Relaxation nuclear magnetic resonance method for detecting antigen content

Abstract

The invention discloses a relaxation nuclear magnetic resonance method for detecting antigen content, which comprises the following steps: combining two probes with an antigen to form a compound, then removing all the compound and capture probes by magnetic separation, and then detecting the transverse relaxation time of residual excessive signal probes, establishing a mathematical mapping model of the antigen concentration and the transverse relaxation time; the method is efficient, low in detection limit and more suitable for detection of low antigen concentration in a biological sample, high-sensitivity (the detection limit is 0.1 pg / mL) detection of the antigen content in a liquid biological sample is achieved, meanwhile, the detection time is greatly shortened, the single-time sample detection time is within 1 min (the time for obtaining nuclear magnetic signals is obtained, and probe capture time is not included), and the method is suitable for large-scale popularization and application. In addition, the CV of an experimental sample is within 10%, and the stability is good.

Description

Technical Field

[0001] The invention relates to a relaxation nuclear magnetic resonance method, in particular to a relaxation nuclear magnetic resonance method for detecting antigen content. Background Art

[0002] Traditional quantitative detection of antigens requires complex physiological and biochemical tests in central laboratories. High performance liquid chromatography-mass spectrometry, ion exchange chromatography-mass spectrometry, and capillary electrophoresis-mass spectrometry based on precision instruments play an important role in antigen detection. They have high sensitivity and accuracy and are currently commonly used methods for clinical detection of target antigens. However, these methods have problems such as expensive instruments, high maintenance costs, long detection time, and the need for highly professional operations. In recent years, the Point of Care Testing (POCT) methods such as enzyme-linked immunosorbent assay and chemifluorescence immunoassay have emerged, but they are greatly affected by background signal interference, slow detection speed, and low efficiency.

[0003] In recent years, researchers have conducted systematic studies on different signal conversion mechanisms. The basic principles mainly include optical, electrochemical and other signal conversion methods. In some studies based on optical signals, the signal conversion and readout process after identification still takes a long time. Although the detection method based on electrical signals has the advantages of fast detection speed and high detection sensitivity, it lacks stability for long-term use, and frequent maintenance will increase costs. Magnetic materials have the advantages of high sensitivity, little background influence, and fast response, and are increasingly used in biomarker detection. However, the use of magnetic nanomaterials in current target antigen sensing methods only stays in the stage of enriching and separating targets. Magnetic labeling is not used for signal characterization, and it still needs to be combined with photoelectric signals for measurement; therefore, more efficient and direct detection methods are urgently needed to be developed. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to further expand the application of relaxation nuclear magnetic resonance technology in the direction of biological detection and provide a relaxation nuclear magnetic resonance method for detecting antigen content.

[0005] Technical solution: The relaxation nuclear magnetic resonance method of the present invention is used to detect antigen content. First, capture probes and signal probes are prepared, and then the capture probes and signal probes are combined with the target antigen to form a complex. Then, the complex and capture probes are removed by magnetic separation, and finally the reduction in the signal probe is detected to construct a mapping relationship between the analyte and the nuclear magnetic relaxation signal.

[0006] Specifically, the following steps are included:

[0007] (1) Preparation of probes: Activate and modify the magnetic nanoparticles. After activation, the modified magnetic nanoparticles are combined with antibodies and blocked to obtain two magnetic probes of different sizes, namely, capture probes and signal probes;

[0008] (2) Construct a mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signal in an aqueous solution of target antigen with known concentration: combine the capture probe and signal probe with the target antigen with known concentration, fully react to form a composite structure, take the supernatant after magnetic separation, and measure the transverse relaxation time of magnetic resonance signal generated by the change of magnetic nanoprobe concentration under different target antigen concentrations. 1 The difference in H-NMR relaxation signals was calculated by calculating the transverse relaxation time change ΔT of the supernatant. 2 , establish a mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signals;

[0009] (3) constructing a mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signal in a diluted serum solution with known antigen concentration: diluting the serum sample and combining it with the above-mentioned magnetic nanoprobe to fully react to form a composite structure, detecting the transverse relaxation time of the supernatant after magnetic separation, and further establishing a mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signal under serum background;

[0010] (4) The antigen content of the biological sample under test is obtained by comparing the measured relaxation time difference with the known data model of the sample type: the serum sample is diluted and combined with the above-mentioned magnetic nanoprobe to fully react, and the transverse relaxation time of the supernatant is detected after magnetic separation. The ΔT of the liquid biological sample under test is substituted into the known data model as the input, and the output obtained is the measured antigen concentration.

