A high-throughput biological macromolecule detection and separation integrated method and system

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a high-throughput biological macromolecule detection and separation integrated method and system, which comprises the following steps: providing a magnetic nanoparticle sample with uniform particle size, connecting biological macromolecules with different molecular weights or conformations on the surface of each magnetic nanoparticle to obtain a to-be-detected sample solution; after a background magnetic field with a specific intensity and direction and a gradient magnetic field with a certain gradient size are applied to the solution, the magnetic nanoparticles with different types of biological macromolecules connected on the surface will present different motion trajectories, so that the effective separation of different biological macromolecules in the liquid can be realized; then, the magnetophoretic velocity of each magnetic nanoparticle is extracted through imaging, and the type of biological macromolecule can be deduced from the velocity. The application can utilize the steady-state magnetophoresis characteristics of the magnetic nanoparticles under the conditions of saturated magnetization and strong gradient magnetic field, realize continuous characterization based on velocity information, and break through the technical bottleneck that the traditional magnetic separation technology only relies on the end-point capture or enrichment result and cannot reflect the difference in dynamic behavior of the magnetic nanoparticles.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and more specifically, relates to a high-throughput integrated method and system for the detection and separation of biological macromolecules. Background Technology

[0002] Magnetic nanoparticles have been widely used in fields such as bioseparation, tissue imaging, in vitro diagnostics, and biomedical analysis due to their excellent biocompatibility, magnetic manipulation, and ease of surface functionalization. In particular, superparamagnetic magnetic nanoparticles exhibit a stable and controllable magnetic response under an applied magnetic field and rapidly demagnetize after the magnetic field is removed, effectively preventing particle aggregation. They have become important functional materials in current magnetic labeling detection technologies.

[0003] In the field of biological detection, existing technologies generally rely on fluorescent labeling, enzyme-linked immunosorbent assays (ELISA), or antibody-specific binding to distinguish and identify different biomolecules. These methods typically require complex labeling steps, and the detection process is affected by factors such as fluorescence bleaching, background noise, and non-specific adsorption. Furthermore, they have certain limitations in terms of high throughput, continuous measurement, and real-time analysis.

[0004] To reduce reliance on optical or chemical labeling, a new approach to biosensor research based on the magnetophoretic behavior of magnetic nanoparticles has emerged in recent years. This method drives magnetic nanoparticles to move in a liquid medium using an external magnetic field and magnetic field gradient, attempting to reflect changes in particle physical properties caused by biomolecule binding. In biosensor scenarios, compared to traditional optical or chemical labeling methods, magnetophoresis detection does not rely on fluorescence or enzyme-linked immunosorbent assay (ELISA) signals, offering advantages such as simplified detection procedures, strong resistance to background interference, and suitability for high-throughput and continuous detection. However, existing research is largely based on overdamped approximation models, primarily focusing on diffusion behavior or average drift characteristics under weak magnetic field conditions. Under strong magnetic fields, magnetic nanoparticles can rapidly reach saturation magnetization. Their force characteristics and steady-state magnetophoretic behavior in strong gradient magnetic fields differ significantly from those in weak magnetic fields, making it difficult for existing models and detection approaches to effectively describe and utilize this process. To date, no systematic research has been found on the steady-state magnetophoretic characteristics of saturated magnetized magnetic nanoparticles in strong gradient magnetic fields, nor has a method been developed to use their steady-state magnetophoretic velocity to characterize the biomolecule-bound parameters on the particle surface. Summary of the Invention

[0005] In view of the above-mentioned defects or improvement needs of the existing technology, the present invention provides a high-throughput integrated method and system for the detection and separation of biological macromolecules. The purpose is to propose an integrated method that combines biological detection and separation based on the steady-state magnetophoresis characteristics of saturated magnetized magnetic nanoparticles in a strong gradient magnetic field.

[0006] To achieve the above objectives, according to one aspect of the present invention, a high-throughput integrated method for the detection and separation of biomacromolecules is provided, comprising:

[0007] A sample of magnetic nanoparticles with uniform particle size was obtained. The magnetic nanoparticles were nanoscale magnetic particles with superparamagnetism. The magnetic nanoparticle sample was dispersed in a liquid medium containing different biomolecules. Different biomolecules to be tested were attached to the surface of different magnetic nanoparticles to form a sample solution to be tested.

[0008] An external DC magnetic field of fixed magnitude and direction is applied to the sample solution to make the magnetic nanoparticles connected with biomolecules reach a saturated magnetization state, ensuring that the direction of their magnetic moment is consistent with the direction of the external DC magnetic field.

[0009] Under the background of an external DC magnetic field, a gradient magnetic field with a constant and controllable gradient is superimposed on the sample solution to apply a stable magnetic driving force to the magnetic nanoparticles connected to biomolecules, causing them to generate magnetophoretic motion along the direction of decreasing magnetic potential energy corresponding to the magnetic field gradient. The gradient magnitude satisfies the following conditions: separation occurs between different biomolecules, and the magnetic nanoparticles reach steady-state uniform magnetophoretic motion after experiencing a ballistic motion phase on the order of nanoseconds. When the steady-state uniform magnetophoretic motion is achieved, each magnetic nanoparticle connected to a biomolecule with an equivalent hydrodynamic particle size smaller than the optical diffraction limit is imaged and detected by interference scattering imaging. The scattering signals of each magnetic nanoparticle connected to a biomolecule are acquired in real time, and the scattering signals are converted into images to be processed by high-speed imaging. Based on each frame of the images to be processed for each magnetic nanoparticle connected to a biomolecule, the motion trajectory of the magnetic nanoparticle is obtained. Based on the motion trajectory, the steady-state magnetophoretic velocity of the magnetic nanoparticle is extracted.

