CN113528119B - Conjugated polymer nanoparticle composite fluorescent probe and preparation method and application thereof - Google Patents
Conjugated polymer nanoparticle composite fluorescent probe and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 11
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- TYKNKGWNSVNROB-UHFFFAOYSA-N O1P(OC1)(=O)OP1(=O)OCO1 Chemical compound O1P(OC1)(=O)OP1(=O)OCO1 TYKNKGWNSVNROB-UHFFFAOYSA-N 0.000 description 1
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention belongs to the technical field of fluorescent probes, and particularly relates to a conjugated polymer nanoparticle composite fluorescent probe, and a preparation method and application thereof. FeOOH materials with broad UV absorption can cause fluorescence quenching of CPNs PBOC-COOH by FRET. In the presence of BChE and Acetylcholinesterase (ATCH), the enzymolysis product (a thiocholine) generated by the BChE and the acetylcholinesterase can effectively degrade FeOOH, so that the fluorescence of CPNs PBOC-COOH is recovered. Experiments prove that the CPNSPBOC-COOH@FeOOH system has ideal fluorescence quenching efficiency. The large surface area and the thin layer structure of FeOOH provide rich contact area for the reaction of thiocholine, so that the sensitivity and the selectivity of the probe can be improved finally, the detection range is wider, the LOD is lower and is 3.5ng/mL, and the detection range is 0.02-2.6 mug/mL.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a conjugated polymer nanoparticle composite fluorescent probe, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Butyrylcholine enzyme (BChE), also known as cholinesterase, pseudocholinesterase and serum cholinesterase, is a nonspecific esterase synthesized by the liver and hydrolyzes many cholinergic biomolecules. BChE is synthesized and secreted into blood by liver cells, and is mainly distributed in liver, serum and lymph fluid, and the content in plasma is higher. The BChE hydrolyzes butyrylcholine to generate choline and acetic acid, the choline can react with a sulfhydryl color developing agent to generate TNB yellow compounds, colorimetric quantification is carried out according to the color shade, and the quantity of the hydrolysis product choline can reflect the activity of cholinesterase. Compared with sister enzyme acetylcholinesterase (AChE), the activity of BChE in serum can be measured to diagnose various diseases such as hepatocellular carcinoma, organophosphorus poisoning, parkinson's disease, alzheimer's disease and the like. Therefore, accurately detecting the content of BChE is of great importance for clinical diagnosis.
However, there are few methods for BChE activity assays compared to various methods for measuring AChE activity. Currently, the most commonly used and commonly used method is Ellman colorimetric method, which is a convenient and easy-to-use high throughput assay. However, the results are often affected by the interference of biomolecules contained in serum samples and the instability of reagents, greatly limiting their practical use. Therefore, the establishment of a new method with stable performance and high anti-interference performance is very promising and valuable. In recent years, high sensitivity, high stability and high selectivity based on fluorescence technology are considered as a promising approach. Heretofore, various materials have been used as effective quenchers, including graphene oxide metal nanoparticles and transition metal chalcogenides. As a two-dimensional nanomaterial, transition metal oxyhydroxide (MOOH) has been receiving a great deal of attention, having excellent light absorption properties, a high specific surface area, and good environmental compatibility. Transition metal oxyhydroxide nanomaterials (MOOHs) have an oxidizing capacity and can be converted to M by reducing species 2+ . When the MOOH encounters a reducing species, the nanomaterial is decomposed, thereby restoring the fluorescence of the probe. These properties make MOOH nanomaterials a very promising fluorescent group nanoquencher.
However, the inventor researches and discovers that the detection limit of the transition metal oxyhydroxide is larger, the detection sensitivity is low, and meanwhile, other noble metal catalysts are needed for auxiliary use, so that the cost is high. In addition, fluorescent probes made of transition metal oxyhydroxide have poor stability.
Disclosure of Invention
Based on the defects in the prior art, the invention provides a conjugated polymer nanoparticle composite fluorescent probe, a preparation method and application thereof, and a FeOOH material with wider ultraviolet absorption, which can quench fluorescence generated by CPNs PBOC-COOH through FRET. In the presence of BChE and Acetylcholinesterase (ATCH), the enzymolysis product (a thiocholine) generated by the BChE and the acetylcholinesterase can effectively degrade FeOOH, so that the fluorescence of CPNs PBOC-COOH is recovered. Experiments prove that the CPNSPBOC-COOH@FeOOH system has ideal fluorescence quenching efficiency. The large surface area and the thin layer structure of FeOOH provide rich contact area for the reaction of thiocholine, so that the sensitivity and the selectivity of the probe can be improved finally, the detection range is wider, the LOD is lower and is 3.5ng/mL, and the detection range is 0.02-2.6 mug/mL.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the present invention, there is provided a conjugated polymer nanoparticle composite fluorescent probe comprising carboxylated conjugated polymer nanoparticle CPNs PBOC -COOH and a transition metal oxyhydroxide.
