Preparation methods and applications of β-cyclodextrin-functionalized covalent organic framework composite materials

Abstract

This invention relates to a method for preparing a β-cyclodextrin functionalized covalent organic framework composite material. The method involves an aldehyde-imine condensation reaction between the aldehyde groups on 2,3,4-trihydroxybenzaldehyde and p-phthalaldehyde and the amino group on hepta(6-amino-6-deoxy)betacyclodextrin to construct a β-CD-COF layer. This one-step method produces a β-cyclodextrin covalent organic framework composite material that exhibits excellent enrichment capacity for phosphopeptides, facilitating purification and extraction. It also shows promising application prospects in mass spectrometry detection and exosome separation.

Description

Technical Field

[0001] This invention relates to the field of advanced nanomaterials technology, and in particular to a method for preparing and applying a β-cyclodextrin-functionalized covalent organic framework composite material. Background Technology

[0002] Exosomes are tiny bilayered phospholipid membrane vesicles (typically 30-150 nm in size). Increasingly recognized as important carriers of intercellular communication, tumorigenesis, signal transduction, and immune responses, exosomes have become a promising source for discovering biomarkers. With the deepening of exosome research, evidence suggests that exosomes participate in the pathological processes of various diseases and even serve as important biomarkers for some diseases, enabling their early detection. To better utilize these unique properties of exosomes for disease diagnosis and treatment, the isolation and capture of exosomes from biological samples is crucial. Ultracentrifugation is a relatively traditional separation method, but it suffers from problems such as cumbersome operation, long processing time, and low efficiency. Therefore, methods for separating exosomes from samples still need further improvement, necessitating the exploration of innovative, low-cost, and efficient methods for separating and capturing exosomes.

[0003] Proteins in exosomes can largely reflect the physiological and pathological state of primitive cells, and the state of post-translational modifications (PTMs) is a key determinant of cell physiology. Comprehensive characterization of PTMs in exosomes is particularly valuable for early diagnosis and disease state monitoring. Protein phosphorylation is one of the most common and important PTMs in eukaryotes. They regulate a variety of cellular responses, including signal transduction, immune responses, and cell proliferation. However, due to the limited number of exosomes, low levels of phosphorylated proteins, and the low abundance and high dynamism of phosphorylated proteins compared to ordinary proteins in complex biological samples, highly specific enrichment of phosphorylated proteomics samples is a crucial step in reducing sample complexity and increasing the number of phosphorylation sites identified. To date, the most commonly used method for separating and enriching phosphopeptides is immobilized metal ion affinity chromatography (IMAC). IMAC is applied to the separation and enrichment of phosphopeptides based on the chelation between the phosphate groups on the phosphorylated peptides and immobilized metal ions. A significant drawback of IMAC is that some peptides containing acidic amino acid residues are also retained, thus interfering with the detection of phosphopeptides. Therefore, there is an urgent need for an innovative method to separate and enrich exosomes and phosphopeptides to facilitate comprehensive research. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a Ti-based... 4+Functionalized β-CD-COF materials can be applied to the specific enrichment of phosphopeptides and the separation of exosomes.

[0005] The technical solution to achieve the objective of this invention is a method for preparing a β-cyclodextrin-functionalized covalent organic framework composite material, comprising the following steps:

[0006] S1. At a certain temperature, 2,3,4-trihydroxybenzaldehyde (THBA), p-phthalaldehyde (TPA) and hepta-(6-amino-6-deoxy)beta-cyclodextrin are used as ligands to form a β-CD-COF layer.

[0007] S2. At a certain temperature, the product obtained in step S1 is dispersed into a titanium sulfate solution for reaction.

[0008] S3. The product obtained in step S2 is thoroughly washed with deionized water and ethanol and then vacuum dried to obtain the β-cyclodextrin-functionalized covalent organic framework composite material.

[0009] Furthermore, the reaction temperature in S1 is 25°C, and the reaction time is 50 hours.

[0010] Furthermore, the reaction temperature in S2 is 25°C, and the reaction time is 4–6 hours.

[0011] Furthermore, in step S1, the mass ratio of 2,3,4-trihydroxybenzaldehyde (THBA), p-phthalaldehyde (TPA), and heptadecano(6-amino-6-deoxy)betacyclodextrin is 1:(1.0-1.2):(1.4-1.5).

