A fluorescent probe, a preparation method and application thereof

Abstract

The application provides a fluorescent probe, a preparation method and application thereof, and relates to the technical field of chemical analysis and detection. The fluorescent probe comprises a compound represented by formula (I) or a salt thereof. The application provides a probe capable of being used for rapid detection and imaging of pancreatic juice leakage in operation and after operation. The probe has good stability and optical performance, high selectivity and sensitivity, and the preparation method is simple.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis detection, in particular to a fluorescent probe, a preparation method and application thereof. BACKGROUND

[0002] Pancreatic surgery is currently the only method to cure pancreatic tumor, but the postoperative complications of pancreatic surgery are many, and the perioperative mortality rate is high. Among all the postoperative complications of pancreatic surgery, postoperative pancreatic fistula is the most common and serious, which is recognized as the main reason for the decrease of postoperative survival rate of patients, and the overall incidence rate remains at 10-30%. Once postoperative pancreatic fistula occurs, pancreatic juice will accumulate in the abdominal cavity, and trypsin will be activated. The activated trypsin can erode and digest the pancreas itself and the surrounding tissues, causing tissue necrosis, hemorrhage and secondary infection, and further leading to more serious complications, and even endangering the life of the patient.

[0003] One of the main reasons for the difficulty in preventing pancreatic fistula is the physical property of colorless and odorless pancreatic juice. Clinically, the quantitative detection of amylase in the drainage fluid is usually used to judge whether pancreatic fistula occurs. However, this method is indirect and not a direct detection of trypsin. The secretion of trypsin is affected by many factors, such as pancreatic diseases, dietary stimulation or hormone inhibition, etc., and simple amylase detection cannot reflect the secretion of trypsin and the severity of pancreatic fistula. In addition, patents CN105334209 and CN111565760 respectively disclose a pancreatic juice color developing agent and a body fluid leakage detection aqueous composition based on pH response, however, these two color developing agents and compositions for detecting pancreatic fistula contain pH indicators such as bromothymol blue, and the sensitivity of these pancreatic juice color developing agents is not high, the color contrast is not obvious, and there is a problem of inaccuracy. In addition, the pH change of body fluid does not always reflect the leakage of pancreatic juice. Patent CN109929548 discloses a new near-infrared fluorescent probe for carboxypeptidase A detection, which is claimed to be used for in vivo fluorescence imaging and has a diagnostic function for pancreatic juice leakage disease. However, carboxypeptidase A is not the main enzyme in pancreatic juice and has a very low content, so the accuracy of this fluorescent probe is not high. Mori and Yamashita et al. reported in the journals of British journal of surgery and Gastroenterology (see 2013, 100: 1220-1228; 2015, 149: 1334-1336) a fluorescent probe glutamylphenylalanine hydroxymethyl rhodamine green (gPhe-HMRG), which is claimed to be able to detect chymotrypsin in pancreatic juice and thus can quickly detect and image the presence of pancreatic juice leakage during and after surgery, but the synthesis of this fluorescent probe is complex and not easy to obtain, making it difficult to apply in practice

[0004] The main reason for the serious consequences of pancreatic juice leakage is that the proteases in the pancreatic juice can erode and destroy the surrounding organs and vascular structures of the pancreas. The proteases in the pancreatic juice include trypsin, chymotrypsin, elastase, carboxypeptidase, etc., among which the content of trypsin is the highest. The technology for detecting trypsin in pancreatic juice will be more targeted and more accurate for pancreatic juice leakage detection. However, there is no report on the fluorescent probe for detecting trypsin in pancreatic juice.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to provide a fluorescent probe, a preparation method and an application thereof. The fluorescent probe of the present application has good selectivity for trypsin and can specifically detect pancreatic juice.

[0007] The technical solutions provided by the present application are as follows:

[0008] In one aspect, the present application provides a fluorescent probe comprising a compound represented by formula (I) or a salt thereof:

[0009]

[0010] In another aspect, the present application provides a preparation method of the fluorescent probe, comprising connecting nile blue as a light-emitting mother nucleus with lysine through a dehydration condensation reaction to form.