In a second aspect of the present invention, there is provided a method for preparing conjugated polymer nanoparticle composite fluorescent probe, wherein CPNs are used as the fluorescent probe PBOC Mixing the-COOH with the transition metal oxyhydroxide at room temperature, and carrying out light-shielding reaction to obtain the catalyst.
In a third aspect of the present invention, there is provided a method for detecting butyrylcholine enzyme, comprising:
CPNs is combined with PBOC -COOH and transition metal oxyhydroxide are incubated at room temperature;
adding butyrylcholine enzyme and acetylcholinesterase for continuous incubation;
and carrying out fluorescence quantitative detection on the solution after the incubation treatment.
In a fourth aspect, the invention provides an application of a conjugated polymer nanoparticle fluorescent probe in detection of butyrylcholine enzyme.
One or more of the technical schemes of the invention has the following beneficial effects:
(1) The invention provides a fluorescent probe based on conjugated polymer nano particles and transition metal oxyhydroxide, which has a novel structure and can be applied as a fluorescent probe for detecting butyrylcholine enzyme; the probe has structural stability and good optical characteristics.
(2) The fluorescent probe provided by the invention can be used for detecting butyrylcholine enzyme and testing various interfering substances. The probe has very good selectivity and sensitivity, so that the probe is particularly suitable for detecting butyrylcholine enzyme, the detection range is wider, the LOD is lower, and the LOD is 3.5ng/mL. Therefore, the method has good practical application value.
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 invention. Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a process and a mechanism diagram for preparing FeOOH composite polymer nanoparticles according to example 4 of the present invention;
FIG. 2 shows CPNs of example 4 of the present invention PBOC Fluorescence intensity patterns after adding butyrylcholine enzyme solution (0-30 mug/mL, the uppermost curve is 0 mug/mL, the lowermost curve is 30 mug/mL, and 1.5 mug/mL is evenly added in the middle) with different concentrations of COOH@FeOOH;
FIG. 3 is a schematic diagram of CPNs of example 4 of the present invention PBOC Fluorescence spectra of different concentrations of FeOOH (0. Mu.g/mL uppermost curve, 130. Mu.g/mL lowermost curve, 10. Mu.g/mL intermediate increase) were added to the-COOH solution (1. Mu.g/mL);
FIG. 4 is a schematic diagram of CPNs of example 4 of the present invention PBOC Fluorescence intensity curves at 550nm with different concentrations of FeOOH added to the-COOH solution (1. Mu.g/mL);
FIG. 5 is a CPNs of example 5 of the present invention PBOC -fluorescence intensity change trend graph of COOH@FeOOH after different concentrations of butyrylcholine enzyme solution (0-30 mug/mL) are added;
FIG. 6 is a graph showing the selectivity of the conjugated polymer nanoparticle fluorescent probe for butyrylcholine enzyme detection according to example 4 of the present invention.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments in accordance with the present disclosure. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The inventor researches and discovers that the detection limit of the prior transition metal oxyhydroxide is larger, the detection sensitivity is low, and the detection sensitivity needs other noble metal catalysts for auxiliary use and has high manufacturing cost. In addition, fluorescent probes made of transition metal oxyhydroxide have poor stability. Therefore, the invention provides a conjugated polymer nanoparticle composite fluorescent probe, a preparation method and application thereof, and FeOOH material with wider ultraviolet absorption can cause CPNs PBOC-COOH to generate fluorescence quenching through FRET. In the presence of BChE and Acetylcholinesterase (ATCH), the enzymolysis product (a thiocholine) generated by the BChE and the acetylcholinesterase can effectively degrade FeOOH, so that the fluorescence of CPNs PBOC-COOH is recovered. Experiments prove that the CPNSPBOC-COOH@FeOOH system has ideal fluorescence quenching efficiency. The large surface area and the thin layer structure of FeOOH provide rich contact area for the reaction of thiocholine, so that the sensitivity and the selectivity of the probe can be improved finally, the detection range is wider, the LOD is lower and is 3.5ng/mL, and the detection range is 0.02-2.6 mug/mL.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the present invention, there is provided a conjugated polymer nanoparticle composite fluorescent probe comprising carboxylated conjugated polymer nanoparticle CPNs PBOC -COOH and a transition metal oxyhydroxide.
