CN115974837A - Ratio type fluorescent probe and preparation method and application thereof - Google Patents
Ratio type fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN115974837A CN115974837A CN202211652481.5A CN202211652481A CN115974837A CN 115974837 A CN115974837 A CN 115974837A CN 202211652481 A CN202211652481 A CN 202211652481A CN 115974837 A CN115974837 A CN 115974837A
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- fluorescent probe
- butyrylcholinesterase
- probe
- ratio
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- 238000002360 preparation method Methods 0.000 title claims abstract description 9
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- 238000001514 detection method Methods 0.000 claims abstract description 9
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- 239000003987 organophosphate pesticide Substances 0.000 claims abstract description 7
- 238000012544 monitoring process Methods 0.000 claims abstract description 6
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 5
- 102100032404 Cholinesterase Human genes 0.000 claims description 57
- 238000002189 fluorescence spectrum Methods 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
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Images
Classifications
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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
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of enzyme activity detection, and particularly relates to a ratio type fluorescent probe and 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 butyrylcholinesterase and monitoring the inhibiting effect of organophosphorus pesticide and carbamate pesticide on butyrylcholinesterase, has the advantages of high precision, high sensitivity, strong selectivity, quick response, good stability and simplicity in operation, particularly analyzes the activity of the butyrylcholinesterase by utilizing the fluorescence intensity ratio of double emission peaks of the ratio-type fluorescent probe, 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
Technical Field
The invention belongs to the technical field of enzyme activity detection, and particularly relates to a ratio type fluorescent probe and a preparation method and application thereof.
Background
Butyrylcholinesterase (BChE) is a serine hydrolase produced by the liver and widely present in blood, liver and glia. The decrease of BChE activity in serum is usually found in the pathogenesis of hepatitis, liver cancer and the like, and the increase of the activity of the BChE activity is found in the pathogenesis of Alzheimer's disease, diabetes, hypertension and the like. Because the organophosphorus pesticide and the carbamate pesticide can inhibit the activity of BChE, the active concentration of BChE in blood is also an important index for diagnosing two types of pesticide poisoning. Therefore, the development of a detection method for BChE activity has important significance 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, namely thiocholine, and have the defects of serious interference of mercaptan, low sensitivity, complex operation and the like. Most of the existing fluorescent probes capable of directly detecting BChE activity are enhanced fluorescent probes, the enzyme activity can be quantified only according to the fluorescence intensity, the detection result is easily influenced by various uncertainty factors such as probe concentration distribution, light stability, microenvironment (pH value, polarity, temperature and the like), instrument stability and the like, and the accuracy is low. In comparison, the ratio type fluorescent probe quantifies the target object by utilizing the ratio of the fluorescence intensity at two (or more) wavelengths, can effectively reduce the interference of factors such as probe concentration, microenvironment, light source intensity and the like in a self-correcting mode, and has more accurate quantification result on the BChE activity.
Therefore, how to design and synthesize an efficient ratiometric fluorescent probe to achieve accurate detection of butyrylcholinesterase 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 an application thereof, wherein the ratio type fluorescent probe is particularly suitable for detecting butyrylcholinesterase 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 ratiometric fluorescent probe having a structure represented by formula (1):
in a second aspect, the present invention provides a method for preparing the ratiometric fluorescent probe, comprising the following steps:
reacting a compound shown in a formula (2) with cyclopropyl formyl chloride in an organic solvent under the condition of alkali to obtain a compound shown in a 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, based on 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 at least one selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, acetonitrile, acetone, N-dimethylformamide, and 1, 2-dichloroethane.
Preferably, the reaction temperature is-20 ℃ to 40 ℃; the reaction time is 2 to 12 hours.
In a third aspect, the invention provides an application of the ratiometric fluorescent probe or the ratiometric fluorescent probe prepared by the preparation method in detecting butyrylcholinesterase activity.
