Sealing agent for magnetic porous materials and use thereof

Abstract

The application discloses a blocking agent for magnetic porous materials and application thereof. The blocking agent comprises the following components: a protein matrix of different species from proteins adsorbed by the magnetic porous materials, a small molecule with a ring structure, a low molecular weight polymer, an amphoteric molecule and a buffer. The application also provides application of the blocking agent in immunodetection. The blocking agent provided by the application can effectively block active sites on the surface of the magnetic porous materials, reduce non-specific reactions in the immunoreaction process and increase detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a sealing agent for magnetic porous materials and its application. Background Technology

[0002] The basic building blocks of living organisms, including carbohydrates, lipids, proteins, and nucleic acids, exhibit important physiological activities during life processes. Biomolecules directly related to physiological or pathological processes are often used as biomarkers to indicate the occurrence and development of diseases. Due to the highly complex composition of living organisms, the extraction and detection of these specific biomolecules is a crucial step in the research of disease biomarkers. For example, blood contains over ten thousand proteins, and variations in their types and abundance contain a wealth of clinical physiological or pathological information. However, due to limitations in detection and identification technologies, only a small fraction can be detected. One major technical barrier is that high-abundance proteins account for 95% of the total blood protein content, posing a significant challenge to the separation, detection, and identification of low-abundance proteins. Magnetic porous materials offer advantages such as light weight, large specific surface area, and ease of separation via magnetic fields, effectively removing high-abundance proteins and significantly increasing the adsorption efficiency of low-abundance biomolecules, making them more suitable for the enrichment and detection of low-abundance substances. However, proteins enriched by these materials typically require complex sample processing and mass spectrometry detection, which is not entirely suitable for the rapid diagnostic needs of clinical applications. In the field of clinical testing, proteins are usually qualitatively and quantitatively detected using immunoassay techniques.

[0003] In the immunoassay of protein molecules enriched on the surface of materials, the strong adsorption capacity of magnetic porous materials can lead to strong adsorption of antibodies, enzymes, and other molecules in the immunoassay reagents during the enrichment of target proteins, resulting in false positives and nonspecific results. During the research, the inventors discovered that conventional immunoblocking reagents, such as BSA and skim milk powder, lack effective blocking of this background signal. This is because conventional blocking reagents can only block nonspecific adsorption sites on conventional substrate surfaces. For substrates with strong adsorption capacity, such as magnetic porous materials, the unique properties of their microporous structure, macroscopic packing structure, and surface potential cause false interference that cannot be effectively blocked by conventional blocking reagents. Summary of the Invention

[0004] Purpose of the invention: To solve the above-mentioned technical problems, the present invention provides a sealing agent for magnetic porous materials and its application.

[0005] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a sealing agent for a magnetic porous material, the sealing agent comprising the following components: a protein matrix of a different species from the protein adsorbed by the magnetic porous material, small molecules with cyclic structures, low molecular weight polymers, amphoteric molecules, and a buffer solution.

[0007] As a specific implementation, the protein matrix is ​​selected from one or more of bovine serum albumin, skim milk powder, casein, and fish glue, with a concentration of 0.5% to 10% (w / v). Preferably, the concentration of the protein matrix is ​​3% to 5% (w / v).

[0008] As a specific implementation, the small molecule with a cyclic structure is selected from one or more of benzoic acid, phenylacetic acid, tyrosine, tryptophan, glucose, sucrose, fructose, and trehalose, and its concentration is 0.1% to 5% (w / v). Preferably, the concentration of the small molecule with a cyclic structure is 1% to 2% (w / v).

[0009] As a specific implementation, the low molecular weight polymer is selected from one or more of polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and polydimethylsiloxane with a molecular weight of less than 4 kDa, and the concentration is 0.1% to 4% (v / v). Preferably, the concentration of the low molecular weight polymer is 0.1% to 0.5% (v / v).

[0010] As a specific implementation, the amphoteric molecule is selected from one or more of lecithin, methyl acrylate, carboxylated betaine, sulfobetaine, polysorbate, polyethylene glycol octylphenyl ether, and polyacrylamide, with a concentration of 0.05% to 1% (w / v or v / v). Preferably, the concentration of the amphoteric molecule is 0.1% to 0.5% (w / v).

