阅读说明：本技术 一种荧光微米探针检测槲皮素的方法和应用 (Method for detecting quercetin by using fluorescent micrometer probe and application ) 是由 王乐 苏稀琪 瞿祎 罗芳芳 于 2021-09-06 设计创作，主要内容包括：本发明公开了一种荧光微米探针检测槲皮素的方法和应用,以姜黄素为靶标、六氯环三磷腈为连接基团形成聚环三磷腈共姜黄素荧光微球,基于荧光微球与铝离子络合形成荧光微米探针,通过槲皮素与金属离子良好的配位能力,与荧光微米探针中铝离子结合释放荧光信号使体系荧光增强,实现对槲皮素的快速检测。本发明的荧光微米探针为聚磷腈超支化微米级荧光微米探针,通过构建“姜黄素-金属离子-槲皮素”微米级反应器提高复杂背景干扰下的荧光信号稳定性和传感性能,对槲皮素小分子的最低检测限为2.32×10～(-8)mol/L,并实现1min内的快速响应,灵敏性高、抗干扰能力强、识别快速且检测结果准确。(The invention discloses a method for detecting quercetin by using a fluorescent micrometer probe and application thereof. The fluorescent micrometer probe is a polyphosphazene hyperbranched micrometer-grade fluorescent micrometer probe, the stability and the sensing performance of a fluorescent signal under the interference of a complex background are improved by constructing a curcumin-metal ion-quercetin micrometer-grade reactor, and the minimum detection limit of a quercetin small molecule is 2.32 multiplied by 10 ‑8 mol/L, and realizes quick response within 1min, and is sensitiveThe method has the advantages of high performance, strong anti-interference capability, quick identification and accurate detection result.)

1. A fluorescent microsphere is characterized in that the fluorescent microsphere is a copolymer formed by curcumin and hexachlorocyclotriphosphazene.

2. The fluorescent microsphere of claim 1, wherein the particle size is 0.5-2 μm.

3. The method for preparing fluorescent microspheres according to claim 1 or 2, comprising the steps of:

(1) dissolving cyclotriphosphazene and curcumin in an organic solvent;

(2) adding triethylamine, and reacting for 4-12 hours.

4. The method for producing fluorescent microspheres according to claim 3,

in the step (1), the molar ratio of hexachlorocyclotriphosphazene to curcumin is 1: 1.5-5;

in the step (2), the molar ratio of hexachlorocyclotriphosphazene to triethylamine is 1: 50-150.

5. A fluorescent microprobe which is a mixture of the fluorescent microsphere of claim 1 or 2 and aluminum ions.

6. The method for preparing a fluorescent microprobe of claim 5, comprising the steps of: the fluorescent microsphere according to claim 1 or 2, which is obtained by dissolving the fluorescent microsphere in an ethanol solution and mixing the solution with an aluminum salt solution.

7. A test strip comprising the fluorescent microsphere of claim 1 or 2 and aluminum ions.

8. Use of the fluorescent microsphere of claim 1 or 2, the fluorescent microprobe of claim 5, or the test strip of claim 7 for detecting quercetin.

9. A method for detecting quercetin, comprising the steps of:

(a) dissolving the fluorescent microspheres of claim 1 or 2 in an ethanol solution, mixing with an aluminum salt solution, adding a sample to be tested, and mixing;

or, the test strip of claim 7 is taken out after being immersed in a sample to be tested;

(b) and measuring the fluorescence emission intensity at 510nm by taking 372nm as an excitation wavelength, and performing qualitative or quantitative detection.

10. The method for detecting quercetin according to claim 9, wherein in the mixed system in step (a), the mass ratio of the fluorescent microspheres to the aluminum ions is 1: 1-2.

Technical Field

The invention relates to a quercetin detection technology, in particular to a method for detecting quercetin by using a fluorescent micrometer probe and application thereof.

Background

The flavonoid quercetin (3 ', 3, 4', 5, 7-pentahydroxyflavone) is widely contained in various Chinese herbal medicines and daily foods, and has antiinflammatory, antioxidant, antitumor, and anticancer effects. Researches prove that the food rich in flavonoids has important biological activity, the activities of various enzymes are enhanced to be beneficial to human cells such as anti-inflammatory, antioxidant, liver protection, antitumor and antibacterial effects, and the detection of quercetin is related to the reduction of certain cancer risks, so that the evaluation of the nutritional quality in food chemistry and the antioxidant activity of quercetin in pharmacology is facilitated.

