Rhodamine derivative fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a rhodamine derivative fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of fluorescence detection. The preparation method comprises the following steps: mixing rhodamine 110 conjugated PEG azide, triethylamine and anhydrous dichloromethane to obtain a mixed solution; and dropwise adding a 2, 4-dinitrobenzenesulfonyl chloride solution into the mixed solution, reacting at low temperature, and reacting at normal temperature in a dark place to obtain the rhodamine derivative fluorescent probe. The rhodamine derivative fluorescent probe shows good stability when being used for detecting the selenocysteine, has low detection limit on the selenocysteine, has high sensitivity and good selectivity, and is suitable for detecting the selenocysteine in livestock and poultry products.

Description

A Rhodamine Derivative Fluorescent Probe, Its Preparation Method and Application Technical Field

[0001] This invention relates to the field of fluorescence detection technology, and in particular to a rhodamine derivative fluorescent probe, its preparation method, and its application. Background Technology

[0002] Selenium (Se), an important micronutrient, plays a crucial role in human physiological and pathological processes. Selenocysteine ​​(Sec), an analogue of cysteine ​​(Cys), has its sulfhydryl group replaced by a selenium-containing selenool group. It serves as the main functional form of various selenium-containing substances in biological systems and is considered a specific building block of selenoproteins (SePs) integrated into enzyme active sites. Furthermore, selenium also plays a role in the prevention and treatment of Keshan disease and Kashin-Beck disease.

[0003] Currently, rapid detection of selenocysteine ​​in agricultural products has become a critical technical problem that urgently needs to be solved. Traditional detection methods for selenocysteine ​​mainly rely on large-scale laboratory instruments such as high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP / MS) or atomic fluorescence spectrometry. These methods involve cumbersome sample preparation and demanding requirements on experimental conditions and personnel qualifications.

[0004] Therefore, it is of great significance to provide a simple and stable method for preparing a rhodamine derivative fluorescent probe for the detection of selenocysteine. Summary of the Invention

[0005] The purpose of this invention is to provide a rhodamine derivative fluorescent probe, its preparation method, and its application, addressing the shortcomings of existing technologies.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a method for preparing a rhodamine derivative fluorescent probe, comprising the following steps: 1) mixing rhodamine 110-conjugated PEG azide, triethylamine and anhydrous dichloromethane and cooling to obtain a mixed solution; 2) adding 2,4-dinitrobenzenesulfonyl chloride solution dropwise to the mixed solution, reacting at low temperature and then reacting in the dark to obtain a rhodamine derivative fluorescent probe.

[0007] Preferably, the molar ratio of the triethylamine and the rhodamine 110 conjugated PEG azide in step 1) is 2~10:1.

[0008] Preferably, the anhydrous dichloromethane in step 1) is anhydrous dichloromethane after nitrogen deoxygenation; the mixture is a mixture of Rhodamine 110 conjugated PEG azide and anhydrous dichloromethane, with triethylamine added to the mixture; when triethylamine is added, the temperature of the mixture is -2~2℃.

[0009] Preferably, the solvent for the 2,4-dinitrobenzenesulfonyl chloride solution in step 2) is anhydrous dichloromethane, which is anhydrous dichloromethane after nitrogen purging to remove oxygen.

[0010] Preferably, the molar ratio of PEG azide conjugated with 2,4-dinitrobenzenesulfonyl chloride and rhodamine 110 is 2.1 to 8:1.

[0011] Preferably, the low-temperature reaction in step 2) is carried out at a temperature of -2 to 2°C for 1 to 3 hours; the light-protected reaction is carried out at room temperature for 10 to 14 hours.

[0012] Preferably, the mixture obtained from the light-shielded reaction is concentrated and purified under reduced pressure to obtain a rhodamine derivative fluorescent probe; the purification is performed by gradient elution using a silica gel chromatography column, with dichloromethane and methanol as the elution reagents, and the molar ratio of dichloromethane to methanol being 4~80:1.

