Magnetic sensitive immune quantitative method and device based on residual magnetic field spatial distribution

Abstract

The invention relates to the technical field of biosensing, and discloses a magnetic sensitive immunoassay quantitative method and device based on residual magnetic field spatial distribution. A weak residual magnetic field generated by aggregation of the magnetic nanoparticles on the marker line is accurately captured and visually presented in the form of a magnetic field distribution curve spectrogram; the equivalent magnetic moment of the magnetic number of the to-be-tested paper strip is obtained by analyzing the magnetic field distribution curve spectrogram, so that the absolute number of the magnetic nanoparticles and the number of the markers in the to-be-tested biological sample are accurately calculated. According to the method, macroscopic physical signals can be accurately and traceably converted into microcosmic absolute quantity of particles, errors possibly caused by quantity and concentration conversion in a traditional method are effectively overcome, the defect that only peak intensity is read and peak shape information is neglected in the traditional method is avoided, and the accuracy and reliability of quantitative analysis are greatly improved.

Description

Technical Field

[0001] This invention relates to the field of biosensing technology and discloses a magnetically sensitive immunoassay quantitative method and device based on the spatial distribution of residual magnetic field. Background Technology

[0002] Lateral flow immunochromatography, commonly known as "immunochromatographic test strips," has gained widespread application in point-of-care testing due to its advantages such as ease of use, low cost, and rapid detection, including early pregnancy testing and infectious disease screening. Traditional immunochromatographic test strips mostly use optical markers such as colloidal gold, with results read by visual observation or ordinary optical readers, and have the following main drawbacks: (1) Optical signals are easily affected by sample background interference, making it difficult to detect trace amounts (i.e., ultra-low concentrations) of analytes; (2) The linear relationship between color intensity and analyte concentration is poor, making it difficult to achieve accurate quantitative analysis. Most results are semi-quantitative or qualitative.

[0003] To overcome the aforementioned shortcomings, researchers introduced magnetic nanoparticles as markers. Magnetic nanoparticles possess excellent biocompatibility and stability, and biological samples exhibit extremely low magnetic background signals, giving magnetic signal detection a naturally high signal-to-noise ratio advantage. Currently, the main magnetic sensors used to read signals from magnetic nanoparticles include: (1) Magnetoresistive sensors (such as GMR and TMR) have high sensitivity, but usually require close contact between the sensor and the test strip. They are not well adapted to large-sized immunoassay strips and have a complex structure. (2) Magnetic induction sensor, which can realize non-contact measurement, but in order to obtain high sensitivity, it is usually necessary to perform complex optimization of the geometry of the induction coil; (3) Superconducting quantum interference device (SQUID) has extremely high sensitivity (fT level), but it requires a cryogenic liquid helium environment to operate. The device is large and expensive, and is not suitable for real-time diagnostic scenarios.

[0004] Furthermore, patent number 201910896195.5 discloses a magnetic immunochromatographic quantitative detection method and system, whose technical solution heavily relies on machine learning models such as convolutional neural networks to fit the relationship between "feature values" and "concentration". Although this "black box" method can obtain seemingly accurate results on specific datasets through training, it lacks clear physical mechanism support and cannot trace the reasoning behind "why this concentration is observed". In particular, when the batch of test strips, magnetic nanoparticle materials, or sample matrix change, the model may fail, requiring extensive retraining, resulting in poor versatility and reliability. Summary of the Invention

[0005] The purpose of this invention is to provide a magnetically sensitive immunoassay quantitative method and device based on the spatial distribution of residual magnetic field, which can accurately and traceably convert macroscopic physical signals into the absolute number of microscopic particles, greatly improving the accuracy and reliability of quantitative analysis.

