Magnetic relaxation veterinary drug residue detection method combined with microfluidics

Abstract

The invention discloses a microfluidic-combined magnetic relaxation veterinary drug residue detection method which comprises the following steps: selecting and preparing a standard solution containing a veterinary drug standard substance, and then preparing a magnetic nanoparticle solution of a coupling antibody antigen corresponding to the standard solution; carrying out magnetic separation on the mixed solution of the three solutions by using a magnetic block, and separating out an antibody magnetic bead solution combined with a target object; after the separated antibody magnetic bead solution combined with the target object flows into a detection cavity, the detection cavity is placed in a uniform magnetic field environment with a magnetic field main direction, a CPMG radio frequency pulse sequence is transmitted to a coil, and the transverse relaxation time of a sample solution is obtained through treatment; the uniform magnetic field environment is passive shimming; and veterinary drug residue detection is carried out according to the transverse relaxation time. According to the invention, the time of magnetic separation is optimized, a reaction system and time parameter optimization oriented to a microfluidic environment are determined, and sample detection is executed and completed at a minimum dosage and a fastest speed.

Description

Technical Field

[0001] This invention belongs to the fields of food safety testing and magnetic relaxation technology, specifically a magnetic relaxation method for detecting veterinary drug residues combined with microfluidics. Background Technology

[0002] With increasing public awareness of health and widespread concern for food safety, the need for rapid and accurate detection of trace veterinary drug residues in animal-derived products during transportation and refrigeration is becoming increasingly urgent. Traditional analytical methods mainly fall into two categories: one is laboratory instrument detection techniques such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS / MS). While these methods possess excellent sensitivity and specificity, their detection procedures are lengthy and pretreatment complex, failing to meet the urgent need for real-time, high-throughput screening of animal-derived products. The other category comprises rapid on-site detection methods such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography. Although these methods are simple to operate, their sensitivity is insufficient, and their reliance on manual interpretation can easily introduce errors. Therefore, there is an urgent need for a technology that can achieve rapid, accurate, and quantitative detection of veterinary drug residues in animal-derived products on-site during cold chain logistics.

[0003] Magnetic relaxation technology, as a rapid non-destructive testing technique, shows promising application prospects in trace substance detection. However, magnetic relaxation detection relies on a stable and uniform static magnetic field environment. While traditional benchtop relaxation sensors can provide high field strength and good uniformity, they are bulky and costly, making them unsuitable for field applications. As instruments become smaller and more portable, limitations such as permanent magnet size and assembly errors reduce the field strength uniformity in the detection area at the magnet's center, leading to problems like T2 fitting deviation and consequently reducing the accuracy and repeatability of quantitative results. Furthermore, in existing technologies, sample pretreatment requires manual mixing of magnetic nanoparticles with the veterinary drug to be tested. This process results in uneven distribution of magnetic particles and insufficient binding with the veterinary drug sample, causing relaxation time measurement errors. Microfluidics, as a novel technology, can precisely control microliters or even nanoliters of fluids, integrating mixing, reaction, and separation operations onto a single chip to automate and miniaturize complex biochemical analysis processes. This not only reduces reagent consumption costs but also controls fluid reaction time, improves fluid mixing efficiency, and significantly enhances the repeatability and reliability of detection. However, existing microfluidic technology solutions are mostly independent of the detection system and lack standardized design that is deeply integrated with portable magnetic relaxation sensing probes (especially their core tuned matching RF coil and shimming detection area) in terms of structure, volume and process.

[0004] Therefore, in response to the two core methodological challenges mentioned above—the degradation of magnetic field performance during portability and the disconnect between microfluidic pretreatment and magnetic relaxation detection—there is an urgent need to develop a portable magnetic relaxation sensing detection method that can organically combine high-performance shimming technology, optimized microfluidic reaction systems, and automated process control to solve the problem of balancing efficiency and accuracy in on-site detection of veterinary drug residues in cold chain logistics. Summary of the Invention

[0005] To address the problems existing in the background technology, this invention provides a magnetic relaxation veterinary drug residue detection method combined with microfluidics. This method combines microfluidics technology with magnetic relaxation sensing technology, enabling not only low-consumption and rapid sample pretreatment, but also achieving high-accuracy detection of trace veterinary drug residues through the setup of main and auxiliary Halbach magnet modules and a passive shimming device. The technical solution includes: Step 1: Select and prepare a standard solution containing veterinary drug standards, and then prepare a magnetic nanoparticle solution of conjugated antibody antigens corresponding to the standard solution; Step 2: Use a magnetic block to perform magnetic separation on the mixture of the three solutions to separate the antibody magnetic bead solution that binds to the target analyte; Step 3: After the antibody magnetic bead solution containing the target analyte is separated and flows into the detection chamber, the detection chamber is placed in a uniform magnetic field environment with a main magnetic field direction. A CPMG radio frequency pulse sequence is emitted to the coil, and the echo signal is read at the pulse interval and processed to obtain the transverse relaxation time of the sample liquid. The uniform magnetic field environment is a passive shim. Step 4: Detect veterinary drug residues based on the transverse relaxation time.

