Synthesis method and application of PBTZM fluorescent probe
By synthesizing PBTZM fluorescent probes and building label-free nucleic acid aptamer sensors, the existing enrofloxacin detection methods are solved, and low-cost, fast and simple detection results are achieved.
Patent Information
- Application Number
- CN202411670000.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-02-25
AI Technical Summary
The existing methods for detecting enrofloxacin are time-consuming, expensive equipment and complex operations, resulting in ineffective detection.
A PBTZM fluorescent probe synthesis method is adopted, and a label-free nucleic acid aptamer sensor is constructed, and detection can be achieved through simple mixing steps.
It realizes low-cost, fast and simple detection of enrofloxacin content, reduces detection costs and improves detection efficiency.
Smart Images
Figure CN119504642A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of detecting enrofloxacin, and in particular to a synthesis method of a PBTZM fluorescent probe and application thereof. Background Art
[0002] Enrofloxacin (ENR), also known as ethylciprofloxacin, is a synthetic third-generation fluoroquinolone antibiotic commonly used to treat various bacterial infections in animals. However, long-term consumption of animal products containing excessive amounts of enrofloxacin poses a potential threat to human health, such as dizziness, dyspnea, insomnia, confusion, liver damage, and even aggravates the severity of collagen-induced arthritis. Therefore, many countries and organizations have established legal standards for the maximum residue limit (MRL) of ENR, which ranges from 100 μg / kg to 500 μg / kg depending on the animal product. So far, many analytical methods have been developed to detect ENR, including high-performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), electrochemical method (EC), chemiluminescence method, and surface plasma co-existence (SPR). Although these methods have advantages, some disadvantages such as time-consuming, expensive equipment, and complex operation limit the detection efficiency. Therefore, there is still an urgent need to develop new detection methods, and therefore a method for synthesizing a PBTZM fluorescent probe and its application are needed. Summary of the invention
[0003] The purpose of the present invention is to solve the problems of the prior art and to propose a synthesis method of a PBTZM fluorescent probe and its application.
[0004] In order to achieve the above object, the present invention adopts the following technical solutions:
[0005] The present invention discloses a PBTZM fluorescent probe, the chemical name of which is: 2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-3-methylbenzo[d]thiazole-3H-iodide. The structure is shown in formula (I):
[0006]
[0007] A method for synthesizing a PBTZM fluorescent probe comprises the following steps:
[0008] (1) 2-methylbenzothiazole and 4-(dimethylamino)cinnamaldehyde are mixed in a molar ratio of 1:1, potassium tert-butoxide and DMF solution are added, reacted at 80° C., extracted with dichloromethane, washed, separated, and the excess solvent is removed by rotary evaporation and purified by column chromatography to obtain 2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-benzothiazole;
[0009] (2) 2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-benzothiazole was dissolved in CH3CN, iodomethane was added, and the reaction was refluxed at 85°C. After the reaction was completed at room temperature, dichloromethane was added for extraction and washing. The liquids were separated, and the excess solvent was removed by rotary evaporation and purified by column chromatography to obtain the compound PBTZM.
[0010] Furthermore, the added mass ratio of 2-methylbenzothiazole to potassium tert-butoxide is 1:0.75.
[0011] Furthermore, the added mass-volume ratio of 2-methylbenzothiazole to the DMF solution was 0.03 g / mL.
[0012] Furthermore, the mass-volume ratio of 2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-benzothiazole to methyl iodide is 0.98 g / mL.
[0013] The invention discloses an application of the PBTZM fluorescent probe in detecting the content of enrofloxacin. The synthetic PBTZM is used as a fluorescent dye and an enrofloxacin nucleic acid aptamer is used as a recognition unit. The enrofloxacin nucleic acid aptamer and PBTZM are added to a Tris-HCl buffer solution. After the analyte enrofloxacin is added, the nucleic acid aptamer structure is induced to change, causing a change in the fluorescence signal of the system. The fluorescence intensity value of the system is detected, and the content of the analyte is obtained by calculation. The nucleic acid aptamer of enrofloxacin is: ENR-aptamer: 5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGGGGA-3.
[0014] Furthermore, the parameters for measuring the fluorescence value of the solution are 554 nm as the excitation wavelength and 594 to 656 nm as the emission wavelength.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] The beneficial effects of the present invention are as follows: the present invention synthesizes a novel fluorescent probe PBTZM and constructs a label-free nucleic acid aptamer sensor for ENR detection. The fluorescent aptamer sensor can be obtained in one step by simple mixing, and no other high-cost materials need to be added, so the detection cost is low. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a diagram showing the mechanism of detecting enrofloxacin using the fluorescent nucleic acid aptamer sensor based on the aptamer and the PBTZM fluorescent probe of the present invention;
[0018] Figure 2 The fluorescence spectra of the aptamer before and after binding with enrofloxacin in the present invention;
[0019] Figure 3 This is a diagram showing the optimization of the buffer solution for the detection system of the present invention. DETAILED DESCRIPTION
[0020] The present invention is described in detail below in conjunction with the embodiments and accompanying drawings. However, it should be understood that the embodiments and drawings are only used to exemplify the present invention and do not constitute any limitation on the protection scope of the present invention.