[0011] In one embodiment, the lysine is double Boc-protected lysine, and the reaction formula of the dehydration condensation reaction is as follows:

[0012]

[0013] In one embodiment, nile blue and an equal amount of double Boc-protected lysine are dissolved in a solvent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are added after the dissolution is completed, and the reaction is carried out at room temperature overnight, then methyl tert-butyl ether is added to precipitate the solid product, and the solid is obtained after filtration and drying; preferably, the solvent is dry N,N-dimethylformamide or chloroform.

[0014] In one embodiment, the method further comprises dissolving the obtained solid in dichloromethane, then adding trifluoroacetic acid and stirring, and then purifying the fluorescent probe after concentration; preferably, the purification comprises purifying by column chromatography; more preferably, purifying by HPLC.

[0015] In another aspect, the present application provides a trypsin detection kit comprising the aforementioned fluorescent probe.

[0016] In another aspect, the present application provides the use of the fluorescent probe in detecting pancreatic juice or trypsin. Further, the use of the fluorescent probe comprises incubating a sample of the biological system with the fluorescent probe, and analyzing the cell biological sample by fluorescence imaging.

[0017] In one embodiment, in the detection, the concentration of the fluorescent probe in the solution is 8-12 μg / mL; preferably 10 μg / mL.

[0018] In one embodiment, in the detection, the fluorescent probe is incubated with the pancreatic juice or trypsin in a water bath at 35-37°C for more than 30 minutes, and then tested by a fluorescence spectrometer.

[0019] In one specific embodiment, the use of the fluorescent probe in the detection of trypsin is as follows: the prepared fluorescent probe is formulated into a stock solution, trypsin is prepared into a stock solution with a certain concentration, the fluorescent probe is diluted with PBS buffer to a certain concentration, and then the trypsin stock solution is added. After water bath shaking at 37°C for more than 30 minutes, it is tested on a fluorescence spectrometer. Specifically, for example, the prepared fluorescent probe is dissolved in deionized water to prepare a 1 mg / mL stock solution, and trypsin is dissolved in 10×PBS to prepare a 50 μg / mL stock solution. The fluorescent probe stock solution is taken into 10×PBS to make the concentration 10 μg / mL, and then the trypsin stock solution is added to make the final concentration 5 μg / mL. After water bath shaking at 37°C for 30 minutes, it is tested on a fluorescence spectrometer. The control group is a 10 μg / mL fluorescent probe solution without trypsin.

[0020] In one specific embodiment, the use of the fluorescent probe in the detection of pancreatic juice is as follows: the prepared fluorescent probe is formulated into a stock solution, the fluorescent probe stock solution is added to the pancreatic juice, and enterokinase solution is added. After water bath shaking at 37°C for more than 30 minutes, it is fully incubated. After incubation, it is water bath shaken at 37°C for 30 minutes, and then tested on a fluorescence spectrometer. Specifically, for example, the prepared fluorescent probe is dissolved in deionized water to prepare a 1 mg / mL stock solution. The fluorescent probe stock solution is added to the pancreatic juice to make the concentration 10 μg / mL, and 1 μL of enterokinase solution is added. The final concentration of enterokinase in the system is 1 U / mL. In addition, a control group with only the fluorescent probe and a blank group with only the pancreatic juice are set. After water bath shaking at 37°C for 30 minutes, it is fully incubated. After incubation, it is water bath shaken at 37°C for 30 minutes, and then tested on a fluorescence spectrometer. Whether there is pancreatic juice can be judged according to the decrease of fluorescence intensity. Further, it can be used for the diagnosis of pancreatic juice leakage in clinic.

[0021] The present application utilizes the specific enzymolysis of lysine by trypsin when designing the fluorescent probe. The -COOH group on lysine and the -NH2 group on nile blue are dehydrated and condensed to form the fluorescent probe LYS-NB. The fluorescent probe has the highest fluorescence intensity at 575nm under excitation at 460nm. However, after the lysine is cut off by trypsin, the fluorescence intensity at 575nm under excitation at 460nm is significantly reduced. Therefore, this phenomenon can be used for specific detection of trypsin and pancreatic juice.

