Method and system for coagulation detection based on magnetic field driven microrobot swarm motion

Abstract

This invention discloses a method and system for coagulation detection based on magnetic field-driven micro-robot swarm motion. The method first mixes and incubates diluted magnetic beads with the target plasma and coagulation reagent to form a coagulation reaction system. Based on the detection requirements, a time-varying magnetic field driving mode is selected and applied. Motion video data of the magnetic bead swarm under magnetic field driving is acquired in real time using an image acquisition device. Time-series analysis of the motion video data is performed using image processing and analysis algorithms to extract dynamic parameters characterizing the swarm motion intensity, generating a motion decay curve that changes over time. Based on the trend of the motion decay curve, the viscoelastic changes reflecting the coagulation process are quantified, determining the coagulation state and corresponding coagulation time parameters. This invention not only detects coagulation time parameters but also continuously reflects the coagulation reaction process at the microscale. Compared with traditional detection methods, it offers higher sensitivity, richer information dimensions, and superior detection accuracy.

Description

Technical Field

[0001] This invention relates to the field of coagulation detection technology, and in particular to a coagulation detection method and system based on magnetic field-driven micro-robot swarm motion. Background Technology

[0002] Currently, commonly used blood coagulation detection methods mainly include optical methods and magnetic bead methods, both of which are based on changes in the physical properties of blood during the coagulation process.

[0003] Optical methods are a widely used detection technique. Their basic principle is to determine the coagulation endpoint by monitoring changes in the optical properties of plasma during the coagulation process (such as changes in transmittance or turbidity). Once the coagulation reaction begins, with the formation of fibrin, light scattering in the sample increases or transmittance decreases. The instrument calculates the coagulation time by detecting the change in the light signal. This method has advantages such as high sensitivity, relatively simple structure, and ease of automation. However, optical methods are highly dependent on the optical properties of the sample and are easily affected by factors such as hemolysis, lipemia, and jaundice. Furthermore, factors such as air bubbles, abnormal sample color, and the optical performance of the reaction vessel can also affect the accuracy and stability of the test results, limiting its application under complex sample conditions.

[0004] Magnetic bead methods are another commonly used coagulation detection method. The principle involves introducing millimeter-diameter steel beads (magnetic beads) into the coagulation reaction system and driving them to move periodically using an external magnetic field. Before the blood coagulates, the beads move freely; as the coagulation reaction proceeds, a fibrin network gradually forms and restricts the bead's movement, altering its vibration amplitude or motion state. By monitoring the changes in the bead's motion in real time, the coagulation endpoint can be determined and the coagulation time calculated. Compared to optical methods, magnetic bead methods do not rely on optical signals and have stronger resistance to interference from hemolysis, lipemia, and other factors. However, magnetic bead methods still have certain limitations. The interaction between the magnetic beads and the inner wall of the reaction container (such as friction or adhesion) can affect their motion behavior, thus reducing detection accuracy. Furthermore, traditional magnetic bead methods typically rely on single or a small number of magnetic particles, which have limited response to the microscopic processes of coagulation, leaving room for improvement in sensitivity and dynamic information acquisition.

[0005] Therefore, there is an urgent need to develop a coagulation detection method based on a novel physical mechanism that can achieve high sensitivity, high stability, and microscopic dynamic sensing capabilities, which is of great significance for improving the level of coagulation detection technology. Summary of the Invention

[0006] The purpose of this invention is to provide a coagulation detection method and system based on magnetic field-driven micro-robot swarm motion, aiming to solve the technical problems of insufficient sensitivity, limited anti-interference ability, and difficulty in capturing the microscopic dynamic process of coagulation in existing coagulation detection methods, so as to achieve accurate and rapid detection of coagulation parameters, while enriching the information dimensions of coagulation detection and improving the accuracy and stability of detection results.

[0007] To achieve the above objectives, this invention provides a coagulation detection method based on magnetic field-driven microrobot swarm motion, comprising the following steps: S1. Mix the diluted magnetic beads with the plasma to be tested and the coagulation reagent, and incubate them in a reaction container to form a coagulation reaction system. S2. Select and apply a time-varying magnetic field driving mode that is compatible with the detection requirements to drive the magnetic beads to generate cluster movement. S3. During the coagulation reaction, the motion video data of the magnetic bead cluster under the drive of the magnetic field is acquired in real time through the image acquisition device; S4. Perform time-series analysis on motion video data using image processing and analysis algorithms, extract dynamic parameters characterizing the intensity of cluster motion, and generate motion decay curves that change over time. S5. Based on the changing trend of the motion decay curve, the viscoelastic changes in the coagulation process are quantitatively reflected, and the coagulation state and corresponding coagulation time parameters are determined.

[0008] Preferably, S1 is as follows: S11. The specific process for diluting the magnetic beads is as follows: First, wash the magnetic bead suspension with a concentration of 10 mg / mL with a 1:1 volume ratio of deionized water and ethanol 3 to 5 times to remove impurities; Then, the cleaned magnetic bead suspension was diluted 15 times by volume and resuspended in deionized water to obtain a magnetic bead solution with a concentration of approximately 0.67 mg / mL. S12. Construct a simulated blood plasma environment: First, mix 8 mL of glycerol with 2 mL of deionized water to obtain the basic solution; Then, add an appropriate amount of deionized water to the base solution to dilute it and obtain a solution with the required percentage of glycerol, wherein the percentages of glycerol are 60%, 40%, 30%, 20% and 10% respectively. Finally, the viscosity of the prepared solution and pure deionized water were measured using a viscometer. S13. Preparation of plasma to be tested: Take blood and add it into a vacuum tube. Add sodium citrate anticoagulant, which is 1 / 10 the volume of blood, into the tube beforehand. Centrifuge it at 3000 rpm or 2500 g for 10 minutes and collect the supernatant liquid, which is platelet-deficient plasma; The plasma was then transferred to plastic tubes for storage.

