Preparation and detection method of fluorescent probe for double-emission visual detection of amino acid

Abstract

The invention discloses a fluorescent probe for visually detecting arginine and histidine in an aqueous solution and a preparation method of the fluorescent probe, a sodium fluorescein modified europium (III)-based metal skeleton is used as a ratio fluorescent probe, specific recognition of a recognition site to amino acid is provided, and high-sensitivity and high-selectivity detection of arginine and histidine is realized. By compounding fluorescein sodium and a europium (III)-based metal skeleton, the probe increases new fluorescence emission at 514 nm, and still retains characteristic fluorescence emission of europium (III) at 619 nm, so that when a target object is detected, the fluorescence color of the probe is transited from red to yellow green, and ratiometric fluorescence is presented. Therefore, the ratiometric fluorescent probe provided by the invention can be used for carrying out visual detection on arginine and histidine by integrating RGB (Red, Green and Blue) values of smart phone applications.

Description

Technical Field

[0001] The invention relates to a ratio fluorescent probe for visualizing and quickly detecting amino acid color in water and application thereof. Background Art

[0002] Amino acids, as the basic building blocks of proteins and polypeptides, participate in a variety of life activities. Among them, arginine can provide nitric oxide to the human body, promote vasodilation, and play a considerable role in improving human immune function and maintaining hormone levels; histidine is an essential amino acid for humans and some mammals. It is a neurotransmitter in the central nervous system and can participate in protein synthesis in organisms. Studies have found that abnormal levels of histidine and arginine can be used as important indicators of basic diseases such as uremia, asthma, and cirrhosis. In addition, appropriate arginine and histidine are beneficial to the growth and development of infants and young children. In view of the special physiological effects of arginine and histidine, it is widely used in medicine, food, pharmaceuticals and other fields. Therefore, it is urgent to explore an effective analytical detection method for arginine and histidine. The currently reported detection methods for arginine and histidine include high performance liquid chromatography, electrochemical method, capillary electrophoresis, mass spectrometry, etc. However, the above-mentioned analytical techniques are usually complicated, time-consuming and expensive.

[0003] Metal-organic frameworks (MOFs) are two-dimensional or three-dimensional porous materials that are self-assembled from metal-centered ions or clusters and organic ligands, extending infinite crystal structures through coordination-type connections. Compared with other MOF materials, lanthanide MOFs (Ln-MOFs) have unique optical properties such as long fluorescence lifetime, sharp emission, and large Stokes shift, and have been widely studied in the field of detection.

[0004] However, sensors based on Ln-MOFs are based on single-emission fluorescence recognition, with low sensitivity and low precision. In order to improve the sensitivity of sensors based on Ln-MOFs, fluorescent dye molecules, carbon quantum dots and other fluorescent substances are usually embedded into the pores of Ln-MOFs to construct ratiometric fluorescence sensors with dual-emission characteristics. Among them, fluorescent dye molecules are favored by researchers because of their easy synthesis and low cost. For example, LJ et al. synthesized FS@UIO-66 composite materials as ratiometric fluorescence sensors for selective detection of antibiotics. In addition, LQ et al. prepared an orange fluorescent dual-emission RhB@Zr-MOF fluorescence sensing system for the detection of Cr 2 O 7 2− and CrO 4 2−ions. The above examples show that encapsulating dye molecules into the pores of MOFs improves the recognition sensitivity of Ln-MOFs-based sensors and reduces the errors caused by external conditions. Moreover, under UV irradiation, not only the change in color brightness can be observed, but also the change in color hue can be observed by the eye, which is more popular in visual detection.