[0013] The present invention also provides a rhodamine derivative fluorescent probe prepared by the aforementioned preparation method.

[0014] The present invention also provides the application of the rhodamine derivative fluorescent probe in the detection of selenocysteine.

[0015] The beneficial effects of the present invention are as follows: The rhodamine derivative fluorescent probe of the present invention exhibits good stability in the detection of selenocysteine, has a low detection limit for selenocysteine, and possesses high sensitivity and good selectivity, making it suitable for the detection of selenocysteine ​​in livestock and poultry products. Attached Figure Description

[0016] Figure 1 shows the fluorescence response of the Rhodamine 110-conjugated PEG azido-dinitrobenzenesulfonamide derivative of Example 1 to different concentrations of selenocysteine; Figure 2 shows the Stern-Volmer curves of the fluorescence intensity ratio at 513 nm of the probe system of Example 1 before and after the addition of selenocysteine ​​versus the concentration of selenocysteine, where F is the fluorescence intensity value after the addition of selenocysteine ​​and F0 is the fluorescence intensity value before the addition of selenocysteine; Figure 3 shows the change in the fluorescence ratio of the probe of Example 1 before and after the addition of the target substance, where the fluorescence value after the addition of the target substance is F and the fluorescence value before the addition of the target substance is F0. Figure 4 shows the fluorescence response of 5-fluorescein-dinitrobenzenesulfonamide as a rapid sensing and recognition probe in Comparative Example 1 to different concentrations of selenocysteine; Figure 5 shows the Stern-Volmer curve of the fluorescence intensity ratio at 513 nm of the probe system in Comparative Example 1 before and after the addition of selenocysteine ​​versus the concentration of selenocysteine, where F is the fluorescence intensity value after the addition of selenocysteine ​​and F0 is the fluorescence intensity value before the addition of selenocysteine; Figure 6 shows the change in the fluorescence ratio of the probe in Comparative Example 1 before and after the addition of the target substance, where the fluorescence value after the addition of the target substance is F and the fluorescence value before the addition of the target substance is F0. Detailed Implementation

[0017] This invention provides a method for preparing a rhodamine derivative fluorescent probe, comprising the following steps: 1) mixing rhodamine 110-conjugated PEG azide, triethylamine and anhydrous dichloromethane and cooling to obtain a mixed solution; 2) adding 2,4-dinitrobenzenesulfonyl chloride solution dropwise to the mixed solution, reacting at low temperature and then reacting in the dark to obtain a rhodamine derivative fluorescent probe.

[0018] In this invention, the structural formula of the rhodamine 110-conjugated PEG azide is as follows:

[0019] In this invention, the molar ratio of triethylamine and Rhodamine 110 conjugated PEG azide in step 1) is preferably 2~10:1, more preferably 3.5~8:1, and even more preferably 5~7:1.

[0020] In this invention, the anhydrous dichloromethane in step 1) is preferably anhydrous dichloromethane after nitrogen deoxygenation; the mixing is preferably a mixture of Rhodamine 110 conjugated PEG azide and anhydrous dichloromethane, with triethylamine added to the mixture; when triethylamine is added, the temperature of the mixture is preferably -2~2℃, and more preferably 0℃.

[0021] In this invention, the solvent for the 2,4-dinitrobenzenesulfonyl chloride solution in step 2) is preferably anhydrous dichloromethane, and the anhydrous dichloromethane is preferably anhydrous dichloromethane after nitrogen purging to remove oxygen.

[0022] In this invention, the molar ratio of PEG azide conjugated with 2,4-dinitrobenzenesulfonyl chloride and Rhodamine 110 is preferably 2.1 to 8:1, more preferably 3 to 6:1, and even more preferably 4 to 5:1.

[0023] In this invention, the temperature of the low-temperature reaction in step 2) is preferably -2~2℃, more preferably 0℃, and the reaction time is preferably 1~3h, more preferably 2h; the light-protected reaction is carried out at room temperature, and the reaction time is preferably 10~14h, more preferably 11~13h, and more preferably 12h.

