Iron-complexed chitosan-based fluorescent probe, preparation method and application thereof in detection of l-cys / dl-hcy
By preparing an iron-composite chitosan-based fluorescent probe, the problems of complexity and low selectivity in existing methods for detecting L-Cys and DL-Hcy were solved, enabling rapid and sensitive fluorescence detection, which is applicable to the field of biochemistry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV OF TECH
- Filing Date
- 2026-03-31
- Publication Date
- 2026-06-05
Smart Images
Figure CN122145668A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescence detection and relates to the application of fluorescent probes for detecting L-Cys and DL-Hcy, specifically an iron-composite chitosan-based fluorescent probe, its preparation method, and its application in detecting L-Cys / DL-Hcy. Background Technology
[0002] Cysteine (Cys) and homocysteine (DL-Hcy) are two important biothiols closely related to human life activities, maintaining normal physiological functions and cellular metabolism. Abnormal Cys levels in the physiological system may lead to growth retardation, neurotoxicity, and leukocyte loss. DL-Hcy has a very similar molecular structure to L-Cys, but it has different functions and indispensable roles in the biological system. An imbalance between L-Cys and DL-Hcy may increase the risk of Alzheimer's disease and cardiovascular disease; therefore, detecting and determining the levels of L-Cys and DL-Hcy is of great significance.
[0003] Currently, various analytical techniques exist for detecting L-Cys and DL-Hcy, such as high-performance liquid chromatography (HPLC), voltammetry, capillary zone electrophoresis (CZE), laser desorption / ionization mass spectrometry (LDI-MS), and liquid chromatography-mass spectrometry (LC-MS). However, most of these methods suffer from limitations such as complex operation and time consumption. Fluorescence spectroscopy, on the other hand, offers advantages such as high sensitivity, good specificity, and fast response time, making it a promising method for detecting L-Cys and DL-Hcy. Therefore, researchers have made considerable efforts to develop fluorescent probes for detecting these biothiols. Reported methods are mainly based on Michael addition, cyclization of thiols with aldehydes, and cleavage of disulfides and sulfonamides by thiols. Despite significant progress in the fluorescence detection of biothiols, many reported probes still suffer from cumbersome synthesis processes, low selectivity, and long response times. Therefore, there is a strong need to develop a simple, highly selective, and fast-responding fluorescent probe for detecting L-Cys and DL-Hcy. Summary of the Invention
[0004] This invention provides an iron-composite chitosan-based fluorescent probe, its preparation method, and its application in the detection of L-Cys / DL-Hcy.
[0005] One objective of this invention is to provide a fluorescent probe with a simple synthetic route, mild reaction conditions, and low cost; another objective is to provide a probe with high sensitivity, good selectivity, strong anti-interference ability, and the ability to accurately detect L-Cys and DL-Hcy in complex systems, with the following structure: CS-Pyr-1-Fe 3+ Where n represents the degree of polymerization, which is 300 to 500.
[0006] The synthetic route and synthetic idea are as follows: The method for preparing the iron-composite chitosan-based fluorescent probe is characterized by comprising the following steps: Chitosan was dissolved in an aqueous acetic acid solution. Then, a solution containing compound P3 was added dropwise to the chitosan solution and reacted at 55-75°C for 7-9 hours. Finally, Fe was added. 3+ After post-processing, an aerogel of chitosan-based fluorescent probe containing iron was obtained. The structure of the compound P3 is as follows: P3.
[0007] The method for preparing the iron-composite chitosan-based fluorescent probe, wherein the ratio of chitosan to aqueous acetic acid solution is 180~220mg:5~15mL; The volume percentage of acetic acid in the acetic acid aqueous solution is 0.5-2%.
[0008] The solvent in the solution containing compound P3 is tetrahydrofuran.
[0009] The post-processing specifically includes: leaving the reaction product exposed for 2-5 days to evaporate the solvent, washing with tetrahydrofuran, and finally freeze-drying to obtain an aerogel of iron-containing chitosan-based fluorescent probe.
[0010] The compound P3 and Fe 3+ The molar ratio is 1:50~70.
