CN115974837B - Ratio type fluorescent probe and preparation method and application thereof - Google Patents

Ratio type fluorescent probe and preparation method and application thereof Download PDF

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CN115974837B
CN115974837B CN202211652481.5A CN202211652481A CN115974837B CN 115974837 B CN115974837 B CN 115974837B CN 202211652481 A CN202211652481 A CN 202211652481A CN 115974837 B CN115974837 B CN 115974837B
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fluorescent probe
probe
ratio
type fluorescent
bche
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CN115974837A (en
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倪梅
聂海亮
王朝俊
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China Fire Rescue College
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China Fire Rescue College
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to the technical field of enzyme activity detection, and particularly relates to a ratio type fluorescent probe, a preparation method and application thereof. The ratio type fluorescent probe has the structure shown in the formula (1), is particularly suitable for detecting the activity of butyrylcholine esterase and monitoring the inhibition effect of organophosphorus pesticides and carbamate pesticides on butyrylcholine esterase, has the advantages of high accuracy, high sensitivity, strong selectivity, quick response, good stability and simplicity in operation, and particularly can be used for analyzing the activity of butyrylcholine esterase by utilizing the fluorescence intensity ratio of double emission peaks, and can effectively reduce the interference of factors such as probe concentration, microenvironment, light source intensity and the like in a self-correcting mode.

Description

Ratio type fluorescent probe and preparation method and application thereof
Technical Field
The invention belongs to the technical field of enzyme activity detection, and particularly relates to a ratio type fluorescent probe, a preparation method and application thereof.
Background
Butyrylcholinesterase (BChE) is a serine hydrolase produced by the liver and is widely found in blood, liver and glia. The decrease of BChE activity in serum is usually seen in the onset of hepatitis, liver cancer, etc., while the increase of the activity is seen in the pre-onset of Alzheimer's disease, diabetes, hypertension, etc. Since organophosphorus and carbamate pesticides can inhibit the activity of BChE, the active concentration of BChE in blood is also an important index for diagnosing poisoning of the two pesticides. Therefore, the development of a detection method for BChE activity is of great importance for early diagnosis of related diseases and pesticide poisoning.
The fluorescent probe has the characteristics of high sensitivity, strong specificity, simple operation, quick response, low cost and the like, and gradually becomes a novel tool for measuring BChE. At present, most of reported BChE fluorescent probes indirectly quantify enzyme activity by measuring the concentration of an enzymolysis product thiocholine, and have the defects of serious interference by mercaptan, low sensitivity, complex operation and the like. Most of the existing fluorescent probes capable of directly detecting BChE activity are enhanced fluorescent probes, enzyme activity can be quantified only according to fluorescence intensity, and detection results are easily affected by various uncertainty factors such as probe concentration distribution, light stability, microenvironment (pH value, polarity, temperature and the like), instrument stability and the like, so that accuracy is low. Compared with the prior art, the ratio type fluorescent probe utilizes the ratio of the fluorescent intensity at two (or more) wavelengths to quantify the target object, and can effectively reduce the interference of factors such as probe concentration, microenvironment, light source intensity and the like in a self-correction mode, so that the quantitative result of the BChE activity is more accurate.
Therefore, how to design and synthesize a high-efficiency ratio-type fluorescent probe to realize accurate detection of butyrylcholine esterase activity is a problem to be solved by those skilled in the art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a ratio type fluorescent probe, a preparation method and application thereof, and the ratio type fluorescent probe is particularly suitable for detecting butyrylcholine esterase activity and has the advantages of high sensitivity, high selectivity and quick response.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides a ratio-type fluorescent probe having a structure represented by formula (1):
in a second aspect, the present invention provides a method for preparing the ratio-type fluorescent probe, comprising the steps of:
reacting a compound of formula (2) with cyclopropylcarbonyl chloride in an organic solvent under base conditions to give a compound of formula (1);
preferably, the amount of the cyclopropylcarbonyl chloride is 1 to 6mmol, the amount of the base is 2 to 12 mmol, and the amount of the organic solvent is 20 to 60ml, relative to 1 mmol of the compound of formula (2).
Preferably, the base is at least one selected from cesium carbonate, potassium carbonate, sodium bicarbonate, pyridine, piperazine, triethylamine, and N, N-dimethylaminopyridine; the organic solvent is selected from at least one of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, acetonitrile, acetone, N-dimethylformamide and 1, 2-dichloroethane.
Preferably, the temperature of the reaction is-20-40 ℃; the reaction time is 2-12 hours.
In a third aspect, the invention provides an application of the ratio-type fluorescent probe or the ratio-type fluorescent probe prepared by the preparation method in detection of butyrylcholinesterase activity.