[0010] Based on the correspondence between the steady-state magnetophoretic velocity of each magnetic nanoparticle and its equivalent hydrodynamic damping, the equivalent hydrodynamic radius of the magnetic nanoparticle connected to the biomolecules can be deduced, thus enabling the differentiation and identification of biomolecules connected to the surface of the magnetic nanoparticles.

[0011] Furthermore, the magnetic nanoparticles are made of iron oxide nanoparticles, or cobalt-based, nickel-based, or cobalt-nickel alloy superparamagnetic nanoparticles that have undergone non-toxic treatment or surface modification.

[0012] Furthermore, the uniform magnetic field source for the applied DC magnetic field adopts a high-stability coil structure or a permanent magnet structure, and the magnetic field strength is adjustable. It is used to provide a background magnetic field with DC magnetic field uniformity better than a preset threshold within the measurement area.

[0013] Furthermore, the magnetic field source of the gradient magnetic field adopts a coil structure containing an iron core. By optimizing the coil arrangement and the current distribution between the coils, the magnetic field gradient intensity is improved, and the generated magnetic field gradient reaches hundreds of Tesla per meter or more.

[0014] Furthermore, the size of the magnetic nanoparticles ranges from 10 nanometers to 50 micrometers.

[0015] Furthermore, in constructing the motion trajectory, the method also includes: adjusting the intensity of the DC magnetic field and the gradient of the gradient magnetic field to precisely control the magnetophore motion state of the magnetic nanoparticles, so that they still exhibit a significantly directional and stable motion trajectory under the background of Brownian motion.

[0016] Furthermore, after distinguishing and recognizing the biomolecules attached to the surface of each magnetic nanoparticle, the method also includes:

[0017] The equivalent hydrodynamic radius of all magnetic nanoparticles connected to biomacromolecules was statistically analyzed to construct a particle size-number distribution spectrum, and the concentration of various biomacromolecules in the sample solution was obtained.

[0018] Furthermore, under the combined action of a DC magnetic field and a gradient magnetic field, the magnetic nanoparticles rapidly reach a state of uniform motion, the expression for which is:

[0019]

[0020] in, The equivalent hydrodynamic radius of the magnetic nanoparticles linked with biomolecules. The equivalent Stokes damping coefficient of the magnetic nanoparticles linked with biomacromolecules. , The solvent viscosity is known. This represents the known saturation magnetization of magnetic nanoparticles. This represents the gradient of a known gradient magnetic field. Represents the radius of a known magnetic nanoparticle. The magnetic force experienced by the magnetic nanoparticles.

[0021] According to another aspect of the present invention, a high-throughput integrated system for detecting and separating biomacromolecules is provided, for implementing the high-throughput integrated method for detecting and separating biomacromolecules as described above, comprising: a sample container, a uniform DC magnetic field source, a gradient magnetic field coil, an interference scattering imaging module, a high-speed camera, and a data processing module.

[0022] The sample container is used to hold the sample solution to be tested, and the uniform DC magnetic field source is used to apply an external DC magnetic field of fixed magnitude and direction to the sample solution to make the magnetic nanoparticles connected with biomacromolecules reach a saturated magnetization state, ensuring that the direction of their magnetic moment is consistent with the direction of the external DC magnetic field.

[0023] Gradient magnetic field coils are used to apply stable magnetic force to magnetic nanoparticles connected with biomacromolecules, causing them to generate magnetophoretic motion along the direction of decreasing magnetic potential energy corresponding to the magnetic field gradient. The gradient magnitude satisfies the following conditions: different biomacromolecules separate and the magnetic nanoparticles reach steady-state uniform magnetophoretic motion after experiencing a ballistic motion phase on the order of nanoseconds.

[0024] The interferometric scattering imaging module is used to image and detect each magnetic nanoparticle connected to a biomolecule when the equivalent hydrodynamic particle size is smaller than the optical diffraction limit during stable uniform magnetophoresis, and to acquire the scattering signal of each magnetic nanoparticle connected to a biomolecule in real time; the high-speed camera is used to convert the scattering signal into an image to be processed.

[0025] The data processing module is used to obtain the motion trajectory of each magnetic nanoparticle based on each frame of the image to be processed, and to extract the steady-state magnetophoretic velocity of the magnetic nanoparticle based on the motion trajectory. Based on the correspondence between the steady-state magnetophoretic velocity of each magnetic nanoparticle and its equivalent hydrodynamic damping, the equivalent hydrodynamic radius of the magnetic nanoparticle connected to the biomolecule is deduced, thereby realizing the differentiation and identification of the biomolecules connected to the surface of the magnetic nanoparticle.