In the prior art, many methods for detecting quercetin are available, such as spectrophotometry, ultraviolet absorption, high performance liquid chromatography, electrochemical methods and the like, fluorescence methods are also concerned due to the advantages of rapidness and simplicity, fluorescent probes can realize visual identification under complex backgrounds by preparing cheap kits, and the method can be used for rapid detection and analysis of quercetin. In recent years, fluorescent detection of quercetin based on fluorescent probes such as small molecules, nanomaterials and polymers has been realized. However, most fluorescent probes have slow response times and poor sensitivity.

Disclosure of Invention

The invention mainly aims to provide a fluorescent micrometer probe.

The invention also aims to provide the application of the fluorescent micrometer probe in detecting the quercetin, and the fluorescent micrometer probe has the advantages of quick response time and high sensitivity.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a fluorescent microsphere, which is prepared by copolymerizing curcumin (Cur) serving as a target and Hexachlorocyclotriphosphazene (HCCP) serving as a connecting group to form polycyclotriphosphazene-curcumin (PC) fluorescent microspheres.

Preferably, the particle size of the fluorescent microsphere is 0.5-2 μm.

The preparation method of the fluorescent microsphere comprises the following steps:

(1) dissolving hexachlorocyclotriphosphazene and curcumin in an organic solvent according to the molar ratio of 1: 1.5-5;

(2) adding triethylamine into the mixture, wherein the molar ratio of hexachlorocyclotriphosphazene to triethylamine is 1: 50-150, reacting for 4-12 hours, centrifuging, filtering or suction filtering after the reaction is finished, collecting precipitate, washing with water and ethanol in sequence, and drying;

the organic solvent is one or the combination of more than two of acetonitrile, toluene, tetrahydrofuran or N, N-dimethylformamide.

Preferably, the organic solvent is acetonitrile.

Preferably, in the step (1), the molar ratio of hexachlorocyclotriphosphazene to curcumin is 1: 2-4.

More preferably, in step (1), the molar ratio of hexachlorocyclotriphosphazene to curcumin is 1: 3.

Preferably, in the step (2), the molar ratio of hexachlorocyclotriphosphazene to triethylamine is 1: 80-100.

More preferably, in step (2), the molar ratio of hexachlorocyclotriphosphazene to triethylamine is 1: 90.

A fluorescent micrometer probe comprises the following components in a mass ratio of 1: 1-2, and aluminum ions, which is obtained by dissolving the fluorescent microspheres in an ethanol solution and then mixing the solution with an aluminum salt solution.

Preferably, the mass ratio of the fluorescent microspheres to the aluminum ions in the fluorescent micrometer probe is 1: 1.7.

A test strip for detecting quercetin comprises the following components in a mass ratio of 1: 1-2, the preparation method of the fluorescent microsphere and the aluminum ions comprises the following steps: the fluorescent microspheres are dissolved in an ethanol solution and then mixed with an aluminum salt solution, a mixed system is diluted by ethanol until the total amount of the fluorescent microspheres and aluminum ions is 2-10 mg/mL, a blank test strip is soaked in the mixed system for 5-20 minutes, and then the test strip is taken out and dried.

Preferably, the mass ratio of the fluorescent microspheres to the aluminum ions in the test strip is 1: 1.7.

The invention provides application of the fluorescent microspheres, the fluorescent micrometer probes or the test paper strips in detecting quercetin (Que).

A method for detecting quercetin comprises the following steps:

(1) dissolving the fluorescent microspheres in an ethanol solution, mixing the solution with an aluminum salt solution, diluting the solution with the ethanol solution until the content of the fluorescent microspheres is 50-100 mu g/ml, and adding a sample to be tested for mixing;

or, the test strip is taken out after being immersed into a sample to be tested;

(2) and measuring the fluorescence emission intensity at 510nm by taking 372nm as an excitation wavelength for qualitative or quantitative detection.

Preferably, the linear range of the concentration of the quercetin is 0-5 × 10^-5mol/L, minimum detection limit of 2.32X 10^{- 8}mol/L。

The mechanism of identifying quercetin by the fluorescent micrometer probe in the invention is as follows: due to the good coordination capacity of the quercetin and the metal ions, the quercetin is combined with Al (III) in the fluorescent micrometer probe to form a PC-Al (III) -Que ternary system, and a fluorescent signal is released to enhance the fluorescence of the system.

Compared with the prior art, the invention has the beneficial effects that:

(1) compared with a fluorescence quenching probe, the fluorescent micrometer probe belongs to a fluorescence enhancement type probe, has good specificity, anti-interference performance and high sensitivity when being used for detecting the quercetin, releases strong green fluorescence through the good coordination capacity of the quercetin and metal ions, and obviously enhances the fluorescence of the system.

