Immune platform for ProGRP detection and preparation method and application thereof

Abstract

The invention relates to the technical field of detection, and discloses an immune platform for ProGRP detection and a preparation method and application thereof.The immune platform comprises magnetic graphene oxide, and the magnetic graphene oxide is formed by in-situ growth of magnetic nanoparticles on the surface of graphene oxide; the surface of the magnetic graphene oxide is connected with a capture antibody capable of specifically recognizing a ProGRP antigen; the immune platform further comprises a detection antibody which is specifically recognized with the ProGRP antigen, and the detection antibody is marked by isoluminol; the capture antibody and the detection antibody are connected through a ProGRP antigen. The platform disclosed by the invention can be used for accurately detecting the content of the gastrin-releasing peptide precursor in a serum sample.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to an immune platform for ProGRP detection and a preparation method and application thereof. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world, with the highest incidence and mortality rates in the world. It is important to distinguish between the two major subtypes of small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Progastrin-releasing peptide (ProGRP) is a peptide isolated from gastric nerve fibers and is a hormone commonly produced by small cell lung cancer cells. Under normal circumstances, the concentration of ProGRP in healthy people is generally <60pg / mL. However, when ProGRP>150pg / mL, the probability of developing small cell lung cancer is as high as 93.7%, which is a more sensitive SCLC biomarker than neuron-specific enolase (NSE). Therefore, ProGRP plays an important role in the early warning of small cell lung cancer. In addition, the increase in serum ProGRP levels can reflect the prognosis and course of SCLC, and has been confirmed to be a highly specific and sensitive tumor marker for SCLC. Serological detection of tumor markers has always been a research hotspot for early clinical screening of tumors due to its advantages such as simple operation and non-invasive screening. However, the concentration of ProGRP in complex matrices is low, and the sensitivity and specificity of serum ProGRP in the diagnosis of benign and malignant diseases remains a challenge. In addition, the cost of the test should be reduced for commercialization.

[0003] Therefore, there is an urgent need to develop a ProGRP bioassay method with high sensitivity, strong specificity, simple operation and low cost. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an immune platform for ProGRP detection, a preparation method and an application thereof. The platform of the present invention can realize accurate detection of the content of gastrin-releasing peptide precursor in serum samples.

[0005] The present invention provides an immune platform for ProGRP detection, the immune platform comprising magnetic graphene oxide (MGO), the magnetic graphene oxide having magnetic nanoparticles in situ grown on the surface of graphene oxide, and the surface of the magnetic graphene oxide being connected with a capture antibody (ProGRPAb1) that can specifically recognize ProGRP antigen; The immune platform also includes a detection antibody (Ab2) that specifically recognizes the ProGRP antigen, and the detection antibody is labeled with isoluminol (ABEI); The capture antibody and the detection antibody are linked via the ProGRP antigen.

[0006] Furthermore, the magnetic nanoparticles are ferroferric oxide.

[0007] Furthermore, the detection antibody is linked to isoluminol via an amide bond.

[0008] Furthermore, the magnetic graphene oxide is connected to the capture antibody via an amide bond.

[0009] Furthermore, the capture antibody and the detection antibody can recognize different antigenic epitopes on the ProGRP antigen.

[0010] Furthermore, the capture antibody is an anti-ProGRP mouse monoclonal antibody, product number MB270103, Ningbo Maiyue Biotechnology Co., Ltd.

[0011] Furthermore, the detection antibody labeled with isoluminol is derived from the luminescent marker in the gastrin-releasing peptide precursor assay kit (magnetic particle chemiluminescence method), Shenzhen New Industry Biomedical Engineering Co., Ltd.

[0012] The present invention also provides a method for preparing the immune platform, which comprises: Step 1: Using a co-precipitation method, magnetic nanoparticles are in situ generated on the surface of graphene oxide to obtain magnetic graphene oxide; Step 2: esterifying the magnetic graphene oxide obtained in step 1 to obtain esterified magnetic graphene oxide, and then incubating it with a capture antibody, so that the capture antibody is connected to the surface of the magnetic graphene oxide; Step 3: Labeling the detection antibody with isoluminol; Step 4: Incubate the solution containing the product of step 2 and the serum sample solution to be tested for a second time, remove the supernatant by magnetic separation, add the solution containing the isoluminol-labeled detection antibody of step 3 to the remaining product after magnetic separation, incubate three times, remove the supernatant by magnetic separation, then add the chemiluminescent substrate solution, detect using a photomultiplier tube, and record the chemiluminescence value.

[0013] Furthermore, in step 1, the coprecipitation method specifically includes: dissolving and dispersing ferric chloride hexahydrate and ferrous chloride tetrahydrate in a solution, adding a solution containing graphene oxide under a nitrogen atmosphere, heating, adjusting the pH value of the reaction environment to 10, reacting, cooling, separating the magnetic graphene oxide, washing, and drying to obtain magnetic graphene oxide.

