particles Biolabel: Streptavidin Amplifier: Texas Instruments INA826 Number of particles: Sample 1: Control - 0 pg of particles Sample 2: 0.5 pg of particles Sample 3: 5 pg of particles Sample 4: 50 pg of particles Sample 5: 500 pg of particles Sample 6: 50,000 pg of particles Sample 7: 500,000 pg of particles Sensor data acquisition: 0.012 seconds per reading 〇1,200 readings per sample Total reading time: approx. 15 seconds

[0251] After being introduced into the microfluidic system, the particles were positioned on the sensor by the microfluidic device. A magnet was activated to bring the magnetizable particles into close proximity to the magnetic sensor. The magnet was then turned off, and a permanent magnet placed below the sensor was used to generate a bias field. The magnetic field sensor measured the change in magnetic field strength over time as the magnetizable particles diffused away from the magnetic sensor. The device determines the amount of analyte in a sample by measuring the net movement of the magnetizable particles relative to the magnetic field sensor.

[0252] Sensor data was acquired for each sample.

[0253] The sensor data was then processed as follows: a moving average filter with a 30 sample window was applied, and the data was averaged over time and normalized to the negative control sample (sample 1).

[0254] FIG. 3 is a graph showing the detected signal, with the vertical axis expressed as signal in arbitrary units (au).

[0255] The amount of particles is expressed as picograms (pg) on ​​the horizontal axis.

[0256] The results demonstrate improved sensitivity and signal acquisition with higher particle numbers.

[0257] The graph in Figure 3 is a sensitivity plot, demonstrating an LoQ of approximately 0.5 pg.

[0258] Example 2: Speed ​​of detection The purpose of this study was to examine the speed of the detection system.

[0259] A specific amount of magnetizable particles was added to the microfluidic system for detection. The system setup is summarized below. Magnetic sensor: Honeywell HMC 1021S magnetometer Magnetic particles: Thermo Fisher Dynaparticles T1 (1 μm) streptavidin particles Biolabel: Streptavidin Amplifier: Texas Instruments INA826 Number of particles: Sample 1: Control - 0 pg of particles Sample 2: 50 pg of particles Sample 3: 500,000 pg of particles Sensor data acquisition: 0.012 seconds per reading 〇1,200 readings per sample Total reading time: approx. 15 seconds

[0260] After being introduced into the microfluidic system, the particles were positioned on the sensor by the microfluidic device. A magnet was activated to bring the magnetizable particles into close proximity to the magnetic sensor. The magnet was then turned off, and a permanent magnet placed below the sensor was used to generate a bias field. The magnetic field sensor measured the change in magnetic field strength over time as the magnetizable particles diffused away from the magnetic sensor. The device determines the amount of analyte in a sample by measuring the net movement of the magnetizable particles relative to the magnetic field sensor.

[0261] Sensor data was acquired for each sample.

[0262] The sensor data were then processed as follows: a moving average filter with a 30 sample window was applied and the data was normalized to the negative control sample (sample 1).

[0263] Shown in FIG. 4 is a graph showing signal acquisition over time, with the vertical axis expressed as signal in arbitrary units (au).

[0264] The time point of each reading was expressed as seconds (s) on the horizontal axis.

[0265] The results demonstrate that the method can acquire sufficient signal within 15 seconds to qualitatively measure and identify the amount of particles present in a sample.

[0266] The graph in Figure 4 is a time versus data acquisition plot, demonstrating fast sample detection and data collection in less than 15 seconds.

[0267] The graph in Figure 4 shows that the above system responds to very small amounts of particles (i.e., picogram range) and can be rapidly detected and identified in a matter of seconds.

[0268] Example 3: Detection of streptavidin protein in a sample The objective of this study was to demonstrate the quantitative detection of streptavidin protein in samples as a target analyte.

[0269] Biotin conjugated to latex particles (non-magnetic particles) was used to capture and associate with a specific amount of streptavidin added to the microfluidic system for detection. The system setup is summarized below. Magnetic sensor: Honeywell HMC 1021S magnetometer Magnetic particles: Thermo Fisher Dynaparticles T1 (1µm) streptavidin particles Biolabel: Streptavidin Amplifier: Texas Instruments INA826 ·sample Sample 1: 0 pmol / ml streptavidin protein bound to magnetizable particles Sample 2: 0.33 pmol / ml streptavidin protein bound to magnetizable particles Sample 3: 3.3 pmol / ml streptavidin protein bound to magnetizable particles Sample 4: 33 pmol / ml streptavidin protein bound to magnetizable particles Sample 5: 330 pmol / ml streptavidin protein bound to magnetizable particles Sensor data acquisition: 0.012 seconds per reading 〇1,200 readings per sample Total reading time: approx. 15 seconds