[0009] Preferably, in S1, the particle size of the magnetic beads is 2~4 mm. Its surface is coated with carboxyl groups, giving it superparamagnetic properties.

[0010] Preferably, in S1, the coagulation reagent includes prothrombin time assay reagent PT, activated partial thromboplastin time assay reagent APTT, and prothrombin time assay reagent TT.

[0011] Preferably, in S1, the reaction vessel is a small, circular dish used to hold coagulation analysis samples, with an effective volume of 150–170 mL. .

[0012] Preferably, in S2, the time-varying magnetic field includes a rotating magnetic field and an oscillating magnetic field; the rotating magnetic field is used to drive the magnetic beads to form a stable rotating cluster motion, and its magnetic field parameter is the magnetic field strength. and magnetic field frequency Among them, magnetic field strength The value ranges from 2 to 6. Magnetic field frequency The value ranges from 1 to 5 Hz; An oscillating magnetic field is used to drive a magnetic bead to reciprocate motion; its magnetic field parameter is the magnetic field frequency. Slew rate Among them, magnetic field frequency The value ranges from 1 to 5 Hz, and the gyroscope ratio is... 0.2~1 .

[0013] Preferably, in S5, the characteristic parameters of the motion decay curve include the rotational speed of the rotating magnetic field and the rotational angle of the oscillating magnetic field.

[0014] The present invention also provides a coagulation detection system based on magnetic field-driven micro-robot swarm motion, including a magnetic control drive module, a coagulation signal detection module, an image analysis module, and a coagulation reaction drive module; the magnetic control drive module includes a magnetic field generating unit, a signal control unit, and a motion observation unit; The magnetic field generating unit uses a triaxial Helmholtz coil structure and an optical imaging unit to generate and control the time-varying magnetic field; the time-varying magnetic field includes a rotating magnetic field and an oscillating magnetic field; the signal control unit is used to generate and amplify the drive current signal to adjust the magnetic field parameters; the motion observation unit is used to observe and record the motion state of the magnetic bead in real time. The coagulation signal detection module is electrically connected to the magnetic control drive module and is used to acquire and identify the motion state of the magnetic beads under the action of the magnetic field. The optical imaging unit acquires the motion process of the magnetic bead cluster, and the magnetic beads identified by the coagulation signal detection module are two to three attached rod-shaped magnetic beads. The image analysis module is electrically connected to the coagulation signal detection module. By preprocessing continuous video frames, tracking particle clusters, and calculating inter-frame correlation, dynamic parameters characterizing the intensity of cluster motion are extracted. The characteristic values ​​of the motion decay curve of the dynamic parameters are correlated with the viscoelastic changes caused by the coagulation reaction, which are used to quantitatively determine coagulation parameters. The coagulation reaction driving module is used to generate a controllable magnetic field and drive the movement of superparamagnetic beads in the coagulation analysis sample through magnetic field regulation, thereby participating in and regulating the coagulation reaction process.

[0015] Preferably, when the magnetic field generating unit generates a rotating magnetic field, the magnetic field strength is... The value ranges from 2 to 6. ,frequency The value ranges from 1 to 5 Hz; when an oscillating magnetic field is generated, the magnetic field frequency is... The value ranges from 1 to 5 Hz, and the gyroscope ratio is... 0.2~1 ; The signal control unit includes a function generator and a power amplifier, which are used to generate and amplify the drive current signal. The input current of each axis coil of the triaxial Helmholtz coil structure is provided by the function generator and amplified by the power amplifier to drive the corresponding coil. The amplitude, frequency, and phase of the current in each channel are controlled by software, thereby enabling real-time adjustment of the magnetic field strength and spatial vector direction. The motion observation unit uses an inverted microscope in conjunction with a CCD camera, with an image acquisition frame rate of 25 frames per second.

[0016] Preferably, the algorithm executed by the image analysis module includes the following steps: Step 1: Preprocess the acquired continuous video frames and perform particle cluster tracking, specifically as follows: First, acquire the video stream to be processed, and render and parse it into a continuous sequence of frame images at a preset frame rate; The image analysis module is used to perform grayscale conversion and noise reduction on each frame in the frame image sequence in order to extract clear target areas; The processed image is subjected to connected component calculation and area calculation to extract the target contour of the rod-shaped magnetic beads; Step 2: Quantify the overall angular velocity change of the cluster by calculating inter-frame correlation, specifically as follows: The orientation angle of each rod-shaped magnetic bead in the current frame is obtained by principal axis analysis, and combined with the inter-frame matching algorithm, the original angle change sequence of each magnetic bead's continuous motion is output. The angular velocity of the rod-shaped magnetic bead is calculated based on the angle of continuous motion. To eliminate the phase abrupt change caused by the 180-degree symmetry of the rod-shaped magnetic bead, let the angular change of the magnetic bead between two adjacent frames be . The time interval is angular velocity The calculation logic is as follows: when When <90°, ; when ≥90° and When >0, ; when and hour, .

