CN116239545A - Fluorescent probe, preparation method and application thereof - Google Patents
Fluorescent probe, preparation method and application thereof Download PDFInfo
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- CN116239545A CN116239545A CN202111489448.0A CN202111489448A CN116239545A CN 116239545 A CN116239545 A CN 116239545A CN 202111489448 A CN202111489448 A CN 202111489448A CN 116239545 A CN116239545 A CN 116239545A
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
- C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/976—Trypsin; Chymotrypsin
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Abstract
The invention 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 invention provides a probe which can be used for rapidly detecting and imaging pancreatic juice leakage during and after operation, and has good stability and optical performance, high selectivity and sensitivity and simple preparation method
Description
Technical Field
The invention relates to the technical field of chemical analysis and detection, in particular to a fluorescent probe, a preparation method and application thereof.
Background
Pancreatic surgery is the only method for radically treating pancreatic tumors at present, but has a plurality of postoperative complications of pancreas surgery and high perioperative mortality rate. Among all postoperative complications of pancreatic surgery, postoperative pancreatic fistulae are the most common and serious, which are recognized as the main cause of the reduction of the postoperative survival rate of patients, and the overall incidence is maintained at 10-30%. Once a post-operative pancreatic fistula occurs, pancreatic juice accumulates in the abdominal cavity and trypsin is activated. Activated trypsin can erode, digest the pancreas itself and surrounding tissue, causing tissue necrosis, bleeding and secondary infection, leading to more serious complications and even endangering the patient's life.
One of the main reasons that pancreatic fistula is difficult to prevent is the colorless and odorless physical properties of pancreatic juice, and it is common practice to quantitatively detect amylase in drainage liquid in clinic to determine whether pancreatic fistula occurs. However, this method is indirect and does not directly detect trypsin. The secretion of trypsin is affected by a variety of factors, such as pancreatic diseases, dietary stimulation or hormonal inhibition, and simple amylase detection cannot respond to the secretion of trypsin and the severity of pancreatic fistulae. Further, patent CN105334209 and CN111565760 disclose pancreatic juice developer and aqueous composition for body fluid leakage detection based on pH response, respectively, however, these two developers and compositions for pancreatic fistula detection contain pH indicators such as bromothymol blue, and these pancreatic juice developers are not high in sensitivity, not obvious in color contrast, have the problem of inaccuracy, and in addition, pH change of body fluid does not always reflect leakage of pancreatic juice. Patent CN109929548 discloses a novel near infrared fluorescent probe for carboxypeptidase a detection, which is said to be useful for in vivo fluorescence imaging and has diagnostic function for pancreatic juice leakage disease. However, carboxypeptidase A is not a major enzyme in pancreatic juice and has a low content, so that the accuracy of the fluorescent probe is not high. Mori and Yamashita et al report on a fluorescent probe, glutamyl phenylalanine hydroxymethylrhodamine green (gPhe-HMRG), in journal British journal of surgery and gastroentomology (see 2013,100:1220-1228;2015, 149:1334-1336), claim that chymotrypsin in pancreatic juice can be detected and thus the presence of pancreatic juice leakage can be detected and imaged rapidly during and after surgery, but the fluorescent probe is complex to synthesize, not easy to obtain and difficult to apply in practice
The major cause of the serious consequences of pancreatic juice leakage is that proteases in pancreatic juice corrode and destroy peripheral organs and vascular structures of the pancreas. The protease in pancreatic juice comprises trypsin, chymotrypsin, elastase, carboxypeptidase, etc., wherein the trypsin content is the highest. Techniques directed to detecting trypsin in pancreatic juice will be more targeted and accurate for pancreatic juice leak detection. However, there is no report on a fluorescent probe for detecting trypsin in pancreatic juice.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a fluorescent probe, a preparation method and application thereof. The fluorescent probe has good selectivity to trypsin, and can be used for specifically detecting pancreatic juice.
The technical scheme provided by the invention is as follows:
in one aspect, the present invention provides a fluorescent probe comprising a compound represented by formula (I):
in another aspect, the invention provides a preparation method of the fluorescent probe, which comprises the steps of taking nile blue as a luminous parent nucleus and connecting the nile blue with lysine through dehydration condensation reaction.