[0006] To achieve the above-mentioned technical effects, the technical solution adopted by the present invention is as follows: A magnetically sensitive immunoassay method based on the spatial distribution of remanent magnetic fields, comprising: The capture antibody of the biomarker to be tested is immobilized on a nitrocellulose membrane test paper, wherein the capture antibody is used to form a marker line of the biomarker to be tested on the nitrocellulose membrane test paper; A detection antibody that specifically binds to the biomarker to be tested and a carboxyl-functionalized magnetic nanoparticle are covalently coupled by a chemical method to form a magnetic nanoparticle probe. The biological sample to be tested is mixed with magnetic nanoparticle probes and incubated to form a first complex; the first complex is dropped onto the nitrocellulose membrane test paper and chromatography is performed under capillary action to enrich the magnetic nanoparticle probes on the marking line to form the test strip. A preset magnetic field is used to magnetize the test strip, so that the magnetic nanoparticles enriched on the marking line of the test strip have remanence. The magnetized test strip is linearly swept across the sensitive area of ​​the probe of the atomic magnetometer at a constant translational speed. The data of magnetic field strength change with scanning position or scanning time are recorded to generate a magnetic field distribution curve spectrum that forms a peak magnetic signal in the marking line area. Based on the magnetic field distribution curve, the equivalent magnetic moment of the magnetic strip number to be tested is obtained; and based on the equivalent magnetic moment of the magnetic strip number and the equivalent magnetic moment of a single magnetic nanoparticle, the absolute number of magnetic nanoparticles in the biological sample to be tested at the marked line region is obtained.

[0007] Furthermore, when the capture antibody of the biomarker to be tested is immobilized on the nitrocellulose membrane test paper, the secondary antibody that identifies the capture antibody is also immobilized on the nitrocellulose membrane test paper. The marking lines include a detection line where the capture antibody forms the biomarker to be tested on the nitrocellulose membrane test paper, and a quality control line where the secondary antibody forms the biomarker to be tested on the nitrocellulose membrane test paper.

[0008] Furthermore, the test strip is linearly translated along a direction perpendicular to the central axis of the atomic magnetometer probe and passing through the extension of the central axis of the atomic magnetometer probe. The equivalent magnetic moment of the magnetic signal... , The permeability of free space, This represents any scan position on the magnetic field distribution curve spectrum. This is the center position of the magnetic dipole. The perpendicular distance between the probe of the atomic magnetometer and the paper strip to be tested is... The scan position obtained by the atomic magnetometer The component of the magnetic flux density along the probe axis at that location. denoted as the magnetic flux density of the background magnetic field.

[0009] Furthermore, the number of biomarkers in the biological sample at the location of the marked line region. ,in The equivalent magnetic moment of the peak magnetic signal corresponding to the marker line. The equivalent magnetic moment of a single magnetic nanoparticle.

[0010] Furthermore, methods for obtaining the equivalent magnetic moment of a single magnetic nanoparticle include: Multiple magnetic nanoparticle suspensions of different known concentrations were dropped onto fixed positions on blank nitrocellulose membrane test paper, and the nitrocellulose membrane test paper was then dried. Each dried nitrocellulose membrane test paper is magnetized using the preset magnetic field, so that the magnetic nanoparticles on the nitrocellulose membrane test paper have remanence; the magnetized nitrocellulose membrane test paper is linearly swept across the sensitive area of ​​the probe of the atomic magnetometer at the preset test conditions with the translational speed to obtain the total magnetic moment of the magnetic nanoparticles on each nitrocellulose membrane test paper. Based on the total magnetic moment of the magnetic nanoparticles on each nitrocellulose membrane test paper, as well as the corresponding number concentration and drop volume of the magnetic nanoparticles on the nitrocellulose membrane test paper, the equivalent magnetic moment of a single magnetic nanoparticle was obtained. ,in For the first The total magnetic moment of a known concentration of magnetic nanoparticle suspension on nitrocellulose membrane test paper. , This represents the number of samples of magnetic nanoparticle suspensions with known concentrations. denoted as the aggregation coefficient of the magnetic nanoparticles. The ratio of the spot radius or equivalent radius of the band formed by the magnetic nanoparticles on the nitrocellulose membrane test paper after addition to the spot radius in an ideal monolayer distribution. This is the temperature influence coefficient. The value range is 0.005 to 0.02. The data represents the temperature change during the drying process of nitrocellulose membrane test paper. The uniformity coefficient of the preset magnetic field is... The deviation value between the actual strength and the nominal strength of the preset magnetic field. For the first The number concentration of magnetic nanoparticles in a known concentration suspension. For the first The volume of a known concentration of magnetic nanoparticle suspension dropped onto nitrocellulose membrane test paper.

[0011] Furthermore, the nominal strength of the preset magnetic field is based on Analysis yielded, among which The nominal strength of the preset magnetic field. This is the lower threshold of the signal-to-noise ratio for atomic magnetometers. The translational velocity, The perpendicular distance between the probe of the atomic magnetometer and the paper strip to be tested is... To test bandwidth, For system constants, The value range is 0.1 to 5. This is the response time of the atomic magnetometer.

