A method for detecting protein polypeptide biomarkers based on pH stimulus-responsive materials

Abstract

The application discloses a method for detecting protein polypeptide biomarkers based on pH stimulus response materials, wherein the pH stimulus response immunofunctionalized mesoporous materials can load more small molecule compounds with strong mass spectrum signals, greatly improving the sensitivity of the analysis method; the pH stimulus response immunofunctionalized mesoporous materials can realize signal conversion, avoid direct mass spectrum analysis of biomacromolecule biomarkers, release small molecule compounds with strong mass spectrum signals under specific conditions, and improve the specificity of the detection method.

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 levels 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 the development of diseases, and evaluating the effectiveness of drug treatment. [[1]StepanH,HundM,AndraczekT.Combining biomarkers to predict pregnancy complicationsand redefine preeclampsia:The angiogenic-placental syndrome[J].Hypertension,2020,75(4):918-926.[2]MacdonaldTM,WalkerSP,HannanNJ,et al.Clinicaltools and biomarkers to predict preeclampsia[J].EBioMedicine,2022,75:103780.[3]ErraniC,TraversariM,CosentinoM,et al.The prognostic value of the serumlevel of C-reactive protein for survival of children with ewing's sarcoma[J].Cancers,2023,15(5):1573.].Biomarkers in organisms include biological macromolecules, small molecule compounds, cells and microbial populations. Among them, protein and peptide biomarkers, as the final products of the "central dogma" process, can be more accurately quantified compared to nucleic acid biomarkers that undergo transcription or translation processes. They have received widespread 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, legumin 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 serve as potential indicators for disease diagnosis or prognosis. Therefore, it is crucial to adopt reliable analytical detection methods to monitor changes in the levels 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 are complex in composition, with 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 1 and 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 its application in biomacromolecule analysis. By leveraging functional materials to construct novel signal conversion and amplification strategies, based on the quantitative relationship between the "signal" molecule and the analyte, the detection signal intensity of the small molecule "signal" molecule is used to indirectly reflect the concentration of the analyte. This can overcome the compatibility issues of LC-MS / MS with biomacromolecules while helping 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 levels of protein and polypeptide biomarkers in the body, thereby effectively monitoring the occurrence, development and treatment efficacy of diseases, and providing guidance for disease prevention and adjustment of treatment plans.

[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 a pH stimulus-responsive material, comprising the following steps:

[0007] In the first step, mesoporous silica nanoparticles were prepared by a hydrothermal method. Small molecule compounds with strong mass spectrometry signals were selected as signal molecules and 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, thereby sealing the channels. Biomarker antibody 1 was further introduced onto the surface of the nanoparticles to prepare a pH-stimulated immune-functionalized mesoporous material.

[0008] The method for preparing mesoporous silica nanoparticles by a 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 cetyltrimethylammonium bromide (CTAB) powder, and ultrasonicate for 10-30 minutes (preferably 30 minutes) until 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 at a mass volume ratio of cetyltrimethylammonium bromide to tetraethyl orthosilicate of 1:1-10 g / mL (preferably 1:5 g / mL), react for 7-12 hours (preferably 9 hours), and centrifuge (condition: 13000 rpm). The supernatant was removed by centrifugation after ultrasonic dispersion with anhydrous ethanol, and the operation was repeated 3 times. The supernatant was removed by centrifugation after ultrasonic dispersion with ultrapure water, and the operation was repeated 3 times. The precipitate was ultrasonically dispersed in an ethanol solution containing 2% concentrated hydrochloric acid, and the reaction was stirred at reflux for 1 to 24 hours (preferably 24 hours) at a temperature of 70 to 90° C. (preferably 80° C.) and the supernatant was removed by centrifugation. The precipitate was repeatedly washed with anhydrous ethanol at least 3 times. After washing, the supernatant was removed, and the precipitate was redispersed in an ethanol solution containing 2% concentrated hydrochloric acid, and the temperature was 70 to 90° C. (preferably 80° C.) and refluxed at a constant temperature for 1 to 24 hours (preferably 24 hours). The supernatant was removed by centrifugation, and the precipitate was repeatedly washed with anhydrous ethanol at least 3 times, and freeze-dried to obtain the mesoporous silica nanoparticles.

