A sialylated glycoprotein detection reagent, its preparation method and application

Abstract

This invention relates to a sialylated glycoprotein detection reagent, its preparation method, and its application, belonging to the field of biodetection technology. The detection reagent uses purified water as a solvent and comprises the following components: surface-modified titanium dioxide nanomaterials, metal ions, a pH adjuster, and a solvent. The surface-modified titanium dioxide nanomaterials are obtained by dispersing titanium dioxide nanoparticles in an organic solvent and reacting them with a silane coupling agent. The pH value of the sialylated glycoprotein detection reagent is ≥ 8.0. The reagent of this invention utilizes the high affinity of modified titanium dioxide nanomaterials for sialic acid to specifically aggregate sialylated glycoproteins in blood into particles, facilitating observation. This reagent exhibits good stability, high sensitivity, and strong specificity, making it suitable for rapid tumor screening of blood smear samples.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a sialylated glycoprotein detection reagent, its preparation method and application, especially suitable for rapid detection by blood smear agglutination method. Background Technology

[0002] Glycosylation modification of proteins, especially sialylation modification, plays a crucial role in vital processes such as cell recognition, immune response, cell adhesion, and signal transduction. The expression of abnormal sialylated glycoproteins has been proven to be closely related to the occurrence, development, invasion, and metastasis of various malignant tumors. Therefore, developing a rapid, simple, and accurate method for detecting sialylated glycoprotein levels in the blood is of great significance for the early screening and auxiliary diagnosis of related diseases.

[0003] Currently, common detection methods include lectin affinity assays and immunoassays. While lectin assays are relatively inexpensive, they suffer from drawbacks such as limited lectin types, easy inactivation, and significant batch-to-batch variability. Titanium dioxide (TiO2) nanomaterials, due to their abundant hydroxyl groups on their surface, exhibit high affinity for phosphate groups and ortho-dihydroxy structures (such as sialic acid terminals), and have become an emerging material in the field of glycoprotein / glycopeptide enrichment. However, nano-TiO2 is highly prone to aggregation and exhibits extremely poor dispersion stability in liquid systems, severely affecting its binding efficiency with target analytes and the reliability of analytical results, thus limiting its direct application in in vitro diagnostic reagents.

[0004] Therefore, there is an urgent need to develop a new detection reagent that can solve the dispersibility problem of nanomaterials and achieve efficient, stable and specific detection of sialylated glycoproteins. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a sialylated glycoprotein detection reagent, its preparation method, and its application. This reagent uses surface-modified titanium dioxide nanomaterials as the core recognition element. By optimizing the surface modification process and reagent formulation, the dispersion problem of the nanomaterials is effectively solved, significantly improving the reagent's stability, sensitivity, and detection accuracy. Furthermore, the detection method is simple, has good reproducibility, and is suitable for large-scale production.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a sialylated glycoprotein detection reagent, comprising the following components: surface-modified titanium dioxide nanomaterials, metal ions, pH adjuster, and solvent;

[0008] The surface-modified titanium dioxide nanomaterial is obtained by dispersing titanium dioxide nanoparticles in an organic solvent and reacting them with a silane coupling agent; it has improved dispersibility and specific binding ability to sialylated glycoproteins.

[0009] The pH value of the sialylated glycoprotein detection reagent is ≥ 8.0, preferably 8.5-9.5.

[0010] In one embodiment of the present invention, the concentration of the surface-modified titanium dioxide nanomaterial is 10 mg / L-100 mg / L, preferably 50 mg / L.

[0011] In one embodiment of the present invention, the concentration of the metal ion is 4 mg / L-20 mg / L; the metal ion is aluminum ion.

[0012] In one embodiment of the present invention, the titanium dioxide nanoparticles have a particle size of 5 nm-25 nm.

[0013] In one embodiment of the present invention, the silane coupling agent comprises 3-aminopropyltriethoxysilane and / or 3-glyceroxypropyltrimethoxysilane.

[0014] In one embodiment of the present invention, the mass ratio of the titanium dioxide nanoparticles to the silane coupling agent is 5:1-15:1.

[0015] In one embodiment of the present invention, the reaction temperature is 60°C-80°C;

[0016] And / or, the reaction time is 2 h-4 h.

[0017] In one embodiment of the present invention, the method for preparing the surface-modified titanium dioxide nanomaterial includes the following steps:

[0018] (1) Titanium dioxide nanoparticles were dispersed in anhydrous ethanol and ultrasonically treated to form a uniform dispersion.