[0270] After being introduced into the microfluidic system, the particles were positioned on the sensor by the microfluidic device. A magnet was activated to bring the magnetizable particles into close proximity to the magnetic sensor. The magnet was then turned off, and a permanent magnet placed below the sensor was used to generate a bias field. The magnetic field sensor measured the change in magnetic field strength over time as the magnetizable particles diffused away from the magnetic sensor. The device determines the amount of analyte in a sample by measuring the net movement of the magnetizable particles relative to the magnetic field sensor.

[0271] Sensor data was acquired for each sample.

[0272] The sensor data was then processed as follows: a moving average filter with a 30 sample window was applied, and the data was averaged over time and normalized to the negative control sample (sample 1).

[0273] Shown in Table 1 is the signal detected via biotin capture and binding of streptavidin protein, and is the signal in arbitrary units (au). [Table 1]

[0274] Table 1 demonstrates that the method of the present invention can detect levels of streptavidin at or below 3.3 pmol / mL.

[0275] Example 4: Sensitivity and Limits of Quantification The aim of this study was to demonstrate the sensitivity and quantitative detection of biomarkers in samples from different species.

[0276] Magnetizable particles were functionalized with recombinant antibodies, each targeting a specific biomarker. The functionalized magnetizable particles were used to capture and correlate specific concentrations of the biomarker in each sample added to the microfluidic system for quantification. The system setup is summarized below. Magnetic sensor: Honeywell HMC 2003 magnetometer Magnetic particles: Nanocs MP25-AV (30 nm diameter) chemically functionalized with antibodies: Anti-human CRP detection antibody (R&D Systems DY1707) Anti-human albumin detection antibody (R&D Systems DY1455) Anti-canine IL-6 detection antibody (R&D Systems DY1609) Anti-canine VEGF-A detection antibody (R&D Systems DY1603) Anti-feline TNFα detection antibody (R&D Systems DY2586) Anti-feline GM-CSF detection antibody (R&D Systems DY987) Anti-equine TNFα detection antibody (R&D Systems DY1814) Amplifier: Honeywell HMC 2003 built-in amplifier Sensor data acquisition: 0.007 seconds per reading 〇1,000 readings per sample Total reading time: approx. 10 seconds Sample (recombinant protein): Human CRP Human albumin Canine IL-6 Canine VEGF-A 〇Feline TNFα 〇Feline GM-CSF Equine TNFα

[0277] After being introduced into the microfluidic system, the particles were positioned on the sensor by the microfluidic device. The magnet was activated to bring the magnetizable particles into close proximity to the magnetic sensor ("capture" step). The magnet was then turned off ("release" step). The magnetic field sensor measured the change in magnetic field strength over time as the magnetizable particles diffused away from the magnetic sensor.

[0278] Sensor data was acquired for each sample.

[0279] The sensor data was then processed as follows: Data was averaged across the first 10 readings and then normalized to each relative negative control sample.

[0280] Shown in Table 2 are the sensor values ​​(in arbitrary units [au]) detected via antibody capture and association with biomarkers for each sample. [Table 2]