[0006] The preparation process of the magnetic nanoparticle solution of the conjugated antibody antigen corresponding to the standard solution is as follows: First, the veterinary drug standard solution and the corresponding magnetic nanoparticle solution of the conjugated antigen are thoroughly mixed to form a mixture of the two solutions; then, the mixture of the two solutions is mixed with the corresponding magnetic nanoparticle solution of the conjugated antibody; the ratio of the veterinary drug standard solution, the magnetic nanoparticle solution of the conjugated antibody, and the magnetic nanoparticle solution of the conjugated antigen in the mixture of the three solutions is 1:1:2.

[0007] The separation time in step 2 is the optimal magnetic separation time T. min After the mixture of the three solutions is completed, it enters the magnetic separation chamber. During the separation process, a magnetic block is used to apply a temporary magnetic field to the outside of the chamber where the solution is stored so as to adsorb and retain the magnetic nanoparticles of the coupled antigen and the magnetic nanoparticles of the antibody antigen on the inner surface of the magnetic separation chamber, thereby separating the antibody magnetic bead solution bound to the target. Optimal magnetic separation time T min The solution formula is:

[0008] Where T2 is the transverse relaxation time of the 500nm magnetic bead; e Where A is the natural constant, and T2 saturation value is the value after magnetic separation, with 95% < T2 saturation value < 99%; k T2 is the growth rate constant, reflecting the aggregation efficiency of the magnetic beads under the action of a magnetic field. The larger the value, the faster T2 approaches saturation. t 0 represents the half-saturation time, which corresponds to the magnetic separation time when T2 reaches 50% of the saturation value A during the magnetic separation process.

[0009] The cross-sectional dimensions of the flow channel used in step 1 are 400 mm × 400 mm. The process of forming a mixture of the two solutions takes place in a flow channel with a Fermat spiral distribution. The flow channel has four triangular baffles spaced at certain intervals from the inlet inwards. The two hypotenuses of the triangular baffles face the inflow and outflow directions, respectively. The four triangular baffles are arranged at equal angles of 45° to enhance the mixing efficiency. The triangular baffles are isosceles triangles with a base length of 200 mm and a height of 200 mm. In step 1, the mixing path of the mixture of the three solutions is S-shaped, with the radius of the S-shaped wave being 1 mm.

[0010] The cross-sectional dimensions of the flow channel used in step 3 are 1.2 mm × 1.2 mm.

[0011] The solution flow processes in steps 1, 2 and 3 are all completed in a plate-shaped microfluidic chip. One end of the microfluidic chip is provided with an extension for accommodating the detection cavity, which is placed in the center of the uniform magnetic field environment in which a magnetic field has a main direction. A uniform magnetic field environment exists in the main magnetic direction, formed by Halbach magnet modules. The assembly process of the Halbach magnet modules is as follows: first, the central permanent magnet group is assembled; then, two coaxial z-axis supplementary permanent magnet groups are assembled at both ends of the central permanent magnet group; finally, the shimming group is installed using a shimming device mounting shell. The central permanent magnet assembly includes: eight trapezoidal cross-section main permanent magnets, which form a hollow strip structure with a regular octagonal cross-section. The hollow strip structure allows the extension part to be inserted and removed. At each end of the length direction of the hollow strip structure, there is a set of z-axis supplementary permanent magnets. The magnetic field direction formed by the two symmetrically arranged z-axis supplementary permanent magnets is the same as the main magnetic field direction. The main permanent magnets contain a field homogenizing group for uniform magnetic field direction. The z-axis supplementary permanent magnet assembly includes: 8 trapezoidal cross-section secondary permanent magnets, which are fixedly installed close to the central permanent magnet assembly. The outer circumference of the z-axis supplementary permanent magnet assembly is larger than that of the central permanent magnet assembly, and the cross-sectional dimensions of the central space of the z-axis supplementary permanent magnet assembly are the same as those of the central permanent magnet assembly. The shimming group is installed in the central permanent magnet group through the shimming device mounting shell. The shimming device mounting shell has an insertion port for the extension part in the center. The shimming group includes: a main shimming block group whose mounting surface passes through the main direction of the magnetic field and a secondary shimming block group symmetrically arranged on both sides of the main shimming block group. A row of main shimming blocks is set inside the two side walls along the main direction of the magnetic field. On the inner wall of the shimming device mounting shell at every 15° angle on both sides of each row of main shimming blocks, there are two rows of secondary shimming blocks. Multiple small shim blocks are arranged at equal intervals in the main shim block group and the secondary shim block group. The arrangement of small shim blocks in the main shim block group and the secondary shim block group is the same, each including six small shim blocks arranged sequentially along the axial direction of the central permanent magnet group. The six small shim blocks are arranged symmetrically along the radial plane of the Halbach magnet where the detection cavity axis is located. The number of small shim blocks in the six small shim blocks are 3, 2, 1, 1, 2, and 3 respectively. The small shim blocks are cylindrical and stacked coaxially.