[0012] Application of a β-cyclodextrin-functionalized covalent organic framework composite material in mass spectrometry detection, wherein the β-cyclodextrin-functionalized covalent organic framework composite material is used for selective enrichment of phosphorylated peptides.

[0013] Furthermore, this includes the following steps:

[0014] A. The β-cyclodextrin-functionalized covalent organic framework composite material was dispersed in 100 μL centrifuge tubes containing phosphorylated peptides, 0.5% trifluoroacetic acid (v / v), and 49.5% acetonitrile (v / v), and enriched at 37 °C for 45 min.

[0015] B. Wash thoroughly with a buffer solution of 0.5% trifluoroacetic acid and 49.5% acetonitrile, and then centrifuge.

[0016] C. Elute with 10 μL of 0.4 M ammonia for 15 min, take 1 μL of the eluent and spot the target, then perform MALDI TOF MS mass spectrometry analysis.

[0017] Application of a β-cyclodextrin-functionalized covalent organic framework composite material in serum exosome separation, wherein the β-cyclodextrin-functionalized covalent organic framework composite material is used to capture and separate exosomes.

[0018] Furthermore, this includes the following steps:

[0019] (1) The β-cyclodextrin-functionalized covalent organic framework composite material was dispersed into a 10 μL centrifuge tube containing serum and enriched at 4℃ for 45 min.

[0020] (2) Wash thoroughly with PBS solution;

[0021] (3) Elute with 20 μL of 0.4 M ammonia for 30 min, and perform relevant analysis on the eluent.

[0022] Furthermore, the correlation analysis described in step (3) is a Western blot analysis.

[0023] After adopting the above technical solution, the present invention has the following positive effects:

[0024] (1) The β-cyclodextrin-functionalized covalent organic framework composite material prepared by this invention is a one-step synthesis of functionalized probes, which is convenient and efficient.

[0025] (2) The β-cyclodextrin-functionalized covalent organic framework composite material prepared in this invention constructs a β-CD-COF layer through an aldehyde-imine condensation reaction between the aldehyde groups on 2,3,4-trihydroxybenzaldehyde and p-phthalaldehyde and the amino group on hepta(6-amino-6-deoxy)betacyclodextrin. This results in a material with a large specific surface area and tunable pore size. The successful grafting of Ti is achieved through the chelating ability of hydroxyl groups on Ti ions.

[0026] (3) The preparation method of the present invention is simple, the reaction is mild and controllable, and the obtained material has a good enrichment ability for phosphopeptides, which is beneficial for purification and extraction.

[0027] (4) By optimizing the raw material concentration, the present invention can adjust the microstructure and size of the β-cyclodextrin functionalized covalent organic framework composite material, thereby obtaining the material with the best performance and greatly improving the ability to enrich phosphopeptides.

[0028] (5) The β-cyclodextrin-functionalized covalent organic framework composite material prepared by this invention is simple to operate and time-saving in mass spectrometry detection and exosome separation applications, and the detection results are accurate and specific, showing great application prospects. Attached Figure Description

[0029] 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...

[0030] Figure 1 A scanning electron microscope image of the β-cyclodextrin-functionalized covalent organic framework composite material prepared in Example 1;

[0031] Figure 2 The infrared spectrum of the β-cyclodextrin-functionalized covalent organic framework composite material prepared in Example 1;

[0032] Figure 3 The elemental analysis diagram is shown for the β-cyclodextrin-functionalized covalent organic framework composite material prepared in Example 1.

[0033] Figure 4 The mass spectrum of phosphorylated peptides in β-casein enriched in the β-cyclodextrin-functionalized covalent organic framework composite material prepared in Example 2 is shown.

[0034] Figure 5 The mass spectrum of the β-cyclodextrin-functionalized covalent organic framework composite material prepared in Example 2 is the detection limit (0.5 fmol) of phosphorylated peptides.

[0035] Figure 6 The mass spectrum of phosphorylated peptides in the β-cyclodextrin-functionalized covalent organic framework composite material of Example 3 is shown.

[0036] Figure 7 The mass spectrum of phosphorylated peptides from human serum, a complex biological sample, enriched by the β-cyclodextrin-functionalized covalent organic framework composite material of Example 4.