[0011] Specific synthesis method: Dissolve 180-220 mg of chitosan in 5-15 mL of 1% acetic acid aqueous solution. Then, slowly add different concentrations of P3 (dissolved in 2 mL of THF) dropwise to the chitosan solution, and finally add Fe. 3+ The reaction was carried out at 65°C for 8 hours. The hydrogel was left exposed for 3 days to allow the solvent to evaporate. It was then washed with THF. Finally, the aerogel was freeze-dried to obtain the corresponding aerogel. Finally, the iron-containing chitosan-based fluorescent probe aerogel was obtained by freeze-drying.
[0012] The application of the iron-composite chitosan-based fluorescent probe in the qualitative and quantitative detection of L-Cys includes: The aerogel of the iron-containing chitosan-based fluorescent probe was dissolved in an aqueous acetic acid solution to prepare the probe solution. A solution containing the L-Cys to be tested was added, and the fluorescence change was observed by fluorescence detection at an excitation wavelength of 270~275nm.
[0013] The application of the iron-composite chitosan-based fluorescent probe in the qualitative and quantitative detection of DL-Hcy includes: The iron-containing chitosan-based fluorescent probe aerogel was prepared in an aqueous acetic acid solution to form a probe solution. A solution containing the target DL-Hcy was added, and fluorescence changes were observed by fluorescence detection at an excitation wavelength of 270-275 nm.
[0014] The application of the iron-composite chitosan-based fluorescent probe in the quantitative detection of L-Cys and DL-Hcy includes: The iron-containing chitosan-based fluorescent probe was aerogelled in an aqueous acetic acid solution to prepare a probe solution. Then, the sample to be tested was added, and the fluorescence intensity was detected. Based on the linear relationship between the fluorescence intensity and the concentration of the sample to be tested, the contents of L-Cys and DL-Hcy were quantitatively calculated.
[0015] Specifically, the fluorescent probe CS-Pyr-1-Fe 3+ Dissolve the probe solution in a 1% acetic acid aqueous solution, then add the sample to be tested, detect the fluorescence intensity, and quantitatively calculate the contents of L-Cys and DL-Hcy based on the linear relationship between fluorescence intensity and the concentration of the added sample.
[0016] The mechanism of the probe of the present invention is as follows: Figure 1 As shown.
[0017] The mechanism of action of the fluorescent probe of the present invention is as follows: in the probe molecule, the stable chelating group CS-Pyr-1-Fe 3+ The composite portion showed no fluorescence. After the addition of L-Cys and DL-Hcy, due to the interaction between L-Cys and DL-Hcy and Fe... 3+ The bonding force between them is greater than that between CS-Pyr-1 and Fe. 3+ The binding force between them allows L-Cys and DL-Hcy to bind Fe 3+ From the originally formed CS-Pyr-1-Fe 3+ The fluorescence was significantly enhanced after the L-Cys and DL-Hcy were substituted out of the complex. 3+ The displacement caused the probe itself to be released, thus the detection system exhibited high selectivity and a low detection limit (9.30 × 10⁻⁶) for L-Cys and DL-Hcy. -7 mol / L, 1.91×10 -6 mol / L).
[0018] This invention provides an iron-based chitosan-based fluorescent probe for detecting L-Cys / DL-Hcy. The probe exhibits fluorescence activation upon the addition of L-Cys and DL-Hcy to a 33% THF solvent. The probe molecule described in this invention can qualitatively and quantitatively detect L-Cys and DL-Hcy in real samples. Furthermore, the probe CS-Pyr-1-Fe... 3+ It features a rapid response (110s) and good stability under a wide pH range (pH=3-10), thus having important application value in fields such as biochemistry.
[0019] The fluorescent probe of this invention has the characteristics of simple synthesis route, fast response and low susceptibility to environmental interference. Attached Figure Description
[0020] Figure 1 This is a schematic diagram of the mechanism of the probe of the present invention.