In a fourth aspect, the invention provides the use of the ratiometric fluorescent probe or the ratiometric fluorescent probe prepared by the preparation method for monitoring the inhibition effect of organophosphorus pesticides and carbamate pesticides on butyrylcholinesterase; preferably, the organophosphorus pesticide is selected from one or a combination of any several of paraoxon, phorate, trichlorfon, methamidophos, dichlorvos, malathion, chlorpyrifos, dimethoate and parathion, and the carbamate pesticide is selected from one or a combination of any several of carbaryl, aldicarb, carbofuran and methomyl.
In a fifth aspect, the present invention provides a method for detecting butyrylcholinesterase activity, comprising the following steps:
adding the ratio-type fluorescent probe or the ratio-type fluorescent probe prepared by the preparation method into a sample to be tested, incubating at a certain temperature, measuring the fluorescence spectrum of the generated fluorescent substance, observing two emission peaks, measuring and calculating the intensity ratio of the two emission peaks, and quantifying the activity of butyrylcholinesterase by utilizing the corresponding relation between the ratio and the concentration of butyrylcholinesterase. In the incubation process, the fluorescent probe reacts with butyrylcholinesterase in a sample to be detected, and then the cyclopropanecarbonyl 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 time is 20-60min;
the fluorescence spectrum of the generated fluorescent substance is measured as follows: excitation of the resulting phosphor with 370-450nm light results in fluorescence emission in the wavelength range of 400-800nm, with two emission peaks observed.
The invention has the following beneficial effects:
(1) The ratio-type fluorescent probe disclosed by the invention analyzes the butyrylcholinesterase activity by utilizing the fluorescence intensity ratio of the double emission peaks, can effectively reduce the interference of factors such as probe concentration, microenvironment and light source intensity in a self-correction mode, and has high accuracy of detection results. In addition, the fluorescent probe has high sensitivity to butyrylcholinesterase and is not interfered by coexisting substances such as ions, amino acids, polysaccharides and proteins.
(2) The ratiometric fluorescent probe disclosed by the invention is simple in synthesis steps, easy to separate and purify, good in stability, suitable for batch production and beneficial to commercial popularization and application.
(3) The ratio-type fluorescent probe can be applied to the detection of the activity of butyrylcholinesterase, can also be applied to the monitoring of the inhibition effect of organophosphorus pesticide and carbamate pesticide on butyrylcholinesterase, and has great market application and popularization values in the diagnosis of organophosphorus poisoning and the detection of pesticide residue.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows a fluorescent probe of the present invention 1 H NMR spectrum.
FIG. 2 shows a fluorescent probe of the present invention 13 C NMR spectrum.
FIG. 3 shows an absorption spectrum (a) and a 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 responsiveness of the fluorescence spectrum of the probe of the present invention to butyrylcholinesterase (a) and the ratio of the fluorescence intensity of its two emission peaks to the active concentration of butyrylcholinesterase.
FIG. 5 shows the fluorescence spectrum (a) and inhibition efficiency curve (b) of the fluorescent probe of the present invention reacted with butyrylcholinesterase after the addition of an inhibitor.
FIG. 6 shows the fluorescence spectrum (a) and inhibition efficiency curve (b) of the fluorescent probe of the present invention reacted with butyrylcholinesterase after the addition of paraoxon.
FIG. 7 shows the fluorescence spectrum (a) and the inhibition efficiency curve (b) of the fluorescent probe of the present invention reacted with butyrylcholinesterase after carbofuran was added.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
4-hydroxy-1, 8-naphthalic anhydride was obtained from Zhengzhou Eiffel Techno chemical Co., ltd, and reagents such as 2- (2-aminoethyl) pyridine, methyl iodide, etc. were obtained from Yinaka Techno Co., ltd, beijing.