In a fourth aspect, the present invention provides an application of the above ratio-based fluorescent probe or the ratio-based fluorescent probe prepared by the above preparation method in monitoring inhibition of butyrylcholine esterase by organophosphorus pesticide and carbamate pesticide; preferably, the organophosphorus pesticide is selected from one or a combination of several of paraoxon, phorate, trichlorfon, methamidophos, dichlorvos, malathion, chlorpyrifos, dimethoate and parathion, and the carbamate pesticide is selected from one or a combination of several of carbaryl, aldicarb, carbofuran and methomyl.
In a fifth aspect, the present invention provides a method for detecting butyrylcholinesterase activity, comprising the steps of:
the ratio type fluorescent probe or the ratio type fluorescent probe prepared by the preparation method is added into a sample to be detected, incubation is carried out at a certain temperature, the fluorescence spectrum of the generated fluorescent substance is measured, two emission peaks can be observed, the intensity ratio of the two emission peaks is measured and calculated, and the activity of butyrylcholine esterase can be quantified by utilizing the corresponding relation between the ratio and the concentration of butyrylcholine esterase. In the incubation process, the fluorescent probe reacts with butyrylcholine esterase in a sample to be detected, and then the cyclopropane formyl group is removed, so that the compound shown in the formula (2) is generated.
Preferably, the incubation temperature is 20-45 ℃, the pH value is 4-8, and the incubation time is 20-60min;
the fluorescence spectrum of the fluorescent substance generated by the measurement is specifically: excitation of the generated fluorescent material with light of 370-450nm, there is fluorescence emission, the emission wavelength range is 400-800nm, and two emission peaks are observed.
The beneficial effects obtained by the invention are as follows:
(1) The ratio type fluorescent probe provided by the invention is used for analyzing butyrylcholine esterase activity by utilizing the fluorescence intensity ratio of the double emission peaks, and the interference of factors such as probe concentration, microenvironment, light source intensity and the like can be effectively reduced by a self-correction mode, so that the accuracy of a detection result is high. In addition, the fluorescent probe has high sensitivity to butyrylcholinesterase and is not interfered by coexisting matters such as ions, amino acids, polysaccharides, proteins and the like.
(2) The ratio type fluorescent probe has simple synthesis steps, is easy to separate and purify, has good stability, is suitable for batch production, and is favorable for commercialization popularization and application.
(3) The ratio type fluorescent probe can be applied to detection of butyrylcholine esterase activity, can also be applied to monitoring of inhibition of organophosphorus pesticides and carbamate pesticides on butyrylcholine esterase, and has great market application and popularization value in diagnosis of organophosphorus poisoning and detection of pesticide residues.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 is a schematic diagram of a fluorescent probe according to the present invention 1 H NMR spectrum.
FIG. 2 is a schematic diagram of a fluorescent probe according to the present invention 13 C NMR spectrum.
FIG. 3 shows the absorption spectrum (a) and the fluorescence spectrum (b) before and after the reaction of the fluorescent probe of the present invention with butyrylcholinesterase.
FIG. 4 is a linear relationship (b) between the fluorescence spectrum of the probe of the present invention and the ratio of fluorescence intensity of the butyrylcholinesterase enzyme response (a) and its dual emission peak and the activity concentration of butyrylcholinesterase enzyme.
FIG. 5 shows fluorescence spectra (a) and inhibition efficiency curves (b) of the fluorescent probe of the present invention and butyrylcholinesterase enzyme after addition of inhibitor.
FIG. 6 shows fluorescence spectra (a) and inhibition efficiency curves (b) of the fluorescent probe and butyrylcholine esterase according to the invention after addition of p-oxyphosphorus reaction.
FIG. 7 shows fluorescence spectra (a) and inhibition efficiency curves (b) of the fluorescent probe of the present invention reacted with butyrylcholinesterase after carbofuran addition.
Detailed Description
The present invention is described in further detail below with reference to specific examples, but is not intended to limit the scope of the present invention.
Compound 1 was prepared by the following method: compound 2 (159.1 mg,0.5 mmol) was dissolved in 25mL toluene and CH was added 3 I (1.25 mL,3 mmol) was stirred at 110℃for 6 hours before the reaction was terminated. After the reaction solution was cooled to room temperature, a precipitate was formed. The precipitate was suction filtered and dried to give compound 1.