[0021] Figure 2 The probe of this invention is CS-Pyr-1-Fe 3+ Fluorescence spectra were recorded at room temperature in a THF:H₂O = 1:2 solution, reacting with control, GSH, Gly, L-Arg, L-Leu, DL-Hcy, L-His, L-Cys, D-Pro, D-Val, D-Ala, D-Ser, D-Asp, D-Met, D-Thr, and D-Glu. The excitation wavelength was 274 nm.
[0022] Figure 3 CS-Pyr-1-Fe in this invention 3+ Fluorescence intensity and linear relationship with different concentrations of L-Cys.
[0023] Figure 4 CS-Pyr-1-Fe in this invention 3+ Fluorescence intensity and linear relationship with different concentrations of DL-Hcy.
[0024] Table 1. L-Cys detection data in food samples. Detailed Implementation
[0025] The present invention will be further described below with reference to examples and accompanying drawings, but the present invention is not limited to the following embodiments.
[0026] Example 1 CS-Pyr-1-Fe 3+ Synthesis of fluorescent probes 200 mg of chitosan was dissolved in 10 mL of 1% acetic acid aqueous solution. Then, different concentrations of P3 (dissolved in 2 mL of THF, respectively) were slowly added dropwise to the chitosan solution. Finally, 100 μM Fe was added. 3+The solution was reacted at 65°C for 8 hours. The hydrogel was left exposed for 3 days to allow the solvent to evaporate. It was then washed with THF. Finally, the aerogel containing the iron-containing chitosan-based fluorescent probe was freeze-dried.
[0027] The structure of the compound P3 is as follows: P3.
[0028] The iron-composite chitosan-based fluorescent probe has the following structural formula: Where n represents the degree of polymerization, which is 300 to 500.
[0029] Example 2 CS-Pyr-1-Fe 3+ The selectivity of the complex for different amino acids Prepare a mixture of the following substances GSH, Gly, L-Arg, L-Leu, DL-Hcy, L-His, L-Cys, D-Pro, D-Val, D-Ala, D-Ser, D-Asp, D-Met, D-Thr, and D-Glu to form 2 × 10⁻⁶ ppm. -2 mol / L of mother liquor.
[0030] Take 16 test tubes. Replace the amino acids with 100 µL of water for the control. Add 1000 µL of ultrapure water and 1000 µL of THF to each tube, then add 400 µL of probe stock solution to each. After the reaction, add 500 µL of amino acid stock solution. Shake each solution well and then perform fluorescence detection (Ex=274 nm). Plot fluorescence intensity on the ordinate and wavelength on the abscissa. Figure 2 .from Figure 2 As can be seen, only L-Cys and DL-Hcy can interact with CS-Pyr-1-Fe. 3+ The complex undergoes a displacement reaction, which allows the fluorescence of the probe to be restored.
[0031] Example 3: CS-Pyr-1-Fe under different concentrations of L-Cys 3+ fluorescence intensity of the complex The concentration was set at 2 × 10⁻⁶. -2 A stock solution of L-Cys at mol / L was used. Similar to Example 3, the final L-Cys concentration was controlled by varying the volume added to the test tubes, resulting in L-Cys contents ranging from 0 to 40 equivalents. 100 µL of the probe stock solution was added to each test tube, followed by different volumes of L-Cys stock solution. After the reaction, fluorescence detection (Ex = 274 nm) was performed, and the fluorescence intensity in each system was measured. A curve was plotted with fluorescence intensity on the ordinate and L-Cys concentration on the abscissa, and a linear relationship was observed. Figure 3It can be seen that the fluorescence intensity of the probe gradually increases with the increase of L-Cys concentration, and the fluorescence of the probe reaches saturation when the L-Cys content reaches 40 equivalents.
[0032] Example 4: CS-Pyr-1-Fe under different concentrations of DL-Hcy 3+ fluorescence intensity of the complex The concentration is configured to be 2×10 -2 A mol / L DL-Hcy stock solution was used. Similar to Example 3, the final DL-Hcy concentration was controlled by varying the volume added to the test tubes, resulting in DL-Hcy contents ranging from 0 to 60 equivalents. 100 µL of probe stock solution was added to each test tube, followed by different volumes of DL-Hcy stock solution. After the reaction, fluorescence detection (Ex = 274 nm) was performed, and the fluorescence intensity in each system was measured. A curve was plotted with fluorescence intensity on the ordinate and DL-Hcy concentration on the abscissa, and a linear relationship was observed. Figure 4 It can be seen that the fluorescence intensity of the probe gradually increases with the increase of DL-Hcy concentration, and the fluorescence of the probe reaches saturation when the DL-Hcy content reaches 60 equivalents.