[0017] Therefore, the coagulation detection method and system based on magnetic field-driven microrobot swarm motion described above have the following beneficial effects: (1) This invention breaks through the limitations of the traditional magnetic bead method which relies on the random motion of a single particle. It is the first to use an external magnetic field to precisely control a large number of micron robots, so that they form a cluster of coordinated motion.

[0018] (2) This invention studies the regulation law of magnetic field parameters (intensity, frequency) on the cluster motion mode, transforms the change of microenvironment viscosity during coagulation into the macroscopic change of cluster cooperative motion dynamics, and realizes the transformation from "single particle" to "cluster" and from "passive measurement" to "active driving and sensing".

[0019] (3) This invention amplifies the detection signal through the quorum effect, thereby improving the detection sensitivity, repeatability and anti-interference ability, and provides a new principle strategy for biochemical analysis.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 This is an overall flowchart of an embodiment of the coagulation detection method based on magnetic field-driven micro-robot swarm motion of the present invention; Figure 2 This is a scanning electron microscope image of the magnetic beads in an embodiment of the coagulation detection method based on magnetic field-driven micro-robot swarm motion according to the present invention; Figure 3 This is an EDS characterization image of the magnetic beads in an embodiment of the coagulation detection method based on magnetic field-driven micro-robot swarm motion of the present invention. Figure 4 This is a particle size distribution diagram of the magnetic beads in an embodiment of the coagulation detection method based on magnetic field-driven micron robot swarm motion of the present invention. Figure 5 This is the hysteresis loop of the magnetic bead in an embodiment of the coagulation detection method based on magnetic field-driven micro-robot swarm motion of the present invention; Figure 6 This is an example of the coagulation detection method based on magnetic field-driven micro-robot swarm motion in a plasma-like environment, showing the motion results of magnetic beads in a rotating magnetic field with different magnetic field parameters. (a) shows the motion results of magnetic beads in a rotating magnetic field with different magnetic field strength parameters, (b) shows the motion results of magnetic beads in a rotating magnetic field with different frequency parameters, and (c) shows the motion results of magnetic beads in a rotating magnetic field with different viscosity ratios. Figure 7 This is a diagram showing the motion results of magnetic beads in an oscillating magnetic field with different frequency parameters in a plasma-like environment, based on an embodiment of the coagulation detection method of the present invention, which is based on the magnetic field-driven micro-robot swarm motion. Figure 8 This is a diagram showing the motion results of magnetic beads in an oscillating magnetic field with different gyroscope ratio parameters in a plasma-like environment, based on an embodiment of the coagulation detection method of the present invention, which is based on the magnetic field-driven micro-robot swarm motion. Figure 9 This is a diagram showing the motion results of magnetic beads with different viscosity ratios in an oscillating magnetic field in an plasma-like environment, based on an embodiment of the coagulation detection method of the present invention, which is based on the magnetic field-driven micro-robot swarm motion. Figure 10 This is the detection result of activated partial thromboplastin time of clinical samples by the coagulation reaction system of the coagulation detection method based on magnetic field-driven micro-robot swarm motion in an embodiment of the present invention. Figure 11 This is a schematic block diagram of the coagulation detection system based on the magnetic field-driven micro-robot cluster motion of the present invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. The terms "first," "second," and similar terms used in this invention do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects.

[0024] like Figure 1As shown, the coagulation detection method based on magnetic field-driven microrobot swarm motion includes the following steps: S1. Mix the diluted magnetic beads with the plasma to be tested and the coagulation reagent, place them in a reaction container and incubate to form a coagulation reaction system and pre-activate it.

[0025] Magnetic beads are magnetic particles with a micron-sized particle size, preferably superparamagnetic microspheres, to avoid irreversible aggregation caused by magnetic residue; for example, the particle size of the magnetic beads is 2~4. Its surface is coated with carboxyl groups, exhibiting superparamagnetism. Therefore, the magnetic bead material is... Nanoparticles or their composites are used to improve magnetic response performance.

[0026] Coagulation reagents include prothrombin time (PT), activated partial thromboplastin time (APTT), and prothrombin time (TT). The reaction vessel is a small, round dish used to hold coagulation analysis samples, with an effective volume of 150–170 mL. .

[0027] S2. Turn on the magnetic field generator to construct a spatially controllable magnetic field environment. Based on the detection requirements, specifically the expected or real-time state of plasma in the coagulation reaction system, select and apply a time-varying magnetic field driving mode that is compatible with it, and adjust the corresponding magnetic field parameters to meet the conditions for driving the particle cluster movement, so as to drive the magnetic beads to generate cluster movement.

[0028] The S1 coagulation reaction system is placed within the effective area of ​​the magnetic field generator. The magnetic field generator is activated, and the external magnetic field drives the magnetic beads to form an ordered cluster movement. Under the action of the external magnetic field, the magnetic beads are arranged in a chain-like pattern. During the coagulation process, a fibrin network is gradually formed, which constrains the movement of particles, thereby participating in and influencing the coagulation process.

[0029] The time-varying magnetic field in this step includes, but is not limited to, a rotating magnetic field and an oscillating magnetic field; the rotating magnetic field is used to drive the magnetic beads to form a stable rotating cluster motion, and its magnetic field parameter is the magnetic field strength. and magnetic field frequency Among them, magnetic field strength The value ranges from 2 to 6. Magnetic field frequency The value ranges from 1 to 5 Hz.

[0030] An oscillating magnetic field is used to drive a magnetic bead to reciprocate motion; its magnetic field parameter is the magnetic field frequency. Slew rate Among them, magnetic field frequency The value ranges from 1 to 5 Hz, and the gyroscope ratio is... 0.2~1 .