[0019] (2) Add silane coupling agent (APTES) to the obtained dispersion and react at a constant temperature of 60℃-80℃ for 2 h-4 h;

[0020] (3) After the reaction is complete, the product is collected by centrifugation and washed several times with alternating ethanol and purified water to remove unreacted coupling agent;

[0021] (4) The washed product was vacuum dried at 50℃-60℃ and then ground to obtain surface-modified titanium dioxide nanomaterials.

[0022] In one embodiment of the present invention, the organic solvent is one or more of ethanol, glycerol and n-butanol.

[0023] In one embodiment of the present invention, the pH adjuster is sodium hydroxide and / or Tris buffer;

[0024] The solvent is water.

[0025] The second objective of this invention is to provide a method for preparing the aforementioned sialylated glycoprotein detection reagent, characterized by comprising the following steps:

[0026] S1. Titanium dioxide nanoparticles are dispersed in an organic solvent, a silane coupling agent is added to react, and after centrifugation, washing, drying and grinding, surface-modified titanium dioxide nanomaterials are obtained.

[0027] S2. Dissolve the metal ion salt in part of the solvent; adjust the pH of the resulting solution to ≥8.0, add the remaining solvent and stir evenly, let stand, and then separate the solid and liquid phases to obtain the liquid phase.

[0028] S3. Mix the liquid phase obtained in step S2 with the surface-modified titanium dioxide nanomaterial obtained in step S1 to obtain the sialylated glycoprotein detection reagent.

[0029] In one embodiment of the present invention, in step S1, the stirring time is 1 h to 3 h.

[0030] In one embodiment of the present invention, in step S2, the metal ion salt is aluminum chloride.

[0031] In one embodiment of the present invention, in step S2, the settling time is ≥6 h.

[0032] In one embodiment of the present invention, in step S2, the solid-liquid separation is performed by using a microporous filter with a pore size of 0.8 μm-1.2 μm.

[0033] In one embodiment of the present invention, in step S3, the mixing method is: ultrasonic treatment (power 300 W) for 10 min-20 min to enhance dispersibility.

[0034] A third objective of this invention is to provide the application of the aforementioned sialylated glycoprotein detection reagent in the detection of sialylated glycoproteins, wherein the application does not involve the diagnosis or treatment of diseases.

[0035] The design principle of this invention is as follows: Utilizing the specific affinity of surface-aminated titanium dioxide nanomaterials for sialic acid residues, abnormal sialylated glycoproteins in blood smears are captured and cross-linked to form visible agglutinated particles, making the agglutination phenomenon easier to observe and identify with the naked eye or microscope. Metal ions (Al) 3+The addition of sialic acid helps maintain the charge stability and steric hindrance of the nanomaterial surface, preventing aggregation during storage and thus significantly improving the shelf life and reproducibility of the reagent. An alkaline environment (pH ≥ 8.0) is beneficial for enhancing the affinity between the TiO2 surface and sialic acid.

[0036] The technical solution of the present invention has the following advantages compared with the prior art:

[0037] (1) High sensitivity and specificity: The surface-modified TiO2 nanomaterial has a stronger enrichment ability for sialylated glycoproteins than traditional lectins, resulting in high detection sensitivity and good specificity.

[0038] (2) Excellent stability: By modifying the surface chemically and adding metal ions, the problem of nanomaterial aggregation is solved, and the shelf life of the reagent is significantly extended to more than 18 months.

[0039] (3) Low cost: Titanium dioxide nanomaterials are widely available and their price is much lower than that of many lectins, which reduces reagent costs.

[0040] (4) Simple operation: The detection process does not require complicated instruments. It only requires adding reagents, spreading, and observing, which is very suitable for large-scale screening in primary medical units. Attached Figure Description

[0041] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0042] Figure 1 This is a comparison diagram of the agglutination effect of the reagents of Comparative Example 1 (unmodified TiO2) and Example 3 (modified TiO2) on blood smears.