[0012] The outer diameter of the thinner part of the central permanent magnet assembly is 60mm, and the inner diameter is 30mm; the inner diameter of the thicker z-axis supplementary permanent magnet assembly at both ends is 30mm, the outer diameter is 84mm, and the thickness is 17mm; the overall axial length of the Halbach magnet module is 112mm; the thickness of the small uniform field block is 1mm and the diameter is 2mm. The Halbach magnet module is set in a 32°C temperature environment to maintain the test environment temperature, thereby ensuring the stability of the magnetic field; The total volume of the mixture of the three solutions is the minimum total volume V. m0 The mixing time of the mixture of the three solutions is the optimal reaction time T under the calibration conditions. ref The concentration gradient of the veterinary drug standard in the standard solution is 0.1~1000 μg / kg, and the veterinary drug standard is a quinolone, sulfonamide or chloramphenicol; Among them, the minimum total volume V m0 =4V, where ;

[0013] Among them, T min The shortest time determined by diffusion kinetics; total reaction volume V o =V m0 T b The optimal reaction time required to achieve immunoadsorption equilibrium.

[0014] In step 4, the measured transverse relaxation time T2 is compared with the pre-established norfloxacin standard curve, and the concentration of norfloxacin in the sample is calculated based on the change in transverse relaxation time DT2.

[0015] The beneficial effects of this invention are as follows: 1. High integration and speed of the detection process: The method of this invention integrates the sample introduction, precise quantitative mixing reaction of immunomagnetic beads, magnetic separation, and magnetic relaxation in situ detection into a microfluidic chip. This not only optimizes the magnetic separation time but also determines the reaction system and time parameter optimization for the microfluidic environment. It enables sample detection to be completed at the lowest dose and fastest speed, avoiding human error and cross-contamination. It truly realizes rapid detection of "sample in, result out", which greatly improves the convenience and efficiency of on-site operation.

[0016] 2. High sensitivity and accuracy of detection results: This method uses a "mix-then-react" mixing approach for the sample, which creates a Dean vortex between the sample and immunomagnetic nanoparticles within a defined flow path, greatly improving mixing efficiency. Existing techniques do not generate a sufficiently large and uniform magnetic field. By using a main and auxiliary Halbach magnet module and a passive shimming device, we significantly improve the magnetic field uniformity in the detection area at the center of the magnet to below 300 ppm, achieving high signal-to-noise ratio quantitative detection of trace veterinary drug residues.

[0017] 3. High versatility and broad application prospects: This method can also be used to rapidly detect pesticide residues in other fields by changing the magnetic nanoparticle solutions with different specific antibodies on their surfaces and related reagents. It also shows broad application potential in food safety monitoring, environmental pollutant screening and even rapid diagnosis of clinical pathogens. Attached Figure Description

[0018] Figure 1 This is a schematic flowchart of an embodiment of a magnetic relaxation veterinary drug residue detection method combining microfluidics according to the present invention; Figure 2 This is a schematic diagram of the microfluidic chip where the microchannel is located in an embodiment of the present invention; Figure 3 This is a schematic diagram of the longitudinal section of the Halbach magnet module in an embodiment of the present invention; Figure 4 This is a schematic diagram of the microfluidic chip and Halbach magnet module in use in an embodiment of the present invention; Figure 5 This is a cloud map showing the simulation results of the magnetic field strength of the central permanent magnet assembly in an embodiment of the present invention. Figure 6 This is a cloud map showing the simulation results of the optimized magnetic field strength of the permanent magnet assembly in an embodiment of the present invention. Figure 7 This is a schematic diagram of the Halbach magnet structure in an embodiment of the present invention; Figure 8This is a schematic diagram of the direction of the Halbach magnet's magnetic field in an embodiment of the present invention; Figure 9 This is a schematic diagram of the field homogenizing device in an embodiment of the present invention; Figure 10 This is a top view of the arrangement of small uniform field blocks in an embodiment of the present invention; Figure 11 This is a schematic diagram of the arrangement of small uniform field blocks in an embodiment of the present invention; Figure 12 This is a standard curve diagram of norfloxacin in an embodiment of the present invention.

[0019] In the figure, 1-fixed base, 2-microfluidic chip, 201-triangular baffle, 202-magnetic separation cavity, 203-extension part, 204-detection cavity, 3-Halbach magnet module, 301-magnet shell, 302-z-axis supplementary permanent magnet group, 303-central permanent magnet group, 304-shielding group, 305-shielding device mounting shell. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings.

