A method for constructing a magnetic relaxation sensor technology detection probe

Abstract

The application relates to a construction method of a magnetic relaxation sensing technology detection probe, which comprises the following steps: mixing paramagnetic graphene quantum dot dispersion liquid and antibody dispersion liquid, and irradiating the mixture with ultraviolet light to obtain a magnetic relaxation sensing technology detection probe. The application has the advantages of rapid construction (within 1 hour), simple operation, no need for complicated activation treatment and impurity removal post-treatment, and the obtained detection probe has the advantages of strong stability and strong specificity. The specific detection probe can be quickly constructed under the condition that there is no specific magnetic relaxation sensing detection probe for a specific marker, and the detection period of the magnetic relaxation sensing technology is shortened.

Description

Technical Field

[0001] This invention belongs to the field of biomarker detection, and specifically relates to a method for constructing a detection probe using magnetic relaxation sensing technology. Background Technology

[0002] Magnetic relaxation sensing is a technique for detecting ex vivo biological samples in a liquid environment using nuclear magnetic resonance relaxation. It belongs to the category of in vitro diagnostic techniques. Magnetic relaxation sensing can perform specific, highly sensitive, and / or quantitative detection of various biomolecules, including proteins, nucleic acids, bacteria, and viruses.

[0003] The implementation process of magnetic relaxation sensing technology is as follows: 1. Connect magnetic nanoparticles to specific antibodies or specific aptamers to obtain detection probes; 2. Mix the detection probes with a sample containing the target biomolecule in a liquid phase environment, where the detection probes generate nanoclusters on the surface of the target biomolecules; 3. Detect the relaxation time of the aforementioned mixed sample in a magnetic resonance system. By comparing the relaxation time with that of a blank sample, it can be determined whether the sample contains the target biomolecule and further determine the content of the target biomolecule. In the above process, the classic connection method commonly used in process 1 is click chemistry. Figure 1 This method involves activating the carboxylic acid surface of magnetic nanoparticles using crosslinking agents such as EDC and Sulfo-NHS, then attaching amino-containing antibodies or aptamers to the surface, and finally obtaining the detection probe through centrifugation and other processes. However, this method for obtaining magnetic relaxation sensing probes is cumbersome and time-consuming (approximately 5 hours), and cannot meet the needs of rapid probe construction in specific application scenarios. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing a detection probe for magnetic relaxation sensing technology, which solves the problem of the cumbersome preparation of detection probes in the prior art.

[0005] This invention provides a method for constructing a detection probe for magnetic relaxation sensing technology, comprising the following steps:

[0006] S1. Prepare a paramagnetic graphene quantum dot dispersion and an antibody dispersion; wherein the antibody is a hemangioma marker antibody; S2. Mix the paramagnetic graphene quantum dot dispersion and the antibody dispersion, and irradiate them with ultraviolet light to obtain a detection probe for magnetic relaxation sensing technology.

[0007] The size of the paramagnetic graphene quantum dots in step S1 is 2-10 nm, and the paramagnetic material contained is gadolinium ions.

[0008] The solvent for the magnetic graphene quantum dot dispersion in step S1 is phosphate buffer solution with a concentration of 1 μg / mL to 1 mg / mL.

[0009] The solvent for the antibody dispersion in step S1 is phosphate buffer with a concentration of 1 μg / mL to 1 mg / mL.

[0010] In step S2, the mixing volume ratio of the paramagnetic graphene quantum dot dispersion and the antibody dispersion is 10:0.1 to 0.1:10.

[0011] The ultraviolet light power in step S2 is 10-100mW, and the irradiation time is 5-60min.

[0012] The magnetic relaxation sensing probe obtained in step S2 is mixed with the sample to be tested. The sample to be tested may contain biomarkers. The relaxation time after mixing is measured as Ta using a magnetic resonance system, and the relaxation time of the blank control sample is measured as Tb. The difference between Ta and Tb is used to determine whether the sample to be tested contains biomarkers.

[0013] In classic magnetic relaxation sensing technology, the magnetic nanoparticles in the detection probe are iron(III) oxide (Fe3O4), which suffers from poor dispersibility and cumbersome preparation steps. The paramagnetic graphene quantum dots in this invention are gadolinium-loaded graphene quantum dots, a nanoscale carbon material with excellent dispersibility, simple preparation steps, and abundant and easily modulated functional groups on its surface. The acidity of the carboxylic acid at the graphene quantum dot interface can be enhanced by ultraviolet light excitation, achieving carboxylic acid activation similar to that of EDC / Sulfo-NHS, thereby enabling the connection between the graphene quantum dots and amino-containing antibodies / aptamers. Using ultraviolet light irradiation as the probe preparation condition and the magnetic nanoparticles contained in the graphene quantum dots as the detection probe, rapid construction of the detection probe in magnetic relaxation sensing technology can be achieved, thus accelerating the detection process of magnetic relaxation sensing technology. Simultaneously, due to the high relaxation rate of graphene quantum dots, the magnetic relaxation sensing technology based on them also has the advantage of high detection sensitivity.

