Method for detecting protein polypeptide biomarker based on pH stimulation response material

Abstract

The invention discloses a method for detecting a protein polypeptide biomarker based on a pH stimulation response material, in the method, a pH stimulation response immunofunctionalized mesoporous material can load more small molecule compounds with strong mass spectrum signals, and the sensitivity of an analysis method is greatly improved; the pH stimuli-responsive immunofunctionalized mesoporous material can achieve the effect of signal conversion, direct analysis of biomacromolecular biomarkers by mass spectrometry is avoided, small molecular compounds with strong mass spectrometry signals are released under specific conditions, and the specificity of the detection method is improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and in particular, relates to a method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials. Background Art

[0002] Detecting the concentration level of biomarkers in the body helps to objectively evaluate the structural and functional changes of various tissues and organs in the body, and is of great significance for predicting the occurrence of diseases, monitoring disease development, and evaluating the effectiveness of drug treatment. [[1]Stepan H, Hund M, Andraczek T. Combining biomarkers to predict pregnancy complications and redefine preeclampsia: The angiogenic-placental syndrome [J]. Hypertension, 2020, 75 (4): 918-926. [2]Macdonald TM, Walker SP, Hannan NJ, et al. Clinical tools and biomarkers to predict preeclampsia [J]. EBioMedicine, 2022, 75: 103780. [3]Errani C, Traversari M, Cosentino M, et al. The prognostic value of the serum level of C-reactive protein for survival of children with ewing's sarcoma[J].Cancers,2023,15(5):1573.].Biomarkers in the body include biological macromolecules, small molecule compounds, cells and microbial populations. Among them, protein and peptide biomarkers, as the final product of the "central dogma" process, can be more accurately quantified compared to nucleic acid biomarkers that undergo transcription or translation processes. They have received extensive attention in the diagnosis of various diseases [[4] Biomarkers Definitions Working Group. Biomarkers and surrogate endpoints: Preferred definitions and conceptual framework [J]. Clinical Pharmacology Therapeutics, 2001, 69(3): 89-95. [5] Choi JH. Proteolytic biosensors with functional nanomaterials: Current approaches and future challenges [J], Biosensors, 2023, 13(2): 171.] For example, legumain is one of the cysteine ​​proteases of the C13 family. It plays an important role in various diseases such as atherosclerosis, inflammation and tumorigenesis, and is a potential biomarker for diseases such as atherosclerosis [[6] Zhao Y, Hai Z, Wang H, et al. Legumain-specific near-infrared fluorescence "turn on" for tumor-targeted imaging [J]. Analytical Chemistry, 2018, 90 (15): 8732-8735.]. Specific proteins or peptides in biological samples can be used as potential indicators for disease diagnosis or prognosis. Therefore, it is crucial to adopt reliable analytical detection methods to monitor the level changes of protein and peptide biomarkers in organisms.

[0003] Currently, the commonly used methods for detecting protein and peptide biomarkers include colorimetry, spectrophotometry, fluorescence, immunoassay and mass spectrometry [[7]Wu H, Randolph T W. Rapid quantification of protein particles in high-concentration antibody formulations [J]. Journal of Pharmaceutical Sciences, 2019, 108 (3): 1110-1116. [8]Hayrapetyan H, Tran T, Tellez-Corrales E, et al. Enzyme-linked immunosorbent assay: Types and applications [J]. Methods in Molecular Biology, 2023, 2612: 1-17. [9]Zhang JH, Shen Q, Zhou YG. Quantification of tumor protein biomarkers from lung patient serum using nanoimpact electrochemistry [J]. ACS sensors, 2021, 6 (6): 2320-2329.

[10] Calderón-Celis F, Encinar JR, Sanz-Medel A. Standardization approaches in absolute quantitative proteomics with mass spectrometry [J]. Mass Spectrometry Reviews, 2018, 37 (6): 715-737.] However, biological samples have complex components, many endogenous interfering substances, and the relative abundance of protein and peptide biomarkers is low. Traditional analytical methods are limited by insufficient sensitivity, unstable detection results, difficulty in achieving simultaneous determination of multiple components, and narrow analysis concentration range. They are not enough to meet the detection needs of protein and peptide biomarkers in vivo [

[11] Rafat N, Brewer L, Das N, et al. Inexpensive high-throughput multiplexed biomarker detection using enzymatic metallization with cellphone-based computer vision [J].ACS Sensors, 2023, 8(2): 534-542.

[12] Cioates Negut C, Stefan-Van Staden RI, Badulescu M, et al.Disposable stochastic sensors obtained by nanolayer deposition of copper, graphene, and copper-graphene composite on silk for the determination of isocitrate dehydrogenases 1and 2[J].Analytical and Bioanalytical Chemistry, 2022, 414(5): 1797-1807.]. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has an absolute advantage in the field of small molecule detection due to its high detection sensitivity. However, conventional mass spectrometry detectors have poor compatibility with protein and peptide biomacromolecules, which greatly limits their application in biomacromolecule analysis. By using functional materials to construct a new signal conversion and amplification strategy, based on the corresponding quantitative relationship between the "signal" molecule and the molecule to be tested, the detection signal intensity of the "signal" molecule of the small molecule compound is used to indirectly reflect the concentration of the analyte to be tested. This can overcome the compatibility of LC-MS / MS with biological macromolecules and help to further improve the sensitivity of the analytical method. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials, which can be used to detect the concentration level of protein and polypeptide biomarkers in the body, thereby effectively monitoring the occurrence, development and treatment efficacy of diseases, and providing guidance for the prevention and treatment of diseases.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials, comprising the following steps:

[0007] In the first step, mesoporous silica nanoparticles were prepared by a hydrothermal method, and small molecule compounds with strong mass spectrometry signals were selected as signal molecules to be loaded into the mesoporous channels of the mesoporous silica nanoparticles. A thin layer of polydopamine was formed by the self-polymerization of dopamine hydrochloride and encapsulated on the surface of the mesoporous silica nanoparticles to close the channels. Biomarker antibody 1 was further introduced to the surface of the nanoparticles to prepare a pH stimulus-responsive immune functionalized mesoporous material.

