Preparation and application of a biotin functionalized composite fluorescent probe

Abstract

The application relates to a kind of biotin functional composite fluorescent probes (denoted as CdTe@cys-biotin) provided by the application and application thereof.Based on chemical oxidation strategy and high affinity between streptavidin and biotin, fluorescence amplification is realized and applied to detection of glycan expression on the surface of exosome for the first time.The fluorescent probe can not only quantitatively detect the glycan on the surface of exosome, but also highly accurately distinguish exosome from normal human and serum of nephropathy patient, and provide a powerful platform for sensitive detection of exosome in cancer diagnosis.

Description

Technical Field

[0001] This invention relates to the field of proteomics analysis, specifically to the preparation and application of a biotin-functionalized composite fluorescent probe. Background Technology

[0002] Exosomes are extracellular vesicles (30-150 nm in diameter) secreted by cells, carrying abundant molecular information, such as RNA and proteins from the mother cell. As specialized secreted vesicles, exosomes naturally exist in bodily fluids such as blood, urine, and cerebrovascular fluid, participating in tumorigenesis, signal transduction, and immune responses, and acting as messengers mediating intercellular communication. Recent studies have shown that abnormal expression of exosomes is often associated with tumorigenesis and development. Furthermore, exosomes have been found to be superior to other substances, such as circulating tumor vesicles and circulating tumor DNA, in terms of abundance and stability. Therefore, the isolation and extraction of exosomes from serum for analysis holds great potential for clinical diagnosis and treatment.

[0003] As one of the most common post-translational modifications, exosome glycosylation is associated with neurotransmission and cancer progression. Therefore, characterizing exosome glycosylation is crucial for understanding the biological significance of exosomes and exploring clinical biomarkers. Although researchers have recognized the importance of exosome glycosylation in physiological processes, the complexity of carbohydrate composition and the diversity of modification sites pose challenges to detecting the degree of glycosylation and conducting functional studies. Currently, several techniques have been reported, including Western blotting, lectin arrays, and mass spectrometry, for analyzing exosome-glycosylated proteins. Recently, fluorescence-based detection methods have attracted attention due to their advantages of rapid response and quantitative measurement. Fluorescent probes not only enhance the cell-target specificity of exosomes but also transform exosome-cell binding events into activatable signals for real-time monitoring. Because fluorescent probes are highly modifiable, designing novel fluorescent probes is of practical significance for identifying and labeling specific targets. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a biotin-functionalized composite fluorescent probe that uses a series of modification methods to achieve fluorescence amplification and applies this strategy to the quantitative detection of exosome surface glycans.

[0005] The technical solution to achieve the objective of this invention is to provide a method for preparing a biotin-functionalized composite fluorescent probe, comprising the following steps:

[0006] S1. Cadmium chloride and sodium telluride were dispersed in 50 mL of distilled water, L-cysteine, trisodium citrate dihydrate and sodium borohydride were added and mixed thoroughly, stirred and refluxed at a certain temperature, rapidly cooled and filtered through a 0.22 micron filter to prepare L-cysteine ​​modified CdTe nanocrystals in one pot.

[0007] S2. Biotin is used as an outer layer modification. The L-cysteine-modified CdTe nanocrystals obtained in step S1 are used as the matrix, and biotin is linked to L-cysteine ​​to form an outer layer modification.

[0008] Furthermore, the reaction temperature in S1 is 100 °C, and the reflux time is 45 minutes.

[0009] Furthermore, the reaction temperature in S2 is room temperature, and the reaction time is 12-13 h.

[0010] Furthermore, in step S1, the mass ratio of cadmium chloride, sodium telluride, and L-cysteine ​​is 1:(0.3~0.4):(1.7~1.8).

[0011] Furthermore, the biotin-functionalized composite fluorescent probe is used in fluorescence signal amplification strategies.

[0012] Further steps include: incubating Fe3O4@TiO2-CD63 aptamer@exosome with NaIO4 solution (1 mM, 500 μL) at 4 °C for 30 min to oxidize the sugars on the exosomes; adding a solution (500 μL) of aniline and biotinylate (10 mM: 100 μM) to the above sample; discarding the supernatant under the action of a magnet and washing the precipitate three times with PBS; finally, adding streptavidin-FITC and the prepared fluorescent probe, and incubating at room temperature for 30 min each.

[0013] Furthermore, the rotation speed during the incubation process is 1250 rpm.

[0014] Furthermore, the biotin-functionalized composite fluorescent probe is used to detect the expression of exosome surface glycans.