[0012] To achieve the above-mentioned technical effects, the present invention also provides a magnetically sensitive immunoassay device based on the spatial distribution of a residual magnetic field, used to implement the aforementioned magnetically sensitive immunoassay method, comprising: The magnetization unit is used to provide a preset magnetic field to magnetize the test strip; A magnetic shielding device is used to isolate the ambient magnetic field. It has a hollow structure inside, and an atomic magnetometer is installed inside the hollow structure. The driving unit is used to drive the test strip marked with magnetic nanoparticles to extend into the magnetic shielding device and move linearly at a constant translational speed along a trajectory perpendicular to the central axis of the atomic magnetometer probe and passing through the extension line of the central axis of the atomic magnetometer probe. The data processing unit is connected in communication with the atomic magnetometer and the drive unit to synchronously record data on the change of magnetic field strength with scanning position or scanning time. The data analysis unit is used to analyze the absolute number of magnetic nanoparticles in the biological sample at the location of the marked line region based on the recorded data of the change of magnetic field strength with scanning position or scanning time, and the equivalent magnetic moment of a single magnetic nanoparticle.

[0013] Furthermore, the data analysis unit includes: The spectrum generation module is used to generate a magnetic field distribution curve spectrum in the marked line area based on the recorded data of magnetic field strength changing with scanning position or scanning time. The equivalent magnetic moment analysis module is used to analyze the magnetic field distribution curve using... Analysis of the equivalent magnetic moment of the magnetic signal , The permeability of free space, This represents any scan position on the magnetic field distribution curve spectrum. This is the center position of the magnetic dipole. The perpendicular distance between the probe of the atomic magnetometer and the paper strip to be tested is... The scan position obtained by the atomic magnetometer The component of the magnetic flux density along the probe axis at that location. The magnetic flux density is the background magnetic field. The quantity analysis module is used to analyze the equivalent magnetic moment of the peak magnetic signal and the equivalent magnetic moment of a single magnetic nanoparticle. Analyze the number of biomarkers in the biological sample at the location of the marked line region. , The equivalent magnetic moment of a single magnetic nanoparticle.

[0014] Compared with the prior art, the beneficial effects of this invention are: 1. This invention can accurately and traceably convert macroscopic physical signals into the absolute number of microscopic particles, effectively overcoming the errors that may be caused by the conversion between "quantity" and "concentration" in traditional methods, and avoiding the shortcomings of traditional methods that only read peak intensity and ignore peak shape information, thus greatly improving the accuracy and reliability of quantitative analysis.

[0015] 2. This invention obtains the magnetic field distribution curve spectrum using an atomic magnetometer and the equivalent magnetic moment of the magnetic signal using an analytical physical model. This establishes a continuous physical traceability chain in the field of magnetic immunoassay, progressively returning from the final particle count (N) to the SI base units (kilogram, meter, second) and fundamental physical constants. This ensures that every measurement in the magnetic immunoassay quantitative method of this invention has a solid physical basis, avoiding the problem in existing technologies where the acquired signal value is a vaguely defined "response value" related to multiple factors, making traceability impossible. It boasts extremely high accuracy and reliability, eliminating reliance on experience and "black box" algorithms.

[0016] 3. The magnetically sensitive immunoassay quantitative device of this invention is a completely non-contact detection method, avoiding sensor contamination and wear. The linear scanning method is highly automated and fast (the entire process can be completed within 15 minutes), enabling high-throughput screening. Attached Figure Description