The fluorescent micrometer probe is a polyphosphazene hyperbranched micrometer-grade fluorescent micrometer probe, the curcumin is used as a fluorophore, the complexation with metal ions is realized through the carbonyl group of the fluorophore to adjust the fluorescent change, the polyphosphazene hyperbranched framework is favorable for constructing a curcumin-metal ion-quercetin micrometer-grade reactor, the fluorescent signal stability and the sensing performance under the interference of complex backgrounds are improved, and the minimum detection limit of quercetin small molecules is 2.32 multiplied by 10^-8mol/L and achieves quick response within 1 min.

(3) The test strip is a visual test strip, is convenient to use, low in cost, simple and convenient in detection process, strong in anti-interference capability, rapid and sensitive, accurate in detection result, good in selectivity and sensitivity for detecting the quercetin, and capable of being used for rapidly detecting the quercetin in the wine or the beverage.

Drawings

FIG. 1 is a fluorescence selectivity diagram of a fluorescent micrometer probe PC-Al (III) in the example, and an excitation wavelength is 372 nm.

FIG. 2 is an anti-interference graph of the fluorescence micrometer probe PC-Al (III) for identifying quercetin in the example, and the excitation wavelength is 372 nm.

FIG. 3 is a graph showing the response time of the optical probe PC-Al (III) to quercetin in the example.

FIG. 4 shows the stability of the fluorescence microprobe PC-Al (III) for identifying quercetin in the examples.

FIG. 5 is a fluorescence titration chart of the fluorescence micrometer probe PC-Al (III) for identifying quercetin in the example, and the excitation wavelength is 372 nm.

FIG. 6 is a graph of the lowest detection limit of quercetin identified by fluorescent micrometer probe PC-Al (III) in the examples, with an excitation wavelength of 372 nm.

FIG. 7 is a graph showing the effect of the test strip on detecting quercetin in the example.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

The chemical reagents and solvents used in the preparation of the fluorescent microprobe PC-Al (III) were purchased from explorations, the metal ions and the like were purchased from Aladdin reagents, and the fluorescence spectra were recorded using an Edinburgh FS-5 fluorescence analyzer, England.

EXAMPLE 1 preparation of fluorescent microspheres and fluorescent microprobes

Preparation of fluorescent microspheres

In a 250mL round bottom flask, 0.10g (0.30mmol) of Hexachlorocyclotriphosphazene (HCCP) and 0.32g (0.90mmol) of curcumin were dissolved in 50mL of acetonitrile, and after the hexachlorocyclotriphosphazene and curcumin were completely dissolved in the acetonitrile solution, 2mL of triethylamine (about 27mmol) was added and stirred at room temperature for 8 h. After the reaction is completed, centrifuging to obtain a lower-layer precipitate, and washing with ethanol and purified water until a supernatant is clear. Vacuum drying the obtained precipitate at 50 ℃ for 12h to obtain light yellow powder with the particle size of 0.5-2 mu m, namely the fluorescent microspheres, wherein the technical route is as follows:

(II) preparation of fluorescent micrometer probe PC-Al (III)

Preparing 12mg/mL PC solution with ethanol solution, and preparing 100mg/mL Al (NO) with purified water₃)₃·9H₂And adding 10 mu L of PC solution and 10 mu L of Al (III) solution into 2mL of ethanol solution of O aqueous solution (the concentration of aluminum ions is 7mg/mL), and uniformly mixing to obtain the fluorescent micrometer probe PC-Al (III).

(III) preparation of test paper strip

Preparing 10mg/mL quercetin ethanol solution, adding 2mL ethanol solution to dilute to 0.5mg/mL, and adding aluminum ion solution with mass concentration 1.7 times that of PC to prepare PC-Al (III) probe solution. The appropriately sized test strips were then cut from ordinary filter paper, immersed in a PC-al (iii) ethanol suspension for 10 minutes, and the strips were removed until completely dry at room temperature.

Example 2 fluorescence specificity selectivity test

Preparing 12mg/mL ethanol solution of PC and 100mg/mL Al (III) water solution, adding 2mL ethanol solution, 10 mu L PC solution and 10 mu L Al (III) water solution into a cuvette, and observing the selectivity of a fluorescent micrometer probe PC-Al (III) on quercetin small molecules by using a fluorescence spectrometer.

The result is shown in figure 1, under the excitation condition of 372nm, the single fluorescent micrometer probe PC-Al (III) has weak fluorescence emission intensity at 510nm in the ethanol solution, after the quercetin is added, the fluorescence emission intensity at 510nm is obviously enhanced, but when other substances are added, the fluorescence emission intensity of the solution system is not obviously changed compared with the fluorescence emission intensity of the single probe system, and the fluorescent micrometer probe PC-Al (III) has better fluorescence specificity selectivity on the quercetin in the ethanol solution.