[0026] In summary, compared with the prior art, the technical solutions conceived by this invention have the following main advantages:

[0027] This invention provides a high-throughput integrated method for the detection and separation of biomacromolecules, specifically involving the detection, resolution, and separation of biomacromolecules (especially proteins) utilizing the motion characteristics of magnetic nanoparticles in a gradient magnetic field. First, a sample of magnetic nanoparticles with uniform particle size is provided. By modifying and attaching biomacromolecules of different molecular weights or conformations to the surface of each magnetic nanoparticle, the magnetic nanoparticles exhibit different hydrodynamic sizes in a liquid medium, thus providing a physical basis for subsequent resolution and quantification based on magnetophoresis velocity. After applying a background magnetic field of specific magnitude and direction, as well as a gradient magnetic field of a certain gradient magnitude, the magnetic nanoparticles attached to different types of biomacromolecules exhibit different trajectories, thereby achieving the separation of different biomacromolecules in the liquid, while also enriching magnetic nanoparticles attached to the same type of biomacromolecule. Subsequently, the magnetophoresis velocity of each magnetic nanoparticle is extracted through imaging. The steady-state magnetophoresis velocity is determined by the magnetic response characteristics of the magnetic nanoparticles and their equivalent hydrodynamic radius in the liquid medium; the type of biomacromolecule can be inferred from the velocity. Therefore, this invention utilizes the steady-state magnetophoretic properties of magnetic nanoparticles under saturated magnetization and strong gradient magnetic field conditions to achieve continuous characterization based on velocity information. This overcomes the technical bottleneck of traditional magnetic separation techniques, which rely solely on endpoint capture or enrichment results and cannot reflect the dynamic differences in magnetic nanoparticle behavior. The method requires no fluorescent labeling or complex biochemical reactions, necessitates relatively simple equipment, and exhibits high detection stability and repeatability. Furthermore, this invention allows for the simultaneous parallel measurement of a large number of magnetic nanoparticles, constructing magnetophoretic velocity spectra for high-throughput analysis. By adjusting the magnetic field parameters and measurement timescale, it enables the differentiation of different biomolecules, even achieving continuous separation over long timescales. It possesses excellent scalability and application prospects, and can be widely applied in protein analysis, biodetection, magnetic labeling separation, and related biomedical and nanotechnology fields. Attached Figure Description

[0028] Figure 1 This is a flowchart of a high-throughput integrated method for detecting and separating biomacromolecules, provided as an embodiment of the present invention.

[0029] Figure 2 This is a schematic diagram of the overall structure of the high-throughput magnetophoresis spectrometer system provided in an embodiment of the present invention.

[0030] Figure 3 This is a schematic diagram of a uniform DC magnetic field source structure provided in an embodiment of the present invention.

[0031] Figure 4 This is a schematic diagram of the magnetic field strength of a uniform DC magnetic field source provided in an embodiment of the present invention.

[0032] Figure 5 A schematic diagram of a gradient magnetic field coil structure provided in an embodiment of the present invention.

[0033] Figure 6This is a schematic diagram of the magnetic field strength of the gradient magnetic field coil provided in an embodiment of the present invention.

[0034] Figure 7 This is a schematic diagram of the magnetophoretic motion of magnetic nanoparticles carrying biomacromolecules of different sizes in a gradient magnetic field, as provided in an embodiment of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0036] Example 1

[0037] A high-throughput integrated method for the detection and separation of biomacromolecules, such as Figure 1 As shown, it includes:

[0038] A sample of magnetic nanoparticles with uniform particle size was obtained. The magnetic nanoparticles were nanoscale magnetic particles with superparamagnetism. The magnetic nanoparticle sample was dispersed in a liquid medium containing different biomolecules. Different biomolecules to be tested were attached to the surface of different magnetic nanoparticles to form a sample solution to be tested.

[0039] An external DC magnetic field of fixed magnitude and direction is applied to the sample solution to make the magnetic nanoparticles connected with biomolecules reach a saturated magnetization state, ensuring that the direction of their magnetic moment is consistent with the direction of the external DC magnetic field.

[0040] Under the background of an external DC magnetic field, a gradient magnetic field with a constant and controllable gradient is superimposed on the sample solution to apply a stable magnetic driving force to the magnetic nanoparticles connected to biomolecules, causing them to generate magnetophoretic motion along the direction of decreasing magnetic potential energy corresponding to the magnetic field gradient. The gradient magnitude satisfies the following conditions: separation occurs between different biomolecules, and the magnetic nanoparticles reach steady-state uniform magnetophoretic motion after experiencing a ballistic motion phase on the order of nanoseconds. When the steady-state uniform magnetophoretic motion is achieved, each magnetic nanoparticle connected to a biomolecule with an equivalent hydrodynamic particle size smaller than the optical diffraction limit is imaged and detected by interference scattering imaging. The scattering signals of each magnetic nanoparticle connected to a biomolecule are acquired in real time, and the scattering signals are converted into images to be processed by high-speed imaging. Based on each frame of the images to be processed for each magnetic nanoparticle connected to a biomolecule, the motion trajectory of the magnetic nanoparticle is obtained. Based on the motion trajectory, the steady-state magnetophoretic velocity of the magnetic nanoparticle is extracted.

[0041] Based on the correspondence between the steady-state magnetophoretic velocity of each magnetic nanoparticle and its equivalent hydrodynamic damping, the equivalent hydrodynamic radius of the magnetic nanoparticle connected to the biomolecules can be deduced, thus enabling the differentiation and identification of biomolecules connected to the surface of the magnetic nanoparticles.