The surface of the transition metal hydroxide has positive charges, and carboxylation can lead the polymer nano-ions to have negative charges, thereby being beneficial to fluorescence resonance energy transfer.
The stability of the probe in the embodiments of the invention is mainly reflected in stable detection value, and the platform is more stable by utilizing positive and negative charge adsorption.
In one or more embodiments of the present invention, the transition metal oxyhydroxide is selected from at least one of CoOOH, niOOH, and FeOOH.
Conjugated polymer nanoparticles CPNs PBOC the-COOH and the transition metal oxyhydroxide have positive and negative charge adsorption, so that the prepared fluorescent probe has high stability and sensitivity.
In a second aspect of the present invention, there is provided a method for preparing conjugated polymer nanoparticle composite fluorescent probe, wherein CPNs are used as the fluorescent probe PBOC Mixing the-COOH with the transition metal oxyhydroxide at room temperature, and carrying out light-shielding reaction to obtain the catalyst.
The light-tight reaction is because the effect of light on the reaction is avoided.
Preferably, the PBOC is of the formula
The n is a natural number greater than 0, preferably greater than 45, more preferably 50, and the PBOC structural unit has a molecular weight of 689.
Polystyrene-maleic anhydride (PSMA) is as follows:
the m range is a natural number greater than 0, preferably a natural number greater than 45, more preferably 50, and the structural unit has a molecular weight of 278.
In one or more embodiments of the present invention, the CPNs PBOC The mass ratio of-COOH to transition metal oxyhydroxide is from 1:0.1 to 130, preferably from 1:35 to 40.
Preferably, the photophobic reaction time is 10-20min, preferably 15min.
Specifically, 1 μg/mL of CPNsPBOC-COOH was mixed with FeOOH (0.1 to 130 μg/mL) at various concentrations, respectively, at room temperature, reacted for 15 minutes in the absence of light, and fluorescence spectra were recorded at λem=430 nm. The efficiency of fluorescence quenching was explored to construct an ideal CPNSPBOC-COOH@FeOOH fluorescent probe.
In one or more embodiments of the present invention, the carboxylated conjugated polymer nanoparticles CPNs PBOC The preparation method of the-COOH comprises the following steps:
mixing conjugated polymer PBOC and PSMA, performing ultrasonic treatment to form homogeneous solution, adding ultrapure water, performing ultrasonic treatment, evaporating, concentrating, and filtering to obtain supernatant.
In one or more embodiments of the invention, the mass ratio of the conjugated polymer PBOC to PSMA is 5:1 to 3, preferably 5:2;
PSMA is polystyrene-maleic anhydride, provides carboxyl groups for PBOC, and provides for fluorescent effects with transition metal oxyhydroxide. The purpose of the ultrapure water addition is to wash the by-products produced during the reaction and the unreacted starting materials. In some embodiments of the invention, less PSMA is used than PBOC in order to avoid PBOC being coated with PSMA, affecting COOH graft modification.
Preferably, the conjugated polymer PBOC concentration is 0.5-2mg/mL, preferably 1mg/mL.
In one or more embodiments of the present invention, the solvent used in the conjugated polymer PBOC and PSMA mixture is tetrahydrofuran;
in order to remove impurities in the reaction system better, the volume ratio of the ultrapure water to the homogeneous solution is 1:1-2, preferably 1:2;
preferably, the ultrasonic time for the homogeneous solution and the addition of ultrapure water is 10min, respectively.
mu.L was removed from 1mg/mL of the mother liquor of the conjugated polymer PBOC. To this was added 100. Mu.L of PSMA mother liquor (1 mg/mL) and the volume was adjusted to 10mL with Tetrahydrofuran (THF) to prepare a mixed solution. It was sonicated for 3 minutes to form a homogeneous solution. Then, 20mL of ultrapure water was rapidly added and sonicated for 10 minutes. The mixed solution was distilled off THF by rotary evaporation and concentrated to 10mL. Finally, the supernatant (CPNs PBOC-COOH) (25. Mu.g/mL) was collected by filtration through an aqueous nanofiltration (0.2 μm).