In one embodiment, the lysine is a double Boc protected lysine and the dehydration condensation reaction is of the formula:
in one embodiment, nile blue and an equivalent amount of double Boc protection lysine are dissolved in a solvent, after the dissolution is completed, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are added, the mixture is reacted at room temperature overnight, then methyl tertiary butyl ether is added to separate out a solid product, and the solid product is obtained after filtration and drying; preferably, the solvent is dried N, N-dimethylformamide or chloroform.
In one embodiment, the method further comprises dissolving the obtained solid in dichloromethane, then adding trifluoroacetic acid and trichloroacetic acid for stirring, concentrating, and purifying to obtain the fluorescent probe; preferably, the purification comprises purification by column chromatography; more preferably, purification is performed by HPLC.
In another aspect, the present invention provides a kit for trypsin detection comprising the fluorescent probe described above.
In another aspect, the invention provides the use of the fluorescent probe for detecting pancreatic juice or trypsin. Further, the application of the fluorescent probe comprises incubating the sample of the biological system with the fluorescent probe, and analyzing the cell biological sample by using a fluorescence imaging method.
In one embodiment, in the detection, the concentration of fluorescent probe in solution is 8 to 12 μg/mL; preferably 10. Mu.g/mL.
In one embodiment, in said detecting, said fluorescent probe and said pancreatic juice or trypsin are subjected to a fluorescence spectrometer test after shaking in a water bath at 35-37 ℃ for more than 30 minutes.
In a specific embodiment, the fluorescent probe is applied to trypsin detection as follows: preparing a prepared fluorescent probe into stock solution, preparing trypsin into the stock solution with a certain concentration, diluting the fluorescent probe to a certain concentration by using PBS buffer solution, adding the trypsin stock solution, oscillating for more than 30 minutes in a water bath at 37 ℃, and testing on a fluorescence spectrometer. Specifically, for example, a stock solution of 1mg/mL was prepared by dissolving the prepared fluorescent probe in deionized water, and a stock solution of 50. Mu.g/mL was prepared by dissolving trypsin in 10 XPBS; stock solutions of fluorescent probes were taken into 10 XPBS to a concentration of 10. Mu.g/mL, followed by addition of stock solutions of trypsin to a final concentration of 5. Mu.g/mL, and were measured on a fluorescence spectrometer after shaking in a water bath at 37℃for 30 minutes. The control group was 10. Mu.g/mL fluorescent probe solution without trypsin.
In a specific embodiment, the fluorescent probe is applied to pancreatic juice detection as follows: the prepared fluorescent probe was prepared as a stock solution, and the stock solution of the fluorescent probe was added to pancreatic juice, and enterokinase solution was added. The incubation was performed by shaking in a water bath at 37℃for more than 30 minutes. After incubation, the incubation was performed on a fluorescence spectrometer after shaking in a water bath at 37℃for 30 minutes. Specifically, for example, the prepared fluorescent probe is dissolved in deionized water to prepare a stock solution of 1 mg/mL; the stock solution of fluorescent probe was added to pancreatic juice to a concentration of 10. Mu.g/mL, and 1. Mu.L of enterokinase solution was added thereto, and the final concentration of enterokinase in the system was 1U/mL. In addition, a control group having only fluorescent probes and a blank group having only pancreatic juice were set. It was incubated well by shaking in a water bath at 37℃for 30 minutes. After incubation, the incubation was performed on a fluorescence spectrometer after shaking in a water bath at 37℃for 30 minutes. Whether pancreatic juice is present or not can be determined based on the decrease in fluorescence intensity. Further, it is clinically useful for diagnosis of pancreatic juice leakage.
In the invention, trypsin can be utilized to specifically cleave lysine when designing the fluorescent probe. By combining-COOH on lysine and-NH on Nile blue 2 The groups are dehydrated and condensed to form fluorescent probe LYS-NB. The fluorescent probe has the highest fluorescence intensity at 575nm under the excitation of 460nm wavelength. However, after the lysine of the fluorescent probe is cut off by trypsin, the fluorescent intensity at 575nm is obviously reduced under the excitation of 460nm wavelength. Therefore, this phenomenon can be used for specific detection of trypsin and pancreatic juice.
