阅读说明：本技术 基于七元瓜环检测l-苯丙氨酸的荧光探针及其检测方法 (Fluorescent probe for detecting L-phenylalanine based on seven-element cucurbituril and detection method thereof ) 是由 肖昕 冯华明 邓浩 韦恺妮 胥卫涛 范颖 于 2019-07-31 设计创作，主要内容包括：本发明公开了一种基于七元瓜环检测L-苯丙氨酸的荧光探针及其检测方法,其分子式为C<Sub>42</Sub>H<Sub>42</Sub>N<Sub>28</Sub>O<Sub>14</Sub>@C<Sub>20</Sub>H<Sub>18</Sub>ClNO<Sub>4</Sub>,化学结构式如附图1所示。其检测方法是将所述荧光探针加水稀释,得探针标准溶液,然后向探针标准溶液中加入待测物水溶液,静置5-20s后以固定激发波长350nm进行荧光发射光谱测定,并绘制激发出的该激光波长处的荧光强度的变化曲线；根据变化曲线计算荧光探针溶液中加入待测物水溶液前后分别对应496.96nm下的荧光发射光谱强度变化,即可对L-苯丙氨酸进行检测。本发明是一种新型的荧光探针,具有能检测水中的L-苯丙氨酸的特点,且具有灵敏度高、检测成本低、样品处理简单、操作方便、测定快速以及实时检测的特点。(The invention discloses a fluorescent probe for detecting L-phenylalanine based on seven-element cucurbituril and a detection method thereof, wherein the molecular formula of the fluorescent probe is C 42 H 42 N 28 O 14 @C 20 H 18 ClNO 4 The chemical structural formula is shown in figure 1. Diluting the fluorescent probe with water to obtain a probe standard solution, adding the water solution to be detected into the probe standard solution, standing for 5-20s, and fixing the excitation wavelengthPerforming fluorescence emission spectrometry at 350nm, and drawing a change curve of the fluorescence intensity at the laser wavelength; and calculating the fluorescence emission spectrum intensity change under 496.96nm respectively before and after the water solution to be detected is added into the fluorescent probe solution according to the change curve, and detecting the L-phenylalanine. The invention is a novel fluorescent probe, has the characteristic of detecting L-phenylalanine in water, and has the characteristics of high sensitivity, low detection cost, simple sample treatment, convenient operation, quick determination and real-time detection.)

1. A fluorescent probe for detecting L-phenylalanine based on seven-element cucurbituril is characterized in that: the molecular formula is C₄₂H₄₂N₂₈O₁₄@C₂₀H₁₈ClNO₄The chemical structural formula is shown in figure 1.

2. The seven-membered cucurbituril-based fluorescent probe for detecting L-phenylalanine according to claim 1, wherein: the fluorescent probe is prepared from seven-element cucurbituril and berberine hydrochloride.

3. The fluorescent probe for detecting L-phenylalanine based on seven-membered cucurbituril according to claim 2, wherein the specific preparation method of the fluorescent probe comprises the following steps:

(1) dissolving seven-element cucurbituril in water to obtain a solution A;

(2) dissolving berberine hydrochloride in water to obtain solution B;

(3) and mixing the solution A and the solution B, and reacting at normal temperature to obtain the fluorescent probe.

4. The seven-membered cucurbituril-based fluorescent probe for detecting L-phenylalanine according to claim 3, wherein: the concentration of the solution A in the step (1) is 1.0 multiplied by 10^-4mol/L。

5. The seven-membered cucurbituril-based fluorescent probe for detecting L-phenylalanine according to claim 3, wherein: the concentration of the solution B in the step (2) is 1.0 multiplied by 10^-3mol/L。

6. The seven-membered cucurbituril-based fluorescent probe for detecting L-phenylalanine according to claim 3, wherein: when the solution A and the solution B in the step (2) are mixed, the molar ratio of the seven-element cucurbituril to the berberine hydrochloride in the mixed solution is 1: 0.5-2.

7. The seven-membered cucurbituril-based fluorescent probe for detecting L-phenylalanine according to claim 6, wherein: the molar ratio of the seven-element cucurbituril to the berberine hydrochloride in the mixed solution is 1: 1.

8. A method for detecting L-phenylalanine using the fluorescent probe as set forth in any one of claims 1 to 7, characterized in that: diluting the fluorescent probe with water to obtain a probe standard solution, adding an aqueous solution to be detected into the probe standard solution, standing for 5-20s, performing fluorescence emission spectrometry at a fixed excitation wavelength of 350nm, and drawing a change curve of the fluorescence intensity at the excited laser wavelength; and calculating the fluorescence emission spectrum intensity change under 496.96nm respectively before and after the water solution to be detected is added into the fluorescent probe solution according to the change curve, and detecting the L-phenylalanine.