[0042] In this embodiment, a sample of magnetic nanoparticles with uniform particle size is first provided. By modifying and connecting (chemically or biologically) biomolecules of different molecular weights or conformations to the surface of each magnetic nanoparticle, the magnetic nanoparticles exhibit different hydrodynamic sizes in the liquid medium, thus providing a physical basis for subsequent resolution and quantification based on magnetophoretic velocity. After applying a background magnetic field of specific magnitude and direction, and a gradient magnetic field of a certain gradient magnitude, the magnetic nanoparticles connected to different types of biomolecules exhibit different trajectories, thereby achieving the separation of different biomolecules in the liquid. Simultaneously, magnetic nanoparticles connected to the same type of biomolecules can also be enriched. Subsequently, the magnetophoretic velocity of each magnetic nanoparticle is extracted through imaging. The steady-state magnetophoretic velocity is jointly determined by the magnetic response characteristics of the magnetic nanoparticles and their equivalent hydrodynamic radius in the liquid medium. The equivalent hydrodynamic radius refers to the radius of an equivalent sphere in the solution with the same diffusion coefficient or viscous resistance as the particle. By employing microscopic imaging, interferometric scattering imaging, or other high-precision optical detection methods, combined with image processing algorithms, high temporal resolution measurements of the positions of magnetic nanoparticles in a preset two-dimensional plane or a preset three-dimensional space can be achieved. This allows for the extraction of the magnetophoretic velocity of each magnetic nanoparticle, which is a high-throughput measurement. The type of biomolecule can be inferred from the velocity. For example, interferometric scattering imaging technology can be used to perform fluorescent label-free, high signal-to-noise ratio imaging detection of the target molecule. A high-speed imaging system can be used to achieve high temporal resolution observation of the magnetophoretic motion of the target molecule, acquire its trajectory in real time, and accurately extract the steady-state magnetophoretic velocity of the target molecule.

[0043] In biomolecular detection scenarios, existing magnetophoresis methods are limited to qualitative discrimination under weak magnetic field control. Furthermore, while existing magnetophoretic separation techniques are simple to operate, they are often limited to simple binary enrichment of target analytes using magnetic fields—essentially "capture" rather than "identify." These methods neglect the dynamic characteristics of magnetic nanoparticles in fluids and cannot detect the minute changes in the hydrodynamic radius of proteins modified on the surface of magnetic nanoparticles, thus lacking the fine spectroscopic resolution of chromatography or electrophoresis. Therefore, there is an urgent need in this field for a technology that utilizes the steady-state magnetophoretic motion characteristics of magnetic nanoparticles under a gradient magnetic field to achieve biomolecular separation. This technology could then convert the size or mass characteristics of biomolecules into quantitatively measurable physical signals by measuring differences in their motion speeds, thereby enabling rapid, high-throughput, and high-resolution magnetophoretic measurement and identification of biomolecules.

[0044] Therefore, addressing the limitations of existing magnetic nanoparticle separation and biodetection methods, which primarily rely on endpoint capture or qualitative discrimination, have limited detection throughput and often depend on fluorescent labeling or specific antibodies, resulting in complex detection systems, this embodiment provides a high-throughput magnetophoresis measurement technique. This invention aims to utilize the steady-state magnetophoretic motion characteristics of magnetic nanoparticles under saturated magnetization conditions, driven by a strong gradient magnetic field. By accurately measuring the magnetophoretic velocity, it achieves effective differentiation, quantitative analysis, and separation of different biomolecules attached to the surface of the magnetic nanoparticles; in other words, it achieves both separation and identification of biomolecules.

[0045] As a preferred embodiment, the magnetic nanoparticles are made of iron oxide nanoparticles, or cobalt-based, nickel-based, or cobalt-nickel alloy superparamagnetic nanoparticles that have undergone non-toxic treatment or surface modification.

[0046] Considering the simple configuration and easy adjustment of the magnetic field strength, it can be used as a preferred implementation method. The uniform magnetic field source of the external DC magnetic field adopts a high-stability coil structure or a permanent magnet structure, and the magnetic field strength is adjustable. It is used to provide a background magnetic field with DC magnetic field uniformity better than a preset threshold in the measurement area.

[0047] As a preferred implementation, the magnetic field source of the gradient magnetic field adopts a coil structure containing an iron core. By optimizing the coil arrangement and the current distribution between the coils, the magnetic field gradient intensity is improved, and the generated magnetic field gradient reaches more than hundreds of Tesla per meter to meet the effective driving requirements of nanoscale magnetic particles.

[0048] As a preferred embodiment, the size of the magnetic nanoparticles ranges from 10 nanometers to 50 micrometers, and the morphology of the magnetic nanoparticles within this range covers both the nanoparticles themselves and the assemblies formed by assembling multiple superparamagnetic nanoparticles through an inorganic coating layer.

[0049] As a preferred embodiment, when constructing the motion trajectory, the method further includes: adjusting the intensity of the DC magnetic field and the gradient of the gradient magnetic field to precisely control the magnetophoretic motion state of the magnetic nanoparticles, so that they still exhibit a significantly directional and stable motion trajectory under the background of Brownian motion, thereby obtaining stable and reliable magnetophoretic velocity measurement results. Based on the measured magnetophoretic velocity, the equivalent hydrodynamic radius of the magnetic nanoparticles connected to the biomacromolecules can be deduced, thereby realizing the accurate detection and identification of changes in characteristic parameters such as particle size and mass of biomacromolecules.

[0050] Considering the comprehensiveness of biomolecular information analysis, as a preferred implementation method, after distinguishing and identifying the biomolecular molecules attached to the surface of each magnetic nanoparticle, the method further includes:

[0051] The equivalent hydrodynamic radius of all magnetic nanoparticles connected to biomacromolecules was statistically analyzed to construct a particle size-number distribution spectrum, and the concentration of various biomacromolecules in the sample solution was obtained.