[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 and loading it into the mesoporous channels of the 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 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 (preferably 1.5:1). Ultrasonication is performed until completely dispersed, and the reaction is stirred at room temperature in the dark for 1 to 24 hours (preferably 24 hours). The reaction is centrifuged (conditions: 13,000 rpm for 4 minutes), and the precipitate obtained by centrifugation is repeatedly washed with ultrapure water, and lyophilized to obtain mesoporous silica loaded with a small molecule compound with a strong mass spectrometry signal.

[0013] The method for preparing the 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 seal 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 (conditions: centrifugation at 13000 rpm for 4 minutes) is performed to remove the supernatant, 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. The mass ratio of biomarker antibody 1 to 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 pipetting and mixing, the mixture is stirred at room temperature for 1 to 12 hours (preferably 6 hours). After the reaction is completed, 1% (v / v) glycerol is added and pipetted to mix. PBS (10 mM, pH 7.4) containing 4 wt% bovine serum albumin (BSA) is continued to be added and reacted 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 of human legumain polyclonal antibody (10 mM, pH 7.4) was allowed to cool to room temperature and mixed by repeated pipetting.

[0021] In the second step, the standard sample is mixed with the immunomagnetic beads and the pH-stimuli-responsive immunofunctionalized mesoporous material, and then incubated. The mixture is then magnetically separated to obtain a complex of the immunomagnetic beads-standard sample-pH-stimuli-responsive immunofunctionalized mesoporous material. The complex is then ultrasonically treated in an acidic buffer solution containing an internal standard. The sample supernatant is detected by LC-MS / MS, and a sample chromatogram is collected to record the chromatographic peak areas of the small molecule compounds with strong mass spectrometry signals and the internal standard. A standard regression curve of the biomarker concentration-f is established, with the biomarker concentration in the standard sample as the horizontal axis and the ratio of the small molecule compound with strong mass spectrometry signals to the internal standard peak area f as the vertical axis.

[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 leguamin monoclonal antibody lyophilized powder, gently pipette until fully dissolved and mixed to obtain a standard solution, divide the solution into portions, and 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) for stepwise dilution 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 were dissolved in PBS containing 2 wt% BSA and diluted at least 10 times. The immunomagnetic beads were mixed with the standard sample and incubated at 25-40°C (preferably 37°C) with constant temperature shaking for 1-3 hours (preferably 2 hours). The supernatant was removed by magnetic separation. The beads were washed repeatedly for 3 times by adding PBS containing 2 wt% BSA. The supernatant was removed by magnetic separation and then dispersed in PBS containing 2 wt% BSA. The pH stimulus response immune function prepared in the first step was added at a concentration of 1-5 mg / mL (preferably 2 mg / mL). The mesoporous material suspension is prepared, and 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). The mixture is repeatedly blown and mixed, and the temperature is 25 to 40°C (preferably 37°C) and the reaction is carried out at a constant temperature of 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-stimulation-responsive immunofunctionalized mesoporous material, and the mixture is incubated. The mixture is then magnetically separated to obtain a complex of immunomagnetic beads-biomarker-pH-stimulation-responsive immunofunctionalized mesoporous material. The complex is then 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 chromatographic peak area of ​​the internal standard 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 suspension of N-hydroxysulfosuccinimidyl magnetic beads (NHS-MBs) at a concentration of 1-20 mg / mL (preferably 10 mg / mL) for washing. The supernatant was discarded after magnetic separation. A solution of biomarker antibody 2 at a concentration of 200-500 μg / mL (preferably 308 μg / mL) was added. The mass ratio of N-hydroxysulfosuccinimidyl magnetic beads (NHS-MBs) to biomarker antibody 2 was 1-100:1 (preferably 25:1). The suspension was gently stirred. Vortex, incubate in the dark for 1 to 3 hours (preferably 2 hours) at room temperature, perform magnetic separation and discard the supernatant, add a 1 to 5 mol / L (preferably 3 mol / L) ethanolamine solution and wash repeatedly for at least 3 times, discard the supernatant, redisperse in a 1 to 5 mol / L (preferably 3 mol / L) ethanolamine solution, shake at room temperature for 1 to 3 hours (preferably 2 hours) and discard the supernatant, wash again with PBS containing 2 wt% BSA at least 3 times and shake for 1 to 6 hours (preferably 4 hours), discard the supernatant, and redisperse in PBS containing 2 wt% BSA to obtain an immunomagnetic bead dispersion system.