[0014] Further, when ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved and dispersed in the solution, the mass concentration of ferric chloride hexahydrate is 1.67 g / 50 mL.

[0015] Further, when ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved and dispersed in the solution, the mass concentration of ferrous chloride tetrahydrate is 0.59 g / 50 mL.

[0016] Furthermore, the solution is deionized water.

[0017] Furthermore, in the solution containing graphene oxide, the mass concentration of graphene oxide is 0.05 g / 40 mL.

[0018] Furthermore, the volume ratio of the solution containing ferric chloride hexahydrate and ferrous chloride tetrahydrate to the solution containing graphene oxide is 5:4.

[0019] Furthermore, the temperature is raised to 80°C.

[0020] Furthermore, the pH value of the reaction environment was adjusted by using ammonia water with a volume fraction of 25%.

[0021] Furthermore, the reaction time is 1 h.

[0022] Furthermore, the specific method for separating the magnetic graphene oxide includes: using a magnet to adsorb the magnetic graphene oxide.

[0023] Furthermore, the specific method of washing includes: washing with deionized water and ethanol in sequence at least three times.

[0024] Furthermore, the drying temperature is 50°C-60°C.

[0025] Furthermore, the specific method for esterifying the magnetic graphene oxide includes: adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to a PBS buffer solution containing magnetic graphene oxide, reacting, washing, and separating.

[0026] Furthermore, the reaction temperature is 36°C-38°C, and the reaction time is 0.8h-1.2h.

[0027] Furthermore, the mass concentration of the magnetic graphene oxide in the PBS buffer solution is 5 mg / mL.

[0028] Furthermore, the mass concentration of N-hydroxysuccinimide (NHS) in the PBS buffer solution is 0.1 mg / mL.

[0029] Furthermore, the mass concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the PBS buffer solution is 0.15 mg / mL.

[0030] Furthermore, the specific method of washing includes: washing at least three times with PBS buffer solution.

[0031] Furthermore, the specific method of separation includes: using a magnet for adsorption.

[0032] Furthermore, the specific method of incubating the esterified magnetic graphene oxide with the capture antibody once includes: dispersing the esterified magnetic graphene oxide in a PBS buffer solution, adding the capture antibody, reacting, washing, separating, and connecting the capture antibody to the magnetic graphene oxide.

[0033] Furthermore, the mass concentration of the esterified magnetic graphene oxide in the PBS buffer solution is 1.25 mg / mL.

[0034] Furthermore, the mass concentration of the capture antibody in the PBS buffer solution is 0.1 mg / mL.

[0035] Furthermore, the reaction temperature is 25° C. and the reaction time is 24 h.

[0036] Furthermore, the specific method of washing includes: washing at least three times with PBS buffer solution.

[0037] Furthermore, the specific method of separation includes: using a magnet for adsorption.

[0038] Furthermore, before the secondary incubation, bovine serum albumin (BSA) needs to be added to the solution containing the magnetic graphene oxide connected with the capture antibody, sealed and separated, and the separated substance is dispersed in a PBS buffer solution to obtain a solution containing the product of step 2 in step 4.

[0039] Furthermore, the method for preparing the solution containing magnetic graphene oxide connected with capture antibodies is as follows: dispersing the magnetic graphene oxide connected with capture antibodies in a PBS buffer solution.

[0040] Furthermore, the mass concentration of magnetic graphene oxide connected with capture antibodies in the solution containing magnetic graphene oxide connected with capture antibodies is 1.25 mg / mL.

[0041] Furthermore, the mass concentration of the bovine serum albumin in the solution containing the magnetic graphene oxide connected with the capture antibody is 5%.

[0042] Furthermore, the sealing treatment time is 2 hours.

[0043] Furthermore, the specific method of separation includes: using a magnet for adsorption.

[0044] Furthermore, the mass concentration of the separated substance in the PBS buffer solution was 1.25 mg / mL.

[0045] Furthermore, in step 3, the specific method of labeling the detection antibody with isoluminol includes: labeling the detection antibody with amino-modified isoluminol.

[0046] Furthermore, in step 4, the ratio of the solution containing the product of step 2 to the serum sample solution to be tested is 10:50 by volume.

[0047] Furthermore, in step 4, the temperature for the secondary incubation is 37° C. and the time is 20 min.

[0048] Furthermore, in step 4, the mass concentration of the remaining product in the solution containing the isoluminol-labeled detection antibody is 2.5 mg / mL.

[0049] Furthermore, in step 4, the mass concentration of the isoluminol-labeled detection antibody in the solution containing the isoluminol-labeled detection antibody in step 3 is 2.5 μg / mL.

[0050] Furthermore, in step 4, the preparation method of the solution containing the isoluminol-labeled detection antibody of step 3 is: dispersing the isoluminol-labeled detection antibody in PBS buffer.

[0051] Furthermore, in step 4, the temperature for three incubations is 37° C. and the time is 20 min.

[0052] Furthermore, in step 4, three incubations are performed, and after the supernatant is removed by magnetic separation, it is necessary to wash at least 5 times with PBS buffer solution.