[0021] like Figure 1 The embodiment of the present invention shown includes: Step 1: Select veterinary drug standard solutions targeting quinolones, sulfonamides, chloramphenicol, etc., and prepare standard solutions with concentration gradients of 0.1~1000μg / kg; then, prepare magnetic nanoparticle solutions of conjugated antibody antigens corresponding to the standard solutions; In this embodiment, the standard solution is a quinolone veterinary drug standard solution; the preparation process of the magnetic nanoparticle solution of the conjugated antibody antigen corresponding to the standard solution is as follows: first, thoroughly mix 300 μL of veterinary drug standard solution (norfloxacin standard solution in this embodiment) and the corresponding magnetic nanoparticle solution of the conjugated antigen (in this embodiment, the corresponding BSA-MNP is selected according to the quinolone veterinary drug standard solution). 500nm The solution, namely the 500 nm magnetic nanoparticle solution of the conjugated antigen, is used to form a mixture of the two solutions; then, the mixture of the two solutions and the corresponding magnetic nanoparticle solution of the conjugated antibody are mixed at a ratio of 3:1 (in this embodiment, the corresponding Ab-MNP is selected based on the quinolone veterinary drug standard solution). 30nm The solution (i.e., a solution of 30 nm magnetic nanoparticles containing conjugated antibodies) was used to form a 400 μL mixture of three solutions in a 1:1:2 ratio. The mixing time for the three solutions was 30 min. The mixture contained veterinary drug standard solution, Ab-MNP... 30nm and BSA-MNP 500nmThe solution ratio is 1:1:2; after the mixing reaction of the three solutions is completed, the mixture enters the magnetic separation chamber 203. Step 2: Use a magnetic block to perform magnetic separation on the mixture of the three solutions to separate the antibody magnetic bead solution bound to the target analyte (Ab-MNP in this example). 30nm -Target solution); the separation time is the optimal magnetic separation time T. min ; During the separation process in step 2, a temporary magnetic field is applied to the outside of the cavity containing the solution using a magnetic block to bind the magnetic nanoparticles of the antigen-coupled substance to the magnetic nanoparticles of the antibody-antigen combination (in this embodiment, BSA-MNP). 500nm BSA-MNP 500nm -Ab-MNP 30nm The antibody magnetic beads (Ab-MNP) solution, which binds to the target analyte, is adsorbed and retained on the inner surface of the magnetic separation chamber 203. 30nm -Target solution) was separated.

[0022] In this embodiment, the optimal magnetic separation time is 2 minutes, and the location is in the magnetic separation cavity 203; This embodiment provides a preferred implementation method. To achieve standardized control and result correction of the magnetic separation condition, a calibration curve of "magnetic separation time - transverse relaxation time T2" ​​is established based on experimental data. Specifically, the magnetic separation time... t Using the measured transverse relaxation time T2 of the 500 nm magnetic bead as the dependent variable, the relationship between "magnetic separation time - T2 time" is modeled using the weighted least squares method, resulting in an empirical fitting formula for predicting T2 time and calibrating magnetic separation parameters, as follows:

[0023] Where T2 is the transverse relaxation time (ms) of the 500nm magnetic bead. t The magnetic separation time (min) is applicable in the range of 0 ≤ t ≤ 6 min; e is the natural constant, and A is the T2 saturation value (ms) after magnetic separation; k T2 growth rate constant (min⁻¹) reflects the aggregation efficiency of magnetic beads under the action of a magnetic field. The larger the value, the faster T2 approaches saturation. t 0 represents the half-saturation time (min), corresponding to the time when T2 reaches 50% of the saturation value A during the magnetic separation process. This characterizes how quickly the magnetic separation process reaches a steady state. After this moment, the rate of increase of T2 gradually slows down and approaches saturation. In this embodiment, the data fitting yielded a value of A of 132.92. k The value is 2.82. t If 0 takes the value 0.72, then the above formula can be written as:

[0024] Based on the above formula and the requirements for rapid on-site testing, the lower threshold for reaching a stable magnetic separation state is defined as 95% of the saturation value in the magnetic separation time, at which point the magnetic separation time is approximately 1.76 minutes. According to the "marginal benefit" principle, the upper threshold for reaching a stable magnetic separation state is defined as 99% of the saturation value in the magnetic separation time, at which point the magnetic separation time is approximately 2.34 minutes. Therefore, the optimal magnetic separation time range for the transverse relaxation time is 1.76-2.34 minutes. Incorporating engineering margins, 2 minutes is used as the specific value for the optimal magnetic separation time in this embodiment. Optimal magnetic separation time T min The solution formula is:

[0025] Where T2 is the transverse relaxation time of the 500nm magnetic bead; e Where A is the natural constant, and T2 saturation value is the value after magnetic separation, with 95% < T2 saturation value < 99%; k T2 is the growth rate constant, reflecting the aggregation efficiency of the magnetic beads under the action of a magnetic field. The larger the value, the faster T2 approaches saturation. t 0 represents the half-saturation time, which corresponds to the magnetic separation time when T2 reaches 50% of the saturation value A during the magnetic separation process.

[0026] Step 3: Separate the antibody magnetic bead solution that binds to the target analyte (Ab-MNP in this example). 30nm After the target solution flows into the detection chamber, the detection chamber is placed in a uniform magnetic field environment with a main magnetic field direction. A CPMG radio frequency pulse sequence is emitted to the coil, and the echo signal is read at the pulse interval and processed to obtain the transverse relaxation time of the sample liquid. The uniform magnetic field environment is a passive uniform field, which is provided by a Halbach magnet module composed of three permanent magnets in this embodiment. Step 4: Detect veterinary drug residues based on the transverse relaxation time. Specifically: compare the measured transverse relaxation time T2 with a pre-established norfloxacin standard curve (e.g., ...). Figure 12 As shown in the figure, the concentration of norfloxacin in the sample is calculated based on the change in transverse relaxation time DT2. This result can be output in real time via a host computer touchscreen interface, enabling rapid quantitative analysis.