[0008] The method for preparing mesoporous silica nanoparticles by hydrothermal method comprises the following steps:

[0009] Add tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer solution with a concentration of 10-80 mmol / L (preferably 50 mmol / L) and a pH of 7-9 (preferably 8.0) to the hexadecyltrimethylammonium bromide (CTAB) powder, and ultrasonicate for 10-30 minutes (preferably 30 minutes) until it is completely dispersed; stir and reflux at a temperature of 50-70°C (preferably 60°C) for 0.5-2 hours (preferably 1 hour), and uniformly add tetraethyl orthosilicate (TEOS) dropwise, the mass volume ratio of hexadecyltrimethylammonium bromide to tetraethyl orthosilicate is 1:1-10 g / mL (preferably 1:5 g / mL), react for 7-12 hours (preferably 9 hours), and centrifuge (condition: 13000 rpm). The method comprises the steps of: ultrasonically dispersing the precipitate in an ethanol solution containing 2% concentrated hydrochloric acid, stirring and reflux for 1 to 24 hours (preferably 24 hours) at a temperature of 70 to 90° C. (preferably 80° C.), washing the precipitate repeatedly with anhydrous ethanol for at least 3 times, removing the supernatant after washing, and re-dispersing the precipitate in an ethanol solution containing 2% concentrated hydrochloric acid, refluxing at a constant temperature for 1 to 24 hours (preferably 24 hours) at a temperature of 70 to 90° C. (preferably 80° C.), centrifuging and removing the supernatant, and repeatedly washing the precipitate with anhydrous ethanol for at least 3 times. After washing, the supernatant is removed, and the precipitate is dispersed in an ethanol solution containing 2% concentrated hydrochloric acid, refluxed at a constant temperature for 1 to 24 hours (preferably 24 hours) at a temperature of 70 to 90° C. (preferably 80° C.), centrifuging and removing the supernatant, and repeatedly washing the precipitate with anhydrous ethanol for at least 3 times. The mesoporous silica nanoparticles are obtained by freeze-drying.

[0010] The small molecule compound with strong mass spectrometry signal is doxorubicin.

[0011] The method of selecting a small molecule compound with a strong mass spectrometry signal as a signal molecule to be loaded into the mesoporous channels of mesoporous silica nanoparticles comprises the following steps:

[0012] A solution of a small molecule compound with a strong mass spectrometry signal at a concentration of 1 to 20 mg / mL (preferably 3 mg / mL) is added to the mesoporous silica powder, the mass ratio of the small molecule compound with a strong mass spectrometry signal to the mesoporous silica is 1 to 20:1 (preferably 1.5:1), ultrasonicated until completely dispersed, stirred at room temperature in the dark for 1 to 24 hours (preferably 24 hours), centrifuged (condition: 13000 rpm for 4 minutes), repeatedly washed with ultrapure water, and freeze-dried to obtain the mesoporous silica loaded with the small molecule compound with a strong mass spectrometry signal.

[0013] The method for preparing a small molecule compound solution with a strong mass spectrometry signal comprises the following steps: dissolving the small molecule compound with a strong mass spectrometry signal in a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution with a concentration of 20 mmol / L and a pH of 7.25 to obtain a small molecule compound solution with a strong mass spectrometry signal with a concentration of 1 to 20 mg / mL (preferably 3 mg / mL).

[0014] The method of utilizing the self-polymerization of dopamine hydrochloride to form a thin layer of polydopamine encapsulated on the surface of mesoporous silica nanoparticles to close the pores comprises the following steps:

[0015] The mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal is added to a tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer solution with a concentration of 5-20 mmol / L (preferably 10 mmol / L) and a pH of 7-9 (preferably 8.5), and ultrasonically dispersed for 1-5 minutes (preferably 3 minutes). The concentration of the mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal in the final system is 0.5-2 mg / mL (preferably 1 mg / mL). Dopamine hydrochloride powder is added, and the mass ratio of the mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal to dopamine hydrochloride is 1-2:1 (preferably 2:1). The reaction is stirred at room temperature in the dark for 3-24 hours (preferably 3 hours). After the reaction is completed, centrifugation (condition: 13000 rpm centrifugation for 4 minutes) is performed to remove the supernatant, and the precipitate is washed with ultrapure water at least three times, and freeze-dried to obtain polydopamine encapsulated mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal.

[0016] The method of further introducing the biomarker antibody 1 onto the surface of the nanoparticles to prepare the pH stimulus responsive immune functionalized mesoporous material comprises the following steps:

[0017] The polydopamine-encapsulated mesoporous silica lyophilized powder loaded with a small molecule compound with a strong mass spectrometry signal is added to PBS (10 mM, pH 7.4) and ultrasonically dispersed, and a biomarker antibody 1 solution with a concentration of 50 to 200 μg / mL (preferably 200 μg / mL) is added, and the mass ratio of the biomarker antibody 1 to the polydopamine-encapsulated mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal is 1:1 to 200 (preferably 1:50). After repeated blowing and mixing, the mixture is stirred at room temperature for reaction for 1 to 12 hours (preferably 6 hours). After the reaction is completed, 1% (v / v) glycerol is added and blown and mixed, and PBS (10 mM, pH 7.4) containing 4 wt% bovine serum albumin (BSA) is continued to be added for reaction for 1 to 3 hours (preferably 2 hours) to obtain the pH stimulus-responsive immune functionalized mesoporous material dispersion system with a concentration of 0.5 to 2 mg / mL (preferably 1 mg / mL).

[0018] The biomarker antibody 1 is selected from human legumain polyclonal antibody (PCAb).

[0019] The method for preparing the biomarker antibody 1 solution comprises the following steps:

[0020] A 0.2 mg / mL PBS solution (10 mM, pH 7.4) of human legumain polyclonal antibody was allowed to cool to room temperature and mixed by repeatedly pipetting.

[0021] In the second step, the standard sample is mixed with the immunomagnetic beads and the pH stimulus responsive immune functionalized mesoporous material, and then magnetically separated to obtain a complex of the immunomagnetic beads-standard sample-pH stimulus responsive immune functionalized mesoporous material; the complex is placed in an acidic buffer solution containing an internal standard for ultrasonic treatment, and the sample supernatant is detected by LC-MS / MS. The sample chromatogram is collected to record the chromatographic peak areas of the small molecule compounds with strong mass spectrometry signals and the internal standard, and the concentration of the biomarker in the standard sample is used as the horizontal coordinate, and the ratio of the peak area of ​​the small molecule compound with strong mass spectrometry signal to the internal standard f is used as the vertical coordinate to establish a standard regression curve of the biomarker concentration-f;

[0022] The preparation method of the standard sample comprises the following steps:

[0023] Mix PBS with a concentration of 5 to 20 mmol / L (preferably 10 mmol / L) and a pH of 7 to 9 (preferably 7.4) with the recombinant human legamin monoclonal antibody lyophilized powder, gently blow until fully dissolved and mixed to obtain a standard solution, divide the solution into portions, add PBS with a concentration of 5 to 20 mmol / L (preferably 10 mmol / L) and a pH of 7 to 9 (preferably 7.4) to dilute step by step to obtain a standard sample.