The beneficial effects are that:
(1) The fluorescent probe provided by the invention has good selectivity to trypsin, can detect trypsin in pancreatic juice, and can detect pancreatic juice leakage more specifically and accurately;
(2) The synthesis method of the fluorescent probe has the advantages of simple operation, mild reaction conditions, wide raw material sources and low cost;
(3) The fluorescent probe obtained by the reaction has high yield and stable performance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a mass spectrum of a novel fluorescent probe of the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of a novel fluorescent probe of the present invention;
FIG. 3 is an excitation spectrum and an emission spectrum of the novel fluorescent probe of the present invention;
FIG. 4 is an emission spectrum of the novel fluorescent probe of the present invention after incubation with trypsin at an excitation wavelength of 460 nm;
FIG. 5 shows the change of fluorescence intensity before and after incubation of the novel fluorescent probe with pancreatic juice;
FIG. 6 shows the result of the novel fluorescent probe of the present invention for pancreatic juice detection;
FIG. 7 shows the change in maximum fluorescence emission intensity of the novel fluorescent probe according to the present invention after laser irradiation in PBS buffer (20 mM, pH=7.4), with the abscissa indicating the illumination time and the ordinate indicating the relative fluorescence intensity, i.e. the ratio of the maximum fluorescence emission intensity to the initial maximum fluorescence emission intensity.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: preparation of novel fluorescent probes
The technical roadmap is as follows:
the preparation method comprises the following steps:
318mg of Nile blue (Compound 1) and 346mg of bis-Boc protected lysine (Compound 2) were dissolved in 5mL of dry N, N-dimethylformamide with magnetic stirring for 2 hours. After completion of the dissolution, 192mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 12mg of 4-dimethylaminopyridine were added and reacted at room temperature for 12 hours.
After the reaction, 15mL of methyl tert-butyl ether was added thereto, and the mixture was left standing for 6 hours. When solid product is precipitated, it is filtered off with a buchner funnel and drained. The resulting solid was dissolved in 5mL of methylene chloride, and 1mL of trifluoroacetic acid was added thereto and stirred for one hour. Preparation of the liquid phase by Waters HPLC (10-60%, acetonitrile vs. water, 0.1% TFA) gave 88mg of fluorescent probe in 13.25% yield.
The molecular weight of the fluorescent probe was measured by using a Waters QuattroMicro API MS/MS mass spectrometer, and the results are shown in FIG. 1. Mass spectrum representative nuclear mass ratio (m/z): 51.1,84.1, 149.4, 215.1,223.6, 318.0, 446.3. The hydrogen spectrum of the fluorescent probe was measured by Bruker 400 NMR using TMS as an internal standard and DMSO-d6 as a solvent, and the results are shown in FIG. 2.
Example 2 spectral scanning of novel fluorescent probes
The probe was prepared into a solution with a concentration of 1. Mu.g/mL using deionized water, and then scanned for excitation and emission spectra using a fluorescence spectrophotometer, and each experiment was repeated twice. In the test, 3mL of sample solution is added into a quartz fluorescence cuvette, the quartz fluorescence cuvette is placed into an FLS1000 type transient fluorescence spectrometer, the excitation wavelength and the emission wavelength are set, the emission spectrum scanning range is from the excitation wavelength of +15nm to the emission wavelength of +160nm, the step length is 1nm, and the scanning speed is 0.3 seconds, and the result is shown in figure 3. The maximum excitation wavelength of the novel fluorescent probe is 460nm, and the maximum emission wavelength is about 575 nm.
Example 3 Spectrum scanning of novel fluorescent probes after incubation with trypsin
The fluorescent probes were prepared in deionized water to a concentration of 10. Mu.g/mL and divided into 2 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. Mu.g/mL. The two sets were then incubated thoroughly by shaking in a water bath at 37℃for 30 minutes. After the incubation, the FLS1000 type transient fluorescence spectrometer is placed, the excitation wavelength is set to be 460nm, the emission wavelength scanning range is 470-700nm, and the result is shown in FIG. 4. The fluorescence intensity of the fluorescent probe at 460nm was 350000, but after trypsin hydrolysis, the fluorescence intensity was 40000 and 89%.