[0027] The cross-sectional dimensions of the flow channel used in step 1 are 400 mm × 400 mm; in this embodiment, 100 μL of Ab-MNP is mixed. 30nm Solution and 200 μL BSA-MNP 500nmThe solution (the process of forming a mixture of two solutions) is located in a flow channel with a Fermat spiral distribution. The flow channel has four triangular baffles 201 arranged at certain intervals from the inlet inward. The two hypotenuses of the triangular baffles 201 face the inflow and outflow directions, respectively. The four triangular baffles are arranged at equal angles of 45° to enhance the mixing efficiency. The triangular baffles are isosceles triangles with a base length of 200 mm and a height of 200 mm. In step 1, to ensure better mixing of the two solutions, the total length of the Fermat spiral is 25 mm, and its parametric equation is: ;in x , y These are the horizontal and vertical coordinates, respectively; In step 1, the mixing path of the three solutions is S-shaped and has a total length of 470 mm (the flow time of this length of mixed solution is the same as the mixing time, which is 30 min), wherein the radius of the S-shaped wave is 1 mm.

[0028] The cross-sectional dimensions of the flow channel used in step 3 are 1.2 mm × 1.2 mm; Considering that small Halbach magnets are sensitive to ambient temperature, in this embodiment, the Halbach magnet module 3 is set in a temperature environment of 32°C to maintain the test environment temperature, thereby ensuring the stability of the magnetic field.

[0029] In this embodiment, the solution flow process in steps 1 to 4 is completed in a plate-shaped microfluidic chip 2. One end of the microfluidic chip 2 is provided with an extension 203 for accommodating the detection cavity 204. The detection cavity 204 is placed in the center of a uniform magnetic field environment with a main magnetic field direction. The Halbach magnet module 3 forms a uniform magnetic field environment with a principal magnetic field direction. The dumbbell-shaped Halbach magnet module 3 includes: a central permanent magnet group 303, two z-axis supplementary permanent magnet groups 302, and a shimming group 304. The assembly process is as follows: first, the central permanent magnet group 303 is assembled; then, two coaxial z-axis supplementary permanent magnet groups 302 are assembled at both ends of the central permanent magnet group 303; finally, the shimming group 304 is installed through the shimming device mounting shell 305. The central permanent magnet assembly 303 includes: eight trapezoidal cross-section main permanent magnets, which form a hollow strip structure with a regular octagonal cross-section. The hollow strip structure allows the insertion part 203 to be inserted and removed. At each end of the length direction of the hollow strip structure, there is a set of z-axis supplementary permanent magnet assemblies. The magnetic field direction formed by the two symmetrically arranged z-axis supplementary permanent magnet assemblies 302 is the same as the main magnetic field direction. The main permanent magnet assembly is equipped with a field homogenizing assembly 304 for uniform magnetic field direction. The z-axis supplementary permanent magnet assembly 302 includes: 8 trapezoidal cross-section secondary permanent magnets, which are fixedly installed close to the central permanent magnet assembly 303. The outer circumferential dimension of the z-axis supplementary permanent magnet assembly 302 is larger than that of the central permanent magnet assembly 303. The cross-sectional dimension of the central space of the z-axis supplementary permanent magnet assembly 302 is the same as that of the central permanent magnet assembly 303. The shimming group 304 is installed in the central permanent magnet group 303 through the shimming device mounting shell 305. The shimming device mounting shell 305 has an insertion port of the extension part 203 in the center. The shimming group 304 includes: a main shimming block group whose mounting surface passes through the main direction of the magnetic field and a secondary shimming block group symmetrically arranged on both sides of the main shimming block group. A row of main shimming blocks is set inside the two side walls along the main direction of the magnetic field. On the inner wall of each 15° angle on both sides of each row of main shimming blocks (the inner wall of the shimming device mounting shell 305), there are two rows of secondary shimming blocks (a total of 4 rows).

[0030] Multiple small shim blocks are arranged at equal intervals in the main and secondary shim blocks groups. The arrangement of small shim blocks in the main and secondary shim blocks groups is the same, each including six small shim blocks arranged sequentially along the axial direction of the central permanent magnet group. The six small shim blocks are symmetrically arranged along the radial plane of the Halbach magnet where the detection cavity 204 axis is located. The number of small shim blocks in the six small shim blocks is 3, 2, 1, 1, 2, and 3 respectively. The small shim blocks are cylindrical and stacked coaxially. In this embodiment, the minimum interval between the main shimming block group and the adjacent secondary shimming block group, or between the secondary shimming block group and the adjacent secondary shimming block group, is 3 mm; the thickness of the small shimming block is 1 mm and the diameter is 2 mm.