[0005] Based on this, the present invention uses pyridine 3,5-dicarboxylic acid (H 2 PMA), pyromellitic acid (H 4 Eu-MOF was constructed by the reaction of BTC) and europium nitrate under solvothermal conditions, and sodium fluorescein (FS) molecules were encapsulated into the pores of Eu-MOF to construct FS@Eu-MOF, a ratiometric fluorescent probe with dual emission. This probe is used for highly sensitive color recognition and visualization detection of arginine and histidine in aqueous solution. It can be visualized by the eye or combined with RGB color analysis technology through a smartphone, and has the advantages of rapid on-site analysis and low cost. Summary of the invention

[0006] The purpose of the present invention is to provide a ratio fluorescent probe for color recognition, visualization and rapid detection of arginine and histidine in aqueous solution and application thereof.

[0007] To achieve the above object, the present invention provides a method for preparing a visualized multicolor ratio fluorescent probe, which comprises the following steps: 2 PMA, H 4 BTC and FS were added to a certain volume of water and N,N-dimethylacetamide solution in the same proportion, mixed evenly, and ultrasonicated for 10-30 min. Then, the mixture was placed in an oven and the temperature was set to 90-120 °C. After the reaction time was 48-96 h, block crystals were obtained. The crystals were repeatedly washed with deionized water to remove impurities, dried in a forced air drying oven, and ground in a mortar to obtain an orange powder.

[0008] The present invention provides an application of ratiometric fluorescent probe in visual detection of amino acids, realizing visual detection of arginine or histidine in aqueous solution, and the specific steps are as follows: firstly, a fluorescent probe suspension is prepared, and arginine or histidine solution of different concentrations is gradually added to the system, and the fluorescence is gradually enhanced. By measuring the fluorescence intensity ratio I 514 / I 619 A standard curve is established with the concentration of arginine or histidine to achieve quantitative detection of arginine or histidine in aqueous solution. Under ultraviolet light at 254 nm, a series of different fluorescence color changes are presented. A standard curve is established with the G / R ratio converted through image acquisition and color output and the concentration of arginine or histidine, thereby achieving visual detection of arginine or histidine in aqueous solution.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] (1) The ratiometric fluorescent probe provided by the present invention realizes the quantitative detection of arginine and histidine by establishing a linear relationship between fluorescence intensity and amino acid concentration. The dual-emission ratiometric fluorescent probe can improve the sensitivity and accuracy of detection and reduce the error caused by external conditions.

[0011] (2) The ratiometric fluorescent probe provided by the present invention can achieve qualitative detection of arginine and histidine through obvious changes in fluorescence color and intensity. Under ultraviolet light, not only can the change in color brightness be observed by the naked eye, but also the change in color hue can be observed, making visual detection more intuitive and convenient.

[0012] The technical solution of the present invention is further described in detail below through the accompanying drawings and implementation modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 (a) is the PXRD pattern of Eu-MOF, FS@Eu-MOF and simulation, (b) is the PXRD pattern of ligand H 2 PMA, H 4 Infrared spectra (FT-IR) of BTC, Eu-MOF and FS@Eu-MOF.

[0014] Figure 2 (a) is the fluorescence spectrum of FS@Eu-MOF in the presence of different concentrations of arginine, and (b) is the linear relationship between arginine concentration and fluorescence response signal.

[0015] Figure 3 (a) is the fluorescence spectrum of FS@Eu-MOF in the presence of different concentrations of histidine, and (b) is the linear relationship between histidine concentration and fluorescence response signal.

[0016] Figure 4 (a) is a visualization detection diagram of arginine, and (b) is a linear relationship diagram between arginine concentration and G / R value.

[0017] Figure 5 (a) is a visualization detection diagram of histidine, and (b) is a linear relationship diagram between histidine concentration and G / R value. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments of the present invention:

[0019] Example 1

[0020] This embodiment provides a method for preparing a ratiometric fluorescent probe, comprising the following steps: Synthesis of FS@Eu-MOF: 0.12 mml Eu(NO)3 6H 2 O, 0.1 mml H 2 PMA, 0.1 mml H 4 BTC and 0.1 mml FS, 4 mL H 2 O and 1 mL DMA were placed in a beaker and ultrasonically stirred until the mixture was uniformly mixed, and then transferred to a reactor and reacted in an oven at 100 °C for 72 h. The mixture was taken out to obtain block crystals, which were then dried in a forced air drying oven and ground to obtain orange powder.

