阅读说明：本技术 一种特异性检测精氨酸的荧光检测剂及检测试纸的制备方法 (Fluorescence detection agent for specifically detecting arginine and preparation method of detection test paper ) 是由 黄潇楠 路昭然 朱晓雨 韩天宇 于 2019-09-23 设计创作，主要内容包括：本发明公开了一种特异性检测精氨酸的荧光检测剂及检测试纸的制备方法。该检测剂((E)-2-((4-(二乙基氨基)亚苄基)氨基)苯甲酸,DBAB)是由4-(二乙基氨基)苯甲醛和2-氨基苯甲酸一步式合成；然后将白色滤纸浸入DBAB溶液中制备得到检测试纸。在溶液中,DBAB对精氨酸表现出特异识别性。比色实验中,DBAB-Arg的混合溶液表现出明显的黄色到无色的颜色变化；荧光实验中,DBAB-精氨酸的混合溶液表现出明显的青色到紫色的荧光变化。在试纸实验中也有相同的现象。根据紫外吸收光谱和荧光光谱显示,DBAB对于精氨酸是具有双吸收信号和双发射信号的多通道传感器。该检测方法具有高选择性和瞬时响应性。(The invention discloses a fluorescence detection agent for specifically detecting arginine and a preparation method of detection test paper. The detector ((E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid, DBAB) was synthesized from 4- (diethylamino) benzaldehyde and 2-aminobenzoic acid in one step; and then, soaking the white filter paper into the DBAB solution to prepare the detection test paper. In solution, DBAB exhibits specific recognition for arginine. In a colorimetric experiment, a mixed solution of DBAB-Arg shows obvious color change from yellow to colorless; in fluorescence experiments, the DBAB-arginine mixed solution showed a clear cyan to violet fluorescence change. The same phenomenon also occurs in the test paper experiment. DBAB is a multichannel sensor with double absorption and double emission signals for arginine, shown by ultraviolet absorption and fluorescence spectroscopy. The detection method has high selectivity and transient responsiveness.)

1. A preparation method of a fluorescence detection agent for specifically detecting arginine is characterized by comprising the following specific steps: dissolving 4- (diethylamino) benzaldehyde in dehydrated ethanol; dissolving 2-aminobenzoic acid in an organic solvent; then adding the 2-aminobenzoic acid solution and acetic acid to a dehydrated ethanol solution of 4- (diethylamino) benzaldehyde to form a mixture; and stirring the mixture at 48-52 ℃ for 4.9-5.1 hours, cooling to room temperature, filtering, and finally washing with ethanol and hexane to obtain the fluorescence detection agent (E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid for specifically detecting arginine.

2. The method according to claim 1, wherein the dehydrated ethanol solution of 4- (diethylamino) benzaldehyde has a concentration ranging from 0.07 to 0.09 mmol/mL.

3. The preparation method according to claim 1, wherein the organic solvent is one or more selected from the group consisting of N, N-dimethylformamide, tetrahydrofuran, and acetone.

4. The method according to claim 1, wherein the concentration of the 2-aminobenzoic acid solution is in the range of 15 to 20 mmol/mL.

5. The process according to claim 1, wherein the molar ratio of 4- (diethylamino) benzaldehyde to 2-aminobenzoic acid is 0.6-0.7:1, and the molar ratio of 4- (diethylamino) benzaldehyde to acetic acid is 6.4-6.5: 1.

6. A preparation method of test paper for specifically detecting arginine is characterized by comprising the following specific steps: cutting white filter paper into uniform paper strips, immersing the paper strips into a tetrahydrofuran solution of (E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid, wherein the concentration of the (E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid is 0165-0.175mmol/mL, immersing for 2-10 minutes, taking out, and ventilating and drying at room temperature to obtain the finished product.

7. The use method of the fluorescence detection agent for specifically detecting arginine is characterized by comprising the following specific steps: and (3) dripping the solution to be detected into a tetrahydrofuran or DMF (dimethyl formamide) solution of (E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid, observing whether the solution changes color, if so, determining that the solution to be detected contains arginine, and if not, determining that the solution to be detected does not contain arginine.

8. The method for using the test paper for specifically detecting arginine, which is prepared according to the method of claim 6, is characterized by comprising the following steps: and dropping the liquid to be detected on the detection test paper, or putting the detection test paper into the liquid to be detected, observing whether the detection test paper changes color, if so, determining that the liquid to be detected contains arginine, and if not, determining that the liquid to be detected does not contain arginine.

Technical Field

The invention belongs to the technical field of amino acid detection, and particularly relates to a fluorescent detection agent for specifically detecting arginine and a preparation method of detection test paper.