[0024] The preparation method of the composite of immunomagnetic beads-standard sample-pH stimulation responsive immune functionalized mesoporous material comprises the following steps:

[0025] The immunomagnetic beads are dissolved in PBS containing 2wt% BSA and diluted at least 10 times, the immunomagnetic beads are mixed with the standard sample, and the temperature is 25-40°C (preferably 37°C) and constant temperature shaking incubation for 1-3h (preferably 2h), and the supernatant is removed by magnetic separation, and PBS containing 2wt% BSA is added to wash repeatedly for 3 times, and the supernatant is removed by magnetic separation, and then dispersed in PBS containing 2wt% BSA, and the pH stimulus response immune function prepared in the first step is added at a concentration of 1-5mg / mL (preferably 2mg / mL). The mesoporous material suspension is prepared, the mass ratio of the immunomagnetic beads to the pH stimulus responsive immune functionalized mesoporous material prepared in the first step is 1 to 10:1 (preferably 5:1), and the mixture is repeatedly blown and beaten to mix well. The reaction is carried out at a constant temperature of 25 to 40°C (preferably 37°C) and rotated for 1 to 3 hours (preferably 2 hours). The supernatant is removed by magnetic separation, and PBS (10 mM, pH 7.40) is added and repeatedly washed for at least 3 times. The supernatant is discarded by applying an external magnetic field to obtain a complex of immunomagnetic beads-standard sample-pH stimulus responsive immune functionalized mesoporous material.

[0026] In the third step, the test sample containing the biomarker is mixed with the immunomagnetic beads and the pH stimulus responsive immunofunctionalized mesoporous material, and then magnetically separated to obtain a complex of immunomagnetic beads-biomarker-pH stimulus responsive immunofunctionalized mesoporous material; the complex is ultrasonically treated in an acidic buffer solution containing an internal standard, and the sample supernatant is detected by LC-MS / MS. The ratio of the small molecule compound with a strong mass spectrometry signal obtained by the detection to the internal standard chromatographic peak area is substituted into the standard regression curve to calculate the concentration of the biomarker in the test sample.

[0027] The preparation method of the immunomagnetic beads comprises the following steps:

[0028] 0.5-2 mmol / L (preferably 1 mmol / L) HCl (4°C) was added to a 1-20 mg / mL (preferably 10 mg / mL) N-hydroxysulfosuccinimidyl magnetic beads (NHS-MBs) suspension for washing, magnetic separation was performed and the supernatant was discarded, and a 200-500 μg / mL (preferably 308 μg / mL) biomarker antibody 2 solution was added, the mass ratio of N-hydroxysulfosuccinimidyl magnetic beads (NHS-MBs) to biomarker antibody 2 was 1-100:1 (preferably 25:1), and the mixture was gently stirred. Vortex, incubate at room temperature in the dark for 1 to 3 h (preferably 2 h), perform magnetic separation and discard the supernatant, add an ethanolamine solution with a concentration of 1 to 5 mol / L (preferably 3 mol / L) to wash repeatedly for at least 3 times and discard the supernatant, disperse in an ethanolamine solution with a concentration of 1 to 5 mol / L (preferably 3 mol / L), oscillate at room temperature for 1 to 3 h (preferably 2 h) and discard the supernatant, wash again with PBS containing 2 wt% BSA at least 3 times and oscillate for 1 to 6 h (preferably 4 h), discard the supernatant, and disperse in PBS containing 2 wt% BSA to obtain an immunomagnetic bead dispersion system.

[0029] The biomarker antibody 2 is selected from recombinant human legamin monoclonal antibody.

[0030] The method for preparing the biomarker antibody 2 solution comprises the following steps:

[0031] A 4-morpholineethanesulfonic acid (MES) buffer solution with a concentration of 50 to 150 mmol / L (preferably 100 mmol / L) and a pH of 4 to 5 (preferably 4.8) is added to the biomarker antibody 2 to obtain the biomarker antibody 2 solution.

[0032] The method for preparing the composite of immunomagnetic beads-biomarker-pH stimulation responsive immune functionalized mesoporous material comprises the following steps:

[0033] The immunomagnetic beads are dissolved in PBS containing 2wt% BSA and diluted at least 10 times, the immunomagnetic beads are mixed with the sample to be tested containing the biomarker, and the temperature is 25-40°C (preferably 37°C) and constant temperature shaking incubation for 1-3h (preferably 2h), and the supernatant is removed by magnetic separation, and PBS containing 2wt% BSA is added to wash repeatedly for 3 times, and the supernatant is removed by magnetic separation, and then dispersed in PBS containing 2wt% BSA, and the pH stimulus response immune prepared in the first step is added at a concentration of 1-5mg / mL (preferably 2mg / mL). The immunofunctionalized mesoporous material suspension is prepared in a mass ratio of 1 to 10:1 (preferably 5:1) between the immunomagnetic beads and the pH stimulus responsive immunofunctionalized mesoporous material prepared in the first step. The mixture is repeatedly blown and beaten to mix well. The reaction is carried out at a constant temperature of 25 to 40°C (preferably 37°C) and rotated for 1 to 3 hours (preferably 2 hours). The supernatant is removed by magnetic separation, and PBS (10 mM, pH 7.40) is added and repeatedly washed for at least 3 times. The supernatant is discarded by applying an external magnetic field to obtain a complex of immunomagnetic beads-biomarkers-pH stimulus responsive immunofunctionalized mesoporous materials.

[0034] The solvent of the pH stimulus-responsive immune-functionalized mesoporous material suspension prepared in the first step is 10 mM PBS containing 2 wt % BSA, and the pH value is 7.4.

[0035] The method of placing the sample in an acidic buffer solution containing an internal standard for ultrasonic treatment and detecting the sample supernatant by LC-MS / MS comprises the following steps:

[0036] Add disodium hydrogen phosphate-citrate buffer solution with a pH of 2 to 7.5 (preferably 2.35) and an internal standard solution to the prepared immunomagnetic beads-biomarker-pH stimulus-responsive immune functionalized mesoporous material complex, vortex for 0.5 to 2 min (preferably 1 min), ultrasonicate for 5 min to 2 h (preferably 1 h) at a temperature of 25 to 40 ° C (preferably 37 ° C), centrifuge (13000 rpm, 5 min), take the supernatant and dilute it with the mobile phase (0.05% formic acid water: methanol = 40:60, v / v), vortex and mix to obtain the supernatant.

[0037] The internal standard solution is an aqueous solution of daunorubicin with a concentration of 1 μg / mL.

[0038] The LC-MS / MS conditions were as follows: ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm), mobile phase: 0.05% formic acid water (A)-methanol (B) at a ratio of 40:60, v / v, isocratic elution, flow rate of 0.3 mL / min, column temperature of 35° C., injection volume of 1 μL, analysis time of 5 min; Shimadzu 8045 triple quadrupole mass spectrometer, ion source of ESI, scanning mode of multiple reaction monitoring (MRM), and detection in positive ion mode.

[0039] Other mass spectrometry parameters were as follows: nebulizing gas flow rate was 3.0 L / min, drying gas flow rate was 10.0 L / min, heating gas flow rate was 10.0 L / min, interface temperature was 300 °C, DL tube temperature was 200 °C, heating block temperature was 400 °C, and collision gas was argon.