[0011] Wherein, step (1) is to prepare two sizes of magnetic nanoparticles into solutions respectively, and adjust the pH. Then, NHS solution is added first, and then EDC solution is added for activation. The role of adding NHS solution is to stabilize the activated product; the role of adding EDC solution is to activate the carboxyl groups on the magnetic beads; antibodies are added for reaction respectively, and then BSA solution is added for blocking.

[0012] Among them, the magnetic beads have particle sizes of 30nm and 1000nm respectively, wherein the detection antibody is coupled with the 30nm magnetic nanoparticles to make a signal probe, and the recognition antibody is coupled with the 1000nm magnetic nanoparticles to make a capture probe; further preferably, the detection antibody is coupled with the 30nm magnetic nanoparticles to make a signal probe, and the recognition antibody is coupled with the 1000nm magnetic nanoparticles to make a capture probe.

[0013] Among them, the pH adjustment in the activation modification is to add MES buffer to adjust the pH to between 5.5 and 6.7; the activation conditions are 24-26°C, 1500r / min, and 30-40min; the pH adjustment in the addition of antibodies is to use PBS solution to adjust the pH to between 7.2 and 7.4; the reaction conditions after adding the antibody are 36-37°C, 1500r / min, and 2-2.5h; the reaction conditions of the blocking link are 36-37°C, 1000r / min, and 30-40min.

[0014] Specifically, step (1) comprises the following steps: firstly adding NHS, then adding EDC to activate the carboxyl groups modified on the surface of the magnetic nanoparticles, reacting at 25° C. and 1500 r / min for 0.5 h, and magnetically separating and washing the activated products; then respectively mixing the carboxyl magnetic nanoparticles of different sizes with the corresponding antibodies, ultrasonically resuspending, and reacting at 37° C. and 1500 r / min for 2 h; then adding 1% BSA solution to seal for half an hour, placing the mixed solution on a magnetic stand and standing, discarding the supernatant after complete precipitation to wash away the unreacted free antibodies and BSA, and obtaining the capture probe and the signal probe after ultrasonic resuspending.

[0015] Wherein, in step (2), the target antigen is an antigen to which the corresponding antibody is easily bound by the imide functional group, and the amino-terminal pro-brain natriuretic peptide is used as an example in the embodiment of this invention.

[0016] Wherein, in step (2) or (3), the transverse relaxation is detected by using a 18-20 MHz low-field nuclear magnetic resonance measuring instrument, with a measuring pulse of CPMG, a scanning number of 1, and a gain of 65.

[0017] Wherein, in step (2) or (3), the signal probe and the capture probe are added in excess compared to the antigen, and the composite structure is a capture probe-antigen-signal probe complex in which the antigen is simultaneously bound to the two probes.

[0018] Wherein, in step (2) or (3), the magnetic separation is allowed to stand for 45s to 1min at a field strength of 300mT. After sufficient reaction to form a composite structure, magnetic separation is performed to ensure that all composite structures and excess capture probes are separated, and only excess residual signal probes are left in the supernatant. The transverse relaxation time of the supernatant can then be measured and a mapping mathematical model can be constructed with the antigen concentration. In this process, the greater the concentration of the antigen, the more composite structures it forms with the two probes, so that the excess residual signal probes are less after the combination, and the longer the transverse relaxation time, so that a mapping mathematical model can be constructed.

[0019] Wherein, in step (3), the dilution factor is 1000 times.

[0020] Among them, in step (4), the detection instrument of the sample to be tested must be consistent with the relaxation nuclear magnetic resonance detection instrument used to establish the data model, have the same proton resonance frequency, test temperature, and use the same measurement pulse sequence, number of scans, echo interval and other measurement parameters.