[0011] In a specific embodiment, the buffer solution serves as the solvent for the blocking agent. The buffer solution is selected from phosphate buffer, citrate buffer, Tris-HCl buffer, or HEPES buffer, with a concentration of 5–150 mM and a pH value of 5.5–9.0. Preferably, the buffer solution is selected from 10 mM phosphate buffer with pH = 7.4, or 50 mM Tris-HCl buffer with pH = 8.0.

[0012] As a preferred embodiment, the sealing agent comprises 5% (w / v) skim milk powder, 1% (w / v) benzoic acid, 0.1% (v / v) polydimethylsiloxane, and 0.1% (w / v) egg yolk lecithin, and the solvent is 10mM phosphate buffer (8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate, 2.4 g / L potassium dihydrogen phosphate, and deionized water).

[0013] Alternatively, the blocking agent comprises 3% (w / v) bovine serum albumin, 2% (w / v) tyrosine, 0.5% (v / v) polyethylene glycol 600, 0.5% (w / v) sulfobetaine, and 50 mmol / L Tris-HCl buffer as the solvent.

[0014] As a specific implementation, the magnetic porous material is selected from one or a mixture of several of magnetic aluminosilicate molecular sieves, magnetic titanium silica molecular sieves, magnetic porous alumina, and magnetic porous silica materials.

[0015] Secondly, the present invention provides a method for preparing a sealing agent for the magnetic porous material, comprising the following steps:

[0016] The protein matrix, small molecules with cyclic structures, low molecular weight polymers, and amphoteric molecules are dissolved in a buffer solution and brought to a final volume.

[0017] Thirdly, the present invention provides a method for sealing a magnetic porous material, comprising using the sealing agent to mix and incubate with the magnetic porous material.

[0018] Preferably, the mixing ratio is 0.1–2 mL of blocking agent per 1 mg of magnetic porous material, the incubation temperature is 4–37 degrees Celsius, the incubation time is 15–180 minutes, and the shaking speed during incubation is 0–1000 rpm. More preferably, 1 mL of blocking reagent is added to each 1 mg of material, and the mixture is incubated at 37 degrees Celsius for 60 minutes.

[0019] Fourthly, the present invention provides the application of the blocking agent in immune detection.

[0020] As a specific implementation, the application includes capturing the antigen to be tested using a magnetic porous material, blocking the magnetic porous material with the blocking agent, and then incubating and detecting it using a labeled antibody.

[0021] Furthermore, the antigen to be tested is selected from one of the following: protein, polypeptide, microvesicle, exosome, microorganism, or cell. Preferably, the antigen to be tested is a protein.

[0022] Furthermore, the labeled antibody is selected from one of biotin-labeled antibodies, peroxidase-labeled antibodies, alkaline phosphatase-labeled antibodies, fluorescently labeled antibodies, and luminescently labeled antibodies. Preferably, a fluorescently labeled antibody is used for specific recognition.

[0023] Furthermore, the detection method is selected from colorimetric method, light absorption method, chemiluminescence method, and fluorescence method. Preferably, a fluorescent microplate reader is used for detection.

[0024] This invention relates to an immunoassay blocking reagent for magnetic porous materials. Based on conventional immunoassay blocking reagents, it adds multiple effective components to block active adsorption sites targeting different properties of the material. The blocking reagent includes conventional protein non-specific site blocking agents (large protein molecules), and adds small molecules with multi-ring structures to block the microporous structure of the material surface. It also adds low molecular weight polymers to block the macroporous structure formed by material accumulation, surfactants and salt ion buffers to neutralize the surface potential of the material, and controls the acidity / alkalinity of the blocking solution to reduce non-specific adsorption.

[0025] Beneficial effects: Compared with the prior art, the blocking reagent provided by the present invention uses a variety of molecular ratios with different properties and functions to target the different inherent characteristics of magnetic porous materials that cause non-specific adsorption, and the resulting comprehensive blocking effect can effectively reduce the adsorption of the material to the detection reagent, reduce the background signal of the immunoassay reaction, and help increase the detection sensitivity and reduce false positives. Detailed Implementation

[0026] The following provides a comprehensive description of the present invention. The embodiments described are the most preferred embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0027] Example 1

[0028] A preferred magnetic porous material sealing agent is prepared, comprising 5% (w / v) skim milk powder, 1% (w / v) benzoic acid, 0.1% (v / v) polydimethylsiloxane, 0.1% (w / v) egg yolk lecithin, 8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate, 2.4 g / L potassium dihydrogen phosphate, and deionized water. The specific preparation method is based on a total volume of 100 mL.

