Ratio type fluorescent probe for detecting sialic acid as well as preparation method and application of ratio type fluorescent probe
By preparing a ratio carbon quantum dot fluorescence probe, the double emission peak fluorescence intensity ratio is used to detect sialic acid, which solves the problem of traditional fluorescence methods being susceptible to environmental interference, and achieves high sensitivity and high accuracy sialic acid detection.
Patent Information
- Application Number
- CN202510488886.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-18
- Publication Date
- 2025-07-18
AI Technical Summary
Traditional fluorescence methods are susceptible to environmental interference when detecting sialic acid, resulting in inaccurate detection.
A ratio carbon quantum dot fluorescent probe was used to prepare hydrothermal reactions between aromatic amine chemicals and phenylboric acid chemicals, and a carbon quantum dot fluorescent probe with double emission peaks was prepared, and the double emission peak fluorescence intensity ratio was used for sialic acid detection.
It realizes high sensitivity and high accuracy sialic acid detection, reducing the influence of environmental and instrument factors, and the detection results are good reproducible.
Smart Images
Figure CN120329945A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a ratiometric fluorescent probe for detecting sialic acid and its application. Background Art
[0002] Sialic acid is ubiquitously present in various types of cells in the human body, and the latter plays a crucial role in signal transduction on the surface of various tissue cells, especially in the central nervous system. For example, most of the sialic acid in the brain tissue binds to gangliosides, accounting for about 65% of the total sialic acid content in the brain tissue; about 32% of the sialic acid binds to glycoproteins, and the rest exists in free form. The high content of sialic acid is an important material basis for the function of the nervous system, which means that it may have unique structures and functions in the nervous system. During the critical period of brain development, the intervention of sialic acid may have long-term irreversible effects on the brain. Secondly, the content of sialic acid affects the biochemical properties and functions of glycoproteins and lipids, masks potential glycans, mediates cell adhesion and migration, and forms sialic acid-binding proteins such as factor H, selectins, and lectins. Factor H can bind to sialic acid on the surface of host cells to avoid complement activation; selectin substances interact with sialic acid during lymphocyte transport and extravasation to the inflammatory site; sialic acid undergoes immunomodulation through interactions with immunoglobulins such as lectins. In addition to its important role in physiological processes, many diseases are related to sialic acid and its biosynthesis. For example, a variety of human pathogens use sialic acid on the surface of host cells as an attachment site or modify their surface with host sialic acid as molecular mimicry to avoid recognition by the immune system. The abnormal expression of sialylglycans accelerates tumor growth, metastasis, and limits the anti-tumor immune response. In addition, genetic defects in sialic acid biosynthesis have been identified and may lead to numerous clinical phenotypes such as myopathy, sialuria, and abnormal neuronal development. The importance of sialic acid expression and recognition in human biology and pathology makes it a potential therapeutic target. Therefore, the detection of sialic acid content in the human body is not only the basis for studying biological functions but also the key to the future application of sialic acid in bioprocesses or therapies.
[0003] With the rapid development of nanotechnology, various methods for detecting sialic acid have been developed, such as electrochemical methods, colorimetric methods, and fluorescence methods. Among them, the fluorescence method has particular advantages due to its high sensitivity. Fluorescent probes are constructed by functionalizing luminescent materials with boric acid. Sialic acid can be specifically recognized by the fluorescent probe, causing an increase or decrease in the signal intensity of the probe. However, it is difficult to use the change in single fluorescence intensity as a reliable quantitative basis for sialic acid detection. In such biomolecule detections, environmental interference is difficult to completely eliminate. It is necessary to introduce additional verification to ensure the accuracy of the detection results.
[0004] Benefiting from the remarkable self-calibration ability, ratiometric fluorescent probes have become a promising tool for detecting biomolecules. The change in the fluorescence intensity ratio serves as a quantitative reference, effectively reducing the influence of environmental and instrumental factors. Carbon quantum dots have high application potential as ratiometric fluorescent probes due to their good water dispersibility and easy surface functionalization. Carbon quantum dots can be further optimized into ratiometric fluorescent probes for the selective recognition of sialic acid through additional boronic acid functionalization and multicolor emission regulation. Summary of the Invention
[0005] The object of the present invention is to solve the problem that traditional fluorescence methods rely on a single peak and are susceptible to environmental interference and inaccurate detection. A ratiometric fluorescent probe for detecting sialic acid is provided, which can achieve highly sensitive, highly accurate, and simple rapid detection of the target sialic acid. Its operation process is simple, the cost is low, and the detection results have good reproducibility.