[0052] Furthermore, statistical analysis can be performed on the magnetophoretic velocities of multiple magnetic nanoparticles to achieve high-throughput measurement and velocity spectrum construction. Magnetic nanoparticles modified with different biomolecules correspond to different velocity ranges or peak positions in the velocity spectrum.

[0053] As a preferred embodiment, under the combined action of a DC magnetic field and a gradient magnetic field, the magnetic nanoparticles rapidly reach a state of uniform motion, the expression for which is:

[0054]

[0055] in, The equivalent hydrodynamic radius of the magnetic nanoparticles linked with biomolecules. The equivalent Stokes damping coefficient of the magnetic nanoparticles linked with biomacromolecules. , The solvent viscosity is known. This represents the known saturation magnetization of magnetic nanoparticles. This represents the gradient of a known gradient magnetic field. This represents the radius of a known magnetic nanoparticle.

[0056] The magnetic moment of magnetic nanoparticles at saturation magnetization is approximately:

[0057]

[0058] in, The saturation magnetization of the known magnetic nanoparticles is determined by the properties of the magnetic nanoparticles themselves. The volume of the magnetic nanoparticle is related to its radius. It is proportional to the cube of the law. Assume the magnetic induction intensity of the gradient magnetic field is... This indicates that the gradient magnetic field direction is along The gradient magnitude is . Then the magnetic force experienced by the magnetic nanoparticles under the gradient magnetic field is:

[0059]

[0060] Therefore, under the combined action of a DC magnetic field and a gradient magnetic field, the magnetic nanoparticles rapidly reach a state of uniform motion, and their velocity expression is as follows:

[0061]

[0062] in, The equivalent hydrodynamic radius of the magnetic nanoparticles linked with biomolecules. The equivalent Stokes damping coefficient of the magnetic nanoparticles linked with biomacromolecules. Given the solvent viscosity. The magnetic force experienced by the magnetic nanoparticles. It is related to the particle size of magnetic nanoparticles, the equivalent hydrodynamic radius, and the magnetic field gradient.

[0063] By adjusting the intensity of the DC magnetic field and the gradient of the gradient magnetic field, the magnetophoretic motion of magnetic nanoparticles can be precisely controlled, ensuring that they exhibit a highly directional and stable trajectory even under Brownian motion, thus obtaining stable and reliable magnetophoretic velocity measurement results. Based on the measured magnetophoretic velocity, the equivalent hydrodynamic radius of the magnetic nanoparticles connected to biomolecules can be deduced, thereby enabling the detection and identification of changes in characteristic parameters such as particle size and mass of biomolecules.

[0064] In summary, this embodiment involves multiple fields such as magnetic nanoparticle manipulation, interferometric scattering imaging technology, biomedical engineering, analytical chemistry, and magnetophoretic detection. The core is still a solution related to the field of biological detection. Specifically, it involves a method for detecting, distinguishing, and separating biomolecules (especially proteins) by utilizing the motion characteristics of magnetic nanoparticles in a gradient magnetic field. By utilizing the steady-state magnetophoretic motion characteristics of magnetic nanoparticles under a gradient magnetic field and measuring the differences in their motion speed, the size or mass characteristics of biomolecules are converted into quantitatively measurable physical signals, thereby achieving rapid, high-throughput, and high-resolution magnetophoretic measurement and identification of biomolecules.

[0065] Example 2

[0066] A high-throughput integrated system for detecting and separating biomacromolecules is provided to implement a high-throughput integrated method for detecting and separating biomacromolecules as described in Example 1. The system includes: a sample container, a uniform DC magnetic field source, a gradient magnetic field coil, an interference scattering imaging module, a high-speed camera, and a data processing module.

[0067] The sample container holds the sample solution to be tested. A uniform DC magnetic field source applies a fixed-magnetic field of magnitude and direction to the sample solution, saturating the magnetic nanoparticles connected to biomolecules and ensuring that their magnetic moment direction is aligned with the applied DC magnetic field. A gradient magnetic field coil applies a stable magnetic force to the magnetic nanoparticles connected to biomolecules, causing them to undergo magnetophore motion along the direction of decreasing magnetic potential energy corresponding to the magnetic field gradient. The gradient magnitude satisfies the following conditions: separation occurs between different biomolecules, and the magnetic nanoparticles reach steady-state uniform magnetophore motion after undergoing a ballistic motion phase on the order of nanoseconds. An interferometric scattering imaging module is used to analyze the equivalent hydrodynamic forces when the stable uniform magnetophore motion is achieved. Imaging detection is performed on magnetic nanoparticles with a particle size smaller than the optical diffraction limit, each connected to a biomolecule, to acquire the scattering signal of each magnetic nanoparticle in real time. A high-speed camera is used to convert the scattering signal into an image to be processed. A data processing module is used to obtain the motion trajectory of each magnetic nanoparticle based on each frame of the image to be processed, and to extract the steady-state magnetophore velocity of the magnetic nanoparticle based on the motion trajectory. Based on the correspondence between the steady-state magnetophore velocity of each magnetic nanoparticle and its equivalent hydrodynamic damping, the equivalent hydrodynamic radius of the magnetic nanoparticle connected to the biomolecule is deduced, thus realizing the differentiation and identification of the biomolecules connected to the surface of the magnetic nanoparticle.

[0068] To better illustrate the present invention, the following examples are provided:

[0069] like Figure 2 As shown, a high-throughput magnetophoresis spectrometer system is provided, which is an integrated system for high-throughput detection and separation of biomacromolecules. The system includes a uniform DC magnetic field source, a gradient magnetic field coil, an interference scattering imaging device, a high-speed camera, and a data processing module.