Example 3 interference immunity test

12mg/mL of PC ethanol solution and 100mg/mL of Al (III) water solution are prepared, 2mL of ethanol solution and 10 μ L of PC solution and 10 μ L of Al (III) solution are respectively added into 26 clean fluorescence cuvettes, and then 10 μ M of quercetin and 100 μ M of other analytes are respectively added, such as:

other classes of small molecules: cysteine (Cys), histidine (His), glucose (Glu), Ascorbic Acid (AA), Tannin (Tannin), Catechol (cathecol), Rutin (Rutin), Daidzein (Daidzein), Baicalein (Baicalein);

cation: na (Na)⁺、K⁺、Ca²⁺、Cu²⁺,Zn²⁺、Cd²⁺、Sn²⁺、Co²⁺、Ni²⁺、Mg²⁺、Fe³⁺；

Anion: cl^-、ACO^-、H₂PO₄ ^-、HCO₃ ^-、CO₃ ^2-、SO₄ ^2-。

Detecting on a fluorescence spectrometer, drawing a histogram of the highest fluorescence intensity corresponding to different analytes to obtain a fluorescence emission histogram, as shown in fig. 2, proving that the identification of the quercetin in the ethanol solution by the fluorescent micrometer probe PC-Al (III) is not interfered by other analytes, and has better anti-interference performance.

Example 4 response time testing

Preparing 12mg/mL of PC ethanol solution and 100mg/mL of Al (III) water solution, adding 2mL of ethanol solution, 10 muL of PC solution and 10 muL of Al (III) solution into a cuvette, detecting the response time of a probe PC-Al (III) to a quercetin small molecule by using a fluorescence spectrometer, and detecting quercetin within 1min, wherein as shown in figure 3, the fluorescence micrometer probe PC-Al (III) is proved to be capable of quickly responding to quercetin and capable of quickly detecting quercetin in the ethanol solution.

Example 5 stability testing

Preparing 12mg/mL of PC ethanol solution and 100mg/mL of Al (III) water solution, adding 2mL of ethanol solution, 10 μ L of PC solution and 10 μ L of Al (III) solution into a cuvette, and keeping the cuvette for 0-120 min to measure the detection effect of the cuvette on quercetin, wherein as shown in FIG. 4, the fluorescence of the fluorescent probe PC-Al (III) and the fluorescence intensity of the fluorescent micrometer probe PC-Al (III) on Que are hardly changed within 2h, and the result shows that the fluorescent micrometer probe PC-Al (III) shows good stability in the ethanol solution system.

Example 6 Linear regression equation

Preparing 12mg/mL PC ethanol solution, 100mg/mL Al (III) water solution and 2mmol/L quercetin solution, adding 2mL ethanol solution, 10 μ L PC and 10 μ L Al (III) water solution into a clean fluorescence cuvette, gradually adding quercetin solution with the volumes of 0, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 45 μ L, 50 μ L, 55 μ L, 60 μ L and 65 μ L respectively, and with 372nm as excitation wavelength, measuring the fluorescence emission intensity at 510nm on a fluorescence spectrometer, and with the concentration of quercetin as a horizontal coordinate and the fluorescence intensity at 510nm as a vertical coordinate to obtain a working curve of the concentration of quercetin, wherein the linear regression equation is as follows: f_510nm＝7.5×10¹⁰The unit of C and C is mol/L, as shown in FIG. 5.

Example 7 lowest detection Limit experiment

Preparing 12mg/mL PC ethanol solution and 100mg/mL Al (III) water solution, adding 2mL ethanol solution, 10 μ L PC and 10 μ L Al (III) water solution into a clean fluorescence cuvette, measuring the response intensity of the cuvette to quercetin with different concentrations, and continuously increasing the fluorescence emission intensity of the system at 510nm with the increase of the concentration of the quercetin to find that the linear range of the fluorescence emission intensity of the solution at the concentration of the quercetin is 0-5 × 10^-5mol/L(R²The detection limit of the probe molecule on quercetin is calculated to be 2.32 multiplied by 10 after (3 sigma/k) is 0.997^-8mol/L (see figure 6), which shows that the fluorescent micrometer probe PC-Al (III) can be used for detecting the quercetin in the drinks such as wine and the like.

Example 8 test strip assay

Quercetin solutions with concentration gradients of 0 μ M, 20 μ M, 40 μ M, 60 μ M, 80 μ M, 100 μ M, 120 μ M, etc. were prepared, the test strips prepared in example 1 were immersed in the above solutions for 1min, then taken out, dried at room temperature, and the fluorescence change of the test strips caused by quercetin with different concentrations was observed under 365nm fluorescence, as shown in fig. 7.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.