[0015] Further steps include: incubating Fe3O4@TiO2-CD63 aptamer@exosome with NaIO4 solution (1mM, 500μL) at 4°C for 30 minutes to oxidize the sugars on the exosomes; adding a solution (500μL) of aniline and biotinylate (10mM:100μM) to the above sample; discarding the supernatant under the action of a magnet and washing the precipitate three times with PBS; finally, adding streptavidin-FITC and the prepared fluorescent probe, and incubating at room temperature for 30 minutes each; dispersing the product in 2mL of water, and measuring and recording the fluorescence value at an excitation wavelength of 450nm.

[0016] Furthermore, the rotation speed during the incubation process is 1250 rpm.

[0017] After adopting the above technical solution, the present invention has the following positive effects:

[0018] (1) Compared with existing technologies, the preparation method of this invention can produce biotin-functionalized composite fluorescent probes. L-cysteine-modified CdTe nanocrystals were synthesized using a one-pot method, yielding uniformly sized nanowires. The excitation and emission wavelengths of the resulting CdTe nanocrystals could be adjusted by controlling the reaction time. In the aqueous synthesis of CdTe nanocrystals, L-cysteine ​​was used as a protective agent and surface coating agent, effectively reducing its toxicity and providing abundant functional groups on the surface for further modification through inter-functional reactions. The carboxyl group of D-biotin was activated by NHS / EDC coupling, and then reacted with an amino group to generate an amide bond, which was used to couple to the surface of CdTe@cys, successfully modifying the CdTe nanocrystals. The formed fluorescent probe can be specifically recognized and fluorescently labeled by biotin, which has an affinity for streptomycin.

[0019] (2) By optimizing the reaction time and raw material concentration, the present invention can adjust the microstructure and size of the biotin-functionalized composite fluorescent probe, thereby obtaining the material with the best performance and greatly improving the fluorescence amplification capability.

[0020] (3) The biotin-functionalized composite fluorescent probe synthesized in this invention has advantages such as simple synthesis route and high specificity in fluorescence amplification strategy and detection of exosome surface glycan expression, and has good application prospects.

[0021] (4) The fluorescent probe synthesized in this invention can not only quantitatively detect the glycans on the surface of exosomes, but also distinguish exosomes from the serum of normal people and patients with kidney disease with high accuracy, providing a powerful platform for sensitive detection of exosomes in cancer diagnosis. Attached Figure Description

[0022] 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...

[0023] Figure 1 X-ray diffraction image of CdTe@cys prepared in Example 1;

[0024] Figure 2 Transmission electron microscope image of the biotin-functionalized composite fluorescent probe prepared in Example 1;

[0025] Figure 3 The fluorescence lifetime diagram of the biotin-functionalized composite fluorescent probe prepared in Example 1;

[0026] Figure 4 Excitation and emission spectra of CdTe@cys nanocrystals prepared in Example 1 before and after biotin modification;

[0027] Figure 5 The spectrum showing the relationship between the fluorescence signal change and exosome concentration of the biotin-functionalized composite fluorescent probe prepared in Example 2 when quantitatively detecting exosome surface glycans derived from HeLa cells;

[0028] Figure 6 The spectrum of the biotin-functionalized composite fluorescent probe prepared in Example 3 for quantitative determination of exosome surface glycan expression levels in clinical serum samples; Detailed Implementation

[0029] Example 1: Preparation method of biotin-functionalized composite fluorescent probe

[0030] Specifically, the following steps are included:

[0031] S1. Disperse cadmium chloride (CdCl2, 29.3 mg) and sodium telluride (Na2TeO3, 8.86 mg) in 50 mL of distilled water, add L-cysteine ​​(50 mg), trisodium citrate dihydrate (100 mg) and sodium borohydride (50 mg) and mix thoroughly.

[0032] S2. Stir the mixture from step S2 at 100°C and reflux for 45 minutes, then rapidly cool and filter through a 0.22-micron filter to obtain a clear CdTe@cys nanocrystal solution.

[0033] S3. The prepared CdTe@cys nanocrystals were stored in the dark.

[0034] S4. Dissolve biotin (161.3 mg) in a solution of N,N-dimethylformamide (DMF, 10 mL) containing N-hydroxysuccinimide (NHS, 75.811 mg) and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, 131.4 mg).

[0035] S5. Stir the mixture from step S4 continuously at room temperature for 30 minutes, then transfer it to a brown bottle. Add the prepared CdTe@cys nanocrystal solution (50 mL) to the bottle and stir together overnight at room temperature. This yields the biotin-functionalized composite fluorescent probe.

[0036] S6. The prepared biotin-functionalized composite fluorescent probe should be stored in the dark.