9. The method for detecting L-phenylalanine with a fluorescent probe according to claim 8, wherein: the concentration of the probe standard solution is 2.0 x 10^-5mol/L。

10. The method for detecting L-phenylalanine with a fluorescent probe according to claim 8, wherein: when the fluorescence emission spectrum intensity of the probe standard solution at 496.96nm is reduced before and after the test object aqueous solution is added, the test object aqueous solution contains L-phenylalanine, otherwise, the test object aqueous solution does not contain L-phenylalanine.

Technical Field

The invention relates to a fluorescent probe for detecting L-phenylalanine and a detection method thereof, in particular to a fluorescent probe for detecting L-phenylalanine based on seven-membered cucurbituril and a detection method thereof.

Background

Amino acids play an extremely important role in the growth and development of organisms: 1. is a constituent of human tissue; 2. various substances constituting the human body; 3. supplying heat; 4. immune regulation; 5. as an important carrier in vivo, plays a role in transportation; 6. and (4) an oxidation function.

The amino acid is the basic composition substance of the protein, the intake of the amino acid is the only way for the human body to obtain the nitrogen source, after the human body absorbs the amino acid, a part of the amino acid is directly used for synthesizing the protein, a part of the amino acid is oxidized and decomposed, the nitrogen-containing part of the amino acid is used for synthesizing other necessary amino acid, and a part of the amino acid is decomposed as energy and is discharged out of the body in the form of urea.

Therefore, it is very important to detect the content and type of amino acids in food, and fluorescent probe is a new type of detection reagent, which is very popular because of its advantages of high sensitivity, low detection cost, simple sample treatment, convenient operation, rapid measurement and real-time detection.

L-Phenylalanine (English name: L-Phenylalanine) is colorless to white flaky crystal or white crystalline powder, is a nutritional supplement, and is one of essential amino acids. Most of the tyrosine is oxidized into tyrosine by the catalysis of phenylalanine hydroxylase in vivo, and the tyrosine synthesize important neurotransmitters and hormones together to participate in sugar metabolism and fat metabolism of the body. Therefore, it is necessary to develop a novel fluorescent probe for detecting L-phenylalanine and a related detection method.

Disclosure of Invention

The invention aims to provide a fluorescent probe for detecting L-phenylalanine based on seven-membered cucurbituril and a detection method thereof. The invention is a novel fluorescent probe, has the characteristic of detecting L-phenylalanine in water, and has the characteristics of high sensitivity, low detection cost, simple sample treatment, convenient operation, quick determination and real-time detection.

The technical scheme of the invention is as follows: fluorescent probe for detecting L-phenylalanine based on seven-element cucurbituril, and molecular formula of fluorescent probe is C₄₂H₄₂N₂₈O₁₄@C₂₀H₁₈ClNO₄The chemical structural formula is shown in figure 1.

The fluorescent probe for detecting L-phenylalanine based on seven-element cucurbituril is prepared from seven-element cucurbituril and berberine hydrochloride.

The specific preparation method of the fluorescent probe for detecting L-phenylalanine based on seven-membered cucurbituril comprises the following steps:

(1) dissolving seven-element cucurbituril in water to obtain a solution A;

(2) dissolving berberine hydrochloride in water to obtain solution B;

(3) and mixing the solution A and the solution B, and reacting at normal temperature to obtain the fluorescent probe.

In the fluorescent probe for detecting L-phenylalanine based on seven-membered cucurbituril, the concentration of the solution A in the step (1) is 1.0X 10^-4mol/L。

The concentration of the solution B in the step (2) is 1.0X 10^-3mol/L。

When the solution A and the solution B in the step (2) are mixed, the molar ratio of the seven-element cucurbituril to the berberine hydrochloride in the mixed solution is 1: 0.5-2.

According to the fluorescent probe for detecting L-phenylalanine based on the seven-element cucurbituril, the molar ratio of the seven-element cucurbituril to the berberine hydrochloride in the mixed solution is 1: 1.

Diluting the fluorescent probe with water to obtain a probe standard solution, adding a water solution to be detected into the probe standard solution, standing for 5-20s, performing fluorescence emission spectrometry at a fixed excitation wavelength of 350nm, and drawing a change curve of fluorescence intensity at the excited laser wavelength; and calculating the fluorescence emission spectrum intensity change under 496.96nm respectively before and after the water solution to be detected is added into the fluorescent probe solution according to the change curve, and detecting the L-phenylalanine.