[0017] Figure 1 This is a flowchart of the magnetically sensitive immunoassay quantitative method in Example 1 or 2; Figure 2 This is a schematic diagram of the magnetically sensitive immunoassay device in Example 1 or 2; Figure 3 This is a schematic diagram of the marking lines on the nitrocellulose membrane test paper in Example 1 or 2; Figure 4 This is the magnetic field distribution curve spectrum in Example 2; The components include: 1. Nitrocellulose membrane test paper; 101. Detection line; 102. Quality control line; 2. Atomic magnetometer; 3. Magnetization unit; 4. Magnetic shielding device; 5. Drive unit; 6. Data processing unit; 7. Data analysis unit; 701. Spectrum generation module; 702. Equivalent magnetic moment analysis module; 703. Quantitative analysis module. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0019] Example 1 See Figures 1 to 3 A magnetically sensitive immunoassay method based on the spatial distribution of remanent magnetic fields, comprising: The capture antibody of the biomarker to be tested is immobilized on nitrocellulose membrane test paper 1, wherein the capture antibody is used to form a marker line of the biomarker to be tested on nitrocellulose membrane test paper 1; A detection antibody that specifically binds to the biomarker to be tested and a carboxyl-functionalized magnetic nanoparticle are covalently coupled by a chemical method to form a magnetic nanoparticle probe. The biological sample to be tested is mixed with magnetic nanoparticle probes and incubated to form a first complex; the first complex is dropped onto the nitrocellulose membrane test paper 1 and subjected to chromatography under capillary action to enrich the magnetic nanoparticle probes on the marked line to form the test strip. A preset magnetic field is used to magnetize the test strip, so that the magnetic nanoparticles enriched on the marking line of the test strip have remanence; the magnetized test strip is linearly swept across the sensitive area of ​​the probe of the atomic magnetometer 2 at a constant translation speed, and the data of magnetic field strength changing with scanning position or scanning time are recorded to generate a magnetic field distribution curve spectrum that forms a peak magnetic signal in the marking line area. Based on the magnetic field distribution curve, the equivalent magnetic moment of the magnetic strip number to be tested is obtained; and based on the equivalent magnetic moment of the magnetic strip number and the equivalent magnetic moment of a single magnetic nanoparticle, the absolute number of magnetic nanoparticles in the biological sample to be tested at the marked line region is obtained.

[0020] In this embodiment, the test strip enriched with magnetic nanoparticle probes on the marked line is magnetized. An atomic magnetometer 2 accurately captures the weak residual magnetic field generated by the aggregation of magnetic nanoparticles on the marked line, presenting it visually as a magnetic field distribution curve. By analyzing the magnetic field distribution curve, the equivalent magnetic moment of the test strip's magnetic signature is obtained, thereby accurately calculating the absolute number of magnetic nanoparticles in the biological sample. Subsequently, the number of biomarkers can be calculated based on the conversion coefficient specifically linking the number of magnetic nanoparticles to the number of biomarkers. This invention can accurately and traceably convert macroscopic physical signals (total magnetic moment) into the absolute number of microscopic particles, effectively overcoming the errors that may arise from the conversion between "quantity" and "concentration" in traditional methods. It also avoids the shortcomings of traditional methods that only read peak intensity while ignoring peak shape information, greatly improving the accuracy and reliability of quantitative analysis and ensuring the repeatability and traceability of the test results.

[0021] In this embodiment, when the capture antibody for the biomarker to be tested is immobilized on the nitrocellulose membrane test paper 1, the secondary antibody that identifies the capture antibody is also immobilized on the nitrocellulose membrane test paper 1. The marking lines include a detection line 101 where the capture antibody forms the biomarker to be tested on the nitrocellulose membrane test paper 1, and a control line 102 where the secondary antibody forms the biomarker to be tested on the nitrocellulose membrane test paper 1. The absolute number of magnetic nanoparticles and the number of biomarkers on the test line 101 and the control line 102 of the biomarker to be tested can be calculated by obtaining the magnetic signals of the biomarker to be tested at the detection line 101 and the control line 102, respectively. Comparing the absolute number of magnetic nanoparticles or the number of biomarkers on the detection line 101 and the control line 102 can further verify the accuracy and reliability of the test results and eliminate false positive or false negative results caused by the test strip itself or other factors during the test process.

[0022] Based on the same inventive concept, this embodiment also provides a magnetically sensitive immunoassay device based on the spatial distribution of a residual magnetic field, used to implement the aforementioned magnetically sensitive immunoassay method, comprising: Magnetization unit 3 is used to provide a preset magnetic field to magnetize the test strip; The magnetic shielding device 4 is used to isolate the ambient magnetic field. Its interior is a hollow structure, and an atomic magnetometer 2 is installed inside the hollow structure. The driving unit 5 is used to drive the test paper strip marked with magnetic nanoparticles to extend into the magnetic shielding device 4 and move linearly along a trajectory perpendicular to the central axis of the atomic magnetometer 2 probe and passing through the extension line of the central axis of the atomic magnetometer 2 probe at a constant translational speed. Data processing unit 6 is communicatively connected to atomic magnetometer 2 and drive unit 5, and is used to synchronously record data on changes in magnetic field strength with scanning position or scanning time; The data analysis unit 7 is used to analyze the absolute number of magnetic nanoparticles and the number of biomarkers in the biological sample at the location of the marked line area based on the recorded data of the change of magnetic field strength with scanning position or scanning time, and the equivalent magnetic moment of a single magnetic nanoparticle.