When the transition metal hydroxide oxide is FeOOH, the preparation method of the FeOOH nano rod is as follows:
2.7g FeCl was added to 100mL of ultrapure water 3 ·6H 2 O and 0.9g of urea. The mixed solution was placed in a polytetrafluoroethylene-lined stainless steel autoclave (100 mL) and placed in an oven at a set temperature of 120 ℃ for 4 hours. After the completion of the reaction, the vessel was cooled to room temperature, and centrifuged with ultrapure water to precipitate several times. Finally, the FeOOH nano rod is obtained through freeze drying.
FeOOH has a wider ultraviolet absorption range, and the surface of FeOOH has a certain positive charge, and can generate very effective FRET action with CPNs PBOC-COOH with negative charge, so that CPNs PBOC Fluorescence quenching of-COOH.
In a third aspect of the present invention, there is provided a method for detecting butyrylcholine enzyme, comprising:
CPNs is combined with PBOC -COOH and transition metal oxyhydroxide are incubated at room temperature;
adding butyrylcholine enzyme and acetylcholinesterase for continuous incubation;
and carrying out fluorescence quantitative detection on the solution after the incubation treatment.
Butyrylcholine and CPNs PBOC -COOH mass ratio of 0.5-3:1, preferably 2:1;
preferably, the specific conditions for the quantitative fluorescence detection are as follows: the fluorescence spectrum measuring range is 440-700 nm, the excitation wavelength is 430nm, and the excitation/emission slit is set to 10nm;
preferably, the incubation temperature is 37 ℃.
Further, CPNs are combined PBOC Incubating a mixed solution of-COOH and FeOOH with butyrylcholinase at room temperature;
BChE solutions of different concentrations (0-30. Mu.g/mL) and ATCH solutions (50. Mu.L, 8 mM) were stirred at 37℃for 15 minutes to obtain enzymatic hydrolysis products. Then, the enzymatic hydrolysate was added to CPNs PBOC -cooh@feooh fluorescent sensing platform. The mixture was fixed to 1mL with PBS buffer (ph=7.4) and stirred well for 15min before fluorescence spectra were recorded at λem=430 nm.
In some embodiments of the invention, the enzymatic products of both BChE and ATCh solutions are fluorescent-recovering substances, which are detected after enzymatic hydrolysis.
In a fourth aspect, the invention provides an application of a conjugated polymer nanoparticle fluorescent probe in detection of butyrylcholine enzyme.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
EXAMPLE 1 Synthesis of conjugated Polymer PBOC
Tetraethyl (2, 5' -di (ethylhexyl) -1,4' -phenylene) bis (methylene) diphosphate (0.5 g,0.74 mmol) was added to THF in water and nitrogen protected, the reaction mixture was kept under a cold bath, then potassium t-butoxide (0.2 g,1.78 mmol) was added to the mixture and kept under nitrogen protection, stirring was continued for 30 minutes and then transferred to an oil bath, N-octyl-3, 6' -dicarboxyl-carbazole (0.3 g,0.96 mmol) was added, nitrogen protection was continued at 25 ℃ for 72 hours, after completion of the reaction, poured into water and extracted with dichloromethane, dichloromethane was removed by distillation under reduced pressure, and the residue was purified by THF and N-hexane fractional precipitation to give an orange solid. The molecular weight of the structural unit is 689, and the polymerization degree is 50.
Example 2 Conjugated Polymer Nanoparticles (CPNs) PBOC -COOH) synthesis
Fluorescent conjugated polymer PBOC is dissolved in THF, functional polymer PSMA is dissolved in ultrapure water to prepare 1 mg.mL respectively -1 Stock solution. THF mixed solution(10 mL) includes 25. Mu.g.mL -1 The PBOC and ultra-pure water solutions (5 mL) contained 20. Mu.g.mL - 1 PEI was sonicated separately for 5 minutes and mixed to form a homogeneous solution, then rapidly added to 15mL of ultrapure water under sonication for 15 minutes and finally the THF was removed by rotary evaporation to 10mL. Finally, filtering the mixture through a 0.2-micron filter to obtain CPNs PBOC -COOH。
EXAMPLE 3 Synthesis of transition metal oxide FeOOH
FeCl is added 3 ·6H 2 O (2.702 g) and 0.9g urea were dissolved in 100mL of ultrapure water. The solution was transferred to a 100mL polytetrafluoroethylene-lined stainless steel autoclave and maintained at 120 ℃ for 4 hours. After cooling to room temperature, feOOH was rinsed three times with ultrapure water. Finally, the product is obtained through freeze drying.