[0031] By adjusting different magnetic field modes and parameters, the movement state of magnetic beads can be precisely controlled, thereby enhancing the sensitivity of detection at different stages of the coagulation process.

[0032] S3. During the coagulation reaction, the motion video data of the magnetic bead cluster under the drive of the magnetic field is acquired in real time through the image acquisition device, and the motion state of the magnetic beads during the coagulation process is recorded.

[0033] Image analysis algorithms are used to extract the motion feature parameters of magnetic beads, and coagulation-related parameters are calculated accordingly to detect coagulation function.

[0034] S4. Perform time-series analysis on motion video data using image processing and analysis algorithms, extract dynamic parameters characterizing the intensity of cluster motion, calculate coagulation-related parameters based on these parameters, generate motion decay curves that change over time, and realize the detection of coagulation function.

[0035] S5. Based on the changing trend of the motion decay curve, including the rotation speed of the rotating magnetic field and the rotation angle of the oscillating magnetic field, the viscoelastic changes of the coagulation process are quantitatively reflected to determine the coagulation state and the corresponding coagulation time parameters.

[0036] The main materials and their sources for the coagulation detection method based on magnetic field-driven micro-robot swarm motion in this invention are as follows: The magnetic beads are carboxyl magnetic beads with a particle size of 3. The concentration was 10 mg / ml, brand BEAVERBIO / BeaverBeads™ Streptavidin, catalog number 70117-5, superparamagnetic microspheres. Prothrombin time (PT) assay kit (phase one method) (R24682), activated partial thromboplastin time (APTT) assay kit (ellagic acid coagulation method) (R41056), and thrombin time (TT) assay kit (R21583) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. Glycerol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0037] Main instruments: Scanning electron microscopy (SEM) was performed on a SU8020 field emission scanning electron microscope (Hitachi, Tokyo, Japan). Energy-dispersive X-ray spectroscopy (EDS) was performed on a FEI Tecnai G2 F20 field emission transmission electron microscope (FEI, USA) equipped with an Oxford XPLORE EDS module. Hysteresis loop (VSM) measurements were performed on a Lake Shore 8604 vibrating sample magnetometer (Lake Shore Corporation, USA). Viscosity measurements were performed on a viscometer (Shanghai Lichen Instrument Technology Co., Ltd.).

[0038] (1) Sample preprocessing, specifically: S11. The specific process for diluting the magnetic beads is as follows: First, the magnetic bead suspension with a concentration of 10 mg / mL was washed 3 to 5 times with a 1:1 volume ratio of deionized water and ethanol to remove impurities.

[0039] Then, the cleaned magnetic bead suspension was diluted 15 times by volume and resuspended in deionized water to obtain a magnetic bead solution with a concentration of approximately 0.67 mg / mL.

[0040] S12. Construct a simulated blood plasma environment: First, mix 8 mL of glycerol with 2 mL of deionized water to obtain the basic solution.

[0041] Then, add an appropriate amount of deionized water to the base solution to dilute it and obtain a solution with the required percentage of glycerol, wherein the percentages of glycerol are 60%, 40%, 30%, 20% and 10%.

[0042] Finally, the viscosity of the prepared solution and pure deionized water was measured using a viscometer.

[0043] S13. Preparation of plasma to be tested: Blood is collected and placed into a vacuum tube, which is pre-filled with sodium citrate anticoagulant at a volume equal to 1 / 10 of the blood volume.

[0044] Centrifuge it at 3000 rpm or 2500 g for 10 minutes and collect the supernatant liquid, which is platelet-deficient plasma.

[0045] The plasma was then transferred to plastic tubes for storage to prevent platelet activation.

[0046] (2) Scanning electron microscopy characterizes the microstructure of the magnetic beads, such as Figure 2 As shown, the magnetic beads are regular spherical in shape, evenly distributed, and have a dense and smooth surface structure with no obvious holes or broken structures.

[0047] EDS characterizes the elemental composition of magnetic beads, such as Figure 3 As shown, the surface of the magnetic bead is mainly composed of iron ( The composition indicates that it is composed of iron(III) oxide (Fe3O4). ) magnetic beads. In addition, the figure also shows oxygen ( This further verified the oxide composition of the magnetic beads.

[0048] like Figure 4 The figure shows the particle size distribution of the magnetic beads. The results in the figure verify that the particle size distribution of the magnetic beads is uniform, indicating that the magnetic beads used have good particle size consistency.

[0049] like Figure 5 The figure shows the hysteresis loop of the magnetic beads, and the results in the figure verify that the magnetic beads used have good superparamagnetic characteristics.

[0050] The following study investigates the clustering motion of magnetic beads in a simulated blood plasma environment: Example 1: Experimental method for the motion of a cluster of magnetic beads under a rotating magnetic field: In this embodiment, the motion characteristics of a magnetic bead cluster under a rotating magnetic field are studied. The specific steps are as follows: Take 10 A magnetic bead suspension with a concentration of 0.67 mg / mL was prepared, and after removing the resuspended portion, 160 mL of [unspecified substance] was added. A pre-prepared aqueous glycerol solution was used as a simulated plasma solution and thoroughly mixed to form the reaction system. The mixture was incubated in a 37°C water bath for 3 minutes to stabilize the system temperature and simulate physiological conditions. After incubation, the mixture was transferred to the reaction vessel using a pipette and gently mixed by pipetting to ensure system homogeneity.