[0023] In this embodiment, the data analysis unit 7 includes: The spectrum generation module 701 is used to generate a magnetic field distribution curve spectrum in which a peak magnetic signal is formed in the marked line area, based on the recorded data of the change of magnetic field strength with scanning position or scanning time. The equivalent magnetic moment analysis module 702 is used to analyze the magnetic field distribution curve using... Analysis of the equivalent magnetic moment of the magnetic signal , The permeability of free space, This represents any scan position on the magnetic field distribution curve spectrum. This is the center position of the magnetic dipole. The perpendicular distance between the probe of atomic magnetometer 2 and the paper strip to be tested is... The scan position measured by atomic magnetometer 2 The component of the magnetic flux density along the probe axis at that location. The magnetic flux density is the background magnetic field. Quantitative analysis module 703 is used to analyze the equivalent magnetic moment of the peak magnetic signal and the equivalent magnetic moment of a single magnetic nanoparticle using... Analyze the number of biomarkers in the biological sample at the location of the marked line region. , The equivalent magnetic moment of a single magnetic nanoparticle.

[0024] Example 2 See Figures 1 to 4 This embodiment uses the detection of carcinoembryonic antigen (CEA) as an example to describe in detail the magnetically sensitive immunoassay quantitative method based on the spatial distribution of the residual magnetic field of the present invention. The specific operation procedure is as follows: Step 1: Immobilize the capture antibody and secondary antibody (goat anti-mouse IgG was used in this example) of carcinoembryonic antigen (CEA) onto nitrocellulose membrane test paper 1 to form detection line 101 (T line) and control line 102 (C line) respectively.

[0025] Step 2: The detection antibody that specifically binds to carcinoembryonic antigen (CEA) and carboxyl-functionalized magnetic nanoparticles (in this example, nano Fe3O4 is used) are covalently coupled by chemical methods (such as EDC / NHS method) to form a magnetic nanoparticle probe (hereinafter referred to as: MNP probe).

[0026] Step 3: Mix and incubate the biological sample to be tested with the magnetic nanoparticle probe to form a first complex; drop the first complex onto the nitrocellulose membrane test paper 1 (hereinafter referred to as: LFIA test strip), and perform chromatography under capillary action to enrich the magnetic nanoparticle probe on the marking line to form the test strip; In this embodiment, a serum sample is used as the test sample. The serum sample is mixed with the MNP probe and incubated to form an "analyte-MNP probe" complex (i.e., the first complex). The complex is then dropped onto the sample pad of the LFIA test strip and subjected to chromatography under capillary action. When the complex liquid flows through the T line, the "analyte-MNP probe" complex is captured by the capture antibody on the T line, forming a "capture antibody-analyte-MNP probe" sandwich structure, which enriches MNP at the T line. Unbound MNP probes continue to migrate and are captured by the secondary antibody on the C line.

[0027] Step 4: Magnetize the test strip using a preset magnetic field, so that the magnetic nanoparticles enriched on the marking lines of the test strip acquire remanence; linearly scan the magnetized test strip across the sensitive area of ​​the probe of the atomic magnetometer 2 at a constant translational speed, and record the data of magnetic field strength changes with scanning position or scanning time to generate a magnetic field distribution curve spectrum showing a peak magnetic signal in the marking line region; the specific steps are as follows: 4.1 Place the LFIA test strip after the reaction is completed on the support of the sample driving unit 5; use an electromagnetic field or permanent magnet to magnetize the test strip (the condition used in this embodiment is 2 minutes, and the specific magnetization time can be optimized and adjusted according to different magnetic nanoparticles) so that the MNPs enriched in the T-line and C-line regions have remanence. To achieve accurate quantification of trace biomarkers, the nominal strength of the preset magnetic field in this embodiment is based on... Analysis yielded, among which The nominal strength of the preset magnetic field. This is the lower threshold of the signal-to-noise ratio for atomic magnetometer 2. The translational velocity, The perpendicular distance between the probe of atomic magnetometer 2 and the paper strip to be tested is... To test bandwidth, For system constants, The value range is 0.1 to 5. The response time of atomic magnetometer 2 is given. By comprehensively considering the limiting value of the detection signal-to-noise ratio and the influencing parameters such as translational speed, vertical distance, and detection bandwidth, a functional analysis model of the nominal strength of the preset magnetic field is constructed. This model is used to set the nominal strength of the preset magnetic field, ensuring that the magnetic nanoparticles enriched on the marking line of the test strip have appropriate remanence during the subsequent magnetization process. This can significantly improve the signal-to-noise ratio while ensuring detection throughput, providing a reliable basis for the accurate quantification of trace biomarkers.