[0024] In this invention, the mixture obtained from the light-shielded reaction is preferably concentrated and purified under reduced pressure to obtain a rhodamine derivative fluorescent probe; the purification is preferably carried out by gradient elution using a silica gel chromatography column, and the reagents for gradient elution are preferably dichloromethane and methanol, with the molar ratio of dichloromethane to methanol preferably being 4~80:1, more preferably 10~70:1, and even more preferably 30~50:1.

[0025] The present invention also provides a rhodamine derivative fluorescent probe prepared by the aforementioned preparation method.

[0026] The present invention also provides the application of the rhodamine derivative fluorescent probe in the detection of selenocysteine.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] 25 mg of rhodamine 110 conjugated PEG azide (0.044 mmol) was added to a 50 mL round-bottom flask, followed by 5 mL of anhydrous dichloromethane after deoxygenation by purging with nitrogen gas to obtain a mixture. The mixture was cooled in an ice bath (0 °C), and 25 μL of triethylamine (0.178 mmol) was added. The mixture was stirred until homogeneous to form a rhodamine mixture.

[0030] 46.3 mg of 2,4-dinitrobenzenesulfonyl chloride (0.174 mmol) was dissolved in 2 mL of anhydrous dichloromethane after deoxygenation by purging with nitrogen to obtain a 2,4-dinitrobenzenesulfonyl chloride solution. The 2,4-dinitrobenzenesulfonyl chloride solution was slowly added dropwise to a rhodamine mixture, and the mixture was stirred until a homogeneous mixture was formed. After reacting the homogeneous mixture at 0°C for 2 h, the ice bath was removed, and the reaction system was allowed to slowly and naturally warm to room temperature. The reaction was then continued at room temperature in the dark for 12 h, during which the reaction process was observed periodically until the starting material spot disappeared using TLC.

[0031] After the reaction was completed, the reaction product was concentrated under reduced pressure by rotary evaporation (500 MPa, 30 min), and then purified by silica gel chromatography using a mixture of dichloromethane and methanol at a molar ratio of 80:1. The collected eluent was concentrated under reduced pressure to obtain a red solid, which was the Rhodamine 110-conjugated PEG azide-dinitrobenzenesulfonamide derivative.

[0032] Example 2

[0033] 25 mg of rhodamine 110 conjugated PEG azide (0.044 mmol) was added to a 50 mL round-bottom flask, followed by 5 mL of anhydrous dichloromethane after deoxygenation by purging with nitrogen gas to obtain a mixture. The mixture was cooled in an ice bath (0 °C), and 50 μL of triethylamine (0.357 mmol) was added. The mixture was stirred until homogeneous to form a rhodamine mixture.

[0034] 80 mg of 2,4-dinitrobenzenesulfonyl chloride (0.300 mmol) was dissolved in 2 mL of anhydrous dichloromethane after deoxygenation by purging with nitrogen to obtain a 2,4-dinitrobenzenesulfonyl chloride solution. The 2,4-dinitrobenzenesulfonyl chloride solution was slowly added dropwise to a rhodamine mixture, and the mixture was stirred until a homogeneous mixture was formed. After reacting the homogeneous mixture at 0 °C for 1.5 h, the ice bath was removed, and the reaction system was allowed to slowly and naturally warm to room temperature. The reaction was then continued at room temperature in the dark for 14 h, during which the reaction process was monitored periodically until the starting material spot disappeared using TLC.

[0035] After the reaction was completed, the reaction product was concentrated under reduced pressure by rotary evaporation (500 MPa, 30 min), and then purified by silica gel chromatography using a gradient elution with a mixture of dichloromethane and methanol at a molar ratio of 80:15. The collected eluent was concentrated under reduced pressure to obtain a red solid, which was the Rhodamine 110-conjugated PEG azide-dinitrobenzenesulfonamide derivative.