[0040] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0041] The present invention provides a method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials, in which the pH stimulus responsive immune functionalized mesoporous material can load a larger number of small molecule compounds with strong mass spectrometry signals, thereby greatly improving the sensitivity of the analysis method; the pH stimulus responsive immune functionalized mesoporous material can achieve the effect of signal conversion, avoiding direct mass spectrometry analysis of biological macromolecule biomarkers, and at the same time release small molecule compounds with strong mass spectrometry signals under specific conditions, thereby improving the specificity of the detection method.

[0042] The method of the present invention targets different biomarkers to be tested, loads different small molecule compounds with strong mass spectrometry signals into pH-stimulated responsive immune functionalized mesoporous materials, and introduces corresponding specific recognition antibodies on the surface of the material to achieve analysis and detection thereof, and has strong universality.

[0043] In summary, the method of the present invention has high sensitivity, good specificity and is easy to operate. By means of magnetic separation, the protein and polypeptide biomarkers to be tested in the sample are quickly and exclusively separated and enriched, and the pH stimulus-responsive immunofunctionalized mesoporous material constructed by the signal conversion and amplification strategy is further used to achieve the exclusive release of small molecule compounds with strong mass spectrometry signals. The small molecule compounds with strong mass spectrometry signals specifically released are detected based on the LC-MS technology, achieving the purpose of signal amplification, significantly improving the sensitivity of detecting biomarkers, and effectively solving the research bottleneck of in vivo analysis of protease biomarkers.

[0044] The method of the present invention is based on immunomagnetic beads, specifically separating and enriching protein and polypeptide biomarkers to be tested in biological samples, and based on the preparation of pH stimulus responsive immune functionalized mesoporous materials by self-polymerization reaction, further specifically identifying and binding to the biomarkers to be tested, and releasing the small molecule compounds with strong mass spectrometry signals loaded thereon under specific pH conditions, thereby achieving highly sensitive detection of protein and polypeptide biomarkers in biological samples by LC-MS / MS, and improving the sensitivity of detection of analytical methods in multiple dimensions. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of SEM and HRTEM characterization results of mesoporous silica.

[0046] Figure 2Schematic diagram of HRTEM and DLS characterization results of pH stimulus responsive immune functionalized mesoporous materials.

[0047] Figure 3 Schematic diagram of the effects of stirring reaction time at room temperature in the dark and doxorubicin solution concentration on the results when preparing doxorubicin-loaded mesoporous silica (DOX-MSNs).

[0048] Figure 4 Schematic diagram of the optimization results of material ratio, reaction time, pH and ultrasonic time conditions for the specific release of signal molecules when preparing polydopamine-encapsulated mesoporous silica loaded with doxorubicin (DOX-MSNs / PDA).

[0049] Figure 5 Schematic diagram of the basic process for the preparation of pH stimulation-responsive immune-functionalized mesoporous materials and the specific release of doxorubicin.

[0050] Figure 6 Schematic diagram of the chromatographic results of blank samples, blank samples plus doxorubicin standard solution, and standard samples containing biomarkers.

[0051] Figure 7 This is a schematic diagram of the results of detecting samples containing caspas-3, matrix metalloproteinases-9 (MMP-9) and Fibroblast activation protein (FAP) and other protein and polypeptide biomarkers, as well as samples containing legumin (LGMN).

[0052] Figure 8 Schematic diagram of the changes in mass spectrometry detection signals for standard samples containing different concentrations of legume protein. DETAILED DESCRIPTION

[0053] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0054] Example 1

[0055] The first step is the preparation of pH stimuli-responsive immune functionalized mesoporous materials

[0056] Place 1.8g of hexadecyltrimethylammonium bromide (CTAB) powder in a 500mL round-bottom flask, add 300mL of tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer solution (concentration of 50mmol / L, pH 8.0), and ultrasonicate for 30 minutes until completely dispersed. Place the round-bottom flask in a 60℃ oil bath and stir under reflux for 1 hour. 9 mL of tetraethyl orthosilicate (TEOS) was added dropwise at a uniform rate, and the reaction was carried out for 9 hours. The obtained milky white product was centrifuged at 13000 rpm for 4 min to obtain a white precipitate. After ultrasonic dispersion with anhydrous ethanol, the supernatant was removed by centrifugation. The operation was repeated 3 times. Ultrasonic dispersion with ultrapure water was centrifuged to remove the supernatant. The operation was repeated 3 times. The precipitate was ultrasonically dispersed in 600 mL of an ethanol solution containing 2% concentrated hydrochloric acid, stirred and refluxed at a temperature of 80°C for 24 hours, and the supernatant was removed by centrifugation. The precipitate was repeatedly washed with anhydrous ethanol 3 times, and the supernatant was removed after washing. The precipitate was dispersed in an ethanol solution containing 2% concentrated hydrochloric acid and refluxed at 80°C for 24 hours. The supernatant was removed by centrifugation, and the precipitate was repeatedly washed with anhydrous ethanol at least 3 times. The precipitate was freeze-dried to obtain 1.486 g of mesoporous silica nanoparticles.

[0057] The prepared mesoporous silica was characterized by surface scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM), and the morphology and distribution of the mesoporous silica used in this method were evaluated.

[0058] The results are as follows Figure 1 As shown, Figure 1 The figure shows the SEM and HRTEM characterization results of mesoporous silica, where A is the SEM characterization result of mesoporous silica; B is the HRTEM characterization result of mesoporous silica. The characterization results show that the MSNs nanoparticles are evenly distributed and the particle size is normally distributed, mainly concentrated between 140 and 200 nm, with an average particle size of about 175 nm.

[0059] A doxorubicin solution with a concentration of 3 mg / mL was prepared by dissolving it in 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution (concentration: 20 mmol / L, pH: 7.25).

[0060] 5 mL of 3 mg / mL doxorubicin solution was added to 10 mg of mesoporous silica powder, and the mixture was completely dispersed by ultrasound. The mixture was stirred at room temperature and protected from light for 24 hours. The mixture was centrifuged at 13000 rpm for 4 minutes. The initial concentration of doxorubicin solution (C0) and the concentration of doxorubicin in the supernatant after 24 hours of reaction (C t ), according to (C0-C t )×total volume of reaction system / mass of added mesoporous silica, the amount of doxorubicin loaded per unit mass of material can be calculated. According to (C0-C t) / C0, the percentage of doxorubicin loaded on mesoporous silica in the total input can be calculated, thereby optimizing the loading conditions. The precipitate obtained by centrifugation was repeatedly washed with ultrapure water for 3 times and freeze-dried to obtain 9.78 mg of mesoporous silica loaded with doxorubicin (DOX-MSNs).