Example 1 Synthesis of fluorescent Probe
The synthetic route for preparing the fluorescent probe in this example is as follows:
the specific synthesis steps are as follows:
compound 1 (167mg, 0.36mmol) is dissolved in 50mL CH 2 Cl 2 Cesium carbonate (704 mg, 2.16mmoL) 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, and the reaction was terminated after stirring was continued for 8 hours. 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 a dichloromethane/methanol mixed solution with the volume ratio of 20.
FIG. 1 shows a ratio type fluorescent probe 1 H NMR(500MHz,DMSO-d 6 ) Spectrum: δ 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 =8.0hz, 1h), 4.47 (t, J =7.0hz, 2h), 4.44 (s, 3H), 3.51 (t, J =7.1hz, 2h), 2.17 (tt, J =7.8,4.7hz, 1h), 1.26-1.15 (m, 4H).
FIG. 2 shows a ratiometric fluorescent probe 13 C NMR(125MHz,DMSO-d 6 ) Spectrum: 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 and 13 the C NMR spectrum corresponds to the chemical structure of the ratiometric fluorescent probe, which indicates that the probe is successfully synthesized.
Experimental example 1: test of capability of fluorescent probe in reaction with butyrylcholine esterase in buffer solution
(1) The fluorescent probe prepared in example 1 was formulated into a DMSO stock solution (concentration of 0.2 mM), added to an EP tube, and then deionized water, PBS buffer (concentration of 200mM, pH 7), and a stock solution of BChE (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 to stand in a constant temperature incubator at 37 ℃ for 1 hour, and then the 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 reacts with BChE, the absorption peak is red shifted from 345nm to 450nm (FIG. 3 a); the fluorescence intensity at 467nm of the emission peak is slightly reduced, and the fluorescence intensity at 555nm is obviously enhanced to form a new emission peak (figure 3 b). The results indicate that the probe has a typical two-channel fluorescence response to BChE.
A series of EP tubes were added with DMSO stock solutions of probe, deionized water, PBS buffer, and different volumes of stock solutions of BChE, respectively. 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 to stand at 37 ℃ for 1 hour in a constant temperature incubator, and then the fluorescence spectrum thereof was measured.
FIG. 4 is a titration experiment of probe versus BChE concentration. With the gradual increase of BChE concentration to 2.0U/mL, the fluorescence intensity of the probe at 467nm emission peak slightly decreased, but the fluorescence intensity at 555nm emission peak gradually increased (FIG. 4 a). Fluorescence intensity ratio F of probe at two emission peaks 550 /F 465 Shows good linear relation 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 accurate calibrationBChE activity was measured.
Experimental example 2: inhibitor characterization of fluorescent probes
Changes in fluorescence spectra were detected after pretreatment with different concentrations of phenylmethylsulfonyl fluoride (PMSF, BChE inhibitor) and BChE (0.2U/mL) for 30 min, followed by incubation with probe (10. Mu.M) for 1h at 37 ℃.
FIG. 5 is a fluorescence spectrum of probe reacted with inhibitor pretreated BChE. As the concentration of PMSF was gradually increased to 200. Mu.M, the fluorescence intensity of the probe at 555nm was gradually decreased (FIG. 5 a), indicating that PMSF has a significant inhibitory effect on BChE, and that the inhibition rate has a good linear relationship with the logarithm of PMSF concentration (. Ltoreq.200. Mu.M) (R is 2 = 0.9939) (fig. 5 b). The result shows that the probe is fluorescence change caused by enzyme catalytic reaction with butyrylcholinesterase.
Experimental example 3: fluorescent probe for monitoring inhibition effect of organophosphorus pesticide on BChE
Changes in fluorescence spectra were detected after pretreatment with BChE (0.2U/mL) for 30 min with various concentrations of paraoxon (paraxon, organophosphorus pesticide) and subsequent incubation with probes (10. Mu.M) for 1h at 37 ℃.