[0037] The X-ray diffraction image (Bruker XRD (D4)) of the prepared CdTe@cys nanocrystals is shown below. Figure 1 As shown, the characteristic peaks of CdTe@cys nanocrystals appear on the (220) and (311) planes of the X-ray diffraction image. Transmission electron microscopy image of the biotin-functionalized composite fluorescent probe (FEI Tecnai F20, USA) is shown below. Figure 2 As shown, the TEM image reveals that the final product is a uniformly sized nanowire. The fluorescence lifetime diagram of the biotin-functionalized composite fluorescent probe is shown below. Figure 3 As shown, the fluorescence intensity decay was measured, and the fluorescence lifetime was 2.59 μs. The excitation and emission spectra of the prepared CdTe@cys nanocrystals before and after biotin modification are shown below. Figure 4 As shown, lines a and b represent the emission and excitation spectra of CdTe@cys, respectively; lines c and d represent the emission and excitation spectra of CdTe@cys-biotin, respectively. It can be seen that biotin coupling leads to a slight decrease in fluorescence intensity and a slight blue shift in the emission wavelength. These experimental results confirm the effective preparation of this biotin-functionalized composite fluorescent probe.

[0038] Example 2: Application of biotin-functionalized composite fluorescent probes in fluorescence signal amplification strategies

[0039] Taking HeLa cell-derived exosomes as an example, the specific steps include:

[0040] (1) Preparation of HeLa cell-derived exosome samples: HeLa cells were cultured in a humidified incubator at 37°C with 5% carbon dioxide in a medium supplemented with 10% FBS and 1% (v / v) penicillin-streptomycin. When the cells reached 70-80% confluence, they were separated from the medium and washed twice with PBS. After culturing the cells in serum-free medium for 24-48 hours, the medium was collected, centrifuged at 1000 g for 10 minutes, and then centrifuged at 4500 g for 10 minutes at 4°C to remove cell debris and large particles. The supernatant was filtered through a 0.22 μm filter, concentrated using a 100 kDa ultrafiltration tube, and finally centrifuged at 100,000 g for 2 hours at 4°C to obtain exosomes. HeLa cell-derived exosomes were stored at -80°C for further use.

[0041] (2) Fluorescence amplification: Fe3O4@TiO2-CD63 aptamer@exosome was incubated with NaIO4 solution (1 mM, 500 μL) at 4 °C for 30 min to oxidize the sugars on the exosomes. A solution of aniline and biotinylate (10 mM: 100 μM) (500 μL) was added to the above sample. The supernatant was discarded under the action of a magnet, and the precipitate was washed three times with PBS. Finally, streptavidin-FITC and the prepared fluorescent probe were added, and incubated at room temperature for 30 min each. The rotation speed during incubation was 1250 rpm.

[0042] (3) Measurement: After washing the above product three times with PBS, the solid was dispersed in 2 mL of water, and the fluorescence value was measured and recorded at an excitation wavelength of 450 nm. In order to quantitatively analyze the exosome surface glycans, the fluorescence signals detected at different concentrations were recorded. Figure 5 The fluorescence intensity increased with increasing exosome concentration. The results indicated a detection limit of 1.10 × 10⁻⁶. 2 particles / mL, at 3.30 × 10 2 - 3.30 × 10 6 A linear relationship can be established for particles / mL. The correlation equation is F = 400.36 + 36.99 log [exosomes] (R0)2 2 = 0.99343), where F is the fluorescence intensity of the corresponding exosome. The results indicate that the method for detecting glycans in complex samples based on high-purity exosome extraction is successful and can significantly increase the detection range of the signal probe.

[0043] Example 3: Application of biotin-functionalized composite fluorescent probe in detecting serum-derived exosome surface glycan expression

[0044] Specifically, the following steps are included:

[0045] (1) Preparation of serum-derived exosome samples: Serum samples were collected from multiple healthy volunteers and volunteers with kidney disease. First, the serum was centrifuged at 3000g for 3 minutes. The supernatant was collected and diluted with an equal volume of PBS, then centrifuged at 12000g for 45 minutes. The supernatant was then ultracentrifuged at 110000g for 2 hours, leaving a precipitate and an equal volume of liquid at the bottom. The mixture was filtered through a 0.22μm filter to remove cells, cell debris, and apoptotic vesicles. All centrifugation steps were performed at 4°C, and the product was stored at -80°C for further use. Serum-derived exosomes were stored at -80°C for further use.

[0046] (2) Fluorescence amplification: Fe3O4@TiO2-CD63 aptamer@exosome was incubated with NaIO4 solution (1 mM, 500 μL) at 4 °C for 30 min to oxidize the sugars on the exosomes. A solution of aniline and biotinylate (10 mM: 100 μM) (500 μL) was added to the above sample. The supernatant was discarded under the action of a magnet, and the precipitate was washed three times with PBS. Finally, streptavidin-FITC and the prepared fluorescent probe were added, and incubated at room temperature for 30 min each. The rotation speed during incubation was 1250 rpm.