[0070] Specifically, the system includes a laser source, an optical isolator, an adjustable attenuator (ATT), a fiber coupling and collimator (fiber coupling + PM fiber + collimator), a mirror (M1), a beam splitter (BS), a beam expander lens group (L1, L2, L3), a converging lens L4, a sample area, an objective lens (BFP), a high-speed camera, and a data processing module. The laser source, optical isolator, ATT, fiber coupling and collimator, mirror (M1), beam splitter (BS), beam expander lens group (L1, L2, L3), converging lens L4, and objective lens (BFP) constitute the interferometric scattering imaging device. The monochromatic, coherent light emitted from the laser source first passes through the optical isolator to prevent reflected light from propagating back, then the optical power is adjusted by the adjustable attenuator, and finally collimated by the fiber coupling and collimator before being introduced into the interferometric imaging optical path. The light beam's focal plane is adjusted by L4, and its direction is changed by mirror M1. It is then split into reference and sample beams by a beam splitter. The sample beam is directed towards the sample through the objective lens, where it merges with the scattered signal generated by the sample and interferes with the reference beam at BS. The resulting interference signal enters the high-speed camera through lens groups L1, L2, and L3. Coil coils on both sides of the sample region generate a uniform magnetic field, while coils in opposite directions generate a gradient magnetic field. The light scattered by the analyte molecule is collected by the objective lens to form an interference scattering signal, which is then acquired by the high-speed camera and transmitted in real-time to the data processing module. This module extracts the particle trajectory and steady-state magnetophoresis velocity information for continuous characterization and analysis of the particle size, mass, and dynamic characteristics of biomolecules attached to the surface of magnetic nanoparticles. The uniform DC magnetic field source consists of two oppositely placed circular coils with precisely designed geometric parameters to create an approximately uniform magnetic field in the central sample region.

[0071] In this example, the sample is an aqueous solution of magnetic nanoparticles dispersed in a liquid medium. The magnetic nanoparticles are superparamagnetic iron oxide particles, and their surfaces are chemically modified to connect biomolecules of different sizes or conformations. To achieve the high-throughput magnetophoresis spectrometer function, the following steps are included:

[0072] Step S1, as follows Figure 3 and Figure 4As shown, the system first provides a uniform DC magnetic field source. This source can be a Helmholtz coil, an electromagnet, or other structures capable of generating a highly uniform magnetic field within the measurement area. In this example, the DC magnetic field source is a Helmholtz coil with adjustable magnetic field strength ranging from 0.05 T to 0.5 T, and a magnetic field uniformity error of less than 1%. Before entering the magnetophoretic measurement process, the magnetic nanoparticles are first subjected to this uniform DC magnetic field, aligning their magnetic moment direction with the applied magnetic field direction. This aligns the magnetic nanoparticles with the applied magnetic field, thereby achieving saturation magnetization and providing stable and repeatable magnetic response conditions for subsequent magnetophoretic motion.

[0073] Specifically, Figure 3 illustrates the structure of a uniform DC magnetic field source. The figure shows a coil pair structure consisting of two precisely designed circular coils placed opposite each other, with optimized spacing and diameter to ensure the magnetic field in the central region is as uniform as possible. The sample area, marked with a blue cube, is located in the uniform magnetic field region at the center of the coils and is used to withstand a constant DC magnetic field, allowing the magnetic nanoparticles to reach saturation magnetization. The three-dimensional coordinates (x, y, z axes) in the figure indicate the spatial position of the sample in the magnetic field and the direction of the magnetic field. The size, spacing, and number of turns of the coils are selected through magnetic field homogenization design, and combined with gradient magnetic field coils, magnetophoresis is achieved. After the current interface is energized, the magnetic field strength can be controlled by adjusting the coil current, thereby ensuring stable magnetophoresis experiments of the magnetic nanoparticles under saturation magnetization conditions.

[0074] Figure 4 illustrates the magnetic field strength of a uniform DC magnetic field source. The figure clearly reflects the variation of the magnetic field strength within a given spatial range. The maximum magnetic field strength is 0.0490575 T, and the minimum magnetic field strength is 0.0488371 T, indicating that the magnetic field source has good uniformity and stability, providing reliable support for achieving high-precision magnetic field control.

[0075] Step S2, as follows Figure 5 and Figure 6 As shown, the system incorporates a gradient magnetic field coil within the magnetophoresis measurement region. The gradient magnetic field coil employs a four-coil structure and includes an iron core to enhance the magnetic field gradient strength. By adjusting the magnitude and direction of the current flowing through the gradient magnetic field coil, a magnetic field gradient of the desired magnitude can be generated within the measurement region. In this example, the gradient magnetic field can reach up to 500 T / m, used to drive the magnetic nanoparticles to undergo magnetophoresis along the magnetic field gradient direction. Since the magnetic nanoparticles are already in a saturated magnetized state, their force in the gradient magnetic field is stable, which is beneficial for forming a measurable magnetophoresis process.

[0076] Figure 5The diagram illustrates a gradient magnetic field coil structure, which includes a magnetic yoke, several pole shoes, and excitation coils corresponding to the pole shoes. The magnetic yoke has a closed frame structure to create a low magnetic reluctance loop and improve magnetic field utilization efficiency. Multiple pole shoes are symmetrically arranged inside the yoke, with their ends facing the magnetic field working area and forming a central magnetic field region. Each excitation coil is wound around the outside of its corresponding pole shoe. By applying currents of predetermined direction and magnitude to different coils, a gradient magnetic field varying along a predetermined direction is superimposed within the central working area. This structure can achieve a high magnetic field gradient within a small spatial scale while ensuring the symmetry and stability of the magnetic field distribution in the working area, making it suitable for magnetophoretic manipulation experiments or microscale magnetic manipulation applications.