[0029] The biomarker antibody 2 is selected from recombinant human leguamin 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 complex of immunomagnetic beads-biomarker-pH stimulation-responsive immune functionalized mesoporous material comprises the following steps:

[0033] The immunomagnetic beads were dissolved in PBS containing 2 wt% BSA and diluted at least 10 times. The immunomagnetic beads were mixed with the sample to be tested containing the biomarker, and the mixture was incubated at a constant temperature of 25-40°C (preferably 37°C) for 1-3 hours (preferably 2 hours). The supernatant was removed by magnetic separation, and the mixture was repeatedly washed three times by adding PBS containing 2 wt% BSA. The supernatant was removed by magnetic separation, and the mixture was dispersed in PBS containing 2 wt% BSA. The pH stimulus-responsive immunobeads prepared in the first step were added at a concentration of 1-5 mg / mL (preferably 2 mg / mL). The immunofunctionalized mesoporous material suspension is prepared, and the mass ratio of the immunomagnetic beads to the pH stimulus responsive immunofunctionalized mesoporous material prepared in the first step is 1 to 10:1 (preferably 5:1). The mixture is repeatedly blown and mixed, and the temperature is 25 to 40°C (preferably 37°C) and the reaction is carried out under constant rotation 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 washed repeatedly 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 material.

[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, with a pH of 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] The prepared immunomagnetic beads-biomarker-pH stimulus-responsive immune functionalized mesoporous material complex is added with sodium hydrogen phosphate-citrate buffer solution with a pH of 2 to 7.5 (preferably 2.35) and an internal standard solution, vortexed for 0.5 to 2 minutes (preferably 1 minute), ultrasonicated at a temperature of 25 to 40°C (preferably 37°C) for 5 minutes to 2 hours (preferably 1 hour), centrifuged (13000 rpm, 5 minutes), and the supernatant is diluted with a mobile phase (0.05% formic acid water: methanol = 40:60, v / v), and vortexed to obtain a 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: an ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm), a mobile phase consisting of 0.05% formic acid in water (A) and methanol (B) in a ratio of 40:60 (v / v), isocratic elution, a flow rate of 0.3 mL / min, a column temperature of 35°C, an injection volume of 1 μL, and an analysis time of 5 min; a Shimadzu 8045 triple quadrupole mass spectrometer was used, with an ESI ion source, multiple reaction monitoring (MRM) scanning, and detection in positive ion mode.

[0039] Other mass spectrometry parameters were as follows: nebulizing gas flow rate of 3.0 L / min, drying gas flow rate of 10.0 L / min, heating gas flow rate of 10.0 L / min, interface temperature of 300 °C, DL tube temperature of 200 °C, heating block temperature of 400 °C, and collision gas of 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 this method, 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 signal conversion, avoiding direct mass spectrometry analysis of biological macromolecule biomarkers, while releasing 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 small molecule compounds with different 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, which has strong universality.

[0043] In summary, the method of the present invention has high sensitivity, good specificity, and is easy to operate. By using magnetic separation to rapidly and exclusively separate and enrich the protein and peptide biomarkers to be detected in the sample, and by constructing a pH-stimulated immune-functionalized mesoporous material with a signal conversion and amplification strategy, the method further enables the exclusive release of small molecule compounds with strong mass spectrometry signals. The specifically released small molecule compounds with strong mass spectrometry signals are then detected using LC-MS technology, achieving the goal of signal amplification, significantly improving the sensitivity of biomarker detection, and effectively resolving 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. It is based on the preparation of pH stimulus-responsive immune functionalized mesoporous materials through 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 sample, blank sample plus doxorubicin standard solution and standard sample containing biomarkers.