[0021] Principle of the invention: The relaxation nuclear magnetic resonance method of the present invention can still perform a target antigen detection method using magnetic signal sensing after achieving specific capture using magnetic materials, so as to give full play to the advantages of magnetic materials, establish a more direct signal transmission path, and greatly improve the detection efficiency. Compared with other existing antigen detection methods based on electrical or optical signal mapping, the mapping relationship between the target antigen and the nuclear magnetic relaxation signal is established by achieving efficient combination of magnetic nanoprobes and target antigens; the two introduced probes and magnetic separation technology are more sensitive to slight changes in antigen concentration; the two probes are combined with the antigen to form a complex and then magnetically separated, and the change in relaxation time is measured by NMR, and then the antigen concentration in the original sample is calculated by the data model.

[0022] The relaxation nuclear magnetic resonance method for detecting antigen content of the present invention respectively develops a small-sized signal probe and a large-sized capture probe, and combines magnetic selection with a magnetic relaxation method to overcome the interference of complex matrix in serum on the signal. In addition to the relaxation sensing principle based on the change of the probe distribution state, the change of the probe concentration can also cause the change of the transverse relaxation time, and the signal transmission path from the antigen concentration to the probe concentration change can be constructed accordingly. The detection is completed by magnetic nanoparticles of different sizes, and the magnetic field sensitivity of large-sized magnetic beads is utilized; that is, the characteristic that they can be attracted by the magnetic field more quickly than small magnetic beads, and they are made into capture probes after the surface modification of recognition antibodies, and at the same time, the characteristic of small-sized magnetic beads with strong dispersion stability is utilized, and they are used as signal probes after the surface modification of detection antibodies. Since the recognition antibody and the detection antibody correspond to different antigen binding sites respectively, the probes made of large magnetic beads and small magnetic beads can be combined with the target marker to form a signal probe-antigen-capture probe complex.

[0023] Due to the different sensitivity of magnetic nanoparticles of different sizes to magnetic fields, large magnetic beads can be separated by a short period of attraction (1 to 10 minutes) under a magnetic field of lower intensity, but small magnetic beads need to be attracted for several hours under a magnetic field of higher intensity to complete separation. Based on this, the excess capture probes and sandwich complexes can be separated in a short time under low field strength conditions to complete the screening of free signal probes. This process is called magnetic separation. After magnetic separation, the transverse relaxation time of the remaining sample is measured by ultrasonic resuspension: the more target antigen content, the more complexes are separated by magnetic separation, the fewer remaining free signal probes, and the lower the concentration of magnetic nanoparticles in the corresponding sample, the higher the measured T 2On the contrary, the less the target antigen content, the fewer sandwich complexes will be separated by magnetic separation, and the more free signal probes will remain. The higher the concentration of magnetic nanoparticles in the corresponding sample, the higher the measured T 2 The present invention can realize highly sensitive quantitative detection of antigens in a short time by efficiently combining the target antigen with the magnetic nanoprobe and utilizing micro-nuclear magnetic resonance technology.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the method of the present invention is efficient and has a low detection limit, and is more suitable for the detection of low antigen concentrations in biological samples. The high-sensitivity (detection limit of 0.1 pg / ml) detection of antigen content in liquid biological samples greatly shortens the detection time. The single sample detection time is within 1 minute (referring to the time for obtaining the nuclear magnetic resonance signal, excluding the probe capture time), and the experimental sample test CV is within 10%, with good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 TEM image (a) and EDS image (b) of the multi-sized magnetic nanoprobe prepared in Example;

[0026] Figure 2 TEM image of a mixed solution of capture probe and signal probe prepared for the example;

[0027] Figure 3 TEM image of a sandwich composite prepared for the example;

[0028] Figure 4 ΔT for the embodiment 2 Example of a data model comparing log concentration of antigen, where (a) is log concentration of antigen - ΔT 2 Curve, (b) is a schematic diagram of linear fitting. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0030] Example 1

[0031] The relaxation nuclear magnetic resonance method for detecting antigen content of the present invention comprises the following steps:

[0032] (1) Preparation of magnetic nanoprobe solution (detection target antigen concentration range 10 4 —10 -4 ng / mL):

[0033] Prepare 100 μl of 1 mg / ml magnetic bead solution of two sizes (30 nm and 1 μm carboxyl magnetic beads from Nanjing Dongna Biotechnology Co., Ltd.), add 900 μl MES buffer, 200 μl EDC solution of 1 mg / ml and 100 μl NHS solution of 10 mg / ml in sequence, react at 25°C, 1500 r / min for 0.5 h, and magnetically separate (500 mT, 4 h at 4°C) and wash the activated product with PBS buffer;