In the method for detecting L-phenylalanine by using the fluorescent probe, the concentration of the standard solution of the probe is 2.0X 10^{- 5}mol/L。

According to the method for detecting L-phenylalanine by using the fluorescent probe, when the fluorescence emission spectrum intensity of the probe standard solution at 496.96nm is reduced before and after the aqueous solution to be detected is added, the aqueous solution to be detected contains the L-phenylalanine, otherwise, the aqueous solution to be detected does not contain the L-phenylalanine.

The invention has the advantages of

The fluorescent probe is a novel fluorescent probe and can detect the L-phenylalanine in the aqueous solution; in addition, the detection limit of the fluorescent probe of the invention is as low as 1.7398X 10^-6mol/L, has the advantage of high sensitivity; meanwhile, the fluorescent probe can be prepared by a simple method only by taking the seven-element cucurbituril and the berberine hydrochloride as raw materials and is detected by the simple method, so that the fluorescent probe has the advantages of low detection cost, simple sample treatment and convenient operation; moreover, the detection operation is simple, so that the detection can be real-time and quick.

In order to verify the beneficial effects of the invention, the inventor carries out a great deal of experimental research, and part of the experimental processes and results are as follows:

experimental example 1A suitable molar ratio of cucurbituril and berberine hydrochloride is explored

In order to explore the proper molar ratio of the probe formed by the seven-element cucurbituril and the berberine hydrochloride, the interaction between a subject and an object is investigated by adopting an ultraviolet absorption spectrometry and a fluorescence spectrometry.

The method for determining the data of the ultraviolet absorption spectrum, the fluorescence spectrum and the fluorescence spectrum among the systems by a molar ratio method comprises the following steps: preparing berberine hydrochloride and seven-element cucurbituril into 1.0mmol/L and 0.1mmol/L aqueous solutions respectively for later use, fixing the concentration of an object to be 0.02mmol/L, changing the concentration of the seven-element cucurbituril, preparing N seven-element cucurbituril/N berberine hydrochloride aqueous solutions of 0, 0.1, 0.2, 0.3, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 and 1.5, and measuring the ultraviolet-visible absorption spectrum of the solution at room temperature; fixing guest concentration of 0.02mmol/L, changing the concentration of seven-membered cucurbituril, preparing aqueous solution of N seven-membered cucurbituril/N berberine hydrochloride of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, measuring the fluorescence emission spectrum of the solution under the conditions of excitation wavelength of 350nm, excitation slit of 10nm, emission slit of 10nm and voltage of 540V, and measuring the ultraviolet absorption spectrum and fluorescence spectrum between systems by using an equimolar continuous variation method (JOB method), wherein the total concentration of the immobilized subject and the immobilized object is 4.0mmol/L, by continuously changing the ratio of the amount of the substances between the subject and the object, a series of solutions to be tested with different molar ratios of N seven-membered cucurbituril/(N seven-membered cucurbituril + N berberine hydrochloride) of 0.1, 0.2 … … 0.8.8, 0.9 and 1.0 are prepared, and the ultraviolet absorption spectrum and the fluorescence spectrum are measured according to the method.

Experimental example 2 quantitative analysis

The concentration of the product obtained by the invention is 2.0X 10^-5The detection results are shown in fig. 4 and 5, and it can be seen that the concentrations of L-phenylalanine in the standard solutions are different after different volume fractions are added, the fluorescent probe solutions have different degrees of fluorescence attenuation due to the different concentrations of L-phenylalanine, and the linear range of the L-phenylalanine response is (1.0-20.0) × 10^-5mol/L, detection limit of 1.5871X 10^-6mol/L (as shown in FIG. 6).

Drawings

FIG. 1 is a molecular structural formula of the fluorescent probe of the present invention;

FIG. 2 is a molar ratio method of seven-element cucurbituril and berberine hydrochloride;

FIG. 3 is a graph showing fluorescence spectra when a probe standard solution is added to a solution containing different L-amino acids;

FIG. 4 is a graph showing fluorescence spectra of a probe standard solution added with solutions containing L-phenylalanine at different concentrations;

FIG. 5 shows fluorescence spectrum intensities of a probe standard solution added with solutions containing L-phenylalanine at different concentrations;

FIG. 6 is a simulation of the detection limit of the fluorescent probe of the present invention;

Detailed Description

The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.

Examples of the invention