[0022] Advantages:

[0023] (1) The fluorescent probe provided by the present application has good selectivity for trypsin, and can detect trypsin in pancreatic juice, so as to more accurately detect pancreatic juice leakage.

[0024] (2) The synthesis method of the fluorescent probe of the present application is simple in operation, mild in reaction conditions, and low in cost.

[0025] (3) The fluorescent probe obtained by the reaction of the present application has high yield and stable performance. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0027] Figure 1 The mass spectrum of the novel fluorescent probe of the present application;

[0028] Figure 2 The nuclear magnetic resonance hydrogen spectrum of the novel fluorescent probe of the present application;

[0029] Figure 3 The excitation spectrum and emission spectrum of the novel fluorescent probe of the present application;

[0030] Figure 4 The emission spectrum of the novel fluorescent probe of the present application after incubation with trypsin under excitation wavelength of 460nm;

[0031] Figure 5 The change of fluorescence intensity of the novel fluorescent probe of the present application before and after incubation with pancreatic juice;

[0032] Figure 6 The results of the novel fluorescent probe of the present application for detection of pancreatic juice;

[0033] Figure 7The change of the maximum fluorescence emission intensity of the novel fluorescent probe in PBS buffer (20 mM, pH = 7.4) after laser irradiation for different time is shown in the figure, in which the abscissa represents the irradiation time, and the ordinate represents the relative fluorescence intensity, i.e. the ratio of the maximum fluorescence emission intensity to the initial maximum fluorescence emission intensity. DETAILED DESCRIPTION

[0034] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0035] Example 1: Preparation of the novel fluorescent probe

[0036] The technical route is shown in the following figure:

[0037]

[0038] The preparation steps are as follows:

[0039] Dissolve 318 mg of Nile blue (compound 1) and 346 mg of Boc-protected lysine (compound 2) in 5 mL of dry N,N-dimethylformamide, and dissolve for 2 hours under magnetic stirring. After the dissolution is completed, add 192 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 12 mg of 4-dimethylaminopyridine, and react at room temperature for 12 hours.

[0040] After the reaction is completed, add 15 mL of methyl tert-butyl ether, and stand for 6 hours. When the solid product is precipitated, filter it with a Buchner funnel, and dry it. Dissolve the obtained solid in 5 mL of dichloromethane, and then add 1 mL of trifluoroacetic acid, and stir for one hour. Use Waters HPLC liquid phase preparation (10-60%, acetonitrile to water, 0.1% TFA) to obtain 88 mg of the fluorescent probe, with a yield of 13.25%.

[0041] Determine the molecular weight of the fluorescent probe by using a Waters QuattroMicro API MS / MS mass spectrometer, and the result is shown in the following figure. Figure 1 The representative mass spectrum (m / z) is 51.1, 84.1, 149.4, 215.1, 223.6, 318.0, and 446.3. Determine the hydrogen spectrum of the fluorescent probe by using a Bruker 400 nuclear magnetic resonance instrument with TMS as an internal standard and DMSO-d6 as a solvent, and the result is shown in the following figure. Figure 2

[0042] Example 2: Spectral scanning of the novel fluorescent probe

[0043] ​The probe was prepared into a solution with a concentration of 1 μg / mL using deionized water, and then its excitation spectrum and emission spectrum were scanned using a fluorescence spectrophotometer, twice for each experiment. During testing, 3 mL of the sample solution was added to a quartz fluorescence cuvette, and then placed into a FLS1000 transient fluorescence spectrometer, and the excitation wavelength and emission wavelength were set. The emission spectrum scanning range was excitation wavelength + 15 nm to emission wavelength + 160 nm, the step was 1 nm, and the scanning speed was 0.3 seconds. The results are shown in Figure 3 The maximum excitation wavelength of the new fluorescent probe was 460 nm, and the maximum emission wavelength was about 575 nm.

[0044] Example 3. Spectral scanning of the new fluorescent probe after incubation with trypsin

[0045] The fluorescent probe was prepared into a solution with a concentration of 10 μg / mL using deionized water, and then divided into two groups. One group was not added with trypsin, and the other group was added with trypsin, so that the final concentration of trypsin in the system was 5 μg / mL. Then the two groups of systems were placed in a 37°C water bath for 30 minutes to fully incubate. After incubation, they were placed into a FLS1000 transient fluorescence spectrometer, and the excitation wavelength was set to 460 nm, and the emission wavelength scanning range was 470-700 nm. The results are shown in Figure 4 The fluorescence intensity of the fluorescent probe at 460 nm was 350000, but after trypsin hydrolysis, its fluorescence intensity decreased to 40000, a decrease of 89%.