[0021] The present invention is further demonstrated below in conjunction with specific embodiments:
[0022] like Figure 1 As shown, the present invention comprises the following steps:
[0023] Example 1 Synthesis of PBTZM
[0024] Compound 1 (2-methylbenzothiazole) (1mmol, 149.21mg), compound 2 (4-(dimethylamino) cinnamaldehyde) (1mmol, 175.23mg, the mass ratio of compound 1 to compound 2 is 1:1.17) and potassium tert-butoxide (1mmol, 112.21mg, the mass ratio of compound 1 to potassium tert-butoxide is 1:0.75) were added to 5mL DMF (the mass-volume ratio of compound 1 to DMF is 0.03g / mL), reacted at 80°C, and monitored by TLC. After the reaction was completed, the reaction was quenched with water, extracted with dichloromethane, washed with water, saturated brine, and saturated sodium bicarbonate solution, separated, and the excess solvent was evaporated, and purified by column chromatography to obtain compound 3.
[0025] Compound 3 (2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-benzothiazole) (1mmol, 306.43mg) was dissolved in CH3CN, methyl iodide (5mmol, the mass-volume ratio of compound 3 to methyl iodide was 0.98g / mL) was added, and the reaction was refluxed at 85°C and monitored by TLC. After the reaction was completed, the reaction was quenched with water, extracted with dichloromethane, washed with water, saturated brine, and saturated sodium bicarbonate solution, separated, and the excess solvent was evaporated, and purified by column chromatography to obtain compound PBTZM.
[0026] The synthesis formula is as follows:
[0027]
[0028] Example 2 Solution Preparation
[0029] 1. Preparation of Enrofloxacin Solution
[0030] Dissolve enrofloxacin solid in 1 mL of glacial acetic acid in a 1.5 mL centrifuge tube to obtain a 100 mM ENR stock solution.
[0031] 2. Preparation of Enrofloxacin Aptamer Solution
[0032] The aptamer was centrifuged at 6000 rpm for 1 min, the tube cover was slowly opened, 1 mL of ultrapure water was added with a pipette and mixed evenly, and the solution was centrifuged again at 6000 rpm for 1 min to prepare a 1 μmol L-1 nucleic acid aptamer solution, which was stored in a 4°C refrigerator.
[0033] 3. PBTZM preparation
[0034] Weigh PBTZM solid and dissolve it in ultrapure water to prepare a 1 mM stock solution, which is then stored in a 4°C refrigerator away from light.
[0035] Example 3 Feasibility Experiment
[0036] (1) Preparation of enrofloxacin solution samples: The enrofloxacin stock solution with a concentration of 100 mmol / L obtained in Example 2 was diluted to 20 μmol / L, 50 μmol / L, and 100 μmol / L with a buffer solution.
[0037] (2) Preparation of PBTZM solution sample: The 1 mmol / L PBTZM stock solution obtained in Example 3 was diluted to 20 μmol / L with ultrapure water. 50 μL of the 20 μmol / L PBTZM solution was added to 150 μL Tris-HCl buffer.
[0038] (3) Preparation of PBTZM / enrofloxacin solution sample: Add 20 μL of 20 μmol / L enrofloxacin solution and 50 μL of 20 μmol / L PBTZM solution to 130 μL Tris-HCl buffer and incubate on a horizontal shaker for 10 min.
[0039] (4) Preparation of PBTZM / aptamer solution samples: Add 30 μL of 1 μmol / L enrofloxacin aptamer solution and 50 μL of 20 μmol / L PBTZM solution to 120 μL Tris-HCl buffer and incubate on a horizontal shaker for 10 min.
[0040] (5) Preparation of PBTZM / aptamer / enrofloxacin solution samples: 50 μL of 2 μmol / L PBTZM solution and 30 μL of 1000 nmol / L enrofloxacin aptamer solution were added to 100 μL Tris-HCl buffer, and the mixture was placed in a horizontal shaker and incubated for 10 min; then 20 μL of the 20 μmol / L, 50 μmol / L, and 100 μmol / L enrofloxacin solutions obtained in Example 1 were added, and the final concentrations of enrofloxacin in the solution were 2 μmol / L, 5 μmol / L, and 10 μmol / L;
[0041] (6) Set 554 nm as the excitation wavelength and 594-656 nm as the emission wavelength. Pour the solution after the reaction into a black 96-well plate to measure its fluorescence value and record the fluorescence intensity.
[0042] The results are as follows Figure 2 As shown in the figure, line a is the fluorescence of PBTZM, line b is the fluorescence of PBTZM+enrofloxacin, and its fluorescence signal is very weak, proving that ENR does not react to PBTZM; line c is PBTZM / aptamer, because PBTZM is inserted into the aptamer, the fluorescence signal is enhanced to a certain extent; lines d, e, and f are PBTZM / aptamer / enrofloxacin, and the addition of enrofloxacin to the PBTZM / aptamer solution will cause the aptamer and enrofloxacin to selectively bind, making PBTZM away from the aptamer and reducing the fluorescence signal. And with the increase of enrofloxacin concentration, the fluorescence signal continues to decrease.