[0006] To achieve the above object, the technical solution of the present invention is as follows: A ratiometric carbon quantum dot fluorescent probe for detecting sialic acid is prepared by the following method:
[0007] 1) Take an aromatic amine chemical substance, dissolve it in a solvent at room temperature, stir slowly, and after the solute is completely dissolved, add a benzene boronic acid-containing chemical substance, and continue to stir slowly until the solution is homogeneous and transparent.
[0008] 2) Transfer the mixed solution to a polytetrafluoroethylene hydrothermal autoclave for hydrothermal reaction. After the reaction is completed and the solution temperature drops to room temperature, centrifuge, transfer the supernatant to a dialysis bag for dialysis, and then perform freeze-drying purification after dialysis is completed.
[0009] Yellow powder carbon quantum dots are obtained.
[0010] For the above ratiometric carbon quantum dot fluorescent probe for detecting sialic acid, in step 1), the mass ratio of the aromatic amine chemical substance to the benzene boronic acid-containing chemical substance is 1:5 - 10:1.
[0011] For the above ratiometric carbon quantum dot fluorescent probe for detecting sialic acid, in step 1), the aromatic amine chemical substance is one of o-phenylenediamine, p-phenylenediamine, m-phenylenediamine, o-toluidine, dimethylaniline, 4-methyl-m-phenylenediamine.
[0012] For the above ratiometric carbon quantum dot fluorescent probe for detecting sialic acid, in step 1), the solvent is one of methanol, ethanol, ether, acetone, N,N-dimethylformamide.
[0013] The above-mentioned ratiometric carbon quantum dot fluorescent probe for detecting sialic acid. In step 1), the boronic acid-containing chemical can be 2-hydroxymethylphenylboronic acid, 2,3-dimethylphenylboronic acid, 3,4-dimethylphenylboronic acid, 3-vinylphenylboronic acid, 2-carbamoylphenylboronic acid, 3,5-dimethoxyphenylboronic acid, 2-butoxyphenylboronic acid.
[0014] The above-mentioned ratiometric carbon quantum dot fluorescent probe for detecting sialic acid. In step 2), the hydrothermal reaction is carried out at 160 - 260 °C for 12 - 48 hours.
[0015] The above-mentioned ratiometric carbon quantum dot fluorescent probe for detecting sialic acid. In step 2), the centrifugation is carried out at 3000 - 8000 r / min for 5 - 30 minutes.
[0016] The above-mentioned ratiometric carbon quantum dot fluorescent probe for detecting sialic acid. In step 2), the dialysis is to take the supernatant and transfer it to a dialysis bag with a molecular weight cut-off of 200 - 1000 Da, and dialyze for 6 - 24 hours.
[0017] Application of the above-mentioned ratiometric carbon quantum dot fluorescent probe in identifying sialic acid molecules.
[0018] The above-mentioned application, the method is as follows:
[0019] 1) Preparation of the ratiometric carbon quantum dot fluorescent probe dispersion: Take the above-mentioned ratiometric carbon quantum dot fluorescent probe, add it to distilled water, and gently shake to make the solution uniform;
[0020] 2) Sialic acid molecule recognition: Mix the solution containing sialic acid molecules to be tested with the fluorescent probe dispersion obtained in step 1, and let it stand at room temperature for 1 - 10 minutes to obtain a mixed fluorescent probe - analyte dispersion;
[0021] 3) Test the emission spectrum of the mixed dispersion obtained in step 2, obtain the fluorescence intensities of the dual emission peaks, and take the ratio of the fluorescence intensity with a smaller wavelength (390 - 440 nm) to the fluorescence intensity with a larger wavelength (550 - 600 nm) as the analysis result. Description of the Drawings
[0022] Figure 1 It is a transmission electron microscope image of dual-emission carbon quantum dots.
[0023] Figure 2 Fluorescence spectrum of carbon quantum dots excited at 360 nm. Detailed Description of the Invention
[0024] Example 1 Preparation of the ratiometric carbon quantum dot fluorescent probe and method for detecting sialic acid
[0025] 1. Preparation method
[0026] 1) Take 1 g of o-phenylenediamine, dissolve it in 10 mL of ethanol at room temperature, stir slowly. After the solute is completely dissolved, add 0.1 g of 2-hydroxymethylphenylboronic acid, and continue to stir slowly until the solution is homogeneous and transparent.