[0029] 1. Prepare 10mM phosphate buffer: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphate, and 0.24g potassium dihydrogen phosphate, dissolve them in 800mL deionized water, adjust the pH to 7.4, and add deionized water to a final volume of 1000mL.

[0030] 2. Weigh 5g of skim milk powder, 1g of benzoic acid, and 0.1g of egg yolk lecithin, add them to 80mL of the phosphate buffer solution prepared in step 1, and dissolve them thoroughly.

[0031] 3. Add 0.1 mL of polydimethylsiloxane and mix well.

[0032] 4. Use the 10mM phosphate buffer prepared in step 1 to bring the volume to 100mL.

[0033] Example 2

[0034] A preferred magnetic porous material sealing agent was prepared, comprising 3% (w / v) bovine serum albumin, 2% (w / v) tyrosine, 0.5% (v / v) polyethylene glycol 600, 0.5% (w / v) sulfobetaine, 50 mmol / L Tris-HCl, and deionized water. The specific preparation method is based on a total volume of 100 mL:

[0035] 1. Prepare 1M Tris-HCl buffer stock solution: Weigh 121.1g Tris, dissolve in 800mL deionized water, add 42mL concentrated hydrochloric acid to adjust the pH to 8.0, and add deionized water to make up to 1000mL.

[0036] 2. Prepare 50mM Tris-HCl buffer: Measure 50mL of the stock solution prepared in step 1 and add deionized water to make up to 1000mL.

[0037] 3. Weigh 3g bovine serum albumin, 2g tyrosine, and 0.5g sulfobetaine, add them to 80mL of Tris-HCl buffer prepared in step 2, and dissolve them thoroughly.

[0038] 4. Add 0.5 mL of polyethylene glycol 600 and mix well.

[0039] 5. Adjust the volume to 100 mL using the Tris-HCl buffer prepared in step 2.

[0040] Example 3

[0041] Antigen detection was performed using the blocking reagent from Example 1. The specific steps were as follows:

[0042] 1. Take three EDTA anticoagulated plasma samples P1, P2 and P3, and use the human PD-L1 ELISA kit (abcamab277712) to determine the PD-L1 protein content.

[0043] 2. Take 12 identical 0.1 mL aqueous solutions of magnetic molecular sieve material (the material disclosed in Example 1 of patent CN202210301054.6) with a concentration of 10 mg / mL. Add 0.1 mL of EDTA anticoagulated plasma sample P1, P2, and P3 to tubes 1-3, 4-6, and 7-9 respectively. Then add 0.5 mL of 10 mM phosphate buffer to each of tubes 1-9 in sequence. Add 0.6 mL of 10 mM phosphate buffer to tubes 10-12 as a negative control. Incubate at 1000 rpm for 30 minutes, then magnetically separate and discard the supernatant.

[0044] 3. Resuspend each sample in 1 mL of 10 mM phosphate buffer, shake for 5 minutes, perform magnetic separation, and discard the supernatant.

[0045] 4. Add 1 mL of the blocking reagent prepared in Example 1 to each of the experimental groups (samples 1, 4, 7, and 10). Add 1 mL of 10 mM phosphate buffer containing 5% BSA (the same buffer prepared in step 1 of Example 1) to each of the control groups 1 (samples 2, 5, 8, and 11). Add 1 mL of 1xPBS solution to each of the control groups 2 (samples 3, 6, 9, and 12). Mix thoroughly and incubate at 37°C for 1 hour. Subsequent procedures were identical for all 12 samples.

[0046] 5. After magnetic separation and discarding the supernatant, add 0.1 mL of FITC-labeled mouse anti-human PD-L1 monoclonal antibody (SolarbioK009977M-FITC) diluted 1:100 and incubate at room temperature for 1.5 hours at 400 rpm.

[0047] 6. Add 1 mL of 10 mM phosphate buffer containing 0.1% Tween-20, mix well by pipetting, let stand for 1 minute, and then magnetically separate and discard the supernatant. Repeat this step three times.