[0051] Figure 7 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 leguminous protein (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. Those skilled in the art should understand 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 stimulus-responsive immune functionalized mesoporous materials

[0056] Place 1.8 g of cetyltrimethylammonium bromide (CTAB) powder in a 500 mL round-bottom flask. Add 300 mL of Tris-HCl buffer (50 mmol / L, pH 8.0). Ultrasonicate for 30 minutes until completely dispersed. Place the round-bottom flask in a 60°C oil bath and stir under reflux for 1 hour. 9 mL of tetraethyl orthosilicate (TEOS) was added dropwise at a uniform rate and reacted for 9 hours. The obtained milky white product was centrifuged at 13000 rpm for 4 min to obtain a white precipitate. The product was ultrasonically dispersed with anhydrous ethanol and the supernatant was removed by centrifugation. The operation was repeated 3 times. The product was ultrasonically dispersed with ultrapure water and the supernatant was removed by centrifugation. 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 80°C for 24 hours, the supernatant was removed by centrifugation, and the precipitate was washed repeatedly with anhydrous ethanol 3 times. After washing, the supernatant was removed and the precipitate was dispersed again 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 washed repeatedly with anhydrous ethanol at least 3 times. The product 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 Schematic diagrams of SEM and HRTEM characterization results of mesoporous silica (A) and (B) show the SEM and HRTEM results. The results show that the MSN nanoparticles are uniformly distributed and have a normal particle size distribution, primarily between 140 and 200 nm, with an average particle size of approximately 175 nm.

[0059] Doxorubicin solution with a concentration of 3 mg / mL was prepared by dissolving 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 ultrasonically dispersed. The mixture was stirred at room temperature in the dark for 24 hours, and 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 (C0) were determined by UV-spectrophotometry. 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 the mesoporous silica relative to the total input can be calculated, thereby optimizing loading conditions. The centrifuged precipitate was washed three times with ultrapure water and lyophilized to yield 9.78 mg of doxorubicin-loaded mesoporous silica (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) and ultrasonically dispersed for 3 minutes. The final DOX-MSNs concentration in the 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, the mixture was centrifuged at 13,000 rpm for 4 minutes, the supernatant was removed, and 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] A 0.2 mg / mL PBS solution (10 mM, pH 7.4) of the purchased human legumain polyclonal antibody (PCAb) was allowed to cool to room temperature and mixed by repeated pipetting.

[0063] 1 mg of DOX-MSNs / PDA lyophilized powder was added to 400 μL of PBS (10 mM, pH 7.4) and ultrasonically dispersed. 100 μL of 200 μg / mL PCAb solution was added, and the mixture was repeatedly pipetted and mixed. The mixture was then placed on a rotary mixer and reacted at room temperature for 6 h. After the reaction, 10 μL of 1% (v / v) glycerol was added and pipetted to mix. 500 μL of PBS (10 mM, pH 7.40) containing 4 wt% bovine serum albumin (BSA) was added and reacted for 2 h (to block the nonspecific adsorption sites on DOX-MSNs / PDA). A pH-stimulated immune-functionalized mesoporous material (DOX-MSNs / PDA@PcAb) dispersion with a concentration of 1 mg / mL was obtained and stored at 4°C for later 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 2Schematic diagrams of the HRTEM and DLS characterization results of pH-responsive immune-functionalized mesoporous materials. Figure A shows the HRTEM characterization results of the pH-responsive immune-functionalized mesoporous materials, and Figure B shows the DLS characterization results of the pH-responsive immune-functionalized mesoporous materials. The results show that the pH-responsive immune-functionalized mesoporous materials prepared by the present invention have uniform particle size, with an average particle size of approximately 185 nm.