[0027] 2) Transfer the mixed solution obtained in step 1 to a polytetrafluoroethylene hydrothermal reactor, and keep it at 160 °C for 12 hours. After the reaction is completed and the solution temperature drops to room temperature, centrifuge at 3000 r / min for 5 minutes, take the supernatant and transfer it to a dialysis bag with a molecular weight cut-off of 200 Da, and dialyze for 6 hours. After dialysis, perform freeze-drying purification, collect the yellow powder to obtain the ratiometric carbon quantum dot fluorescent probe. The probe can be observed as a spherical shape under a transmission electron microscope (see Figure 1 ), and the size is below 10 nm. Disperse the probe in deionized water, and obvious dual-peak emission can be observed by spectroscopic testing (see Figure 2 ). Immediately seal and store it refrigerated after collection.
[0028] 2. Sialic acid detection
[0029] The ratiometric carbon quantum dot fluorescent probe is used to detect sialic acid molecules, and the specific steps are as follows:
[0030] 1) Preparation of the ratiometric carbon quantum dot fluorescent probe dispersion: Take 0.002 g of the ratiometric carbon quantum dot fluorescent probe described in claim 1, add 1 mL of distilled water, and gently shake to make the solution homogeneous;
[0031] 2) Recognition of sialic acid molecules: Mix 1 mL of the solution containing sialic acid molecules to be tested with the fluorescent probe dispersion obtained in step 1, and let it stand at room temperature for 1 minute to obtain the mixed fluorescent probe-analyte dispersion;
[0032] 3) Test the emission spectrum of the mixed dispersion obtained in step 2, obtain the fluorescence intensities of the dual emission peaks, and take the ratio of the peak intensity with a smaller wavelength (390 - 440 nm) to the peak intensity with a larger wavelength (550 - 600 nm) as the test result for analyzing the sialic acid content. The peak intensity ratio has a linear relationship with the sialic acid concentration, and the relationship expression between the two is y = 0.693x - 0.564.
[0033] Where the sialic acid concentration is x and the dual emission peak intensity ratio is y.
[0034] Example 2 Preparation of the ratiometric carbon quantum dot fluorescent probe and method for detecting sialic acid
[0035] Change the mass ratio of the two solutes in step 1 of the preparation of the ratiometric carbon quantum dot fluorescent probe in Example 1 to 1:2, and keep the other steps unchanged.
[0036] Preparation of Ratio-Type Carbon Quantum Dot Fluorescent Probe and Detection Method for Sialic Acid
[0037] Replace the hydrothermal reaction temperature in step 2 of the preparation of the ratio-type carbon quantum dot fluorescent probe in Example 1 with 200 °C, and keep the other steps unchanged.
[0038] Example 4 Preparation of Ratio-Type Carbon Quantum Dot Fluorescent Probe and Detection Method for Sialic Acid
[0039] Replace the hydrothermal reaction time in step 2 of the preparation of the ratio-type carbon quantum dot fluorescent probe in Example 1 with 24 hours, and keep the other steps unchanged.
[0040] Example 5 Preparation of Ratio-Type Carbon Quantum Dot Fluorescent Probe and Detection Method for Sialic Acid
[0041] Replace the dialysis time in step 2 of the preparation of the ratio-type carbon quantum dot fluorescent probe in Example 1 with 12 hours, and keep the other steps unchanged.
[0042] Example 6 Preparation of Ratio-Type Carbon Quantum Dot Fluorescent Probe and Detection Method for Sialic Acid
[0043] Replace the solvent volume in step 2 of the preparation of the ratio-type carbon quantum dot fluorescent probe in Example 1 with 20 mL, and keep the other steps unchanged.
[0044] Example 7 Preparation of Ratio-Type Carbon Quantum Dot Fluorescent Probe and Detection Method for Sialic Acid
[0045] Replace the centrifugation time in step 2 of the preparation of the ratio-type carbon quantum dot fluorescent probe in Example 1 with 30 minutes, and keep the other steps unchanged.
[0046] Example 8 Preparation of Ratio-Type Carbon Quantum Dot Fluorescent Probe and Detection Method for Sialic Acid
[0047] Replace the centrifugation speed in step 2 of the preparation of the ratio-type carbon quantum dot fluorescent probe in Example 1 with 5000 r / min, and keep the other steps unchanged.