[0048] 7. Add 1 mL of 10 mM phosphate buffer, mix well by pipetting, let stand for 1 minute, then magnetically separate and discard the supernatant. Repeat this step twice.

[0049] 8. Resuspend the sample in 0.1 mL of 10 mM phosphate buffer. Detect the fluorescence using a fluorescence microplate reader. The excitation wavelength is 488 nm, and the emission wavelength is 530 nm. The fluorescence intensity is shown in the table below:

[0050] Table 1. Fluorescence intensity at 530 nm for three groups of samples

[0051] PD-L1 concentration experimental group Control group 1 Control group 2 Blocking reagent — The sealing agent of the present invention 5% BSA 10mM phosphate buffer Plasma P1 (numbers 1-3) 181 pg / mL 105.7 171.5 197.4 Plasma P2 (number 4-5) 243 pg / mL 153.5 188.3 196.0 Plasma P3 (7-9) 33 pg / mL 51.9 179.5 191.6 Negative controls (numbers 10-12) — 20.6 170.2 190.1

[0052] in conclusion:

[0053] Control group 2 did not use a blocking agent, control group 1 used 5% bovine serum albumin (BSA) as a conventional blocking agent for immunoassays, and the experimental group used the blocking agent prepared in Example 1. The detection values ​​of samples in control group 1 were all lower than those in control group 2, indicating that BSA has a certain blocking effect on the substrate. However, the negative control response signal in control group 1 was still high, indicating that BSA cannot completely block the non-specific response signal of the substrate material, severely reducing the detection sensitivity. Comparing the experimental group with control groups 1 and 2, the response value of the negative control sample significantly decreased from 170.2 and 190.1, which were close to the instrument saturation value, to 20.6, indicating that the blocking agent provided by this invention can effectively reduce the background signal. The response values ​​of the three plasma samples tested in the experimental group were clearly distinguishable from the negative control, and the response values ​​were basically consistent with the detection results of commercial ELISA kits. These results indicate that the blocking agent has a good blocking effect on magnetic porous materials.

[0054] Example 4

[0055] The blocking reagent from Example 2 was used for antigen detection. The specific steps are as follows:

[0056] 1. Take 5 serum samples (S1, S2, S3, S4, S5) and use the Roche E411 instrument (reagent catalog number 05109442190).

[0057] The level of interleukin-6 was measured.

[0058] 2. Take 18 identical 10 μL aqueous solutions of 50 mg / mL magnetic nanocomposite Fe3O4@TS-1 molecular sieve (i.e., the material disclosed in Example 1 of patent application number 202310553103.X). Add 200 μL of serum samples S1, S2, S3, S4, and S5 from step 1 to tubes 1-3, 4-6, 7-9, 10-12, and 13-15 respectively. Then add 0.5 mL of 50 mM Tris-HCl buffer (pH 8.0) to tubes 1-15 respectively, and add 0.6 mL of 50 mM Tris-HCl buffer (pH 8.0) to tubes 16-18 respectively. Incubate with shaking at 1000 rpm for 5 minutes, perform magnetic separation, and discard the supernatant.

[0059] 3. Add 1 mL of the blocking reagent prepared in Example 2 to each of the experimental groups (samples 1, 4, 7, 10, 13, and 16). Add 1 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 5% BSA to each of the control groups 1 (samples 2, 5, 8, 11, 14, and 17). Add 1 mL of 50 mM Tris-HCl buffer (pH 8.0) to each of the control groups 2 (samples 3, 6, 9, 12, 15, and 18). After mixing thoroughly, incubate overnight at 4°C.

[0060] 4. After magnetic separation and discarding the supernatant, add 0.1 mL of recombinant rabbit anti-human IL-6 monoclonal antibody (abcamab233706) diluted 1:500 and incubate at room temperature for 1 hour at 400 rpm.

[0061] 5. Add 1 mL of Tris-HCl buffer containing 0.1% Tween-20, mix well by pipetting, let stand for 1 minute, and then magnetically separate and discard the supernatant. Repeat this step three times.

[0062] 6. Add 0.1 mL of Alexa diluted 1:2000. 488-labeled goat anti-rabbit IL-6 monoclonal antibody (abcamab150077) was incubated at room temperature for 1 hour at 400 rpm.