[0043] This proves that the fluorescence analysis method for detecting the fluorescence signal intensity of enrofloxacin content, which uses PBTZM as a fluorescent probe and enrofloxacin nucleic acid aptamer as a recognition element, is feasible.
[0044] Example 4 Optimization of the optimal buffer solution for the investigation system by different experimental conditions
[0045] Four different buffer solutions (pure water, Tris-HCl buffer (pH 7.5), PBS buffer (pH 7.4), MOPS buffer (pH 7.0)) were selected and added with nucleic acid aptamer and fluorescent probe PBTZM, mixed and allowed to stand for 10 minutes, and finally ENR was added to measure the fluorescence intensity at a wavelength of 610 nm. A blank solution was prepared in the same way.
[0046] Depend on Figure 3 It can be seen that the most significant fluorescence intensity change can be observed in the reaction medium of Tris-HCl buffer. Therefore, Tris-HCl buffer was selected as the buffer solution for subsequent experimental studies.
[0047] Aptamer fluorescence sensor: Fluorescence is the most commonly used optical detection method in aptamer sensing systems. Fluorescence is easy to operate, low in cost, fast in response, and the interaction between aptamers and fluorescent substances in fluorescence detection technology is flexible and diverse. The fluorescent aptamer sensing platform detects by monitoring the changes in fluorescence intensity or fluorescence anisotropy caused by changes in the microenvironment of the system. Most fluorescent aptamer sensing platforms detect target molecules based on changes in fluorescence intensity caused by the affinity between the target molecule and the aptamer; or fluorescent groups and quenching groups are marked on the ends of the aptamer chains, respectively, and the analyte is quantitatively analyzed by introducing the target molecule into the system to induce changes in the fluorescence intensity signal.
[0048] Fluorescent aptamer sensing technology is an analytical method established by inducing the change of aptamer configuration by target molecules and combining with fluorescent probes to cause changes in fluorescent signals. It has the advantages of simple operation, rapid response, and high sensitivity. A label-free nucleic acid aptamer sensor was constructed for ENR detection. The fluorescent aptamer sensor can be obtained in one step with simple mixing, and no other high-cost materials need to be added, so the detection cost is low.
[0049] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A PBTZM fluorescent probe, characterized in that: The chemical name of the fluorescent probe is: 2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-3-methylbenzo[d]thiazole-3H-iodide. The structure is shown in the formula:
2. A method for synthesizing a PBTZM fluorescent probe, characterized in that: (1) 2-methylbenzothiazole and 4-(dimethylamino)cinnamaldehyde are mixed in a molar ratio of 1:1, potassium tert-butoxide and DMF solution are added, reacted at 80° C., extracted with dichloromethane, washed, separated, and the excess solvent is removed by rotary evaporation and purified by column chromatography to obtain 2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-benzothiazole; (2) 2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-benzothiazole was dissolved in CH3CN, iodomethane was added, and the reaction was refluxed at 85°C. After the reaction was completed at room temperature, dichloromethane was added for extraction and washing. The liquids were separated, and the excess solvent was removed by rotary evaporation and purified by column chromatography to obtain the compound PBTZM.
3. The method for synthesizing a PBTZM fluorescent probe according to claim 2, characterized in that: The added mass ratio of 2-methylbenzothiazole to potassium tert-butoxide is 1:0.
75.
4. The method for synthesizing a PBTZM fluorescent probe according to claim 2, characterized in that: The added mass-volume ratio of 2-methylbenzothiazole to the DMF solution was 0.03 g / mL.
5. The method for synthesizing a PBTZM fluorescent probe according to claim 2, characterized in that: The mass-volume ratio of 2-[4-(4-dimethylaminophenyl)-1,3-butadienyl]-benzothiazole to methyl iodide is 0.98 g / mL.
6. Use of the PBTZM fluorescent probe as claimed in claim 1 in detecting the content of enrofloxacin, characterized in that: The synthesized PBTZM is used as a fluorescent dye and the enrofloxacin nucleic acid aptamer is used as a recognition unit. The enrofloxacin nucleic acid aptamer and PBTZM are added to a Tris-HCl buffer solution. After the analyte enrofloxacin is added, the nucleic acid aptamer structure is induced to change, causing a change in the fluorescence signal of the system. The fluorescence intensity value of the system is detected, and the content of the analyte is calculated. The nucleic acid aptamer of enrofloxacin is: ENR-aptamer: 5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCCCGG GTT ATT TCA GGG GGA-3.
7. The reference according to claim 6, characterized in that: The parameters for measuring the fluorescence value of the solution are 554nm as the excitation wavelength and 594-656nm as the emission wavelength.