[0281] The results in Table 2 demonstrate limits of quantitation in the range of 0.1 pg / mL across a range of biomarkers for various species. The results also demonstrate biomarker detection across six orders of magnitude, from 0.1 to 10,000 pg / mL. According to aspect (1), there is provided a method for detecting an analyte in a sample, comprising: contacting the sample containing the target analyte with magnetizable particles, the particles being coated with binding molecules complementary to the target analyte, resulting in bound and unbound binding agent complexes; placing the magnetizable particles, including both the bound and the unbound binding agent complexes, in proximity to a magnetic field sensor; varying the magnetic field sufficiently to release at least a portion of the magnetizable particles, including both the bound and the unbound binding agent complexes, from proximity to the magnetic field sensor; measuring a change in the detected magnetic signal from a net movement, either translational or rotational, of the magnetizable particle relative to the magnetic sensor. According to aspect (), the method comprises: a sample well or sample reservoir; one or more magnets for generating the magnetic field in the sample well or the sample reservoir; and a magnetic field sensor for measuring changes in the magnetic field over time in the sample well or sample reservoir. According to aspect (3), providing the sample inspection device further includes varying the magnetic field sufficient to release at least a portion of the magnetizable particles from proximity to the magnetic sensor. According to embodiment (4), the detection and quantification of the analyte in the sample depends on the amount of the magnetizable particles detected via the magnetic field sensor. According to embodiment (5), the magnetizable particles are functionalized with molecules that specifically bind to the analyte. According to aspect (6), the sample and the magnetizable particles are processed by a microfluidic device. According to aspect (7), the microfluidic device promotes binding between the magnetizable particles and the analyte. According to aspect (8), the magnetizable particles are magnetizable particles. According to embodiment (9), the magnetizable particles are paramagnetic or ferromagnetic. According to embodiment (10), the magnetizable particles have an average particle size of about 5 to about 5000 nm. According to aspect (11), the microfluidic device positions the magnetizable particles and the analyte in close proximity to the magnetic sensor. According to aspect (12), the magnetizable particles and the analyte are brought within 1 to 5,000 μm of the sensing element of the magnetic sensor. According to aspect (13), the one or more magnets (or electromagnets) align the magnetizable particles. According to aspect (14), the one or more magnets generate the magnetic field that changes over time. According to aspect (15), the magnetic field generator can generate a continuum of magnitudes. According to aspect (16), the magnetic field generator can alternately switch the magnetic field between on and off. According to aspect (17), the magnetic field is generated and positioned to maximize its influence on the magnetizable particles but minimize its influence on the magnetic sensor. According to aspect (18), the magnetic field sensor measures changes over time in the magnetic field strength generated by the magnetizable particles. According to aspect (19), the magnetic field sensor is adapted to maximize sensing of the magnetizable particles and minimize sensing from the magnet. According to aspect (20), data acquisition by the sensor is synchronized with the microfluidic device. According to aspect (21), data is continuously acquired from the sensor. According to aspect (22), the acquired data is flagged as either (1) environmental and / or ambient, or (2) sample data. According to aspect (23), the method is calibrated based on synchronization of the signal acquisition with the operation of the microfluidic device. According to aspect (24), the data is acquired over a period of about 1 second to about 60 seconds. According to aspect (25), the signal output from the magnetic field sensor is increased by a signal amplifier. According to aspect (26), the signal output from the magnetic field sensor is a voltage reading proportional to the magnetic field strength it senses. According to aspect (27), the voltage from the magnetic field sensor is increased in magnitude to a higher voltage while keeping all changes proportional to the original signal, within a range compatible with the data processing and acquisition electronics. According to aspect (28), the amplified signal is converted from a voltage reading to a digital bit stream and recorded and / or analyzed by a computer. According to aspect (29), the conversion is performed by an analog-to-digital converter (ADC). According to aspect (30), The method comprises: a) generating within 15 seconds a magnetic signal sufficient to detect and / or measure the amount of said target analyte in said sample; or b) has a limit of detection (LOD) of at least about 0.05 pg / mL; or c) has a limit of quantitation (LOQ) of at least about 0.1 pg / mL; or d) One or more of (a) to (c). According to aspect (31), there is provided a device for carrying out any one of the methods of aspects (1) to (30), the device comprising the magnetic field sensor, one or more magnets for generating the magnetic field, and a sample well or sample reservoir. According to aspect (30), there is provided a device for detecting an analyte in a sample, comprising: a sample well separate from or integrated into the microfluidic device; one or more magnets for generating a magnetic field in the sample well; a magnetic field sensor for measuring changes in the magnetic field over time proximate the sample well; a device wherein the one or more magnets are adapted to control the position of magnetizable particles relative to the magnetic field sensor, and the magnetic sensor is adapted for use in detecting changes in net motion, either translation or rotation, of the magnetizable particles relative to the magnetic sensor. According to aspect (33), the device is operable in any orientation.

Claims

1. 1. A method for detecting an analyte in a sample, comprising: - taking a baseline reading of magnetic field strength in the absence of said sample; providing the sample, which comprises a target analyte and magnetizable particles coated with binding molecules complementary to the target analyte, to a microfluidic device; contacting the sample containing the target analyte with the magnetizable particles, resulting in bound and unbound binding agent complexes; placing the magnetizable particles, including both the bound and the unbound binding agent complexes, in proximity to a magnetic field sensor; - varying the magnetic field sufficiently to release at least a portion of the magnetizable particles, including both the bound and the unbound binding agent complexes, from proximity to the magnetic field sensor; - Determining the amount of analyte in the sample by measuring a change in magnetic signal detected from a net movement, selected from translational or rotational movement, of the magnetizable particles relative to the magnetic field sensor.