Example 4 Synthesis of fluorescent probes
As shown in fig. 1, CPNs PBOC COOH solutions (100. Mu.L, 10. Mu.g.mL) -1 ) Butyrylcholine enzyme solution (100. Mu.L, 20. Mu.g.mL) -1 ) Incubating for 10min at room temperature, and adding FeOOH solution (200. Mu.L, 0.44. Mu.M) thereto for further incubation for 15min to give fluorescent probes (CPNs) with significant quenching PBOC -cooh@feooh). The fluorescence test shows that the three materials have the best fluorescence quenching effect under the proportion, namely the best combination of fluorescent probes.
As shown in FIG. 2, CPNs of this embodiment PBOC Fluorescence intensity profile of-COOH@FeOOH after addition of butyrylcholine enzyme solutions of different concentrations (0-30. Mu.g/mL).
As shown in FIG. 3, CPNs prepared in example 2 PBOC Fluorescence spectra of FeOOH added to the-COOH solution (1. Mu.g/mL) at various concentrations, as shown, decreased peak intensity at 550nm with increasing content due to electrostatic adsorption of positive and negative ions between the various components, with increasing concentrations being self-extinguishing.
As shown in FIG. 4, the fluorescence intensity at 550nm significantly decreased as the FeOOH concentration increased when the FeOOH concentration was varied in the range of 0-30. Mu.g/mL, the fluorescence intensity at 550nm slowly decreased as the FeOOH concentration increased when the FeOOH concentration was varied in the range of 30-40. Mu.g/mL, and the fluorescence intensity at 550nm moderately decreased as the FeOOH concentration increased when the FeOOH concentration was varied in the range of 40-150. Mu.g/mL. The reason for this is that, initially, based on FRET, the positive and negative charge attraction effect is remarkable, gradually reaches a saturated state, and finally, self-extinguishing occurs.
Example 5 detection of fluorescent probes for BChE
BChE solutions of different concentrations (0-30. Mu.g/mL) and ATCH solutions (50. Mu.L, 8 mM) were stirred at 37℃for 15 minutes to obtain enzymatic hydrolysis products. Then, the enzymatic hydrolysate was added to CPNs described in example 4 PBOC -cooh@feooh fluorescent sensing platform. Incubation (37 ℃) for 30 min before fluorescence measurement was performed, the volume was fixed to 1mL with PBS buffer (ph=7.4) and stirred well for 15min before fluorescence spectra were recorded at λem=430 nm. The fluorescence spectrum showing fluorescence change with the addition of BChE solution with different concentrations can be obtained. The fluorescence spectrum measurement range is 440-700 nm, the excitation wavelength is 430nm, and the excitation/emission slit is set to 10nm. The detection system has fluorescence recovery in the presence of BChE and has good linear detection relation. The detection range is wider (0.02-2.6 mug/mL), and the LOD is lower (3.5 ng/mL).
FIG. 5 shows CPNs of the present embodiment PBOC -cooh@feooh fluorescence intensity trend graph after adding butyrylcholine enzyme solution (0-30 μg/mL) with different concentrations. As shown in the figure, when the concentration of butyrylcholine enzyme solution is changed from 0 to 2.5 mug/mL, the fluorescence intensity is changed more severely, which indicates that the fluorescence probe of the embodiment has higher detection sensitivity.
Example 6 Selective detection of fluorescent probes for BChE
FIG. 6 shows a graph of the selectivity of the conjugated polymer nanoparticle fluorescent probe prepared in example 4 of the present invention to butyrylcholine enzyme detection, wherein the selectivity of the fluorescent probe prepared in example 4 to BChE is higher on the premise that other components exist in the detection system.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. A conjugated polymer nanoparticle composite fluorescent probe is characterized by comprising carboxylated conjugated polymer nanoparticle CPNs PBOC -COOH and transition metal oxyhydroxide;
the surface of the transition metal hydroxide has positive charges, and carboxylation can lead conjugated polymer nano ions to have negative charges, so that conjugated polymer nano particles CPNs PBOC Positive and negative charge adsorption of-COOH and transition metal oxyhydroxide to CPNs PBOC -COOH produces fluorescence quenching;
when the fluorescent probe detects butyrylcholine enzyme, the detection range is 0.02-2.6 mu g/mL, and the LOD is 3.5 ng/mL;
the transition metal oxyhydroxide is FeOOH;
the PBOC structural formula is
；
N is a natural number greater than 0;
the carboxylated conjugated polymer nanoparticles CPNs PBOC The preparation method of the-COOH comprises the following steps:
mixing conjugated polymer PBOC with polystyrene-maleic anhydride, performing ultrasonic treatment to form a homogeneous solution, adding ultrapure water, performing ultrasonic treatment, evaporating, concentrating, and filtering to obtain supernatant;
the mass ratio of the conjugated polymer PBOC to the polystyrene-maleic anhydride is 5:1-3;
the concentration of the conjugated polymer PBOC is 0.5-2mg/mL;
the solvent used for the mixing of the conjugated polymer PBOC and the polystyrene-maleic anhydride is tetrahydrofuran.