[0051] Subsequently, the reaction vessel was placed in the central region of the 3D-printed Helmholtz coil device to create a rotating magnetic field environment, and the magnetic field parameters were set to magnetic field strength. =3mT, magnetic field frequency =2Hz, to drive the magnetic beads to form a rod-shaped cluster and generate rotational motion.

[0052] Based on this, the following variable experimental studies were conducted: Effect of viscosity on magnetic bead motion: By adjusting the ratio of glycerol aqueous solution, simulated plasma environments with different viscosities were constructed. The viscosity gradients were set to 0.89 (corresponding to 0% glycerol aqueous solution), 1.18 (corresponding to 10% glycerol aqueous solution), 1.70 (corresponding to 20% glycerol aqueous solution), 2.25 (corresponding to 30% glycerol aqueous solution), and 3.40 (corresponding to 40% glycerol aqueous solution). Under the same magnetic field conditions, the motion speed of rod-shaped magnetic beads was measured to analyze the relationship between viscosity and the motion behavior of the magnetic beads.

[0053] The effect of magnetic field strength on the motion of magnetic beads: with the viscosity of the glycerol aqueous solution fixed at 1.70. Under these conditions, maintain the magnetic field frequency With the frequency constant at 2Hz, the magnetic field strength was adjusted to create a gradient change, and the influence of the magnetic field strength on the speed of the magnetic bead was studied.

[0054] The effect of magnetic field frequency on the motion of magnetic beads: with the viscosity of the glycerol aqueous solution fixed at 1.70. And magnetic field strength Under the condition of 3mT, the magnetic field frequency is adjusted to form a gradient change, and the influence of the magnetic field frequency on the motion characteristics of the magnetic bead is analyzed.

[0055] During the above experiment, the motion of the magnetic beads was captured by an imaging system. Each video segment lasted 2 to 3 seconds. The rotational angular velocity of the rod-shaped magnetic beads was calculated and analyzed using a rotating magnetic field coagulation analysis unit.

[0056] Example 2: Experimental method for the motion of magnetic beads clusters under an oscillating magnetic field: In this embodiment, the dynamic behavior of the magnetic bead cluster under oscillating magnetic field conditions is further studied. The specific steps are as follows: Take 10 A magnetic bead suspension with a concentration of 0.67 mg / mL was prepared, and after removing the resuspended portion, 160 mL of [unspecified substance] was added. The glycerol aqueous solution was mixed and incubated in a 37°C water bath for 3 minutes. After incubation, the mixture was transferred to a reaction vessel and gently pipetted to mix thoroughly.

[0057] The reaction vessel was placed in the central region of a Helmholtz coil, and an oscillating magnetic field was applied. The initial magnetic field parameters were set to magnetic field strength B = 3 mT and magnetic field frequency. =2Hz, to drive the magnetic beads to produce periodic oscillation and cluster reconfiguration behavior.

[0058] Under these conditions, the following variable analysis will be conducted: The effect of viscosity on the oscillation motion of magnetic beads: Adjust the viscosity of the glycerol aqueous solution and set the viscosity gradient to 3.40 (corresponding to 40% glycerol aqueous solution), 7.80 (corresponding to 60% glycerol aqueous solution), and 55 (corresponding to 80% glycerol aqueous solution). Under the same magnetic field parameters, the motion characteristics of magnetic beads in environments with different viscosities were analyzed.

[0059] The effect of magnetic field gyrometry on the oscillating motion of magnetic beads: with the viscosity of the glycerol aqueous solution fixed at 3.40. Magnetic field frequency Adjusting the gyrometry of the magnetic field at 2Hz The gradient change was formed to study its effect on the speed and motion pattern of the magnetic beads.

[0060] The effect of magnetic field frequency on the oscillating motion of magnetic beads: with the viscosity of the glycerol aqueous solution fixed at 3.40. Magnetic field gyrometry =0.6 Under certain conditions, the frequency of the magnetic field is adjusted to create a gradient change in order to analyze the regulatory effect of frequency change on the oscillation behavior of the magnetic bead.

[0061] During data acquisition, the movement of the magnetic beads was recorded on video, with each video segment lasting 2–3 seconds. The video was then converted into a continuous image sequence at 25 frames per second and input into an oscillating magnetic field coagulation analysis unit for processing. The analysis unit uses a horizontal line as a reference point to calculate the instantaneous angle of the rod-shaped magnetic bead cluster frame by frame, thereby obtaining the variation pattern of its oscillation angle.

[0062] The effect of viscosity on the motion characteristics of magnetic beads was studied under a rotating magnetic field. Experimental results show that the motion speed of the rod-shaped magnetic bead cluster gradually decreases with increasing viscosity of the glycerol aqueous solution. When the solution viscosity increases to approximately 3.40... At that time, the movement of the magnetic beads slowed down significantly, the cluster movement became restricted, and it was difficult to maintain stable rotational behavior. This indicates that in a high viscosity environment, the magnetic field driving effect is significantly suppressed, which is not conducive to the effective movement of the magnetic beads.

[0063] Furthermore, in investigating the influence of magnetic field parameters on the motion of magnetic beads, it was found that, keeping other conditions constant, the motion speed of the rod-shaped magnetic bead cluster gradually increased with the increase of magnetic field strength and frequency. This indicates that enhancing the driving force of the magnetic field helps to improve the motion response and efficiency of the magnetic beads. Figure 6 As shown.