[0033] Example 5 CS-Pyr-1-Fe 3+ Detection of L-Cys in real samples using the complex Three representative food products from the market were selected as actual samples. All functional food samples were diluted with ultrapure water (c = 4 × 10⁻⁶). -4 mol / L).
[0034] For actual sample testing, 100 µL of CS-Pyr-1-Fe was taken. 3+ The mother liquor was mixed with 1500 µL of ultrapure water and 1000 mL of THF. After the reaction, 300 μL of food sample was added dropwise, and the reaction was repeated. Each solution was shaken well and subjected to fluorescence detection (Ex = 274 nm). The recovery rate and RSD were calculated. Table 1 is plotted with the actual sample as the ordinate and L-Cys concentration, recovery rate, and RSD as the abscissa.
[0035] Table 1. L-Cys detection data in food samples
Claims
1. An iron-composite chitosan-based fluorescent probe, characterized in that, It has the following structural formula: Where n represents the degree of polymerization, which is 300 to 500.
2. The method for preparing the iron-composite chitosan-based fluorescent probe according to claim 1, characterized in that, Includes the following steps: Chitosan is dissolved in acetic acid aqueous solution, then a solution containing compound P3 is dropped into the chitosan solution at 55~75℃ for 7~9 hours, finally Fe 3+ is added, and an aerogel containing iron-complexed chitosan-based fluorescent probe is obtained after post-processing. The structure of the compound P3 is as follows: P3。 3. The method for preparing the iron-composite chitosan-based fluorescent probe according to claim 2, characterized in that, The ratio of chitosan to aqueous acetic acid is 180-220 mg: 5-15 mL; The volume percentage of acetic acid in the acetic acid aqueous solution is 0.5-2%.
4. The method for preparing the iron-composite chitosan-based fluorescent probe according to claim 2, characterized in that, The solvent in the solution containing compound P3 is tetrahydrofuran.
5. The method for preparing the iron-composite chitosan-based fluorescent probe according to claim 2, characterized in that, The post-processing specifically includes: leaving the reaction product exposed for 2-5 days to evaporate the solvent, washing with tetrahydrofuran, and finally freeze-drying to obtain an aerogel of iron-containing chitosan-based fluorescent probe.
6. The method for preparing the iron-composite chitosan-based fluorescent probe according to claim 2, characterized in that, The compound P3 and Fe 3+ The molar ratio is 1:50~70.
7. The application of the iron-composite chitosan-based fluorescent probe according to claim 1 in the qualitative and quantitative detection of L-Cys, characterized in that, include: The aerogel of the iron-containing chitosan-based fluorescent probe was dissolved in an aqueous acetic acid solution to prepare the probe solution. A solution containing the L-Cys to be tested was added, and the fluorescence change was observed by fluorescence detection at an excitation wavelength of 270~275nm.
8. The application of the iron-composite chitosan-based fluorescent probe according to claim 1 in the qualitative and quantitative detection of DL-Hcy, characterized in that, include: The iron-containing chitosan-based fluorescent probe aerogel was prepared in an aqueous acetic acid solution to form a probe solution. A solution containing the target DL-Hcy was added, and fluorescence changes were observed by fluorescence detection at an excitation wavelength of 270-275 nm.
9. The application of the iron-composite chitosan-based fluorescent probe according to claim 1 in the quantitative detection of L-Cys and DL-Hcy, characterized in that, include: The iron-containing chitosan-based fluorescent probe was aerogelled in an aqueous acetic acid solution to prepare a probe solution. Then, the sample to be tested was added, and the fluorescence intensity was detected. Based on the linear relationship between the fluorescence intensity and the concentration of the sample to be tested, the contents of L-Cys and DL-Hcy were quantitatively calculated.