[0043] Figure 2 This refers to the dispersion stability of different reagents in this invention. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0046] Example 1: Preparation of surface-modified titanium dioxide nanomaterials

[0047] Weigh 1.0 g of titanium dioxide nanoparticles (P25, particle size approximately 21 nm) and disperse them in 100 mL of anhydrous ethanol. Sonicate the solution for 30 min. Slowly add 10 mL of an ethanol solution containing 0.1 g of APTES. Transfer the mixture to a three-necked flask and reflux in an oil bath at 70 °C for 3 h. After the reaction is complete, collect the solid product by centrifugation and wash it three times alternately with anhydrous ethanol and purified water. Dry the product overnight in a vacuum oven at 60 °C, then lightly grind it to obtain surface-aminated titanium dioxide nanomaterials.

[0048] Example 2: Preparation of sialylated glycoprotein detection reagent

[0049] This embodiment provides a sialylated glycoprotein detection reagent, the specific formula of which is as follows:

[0050] Surface-modified titanium dioxide nanomaterials prepared in Example 1: 30 mg / L;

[0051] Aluminum chloride (providing Al) 3+ ): 10 mg / L;

[0052] pH adjuster (sodium hydroxide): Adjust to pH 9.0;

[0053] Solvent: Purified water.

[0054] The preparation method of the sialylated glycoprotein detection reagent in this embodiment is as follows:

[0055] S1: Accurately place 10 mg of aluminum chloride in a beaker, add 800 mL of purified water, and stir magnetically for 2 h.

[0056] S2: Slowly adjust the pH of the above solution to 9.0 with 1 M NaOH solution, add purified water to a total volume of 1000 mL, and continue stirring for 30 min. Transfer the solution to a reagent bottle and let it stand for 8 h to mature.

[0057] S3: Filter the above solution using a 1.0 μm microporous membrane. Under aseptic conditions, add 30 mg of the surface-modified titanium dioxide nanomaterial prepared in Example 1 to the filtrate and stir magnetically for 1 h to disperse it evenly. Then, sonicate (300 W, 15 min) to obtain the final detection reagent, aliquot it, and store it at 4°C.

[0058] Example 3: Preparation of sialylated glycoprotein detection reagent

[0059] This embodiment provides a sialylated glycoprotein detection reagent, the specific formula of which is as follows:

[0060] Surface-modified titanium dioxide nanomaterials prepared in Example 1: 50 mg / L;

[0061] Aluminum chloride: 20 mg / L;

[0062] pH adjuster: Adjust to pH 10.0;

[0063] Solvent: Purified water.

[0064] The preparation method of the sialylated glycoprotein detection reagent in this embodiment is the same as that in Example 2.

[0065] Comparative Example 1:

[0066] This comparative example provides a sialylated glycoprotein detection reagent, which is similar to Example 2, except that the surface-modified titanium dioxide nanomaterials prepared in Example 1 are replaced with unmodified titanium dioxide nanopowder (P25).

[0067] Comparative Example 2:

[0068] This comparative example provides a sialylated glycoprotein detection reagent, which is similar to Example 2, except that aluminum chloride is not added.

[0069] Performance testing

[0070] (1) Comparison of the agglutination effects of reagents from Comparative Example 1 (unmodified TiO2) and Example 3 (modified TiO2) on blood smears is shown in the figure below. Figure 1 As shown, it can be seen that the agglomerate area in the negative sample of Example 3 is smaller than that in the positive sample; in Comparative Example 1, agglomerates with larger areas can be observed in both the negative and positive samples, indicating that Example 3 has a better application scenario.

[0071] (2) Dispersion stability test: The reagents prepared in Examples 2 and 3, and Comparative Examples 1 and 2 were stored at 4°C and observed at 0, 1, 3, and 6 months. The reagents in Examples 2 and 3 remained in a uniform suspension without precipitation. The reagent in Comparative Example 1 showed obvious precipitation within 1 month, and the particles were coarse after shaking. The reagent in Comparative Example 2 showed slight aggregation after 3 months, as shown in the results. Figure 2 As shown, after 18 months of storage, there was still no obvious coagulation and sedimentation phenomenon in Examples 2 and 3, indicating good stability.

[0072] (3) Detection performance test: Whole blood samples were collected from 20 clinically diagnosed cancer patients (positive samples) and 20 healthy volunteers (negative samples), and dried blood smears were prepared. 50 μL of the test reagent was added to each smear, spread evenly, and allowed to dry. The morphology and area of ​​the agglomerated particles were observed under an optical microscope. The results are shown in Tables 1 and 2. At the same time, the test effect after 6 months of reagent storage was compared and examined. The results are shown in Tables 3 and 4.