[0053] Furthermore, in step 4, the chemiluminescent substrate solution is prepared by dispersing sodium hydroxide and hydrogen peroxide solution in deionized water.

[0054] Furthermore, the mass fraction of the sodium hydroxide is 1.5%.

[0055] Furthermore, the volume fraction of the hydrogen peroxide solution is 0.18%.

[0056] The present invention also provides an application of the immune platform, which includes using the immune platform to detect ProGRP standard solutions with different standard concentrations and a test solution containing an unknown ProGRP concentration, establishing a linear regression equation between the concentration and chemiluminescence intensity of the ProGRP standard solution, and substituting the chemiluminescence intensity obtained from the test solution into the linear regression equation to obtain the ProGRP concentration in the test solution.

[0057] The embodiments of the present invention have the following technical effects: 1. The platform of the present invention adopts a large number of capture antibodies connected to the surface of magnetic graphene oxide, which is conducive to maximally capturing the antigen in the test solution, laying the foundation for accurately detecting low-concentration ProGRP, and through the detection antibody, the content of the target is converted into chemiluminescence for detection. The chemiluminescence method has high sensitivity and can play a signal amplification role, thereby achieving accurate detection of ProGRP. Therefore, the platform of the present invention is particularly suitable for the detection of test solutions containing low-concentration ProGRP.

[0058] 2. In the present invention, the magnetic graphene oxide is esterified and then connected to the capture antibody through an amide bond, which is beneficial to the stable connection of the capture antibody during the magnetic attraction process or the preparation process, thereby paving the way for capturing the ProGRP antigen; secondly, the detection antibody is connected to isoluminol through an amide bond, which is the basis for converting the ProGRP antigen concentration into chemiluminescence intensity.

[0059] 3. In the present invention, magnetic nanoparticles are grown in situ on the surface of graphene oxide, which can reduce the influence of other substances on the test results except the target through the effect of magnets during the preparation process, thereby facilitating the accuracy of the test. In addition, the use of a blocking agent to block the non-specific sites on the surface of magnetic graphene oxide also further improves the accuracy of the test.

[0060] 4. The sandwich chemiluminescent immunoassay platform for ProGRP detection provided by the present invention is not only inexpensive, but also has a large specific surface area and can be used as a solid phase carrier to load more detection antibodies. At the same time, due to its excellent magnetic responsiveness, it can be used as a capture probe and enrichment tool for ProGRP. The preparation method of the constructed chemiluminescent immunoassay platform is simple, and has good accuracy, selectivity and sensitivity. This chemiluminescent immunoassay platform is used for the first time to detect ProGRP in serum samples.

[0061] 5. The MGO immunosubstrate used in the present invention has unique superparamagnetism and improved surface area to volume ratio, has good prospects in detection, and provides a method for establishing an effective tumor biomarker enrichment and detection system. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0063] Figure 1It is a schematic diagram of the preparation principle of the magnetic graphene oxide immobilized capture antibody ProGRPAb1 (MGO@ProGRPAb1) provided in an embodiment of the present invention.

[0064] Figure 2 It is a working principle diagram of the immune platform provided by an embodiment of the present invention.

[0065] Figure 3 This is a morphology diagram of magnetic graphene oxide provided by an embodiment of the present invention.

[0066] Figure 4 It is an infrared spectrum diagram of graphene oxide and magnetic graphene oxide provided by the embodiments of the present invention.

[0067] Figure 5 1 is an XRD diagram of the magnetic graphene oxide provided by an embodiment of the present invention.

[0068] Figure 6 It is a thermogravimetric curve diagram of the magnetic graphene oxide provided by an embodiment of the present invention.

[0069] Figure 7 is a solution of magnetic graphene oxide in different states provided by an embodiment of the present invention, wherein Figure 7 a is the solution without an external magnetic field. Figure 7 In b, the solution is under an external magnetic field.

[0070] Figure 8 It is the hysteresis loop of the magnetic graphene oxide provided by the embodiment of the present invention.

[0071] Fig. 9 It is the standard curve provided by the embodiment of the present invention.

[0072] Fig.10 It is the fitting data of the test results of the embodiments and comparative examples provided by the present invention. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0074] In a first aspect, some embodiments of the present invention provide an immune platform for ProGRP detection, the immune platform comprising magnetic graphene oxide, wherein the magnetic graphene oxide is graphene oxide with magnetic nanoparticles grown in situ on its surface, and the surface of the magnetic graphene oxide is connected to a capture antibody that can specifically recognize the ProGRP antigen; The immune platform also includes a detection antibody that specifically recognizes the ProGRP antigen, and the detection antibody is labeled with isoluminol; The capture antibody and the detection antibody are linked via the ProGRP antigen.

[0075] In the present invention, the magnetic graphene oxide can form a stable connection relationship with the capture antibody, so that in the process of preparing the platform, the capture antibody can be stably connected to the surface of the magnetic graphene oxide, which is conducive to better capture of the ProGRP antigen.