[0028] 4.2 Activate the sample driving unit 5 to linearly scan the LFIA test strip across the sensitive area of ​​the SERF atomic magnetometer 2 at a constant linear velocity (generally 1 mm / s to 10 mm / s, 2.5 mm / s in this embodiment). (In this embodiment, the test strip is kept linearly translated along a direction perpendicular to the central axis of the atomic magnetometer 2 probe and passing through the extension line of the central axis of the atomic magnetometer 2 probe). The atomic magnetometer 2 continuously detects the magnetic flux density component (Bz component perpendicular to the scanning direction) of the spatial point directly below it in real time. The data processing unit 6 synchronously records the data of magnetic field strength changing with scanning position (time), generating a magnetic field distribution curve spectrum containing two peaks, such as... Figure 4 The solid line in the middle; Figure 4 The dashed line represents the magnetic field distribution curve obtained from testing an LFIA test strip containing only the T and C lines and without any added complex. Figure 4 Each peak corresponds to the magnetic signal in the T-line and C-line regions, respectively.

[0029] Step 5: Based on the magnetic field distribution curve, analyze and obtain the equivalent magnetic moment of the magnetic strip number to be tested; and based on the equivalent magnetic moment of the magnetic strip number and the equivalent magnetic moment of a single magnetic nanoparticle, analyze and obtain the absolute number of magnetic nanoparticles and the number of markers in the biological sample at the marked line region; specifically including: 5.1 Calculation of the equivalent magnetic moment of a single magnetic nanoparticle 5.1.1 Multiple magnetic nanoparticle suspensions of different known concentrations were dropped onto fixed positions on blank nitrocellulose membrane test paper 1, and the nitrocellulose membrane test paper 1 was dried. 5.1.2 The preset magnetic field is used to magnetize each dried nitrocellulose membrane test paper 1, so that the magnetic nanoparticles on the nitrocellulose membrane test paper 1 have remanence; the magnetized nitrocellulose membrane test paper 1 is linearly swept across the sensitive area of ​​the probe of the atomic magnetometer 2 at the preset test conditions with the translational speed to obtain the total magnetic moment of the magnetic nanoparticles on each nitrocellulose membrane test paper 1. 5.1.3 Based on the total magnetic moment of the magnetic nanoparticles on each nitrocellulose membrane test paper 1, and the corresponding number concentration and drop volume of the magnetic nanoparticles on the nitrocellulose membrane test paper 1, the equivalent magnetic moment of a single magnetic nanoparticle is analyzed and obtained. ,in For the first The total magnetic moment of the magnetic nanoparticles in a known concentration suspension on nitrocellulose membrane test paper 1. , This represents the number of samples of magnetic nanoparticle suspensions with known concentrations. denoted as the aggregation coefficient of the magnetic nanoparticles. The ratio of the actual spot radius or equivalent radius of the band formed by the magnetic nanoparticles on the nitrocellulose membrane test paper 1 after the magnetic nanoparticle suspension is added to the spot radius when the magnetic nanoparticles are ideally distributed in a monolayer. The value ranges from 1.0 to 3.0 (1.0 represents an ideal monolayer uniform distribution, and values ​​greater than 1.5 indicate particle diffusion due to aggregation, coffee ring effect, etc.), within a certain concentration range. The changes are not significant, so a representative concentration (such as a magnetic nanoparticle suspension sample corresponding to a known median concentration) can be used. As a fixed value; This is the temperature influence coefficient. The value range is 0.005 to 0.02. The data represents the temperature change during the drying process of nitrocellulose membrane test paper 1. The uniformity coefficient of the preset magnetic field is... The deviation value between the actual strength and the nominal strength of the preset magnetic field. For the first The number concentration of magnetic nanoparticles in a known concentration suspension. For the first The volume of a known concentration of magnetic nanoparticle suspension dropped onto nitrocellulose membrane test paper 1.