[0046] Example 4. The new fluorescent probe for the detection of trypsin

[0047] Solutions with trypsin concentrations of 0, 1, 2, 3, 4, and 5 μg / mL were prepared, and then the fluorescent probe was added, so that the final concentration of the probe in the system was 10 μg / mL. Each group of systems was placed in a 37°C water bath for 30 minutes to fully incubate. After incubation, they were placed into a FLS1000 transient fluorescence spectrometer, and the excitation wavelength was set to 460 nm. The fluorescence intensity at an emission wavelength of 575 nm was measured, and the results are shown in Figure 5 With the increase of the concentration of trypsin in the system, the fluorescence intensity of the fluorescent probe showed a linear decrease. The new fluorescent probe can be used for the detection of trypsin.

[0048] Example 5. The new fluorescent probe for the detection of pancreatic juice

[0049] The fluorescent probe was prepared into a solution with a concentration of 10 μg / mL with deionized water and was divided into two groups. One group was not added with pancreatic juice, and the other group was added with pancreatic juice and 1 μL of enterokinase solution. In addition, a pancreatic juice blank group was set. The three groups of systems were placed in a 37°C water bath for 30 minutes to fully incubate. After incubation, they were placed in a FLS1000 type transient fluorescence spectrometer, the excitation wavelength was set to 460 nm, and the fluorescence intensity at an emission wavelength of 575 nm was measured, and the results are shown in Figure 6 After the fluorescent probe was incubated with pancreatic juice, the fluorescence intensity was significantly reduced. Therefore, the presence or absence of pancreatic juice can be determined according to the reduction of fluorescence intensity. Further, it can be used for the diagnosis of pancreatic juice leakage in clinical practice.

[0050] Example 6. Light stability test of the new fluorescent probe

[0051] The new fluorescent probe and the fluorescein dye were respectively taken in PBS buffer, and a fluorescent probe test solution with a concentration of 10 μg / mL was prepared, and was continuously irradiated under a 200W tungsten lamp, the light source distance from the sample was 50 cm, and after the temperature of the test solution was stabilized at 25°C, the fluorescence spectrum test was carried out. 0, 0.5, 1, 1.5, 2, 4, 6, and 8 hours were taken as time points, and the fluorescence intensity of the new fluorescent probe and the fluorescein dye at 575 nm and 519 nm was tested respectively. The ratio of the maximum fluorescence emission intensity of the new fluorescent probe after different time laser irradiation to the initial maximum fluorescence emission intensity is shown in Figure 7 After 8 hours of continuous irradiation, the fluorescence emission intensity of the new fluorescent probe only decreased by 20% compared with the initial maximum fluorescence emission intensity, while the emission intensity of the fluorescein dye decreased by nearly 80%, which proves that the new fluorescent probe has good light stability.

[0052] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A fluorescent probe, characterized in that, Includes the compound or its salt represented by formula (I): （I）； The anion of the fluorescent probe is trifluoroacetate ion.

2. A method for preparing the fluorescent probe according to claim 1, characterized in that, Nile blue and bis-Boc protected lysine were dissolved in a solvent. After dissolution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added and reacted at room temperature. Then, methyl tert-butyl ether was added to precipitate a solid product. After filtration and drying, a solid was obtained. The obtained solid was dissolved in dichloromethane, then trifluoroacetic acid was added and stirred. After concentration, the solution was purified to obtain the fluorescent probe.

3. The preparation method according to claim 2, characterized in that, The solvent is dry N,N-dimethylformamide or chloroform.

4. The preparation method according to claim 2, characterized in that, The purification includes purification using column chromatography.

5. The preparation method according to claim 4, characterized in that, Purification was performed by HPLC.

6. A reagent kit, characterized in that, It includes the fluorescent probe of claim 1.

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