[0066] Figure 3 This is a schematic diagram showing the effects of room temperature dark-shielded stirring reaction time and doxorubicin solution concentration on the results when preparing doxorubicin-loaded mesoporous silica (DOX-MSNs). A is a schematic diagram showing the effects of room temperature dark-shielded stirring reaction time on the amount of doxorubicin loaded per unit mass of material (DLC (%)) when preparing doxorubicin-loaded mesoporous silica (DOX-MSNs). DLC (%) is calculated according to (initial doxorubicin concentration of reaction - end doxorubicin concentration of reaction) × total volume of reaction system / mass of added mesoporous silica. The larger the DLC (%), the more doxorubicin loaded per unit mass of mesoporous silica, which is more conducive to the subsequent mass spectrometry signal amplification. B is a schematic diagram showing the effect of doxorubicin solution concentration on the percentage of loaded doxorubicin (DLE) (%) in the preparation of doxorubicin-loaded mesoporous silica (DOX-MSNs). DLE (%) is calculated based on (initial doxorubicin concentration at the start of the reaction - doxorubicin concentration at the end of the reaction) / initial doxorubicin concentration. A larger DLE (%) indicates a higher 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 ultrasound time for the 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 at a pH of 2.35 (sodium hydrogen phosphate-citric acid buffer, 20mM) with a mass ratio of DOX-MSNs to dopamine hydrochloride of 2:1, 1:1, and 1:2; B is a schematic diagram of the specific release results of doxorubicin when DOX-MSNs / PDA is prepared at a pH of 5.02 (sodium hydrogen phosphate-citric acid buffer, 20mM) with a mass ratio of DOX-MSNs to dopamine hydrochloride of 2:1, 1:1, and 1:2; C is a schematic diagram of the specific release results of doxorubicin when DOX-MSNs / PDA is prepared at a pH of 7.43 (sodium hydrogen phosphate-citric acid buffer, 20mM). A is a schematic diagram of the specific release results of doxorubicin when DOX-MSNs / PDA was prepared with a mass concentration ratio of DOX-MSNs to dopamine hydrochloride of 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 with a mass concentration ratio of DOX-MSNs to dopamine hydrochloride of 2:1 under different ultrasound times at pH 2.35 (disodium hydrogen phosphate-citrate buffer, 20 mM); 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] As shown in Figures AC, the release rate of doxorubicin from the pH-responsive immune-functionalized mesoporous material gradually decreases with decreasing DOX-MSNs to dopamine hydrochloride concentration ratio. When the DOX-MSNs to dopamine hydrochloride concentration ratio is 2:1, the pH-responsive immune-functionalized mesoporous material exhibits improved pH responsiveness. The release rate of doxorubicin from the pH-responsive immune-functionalized mesoporous material gradually increases with decreasing pH of the dispersion system, and exhibits even better pH responsiveness when the pH of the dispersion system is 2.35. Furthermore, the release rate of doxorubicin from the pH-responsive immune-functionalized mesoporous material increases with increasing ultrasound time. To improve the efficiency of the analytical method, a 3-hour reaction with stirring at room temperature in the dark was selected. The above optimization results indicate that a 2:1 DOX-MSNs to dopamine hydrochloride concentration ratio, a 3-hour reaction with stirring at room temperature in the dark, and a pH 2.35 sodium hydrogen phosphate-citrate buffer solution exhibit excellent pH responsiveness. Figure 5 Schematic diagram of the basic process of preparing pH stimulus-responsive immune functionalized mesoporous materials and their specific release of doxorubicin.