2. The conjugated polymer nanoparticle composite fluorescent probe of claim 1, wherein n is a natural number greater than 45.
3. The conjugated polymer nanoparticle composite fluorescent probe of claim 2, wherein n is 50.
4. The conjugated polymer nanoparticle composite fluorescent probe of claim 1, wherein the PBOC structural unit has a molecular weight of 689.
5. The conjugated polymer nanoparticle composite fluorescent probe of claim 1, wherein the mass ratio of the conjugated polymer PBOC to polystyrene-maleic anhydride is 5:2;
the conjugated polymer PBOC concentration was 1mg/mL.
6. The conjugated polymer nanoparticle composite fluorescent probe of claim 1, wherein the carboxylated conjugated polymer nanoparticles CPNs PBOC In the preparation method of the-COOH,
the volume ratio of the ultrapure water to the homogeneous solution is 1:1-2;
the ultrasonic time for forming a homogeneous solution and adding ultrapure water was 10min, respectively.
7. The conjugated polymer nanoparticle composite fluorescent probe of claim 6, wherein the volume ratio of ultrapure water to homogeneous solution is 1:2.
8. The method for preparing conjugated polymer nanoparticle composite fluorescent probe according to claim 1, wherein CPNs are used for preparing the conjugated polymer nanoparticle composite fluorescent probe PBOC mixing-COOH and FeOOH at room temperature, and reacting in dark place.
9. The method for preparing conjugated polymer nanoparticle composite fluorescent probe of claim 8, wherein the CPNs PBOC The mass ratio of-COOH to FeOOH is 1:0.1-130；
The light-shielding reaction time is 10-20min.
10. The method for preparing the conjugated polymer nanoparticle composite fluorescent probe according to claim 9, wherein the photophobic reaction time is 15min.
11. A method for detecting butyrylcholine enzyme, comprising:
CPNs is combined with PBOC -COOH and FeOOH incubation at room temperature;
adding butyrylcholine enzyme and acetylcholinesterase for continuous incubation;
carrying out fluorescence quantitative detection on the solution after incubation treatment;
the PBOC structural formula is
；
N is a natural number greater than 0;
the carboxylated conjugated polymer nanoparticles CPNs PBOC The preparation method of the-COOH comprises the following steps:
mixing conjugated polymer PBOC with polystyrene-maleic anhydride, performing ultrasonic treatment to form a homogeneous solution, adding ultrapure water, performing ultrasonic treatment, evaporating, concentrating, and filtering to obtain supernatant;
the mass ratio of the conjugated polymer PBOC to the polystyrene-maleic anhydride is 5:1-3;
the concentration of the conjugated polymer PBOC is 0.5-2mg/mL;
the solvent used for the mixing of the conjugated polymer PBOC and the polystyrene-maleic anhydride is tetrahydrofuran.
12. The method for detecting butyrylcholine enzyme according to claim 11, wherein butyrylcholine enzyme and CPNs PBOC -COOH in a mass ratio of 0.5-3:1.
13. The butyrylcholine enzyme detection method of claim 12, which is specificCharacterized in that butyrylcholine enzyme and CPNs PBOC -COOH in a mass ratio of 2:1.
14. The butyrylcholine enzyme detection method according to claim 11, wherein the specific conditions for the fluorescent quantitative detection are: the fluorescence spectrum measurement range is 440-700 nm, the excitation wavelength is 430nm, and the excitation/emission slit is set to 10nm.
15. The method for detecting butyrylcholinase according to claim 11, wherein the incubation temperature for adding butyrylcholinase and acetylcholinesterase is 37 ℃.
16. The use of the conjugated polymer nanoparticle composite fluorescent probe according to claim 1 for detecting butyrylcholine enzyme.
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