[0014] Beneficial effects

[0015] This invention offers advantages such as rapid construction (within 1 hour), simple operation, and no need for cumbersome activation and impurity removal post-processing. The resulting detection probe exhibits high stability and specificity. It can rapidly construct specific detection probes even when no specific magnetic relaxation sensing probes for a particular marker are available, thus shortening the detection cycle of magnetic relaxation sensing technology. Attached Figure Description

[0016] Figure 1 These are detection probes obtained by linking existing magnetic nanoparticles with specific antibodies or specific aptamers.

[0017] Figure 2 This is a flowchart illustrating the preparation and application of the magnetic relaxation sensing probe of the present invention.

[0018] Figure 3 This invention provides a comparison and trend of relaxation time of the detection probe in the magnetic relaxation sensing technology of Embodiment 1.

[0019] Figure 4 This is a comparison of the relaxation time of the magnetic relaxation sensing probe in Embodiment 2 of the present invention when detecting different proteins;

[0020] Figure 5 The relaxation time of the detection probe in Embodiment 2 of the present invention is measured by magnetic relaxation sensing technology at different storage times.

[0021] Figure 6 This is a comparison of the relaxation time of the magnetic relaxation sensing probe in Embodiment 3 of the present invention when detecting different concentrations of the analyte CD105.

[0022] Figure 7 This illustrates the linear relationship between the relaxation time of the magnetic relaxation sensing probe in Embodiment 3 of the present invention and the concentration of CD105 at different concentrations of the analyte. Detailed Implementation

[0023] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0024] Example 1

[0025] Combination Figure 2 This embodiment provides a method for constructing a detection probe for magnetic relaxation sensing technology, including the following steps:

[0026] S1. Provide a paramagnetic graphene quantum dot dispersion, wherein the size of the paramagnetic graphene quantum dots is 10 nm, the paramagnetic material contained therein is gadolinium ion, the dispersion is phosphate buffer, and the concentration is 1 mg / mL; provide an antibody dispersion, wherein the antibody is CD105 antibody, and the concentration is 1 μg / mL.

[0027] S2. Mix the paramagnetic graphene quantum dot dispersion with the antibody dispersion at room temperature. The mixing ratio is 0.1:10 (liquid volume ratio). Irradiate the mixture with ultraviolet light at a power of 100mW for 0–60 min to obtain multiple magnetic relaxation sensing probes with different ultraviolet irradiation times.

[0028] Figure 3The relaxation time (Tb) of the detection probe after UV irradiation of the mixture of paramagnetic graphene quantum dot dispersion and antibody dispersion for 0, 5, 10, 15, 20, 30, 35, 40, 45, 50, 55, and 60 min is shown. It can be seen that with increasing UV irradiation time, the relaxation time of the detection probe gradually shortens and stabilizes around 160 ms, indicating that the optimal UV irradiation time for the detection probe under the above conditions is 50 min. In subsequent examples, the testing process for the optimal UV irradiation time will not be listed again; only the optimal UV irradiation time under the conditions described in this example will be listed.

[0029] Example 2

[0030] Combination Figure 2 This embodiment provides a method for constructing a detection probe for magnetic relaxation sensing technology, including the following steps:

[0031] S1. Provide a paramagnetic graphene quantum dot dispersion, wherein the size of the paramagnetic graphene quantum dots is 2nm, the paramagnetic substance contained therein is gadolinium ion, the dispersion is phosphate buffer, and the concentration is 10μg / mL; provide an antibody dispersion, wherein the antibody is IgG, and the concentration is 0.1mg / mL.

[0032] S2. Mix the paramagnetic graphene quantum dot dispersion with the antibody dispersion at room temperature. The mixing ratio is 1:1 (liquid volume ratio). Irradiate the mixture with ultraviolet light at a power of 50mW for 20min to obtain the magnetic relaxation sensing probe.

[0033] S3. Apply the magnetic relaxation sensing probe to the magnetic relaxation sensing technology. The magnetic relaxation sensing probe without the added biomarker Protein G is recorded as the blank sample, and its relaxation time in the magnetic resonance system is Tb. The magnetic relaxation sensing probe with the added biomarker Protein G (0.05 mg / mL) is recorded as the experimental sample, and its relaxation time in the magnetic resonance system is Ta.