[0064] The effects of magnetic field gyrometry, driving frequency, and solution viscosity on the motion behavior of magnetic beads under oscillating magnetic field conditions were systematically analyzed. For example... Figure 8 As shown, the experimental results indicate that as the gyrometry ratio of the magnetic field increases, the oscillation amplitude of the rod-shaped magnetic bead gradually increases, indicating that the gyrometry ratio of the magnetic field has a significant regulatory effect on the trajectory and amplitude of the magnetic bead.

[0065] In addition, such as Figure 7 As shown, in the process of investigating the influence of magnetic field frequency on the oscillation behavior of magnetic beads, it was found that the swing amplitude of the rod-shaped magnetic beads first increases and then decreases with the increase of magnetic field frequency, indicating that there is an optimal frequency range under the drive of the oscillating magnetic field, within which the magnetic beads can obtain a large oscillation response.

[0066] On the other hand, such as Figure 9 As the viscosity of the glycerol aqueous solution increases, the oscillation amplitude of the rod-shaped magnetic beads generally decreases. This is because the increased viscosity of the system leads to enhanced fluid damping, thus limiting the movement amplitude of the magnetic beads. However, this mode still applies at higher viscosities. Under lower viscosity conditions, the oscillation amplitude of the magnetic beads is relatively large, but excessively large oscillation amplitude may reduce its sensitivity to changes in system viscosity, thus hindering the accurate characterization of subtle rheological changes during the coagulation reaction and consequently affecting detection sensitivity.

[0067] As the viscosity increases appropriately, the oscillation of the magnetic beads gradually stabilizes, and their motion becomes more sensitive to changes in viscosity, which helps to improve the resolution of the detection signal.

[0068] In summary, under oscillating magnetic field conditions, the movement of magnetic beads is influenced by the coupling effect of frequency and viscosity, resulting in an optimal operating range that balances oscillation amplitude and detection sensitivity. Therefore, in practical applications, it is necessary to comprehensively adjust the magnetic field frequency and system viscosity to obtain the best detection results. Compared to rotating magnetic fields, oscillating magnetic fields are less suitable for low-viscosity environments.

[0069] (3) Validation of coagulation parameters based on clinical samples: Clinical samples were obtained from the First Affiliated Hospital of Chongqing Medical University, including 10 healthy individuals and 30 patients with reduced activated partial thromboplastin time (APTT). To verify the effectiveness of the method in actual samples, activated partial thromboplastin time (APTT) was measured using clinical plasma samples.

[0070] In this embodiment, 6 is taken. Magnetic microbeads were mixed with plasma, APTT ellagic acid solution, and CaCl2 solution at a volume ratio of 50:55:55, and gently blown to mix thoroughly to form a homogeneous reaction system. The mixture was then incubated at 37°C to initiate the coagulation reaction. After incubation, the reaction system was transferred to a reaction vessel and placed on a magnetic rack for 4–6 seconds, allowing the magnetic microbeads to adhere to the bottom of the vessel under the influence of the magnetic field. The reaction vessel was then rapidly transferred to the central region of a Helmholtz coil, where the magnetic beads were driven to form a rod-like cluster and oscillate periodically under the influence of the oscillating magnetic field.

[0071] The oscillating magnetic field parameters are set as follows: magnetic field strength The value is 3mT, the driving frequency is 3Hz, and the slew rate is... It is 0.8 Under these conditions, the magnetic beads can form a stable and repeatable oscillating motion in the system.

[0072] Plasma samples from 10 healthy individuals and 30 patients with abnormal activated partial thromboplastin time (APTT) were selected and tested using the above method. The change in the amplitude of the magnetic bead's oscillation over time was monitored in real time to determine the point at which the bead oscillation tended to stop, and this point was used as the APTT test result.

[0073] Experimental results are as follows Figure 10 As shown, under the above detection conditions, there are significant differences between healthy samples and abnormal APTT samples in terms of magnetic bead movement behavior and corresponding coagulation time parameters. The method of the present invention can effectively distinguish between normal and abnormal APTT states, demonstrating good discriminative ability and clinical application potential.

[0074] like Figure 11 The present invention also provides a coagulation detection system based on magnetic field-driven micro-robot swarm motion, including a magnetic control drive module, a coagulation signal detection module, an image analysis module, and a coagulation reaction drive module; the magnetic control drive module includes a magnetic field generating unit, a signal control unit, and a motion observation unit.

[0075] The magnetic field generating unit employs a triaxial Helmholtz coil structure and an optical imaging unit to generate and modulate a time-varying magnetic field. This magnetic field control drives the movement of superparamagnetic beads in the coagulation analysis sample, thereby participating in and regulating the coagulation reaction process. The time-varying magnetic field includes a rotating magnetic field and an oscillating magnetic field; when the magnetic field generating unit generates the rotating magnetic field, the magnetic field strength... The value ranges from 2 to 6. Magnetic field frequency The value ranges from 1 to 5 Hz; when an oscillating magnetic field is generated, the magnetic field frequency is... The value ranges from 1 to 5 Hz, and the gyroscope ratio is... 0.2~1 .

[0076] The signal control unit generates and amplifies the drive current signal to adjust the magnetic field parameters. The signal control unit includes a function generator and a power amplifier. The input current for each axis coil of the triaxial Helmholtz coil structure is provided by the function generator and amplified by the power amplifier to drive the corresponding coil. Software control is used to schedule and control the amplitude, frequency, and phase of the current in each channel, achieving real-time adjustment of the magnetic field strength and spatial vector direction.