[0037] Figure 8 Transmission electron microscopy image of serum exosomes captured by the β-cyclodextrin-functionalized covalent organic framework composite material in Example 5;

[0038] Figure 9 The results of Western blot analysis of serum exosomes captured by the β-cyclodextrin-functionalized covalent organic framework composite material in Example 5 are shown. Detailed Implementation

[0039] Example 1: Preparation of β-cyclodextrin-functionalized covalent organic framework composite materials

[0040] Includes the following steps:

[0041] S1. 2,3,4-Trihydroxybenzaldehyde (THBA), p-phthalaldehyde (TPA) and heptadecano(6-amino-6-deoxy)betacyclodextrin were used as ligands in a mass ratio of 1:(1.0~1.2):(1.4~1.5) and subjected to an aldimine condensation reaction at 25°C for 50 h to form a β-CD-COF layer.

[0042] S2. Disperse the product obtained in step S1 into a titanium sulfate solution and react for 6 hours at 25°C.

[0043] S3. The product obtained in step S2 is thoroughly washed with deionized water and ethanol and then vacuum dried to obtain the β-cyclodextrin-functionalized covalent organic framework composite material.

[0044] Scanning electron microscope (SEM) images of the prepared β-cyclodextrin-functionalized covalent organic framework composite material (20 kV, Philips XL30 electron microscope, Netherlands) are shown below. Figure 1 As shown, the SEM image reveals that the final product is a uniform sphere with a size of 400-500 nm, giving the material a large specific surface area and tunable pore size. The Fourier transform infrared spectrum (Thermo Fisher Scientific NIcoleti S10, USA) is shown below. Figure 2 As shown, the infrared spectrum of 2,3,4-trihydroxybenzaldehyde (THBA) shows that the stretching vibration of C=O occurs at 1785 cm⁻¹. -1 A band appears at 2775cm. -1 and 2810cm -1 The vibration at this point is related to the -CH stretching of the aldehyde group. FT-IR spectroscopy shows that the stretching vibration of p-phthalaldehyde (TPA) at 1693 cm⁻¹ is due to the C=O stretching vibration. -1 and 1769cm -1 An absorption peak appears at 1597 cm⁻¹. The infrared spectrum of β-CD COF shows an absorption peak at 1597 cm⁻¹. -1 and 3322cm -1 A new absorption peak was observed, confirming the chemical reaction between the material's ligands. The above analysis demonstrates the chemical modification process of this material. The elemental analysis diagram is shown below. Figure 3 As shown, the presence of C, N, O and Ti elements was observed, indicating that the β-cyclodextrin-functionalized covalent organic framework composite material was successfully synthesized.

[0045] Example 2: Application of β-cyclodextrin-functionalized covalent organic framework composite materials in enriching phosphorylated peptides in β-casein

[0046] It includes the following steps:

[0047] (1) Sample preparation: β-casein was enzymatically hydrolyzed in 25 mmol / L NH4HCO3 solution at 37℃ for 16 h.

[0048] (2) Enrichment: 0.5 mg of the β-cyclodextrin-functionalized covalent organic framework composite material obtained in Example 1 was dispersed into 100 μL of centrifuge tube containing 0.5% v / v trifluoroacetic acid and 49.5% v / v acetonitrile, and then 1 μL of the sample prepared in step (1) was added. The mixture was enriched at 37 °C for 45 min. The mixture was washed and separated three times with a buffer solution containing 0.5% v / v trifluoroacetic acid and 49.5% v / v acetonitrile. The mixture was then eluted with 10 μL of 0.4 M ammonia for 30 min and the supernatant was obtained.

[0049] (3) Mass spectrometry analysis: Take 1 μL of the supernatant obtained in step (2) and spot it onto the target for mass spectrometry analysis. The mass spectrum is shown below. Figure 4 As shown, adding 1 μL of β-casein for enrichment resulted in the capture of 13 peaks belonging to phosphopeptides. To further explore the detection limit performance of the material for phosphopeptides, the concentration of β-casein was varied in the experiment. Figure 5 The detection limit (0.5 fmol) for enriching phosphorylated peptides in β-casein using a β-cyclodextrin-functionalized covalent organic backbone indicates that a single peak belonging to a phosphopeptide can still be detected even at a β-casein concentration of 0.5 fmol. This experimental result demonstrates that the synthesized IMAC affinity material exhibits excellent enrichment ability and affinity for phosphopeptides.