2. The method comprises: a sample well or sample reservoir; one or more magnets for generating the magnetic field in the sample well or the sample reservoir; 10. The method of claim 1, further comprising providing a sample testing device comprising: a magnetic field sensor for measuring changes in the magnetic field over time in the sample well or sample reservoir.

3. 3. The method of claim 2, further comprising providing the sample inspection device including varying the magnetic field sufficient to release at least a portion of the magnetizable particles from proximity to the magnetic field sensor.

4. The method of any one of claims 1 to 3, wherein the detection and quantification of the analyte in the sample depends on the amount of the magnetizable particles detected via the magnetic field sensor.

5. The method of any one of claims 1 to 4, wherein the magnetizable particles are functionalized with molecules that specifically bind to the analyte.

6. The method of any one of claims 1 to 5, wherein the sample and the magnetizable particles are processed by a microfluidic device.

7. The method of claim 6 , wherein the microfluidic device facilitates binding between the magnetizable particles and the analyte.

8. The method of any one of claims 1 to 7, wherein the magnetizable particles are magnetic particles.

9. The method of any one of claims 1 to 8, wherein the magnetizable particles are paramagnetic or ferromagnetic.

10. The method of any one of claims 1 to 9, wherein the magnetizable particles have an average particle size of about 5 to about 5000 nm.

11. 8. The method of claim 6 or claim 7, wherein the microfluidic device places the magnetizable particles and the analyte in close proximity to the magnetic field sensor.

12. The method of any one of claims 1 to 11, wherein the magnetizable particles and the analyte are brought within 1 to 5,000 μm of a sensing element of the magnetic field sensor.

13. The method of claim 2 , wherein the one or more magnets (or electromagnets) align the magnetizable particles.

14. 14. The method of claim 2 or claim 13, wherein the one or more magnets generate the magnetic field that varies over time.

15. A method according to any one of the preceding claims, wherein the magnetic field generator is capable of switching the magnetic field alternately between on and off.

16. A method according to any preceding claim, wherein the magnetic field is generated and arranged to maximise its effect on the magnetisable particles but minimise its effect on the magnetic field sensor.

17. A method according to any preceding claim, wherein the magnetic field sensor measures changes over time in the magnetic field strength generated by the magnetisable particles.

18. 15. The method of any one of claims 2, 13 and 14, wherein the magnetic field sensor is adapted to maximize sensing of the magnetizable particles and minimize sensing from the magnet.

19. 12. The method of claim 6, claim 7, or claim 11, wherein data acquisition by the magnetic field sensor is synchronized with the microfluidic device.

20. The method of any one of claims 1 to 19, wherein data is acquired continuously from the magnetic field sensor.

21. 20. The method of claim 19, wherein the method is calibrated based on synchronization of data acquisition by the magnetic field sensor with operation of the microfluidic device.

22. The method of any one of claims 19 to 21, wherein the data is acquired over a period of from about 1 second to about 60 seconds.

23. A method according to any preceding claim, wherein the signal output from the magnetic field sensor is increased by a signal amplifier.

24. A method according to any preceding claim, wherein the signal output from the magnetic field sensor is a voltage reading proportional to the magnetic field strength it senses.

25. 25. A method according to any one of claims 1 to 24, wherein the voltage from the magnetic field sensor is increased in magnitude to a higher voltage whilst keeping all changes proportional to the original signal, within a range compatible with data processing and acquisition electronics.

26. A method described in any one of claims 1 to 25, wherein the amplified signal is converted from a voltage reading into a digital bit stream and recorded and / or analyzed by a computer.

27. 27. The method of claim 26, wherein the conversion is performed by an analog-to-digital converter (ADC).

28. A device for detecting an analyte in a sample implemented by the method for detecting an analyte in a sample of claim 1, comprising: a passive microfluidic device comprising a sample insertion region, a non-sample well region, and a sample well region located therebetween, the sample well region comprising one or more sample wells and one or more channels connected to one or more of the sample wells, and the non-sample well region having one or more channels and no sample wells; one or more magnets for generating a magnetic field in the sample well; the magnetic field sensor disposed in the sample well to measure changes over time in the magnetic field proximate the sample well; - A device wherein the one or more magnets are adapted to control the position of the magnetizable particles relative to the magnetic field sensor, and the magnetic field sensor is adapted for use in detecting changes in net motion, either translation or rotation, of the magnetizable particles relative to the magnetic field sensor.

29. 30. The device of claim 28, wherein the device is operable in any orientation.

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