FIG. 6 is the fluorescence spectra of the probe after reaction with organophosphorus pretreated BChE. As the concentration of paraoxon was gradually increased to 1000ng/mL, the fluorescence intensity of the probe at 555nm was gradually decreased (FIG. 6 a), indicating that paraoxon has a significant inhibitory effect on BChE. The inhibition rate and the logarithm of paraoxon concentration (less than or equal to 200 ng/mL) show good linear relation (R) 2 = 0.9946) (fig. 6 b), applicable to quantitative analysis of paraoxon.
Experimental example 4: fluorescent probe for monitoring inhibition effect of carbamate pesticide on BChE
Changes in fluorescence spectra were detected after pretreatment with BChE (0.2U/mL) for 30 min with varying concentrations of carbofuran (carbamate pesticide) and subsequent incubation with probes (10 μ M) for 1h at 37 ℃.
FIG. 7 is the fluorescence spectra of the probe after reaction with carbamate pretreated BChE. As the concentration of carbofuran was gradually increased to 1400ng/mL, the fluorescence intensity of the probe at 555nm gradually decreased (FIG. 7 a), indicating that carbofuran has a significant effect on BChEThe inhibitory action of (2). The inhibition rate and logarithm of carbofuran concentration (less than or equal to 1200 ng/mL) show good linear relation (R) 2 = 0.9979) (fig. 7 b), applicable to quantitative analysis of carbofuran.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto without departing from the scope of the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Claims (10)
1. A ratiometric fluorescent probe having a structure represented by formula (1):
2. the method for preparing a ratiometric fluorescent probe of claim 1, comprising the steps of:
reacting a compound shown in a formula (2) with cyclopropyl formyl chloride in an organic solvent under the condition of alkali to obtain a compound shown in a formula (1);
3. the 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, based on 1 mmol of the compound of formula (2).
4. The production method according to claim 2 or 3, characterized in that the base is selected from at least one of cesium carbonate, potassium carbonate, sodium hydrogen carbonate, pyridine, piperazine, triethylamine, N-dimethylaminopyridine; the organic solvent is at least one selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, acetonitrile, acetone, N-dimethylformamide, and 1, 2-dichloroethane.
5. The method according to claim 2 or 3, wherein the reaction temperature is-20 ℃ to 40 ℃; the reaction time is 2 to 12 hours.
6. Use of the ratiometric fluorescent probe of claim 1 or prepared by the method of any one of claims 2 to 5 for detecting butyrylcholinesterase activity.
7. Use of a ratiometric fluorescent probe of claim 1, or prepared by the method of any one of claims 2 to 5, for monitoring the inhibitory effect of organophosphorus pesticides and carbamate pesticides on butyrylcholinesterase.
8. The use according to claim 7, wherein the organophosphorus pesticide is selected from one or a combination of any of paraoxon, phorate, trichlorfon, methamidophos, dichlorvos, malathion, chlorpyrifos, dimethoate and parathion;
the carbamate pesticide is one or the combination of any more of carbaryl, aldicarb, carbofuran and methomyl.
9. A method for detecting butyrylcholine esterase activity is characterized by comprising the following steps:
the ratiometric fluorescent probe of claim 1 or the ratiometric fluorescent probe prepared by the preparation method of any one of claims 2 to 5 is added to a sample to be tested, 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 butyrylcholinesterase can be quantified by utilizing the corresponding relationship between the ratio and the concentration of butyrylcholinesterase.
10. The detection method according to claim 9, wherein the incubation temperature is 20-45 ℃, the pH is 4-8, and the time is 20-60min;
the fluorescence spectrum of the generated fluorescent substance is measured as follows: the resulting fluorescent substance is excited with light in the range of 370-450nm, and there is fluorescence emission in the wavelength range of 400-800nm, and two emission peaks are observed.
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| 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 |
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| 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)
| Title |
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| TIANJIAO SHEN, ET AL.: "A ratiometric fluorescent probe for mitochondrial esterase specific detection in living cells", DYES AND PIGMENTS, vol. 178, pages 108345 * |
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