[0050] Example 3: Application of β-cyclodextrin-functionalized covalent organic framework composite material in enriching phosphorylated peptides in mixed proteins

[0051] It includes the following steps:

[0052] (1) Sample preparation: Bovine serum albumin (BSA) was first reduced and alkylated with dithiothreitol and iodoacetamide, and then enzymatically digested at 37°C for 16 h; β-casein was enzymatically digested in 25 mM NH4HCO3 solution at 37°C for 16 h; Bovine serum albumin (BSA) and β-casein were added to centrifuge tubes containing 0.5% trifluoroacetic acid and 49.5% acetonitrile at a molar ratio of 1000:1.

[0053] (2) Enrichment: 0.5 mg of the β-cyclodextrin-functionalized covalent organic framework composite material obtained in Example 1 was dispersed into 100 μL of centrifuge tube containing the phosphorylated peptide from step (1) in 0.5% v / v trifluoroacetic acid and 49.5% v / v acetonitrile, and enriched at 37°C for 45 min; washed and separated 3 times with buffer of 0.5% v / v trifluoroacetic acid and 49.5% v / v acetonitrile; eluted with 10 μL of 0.4 M v / v ammonia for 30 min.

[0054] (3) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (2) and spot it onto the target. Use DHB as the matrix for mass spectrometry analysis. The mass spectrum of phosphorylated peptides in the β-cyclodextrin-functionalized covalent organic framework composite protein mixture (non-phosphorylated protein BSA: phosphorylated protein β-casein = 1000:1) is shown below. Figure 6 As shown, the β-cyclodextrin-functionalized covalent organic framework composite material can still capture signals from peptides belonging to phosphorylated proteins even in the presence of interfering proteins, indicating that the material has excellent selectivity for phosphopeptides.

[0055] Example 4: Application of β-cyclodextrin-functionalized covalent organic framework composite material in the enrichment of phosphorylated peptides in human serum

[0056] It includes the following steps:

[0057] (1) Sample preparation: The obtained human serum (1.5 mL) was added to a mixed solution of 0.2% TFA and 37% hydrochloric acid (0.1 mL / 0.1 mL, v / v), and then the supernatant was collected by centrifugation at 3000 rpm for 7 min. The collected supernatant (1.5 mL) was further diluted with deionized water (3 mL) and stored at -20℃.

[0058] (2) Enrichment: 0.5 mg of the β-cyclodextrin-functionalized covalent organic framework composite material obtained in Example 1 was dispersed into 100 μL of centrifuge tube containing the phosphorylated peptide from step (1) in 0.5% v / v trifluoroacetic acid and 49.5% v / v acetonitrile, and enriched at 37°C for 45 min; washed and separated 3 times with buffer of 0.5% v / v trifluoroacetic acid and 49.5% v / v acetonitrile; eluted with 10 μL of 0.4 M v / v ammonia for 30 min.

[0059] (3) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (2) and spot it onto the target. Use DHB as the matrix for mass spectrometry analysis. The mass spectrum is shown below. Figure 7 As shown, human serum was selected as the actual sample to verify whether the material can analyze complex biological samples. After enrichment with the material, four characteristic phosphopeptide peaks were captured in human serum, indicating that the material has great potential for practical applications.

[0060] Example 5: Application of β-cyclodextrin-functionalized covalent organic framework composite material in the capture and separation of exosomes.

[0061] It includes the following steps:

[0062] (1) Sample preparation: Dilute the serum sample with an equal volume of PBS buffer solution to reduce viscosity; then centrifuge the diluted serum sample at 3000×g for 3 minutes at 4°C; transfer the supernatant to a centrifuge tube and centrifuge at 12000×g for 45 minutes at 4°C; then filter the supernatant through a 0.22μm filter.

[0063] (2) Enrichment: 10 μL of serum was added to 3 mg of the β-cyclodextrin-functionalized covalent organic framework composite material obtained in Example 1; the mixture was shaken at 4 °C for 45 min; it was washed thoroughly with PBS solution, and finally eluted at 4 °C for 30 min with 20 μL of 0.4 M NH3·H2O; the transmission electron microscope image of the serum exosomes captured by the composite material is shown below. Figure 8 As shown, TEM images reveal that exosomes are vesicles with a diameter of approximately 30-150 nm, indicating the successful capture and separation of exosomes by the β-cyclodextrin-functionalized covalent organic framework composite material.