[0030] In this embodiment, the clustering coefficient This can be achieved by establishing magnetic nanoparticles on each nitrocellulose membrane test paper 1. The linear relationship between the measured magnetic moment and the actual magnetic moment is represented by the slope of the coefficient of convergence. The relative deviation of the magnetic moment is the total magnetic moment actually measured by an atomic magnetometer on a sample of magnetic nanoparticle suspension with a known concentration. Compared with the theoretically expected magnetic moment calculated based on the ideal uniform distribution model M theoretical The difference between them. Uniformity coefficient. The standard deviation of the measured magnetic field strength can be calculated by measuring the magnetic field strength at multiple points within the magnetized region. Compared with the average The ratio of these two values ​​is the uniformity coefficient. , Typical value ranges are: <0.1 for permanent magnets; <0.05 for optimized electromagnets. The deviation between the actual and nominal strength of the preset magnetic field is the data value. The average magnetic field strength measured at multiple points within the magnetized region is compared with the nominal strength of the preset magnetic field. The difference needs to be controlled in this embodiment. Within ±5% of the nominal value.

[0031] 5.2 Calculation of equivalent magnetic moment of magnetic signal In this embodiment, the equivalent magnetic moment of the magnetic signal , The permeability of free space, This represents any scan position on the magnetic field distribution curve spectrum. This is the center position of the magnetic dipole. The perpendicular distance between the probe of atomic magnetometer 2 and the paper strip to be tested is... The scan position measured by atomic magnetometer 2 The component of the magnetic flux density along the probe axis at that location. denoted as the magnetic flux density of the background magnetic field.

[0032] 5.3 Calculation of the number of markers In this embodiment, the number of biomarkers in the biological sample to be tested at the location of the marked line area is... ,in The equivalent magnetic moment of the peak magnetic signal corresponding to the marker line. The equivalent magnetic moment of a single magnetic nanoparticle.

[0033] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A magnetically sensitive immunoassay method based on the spatial distribution of remanent magnetic fields, characterized in that, include: The capture antibody of the biomarker to be tested is immobilized on a nitrocellulose membrane test paper, wherein the capture antibody is used to form a marker line of the biomarker to be tested on the nitrocellulose membrane test paper; A detection antibody that specifically binds to the biomarker to be tested and a carboxyl-functionalized magnetic nanoparticle are covalently coupled by a chemical method to form a magnetic nanoparticle probe. The biological sample to be tested is mixed with magnetic nanoparticle probes and incubated to form a first complex; the first complex is dropped onto the nitrocellulose membrane test paper and chromatography is performed under capillary action to enrich the magnetic nanoparticle probes on the marking line to form the test strip. A preset magnetic field is used to magnetize the test strip, so that the magnetic nanoparticles enriched on the marking line of the test strip have remanence. The magnetized test strip is linearly swept across the sensitive area of ​​the probe of the atomic magnetometer at a constant translational speed. The data of magnetic field strength change with scanning position or scanning time are recorded to generate a magnetic field distribution curve spectrum that forms a peak magnetic signal in the marking line area. Based on the magnetic field distribution curve, the equivalent magnetic moment of the magnetic strip number to be tested is obtained; and based on the equivalent magnetic moment of the magnetic strip number and the equivalent magnetic moment of a single magnetic nanoparticle, the absolute number of magnetic nanoparticles in the biological sample to be tested at the marked line region is obtained.

2. The magnetically sensitive immunoassay method according to claim 1, characterized in that, When the capture antibody for the biomarker to be tested is immobilized on the nitrocellulose membrane test paper, the secondary antibody that identifies the capture antibody is also immobilized on the nitrocellulose membrane test paper. The marking lines include the detection line where the capture antibody forms the biomarker to be tested on the nitrocellulose membrane test paper, and the quality control line where the secondary antibody forms the biomarker to be tested on the nitrocellulose membrane test paper.

3. The magnetically sensitive immunoassay quantitative method according to claim 1, characterized in that, The test strip is linearly translated along a direction perpendicular to the central axis of the atomic magnetometer probe and passing through the extension of the central axis of the atomic magnetometer probe. The equivalent magnetic moment of the magnetic sign... , The permeability of free space, This represents any scan position on the magnetic field distribution curve spectrum. This is the center position of the magnetic dipole. The perpendicular distance between the probe of the atomic magnetometer and the paper strip to be tested is... The scan position obtained by the atomic magnetometer The component of the magnetic flux density along the probe axis at that location. denoted as the magnetic flux density of the background magnetic field.

4. The magnetically sensitive immunoassay method according to claim 1, characterized in that, The number of biomarkers in the biological sample at the location of the marked line region ,in The equivalent magnetic moment of the peak magnetic signal corresponding to the marker line. The equivalent magnetic moment of a single magnetic nanoparticle.