[0061] 10 mg of DOX-MSNs solid was added to 10 mL of Tris-HCl buffer solution (concentration of 10 mmol / L, pH 8.5), ultrasonically dispersed for 3 minutes, and the concentration of DOX-MSNs in the final system was 1 mg / mL. 5 mg of dopamine hydrochloride powder was added and stirred at room temperature in the dark for 3 hours. After the reaction was completed, centrifuged at 13000 rpm for 4 minutes, the supernatant was removed, the precipitate was washed three times with ultrapure water, and lyophilized to obtain 10.63 mg of polydopamine encapsulated mesoporous silica loaded with doxorubicin (DOX-MSNs / PDA).

[0062] The purchased 0.2 mg / mL human legumain polyclonal antibody (PCAb) in PBS solution (10 mM, pH 7.4) was placed at room temperature and mixed by repeatedly pipetting.

[0063] 1 mg of DOX-MSNs / PDA lyophilized powder was added to 400 μL PBS (10 mM, pH 7.4) and ultrasonically dispersed, and 100 μL of 200 μg / mL PCAb solution was added. After repeated blowing and mixing, it was placed on a rotary mixer for reaction at room temperature for 6 h. After the reaction, 10 μL of 1% (v / v) glycerol was added, and the mixture was mixed by blowing and mixing. 500 μL of PBS (10 mM, pH 7.40) containing 4 wt% bovine serum albumin (BSA) was added for reaction for 2 h (blocking the nonspecific adsorption sites on DOX-MSNs / PDA), and a pH stimulus-responsive immune functionalized mesoporous material (DOX-MSNs / PDA@PcAb) dispersion system with a concentration of 1 mg / mL was obtained, which was stored at 4 °C for future use.

[0064] The prepared pH stimulus responsive immune functionalized mesoporous material (DOX-MSNs / PDA@PcAb) was characterized by HRTEM and Dynamic Light Scattering (DLS), and its morphology and distribution were evaluated.

[0065] The results are as follows Figure 2 As shown, Figure 2The HRTEM characterization and DLS characterization results of the pH stimulus responsive immune functionalized mesoporous material are shown in Figure 1. A is a schematic diagram of the HRTEM characterization results of the pH stimulus responsive immune functionalized mesoporous material, and B is a schematic diagram of the DLS characterization results of the pH stimulus responsive immune functionalized mesoporous material. The results show that the pH stimulus responsive immune functionalized mesoporous material prepared by the present invention has a uniform particle size, and the average particle size is about 185 nm.

[0066] Figure 3 This is a schematic diagram of the effects of the room temperature light-shielded stirring reaction time and the doxorubicin solution concentration on the results when preparing doxorubicin-loaded mesoporous silica (DOX-MSNs), wherein A is a schematic diagram of the effects of the room temperature light-shielded stirring reaction time on the amount of doxorubicin loaded per unit mass of material DLC (%) when preparing doxorubicin-loaded mesoporous silica (DOX-MSNs). The DLC (%) is calculated according to (initial doxorubicin concentration of the reaction - doxorubicin concentration at the end of the reaction) × total volume of the reaction system / mass of mesoporous silica added. The larger the DLC (%), the more doxorubicin is loaded per unit mass of the mesoporous silica, which is more conducive to the subsequent mass spectrometry signal amplification. B is a schematic diagram of the effect of the concentration of doxorubicin solution on the percentage of doxorubicin loaded in the total input DLE (%) when preparing doxorubicin-loaded mesoporous silica (DOX-MSNs). The DLE (%) is calculated based on (initial doxorubicin concentration of the reaction - doxorubicin concentration at the end of the reaction) / initial doxorubicin concentration of the reaction. The larger the DLE (%), the higher the proportion of doxorubicin loaded into the mesoporous silica.

[0067] As can be seen from the figure, with the extension of time, the amount of doxorubicin loaded on unit mass of mesoporous silica shows an increasing trend, and 24 hours is the optimal reaction time; and when the concentration of doxorubicin solution is less than 3 mg / mL, with the increase of the concentration of doxorubicin solution, the amount of doxorubicin loaded on unit mass of mesoporous silica shows an increasing trend. When the concentration of doxorubicin solution is greater than 3 mg / mL, the amount of doxorubicin loaded on unit mass of mesoporous silica remains the same or even decreases. It can be seen that the DOX concentration of 3 mg / mL is the optimal loading concentration.

[0068] Figure 4Schematic diagram of the optimization results of material ratio, reaction time, pH and ultrasonic time conditions for specific release of signal molecules when preparing polydopamine encapsulated mesoporous silica loaded with doxorubicin (DOX-MSNs / PDA). A is a schematic diagram of the specific release results of doxorubicin when DOX-MSNs / PDA is prepared when the mass concentration ratio of DOX-MSNs to dopamine hydrochloride is 2:1, 1:1, and 1:2 at pH 2.35 (disodium hydrogen phosphate-citric acid buffer, 20mM); B is a schematic diagram of the specific release results of doxorubicin when DOX-MSNs / PDA is prepared when the mass concentration ratio of DOX-MSNs to dopamine hydrochloride is 2:1, 1:1, and 1:2 at pH 5.02 (disodium hydrogen phosphate-citric acid buffer, 20mM); C is a schematic diagram of the specific release results of doxorubicin when DOX-MSNs / PDA is prepared when the mass concentration ratio of DOX-MSNs to dopamine hydrochloride is 2:1, 1:1, and 1:2 at pH 7.43 (disodium hydrogen phosphate-citric acid buffer, 20mM) A is a schematic diagram of the specific release results of doxorubicin when DOX-MSNs / PDA was prepared when the mass concentration ratio of DOX-MSNs to dopamine hydrochloride was 2:1, 1:1, and 1:2, respectively; D is a schematic diagram of the specific release results of doxorubicin when DOX-MSNs / PDA was prepared when the mass concentration ratio of DOX-MSNs to dopamine hydrochloride was 2:1 under different ultrasonic times under the condition of pH 2.35 (disodium hydrogen phosphate-citrate buffer, 20mM); E is a schematic diagram of the specific release results of doxorubicin from DOX-MSNs and DOX-MSNs / PDA at pH 2.35, 5.02, and 7.43, respectively.

[0069] It can be seen from AC that as the mass concentration ratio of DOX-MSNs to dopamine hydrochloride decreases, the release rate of doxorubicin from the pH-stimulated responsive immune-functionalized mesoporous material gradually decreases. When the preparation condition is that the mass concentration ratio of DOX-MSNs to dopamine hydrochloride is 2:1, the pH-stimulated responsive immune-functionalized mesoporous material exhibits better pH responsiveness. As the pH of the dispersion system decreases, the release rate of doxorubicin from the pH-stimulated responsive immune-functionalized mesoporous material gradually increases. When the pH of the dispersion system is 2.35, the pH-stimulated responsive immune-functionalized mesoporous material exhibits better pH responsiveness. In addition, the longer the ultrasonic time, the higher the doxorubicin release rate of the pH-stimulated responsive immune-functionalized mesoporous material. In order to improve the efficiency of the analytical method, the reaction was finally selected to be stirred at room temperature in the dark for 3h. In summary, the optimization results show that when the preparation conditions are that the mass concentration ratio of DOX-MSNs to dopamine hydrochloride is 2:1, the reaction is stirred at room temperature in the dark for 3h, and the pH is 2.35. Sodium hydrogen phosphate-citric acid buffer solution exhibits good pH responsiveness. Figure 5 Schematic diagram of the basic process of preparation of pH stimulus-responsive immune functionalized mesoporous materials and their specific release of doxorubicin.