Example 4 novel fluorescent probes for trypsin detection
Solutions with trypsin concentrations of 0,1,2,3,4 and 5 mug/mL are prepared, fluorescent probes are added to the solutions respectively, the final concentration of the probes in the system is 10 mug/mL, and each group of systems is placed in a water bath at 37 ℃ to oscillate for 30 minutes so as to be fully incubated. After the incubation, the sample was placed in an FLS1000 type transient fluorescence spectrometer, the excitation wavelength was set at 460nm, and the fluorescence intensity at the emission wavelength of 575nm was measured, and the result was shown in FIG. 5. As the trypsin concentration in the system increases, the fluorescence intensity of the fluorescent probe shows an off-line decreasing relationship. The novel fluorescent probe can be used for detecting trypsin.
Example 5 novel fluorescent probes for detection of pancreatic juice
The fluorescent probes were prepared in deionized water to a concentration of 10. Mu.g/mL and divided into 2 groups. One group was not added with pancreatic juice, the other group was added with pancreatic juice, and 1. Mu.L of enterokinase solution was added. In addition, a pancreatic juice blank group was set. The three systems were incubated thoroughly by shaking in a water bath at 37℃for 30 minutes. After the incubation, the sample was placed in an FLS1000 type transient fluorescence spectrometer, the excitation wavelength was set at 460nm, and the fluorescence intensity at the emission wavelength of 575nm was measured, and the result is shown in FIG. 6. The fluorescence intensity of the fluorescent probe is obviously reduced after the fluorescent probe is incubated with pancreatic juice. Therefore, it is possible to determine whether pancreatic juice is present or not based on the decrease in fluorescence intensity. Further, it is clinically useful for diagnosis of pancreatic juice leakage.
Example 6 light stability test of novel fluorescent probes
The novel fluorescent probe and the fluorescein dye are respectively prepared into 10 mug/mL fluorescent probe test solution in PBS buffer solution, and continuously irradiated under a 200W tungsten lamp, the light source is 50cm away from the sample, and the fluorescent spectrum test is carried out after the temperature of the test solution is stabilized at 25 ℃ each time. Taking 0,0.5,1,1.5,2,4,6 and 8 hours as time points, respectively testing the fluorescence intensities of the novel fluorescent probe and the fluorescein dye at 575nm and 519 nm. The ratio of the maximum fluorescence emission intensity to the initial maximum fluorescence emission intensity of the novel fluorescent probe after laser irradiation at different times is shown in fig. 7, and after continuous irradiation for 8 hours, the fluorescence emission intensity of the novel fluorescent probe is reduced by only 20% compared with the initial maximum fluorescence emission intensity, and the emission intensity of the fluorescein dye is reduced by nearly 80%, so that the novel fluorescent probe has good light stability.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A fluorescent probe comprising a compound represented by the formula (I):
2. a method for preparing a fluorescent probe according to claim 1, wherein the fluorescent probe is formed by connecting nile blue and lysine through a dehydration condensation reaction; the lysine is double Boc protection lysine.
3. The preparation method according to claim 2, wherein nile blue and di-Boc protected lysine are dissolved in a solvent, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are added after the dissolution is completed, the reaction is carried out at room temperature, then methyl tertiary butyl ether is added to separate out a solid product, and the solid product is obtained after filtration and drying.
4. A method of preparation according to claim 3, wherein the solvent is dried N, N-dimethylformamide or chloroform.
5. The method according to claim 3, wherein the method further comprises dissolving the obtained solid in methylene chloride, adding trifluoroacetic acid or trichloroacetic acid, stirring, concentrating, and purifying to obtain the fluorescent probe.
6. The method of claim 5, wherein the purifying comprises purifying with column chromatography; preferably, purification is performed by HPLC.
7. A kit comprising the fluorescent probe of claim 1.
8. Use of the fluorescent probe of claim 1 or the kit of claim 7 for detecting pancreatic juice or trypsin.
9. The use according to claim 8, wherein in the detection the concentration of fluorescent probe in solution is 8-12 μg/mL; preferably 10. Mu.g/mL.
10. The use according to claim 8, wherein in said detection, said fluorescent probe and said pancreatic juice or trypsin are subjected to a fluorescence spectrometer test after shaking in a water bath at 35-37 ℃ for more than 30 minutes.
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