[0077] The motion observation unit is used to observe and record the motion state of the magnetic beads in real time. The motion observation unit uses an inverted microscope in conjunction with a CCD camera, with an image acquisition frame rate of 25 frames per second to obtain the complete dynamic motion process.

[0078] The coagulation signal detection module is electrically connected to the magnetic control drive module and is used to acquire and identify the motion state of the magnetic beads under the action of the magnetic field. The optical imaging unit acquires the motion process of the magnetic bead cluster and extracts motion parameters through image processing and analysis algorithms to obtain characteristic signals reflecting the coagulation process. The magnetic beads identified by the coagulation signal detection module are two to three attached rod-shaped magnetic beads.

[0079] The image analysis module is electrically connected to the coagulation signal detection module. By preprocessing continuous video frames, tracking particle clusters, and calculating inter-frame correlation, dynamic parameters characterizing the intensity of cluster motion are extracted. The characteristic values ​​of the motion decay curve of the dynamic parameters are correlated with the viscoelastic changes caused by the coagulation reaction, which are used to quantitatively determine coagulation parameters. The coagulation reaction driving module is used to generate a controllable magnetic field and drive the movement of superparamagnetic beads in the coagulation analysis sample through magnetic field regulation, thereby participating in and regulating the coagulation reaction process.

[0080] The algorithm executed by the image analysis module includes the following steps: Step 1: Preprocess the acquired continuous video frames and perform particle cluster tracking, specifically as follows: Video parsing: Acquire the video stream to be processed, render it at a preset frame rate (e.g., 25 frames / second), and parse it into a continuous sequence of frame images.

[0081] Image processing utilizes the image analysis module to sequentially perform grayscale conversion and noise reduction on each frame in the frame image sequence in order to extract clear target regions.

[0082] Feature extraction and matching: Connected component calculation and area calculation are performed on the processed image to extract the target contour of the rod-shaped magnetic beads.

[0083] Step 2: Quantify the overall angular velocity change of the cluster by calculating inter-frame correlation, specifically as follows: By analyzing the main axis, the orientation angle of each rod-shaped magnetic bead in the current frame is obtained, and combined with the inter-frame matching algorithm, the original angle change sequence of each magnetic bead's continuous motion is output.

[0084] Angular velocity calculation: Calculate the angular velocity of the rod-shaped magnetic bead based on the continuous motion angle. To eliminate the phase abrupt change caused by the 180-degree symmetry of the rod-shaped magnetic bead, let the angular change of the magnetic bead between two adjacent frames be . The time interval is angular velocity The calculation logic is as follows: when When <90°, ; when ≥90° and When >0, ; when and hour, .

[0085] Therefore, this invention employs the aforementioned coagulation detection method and system based on magnetic field-driven microrobot swarm motion. By driving the microrobot swarm motion with a magnetic field, the viscoelastic changes in the coagulation process are transformed into quantifiable swarm motion parameters. This not only achieves accurate detection of coagulation time parameters but also continuously reflects the dynamic process of the coagulation reaction at the microscale. The detection sensitivity, stability, and accuracy are all superior to traditional methods. Furthermore, the detection system of this invention has controllable parameters, is easy to operate, and adapts to different coagulation detection needs. Clinical sample validation has demonstrated its good practicality and clinical application potential, providing reliable technical support for clinical coagulation function testing and promoting technological progress and innovation in the field of coagulation detection.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for coagulation detection based on magnetic field-driven micro-robot swarm motion, characterized in that, Includes the following steps: S1. Mix the diluted magnetic beads with the plasma to be tested and the coagulation reagent, and incubate them in a reaction container to form a coagulation reaction system. S2. Select and apply a time-varying magnetic field driving mode that is compatible with the detection requirements to drive the magnetic beads to generate cluster movement. S3. During the coagulation reaction, the motion video data of the magnetic bead cluster under the drive of the magnetic field is acquired in real time through the image acquisition device; S4. Perform time-series analysis on motion video data using image processing and analysis algorithms, extract dynamic parameters characterizing the intensity of cluster motion, and generate motion decay curves that change over time. S5. Based on the changing trend of the motion decay curve, the viscoelastic changes in the coagulation process are quantitatively reflected, and the coagulation state and corresponding coagulation time parameters are determined.

2. The coagulation detection method based on magnetic field-driven microrobot swarm motion according to claim 1, characterized in that, S1 specifically refers to: S11. The specific process for diluting the magnetic beads is as follows: First, wash the magnetic bead suspension with a concentration of 10 mg / mL with a 1:1 volume ratio of deionized water and ethanol 3 to 5 times to remove impurities; Then, the cleaned magnetic bead suspension was diluted 15 times by volume and resuspended in deionized water to obtain a magnetic bead solution with a concentration of approximately 0.67 mg / mL. S12. Construct a simulated blood plasma environment: First, mix 8 mL of glycerol with 2 mL of deionized water to obtain the basic solution; Then, add an appropriate amount of deionized water to the base solution to dilute it and obtain a solution with the required percentage of glycerol, wherein the percentages of glycerol are 60%, 40%, 30%, 20% and 10% respectively. Finally, the viscosity of the prepared solution and pure deionized water were measured using a viscometer. S13. Preparation of plasma to be tested: Take blood and add it into a vacuum tube. Add sodium citrate anticoagulant, which is 1 / 10 the volume of blood, into the tube beforehand. Centrifuge it at 3000 rpm or 2500 g for 10 minutes and collect the supernatant liquid, which is platelet-deficient plasma; The plasma was then transferred to plastic tubes for storage.