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

[0071] Take 100 μL of 10 mg / mL N-hydroxysulfosuccinimidyl magnetic beads (NHS-MBs) suspension (solvent is N,N-dimethylacetamide), add 200 μL of 1 mmol / L HCl (4°C) to wash, magnetic separation and discard the supernatant, add 130 μL of biomarker antibody 2 recombinant human leguamin monoclonal antibody solution (100 mmol / L 4-morpholineethanesulfonic acid (MES) buffer solution, containing 40 μg biomarker antibody 2 recombinant human leguamin monoclonal antibody, pH 4.8), gently vortex for 15 seconds, incubate at room temperature in the dark for 2 hours, magnetic separation 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 hours and discard the supernatant. Then, the beads were washed again four 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 of biomarker standard solutions: Mix 200 μL of PBS (10 mM, pH 7.4) with 20 ng of lyophilized recombinant human leguamin monoclonal antibody powder. Gently pipette until fully dissolved and mix thoroughly to obtain a 100 ng / mL standard solution. Aliquot into 100 μL centrifuge tubes and store at -80°C until needed. Remove an aliquot of recombinant human leguamin monoclonal antibody (100 ng / mL) and add PBS (10 mM, pH 7.4) for serial dilution to obtain a series of standard solutions: 50 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, and 0.2 ng / mL.

[0073] Preparation of biomarker-containing standard samples and test samples: Precision pipette 10 μL of the biomarker standard solution into 190 μL of blank serum and mix thoroughly 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. Fresh human blood samples were collected, allowed to stand for 30 minutes, and then centrifuged (1000 × g, 10 minutes). The supernatant was removed to obtain the test samples.

[0074] Take 10 μL of the prepared immunomagnetic bead dispersion (10 mg / mL), add 90 μL of PBS containing 2 wt% BSA, and dilute 10-fold by pipetting thoroughly. Pipette 5 μL of the diluted immunomagnetic bead dispersion (1 mg / mL) and mix with 150 μL of the test sample or standard sample containing the biomarker. Incubate at 37°C with shaking for 2 hours. Remove the supernatant by magnetic separation, add 800 μL of PBS containing 2 wt% BSA and wash repeatedly three times. Remove the supernatant by magnetic separation and disperse in 140 μL of PBS containing 2 wt% BSA. Add 10 μL of the pH-stimulated immune-functionalized mesoporous material (DOX-MSNs / PDA@PcAb) suspension prepared in the first step at a concentration of 2 mg / mL (the solvent is 10 mM PBS containing 2 wt% BSA, pH 7.4), mix repeatedly by pipetting, and react at 37°C with rotation for 2 hours. The supernatant was removed by magnetic separation, and 800 μL PBS (10 mM, pH 7.4) was added and washed repeatedly for 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 test samples 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, sonicated 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 in water (A)-methanol (B) (40:60, v / v), isocratic elution, flow rate 0.3 mL / min, column temperature 35°C, injection volume 1 μL, analysis time 5 min. A Shimadzu 8045 triple quadrupole mass spectrometer was used, with an ESI source and multiple reaction monitoring (MRM) scanning mode in positive ion mode. The m / z of the doxorubicin precursor and daughter ions were 544.10 and 397.05, respectively, with a collision energy of -13 eV. The m / z of the internal standard precursor and daughter ions were 528.15 and 321.05, respectively, with a CE of -22 eV. Other mass spectrometry parameters are shown in Table 1:

[0078] Table 1

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

[0080] The amount of doxorubicin in the injected supernatant is directly proportional to the biomarker concentration. A standard regression curve (biomarker concentration - f) was constructed, with the biomarker concentration of the standard sample as the horizontal axis and the peak area ratio of doxorubicin to the internal standard (abbreviated as f) as the vertical axis. The peak area ratio of doxorubicin to the internal standard in the test sample was substituted into the standard curve to calculate the biomarker concentration in the test sample.

[0081] Figure 6 Schematic diagrams of the chromatographic results for a blank sample, a blank sample with doxorubicin standard solution, and a standard sample containing a biomarker. Figure A is a schematic diagram of the chromatographic results for a blank sample, Figure B is a schematic diagram of the chromatographic results for a blank sample with doxorubicin standard solution, and Figure C is a schematic diagram of the chromatographic results for a standard sample containing a biomarker. As can be seen from the figure, the chromatographic peak shape of the standard sample containing a biomarker at 1 ng / mL is good, with no other interfering impurity peaks, indicating that the analytical method of the present invention has good specificity and sensitivity that meets the requirements for detecting the biomarker legumain.