[0031] In this embodiment, the main permanent magnet, the secondary permanent magnet, and the small uniform field block are all made of N52 neodymium iron boron. Each magnetic block is appropriately magnetized and oriented, and each Halbach magnet generates a uniform main magnetic field B0 perpendicular to the cross-section of the magnetic ring in the central region, with the direction as shown in the figure. Figure 12 As shown by the middle arrow, the central magnetic field is superimposed and increases to form a main magnetic field direction, while the external magnetic fields cancel each other out; the main permanent magnet and the auxiliary permanent magnet are fixedly installed in the magnet housing 301, thereby achieving the fixation of their relative positions with other equipment; In this embodiment, the outer diameter of the thinner portion of the central permanent magnet assembly 303 (enclosed by the main permanent magnets) is 60mm, and the inner diameter is 30mm. Figure 8The short side a of each main permanent magnet shown on the right is 12.43 mm, the long side b is 28.99 mm, and the height h is 20 mm. The inner diameter of the supplementary permanent magnet group 302 (enclosed by the secondary permanent magnets), which has thicker z-axis sections at both ends, is the same as the middle region (30 mm), but its outer diameter is 84 mm and its thickness is 17 mm. The overall axial length of the Halbach magnet module 3 is 112 mm. The multi-layered magnetic rings improve the central field strength while also optimizing the magnetic field uniformity.

[0032] In step 3, to determine the magnetic field performance of a uniform magnetic field environment with a principal magnetic field direction, a three-dimensional finite element simulation of the magnetic field is performed using COMSOL simulation software, with the target region set at the center of the magnetic field. f The simulation results for a 5mm × 5mm cylindrical region are shown in the figure. Figure 5 Images (a) through (d) show the three-dimensional cloud map of the magnetic field distribution in the target region, the magnetic field intensity cloud map in the xoy plane, the magnetic field intensity cloud map in the xoz plane, and the magnetic field intensity cloud map in the yoz plane, respectively. Figure 5 It can be seen that the magnetic field strength in the central region is uniformly distributed and symmetrically distributed along each plane. The average magnetic field strength in the target region of the magnetic field center is 1.0707 T, and the magnetic field uniformity is 373 ppm. However, the magnetic field strength still has a significant tendency to decrease at both ends along the z-axis, so further optimization of the structure is needed. In this embodiment, two sets of z-axis supplementary permanent magnet groups 302 are used to form a "dumbbell" Halbach-type magnet structure, and a set of shimming group 304 is added in the central region. The permanent magnet material used is N52 neodymium iron boron. The size of the thinner part in the middle is the same as before optimization. Figure 5 The dimensions are the same. The thicker portions at both ends are symmetrically distributed along the central plane of the magnet. Their inner diameter remains the same as before optimization (15 mm), but the outer diameter is increased to 42 mm, and the thickness is 16 mm. However, the overall height of the magnet remains unchanged at 112 mm. The optimized Halbach-type magnet was simulated using COMSOL simulation software. The target area is the same as before optimization. The simulation results are as follows: Figure 6 As shown in Table 1, the simulation results show that the average magnetic field strength in the target area at the center of the magnetic field is 1.0858 T and the magnetic field uniformity is 283 ppm. The comparison with the magnet performance before optimization is shown in Table 1. It can be seen that the magnetic performance of the optimized Halbach type magnet has been improved accordingly. Table 1 Comparison of Halbach magnet performance before and after optimization.

[0033] The portable magnetic relaxation detection described in this application is based on a static magnetic field provided by a Halbach permanent magnet. BThe CPMG echo signal is acquired by a coil within the uniform field region at the center of the magnet to calculate the transverse relaxation time T2. The magnetic field uniformity in the detection region at the center of the magnet is a key factor affecting detection accuracy and repeatability. Increased magnetic field inhomogeneity in the central region leads to abnormal echo attenuation, baseline drift, and increased equivalent T2 deviation during relaxation detection, directly reducing the accuracy of quantitative results. This is especially true when using immunomagnetic beads to amplify the signal, where detection sensitivity is closely related to the amount of beads used and the abundance of the target analyte. In scenarios involving trace veterinary drug residues, the amount of immunomagnetic beads used is relatively low, resulting in a decreased echo signal-to-noise ratio (SNR) and greater sensitivity to external disturbances in T2 fitting. This makes it easier for detection results to deviate or repeatability to deteriorate, thus placing higher demands on the uniformity of the central magnetic field.

[0034] Therefore, by configuring a passive shimming structure in the central region of the Halbach magnet (e.g., using an equally spaced array of small shimming blocks with temperature control for stabilization), the magnetic field uniformity in the central detection region is significantly improved. This allows for a higher signal-to-noise ratio and more stable T2 readings even under conditions of low bead quantity / weak signal, thus enhancing the accuracy and repeatability of trace detection. These techniques are not optional but crucial improvements necessary for achieving reliable quantification in portable permanent magnet NMR relaxation detection under field application conditions.

[0035] Regarding the selection of the total volume and ratio of the mixture of the three solutions in step 1: Since the uniform field region at the center of the magnet affects the total amount of standard solution in step 1, a more uniform magnetic field range and uniformity will result in a smaller total amount of standard solution required, thereby reducing mixing time and mixing ratio; and increasing the overall detection speed and portability.