Compound 2 was prepared by the following method: 4-hydroxy-1, 8-naphthalic anhydride (0.428 g,2 mmol) and 2- (2-aminoethyl) pyridine (263. Mu.L, 2.2 mmol) were added to absolute ethanol (60 mL), heated to reflux, reacted for 12 hours, and then cooled to room temperature naturally. The reaction solution was concentrated under reduced pressure to about 20mL, and a white precipitate was formed, and the precipitate was filtered off to obtain Compound 2.
4-hydroxy-1, 8-naphthalene dicarboxylic anhydride was purchased from Eiffel chemical Co., ltd. In Zhengzhou, and reagents such as 2- (2-aminoethyl) pyridine and methyl iodide were purchased from Beijing enokawa technology Co., ltd.
Example 1 Synthesis of fluorescent probes
The synthetic route for preparing the fluorescent probe in this example is as follows:
the specific synthesis steps are as follows:
compound 1 (167 mg,0.36 mmol) was dissolved in 50mL CH 2 Cl 2 Cesium carbonate (704 mg,2.16 mmoL) was added thereto, and after stirring in an ice-water bath for 30 minutes, cyclopropylcarbonyl chloride (196. Mu.l, 2.16 mmoL) was added dropwise thereto, and after stirring was continued for 8 hours, the reaction was terminated. The reaction solution is decompressed and concentrated to remove the solvent, and the residue is separated by a column (silica gel, 200-300 meshes, eluent is methylene dichloride/methanol mixed solution with the volume ratio of 20:1) to obtain the ratio type fluorescent probe for detecting butyrylcholine esterase activity.
FIG. 1 is a schematic diagram of a ratio-type fluorescent probe 1 H NMR(500MHz,DMSO-d 6 ) Spectrogram of the graph: delta ppm 9.04 (dd, j=6.2, 1.5hz, 1H), 8.56-8.47 (m, 2H), 8.44-8.33 (m, 2H), 8.03 (dd, j=8.0, 1.6hz, 1H), 8.01-7.92 (m, 2H), 7.73 (d, j=8.0 hz, 1H), 4.47 (t, j=7.0 hz, 2H), 4.44 (s, 3H), 3.51 (t, j=7.1 hz, 2H), 2.17 (tt, j=7.8, 4.7hz, 1H), 1.26-1.15 (m, 4H).
FIG. 2 is a schematic diagram of a ratio-type fluorescent probe 13 C NMR(125MHz,DMSO-d 6 ) Spectrogram of the graph: delta ppm 173.91,163.98,163.42,155.90,151.96,147.37,145.71,132.02,129.54,129.14,128.51,126.41,125.43,122.81,120.68,45.84,37.65,31.57,13.20,10.15.
1 H NMR 13 The C NMR spectrum corresponds to the chemical structure of the ratio type fluorescent probe, which shows that the probe is successfully synthesized.
Experimental example 1: test of ability of fluorescent Probe to react with butyrylcholinesterase in buffer solution
(1) The fluorescent probe prepared in example 1 was formulated as a stock solution of DMSO (at a concentration of 0.2 mM), added to an EP tube, and then deionized water, PBS buffer (at a concentration of 200mM, pH 7) and a stock solution of BChE (at a concentration of 1U/mL) were sequentially added to the tube. The total volume of the solution was 1mL, the final concentration of the probe was 10. Mu.M, the final concentration of PBS was 20mM, and the final concentration of BChE was 0.2U/mL. The above solution was left in a constant temperature incubator at 37℃for 1 hour, and then its absorption spectrum and fluorescence spectrum were measured.
FIG. 3 shows the absorption spectrum (a) and fluorescence emission spectrum (b) before and after the reaction of the probe with BChE. After the probe reacted with BChE, its absorbance peak red shifted from 345nm to 450nm (fig. 3 a); the fluorescence intensity at 467nm was slightly decreased and the fluorescence intensity at 555nm was significantly increased to form a new emission peak (FIG. 3 b). The results indicate that the probe has a typical two-channel fluorescent response to BChE.
Series of EP tubes were taken and probe DMSO stock, deionized water, PBS buffer and stock of BChE of different volumes were added separately. The total volume of the solution was 1mL, the final concentration of the probe was 10. Mu.M, the final concentration of PBS was 20mM, and the final concentration of BChE was 0-0.2U/mL. The above solution was left in a constant temperature incubator at 37℃for 1 hour, and then its fluorescence spectrum was measured.
Fig. 4 is a titration experiment of probe versus BChE concentration. As the BChE concentration was increased to 2.0U/mL, the fluorescence intensity of the probe at the 467nm emission peak was slightly decreased, but the fluorescence intensity at the 555nm emission peak was gradually increased (FIG. 4 a). Fluorescence intensity ratio F of the probe at two emission peaks 550 /F 465 Shows good linear relationship with BChE concentration in the range of 0-0.07U/mL (R 2 = 0.9986) (fig. 4 b), indicating that the probe is capable of accurately quantifying BChE activity.