[0076] In some embodiments, the magnetic nanoparticles are ferroferric oxide.

[0077] In some embodiments, the detection antibody is linked to isoluminol via an amide bond.

[0078] In some embodiments, the magnetic graphene oxide is linked to the capture antibody via an amide bond.

[0079] In some embodiments, the capture antibody is an anti-ProGRP mouse monoclonal antibody.

[0080] In some embodiments, the detection antibody is an anti-ProGRP mouse monoclonal antibody.

[0081] In some embodiments, the capture antibody and the detection antibody recognize different epitopes on the ProGRP antigen.

[0082] In the present invention, the capture antibody and the detection antibody have different antigenic epitopes on the surface of the ProGRP antigen. Therefore, those skilled in the art can select different capture antibodies or detection antibodies according to actual needs.

[0083] In a second aspect, some embodiments of the present invention also provide a method for preparing the immune platform, the preparation method comprising: Step 1: Using a co-precipitation method, magnetic nanoparticles are in situ generated on the surface of graphene oxide to obtain magnetic graphene oxide; Step 2: esterifying the magnetic graphene oxide obtained in step 1 to obtain esterified magnetic graphene oxide, and then incubating it with a capture antibody, so that the capture antibody is connected to the surface of the magnetic graphene oxide; Step 3: Labeling the detection antibody with isoluminol; Step 4: Incubate the solution containing the product of step 2 and the serum sample solution to be tested for a second time, remove the supernatant by magnetic separation, add the solution containing step 3 to the remaining product after magnetic separation, incubate three times, remove the supernatant by magnetic separation, then add chemiluminescent substrate solution, detect using a photomultiplier tube, and record the chemiluminescence value.

[0084] In some embodiments, in step 1, the co-precipitation method specifically includes: dissolving and dispersing ferric chloride hexahydrate and ferrous chloride tetrahydrate in a solution, adding a solution containing graphene oxide under a nitrogen atmosphere, heating, adjusting the pH value of the reaction environment to 10, reacting, cooling, separating the magnetic graphene oxide, washing, and drying to obtain magnetic graphene oxide.

[0085] In some embodiments, when ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved and dispersed in a solution, the mass concentration of ferric chloride hexahydrate is 1.67 g / 50 mL.

[0086] In some embodiments, when ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved and dispersed in a solution, the mass concentration of ferrous chloride tetrahydrate is 0.59 g / 50 mL.

[0087] In some embodiments, the solution is deionized water.

[0088] In some embodiments, in the solution containing graphene oxide, the mass concentration of graphene oxide is 0.05 g / 40 mL.

[0089] In some embodiments, the volume ratio of the solution containing ferric chloride hexahydrate and ferrous chloride tetrahydrate to the solution containing graphene oxide is 5:4.

[0090] In some embodiments, the temperature is increased to 80°C.

[0091] In some embodiments, the pH value of the reaction environment is adjusted using 25% ammonia water by volume.

[0092] In some embodiments, the reaction time is 1 hour.

[0093] In some embodiments, the specific method of separating the magnetic graphene oxide includes: using a magnet to adsorb the magnetic graphene oxide.

[0094] In some embodiments, the specific method of washing includes: washing with deionized water and ethanol in sequence at least three times.

[0095] In some embodiments, the drying temperature is 50°C-60°C.

[0096] In some embodiments, the specific method for esterifying the magnetic graphene oxide includes: adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to a PBS buffer solution containing magnetic graphene oxide, reacting, washing, and separating.

[0097] In the present invention, during the reaction process, EDC reacts with the carboxyl groups on the magnetic graphene oxide to generate an unstable intermediate product O-acylurea, which is very easy to hydrolyze, and the added NHS can react with the O-acylurea to generate sulfo-NHS ester with amino reaction activity, thereby reducing the occurrence of hydrolysis and improving the reaction efficiency, thereby obtaining esterified magnetic graphene oxide.

[0098] In some embodiments, the reaction temperature is 36° C.-38° C., and the reaction time is 0.8 h-1.2 h.

[0099] In some embodiments, the mass concentration of the magnetic graphene oxide in the PBS buffer solution is 5 mg / mL.

[0100] In some embodiments, the mass concentration of N-hydroxysuccinimide (NHS) in the PBS buffer solution is 0.1 mg / mL.

[0101] In some embodiments, the mass concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in the PBS buffer solution is 0.15 mg / mL.

[0102] In some embodiments, the specific method of washing includes: washing at least three times with PBS buffer solution.

[0103] In some embodiments, the specific method of separation includes: using a magnet for adsorption.

[0104] In some embodiments, the specific method of incubating the esterified magnetic graphene oxide with the capture antibody once includes: dispersing the esterified magnetic graphene oxide in a PBS buffer solution, adding the capture antibody, reacting, washing, separating, and connecting the capture antibody to the magnetic graphene oxide.