[0077] Figure 6 The magnetic field strength of the gradient magnetic field coil is illustrated. The distribution of magnetic induction intensity of the gradient magnetic field coil in the central working area is represented by a color cloud map. The colors from cool to warm represent the trend of magnetic induction intensity from low to high. The central region exhibits an approximately axially symmetric magnetic field distribution. The magnetic field gradient changes uniformly within the predetermined range, which meets the requirements of the magnetophoresis experiment for a stable gradient magnetic field. Figure 6 The coordinate directions and scale bars marked in the figure illustrate the directionality and distribution scale of the magnetic field in space. The results show that the gradient magnetic field coil can provide a continuous, smooth, and consistent magnetic field gradient in the central region, which is beneficial for the controllable manipulation of magnetic particles or magnetically labeled biological samples. In the figure, B represents magnetic flux density, and tesla represents the unit of B, Tesla.

[0078] Step S3: Under the influence of a gradient magnetic field, the magnetic nanoparticles first undergo a brief period of unsteady motion, followed by a steady-state uniform magnetophoretic motion. This steady-state magnetophoretic velocity is determined by the magnetic response characteristics of the magnetic nanoparticles and their equivalent hydrodynamic radius in the liquid medium. Since different biomolecules attached to the surface of the magnetic nanoparticles alter their equivalent hydrodynamic radius, different magnetic nanoparticles will exhibit different steady-state magnetophoretic velocities under the same magnetic field conditions.

[0079] Step S4: The system monitors the magnetophore motion of magnetic nanoparticles in real time using a magnetic nanoparticle motion detection system. The detection system can employ optical microscopy, interferometric scattering imaging, or other high-resolution detection methods to record the two-dimensional or three-dimensional position changes of the magnetic nanoparticles over time. By analyzing the trajectory of the magnetic nanoparticles, their steady-state magnetophore velocity information can be extracted, and further, velocity distribution data of multiple magnetic nanoparticles can be obtained.

[0080] Step S5, as follows Figure 7As shown, the system performs statistical analysis on the acquired magnetophoretic velocity data to construct a magnetophoretic velocity spectrum. Magneto nanoparticles modified with different biomolecules correspond to different velocity ranges or peak positions in the velocity spectrum, thereby enabling the differentiation, recognition, or separation of biomolecules.

[0081] Figure 7 The magnetic nanoparticles carrying biomolecules of different sizes are shown to exhibit magnetophoretic motion in a gradient magnetic field. The surface of the magnetic nanoparticles is loaded with biomolecules of different sizes or molecular weights through a specific binding method. Under the action of the gradient magnetic field, due to the different volumes, masses and fluid resistance of the biomolecules they carry, the magnetic nanoparticles exhibit different force balance states and motion trajectories in the direction of the magnetic field gradient. Figure 7 The diagram visually illustrates the migration path and separation trend of magnetic nanoparticles in a gradient magnetic field using schematic symbols and dashed trajectories, thereby enabling the differentiation, sorting, or enrichment of different biomolecules. This magnetophoretic separation mechanism requires no complex labeling or external mechanical actuation, making it suitable for high-sensitivity manipulation applications in biological detection, molecular separation, and microfluidic systems.

[0082] In another example, a high-throughput magnetophoresis system can be combined with microfluidic channels or multi-channel sample structures to achieve parallel magnetophoretic measurements of multiple samples. Magnetic nanoparticles in each channel undergo synchronous magnetophoretic motion under the same or different magnetic field gradients. The system acquires and processes the motion data of the magnetic nanoparticles in each channel in parallel, thereby significantly improving measurement throughput and statistical accuracy.

[0083] Through the technical solutions described in the above embodiments, this invention enables high-precision measurement of the steady-state magnetophoretic velocity of magnetic nanoparticles under saturated magnetization and strong gradient magnetic field conditions. This method eliminates the need for fluorescent labeling or complex biochemical reactions, enabling the differentiation and analysis of different biomolecules based on differences in magnetophoretic velocity. It offers advantages such as a clear system structure, stable measurement, high throughput, and strong scalability.

[0084] The relevant technical solutions are the same as above, and will not be repeated here.