[0034] In this embodiment, the probe construction time was 20 minutes. The relaxation time Tb of the magnetic relaxation sensing probe constructed in this embodiment before detecting Protein G was 265.7 ± 5.9 ms, and the relaxation time Ta after detecting Protein G was 568.1 ± 15.3 ms. The significant difference between Ta and Tb indicates that the magnetic relaxation sensing probe constructed in this embodiment can be used for biomolecule detection. Figure 4As shown, the relaxation time Ta of the detection probe constructed in this embodiment is compared when detecting different biomarkers. When detecting bovine serum albumin (BSA) and hemangioma marker protein CD105, there is no significant difference between Ta and Tb. Only when detecting Protein G is there a significant difference between Ta and Tb, indicating that the magnetic relaxation sensing technology detection probe constructed in this invention has excellent specificity. Figure 5 As shown, the relaxation time Tb of the magnetic relaxation sensing probe constructed in this embodiment was tested at different storage time points. It can be seen that the relaxation time of the probe did not change significantly within the storage time of 1 to 500 hours, indicating that the magnetic relaxation sensing probe constructed rapidly by the present invention has excellent stability.

[0035] Example 3

[0036] Combination Figure 2 This embodiment provides a method for constructing a detection probe for magnetic relaxation sensing technology, including the following steps:

[0037] S1. Provide a paramagnetic graphene quantum dot dispersion, wherein the size of the paramagnetic graphene quantum dots is 5 nm, the paramagnetic material contained therein is gadolinium ion, the dispersion is phosphate buffer, and the concentration is 1 μg / mL; provide an antibody dispersion, wherein the antibody is CD105 antibody, and the concentration is 1 mg / mL.

[0038] S2. Mix the paramagnetic graphene quantum dot dispersion with the antibody dispersion at room temperature. The mixing ratio is 10:0.1 (liquid volume ratio). Irradiate the mixture with ultraviolet light at a power of 10mW for 60min to obtain the magnetic relaxation sensing probe.

[0039] S3. Apply the magnetic relaxation sensing probe to the magnetic relaxation sensing technology. The magnetic relaxation sensing probe without the added analyte CD105 is recorded as the blank sample, and its relaxation time in the magnetic resonance system is Tb. The magnetic relaxation sensing probe with the added analyte Protein G (1ng / mL~1mg / mL) is recorded as the experimental sample, and its relaxation time in the magnetic resonance system is Ta.

[0040] The probe construction time in this embodiment was 60 min. The relaxation time Tb of the magnetic relaxation sensing probe constructed in this embodiment before detecting CD105 was 345.4 ± 8.7 ms. Ta differed from Tb when detecting different concentrations of CD105. Figure 6 Furthermore, the higher the CD105 concentration, the more significant the difference between Ta and Tb, indicating that the magnetic relaxation sensing probe constructed in this embodiment can be used for the detection of multiple concentrations of biomolecules. Figure 7As shown, a linear fit was performed between Ta with CD105 concentrations ranging from 10 ng / mL to 10 μg / mL and the logarithm of CD105 concentration, yielding y = 64.3x + 307.2, with a fitting R² value. 2 =0.99264. The concentration of CD105 can be determined based on the above curve, given the known Ta, thus enabling quantitative detection.

[0041] The above are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a magnetic relaxation sensing technology detection probe, comprising the following steps: S1, providing paramagnetic graphene quantum dot dispersion, the size of the paramagnetic graphene quantum dot is 2 nm, the paramagnetic substance contained therein is gadolinium ion, the dispersion thereof is phosphate buffer, and the concentration thereof is 10 μg / mL; providing antibody dispersion, the antibody is IgG, and the concentration thereof is 0.1 mg / mL; S2, mixing the paramagnetic graphene quantum dot dispersion and the antibody dispersion, the mixing condition is room temperature, the mixing ratio is 1:1 in terms of liquid volume, the mixed solution is irradiated by using ultraviolet light, the ultraviolet light irradiation power is 50 mW, the irradiation time is 20 min, and finally a magnetic relaxation sensing detection probe is obtained.

2. The construction method of claim 1, wherein: The magnetic relaxation sensing technology detection probe obtained in the step S2 is mixed with a sample to be detected, the sample to be detected may contain a marker, a magnetic resonance system is used to measure the relaxation time of the mixture as Ta, the relaxation time of a blank control sample is Tb, and whether the sample to be detected contains the marker is determined according to the difference between Ta and Tb.

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