Experimental example 2: inhibitor characterization of fluorescent probes
The fluorescence spectrum changes were detected after pretreatment with different concentrations of phenylmethylsulfonyl fluoride (PMSF, BChE inhibitor) and BChE (0.2U/mL) for 30 minutes and then incubation with probe (10. Mu.M) for 1 hour at 37 ℃.
FIG. 5 is a fluorescence spectrum after probe-and inhibitor-pretreated BChE reaction. As the concentration of PMSF gradually increased to 200. Mu.M, the fluorescence intensity of the probe at 555nm gradually decreased (FIG. 5 a), indicating that PMSF has a significant inhibitory effect on BChE, and that the inhibition rate shows a good linear relationship with the logarithm of PMSF concentration (. Ltoreq.200. Mu.M) (R 2 = 0.9939) (fig. 5 b). The results indicate that the probe is a change in fluorescence resulting from an enzymatic reaction with butyrylcholinesterase.
Experimental example 3: fluorescent probe for monitoring inhibition of organophosphorus pesticide on BChE
The fluorescence spectrum changes were detected after pretreatment with various concentrations of paraoxon (organophosphorus pesticides) with BChE (0.2U/mL) for 30 min and then incubation with probe (10 μm) for 1 hour at 37 ℃.
FIG. 6 is a fluorescence spectrum of probe after reaction with organophosphorus pretreated BChE. As the concentration of paraoxon gradually increased to 1000ng/mL, the fluorescence intensity of the probe at 555nm gradually decreased (fig. 6 a), indicating a significant inhibition of BChE by paraoxon. The inhibition rate and the logarithmic phase of the phosphorus oxide concentration (less than or equal to 200 ng/mL) show good linear relation (R) 2 = 0.9946) (fig. 6 b), is suitable for quantitative analysis of phosphorus oxide.
Experimental example 4: fluorescent probe for monitoring inhibition of carbamate pesticide on BChE
The fluorescence spectrum changes were detected after pretreatment with various concentrations of carbofuran with BChE (0.2U/mL) for 30 minutes and then incubation with probe (10. Mu.M) for 1 hour at 37 ℃.
FIG. 7 is a fluorescence spectrum of probe after reaction with carbamate-pretreated BChE. As the concentration of carbofuran gradually increased to 1400ng/mL, the fluorescence intensity of the probe at 555nm gradually decreased (fig. 7 a), indicating that carbofuran has a significant inhibitory effect on BChE. The inhibition rate and the logarithmic expression of carbofuran concentration (less than or equal to 1200 ng/mL) show good linear relation (R) 2 0.9979) (fig. 7 b), is suitable for quantitative analysis of carbofuran.
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (5)

1. A ratio-type fluorescent probe is characterized by having a structure represented by formula (1):
2. the method for preparing the ratio-type fluorescent probe according to claim 1, comprising the steps of:
reacting a compound of formula (2) with cyclopropylcarbonyl chloride in an organic solvent under base conditions to give a compound of formula (1);
3. the preparation method according to claim 2, wherein the amount of the cyclopropylcarbonyl chloride is 1 to 6mmol, the amount of the base is 2 to 12 mmol, and the amount of the organic solvent is 20 to 60ml, relative to 1 mmol of the compound of formula (2).
4. A method of preparation according to claim 2 or 3, wherein the base is selected from at least one of cesium carbonate, potassium carbonate, sodium bicarbonate, pyridine, piperazine, triethylamine, N-dimethylaminopyridine; the organic solvent is selected from at least one of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, acetonitrile, acetone, N-dimethylformamide and 1, 2-dichloroethane.
5. A method of preparation according to claim 2 or 3, wherein the temperature of the reaction is from-20 ℃ to 40 ℃; the reaction time is 2-12 hours.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108192597A (en) * 2016-12-08 2018-06-22 华中师范大学 Half cyanines class fluorescence probe of near-infrared for detecting butyrylcholine esterase and its preparation method and application
CN111592504A (en) * 2020-06-12 2020-08-28 青岛科技大学 Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108192597A (en) * 2016-12-08 2018-06-22 华中师范大学 Half cyanines class fluorescence probe of near-infrared for detecting butyrylcholine esterase and its preparation method and application
CN111592504A (en) * 2020-06-12 2020-08-28 青岛科技大学 Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
A ratiometric fluorescent probe for mitochondrial esterase specific detection in living cells;Tianjiao Shen, et al.;Dyes and Pigments;第178卷;108345 *

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