[0048] Example 9 Preparation of Ratio-Type Carbon Quantum Dot Fluorescent Probe and Detection Method for Sialic Acid
[0049] Replace the molecular weight cut-off of the dialysis bag in step 2 of the preparation of the ratio-type carbon quantum dot fluorescent probe in Example 1 with 1000 Da, and keep the other steps unchanged.
[0050] Example 10 Preparation of Ratio-Type Carbon Quantum Dot Fluorescent Probe and Detection Method for Sialic Acid
[0051] Replace the standing time after mixing the solutions in step 2 of the detection of the ratio-type carbon quantum dot fluorescent probe in Example 1 with 10 minutes, and keep the other steps unchanged.
Claims
1. A ratiometric carbon quantum dot fluorescent probe for detecting sialic acid, characterized in that, It is prepared by the following method: 1) Take an aromatic amine chemical substance, dissolve it in a solvent at room temperature, stir slowly. After the solute is completely dissolved, add a benzene boronic acid chemical substance, and continue to stir slowly until the solution is homogeneous and transparent; 2) Transfer the mixed solution to a polytetrafluoroethylene hydrothermal reactor for hydrothermal reaction. After the reaction is completed and the solution temperature drops to room temperature, centrifuge, take the supernatant and transfer it to a dialysis bag for dialysis. After dialysis is completed, perform freeze-drying purification to obtain yellow powder carbon quantum dots.
2. The ratiometric carbon quantum dot fluorescent probe for detecting sialic acid according to claim 1, wherein In step 1), the mass ratio of the aromatic amine chemical substance to the benzene boronic acid chemical substance is 1:5 - 10:
1.
3. A ratiometric carbon quantum dot fluorescent probe for detecting sialic acid according to claim 1, characterized in that, In step 1), the aromatic amine chemical substance is one of o-phenylenediamine, p-phenylenediamine, m-phenylenediamine, o-toluidine, dimethylaniline, 4-methyl-m-phenylenediamine.
4. A ratiometric carbon quantum dot fluorescent probe for detecting sialic acid according to claim 1, characterized in that, In step 1), the solvent is one of methanol, ethanol, ether, acetone, N,N-dimethylformamide.
5. The ratiometric carbon quantum dot fluorescent probe for detecting sialic acid according to claim 1, wherein In step 1), the benzene boronic acid chemical drugs are one or more of 2-hydroxymethylphenylboronic acid, 2,3-dimethylphenylboronic acid, 3,4-dimethylphenylboronic acid, 3-vinylphenylboronic acid, 2-carbamoylphenylboronic acid, 3,5-dimethoxyphenylboronic acid, 2-butoxyphenylboronic acid.
6. The ratiometric carbon quantum dot fluorescent probe for detecting sialic acid according to claim 1, wherein In step 2), the hydrothermal reaction is carried out at 160 - 260 °C for 12 - 48 hours.
7. A ratiometric carbon quantum dot fluorescent probe for detecting sialic acid according to claim 1, characterized in that, In step 2), the centrifugation is carried out at 3000 - 8000 r / min for 5 - 30 minutes.
8. The ratiometric carbon quantum dot fluorescent probe for detecting sialic acid according to claim 1, wherein In step 2), the dialysis is to take the supernatant and transfer it to a dialysis bag with a molecular weight cut-off of 200 - 1000 Da for dialysis for 6 - 24 hours.
9. Application of the ratiometric carbon quantum dot fluorescence probe described in claim 1 in identifying sialic acid molecules.
10. The application according to claim 9, wherein The method is as follows: 1) Preparation of the ratiometric carbon quantum dot fluorescence probe dispersion: Take the ratiometric carbon quantum dot fluorescence probe described in claim 1, add it to distilled water, and gently shake to make the solution homogeneous; 2) Sialic acid molecule recognition: Mix the solution containing sialic acid molecules to be tested with the fluorescence probe dispersion obtained in step 1, and let it stand at room temperature for 1 - 10 minutes to obtain a mixed fluorescence probe - analyte dispersion; 3) Test the emission spectrum of the mixed dispersion obtained in step 2, obtain the fluorescence intensities of the dual emission peaks, and take the ratio of the fluorescence intensity with a smaller wavelength of 390 - 440 nm to the fluorescence intensity with a larger wavelength of 550 - 600 nm as the analysis result.