[0034] Take the target antigen antibody that has been packaged at 10 μg / ml, thaw it at 4°C, take the activated carboxyl magnetic bead solution with a concentration of 100 μg / ml of two sizes, add them to the antibody respectively, 30 nm corresponds to Antibody-T, 1 μm corresponds to Antibody-C, resuspend by ultrasound, react at 37°C, 1500 r / min for 2 hours, and then add 1% BSA solution to block for 0.5 hours;

[0035] The mixed solution after sufficient reaction was placed on a magnetic rack and allowed to stand. After complete precipitation, the supernatant was discarded and ultrasonic resuspended to wash away the unreacted free antibodies and BSA to obtain a magnetic nanoprobe solution. The prepared probe can be stored at 4°C for one week. The TEM and EDS images of the prepared multi-size magnetic nanoprobes are shown in Figure 1 shown.

[0036] (2) Detection of standard biological fluid samples for target antigen magnetic relaxation (taking amino-terminal pro-brain natriuretic peptide as an example):

[0037] The serum samples were diluted 1000 times to prepare the standard samples, and the concentration of amino-terminal pro-brain natriuretic peptide was increased by 10 times. -4 ~10 4 ng / mL of the test samples (including a set of blank controls), each concentration gradient contained three 500 μL parallel samples;

[0038] Add 100 μL of each of the two magnetic nanoprobe solutions to the above-mentioned sample to be tested, shake and mix, and react at 37°C and 1500 r / min for 2 h to obtain a sandwich complex of signal probe-antigen-capture probe, the TEM image of which is shown in FIG. Figure 2 and Figure 3 As shown;

[0039] The reaction solution was placed under a field strength of 300 mT, and the supernatant was taken to complete magnetic separation. After magnetic separation, 100 μL of each group of supernatant was put into a nuclear magnetic resonance test tube, and 3 tubes were assembled at a time. The transverse relaxation time of the supernatant was measured using an 18-20 MHz low-field nuclear magnetic resonance measuring instrument, with a measuring pulse of CPMG, a scanning number of 1, and a gain of 65.

[0040] (3) A mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signals is constructed in a diluted serum solution of amino-terminal pro-brain natriuretic peptide with known concentration: the transverse relaxation time of each group of samples measured in step (2) is averaged, and then the vertical axis is used as the vertical axis after subtracting the blank group reference value, and a linear fit is performed with the logarithmic concentration value of the antigen on the horizontal axis, such as Figure 4 The results show that as the concentration of the target increases, the ΔT2 value of the sample gradually increases. -3 The linear relationship between the concentration logarithm and the concentration range was good in the range of 10 to 100 ng / mL, the detection limit was 0.1 pg / mL, and the CV of the prepared solution sample test was within 10%. -3 ~100ng / mL is used as the effective detection range.

[0041] (4) The antigen content of the biological sample to be tested is determined by comparing the measured relaxation time difference with the known data model of the sample type: the serum sample to be tested is diluted 1000 times and three parallel samples of 500 μL are taken. Then, 100 μL of each of the two magnetic nanoprobe solutions are added to the above-mentioned sample to be tested. The mixture is shaken and reacted at 37°C and 1500 r / min for 2 h to obtain a sandwich complex of signal probe-antigen-capture probe.

[0042] The reaction solution was placed under a field strength of 300 mT, and the supernatant was taken to complete magnetic separation. After magnetic separation, 100 μL of the supernatant was put into a nuclear magnetic resonance test tube, and 3 tubes were assembled at a time. The transverse relaxation time of the supernatant was measured using an 18-20 MHz low-field nuclear magnetic resonance measuring instrument, with a measuring pulse of CPMG, a scanning number of 1, and a gain of 65.

[0043] The average of the measured transverse relaxation times is used as input to the known data model, and the output obtained is the measured antigen concentration.

Claims

1. A relaxation nuclear magnetic resonance method for detecting antigen content, characterized in that: The method comprises the following steps: firstly preparing a capture probe and a signal probe, then combining the capture probe and the signal probe with a target antigen to form a complex, then removing the complex and the capture probe by magnetic separation, and finally detecting the reduced amount of the signal probe to construct a mapping relationship between the analyte and the nuclear magnetic relaxation signal.