[0036] Example 3

[0037] 25 mg of rhodamine 110 conjugated PEG azide (0.044 mmol) was added to a 50 mL round-bottom flask, followed by 5 mL of anhydrous dichloromethane after deoxygenation by purging with nitrogen to obtain a mixture. The mixture was cooled in an ice bath (0 °C), and 37 μL of triethylamine (0.264 mmol) was added. The mixture was stirred until homogeneous to form a rhodamine mixture.

[0038] 70.4 mg of 2,4-dinitrobenzenesulfonyl chloride (0.264 mmol) was dissolved in 2 mL of anhydrous dichloromethane after deoxygenation by purging with nitrogen to obtain a 2,4-dinitrobenzenesulfonyl chloride solution. The 2,4-dinitrobenzenesulfonyl chloride solution was slowly added dropwise to the rhodamine mixture, and the mixture was stirred until a homogeneous mixture was formed. After reacting the homogeneous mixture at 0 °C for 1.5 h, the ice bath was removed, and the reaction system was allowed to slowly and naturally warm to room temperature. The reaction was then continued at room temperature in the dark for 14 h, during which the reaction process was observed periodically, and the reaction continued until the starting material spot disappeared using TLC monitoring.

[0039] After the reaction was completed, the reaction product was concentrated under reduced pressure by rotary evaporation (500 MPa, 30 min), and then purified by silica gel column chromatography using a gradient elution with a mixture of dichloromethane and methanol at a molar ratio of 80:5. The collected eluent was concentrated under reduced pressure to obtain a red solid, which was the Rhodamine 110-conjugated PEG azide-dinitrobenzenesulfonamide derivative.

[0040] Application Example 1: The response of Rhodamine 110-conjugated PEG azido-dinitrobenzenesulfonamide derivatives to selenocysteine.

[0041] The Rhodamine 110-conjugated PEG-azido-dinitrobenzenesulfonamide derivative prepared in Example 1 was used as a rapid sensing and recognition probe for the rapid recognition of selenocysteine. The recognition probe and selenocysteine ​​(Sec) stock solution (due to the instability of Sec, it needs to be incubated with an equimolar amount of DTT for 2 hours beforehand and should be prepared fresh) were added to a 2 mL cuvette. Finally, the volume was adjusted with PBS buffer (pH=6.8) to ensure that the probe concentration was 1 µM and the Sec concentrations were 0 µM, 0.01 µM, 0.05 µM, 0.1 µM, 0.5 µM, 1 µM, 2 µM, 5 µM, 10 µM, 15 µM, 20 µM, 25 µM, 30 µM, 40 µM, and 50 µM, respectively.

[0042] The fluorescence response of the Rhodamine 110-conjugated PEG azido-dinitrobenzenesulfonamide derivative of Example 1 to different concentrations of selenocysteine ​​is shown in Figure 1. As can be seen from Figure 1, within the range of 0–50 µM, the fluorescence intensity of the probe system in Example 1 at 513 nm increases with increasing Sec concentration, indicating that the probe system is feasible for recognizing selenocysteine.

[0043] Figure 2 shows the Stern-Volmer curves of the fluorescence intensity ratio at 513 nm before and after the addition of selenocysteine ​​to the selenocysteine ​​concentration for the probe system of Example 1. Here, F represents the fluorescence intensity value after the addition of selenocysteine, and F0 represents the fluorescence intensity value before the addition of selenocysteine. As can be seen from Figure 2, within the concentration range of 0.01–50 µM, the fluorescence ratio exhibits a good linear relationship with the selenocysteine ​​concentration, with the linear equation being Y = 0.279C + 1.042 and the linear correlation coefficient being R. 2 =0.9972, and the detection limit (LOD) calculated using the 3σ / k detection limit method is 3.3 nmol / L. The probe system of Example 1 exhibits high stability; in three repeated experiments, the RSD values ​​of the fluorescence ratio data were all less than 4%. In summary, the fluorescent probe obtained in Example 1 combines high stability and high sensitivity and can be used to detect selenocysteine.