[0105] In the present invention, during the reaction between the esterified magnetic graphene oxide and the capture antibody, the ester group is hydrolyzed, thereby forming an amide bond connection with the capture antibody.

[0106] In some embodiments, the mass concentration of the esterified magnetic graphene oxide in the PBS buffer solution is 1.25 mg / mL.

[0107] In some embodiments, the mass concentration of the capture antibody in the PBS buffer solution is 0.1 mg / mL.

[0108] In some embodiments, the reaction temperature is 25° C. and the reaction time is 24 h.

[0109] In some embodiments, the specific method of washing includes: washing at least three times with PBS buffer solution.

[0110] In some embodiments, the specific method of separation includes: using a magnet for adsorption.

[0111] In some embodiments, before the secondary incubation, bovine serum albumin (BSA) is added to the solution containing the magnetic graphene oxide connected with the capture antibody, sealed and separated, and the separated substance is dispersed in a PBS buffer solution to obtain a solution containing the product of step 2 in step 4.

[0112] In some embodiments, the mass concentration of magnetic graphene oxide linked to capture antibodies in the solution containing magnetic graphene oxide linked to capture antibodies is 1.25 mg / mL.

[0113] In some embodiments, the mass concentration of bovine serum albumin in the solution containing magnetic graphene oxide linked to capture antibodies is 5%.

[0114] In some embodiments, the sealing treatment time is 2 hours.

[0115] In some embodiments, the specific method of separation includes: using a magnet for adsorption.

[0116] In some embodiments, the mass concentration of the separated substance in the PBS buffer solution is 1.25 mg / mL.

[0117] In some embodiments, in step 3, the specific method of labeling the detection antibody with isoluminol includes: activating the amino group in the isoluminol molecule and then labeling the detection antibody.

[0118] In the present invention, the isoluminol-labeled detection antibody can also be obtained by commercial methods, derived from the luminescent marker in the gastrin-releasing peptide precursor assay kit (magnetic particle chemiluminescence method), Shenzhen New Industry Biomedical Engineering Co., Ltd.

[0119] In some embodiments, in step 4, the ratio of the solution containing the product of step 2 to the serum sample solution to be tested is 10:50 by volume.

[0120] In some embodiments, in step 4, the temperature for the secondary incubation is 37° C. and the time is 20 min.

[0121] In some embodiments, in step 4, the mass concentration of the remaining product in the solution containing step 3 is 2.5 mg / mL.

[0122] In some embodiments, in step 4, the mass concentration of the isoluminol-labeled detection antibody in the solution of step 3 is 2.5 μg / mL.

[0123] In some embodiments, in step 4, the temperature for three incubations is 37° C. and the time is 20 min.

[0124] In some embodiments, in step 4, three incubations are performed, and after the supernatant is removed by magnetic separation, it is necessary to wash at least 5 times with PBS buffer solution.

[0125] In some embodiments, in step 4, the chemiluminescent substrate solution is prepared by dispersing sodium hydroxide and hydrogen peroxide solution in deionized water.

[0126] In some embodiments, the mass fraction of the sodium hydroxide is 1.5%.

[0127] In some embodiments, the volume fraction of the hydrogen peroxide solution is 0.18%.

[0128] In a third aspect, some embodiments of the present invention also provide an application of the immune platform, which includes using the immune platform to detect ProGRP standard solutions of different standard concentrations and test solutions containing unknown ProGRP concentrations, establishing a linear regression equation between the concentration and chemiluminescence intensity of the ProGRP standard solution, substituting the chemiluminescence intensity obtained from the test solution into the linear regression equation, and obtaining the ProGRP concentration in the test solution.

[0129] The following is further described in conjunction with specific embodiments: The capture antibody (ProGRPAb1) is an anti-ProGRP mouse monoclonal antibody, product number MB270103, Ningbo Maiyue Biotechnology Co., Ltd. The isoluminol-labeled detection antibody is derived from the luminescent marker in the gastrin-releasing peptide precursor assay kit (magnetic microparticle chemiluminescence method), Shenzhen New Industry Biomedical Engineering Co., Ltd.

[0130] Magnetic graphene oxide (MGO) was obtained by in-situ generation of magnetic nanoparticles (Fe3O4) on the surface of graphene oxide (GO) by coprecipitation method. A solution containing MGO nanomaterials was esterified and co-incubated with ProGRPAb1 capture antibody to obtain the MGO immunosubstrate loaded with capture antibody (MGO@ProGRPAb1).