Background

Of the 20 common amino acids in humans, arginine is of great interest because of its important effects on human brain chemistry, including vasodilation, immune response, and neurotransmission. Arginine (Arg), also called proteinogenic amino acid, is the most basic amino acid, and has the highest isoelectric point (pI) of 10.76, compared to the other 20 amino acids. Arginine plays an important role in various biological functions such as cell division, wound healing, protein production, immune response, and the like. In addition, arginine participates in ornithine circulation in a human body, promotes the formation of urea, converts ammonia generated in the human body into non-toxic urea through the ornithine circulation, and then is discharged from urine, so that the blood ammonia concentration is reduced; arginine is the main component of sperm protein and has the functions of promoting sperm generation and providing sperm movement energy; arginine is also a physiological precursor of Nitric Oxide (NO), which plays an important role as a key mediator in vascular homeostasis. In plasma sample analysis, arginine derivative species can be unhealthy or even life-threatening when the number is abnormal. Arginine is therefore useful as an indicator for the diagnosis of certain diseases. Therefore, it is highly desirable to develop a feasible arginine detection method. Although many methods for determining arginine have been reported, such as High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and capillary electrophoresis, most of these methods require specialized experimental equipment and complicated procedures, and are expensive. Therefore, it is necessary to develop a simple, cheap, highly sensitive and highly selective method for detecting arginine. One such detection method is a fluorescence chemical sensor, which has many advantages over other methods, such as ease of operation, high sensitivity, and significant selectivity and rapid response.

Disclosure of Invention

The invention provides a fluorescence detection agent ((E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid and DBAB) for specifically detecting arginine and a preparation method of detection test paper. The detection agent is synthesized by 4- (diethylamino) benzaldehyde and 2-aminobenzoic acid in a one-step mode; then, the white filter paper is immersed in a THF (tetrahydrofuran) solution of DBAB to prepare the test paper.

The preparation method of the fluorescence detection agent for specifically detecting arginine comprises the following steps: dissolving 4- (diethylamino) benzaldehyde in dehydrated ethanol; dissolving 2-aminobenzoic acid in an organic solvent; then adding the 2-aminobenzoic acid solution and acetic acid to a dehydrated ethanol solution of 4- (diethylamino) benzaldehyde to form a mixture; and stirring the mixture at 48-52 ℃ for 4.9-5.1 hours, cooling to room temperature, filtering, and finally washing with ethanol and hexane to obtain the fluorescence detection agent (E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid for specifically detecting arginine.

The concentration range of the dehydrated ethanol solution of the 4- (diethylamino) benzaldehyde is 0.07-0.09 mmol/mL.

The organic solvent is one or more selected from N, N-dimethylformamide, tetrahydrofuran and acetone.

The concentration range of the 2-aminobenzoic acid solution is 15-20 mmol/mL.

The molar ratio of the 4- (diethylamino) benzaldehyde to the 2-aminobenzoic acid is 0.6-0.7:1, and the molar ratio of the 4- (diethylamino) benzaldehyde to the acetic acid is 6.4-6.5: 1.

The preparation method of the test paper for specifically detecting arginine comprises the following steps: cutting white filter paper into uniform paper strips, immersing the paper strips into a tetrahydrofuran solution of (E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid, wherein the concentration of the (E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid is 0165-0.175mmol/mL, immersing for 2-10 minutes, taking out, and ventilating and drying at room temperature to obtain the finished product.

The application method of the fluorescence detection agent for specifically detecting arginine comprises the following steps: and (3) dripping the solution to be detected into a tetrahydrofuran or DMF (dimethyl formamide) solution of (E) -2- ((4- (diethylamino) benzylidene) amino) benzoic acid, observing whether the solution changes color, if so, determining that the solution to be detected contains arginine, and if not, determining that the solution to be detected does not contain arginine.

The using method of the test paper for specifically detecting arginine comprises the following steps: and dropping the liquid to be detected on the detection test paper, or putting the detection test paper into the liquid to be detected, observing whether the detection test paper changes color, if so, determining that the liquid to be detected contains arginine, and if not, determining that the liquid to be detected does not contain arginine.

The invention takes the fluorescent Schiff base as the base material, and the arginine detection test paper which is portable, low in cost and quick in response can be prepared by loading the solution on the filter paper. In solution, DBAB exhibits specific recognition for arginine. DBAB emits bluish green light at high concentration due to the aggregation-induced emission principle, and interacts with carboxyl groups of DBAB molecules due to the basicity of the guanidino group of arginine and hydrogen bonding, thereby destroying intermolecular aggregation and emitting violet light. In a colorimetric experiment, a mixed solution of DBAB-Arg shows obvious color change from yellow to colorless; in fluorescence experiments, the DBAB-arginine mixed solution shows obvious blue to purple fluorescence change. The same phenomenon also occurs in the test paper experiment. DBAB is a multichannel sensor with double absorption and double emission signals for arginine, shown by ultraviolet absorption and fluorescence spectroscopy. The detection method has high selectivity and transient response, and the detection limit of arginine is 0.3 mmol/mL.

Drawings

FIG. 1 is a schematic diagram of the fluorescence detection agent for specifically detecting arginine according to the present invention.

FIG. 2 is a diagram of the detection agent DBAB synthesized in example 1¹H NMR and¹³c NMR chart.

FIG. 3 is an ESI MS plot of DBAB synthesized in example 1.

FIG. 4 is a graph of the ambient light, UV absorption spectrum and fluorescence spectrum of DBAB in example 1 for detecting arginine in solution.

FIG. 5 is a graph showing the reaction time of DBAB to detect arginine in example 1.

FIG. 6 is a graph of the test paper application in example 2.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.