[0044] Application Example 2: The performance of the Rhodamine 110-conjugated PEG-azido-dinitrobenzenesulfonamide derivative probe from Example 1 was tested using egg samples.

[0045] Egg extract: Weigh 2g of egg liquid into a 50mL centrifuge tube and mix well. Add 0.5mL of ascorbic acid aqueous solution (concentration 0.2mol / L) and 10mL of Tris-HCl buffer solution (concentration 10mmol / L, pH=7.5). Extract by sonication at 400W for 30min, homogenize at 1500r / min for 1min, then add 20mg of proteinase XIV, vortex to mix, and shake at 37℃ for 12h on a constant temperature shaker. Centrifuge at 12000r / min for 10min. Filter the extract through a 0.22µm hydrophilic membrane and transfer to a 15mL centrifuge tube. Transfer 5.0mL of the filtrate to an ultrafiltration centrifuge tube and centrifuge at 5000r / min for 10min to obtain the egg extract.

[0046] Spiking experiment: Using the egg extract as the research object, a spiked recovery experiment was conducted. The recognition probe, egg extract, and selenocysteine ​​stock solution were added to a 2 mL cuvette, and finally, PBS buffer solution (pH=6.8) was added to bring the volume to a final concentration, ensuring the probe concentration was 1 µmol / L. The sec concentrations were 0 µmol / L, 5 µmol / L, 10 µmol / L, and 15 µmol / L, with each sample measured in triplicate.

[0047] Table 1. Determination of Sec in egg samples

[0048] As shown in Table 1, the spiked recoveries of the probe system in egg samples ranged from 95% to 107%, with RSDs all less than 5.0%, confirming that the probe is accurate and reliable and can be used to determine the selenocysteine ​​content in livestock and poultry products.

[0049] Application Example 3: Selectivity of the Rhodamine 110-conjugated PEG azide-dinitrobenzenesulfonamide derivative probe from Example 1

[0050] The selectivity of the probe system in Example 1 was investigated, and seven interfering substances were screened as target substances to test their effect on the fluorescence ratio (F / F0). The probe was added to a 2 mL cuvette and diluted with PBS buffer (pH=6.8) to ensure a probe concentration of 1 µM, thus obtaining the probe system. Selenocysteine ​​(Sec), selenomethionine (SeMet), glutathione (GSH), cysteine ​​(Cys), sodium selenite (Se(IV)), selenium standard solution, sodium sulfite (Na2SO3), and ascorbic acid (VC) were added to the probe system, each at a concentration of 40 μmol / L. The changes in the fluorescence ratio of the probe in Example 1 before and after the addition of the target substances are shown in Figure 3. The fluorescence value after the addition of the target substances is F, and the fluorescence value before the addition of the target substances is F0. Figure 3 shows that several common interfering ions have no significant interference with the detection system, indicating that the method established in this invention has good selectivity.

[0051] Comparative Example 1

[0052] Add 70 mg of 5-aminofluorescein (0.2 mmol) to a 25 mL round-bottom flask, then add 5 mL of anhydrous dichloromethane after deoxygenation by purging with nitrogen to obtain a mixture. Cool the mixture in an ice bath (0 °C), then add 56 μL of triethylamine (0.4 mmol) and stir until homogeneous to form a rhodamine mixture.

[0053] 107 mg of 2,4-dinitrobenzenesulfonyl chloride (0.4 mmol) was dissolved in 2 mL of anhydrous dichloromethane after deoxygenation by purging with nitrogen to obtain a 2,4-dinitrobenzenesulfonyl chloride solution. The 2,4-dinitrobenzenesulfonyl chloride solution was slowly added dropwise to a 5-aminofluorescein mixture, and the mixture was stirred until a homogeneous mixture was formed. The homogeneous mixture was reacted at 0°C for 1.5 h, then the ice bath was removed, and the reaction system was allowed to slowly and naturally warm to room temperature. The reaction was then continued at room temperature in the dark for 4 h, with the reaction progress observed periodically until the starting material spot disappeared using TLC monitoring.