[0131] The specific preparation process is: (1) 0.05 g of graphene oxide was ultrasonically dispersed in 40 mL of ultrapure water to obtain a solution containing graphene oxide; (2) Dissolve 1.67 g of ferric chloride hexahydrate (FeCl3·6H2O) and 0.59 g of ferrous chloride tetrahydrate (FeCl2·4H2O) in 50 mL of ultrapure water. After the solution is fully dissolved, add the solution to the graphene oxide solution obtained in step (1) under nitrogen protection, stir evenly by mechanical stirring, turn on the heating switch, control the temperature to 80°C, add 25% by volume of ammonia water to adjust the pH of the reaction environment to 10, and continue the coprecipitation reaction for 1 hour; after the reaction is completed, cool to room temperature (25°C), separate the magnetic graphene oxide (MGO) with a magnet, wash it with deionized water and ethanol for 5 times in sequence, and dry it in a vacuum drying oven at 60°C to obtain magnetic graphene oxide powder; (3) dispersing 5 mg of magnetic graphene oxide powder obtained in step (2) in 1 mL of PBS buffer solution by ultrasonic treatment, and adding 0.1 mg of NHS and 0.15 mg of EDC to react at 37° C. for 1 h to obtain esterified magnetic graphene oxide; (4) The esterified magnetic graphene oxide obtained in step (3) was washed three times with PBS buffer solution, collected by magnetic separation, and 1.25 mg of the esterified magnetic graphene oxide was redispersed in 1 mL of PBS buffer solution. Then, 0.1 mg of the capture antibody ProGRPAb1 was added to the above solution and reacted at 25°C for 24 hours, and then washed three times with PBS buffer solution to remove the free ProGRPAb1 not attached to the surface of the magnetic graphene, to obtain the magnetic graphene oxide immobilized capture antibody ProGRPAb1 (MGO@ProGRPAb1). The preparation principle diagram is shown in Figure 1 ; (5) Add bovine serum albumin (BSA) as a blocking solution to the solution containing magnetic graphene oxide connected with capture antibodies prepared in step (4) with a mass concentration of 1.25 mg / mL and incubate for 2 hours, wherein the mass concentration of bovine serum albumin in the solution containing magnetic graphene oxide connected with capture antibodies is 5%. Block the possible active sites to prevent nonspecific adsorption. Finally, the blocked MGO@ProGRPAb1 is separated and dispersed in 1 mL of PBS buffer solution. The mass concentration of the separated material in the PBS buffer solution is 1.25 mg / mL and stored at 4°C for further use.

[0132] (6) Disperse the commercially available isoluminol-labeled detection antibody (Ab2-ABEI) in PBS buffer. The mass concentration of the isoluminol-labeled detection antibody is 2.5 μg / mL.

[0133] (7) The sealed MGO@ProGRPAb1 solution (10 μL) and ProGRP standard solution (50 μL) in (5) were incubated at 37°C for 20 min. After magnetic separation, a solution of Ab2-ABEI (2.5 μL) with a mass concentration of 2.5 μg / mL was added and incubated at 37°C for 20 min. Magnetic separation was performed again, and the formed (MGO@ProGRPAb1)-ProGRP-(Ab2-ABEI) sandwich complex was washed 5 times with PBS buffer, and 200 μL of chemiluminescent substrate with a mass fraction of 1.5% sodium hydroxide catalyst and a volume fraction of 0.18% hydrogen peroxide solution was added (preparation of chemiluminescent substrate solution: sodium hydroxide and hydrogen peroxide solution were dispersed in deionized water, the mass fraction of sodium hydroxide was 1.5%, and the volume fraction of hydrogen peroxide solution was 0.18%). The chemiluminescent reaction was started, and the CL signal (RLU) was measured by a photomultiplier tube. The detection principle is as follows: Figure 2 shown.

[0134] Among them, the preparation method of ProGRP standard solution is to dilute the working solution with a concentration of 2298.85 pg / mL with normal saline to obtain standard solutions with concentrations of 8.98 pg / mL, 17.96 pg / mL, 35.92 pg / mL, 71.84 pg / mL, 143.68 pg / mL, 287.36 pg / mL, 574.71 pg / mL, 1149.43 pg / mL and 2298.85 pg / mL.

[0135] Establish a linear regression equation between the concentration and chemiluminescence intensity of the ProGRP standard solution (e.g. Fig. 9 As shown), the serum sample to be tested is detected by the above method, and the chemiluminescence intensity of the serum sample obtained is put into the linear regression equation to obtain the concentration of the serum sample to be tested.

[0136] Results and Analysis: exist Figure 3 It can be found that the obtained magnetic graphene oxide presents a relatively dispersed flake morphology, and the Fe3O4 nanoparticles are dispersed on the surface of graphene oxide in spherical or ellipsoidal shapes. Figure 4 The infrared spectra of graphene oxide (GO) and magnetic graphene oxide (MGO) are shown in Figure 1. The 3433 cm -1 , 1632cm -1 The peaks near 1429cm are due to the bending vibration of OH bond and C=C bond; -1 and 1086cm -1The characteristic peaks near 567cm-1 are attributed to the carboxyl CO stretching vibration and CO vibration of the epoxy group. Similar data are also found in the curve of MGO, and -1 Characteristic peaks caused by the stretching vibration of the Fe-O bond were found nearby, indicating that Fe3O4 nanoparticles were successfully grown in situ on the surface of graphene oxide. The crystal structure of MGO was determined by X-ray powder diffractometer. The results are as follows Figure 5 As shown, in Figure 5 The diffraction peaks of MGO at 35.1°, 41.5°, 50.6°, 63.2°, 67.5° and 74.2° correspond to the diffraction peaks of the (220), (311), (400), (422), (511) and (440) planes of inverse spinel Fe3O4, respectively, which further verifies that Fe3O4 nanoparticles are successfully grown in situ on the surface of graphene oxide.