[0085] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A high-throughput integrated method for the detection and separation of biological macromolecules, characterized in that, include: A sample of magnetic nanoparticles with uniform particle size was obtained. The magnetic nanoparticles were nanoscale magnetic particles with superparamagnetism. The magnetic nanoparticle sample was dispersed in a liquid medium containing different biomolecules. Different biomolecules to be tested were attached to the surface of different magnetic nanoparticles to form a sample solution to be tested. An external DC magnetic field of fixed magnitude and direction is applied to the sample solution to make the magnetic nanoparticles connected with biomolecules reach a saturated magnetization state, ensuring that the direction of their magnetic moment is consistent with the direction of the external DC magnetic field. Under the background of an external DC magnetic field, a gradient magnetic field with a constant and controllable gradient is superimposed on the sample solution to apply a stable magnetic driving force to the magnetic nanoparticles connected to biomolecules, causing them to generate magnetophoretic motion along the direction of decreasing magnetic potential energy corresponding to the magnetic field gradient. The gradient magnitude satisfies the following conditions: separation occurs between different biomolecules, and the magnetic nanoparticles reach steady-state uniform magnetophoretic motion after experiencing a ballistic motion phase on the order of nanoseconds. When the steady-state uniform magnetophoretic motion is achieved, each magnetic nanoparticle connected to a biomolecule with an equivalent hydrodynamic particle size smaller than the optical diffraction limit is imaged and detected by interference scattering imaging. The scattering signals of each magnetic nanoparticle connected to a biomolecule are acquired in real time, and the scattering signals are converted into images to be processed by high-speed imaging. Based on each frame of the images to be processed for each magnetic nanoparticle connected to a biomolecule, the motion trajectory of the magnetic nanoparticle is obtained. Based on the motion trajectory, the steady-state magnetophoretic velocity of the magnetic nanoparticle is extracted. Based on the correspondence between the steady-state magnetophoretic velocity of each magnetic nanoparticle and its equivalent hydrodynamic damping, the equivalent hydrodynamic particle size of the magnetic nanoparticles connected to biomolecules can be deduced, thus enabling the differentiation and identification of biomolecules connected to the surface of magnetic nanoparticles. Under the combined action of a DC magnetic field and a gradient magnetic field, the magnetic nanoparticles rapidly reach a state of uniform motion, the expression for which is: in, The equivalent hydrodynamic radius of the magnetic nanoparticles linked with biomolecules. The equivalent Stokes damping coefficient of the magnetic nanoparticles linked with biomacromolecules. , The solvent viscosity is known. This represents the known saturation magnetization of magnetic nanoparticles. This represents the gradient of a known gradient magnetic field. Represents the radius of a known magnetic nanoparticle. The magnetic force experienced by the magnetic nanoparticles.

2. The integrated method for high-throughput detection and separation of biomacromolecules as described in claim 1, characterized in that, The magnetic nanoparticles are made of iron oxide nanoparticles, or cobalt-based, nickel-based, or cobalt-nickel alloy superparamagnetic nanoparticles that have undergone non-toxic treatment or surface modification.

3. The integrated method for high-throughput detection and separation of biomacromolecules as described in claim 1, characterized in that, The uniform magnetic field source with an external DC magnetic field adopts a high-stability coil structure or a permanent magnet structure, and the magnetic field strength is adjustable. It is used to provide a background magnetic field with DC magnetic field uniformity better than a preset threshold in the measurement area.

4. The integrated method for high-throughput detection and separation of biomacromolecules as described in claim 1, characterized in that, The magnetic field source of the gradient magnetic field adopts a coil structure containing an iron core. By optimizing the coil arrangement and the current distribution between the coils, the magnetic field gradient intensity is improved, and the generated magnetic field gradient reaches hundreds of Tesla per meter or more.

5. The integrated method for high-throughput detection and separation of biomacromolecules as described in claim 1, characterized in that, In constructing the motion trajectory, the method also includes: adjusting the intensity of the DC magnetic field and the gradient of the gradient magnetic field to precisely control the magnetophore motion state of the magnetic nanoparticles, so that they still exhibit a significantly directional and stable motion trajectory in the context of Brownian motion.

6. The integrated method for high-throughput detection and separation of biomacromolecules as described in claim 1, characterized in that, After distinguishing and recognizing the biomolecules attached to the surface of each magnetic nanoparticle, the method also includes: The equivalent hydrodynamic particle size of all magnetic nanoparticles connected to biomolecules was statistically analyzed to construct a particle size-number distribution spectrum, and the concentration of various biomolecules in the sample solution was obtained.

7. A high-throughput integrated system for the detection and separation of biological macromolecules, characterized in that, The method for implementing a high-throughput integrated detection and separation method for biomacromolecules as described in any one of claims 1 to 6 includes: a sample container, a uniform DC magnetic field source, a gradient magnetic field coil, an interference scattering imaging module, a high-speed camera, and a data processing module. The sample container is used to hold the sample solution to be tested, and the uniform DC magnetic field source is used to apply an external DC magnetic field of fixed magnitude and direction to the sample solution to make the magnetic nanoparticles connected with biomacromolecules reach a saturated magnetization state, ensuring that the direction of their magnetic moment is consistent with the direction of the external DC magnetic field. Gradient magnetic field coils are used to apply a stable magnetic force to magnetic nanoparticles connected with biomacromolecules, causing them to generate magnetophoretic motion along the direction of decreasing magnetic potential energy corresponding to the magnetic field gradient. The gradient magnitude satisfies the following conditions: different biomacromolecules separate and the magnetic nanoparticles reach steady-state uniform magnetophoretic motion after experiencing a ballistic motion phase on the order of nanoseconds. The interferometric scattering imaging module is used to image and detect each magnetic nanoparticle connected to a biomolecule that is below the optical diffraction limit when a stable uniform magnetophoretic motion is achieved, and to acquire the scattering signal of each magnetic nanoparticle connected to a biomolecule in real time; the high-speed camera is used to convert the scattering signal into an image to be processed. The data processing module is used to obtain the motion trajectory of each magnetic nanoparticle based on each frame of the image to be processed, and to extract the steady-state magnetophoretic velocity of the magnetic nanoparticle based on the motion trajectory. Based on the correspondence between the steady-state magnetophoretic velocity of each magnetic nanoparticle and its equivalent hydrodynamic damping, the equivalent hydrodynamic radius of the magnetic nanoparticle connected to the biomolecule is deduced, thereby realizing the differentiation and identification of the biomolecules connected to the surface of the magnetic nanoparticle.

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