2. The relaxation nuclear magnetic resonance method according to claim 1, characterized in that The specific steps include: (1) Probe preparation: Activate and modify the magnetic nanoparticles. Then, activate the modified magnetic nanoparticles, bind them to antibodies, and block them to obtain two magnetic probes of different sizes, namely, capture probes and signal probes; (2) Construct a mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signal in an aqueous solution of target antigen with known concentration: combine the capture probe and signal probe with the target antigen with known concentration, fully react to form a composite structure, take the supernatant after magnetic separation, and measure the transverse relaxation time of magnetic resonance signal generated by the change of magnetic nanoprobe concentration under different target antigen concentrations. 1 The difference in H-NMR relaxation signals was used to calculate the transverse relaxation time change ΔT2 of the supernatant and establish a mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signals. (3) constructing a mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signal in a diluted serum solution with known antigen concentration: diluting the serum sample and combining it with the above-mentioned magnetic nanoprobe to fully react to form a composite structure, detecting the transverse relaxation time of the supernatant after magnetic separation, and further establishing a mathematical model for mapping antigen concentration and transverse relaxation time of magnetic resonance signal under serum background; (4) The antigen content of the biological sample under test is obtained by comparing the measured relaxation time difference with the known data model of the sample type: the serum sample is diluted and combined with the above-mentioned magnetic nanoprobe to fully react, and the transverse relaxation time of the supernatant is detected after magnetic separation. The ΔT of the liquid biological sample under test is substituted into the known data model as the input, and the output obtained is the measured antigen concentration.

3. The relaxation nuclear magnetic resonance method according to claim 2, characterized in that Step (1) specifically comprises preparing two sizes of magnetic nanoparticles into solutions, adjusting the pH, adding NHS solution and EDC solution for activation modification, then re-adjusting the pH of the modified magnetic nanoparticle solution and adding antibodies for reaction, and finally adding BSA solution for blocking.

4. The relaxation nuclear magnetic resonance method according to claim 3, characterized in that The magnetic beads have particle sizes of 30nm and 1000nm respectively, wherein the detection antibody is coupled with the 30nm magnetic nanoparticles to form a signal probe, and the recognition antibody is coupled with the 1000nm magnetic nanoparticles to form a capture probe.

5. The relaxation nuclear magnetic resonance method according to claim 3, characterized in that The pH adjustment described in the activation modification is to add MES buffer to adjust the pH to between 5.5 and 6.7; the activation conditions are 24-26°C, 1500r / min, and 30-40min; the pH adjustment described in the addition of antibodies is to use PBS solution to adjust the pH to between 7.2 and 7.4; the reaction conditions after adding the antibody are 36-37°C, 1500r / min, and 2-2.5h; the reaction conditions of the blocking link are 36-37°C, 1000r / min, and 30-40min.

6. The relaxation nuclear magnetic resonance method according to claim 2, characterized in that: In step (2), the target antigen is an antigen to which the corresponding antibody is easily bound by the imide functional group, and the amino-terminal pro-brain natriuretic peptide is used as an example in the embodiment.

7. The relaxation nuclear magnetic resonance method according to claim 2, characterized in that: In step (2) or (3), the transverse relaxation is detected by using a 18-20 MHz low-field nuclear magnetic resonance measuring instrument, and the measuring pulse is CPMG.

8. The relaxation nuclear magnetic resonance method according to claim 2, characterized in that: In step (2) or (3), the signal probe and the capture probe are added in excess compared to the antigen, and the composite structure is a capture probe-antigen-signal probe complex in which the antigen is simultaneously bound to the two probes.

9. The relaxation nuclear magnetic resonance method according to claim 2, characterized in that: In step (2) or (3), the magnetic separation is allowed to stand for 45 seconds to 1 minute at a field strength of 300 mT.

10. The relaxation nuclear magnetic resonance method according to claim 2, characterized in that: In step (4), the detection instrument of the sample to be tested must be consistent with the relaxation nuclear magnetic resonance detection instrument used to establish the data model, have the same proton resonance frequency, test temperature, and use the same measurement pulse sequence, number of scans, and echo interval.

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