[0082] Figure 7 The results are shown schematically for the detection of samples containing caspas-3, matrix metalloproteinases-9 (MMP-9), and fibroblast activation protein (FAP), as well as legumain (LGMN), to assess the specificity of the method. In the figure, BLANK represents a blank serum sample; CASP-3 represents a blank serum sample containing 100 ng / mL caspas-3; MMP-9 represents a blank serum sample containing 100 ng / mL MMP-9; FAP represents a blank serum sample containing 100 ng / mL FAP; and LGMN represents a serum sample containing 0.01 ng / mL legumain (LGMN). As can be seen from the figure, there is no significant difference in the detection response between the samples spiked with high concentrations (100 ng / mL) of the other protein and peptide biomarkers and the blank samples, while the sample containing 0.01 ng / mL legumain exhibits a sensitive response, demonstrating the high specificity of the analytical method of the present invention.

[0083] Figure 8 Schematic diagram of the change in mass spectrometry signal of standard samples containing different concentrations of legumin. 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 legume protein concentration 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. The figure shows a good linear relationship between the mass spectrometry signal intensity and the legumain concentration within the concentration range of 0.01 to 2.5 ng / mL, effectively enabling accurate quantitative analysis of samples at varying concentration levels.

[0084] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still 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. Small molecule compounds with strong mass spectrometry signals were selected as signal molecules and 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, thereby sealing the channels. Biomarker antibody 1 was further introduced onto the surface of the nanoparticles to prepare a pH-stimulated immune-functionalized mesoporous material. The small molecule compound with a strong mass spectrometry signal is doxorubicin; The biomarker antibody 1 is selected from human legumain polyclonal antibody; In the second step, the standard sample is mixed with the immunomagnetic beads and the pH stimulus-responsive immune functionalized mesoporous material, and then incubated, followed by 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. The sample supernatant was detected by LC-MS / MS. The sample chromatogram was collected and the chromatographic peak areas of the small molecule compound with strong mass spectrometry signals and the internal standard were recorded respectively. The standard regression curve of biomarker concentration-f was established with the biomarker concentration in the standard sample as the horizontal axis and the ratio of the peak area of ​​the small molecule compound with strong mass spectrometry signals to the internal standard as the vertical axis. The third step is to mix the sample containing the biomarker with the immunomagnetic beads and the pH-stimulation-responsive immune-functionalized mesoporous material, incubate the mixture, and then perform magnetic separation to obtain a complex of the immunomagnetic beads-biomarker-pH-stimulation-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 chromatographic peak area of ​​the small molecule compound with a strong mass spectrometry signal to the internal standard 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 a hydrothermal method comprises the following steps: The tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a concentration of 10-80 mmol / L and a pH of 7-9 was added to the hexadecyltrimethylammonium bromide powder, and ultrasonicated for 10-30 minutes until 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 1:1-10 g / mL, and the mixture was reacted for 7-12 hours, centrifuged, and ultrasonically dispersed with anhydrous ethanol, and then the supernatant was removed by centrifugation. The operation was repeated 3 times, and the mixture was ultrapure water was used for ultrapure water. The supernatant is removed by acoustic dispersion centrifugation, and the operation is repeated three times. The precipitate is then ultrasonically dispersed in an ethanol solution containing 2% concentrated hydrochloric acid, stirred and refluxed at a temperature of 70 to 90° C. for 1 to 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 redispersed in an ethanol solution containing 2% concentrated hydrochloric acid, refluxed at a constant temperature of 70 to 90° C. for 1 to 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 and loading it into the mesoporous channels of the 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, and the mixture is ultrasonically dispersed until completely dispersed. The mixture is 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 lyophilized 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 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 to 20 mmol / L and a pH of 7 to 9, and ultrasonically dispersed for 1 to 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 to 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 to 2:

1. The reaction is stirred at room temperature in the dark for 3 to 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 freeze-dried powder loaded with 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 loaded with a small molecule compound with a strong mass spectrometry signal is 1:1 to 200. After repeated pipetting and mixing, the mixture is stirred at room temperature for 1 to 12 hours. After the reaction is completed, 1% glycerol is added and pipetted to mix, and PBS containing 4 wt% bovine serum albumin is continued to be added to react for 1 to 3 hours to obtain the 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 5-20 mmol / L and a pH of 7-9 with the recombinant human leguamin monoclonal antibody lyophilized powder, gently pipette until fully dissolved and mixed to obtain a standard solution, divide the solution into portions, and add PBS with a concentration of 5-20 mmol / L and a pH of 7-9 for graded dilution 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 are mixed with the standard sample, incubated at a constant temperature of 25-40°C for 1-3 hours, magnetic separation is performed to remove the supernatant, and PBS containing 2wt% BSA is added to wash repeatedly for 3 times. The supernatant is magnetic separation is removed, and then dispersed in PBS containing 2wt% BSA, and the pH stimulus-responsive immune functionalized mesoporous material suspension prepared in the first step is added with a concentration of 1-5 mg / mL. 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 pipetted and mixed, and the reaction is carried out at a constant temperature of 25-40°C and rotated for 1-3 hours. The supernatant is magnetic separation to remove the supernatant, and PBS is added to wash repeatedly for at least 3 times. An external magnetic field is applied to discard the supernatant 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: A suspension of N-hydroxysulfosuccinimidyl magnetic beads at a concentration of 1 to 20 mg / mL was washed with 0.5 to 2 mmol / L HCl, magnetically separated and the supernatant discarded, a solution of biomarker antibody 2 at a concentration of 200 to 500 μg / mL was added, the mass ratio of N-hydroxysulfosuccinimidyl magnetic beads to biomarker antibody 2 being 1 to 100:1, the beads were gently vortexed, and the beads were incubated at room temperature in the dark for 1 to 3 hours. The beads were magnetically separated and the supernatant discarded, the beads were repeatedly washed at least three times with a 1 to 5 mol / L ethanolamine solution, the supernatant discarded, the beads were redispersed in a 1 to 5 mol / L ethanolamine solution, the beads were shaken at room temperature for 1 to 3 hours, the supernatant discarded, the beads were washed again at least three times with PBS containing 2 wt% BSA and shaken for 1 to 6 hours, the supernatant discarded, and the beads were redispersed in PBS containing 2 wt% BSA to obtain an immunomagnetic bead dispersion system; The biomarker antibody 2 is selected from recombinant human leguamin 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 complex 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 are mixed with a sample to be tested containing a biomarker, incubated at a constant temperature of 25-40°C with shaking for 1-3 hours, magnetically separated to remove the supernatant, and repeatedly washed three times with PBS containing 2wt% BSA. The supernatant is magnetically separated to remove the supernatant, and then dispersed in PBS containing 2wt% BSA. The pH stimulus-responsive immune functionalized mesoporous material suspension prepared in the first step is added at a concentration of 1-5 mg / mL. 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 pipetted and mixed, and the reaction is carried out at a constant temperature of 25-40°C with rotation for 1-3 hours. The supernatant is magnetically separated to remove the supernatant, and repeatedly washed at least three times with PBS. An external 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 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, sonicate at a temperature of 25 to 40°C for 5 minutes to 2 hours, centrifuge, and 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: an ACQUITY UPLC BEH C18 column, a mobile phase consisting of 0.05% formic acid in water and methanol in a ratio of 40:60 (v / v), isocratic elution, a flow rate of 0.3 mL / min, a column temperature of 35°C, an injection volume of 1 μL, and an analysis time of 5 min; a Shimadzu 8045 triple quadrupole mass spectrometer, an ESI source, and multiple reaction monitoring scanning, with detection in positive ion mode. Other mass spectrometry parameters were as follows: nebulizing gas flow rate of 3.0 L / min, drying gas flow rate of 10.0 L / min, heating gas flow rate of 10.0 L / min, interface temperature of 300 °C, DL tube temperature of 200 °C, heating block temperature of 400 °C, and collision gas of argon.

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