5. The magnetically sensitive immunoassay method according to claim 1, characterized in that, Methods for obtaining the equivalent magnetic moment of a single magnetic nanoparticle include: Multiple magnetic nanoparticle suspensions of different known concentrations were dropped onto fixed positions on blank nitrocellulose membrane test paper, and the nitrocellulose membrane test paper was then dried. Each dried nitrocellulose membrane test paper is magnetized using the preset magnetic field, so that the magnetic nanoparticles on the nitrocellulose membrane test paper have remanence; the magnetized nitrocellulose membrane test paper is linearly swept across the sensitive area of ​​the probe of the atomic magnetometer at the preset test conditions with the translational speed to obtain the total magnetic moment of the magnetic nanoparticles on each nitrocellulose membrane test paper. Based on the total magnetic moment of the magnetic nanoparticles on each nitrocellulose membrane test paper, as well as the corresponding number concentration and drop volume of the magnetic nanoparticles on the nitrocellulose membrane test paper, the equivalent magnetic moment of a single magnetic nanoparticle was obtained. ,in For the first The total magnetic moment of a known concentration of magnetic nanoparticle suspension on nitrocellulose membrane test paper. , This represents the number of samples of magnetic nanoparticle suspensions with known concentrations. denoted as the aggregation coefficient of the magnetic nanoparticles. The ratio of the spot radius or equivalent radius of the band formed by the magnetic nanoparticles on the nitrocellulose membrane test paper after addition to the spot radius in an ideal monolayer distribution. This is the temperature influence coefficient. The value range is 0.005 to 0.

02. The data represents the temperature change during the drying process of nitrocellulose membrane test paper. The uniformity coefficient of the preset magnetic field is... The deviation value between the actual strength and the nominal strength of the preset magnetic field. For the first The number concentration of magnetic nanoparticles in a known concentration suspension. For the first The volume of a known concentration of magnetic nanoparticle suspension dropped onto nitrocellulose membrane test paper.

6. The magnetically sensitive immunoassay method according to claim 5, characterized in that, The nominal strength of the preset magnetic field is based on Analysis yielded, among which The nominal strength of the preset magnetic field. This is the lower threshold of the signal-to-noise ratio for atomic magnetometers. The translational velocity, The perpendicular distance between the probe of the atomic magnetometer and the paper strip to be tested is... To test bandwidth, For system constants, The value range is 0.1 to 5. This is the response time of the atomic magnetometer.

7. A magnetically sensitive immunoassay device based on the spatial distribution of a residual magnetic field, used to implement the magnetically sensitive immunoassay method according to any one of claims 1-6, characterized in that, include: The magnetization unit is used to provide a preset magnetic field to magnetize the test strip; A magnetic shielding device is used to isolate the ambient magnetic field. It has a hollow structure inside, and an atomic magnetometer is installed inside the hollow structure. The driving unit is used to drive the test strip marked with magnetic nanoparticles to extend into the magnetic shielding device and move linearly at a constant translational speed along a trajectory perpendicular to the central axis of the atomic magnetometer probe and passing through the extension line of the central axis of the atomic magnetometer probe. The data processing unit is connected in communication with the atomic magnetometer and the drive unit to synchronously record data on the change of magnetic field strength with scanning position or scanning time. The data analysis unit is used to analyze the absolute number of magnetic nanoparticles in the biological sample at the location of the marked line region based on the recorded data of the change of magnetic field strength with scanning position or scanning time, and the equivalent magnetic moment of a single magnetic nanoparticle.

8. The magnetically sensitive immunoassay device according to claim 7, characterized in that, The data analysis unit includes: The spectrum generation module is used to generate a magnetic field distribution curve spectrum in the marked line area based on the recorded data of magnetic field strength changing with scanning position or scanning time. The equivalent magnetic moment analysis module is used to analyze the magnetic field distribution curve using... Analysis of the equivalent magnetic moment of the magnetic signal , The permeability of free space, This represents any scan position on the magnetic field distribution curve spectrum. This is the center position of the magnetic dipole. The perpendicular distance between the probe of the atomic magnetometer and the paper strip to be tested is... The scan position obtained by the atomic magnetometer The component of the magnetic flux density along the probe axis at that location. The magnetic flux density is the background magnetic field. The quantity analysis module is used to analyze the equivalent magnetic moment of the peak magnetic signal and the equivalent magnetic moment of a single magnetic nanoparticle. Analyze the number of biomarkers in the biological sample at the location of the marked line region. , The equivalent magnetic moment of a single magnetic nanoparticle.

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