3. The coagulation detection method based on magnetic field-driven microrobot swarm motion according to claim 2, characterized in that, In S1, the particle size of the magnetic beads is 2~4 mm. Its surface is coated with carboxyl groups, giving it superparamagnetic properties.

4. The coagulation detection method based on magnetic field-driven microrobot swarm motion according to claim 1, characterized in that, In S1, the coagulation reagents include prothrombin time assay reagent PT, activated partial thromboplastin time assay reagent APTT, and prothrombin time assay reagent TT.

5. The coagulation detection method based on magnetic field-driven microrobot swarm motion according to claim 1, characterized in that, In S1, the reaction vessel is a small, circular dish used to hold coagulation analysis samples, with an effective volume of 150–170 mL. .

6. The coagulation detection method based on magnetic field-driven microrobot swarm motion according to claim 1, characterized in that, In S2, the time-varying magnetic field includes a rotating magnetic field and an oscillating magnetic field; the rotating magnetic field is used to drive the magnetic beads to form a stable rotating cluster motion, and its magnetic field parameter is the magnetic field strength. and magnetic field frequency Among them, magnetic field strength The value ranges from 2 to 6. Magnetic field frequency The value ranges from 1 to 5 Hz; An oscillating magnetic field is used to drive a magnetic bead to reciprocate motion; its magnetic field parameter is the magnetic field frequency. Slew rate Among them, magnetic field frequency The value ranges from 1 to 5 Hz, and the gyroscope ratio is... 0.2~1 .

7. The coagulation detection method based on magnetic field-driven microrobot swarm motion according to claim 1, characterized in that, In S5, the characteristic parameters of the motion decay curve include the rotational speed of the rotating magnetic field and the rotational angle of the oscillating magnetic field.

8. A coagulation detection system based on magnetic field-driven microrobot swarm motion, used to implement the method described in any one of claims 1-7, characterized in that, It includes a magnetic drive module, a coagulation signal detection module, an image analysis module, and a coagulation reaction drive module; the magnetic drive module includes a magnetic field generation unit, a signal control unit, and a motion observation unit; The magnetic field generating unit employs a triaxial Helmholtz coil structure and an optical imaging unit to generate and control a time-varying magnetic field; the time-varying magnetic field includes a rotating magnetic field and an oscillating magnetic field; the signal control unit is used to generate and amplify the drive current signal to adjust the magnetic field parameters; The motion observation unit is used to observe and record the motion state of the magnetic beads in real time; The coagulation signal detection module is electrically connected to the magnetic control drive module and is used to acquire and identify the motion state of the magnetic beads under the action of the magnetic field. The optical imaging unit acquires the motion process of the magnetic bead cluster, and the magnetic beads identified by the coagulation signal detection module are two to three attached rod-shaped magnetic beads. The image analysis module is electrically connected to the coagulation signal detection module. By preprocessing continuous video frames, tracking particle clusters, and calculating inter-frame correlation, dynamic parameters characterizing the intensity of cluster motion are extracted. The characteristic values ​​of the motion decay curve of the dynamic parameters are correlated with the viscoelastic changes caused by the coagulation reaction, which are used to quantitatively determine coagulation parameters. The coagulation reaction driving module is used to generate a controllable magnetic field and drive the movement of superparamagnetic beads in the coagulation analysis sample through magnetic field regulation, thereby participating in and regulating the coagulation reaction process.

9. The coagulation detection system based on magnetic field-driven microrobot swarm motion according to claim 8, characterized in that, When the magnetic field generating unit generates a rotating magnetic field, the magnetic field strength The value ranges from 2 to 6. ,frequency The value ranges from 1 to 5 Hz; when an oscillating magnetic field is generated, the magnetic field frequency is... The value ranges from 1 to 5 Hz, and the gyroscope ratio is... 0.2~1 ; The signal control unit includes a function generator and a power amplifier, which are used to generate and amplify the drive current signal. The input current of each axis coil of the triaxial Helmholtz coil structure is provided by the function generator and amplified by the power amplifier to drive the corresponding coil. The amplitude, frequency, and phase of the current in each channel are controlled by software, thereby enabling real-time adjustment of the magnetic field strength and spatial vector direction. The motion observation unit uses an inverted microscope in conjunction with a CCD camera, with an image acquisition frame rate of 25 frames per second.

10. The coagulation detection system based on magnetic field-driven microrobot swarm motion according to claim 9, characterized in that, The algorithm executed by the image analysis module includes the following steps: Step 1: Preprocess the acquired continuous video frames and perform particle cluster tracking, specifically as follows: First, acquire the video stream to be processed, and render and parse it into a continuous sequence of frame images at a preset frame rate; The image analysis module is used to perform grayscale conversion and noise reduction on each frame in the frame image sequence in order to extract clear target areas; The processed image is subjected to connected component calculation and area calculation to extract the target contour of the rod-shaped magnetic beads; Step 2: Quantify the overall angular velocity change of the cluster by calculating inter-frame correlation, specifically as follows: The orientation angle of each rod-shaped magnetic bead in the current frame is obtained by principal axis analysis, and combined with the inter-frame matching algorithm, the original angle change sequence of each magnetic bead's continuous motion is output. The angular velocity of the rod-shaped magnetic bead is calculated based on the angle of continuous motion. To eliminate the phase abrupt change caused by the 180-degree symmetry of the rod-shaped magnetic bead, let the angular change of the magnetic bead between two adjacent frames be . The time interval is angular velocity The calculation logic is as follows: when When <90°, ; when ≥90° and When >0, ; when and hour, .

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