[0064] Western blot analysis: First, three identical samples containing exosome eluent were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a Hepes-Tris gel and then transferred to polyvinylidene fluoride (PVDF) microporous membranes. The membranes were then cut into three slices, one sample per slice, and treated with an inhibitor for 1 hour. After washing with 1×TBST, the three membranes were incubated overnight in dilutions of CD9, CD63, and TSG101 primary antibodies, respectively. They were then washed four times with diluted Tris-buffered saline (1×TBST), and all membranes were incubated for 2 hours in dilution of secondary antibody (i.e., anti-rabbit antibody labeled with hymenoplastin). Finally, the membranes were treated with an electrochemiluminescence (ECL) reagent and imaged using a ChemiDoc™ touch imaging system (Bio-Rad Laboratories). The resulting blots of the protein samples are shown below. Figure 9 As shown, after separation by material capture, the exosome marker protein bands TSG101, CD63, and CD9 can be clearly observed, providing a practical approach for developing novel and convenient separation strategies.

[0065] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a β-cyclodextrin-functionalized covalent organic framework composite material in mass spectrometry detection, characterized in that, The β-cyclodextrin-functionalized covalent organic framework composite material is used for the selective enrichment of phosphorylated peptides; The preparation method of the β-cyclodextrin-functionalized covalent organic framework composite material includes the following steps: S1. At a certain temperature, 2,3,4-trihydroxybenzaldehyde, terephthalaldehyde and hepta(6-amino-6-deoxy)betacyclodextrin are used as ligands to form a β-CD-COF layer; S2. At a certain temperature, the product obtained in step S1 is dispersed into a titanium sulfate solution for reaction. S3. The product obtained in step S2 is thoroughly washed with deionized water and ethanol and then vacuum dried to obtain the β-cyclodextrin-functionalized covalent organic framework composite material.

2. The application of a β-cyclodextrin-functionalized covalent organic framework composite material in serum exosome separation, characterized in that, The β-cyclodextrin-functionalized covalent organic framework composite material is used to capture and separate exosomes; The preparation method of the β-cyclodextrin-functionalized covalent organic framework composite material includes the following steps: S1. At a certain temperature, 2,3,4-trihydroxybenzaldehyde, terephthalaldehyde and hepta(6-amino-6-deoxy)betacyclodextrin are used as ligands to form a β-CD-COF layer; S2. At a certain temperature, the product obtained in step S1 is dispersed into a titanium sulfate solution for reaction. S3. The product obtained in step S2 is thoroughly washed with deionized water and ethanol and then vacuum dried to obtain the β-cyclodextrin-functionalized covalent organic framework composite material.

3. The application according to claim 1 or 2, characterized in that, The reaction temperature in S1 is 25°C, and the reaction time is 50 hours.

4. The application according to claim 1 or 2, characterized in that, The reaction temperature in S2 is 25℃, and the reaction time is 4 to 6 hours.

5. The application according to claim 1 or 2, characterized in that, In step S1, the mass ratio of 2,3,4-trihydroxybenzaldehyde, terephthalaldehyde, and hepta(6-amino-6-deoxy)betacyclodextrin is 1:(1.0-1.2):(1.4-1.5).

6. The application of the β-cyclodextrin-functionalized covalent organic framework composite material according to claim 1 in mass spectrometry detection, characterized in that, Includes the following steps: A. The β-cyclodextrin-functionalized covalent organic framework composite material was dispersed in 100 μL centrifuge tubes containing phosphorylated peptides, 0.5% trifluoroacetic acid (v / v), and 49.5% acetonitrile (v / v), and enriched at 37 °C for 45 min. B. Wash thoroughly with a buffer solution of 0.5% trifluoroacetic acid and 49.5% acetonitrile, and then centrifuge. C. Elute with 10 μL of 0.4 M ammonia for 15 min, take 1 μL of the eluent and spot the target for MALDI TOFMS mass spectrometry analysis.

7. The application of the β-cyclodextrin-functionalized covalent organic framework composite material according to claim 2 in serum exosome separation, characterized in that, Includes the following steps: (1) The β-cyclodextrin-functionalized covalent organic framework composite material was dispersed into a 10 μL centrifuge tube containing serum and enriched at 4 °C for 45 min; (2) Wash thoroughly with PBS solution; (3) Elute with 20 μL of 0.4 M ammonia for 30 min and perform relevant analysis on the eluent.

8. The application of the β-cyclodextrin-functionalized covalent organic framework composite material according to claim 7 in serum exosome separation, characterized in that, The correlation analysis mentioned in step (3) is a Western blot analysis.

Images