[0047] (3) Measurement: After washing the above product three times with PBS, the solid was dispersed in 2 mL of water, and the fluorescence value was measured and recorded at an excitation wavelength of 450 nm. In the comparative experiment, the conditions remained unchanged except for the sample source. It can be observed that in all three experimental groups, the fluorescence detection value of the uremia and diabetic nephropathy groups was higher than that of the healthy individuals group. Figure 6 We also conducted related experiments on serum samples from patients with clear cell renal cell carcinoma and renal tumors, and the results similarly showed that the expression of exosome surface glycans in patients was significantly higher than in healthy individuals. This may be due to the high expression of glycans on exosomes caused by the disease. The feasibility of this assay for quantitative detection of exosome surface glycans and the success of the initial sequential labeling process are discussed.

[0048] 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 biotin-functionalized composite fluorescent probe in a fluorescence signal amplification strategy, characterized in that, The biotin-functionalized composite fluorescent probe described herein is used for a fluorescence signal amplification strategy, comprising the following steps: Fe3O4@TiO2-CD63aptamer@exosome is incubated with 1mM, 500μL NaIO4 solution at 4°C for 30 minutes to oxidize the sugars on the exosomes; 500μL of a 10mM:100μM solution of aniline and biotinylate is added to the above sample; the supernatant is discarded under the action of a magnet, and the precipitate is washed three times with PBS; finally, streptavidin-FITC and the prepared fluorescent probe are added, and incubated at room temperature for 30 minutes each. The preparation method of biotin-functionalized composite fluorescent probe includes the following steps: S1. Cadmium chloride and sodium telluride were dispersed in 50 mL of distilled water, L-cysteine, trisodium citrate dihydrate and sodium borohydride were added and mixed thoroughly. The mixture was stirred and refluxed at a certain temperature, rapidly cooled and filtered through a 0.22 μm filter to prepare L-cysteine-modified CdTe nanocrystals in one pot. S2. Biotin is used as an outer layer modification. The L-cysteine-modified CdTe nanocrystals obtained in step S1 are used as the matrix, and biotin is linked to L-cysteine ​​to form an outer layer modification. The application is not for disease diagnosis or treatment.

2. The application of the biotin-functionalized composite fluorescent probe according to claim 1 in a fluorescence signal amplification strategy, characterized in that, The rotation speed during the incubation process is 1250 rpm.

3. The application of a biotin-functionalized composite fluorescent probe in the detection of exosome surface glycan expression, characterized in that, The biotin-functionalized composite fluorescent probe is used to detect the expression of exosome surface glycans; the steps include: incubating Fe3O4@TiO2-CD63aptamer@exosome with 1mM, 500μL NaIO4 solution at 4℃ for 30 minutes to oxidize the sugars on the exosomes; adding 500μL of a 10mM:100μM solution of aniline and biotinylate to the above sample; discarding the supernatant under the action of a magnet, and washing the precipitate three times with PBS; finally, adding streptavidin-FITC and the prepared fluorescent probe, and incubating at room temperature for 30 minutes each; dispersing the product in 2mL of water, and measuring and recording the fluorescence value at an excitation wavelength of 450nm; The preparation method of biotin-functionalized composite fluorescent probe includes the following steps: S1. Cadmium chloride and sodium telluride were dispersed in 50 mL of distilled water, L-cysteine, trisodium citrate dihydrate and sodium borohydride were added and mixed thoroughly. The mixture was stirred and refluxed at a certain temperature, rapidly cooled and filtered through a 0.22 μm filter to prepare L-cysteine-modified CdTe nanocrystals in one pot. S2. Biotin is used as an outer layer modification. The L-cysteine-modified CdTe nanocrystals obtained in step S1 are used as the matrix, and biotin is linked to L-cysteine ​​to form an outer layer modification. The application is not for disease diagnosis or treatment.

4. The application of the biotin-functionalized composite fluorescent probe according to claim 3 in the detection of exosome surface glycan expression, characterized in that, The rotation speed during the incubation process is 1250 rpm.

5. The application according to claim 1 or claim 3, characterized in that, The reaction temperature in S1 is 100°C, and the reflux time is 45 minutes.

6. The application according to claim 1 or claim 3, characterized in that, The reaction temperature in S2 is room temperature, and the reaction time is 12-13 hours.

7. The application according to claim 1 or claim 3, characterized in that, In step S1, the mass ratio of cadmium chloride, sodium telluride, and L-cysteine ​​is 1:(0.3-0.4):(1.7-1.8).

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