[0137] The thermogravimetric curve of MGO is as follows Figure 6 As shown in the figure, the mass loss within 100℃ is mainly caused by the loss of water molecules; the mass loss at 160℃-200℃ may be caused by the thermal decomposition of oxygen-containing groups in graphene oxide and Fe3O4, generating CO, CO2 and H2O. The further weight loss in the high temperature area is due to the destruction of the GO skeleton. The total weight loss rate of MGO in the range of 25℃ to 800℃ is 52.6%, which shows that MGO was successfully synthesized and has thermal stability from the composition.

[0138] Particle size distribution and Zeta potential are important parameters for determining whether nanoformulations are stable. The average particle size of GO is about 210.7nm, and the average Zeta potential is -18.21mV. The average particle size of MGO is 281.1nm, and the Zeta potential is -8.904mV, both of which are larger than the Zeta potential and hydrodynamic diameter of GO, indicating that Fe3O4 is successfully modified onto the GO surface, and negatively charged nanomaterials are more stable in blood samples.

[0139] Figure 7 The magnetic properties of MGO in aqueous solution are as follows: MGO aqueous solution is a suspension, which is evenly dispersed. MGO can be separated from its aqueous dispersion within 20 seconds under the action of an external magnetic field, and can be collected by a handheld magnet, indicating that MGO has good magnetic properties in aqueous solution. The magnetization intensity is measured by VSM to determine the magnetic strength of MGO. The results are shown in Figure 8 As shown in the figure, the hysteresis loop of MGO is an S-shaped curve. There is no magnetization when no external magnetic field is applied, showing superparamagnetism. The saturation magnetization intensity is about 56.76emu / g, indicating that MGO has excellent magnetic responsiveness. The above results show that the construction of MGO is successful in terms of morphology, characteristic functional groups, crystal structure, composition, potential and superparamagnetism.

[0140] The CL curves of ProGRP at different standard concentrations (8.98 pg / mL-2298.85 pg / mL) were recorded using the MGO chemiluminescent immunoassay platform. Fig. 9 As shown in Figure 2, within the range of 8.98 pg / mL-2298.85 pg / mL, the chemiluminescence intensity was linearly related to the concentration of ProGRP, and the linear regression equation was Y=0.00504X+7.13055 (R 2 =0.99344, X represents the concentration of ProGRP, and Y represents the chemiluminescence intensity). In addition, 6 serum samples from lung cancer patients were analyzed to evaluate the performance of the proposed immune platform in clinical analysis. The accuracy of the method of the present invention was tested by comparing the analysis results with those of a magnetic microparticle chemiluminescence kit (Shenzhen New Industry Biomedical Engineering Co., Ltd.). The results are shown in Table 1. Fig.10 As shown. Fig.10 In ProGRP a The concentration was determined by the magnetic microparticle chemiluminescence method (Shenzhen New Industry Biomedical Engineering Co., Ltd.) in the prior art. b The concentration was determined using the MGO chemiluminescent immunoassay platform of the present invention, and the results of the two methods were fitted in Fig.10 When the two test results are consistent, Fig.10 The fitted content points will appear on the line Y=X. Fig.10 It was found that when the ProGRP content was lower than 1000 pg / mL, the test results of the present invention were consistent with the results of the prior art, which shows that the method of the present invention is accurate. The ProGRP content in serum is much lower than 1000 pg / mL. Therefore, the method of the present invention is accurate and practical in detecting the ProGRP content in serum.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. An immune platform for ProGRP detection, characterized in that: The immune platform comprises magnetic graphene oxide, wherein magnetic nanoparticles are in situ grown on the surface of graphene oxide, and capture antibodies that can specifically recognize ProGRP antigens are connected to the surface of the magnetic graphene oxide; The immune platform also includes a detection antibody that specifically recognizes the ProGRP antigen, and the detection antibody is labeled with isoluminol; The capture antibody and the detection antibody are linked via the ProGRP antigen.

2. The immune platform according to claim 1, characterized in that The magnetic nanoparticles are ferroferric oxide; The detection antibody is connected to isoluminol via an amide bond; The magnetic graphene oxide is connected to the capture antibody via an amide bond; The capture antibody and the detection antibody can recognize different antigenic epitopes on the ProGRP antigen.