[0054] After the reaction was completed, the reaction product was concentrated under reduced pressure by rotary evaporation (500 MPa, 30 min), and then purified by silica gel chromatography using a gradient elution with a mixture of dichloromethane and methanol at a molar ratio of 180:1. The collected eluent was concentrated under reduced pressure to obtain a red solid, which is 5-fluorescein-dinitrobenzenesulfonamide.

[0055] 5-Fluorescence-dinitrobenzenesulfonamide (FFI) from Comparative Example 1 was used as a rapid sensing and recognition probe for the rapid identification of selenocysteine. Referring to Application Examples 1 and 2, the sensitivity and selectivity of the fluorescein probe system were investigated. Figure 4 shows that the fluorescent probe synthesized from 5-aminofluorescein also responded to Sec, and the fluorescence intensity increased with increasing Sec concentration. Figure 5 shows that within the concentration range of 0.01–30 µM, the fluorescence ratio exhibited a good linear relationship with the selenocysteine ​​concentration, with the linear equation Y = 0.476C + 0.793 and a linear correlation coefficient R. 2 =0.9861; As shown in Figure 6, although the probe is sensitive to selenocysteine, it also responds to several interfering substances. We can speculate that these interfering substances will interfere with the sensing system in practical applications. Therefore, compared with 5-fluorescein-dinitrobenzenesulfonamide, the rhodamine 110-conjugated azide-dinitrobenzenesulfonamide probe synthesized in this invention has the characteristics of excellent performance and good selectivity.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a rhodamine derivative fluorescent probe, characterized in that, The process includes the following steps: 1) Mixing Rhodamine 110-conjugated PEG azide, triethylamine, and anhydrous dichloromethane to obtain a mixed solution; 2) Adding 2,4-dinitrobenzenesulfonyl chloride solution to the mixed solution, reacting at low temperature and then in the dark to obtain a Rhodamine derivative fluorescent probe.

2. The preparation method according to claim 1, characterized in that, In step 1), the molar ratio of the triethylamine and the rhodamine 110 conjugated PEG azide is 2~10:

1.

3. The preparation method according to claim 1 or 2, characterized in that, Step 1) The anhydrous dichloromethane is anhydrous dichloromethane after nitrogen purging and deoxygenation; the mixture is a mixture of Rhodamine 110 conjugated PEG azide and anhydrous dichloromethane, with triethylamine added to the mixture; when triethylamine is added, the temperature of the mixture is -2~2℃.

4. The preparation method according to claim 3, characterized in that, In step 2), the solvent for the 2,4-dinitrobenzenesulfonyl chloride solution is anhydrous dichloromethane, which is anhydrous dichloromethane after nitrogen purging to remove oxygen.

5. The preparation method according to claim 4, characterized in that, The molar ratio of PEG azide conjugated with 2,4-dinitrobenzenesulfonyl chloride and rhodamine 110 is 2.1 to 8:

1.

6. The preparation method according to claim 4 or 5, characterized in that, Step 2) The low-temperature reaction is carried out at -2~2℃ for 1~3h; the light-protected reaction is carried out at room temperature for 10~14h.

7. The preparation method according to claim 6, characterized in that, The mixture obtained from the light-shielded reaction was concentrated and purified under reduced pressure to obtain a rhodamine derivative fluorescent probe. The purification was carried out by gradient elution using a silica gel column. The elution reagents were dichloromethane and methanol, with a molar ratio of dichloromethane to methanol of 4-80:

1.

8. The rhodamine derivative fluorescent probe prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the rhodamine derivative fluorescent probe according to claim 8 in the detection of selenocysteine.

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