[0036] To obtain a larger dynamic range of magnetic relaxation response while also considering on-site detection efficiency, this embodiment first optimizes the ratio, added volume, and reaction time of the three solutions. The operation process is as follows: first, the optimal ratio is selected from multiple sets of ratios through experiments, and then the minimum and maximum volumes are analyzed. The transverse relaxation time T2 signal change amplitude corresponding to multiple volume ratios (e.g., 1:1:1, 1:1:2, 2:2:2, all based on 100 µL) of the antibody-conjugated magnetic bead solution (Ab-MNP 30 nm), the test sample / standard solution, and the antigen-conjugated magnetic bead solution (BSA-MNP 500 nm) is used to determine the ratio that results in a larger T2 change and better repeatability. In this embodiment, the preferred volume ratio is Ab-MNP 30 nm : test sample / standard : BSA-MNP 500 nm = 1:1:2. Furthermore, assuming the above proportions are fixed, let V (unit: µL) be the volume added for each "1 part". Then, if V, V, and 2V are added to the three solutions respectively, the total mixed volume will be V. o=4V.

[0037] In volume optimization, V was adjusted within the range of 50–150 µL, and the corresponding T2 signals were acquired. Experiments revealed that when V was too small (e.g., in the 50 µL range), the equivalent concentration of magnetic beads in the system was too high, easily leading to local aggregation and saturation effects, thus compressing the dynamic range of the T2 response. When V was too large (e.g., in the 150 µL range), the system dilution factor was too large, reducing the effective interaction per unit volume and causing a decrease in the T2 response amplitude. Therefore, an equivalent concentration coefficient V was defined. d =100 / V, and set the coefficient threshold that can maintain a significant T2 dynamic range to 0.83≤V. d ≤1.25, therefore the preferred addition volume range is: 80≤V≤120 (mL), thus the minimum total volume range is 320≤V m0 =4V≤480 (mL); While maintaining a 1:1:2 ratio, the preferred addition volume range is 80:160:80 to 120:240:120 (µL). Within this range, the T2 signal exhibits good discriminative power and repeatability. Further, V = 100 µL (i.e., 100:100:200) is preferred as the optimal addition amount. The specific minimum total volume V used in this embodiment... m0 =4V=400µL.

[0038] The optimal reaction time was used for mixing the three solutions in step 1 because: After obtaining the optimal volume, while keeping the component ratio (1:1:2) constant, the total reaction volume V o The optimal reaction time T required to reach immunoadsorption equilibrium b Following a diffusion-controlled kinetic model, changes in volume directly affect the diffusion process, thus influencing the optimal reaction time required to reach binding equilibrium. Based on this mechanism, we established a correlation model that can guide practical operations:

[0039] Among them, T min The shortest time determined by diffusion dynamics; T ref The optimal reaction time under calibration conditions; in this embodiment, T is the optimal reaction time. min The value is 20 min, T ref The value is set to 30 min, thus obtaining the optimal time expression for this embodiment: T b = 20 + 0.025V o .

[0040] In summary, the above-mentioned volume ranges are included. Based on the time relationship, the optimal reaction time range is 28–32 min; further preferred is an addition volume V = 100 µL, which corresponds to an optimal reaction time of 30 min used in this embodiment.

Claims

1. A method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics, characterized in that, include: Step 1: Select and prepare a standard solution containing veterinary drug standards, and then prepare a magnetic nanoparticle solution of conjugated antibody antigens corresponding to the standard solution; Step 2: Use a magnetic block to perform magnetic separation on the mixture of the three solutions to separate the antibody magnetic bead solution that binds to the target analyte; Step 3: After the antibody magnetic bead solution containing the target analyte is separated and flows into the detection chamber, the detection chamber is placed in a uniform magnetic field environment with a main magnetic field direction. A CPMG radio frequency pulse sequence is emitted to the coil, and the echo signal is read at the pulse interval and processed to obtain the transverse relaxation time of the sample liquid. The uniform magnetic field environment is a passive shim. Step 4: Detect veterinary drug residues based on the transverse relaxation time.

2. The method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics according to claim 1, characterized in that, The preparation process of the magnetic nanoparticle solution of the conjugated antibody antigen corresponding to the standard solution is as follows: First, the veterinary drug standard solution and the corresponding magnetic nanoparticle solution of the conjugated antigen are thoroughly mixed to form a mixture of the two solutions; then, the mixture of the two solutions is mixed with the corresponding magnetic nanoparticle solution of the conjugated antibody. The ratio of the veterinary drug standard solution, the magnetic nanoparticle solution of the antibody-conjugated solution, and the magnetic nanoparticle solution of the antigen-conjugated solution in the mixture of the three solutions is 1:1:

2.

3. The method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics according to claim 2, characterized in that, The separation time in step 2 is the optimal magnetic separation time T. min After the mixture of the three solutions is completed, it enters the magnetic separation chamber; During the separation process, a temporary magnetic field is applied to the outside of the cavity where the solution is stored using a magnetic block to adsorb and retain the magnetic nanoparticles that are coupled with the antigen and the magnetic nanoparticles that are bound to the antibody antigen on the inner surface of the magnetic separation cavity, thereby separating the antibody magnetic bead solution that is bound to the target analyte. Optimal magnetic separation time T min The solution formula is: ， Where T2 is the transverse relaxation time of the 500nm magnetic bead; e is the natural constant; A is the T2 saturation value after magnetic separation, 95% < T2 saturation value < 99%; k is the T2 growth rate constant, reflecting the aggregation efficiency of the magnetic bead under the action of the magnetic field, the larger the value, the faster T2 approaches saturation; t0 is the half-saturation time, corresponding to the magnetic separation time when T2 reaches 50% of the saturation value A during the magnetic separation process.