[0063] 7. Add 1 mL of Tris-HCl buffer containing 0.1% Tween-20, mix well by pipetting, let stand for 1 minute, and then magnetically separate and discard the supernatant. Repeat this step three times.

[0064] 8. Add 1 mL of 10 mM phosphate buffer, mix well by pipetting, let stand for 1 minute, and then magnetically separate and discard the supernatant. Repeat this step twice.

[0065] 9. Resuspend the sample in 0.1 mL of 10 mM phosphate buffer. Detect the fluorescence using a fluorescence microplate reader. The excitation wavelength is 488 nm, and the emission wavelength is 530 nm. The fluorescence intensity is shown in the table below:

[0066] Table 1. Fluorescence intensity at 530 nm for three groups of samples

[0067]

[0068]

[0069] in conclusion:

[0070] Control group 2, which did not use a blocking agent, showed near-saturation signals for all samples, indicating that the magnetic TS-1 molecular sieve material has a significant non-specific adsorption capacity for fluorescently labeled antibodies. Control group 1, blocked with 5% bovine serum albumin (BSA), a standard blocking agent for immunoassays, showed significantly lower detection values ​​compared to control group 2 without a blocking agent. However, its negative control signal remained high, and the test sample was not clearly distinguishable from the negative control, indicating that BSA cannot completely block the non-specific response signal of the substrate material. After using the blocking agent prepared in Example 2, the negative control detection value in the experimental group was significantly lower, and the test serum sample was significantly higher than the negative control. The serum IL-6 content measured by immunofluorescence was consistent with the trend of the detection results of commercial equipment. Overall, this blocking agent has a good blocking effect on magnetic porous materials, reducing background signals, increasing detection sensitivity, and reducing false positives. The combined detection results of Examples 3 and 4 show that the blocking agent disclosed in this invention can effectively reduce the background signal of magnetic porous materials during immunoassay, increase detection sensitivity, and reduce false positives.

Claims

1. A sealing agent for sealing magnetic porous materials, characterized in that, The blocking agent comprises the following components: a protein matrix from a different species than the protein adsorbed by the magnetic porous material, small molecules with cyclic structures, low molecular weight polymers, amphoteric molecules, and a buffer solution; the protein matrix is ​​selected from bovine serum albumin and skim milk powder, with a concentration of 3%–5% w / v; the small molecules with cyclic structures are selected from benzoic acid and tyrosine, with a concentration of 1%–2% w / v; the low molecular weight polymer is selected from polyethylene glycol 600 and polydimethylsiloxane with a molecular weight of less than 4 kDa, with a concentration of 0.1%–0.5% v / v; the amphoteric molecules are selected from lecithin and sulfobetaine, with a concentration of 0.1%–0.5% w / v; and the buffer solution is selected from phosphate buffer or Tris-HCl buffer, with a concentration of 10–150 mM and a pH of 5.5–9.

0.

2. The application according to claim 1, characterized in that, The magnetic porous material is selected from one or a mixture of several of the following: magnetic aluminosilicate molecular sieves, magnetic titanium silica molecular sieves, magnetic porous alumina, and magnetic porous silica materials.

3. A method for sealing a magnetic porous material, characterized in that, This includes using the sealing agent described in claim 1 or 2, and mixing and incubating it with a magnetic porous material.

4. The sealing method for magnetic porous materials according to claim 3, characterized in that, The mixing and incubation process involves adding 0.1–2 mL of sealing agent to every 1 mg of magnetic porous material, incubating at a temperature of 4–37 degrees Celsius, incubating for 15–180 minutes, and shaking at a speed of 0–1000 rpm during incubation.

5. The application of the blocking agent as described in claim 1 or 2 in immunoassay, characterized in that, The method includes capturing the antigen to be tested using a magnetic porous material, blocking the magnetic porous material with the blocking agent, and then incubating and detecting it using a labeled antibody.

6. The application according to claim 5, characterized in that, The antigen to be tested is selected from one of proteins, peptides, microvesicles, exosomes, or cells; the labeled antibody is selected from one of biotin-labeled antibodies, peroxidase-labeled antibodies, alkaline phosphatase-labeled antibodies, fluorescent group-labeled antibodies, and luminescent group-labeled antibodies; the detection method is selected from one of colorimetric method, absorption method, chemiluminescence method, and fluorescence method.

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