3. A method for preparing an immune platform for ProGRP detection, preparing the immune platform according to any one of claims 1-2, characterized in that: The preparation method comprises: Step 1: Using a co-precipitation method, magnetic nanoparticles are in situ generated on the surface of graphene oxide to obtain magnetic graphene oxide; Step 2: esterifying the magnetic graphene oxide obtained in step 1 to obtain esterified magnetic graphene oxide, and then incubating it with a capture antibody, so that the capture antibody is connected to the surface of the magnetic graphene oxide; Step 3: Labeling the detection antibody with isoluminol; Step 4: Incubate the solution containing the product of step 2 and the serum sample solution to be tested for a second time, remove the supernatant by magnetic separation, add the solution containing the isoluminol-labeled detection antibody of step 3 to the remaining product after magnetic separation, incubate three times, remove the supernatant by magnetic separation, then add the chemiluminescent substrate solution, detect using a photomultiplier tube, and record the chemiluminescence value.

4. The preparation method according to claim 3, characterized in that: In the step 1, the coprecipitation method specifically comprises: dissolving ferric chloride hexahydrate and ferrous chloride tetrahydrate in a solution, adding a solution containing graphene oxide under a nitrogen atmosphere, heating, adjusting the pH value of the reaction environment to 10, reacting, cooling, separating the magnetic graphene oxide, washing, and drying to obtain the magnetic graphene oxide; When ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved and dispersed in the solution, the mass concentration of ferric chloride hexahydrate is 1.67 g / 50 mL; When ferric chloride hexahydrate and ferrous chloride tetrahydrate are dissolved and dispersed in the solution, the mass concentration of ferrous chloride tetrahydrate is 0.59 g / 50 mL; In the solution containing graphene oxide, the mass concentration of graphene oxide is 0.05 g / 40 mL; The volume ratio of the solution containing ferric chloride hexahydrate and ferrous chloride tetrahydrate to the solution containing graphene oxide is 5:4; The heating is increased to a temperature of 80°C; The pH value of the reaction environment was adjusted using 25% ammonia water by volume; The reaction time is 1 h.

5. The preparation method according to claim 3, characterized in that: The specific method for esterifying the magnetic graphene oxide comprises: adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a PBS buffer solution containing the magnetic graphene oxide, reacting, washing, and separating; The reaction temperature is 36°C-38°C, and the reaction time is 0.8h-1.2h; The mass concentration of the magnetic graphene oxide in the PBS buffer solution is 5 mg / mL; The mass concentration of the N-hydroxysuccinimide in the PBS buffer solution is 0.1 mg / mL; The mass concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the PBS buffer solution is 0.15 mg / mL.

6. The preparation method according to claim 3, characterized in that: The specific method of incubating the esterified magnetic graphene oxide with the capture antibody once includes: dispersing the esterified magnetic graphene oxide in a PBS buffer solution, adding the capture antibody, reacting, washing, separating, and connecting the capture antibody to the magnetic graphene oxide; The mass concentration of the esterified magnetic graphene oxide in the PBS buffer solution is 1.25 mg / mL; The mass concentration of the capture antibody in the PBS buffer solution is 0.1 mg / mL; The reaction temperature is 25°C and the reaction time is 24h.

7. The preparation method according to claim 3, characterized in that: Before the secondary incubation, bovine serum albumin is added to the solution containing the magnetic graphene oxide connected with the capture antibody, and the solution is blocked, washed, separated, and the separated substance is dispersed in a PBS buffer solution to obtain a solution containing the product of step 2 in step 4; The mass concentration of magnetic graphene oxide linked to capture antibodies in the solution containing magnetic graphene oxide linked to capture antibodies is 1.25 mg / mL; The mass concentration of the bovine serum albumin in the solution containing the magnetic graphene oxide connected with the capture antibody is 5%; The sealing treatment time is 2h; The mass concentration of the separated substance in the PBS buffer solution was 1.25 mg / mL.

8. The preparation method according to claim 3, characterized in that: In step 3, the specific method of labeling the detection antibody with isoluminol includes: labeling the detection antibody with amino-modified isoluminol.

9. The preparation method according to claim 3, characterized in that: In step 4, the ratio of the solution containing the product of step 2 to the serum sample solution to be tested is 10:50 by volume; In step 4, the secondary incubation temperature is 37° C. and the time is 20 min; In step 4, the mass concentration of the remaining product in the solution containing step 3 is 2.5 mg / mL; In step 4, the mass concentration of the isoluminol-labeled detection antibody in the solution containing step 3 is 2.5 μg / mL; In step 4, the temperature for three incubations is 37° C. and the time is 20 min; In step 4, the chemiluminescent substrate solution is prepared by dispersing sodium hydroxide and hydrogen peroxide solution in deionized water; the mass fraction of the sodium hydroxide is 1.5%, and the volume fraction of the hydrogen peroxide solution is 0.18%.

10. Application of the immune platform obtained by the preparation method according to any one of claims 3 to 9, characterized in that: The application includes using the immune platform to detect ProGRP standard solutions with different standard concentrations and a test solution containing an unknown ProGRP concentration, establishing a linear regression equation between the concentration and chemiluminescence intensity of the ProGRP standard solution, and substituting the chemiluminescence intensity obtained from the test solution into the linear regression equation to obtain the ProGRP concentration in the test solution.

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