[0070] In the second step, the biomarkers in the samples were specifically pretreated based on the pH stimulation-responsive immune functionalized mesoporous materials (DOX-MSNs / PDA@PcAb).

[0071] Take 100 μL of 10 mg / mL N-hydroxysulfosuccinimidyl magnetic beads (NHS-MBs) suspension (the solvent is N,N-dimethylacetamide), add 200 μL of 1 mmol / L HCl (4°C) to wash, magnetically separate and discard the supernatant, add 130 μL of biomarker antibody 2 recombinant human legamin monoclonal antibody solution (100 mmol / L 4-morpholineethanesulfonic acid (MES) buffer solution, containing 40 μg of biomarker antibody 2 recombinant human legamin monoclonal antibody, pH 4.8), gently vortex for 15 s, incubate at room temperature in the dark for 2 h, magnetically separate and discard the supernatant, add 200 μL of 3 mol / L ethanolamine solution to wash repeatedly 4 times and discard the supernatant, then disperse in 100 μL of 3 mol / L ethanolamine solution, shake at room temperature for 2 h and discard the supernatant. Then, the beads were washed again 4 times with 0.5 mL of PBS containing 2 wt % BSA and shaken for 4 h, the supernatant was discarded, and the beads were dispersed in 100 μL of PBS containing 2 wt % BSA to obtain an immunomagnetic bead dispersion system, which was stored at 4° C. for future use.

[0072] Preparation method of biomarker standard solution: Mix 200 μL PBS (10 mM, pH 7.4) with 20 ng of recombinant human legamin monoclonal antibody lyophilized powder, gently pipette until fully dissolved and mixed to obtain a 100 ng / mL standard solution, dispense into 100 μL centrifuge tubes, and store at -80°C for later use. Take out a dispensed recombinant human legamin monoclonal antibody (100 ng / mL), add PBS (10 mM, pH 7.4) and dilute stepwise to obtain a series of standard solutions of 50 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, and 0.2 ng / mL.

[0073] Preparation method of standard samples containing biomarkers and test samples: accurately pipette 10 μL of biomarker standard solution into 190 μL blank serum and mix well to obtain standard samples with concentrations of 2.5 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, 0.1 ng / mL, and 0.01 ng / mL, respectively. Take fresh human blood sample, let it stand for 30 minutes, centrifuge (1000×g, 10 minutes) and take out the supernatant to obtain the test sample.

[0074] Take 10 μL of the prepared immunomagnetic bead dispersion system (10 mg / mL), add 90 μL of PBS containing 2 wt% BSA, and dilute 10 times by blowing and mixing with a pipette, take 5 μL of the diluted immunomagnetic bead dispersion system (1 mg / mL) and mix with 150 μL of the test sample or standard sample containing the biomarker, incubate at 37°C for 2 hours, remove the supernatant by magnetic separation, add 800 μL of PBS containing 2 wt% BSA and wash repeatedly for 3 times, remove the supernatant by magnetic separation, and disperse in 140 μL of PBS containing 2 wt% BSA. Add 10 μL of the pH stimulus-responsive immune functionalized mesoporous material (DOX-MSNs / PDA@PcAb) suspension prepared in the first step with a concentration of 2 mg / mL (the solvent is 10 mM PBS containing 2 wt% BSA, pH 7.4), blow and mix repeatedly, and react at 37°C for 2 hours. The supernatant was removed by magnetic separation, and 800 μL PBS (10 mM, pH 7.4) was added for repeated washing 4 times. The supernatant was discarded by applying an external magnetic field to obtain a complex of immunomagnetic beads-biomarkers-pH stimulation-responsive immune functionalized mesoporous materials.

[0075] The third step is to quantitatively analyze the concentration level of biomarkers in the sample by LC-MS / MS.

[0076] 95 μL of disodium hydrogen phosphate-citrate buffer (pH 2.35) and 5 μL of internal standard solution (aqueous solution of 1 μg / mL daunorubicin) were added to the complex of immunomagnetic beads-biomarkers-pH stimulus-responsive immune functionalized mesoporous materials prepared in the second step, vortexed for 1 min, ultrasonicated at 37°C for 1 h, centrifuged (13000 rpm, 5 min), and the supernatant was diluted 5 times with the mobile phase (0.05% formic acid water: methanol = 40:60, v / v), vortexed to mix, and transferred to an injection vial. LC-MS / MS method was used for detection and analysis, and chromatograms were collected to record the chromatographic peak areas of doxorubicin and internal standard daunorubicin, respectively.

[0077] LC-MS / MS conditions: ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm), mobile phase: 0.05% formic acid water (A)-methanol (B) (40:60, v / v), isocratic elution, flow rate of 0.3 mL / min, column temperature of 35°C, injection volume of 1 μL, analysis time of 5 min. Shimadzu 8045 triple quadrupole mass spectrometer, ion source of ESI, scanning mode of multiple reaction monitoring (MRM), detection in positive ion mode, m / z of doxorubicin parent ion and daughter ion are 544.10 and 397.05, respectively, collision energy is -13 eV, m / z of internal standard parent ion and daughter ion are 528.15 and 321.05, respectively, CE is -22 eV. Other mass spectrometry parameters are shown in Table 1:

[0078] Table 1

[0079] Parameter (unit) Numeric Atomizing gas flow rate (L / min) 3.0 Drying gas flow (L / min) 10.0 Heating gas flow (L / min) 10.0 Interface temperature (℃) 300 DL tube temperature (℃) 200 Heating block temperature (℃) 400 Collision gas Argon

[0080] The amount of doxorubicin in the injected supernatant is proportional to the concentration of the biomarker. The biomarker concentration of the standard sample is used as the horizontal coordinate, and the peak area ratio of doxorubicin to the internal standard (abbreviated as f) of the standard sample is used as the vertical coordinate to establish a standard regression curve of biomarker concentration-f. The peak area ratio of doxorubicin to the internal standard of the sample to be tested is substituted into the standard curve to calculate the concentration of the biomarker in the sample to be tested.