[0073] Table 1. Performance testing of different reagents for negative samples

[0074]

[0075] Table 2 Performance tests of different reagents for positive samples

[0076]

[0077] Table 3. Detection performance test of negative samples after 6 months of storage with different reagents.

[0078]

[0079] Table 4. Detection performance test of positive samples after 6 months of storage with different reagents.

[0080]

[0081] Results: The reagents of Examples 2 and 3 produced a large number of characteristic agglomerated particles in positive samples, while in negative samples they only showed a uniform colored background with no particles or only a very small number of particles, and the positive and negative concordance rates were both higher than 95%. The reagent of Comparative Example 1 produced non-uniform, non-specific large aggregates in both positive and negative samples, and could not effectively distinguish them. The reagent of Comparative Example 2 had similar detection performance to that of Example 2 in the initial stage, but after 6 months of storage, its agglomeration efficiency in positive samples decreased significantly, resulting in false negative results.

[0082] (4) Anti-interference test: Common interfering substances (such as bilirubin and triglycerides) were added to the samples. The detection results of Examples 2 and 3 were not significantly affected. The results are shown in Tables 5 and 6.

[0083] Table 5. Anti-dryness test of negative samples

[0084]

[0085] Table 6. Anti-dryness test of positive samples

[0086]

[0087] As can be seen from Tables 5 and 6, the modified titanium dioxide reagent can effectively prevent large bilirubin-albumin complexes and triglyceride chylomicrons from approaching and contacting the internal titanium dioxide surface. Therefore, it can effectively reduce the interference of such substances during the test.

[0088] The above results demonstrate that, through surface modification technology and formulation optimization, this invention has successfully prepared a high-performance sialylated glycoprotein detection reagent, which is significantly superior to the unmodified or unstabilized control group in terms of stability, sensitivity, and specificity.

[0089] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A reagent for detecting sialylated glycoproteins, characterized in that, It includes the following components: surface-modified titanium dioxide nanomaterials, metal ions, pH adjusters, and solvents; The surface-modified titanium dioxide nanomaterial is obtained by dispersing titanium dioxide nanoparticles in an organic solvent and reacting them with a silane coupling agent. The pH value of the sialylated glycoprotein detection reagent is ≥ 8.

0.

2. The sialylated glycoprotein detection reagent according to claim 1, characterized in that, The concentration of the surface-modified titanium dioxide nanomaterial is 10 mg / L-100 mg / L.

3. The sialylated glycoprotein detection reagent according to claim 1, characterized in that, The concentration of the metal ions is 4 mg / L-20 mg / L; The metal ion is an aluminum ion.

4. The sialylated glycoprotein detection reagent according to claim 1, characterized in that, The titanium dioxide nanoparticles have a particle size of 5 nm-25 nm.

5. The sialylated glycoprotein detection reagent according to claim 1, characterized in that, The silane coupling agent includes 3-aminopropyltriethoxysilane and / or 3-glyceroxypropyltrimethoxysilane.

6. The sialylated glycoprotein detection reagent according to claim 1, characterized in that, The mass ratio of the titanium dioxide nanoparticles to the silane coupling agent is 5:1-15:

1.

7. The sialylated glycoprotein detection reagent according to claim 1, characterized in that, The reaction temperature is 60℃-80℃; And / or, the reaction time is 2 h-4 h.

8. The sialylated glycoprotein detection reagent according to claim 1, characterized in that, The organic solvent is one or more selected from ethanol, glycerol and n-butanol; And / or, the pH adjuster is sodium hydroxide and / or Tris buffer.

9. The method for preparing the sialylated glycoprotein detection reagent according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Titanium dioxide nanoparticles are dispersed in an organic solvent, a silane coupling agent is added to react, and after centrifugation, washing, drying and grinding, surface-modified titanium dioxide nanomaterials are obtained. S2. Dissolve the metal ion salt in part of the solvent; adjust the pH of the resulting solution to ≥8.0, add the remaining solvent and stir evenly, let stand, and then separate the solid and liquid phases to obtain the liquid phase. S3. Mix the liquid phase obtained in step S2 with the surface-modified titanium dioxide nanomaterial obtained in step S1 to obtain the sialylated glycoprotein detection reagent.

10. The use of the sialylated glycoprotein detection reagent according to any one of claims 1-8 in the detection of sialylated glycoproteins, wherein the use does not relate to the diagnosis and treatment of diseases.

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