4. A method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics according to claim 2 or 3, characterized in that, The cross-sectional dimensions of the flow channel used in step 1 are 400 μm × 400 μm. The process of forming a mixture of the two solutions takes place in a flow channel with a Fermat spiral distribution. This flow channel has four triangular baffles spaced at certain intervals from the inlet inwards. The two hypotenuses of the triangular baffles face the inflow and outflow directions, respectively. The four triangular baffles are arranged at equal angles of 45° to enhance mixing efficiency. The triangular baffles are isosceles triangles with a base length of 200 μm and a height of 200 μm. In step 1, the mixing path of the mixture of the three solutions is S-shaped, with the radius of the S-shaped wave being 1 mm.

5. The method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics according to claim 2, characterized in that, The cross-sectional dimensions of the flow channel used in step 3 are 1.2 mm × 1.2 mm.

6. The method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics according to claim 2, characterized in that, The solution flow process in steps 1, 2 and 3 is completed in a plate-shaped microfluidic chip. One end of the microfluidic chip is provided with an extension for accommodating the detection cavity, which is placed in the center of the uniform magnetic field environment in which a magnetic field has a main direction. A uniform magnetic field environment exists in the main magnetic direction, formed by Halbach magnet modules. The assembly process of the Halbach magnet modules is as follows: first, the central permanent magnet group is assembled; then, two coaxial z-axis supplementary permanent magnet groups are assembled at both ends of the central permanent magnet group; finally, the shimming group is installed using a shimming device mounting shell. The central permanent magnet assembly includes: eight trapezoidal cross-section main permanent magnets, which form a hollow strip structure with a regular octagonal cross-section. The hollow strip structure allows the extension part to be inserted and removed. At each end of the length direction of the hollow strip structure, there is a set of z-axis supplementary permanent magnets. The magnetic field direction formed by the two symmetrically arranged z-axis supplementary permanent magnets is the same as the main magnetic field direction. The main permanent magnets contain a field homogenizing group for uniform magnetic field direction. The z-axis supplementary permanent magnet assembly includes: 8 trapezoidal cross-section secondary permanent magnets, which are fixedly installed close to the central permanent magnet assembly. The outer circumference of the z-axis supplementary permanent magnet assembly is larger than that of the central permanent magnet assembly, and the cross-sectional dimensions of the central space of the z-axis supplementary permanent magnet assembly are the same as those of the central permanent magnet assembly. The shimming group is installed in the central permanent magnet group through the shimming device mounting shell. The shimming device mounting shell has an insertion port for the extension part in the center. The shimming group includes: a main shimming block group whose mounting surface passes through the main direction of the magnetic field and a secondary shimming block group symmetrically arranged on both sides of the main shimming block group.

7. The method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics according to claim 6, characterized in that, A row of main shimming blocks is set inside the two side walls along the main direction of the magnetic field. On the inner wall of the shimming device mounting shell at every 15° angle on both sides of each row of main shimming blocks, there are two rows of secondary shimming blocks. Multiple small shim blocks are arranged at equal intervals in the main shim block group and the secondary shim block group. The arrangement of small shim blocks in the main shim block group and the secondary shim block group is the same, each including six small shim blocks arranged sequentially along the axial direction of the central permanent magnet group. The six small shim blocks are arranged symmetrically along the radial plane of the Halbach magnet where the detection cavity axis is located. The number of small shim blocks in the six small shim blocks are 3, 2, 1, 1, 2, and 3 respectively. The small shim blocks are cylindrical and stacked coaxially.

8. A method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics according to claim 6 or 7, characterized in that, The outer diameter of the thinner part of the central permanent magnet assembly is 60mm, and the inner diameter is 30mm; the inner diameter of the thicker z-axis supplementary permanent magnet assembly at both ends is 30mm, the outer diameter is 84mm, and the thickness is 17mm; the overall axial length of the Halbach magnet module is 112mm; the thickness of the small uniform field block is 1mm and the diameter is 2mm. The Halbach magnet module is set in a 32°C environment to maintain the test environment temperature and thus ensure the stability of the magnetic field.

9. A method for detecting veterinary drug residues using magnetic relaxation combined with microfluidics according to claim 6 or 7, characterized in that, The total volume of the mixture of the three solutions is the minimum total volume V. m0 The mixing time of the mixture of the three solutions is the optimal reaction time T under the calibration conditions. ref The concentration gradient of the veterinary drug standard in the standard solution is 0.1~1000 μg / kg, and the veterinary drug standard is a quinolone, sulfonamide or chloramphenicol; Among them, the minimum total volume V m0 =4V, 80≤V≤120; ， Among them, T min The shortest time determined by diffusion kinetics; total reaction volume V o =V m0 T b The optimal reaction time required to achieve immunoadsorption equilibrium.

10. The method for detecting magnetically relaxed veterinary drug residues combined with microfluidics according to claim 9, characterized in that, In step 4, the measured transverse relaxation time T2 is compared with the pre-established norfloxacin standard curve, and the concentration of norfloxacin in the sample is calculated based on the change in transverse relaxation time ΔT2.

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