[0081] Figure 6 It is a schematic diagram of the chromatographic results of a blank sample, a blank plus doxorubicin standard solution sample, and a standard sample containing a biomarker. Among them, A is a schematic diagram of the chromatographic results of a blank sample, B is a schematic diagram of the chromatographic results of a blank plus doxorubicin standard solution sample, and C is a schematic diagram of the chromatographic results of a standard sample containing a biomarker. It can be seen from the figure that the chromatographic peak shape of the standard sample containing a biomarker containing 1ng / mL is good, and there are no other interfering impurity peaks, indicating that the analytical method of the present invention has good specificity and sensitivity that can meet the detection requirements of the biomarker legumain.

[0082] Figure 7 The results of the detection of samples containing caspas-3, matrix metalloproteinases-9 (MMP-9) and Fibroblast activation protein (FAP) and samples containing legumain (LGMN) are shown to investigate the specificity of the method. In the figure, BLANK represents a blank serum sample; CASP-3 represents a blank serum sample containing 100ng / mL caspas-3; MMP-9 represents a blank serum sample containing 100ng / mL MMP-9; FAP represents a blank serum sample containing 100ng / mL FAP; LGMN represents a serum sample containing 0.01ng / mL legumain (LGMN). As can be seen from the figure, there is no significant difference in the detection response between the samples containing other protein polypeptide biomarkers with high concentrations (100ng / mL) and the blank samples, while the samples containing 0.01ng / mL legumain respond sensitively, indicating that the analytical method of the present invention has a high degree of specificity.

[0083] Figure 8 Schematic diagram of the change in mass spectrometry signal of standard samples containing different concentrations of legume protein. A is the peak area ratio of doxorubicin to internal standard (A) in the concentration range of 0.001 to 5 ng / mL. DOX / ADAU ) is a schematic diagram of the trend of the change of the concentration of legume protein in the sample; B is the peak area ratio of doxorubicin to internal standard (A) in the concentration range of 0.01 to 2.5 ng / mL. DOX / A DAU ) and the legumain concentration in the sample. As can be seen from the figure, within the concentration range of 0.01 to 2.5 ng / mL, the mass spectrometry detection signal intensity of the sample and the legumain concentration in the sample have a good linear relationship, which can effectively meet the accurate quantitative analysis of samples at different concentration levels.

[0084] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials, characterized in that: The following steps are involved: In the first step, mesoporous silica nanoparticles were prepared by a hydrothermal method, and small molecule compounds with strong mass spectrometry signals were selected as signal molecules to be loaded into the mesoporous channels of the mesoporous silica nanoparticles. A thin layer of polydopamine was formed by the self-polymerization of dopamine hydrochloride and encapsulated on the surface of the mesoporous silica nanoparticles to close the channels. Biomarker antibody 1 was further introduced to the surface of the nanoparticles to prepare a pH stimulus-responsive immune functionalized mesoporous material. The small molecule compound with strong mass spectrometry signal is doxorubicin; The biomarker antibody 1 is selected from human legumain polyclonal antibody; The second step is to mix and incubate the standard sample with the immunomagnetic beads and the pH stimulus responsive immune functionalized mesoporous material, and then perform magnetic separation to obtain a complex of the immunomagnetic beads-standard sample-pH stimulus responsive immune functionalized mesoporous material; The sample was placed in an acidic buffer solution containing an internal standard for ultrasonic treatment, and the sample supernatant was detected by LC-MS / MS. The sample chromatogram was collected to record the chromatographic peak areas of the small molecule compound with strong mass spectrometry signals and the internal standard, and the concentration of the biomarker in the standard sample was used as the horizontal coordinate, and the ratio of the peak area of ​​the small molecule compound with strong mass spectrometry signals to the internal standard f was used as the vertical coordinate to establish a standard regression curve of the biomarker concentration-f; The third step is to mix and incubate the sample containing the biomarker with the immunomagnetic beads and the pH stimulus responsive immune functionalized mesoporous material, and then perform magnetic separation to obtain a complex of immunomagnetic beads-biomarker-pH stimulus responsive immune functionalized mesoporous material; The sample was ultrasonically treated in an acidic buffer solution containing an internal standard, and the sample supernatant was detected by LC-MS / MS. The ratio of the small molecule compound with a strong mass spectrometry signal to the internal standard chromatographic peak area was substituted into the standard regression curve to calculate the concentration of the biomarker in the sample to be tested.

2. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The method for preparing mesoporous silica nanoparticles by hydrothermal method comprises the following steps: The hexadecyltrimethylammonium bromide powder was added with a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a concentration of 10-80mmol / L and a pH of 7-9, and ultrasonicated for 10-30 minutes until it was completely dispersed; the mixture was stirred and refluxed at a temperature of 50-70°C for 0.5-2 hours, tetraethyl orthosilicate was added dropwise at a uniform rate, and the mass volume ratio of hexadecyltrimethylammonium bromide to tetraethyl orthosilicate was 1:1-10g / mL, and the mixture was reacted for 7-12 hours, and centrifuged. After ultrasonic dispersion with anhydrous ethanol, the supernatant was removed by centrifugation, and the operation was repeated 3 times, and the mixture was ultrapure water was ultrapurified. The supernatant is removed by acoustic dispersion centrifugation, and the operation is repeated 3 times. Then, the precipitate is ultrasonically dispersed in an ethanol solution containing 2% concentrated hydrochloric acid, stirred and refluxed for reaction at a temperature of 70-90°C for 1-24 hours, the supernatant is removed by centrifugation, and the precipitate is repeatedly washed with anhydrous ethanol for at least 3 times. After washing, the supernatant is removed, and the precipitate is dispersed again in an ethanol solution containing 2% concentrated hydrochloric acid, refluxed at a constant temperature of 70-90°C for 1-24 hours, the supernatant is removed by centrifugation, and the precipitate is repeatedly washed with anhydrous ethanol for at least 3 times, and freeze-dried to obtain the mesoporous silica nanoparticles.

3. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The method of selecting a small molecule compound with a strong mass spectrometry signal as a signal molecule to be loaded into the mesoporous channels of mesoporous silica nanoparticles comprises the following steps: A solution of a small molecule compound with a strong mass spectrometry signal at a concentration of 1 to 20 mg / mL is added to mesoporous silica powder, wherein the mass ratio of the small molecule compound with a strong mass spectrometry signal to the mesoporous silica is 1 to 20:1, ultrasonicated until completely dispersed, stirred at room temperature in the dark for 1 to 24 hours, centrifuged, and the precipitate obtained by centrifugation is repeatedly washed with ultrapure water, and freeze-dried to obtain mesoporous silica loaded with the small molecule compound with a strong mass spectrometry signal; Alternatively, the method of utilizing the self-polymerization of dopamine hydrochloride to form a thin layer of polydopamine to be encapsulated on the surface of mesoporous silica nanoparticles to seal the pores comprises the following steps: The mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal is added to a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a concentration of 5-20 mmol / L and a pH of 7-9, and ultrasonically dispersed for 1-5 minutes. The concentration of the mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal in the final system is 0.5-2 mg / mL. Dopamine hydrochloride powder is added, and the mass ratio of the mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal to dopamine hydrochloride is 1-2:

1. The reaction is stirred at room temperature in the dark for 3-24 hours. After the reaction is completed, the supernatant is removed by centrifugation, the precipitate is washed with ultrapure water at least three times, and freeze-dried to obtain polydopamine-encapsulated mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal.

4. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The method of further introducing the biomarker antibody 1 onto the surface of the nanoparticles to prepare the pH stimulus responsive immune functionalized mesoporous material comprises the following steps: The polydopamine-encapsulated mesoporous silica lyophilized powder of a small molecule compound with a strong mass spectrometry signal is added to PBS and ultrasonically dispersed, and a biomarker antibody 1 solution with a concentration of 50 to 200 μg / mL is added, and the mass ratio of the biomarker antibody 1 to the polydopamine-encapsulated mesoporous silica with a small molecule compound with a strong mass spectrometry signal is 1:1 to 200. After repeated blowing and mixing, the mixture is stirred at room temperature for 1 to 12 hours. After the reaction is completed, 1% glycerol is added and blown and mixed, and PBS containing 4 wt% bovine serum albumin is continued to be added to react for 1 to 3 hours to obtain a pH stimulus-responsive immune functionalized mesoporous material dispersion system with a concentration of 0.5 to 2 mg / mL.

5. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The preparation method of the standard sample comprises the following steps: Mix PBS with a concentration of 5 to 20 mmol / L and a pH of 7 to 9 with the recombinant human leguamin monoclonal antibody lyophilized powder, gently blow until fully dissolved and mixed to obtain a standard solution, divide it into portions, add PBS with a concentration of 5 to 20 mmol / L and a pH of 7 to 9 to dilute it step by step to obtain a standard sample.

6. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The preparation method of the composite of immunomagnetic beads-standard sample-pH stimulation responsive immune functionalized mesoporous material comprises the following steps: The immunomagnetic beads are dissolved in PBS containing 2wt% BSA and diluted at least 10 times, the immunomagnetic beads and the standard sample are mixed, the temperature is 25-40°C and oscillated for 1-3 hours, the supernatant is removed by magnetic separation, PBS containing 2wt% BSA is added and washed repeatedly for 3 times, the supernatant is removed by magnetic separation, and then dispersed in PBS containing 2wt% BSA, and the pH stimulus responsive immune functionalized mesoporous material suspension prepared in the first step with a concentration of 1-5 mg / mL is added, the mass ratio of the immunomagnetic beads to the pH stimulus responsive immune functionalized mesoporous material prepared in the first step is 1-10:1, the mixture is repeatedly blown and mixed, the temperature is 25-40°C and rotated for 1-3 hours, the supernatant is removed by magnetic separation, and PBS is added and washed repeatedly for at least 3 times, and the supernatant is discarded by applying an external magnetic field to obtain a complex of immunomagnetic beads-standard sample-pH stimulus responsive immune functionalized mesoporous material.

7. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The preparation method of the immunomagnetic beads comprises the following steps: Add 0.5-2 mmol / L HCl to a 1-20 mg / mL N-hydroxysulfosuccinimidyl magnetic bead suspension for washing, perform magnetic separation and discard the supernatant, add 200-500 μg / mL biomarker antibody 2 solution, the mass ratio of N-hydroxysulfosuccinimidyl magnetic beads to biomarker antibody 2 is 1-100:1, gently vortex, incubate at room temperature in the dark for 1-3 hours, perform magnetic separation and discard the supernatant, add 1-5 mol / L ethanolamine solution for repeated washing at least 3 times, discard the supernatant, disperse in 1-5 mol / L ethanolamine solution, shake at room temperature for 1-3 hours and discard the supernatant, wash again at least 3 times with PBS containing 2 wt% BSA and shake for 1-6 hours, discard the supernatant, and disperse in PBS containing 2 wt% BSA to obtain an immunomagnetic bead dispersion system; The biomarker antibody 2 is selected from recombinant human legamin monoclonal antibody.

8. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The method for preparing the composite of immunomagnetic beads-biomarker-pH stimulation responsive immune functionalized mesoporous material comprises the following steps: The immunomagnetic beads are dissolved in PBS containing 2wt% BSA and diluted at least 10 times, the immunomagnetic beads and the sample to be tested containing the biomarker are mixed, the temperature is constant at 25-40°C and oscillated for incubation for 1-3 hours, the supernatant is removed by magnetic separation, PBS containing 2wt% BSA is added and repeatedly washed for 3 times, the supernatant is removed by magnetic separation, and then dispersed in PBS containing 2wt% BSA, and the pH stimulus responsive immune functionalized mesoporous material suspension prepared in the first step with a concentration of 1-5mg / mL is added, the mass ratio of the immunomagnetic beads to the pH stimulus responsive immune functionalized mesoporous material prepared in the first step is 1-10:1, and the mixture is repeatedly blown and mixed, the temperature is constant at 25-40°C and rotated for reaction for 1-3 hours, the supernatant is removed by magnetic separation, and PBS is added and repeatedly washed for at least 3 times, and a magnetic field is applied to discard the supernatant to obtain a complex of immunomagnetic beads-biomarker-pH stimulus responsive immune functionalized mesoporous material.

9. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The method of placing the sample in an acidic buffer solution containing an internal standard for ultrasonic treatment and detecting the sample supernatant by LC-MS / MS comprises the following steps: Add disodium hydrogen phosphate-citrate buffer solution with a pH of 2 to 7.5 and an internal standard solution to the prepared immunomagnetic beads-biomarker-pH stimulus-responsive immune functionalized mesoporous material complex, vortex for 0.5 to 2 minutes, ultrasonicate for 5 minutes to 2 hours at a temperature of 25 to 40° C., centrifuge, take the supernatant, dilute it with the mobile phase, and vortex to mix to obtain a supernatant.

10. The method for detecting protein and polypeptide biomarkers based on pH stimulus responsive materials according to claim 1, characterized in that: The LC-MS / MS conditions were as follows: ACQUITY UPLC BEH C18 column, mobile phase: 0.05% formic acid water-methanol 40:60, v / v, isocratic elution, flow rate 0.3 mL / min, column temperature 35°C, injection volume 1 μL, analysis time 5 min; Shimadzu 8045 triple quadrupole mass spectrometer, ion source ESI, scanning mode multiple reaction monitoring, detection in positive ion mode; Other mass spectrometry parameters were as follows: nebulizing gas flow rate was 3.0 L / min, drying gas flow rate was 10.0 L / min, heating gas flow rate was 10.0 L / min, interface temperature was 300 °C, DL tube temperature was 200 °C, heating block temperature was 400 °C, and collision gas was argon.

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