[0021] like Figure 1 (a) shows the PXRD patterns of Eu-MOF, FS@Eu-MOF and simulation. The PXRD pattern of the newly synthesized Eu-MOF shows sharp diffraction peaks, which are almost the same as the simulation pattern obtained from the single crystal structure data. The PXRD pattern of FS@Eu-MOF is consistent with that of Eu-MOF, indicating that the structure of Eu-MOF is not damaged by the encapsulation of sodium fluorescein molecules, proving the successful synthesis of FS@Eu-MOF. Figure (b) shows the PXRD pattern of the newly synthesized Eu-MOF with ligand H 2 PMA, H 4 Infrared spectra (FT-IR) of BTC, Eu-MOF and FS@Eu-MOF. -1 and 3331 cm -1 The absorption peak at 1665 cm is due to the stretching vibration of -OH. -1 and 1452 cm -1 There are two strong absorption peaks at , which are attributed to the symmetric and antisymmetric vibration absorption peaks of C=O. 2 PMA, H 4 1624 cm in BTC −1 and 1614 cm -1 The absorption peaks are the asymmetric stretching vibration peaks of C=O. These characteristic peaks are shifted to 1608 cm in Eu-MOF. −1 , indicating that the -COOH group in the ligand binds to Eu 3+ In addition, at 1221 cm −1 and 1263 cm −1 and 1362 cm −1 and 3084 cm -1 The absorption peak disappears, which also shows that 3+ The above results all confirm the successful synthesis of Eu-MOF and FS@Eu-MOF.

[0022] Example 2

[0023] In this example, a linear relationship between the fluorescence intensity and the amino acid concentration of the prepared fluorescent probe is established to achieve quantitative detection of arginine or histidine.

[0024] The fluorescent probe prepared in Example 1 was prepared into a 2 mg / 3 mL suspension, and different concentrations of arginine or histidine were added to the probe suspension to obtain final mixture concentrations of 5, 10, 20, 30, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 800 and 1000 μM, respectively. 3+ The intensity of the characteristic fluorescence emission peak of the ion hardly changes at low concentrations, but gradually decreases as the concentration increases. The intensity of the fluorescence emission peak at 514 nm gradually increases and gradually shifts to 524 nm and 521 nm, and the final intensity is higher than the emission peak at 619 nm. Therefore, the fluorescence intensity ratio I 514 / I 619 As the response signal for quantitative analysis, under the condition of excitation wavelength of 307 nm, the fluorescence spectrum of the mixed system at 450-700 nm was recorded, such as Figure 3 (a) and 4(a). As the concentrations of arginine and histidine increase, the fluorescence intensity gradually increases. Figure 3 (b) and 4(b) are the fluorescence response signal diagrams between the concentration of arginine and histidine and the intensity ratio of the ratio fluorescence probe. The arginine concentration and the response signal I 514 / I 619 The linear relationship is Y=0.053X-0.0306, R 2 The calculated value is 0.9932, and the detection limit LOD is 13.55 μM. Histidine concentration and response signal I 514 / I 619 The linear relationship is Y=0.0039X+0.0997, R 2 The calculated value is 0.9936, and the detection limit LOD is 18.13 μM. There is a good linear relationship between the fluorescence intensity ratio and the concentration of arginine or histidine, which enables quantitative detection of arginine or histidine.

[0025] Example 3

[0026] This embodiment provides a method for visual detection of ratiometric fluorescent probes to achieve rapid on-site detection of amino acids.

[0027] The concentrations of the mixture prepared according to Example 2 were 0, 50, 100, 150, 200, 300, 400, 600, 800 and 1000 μM, respectively. Subsequently, under 254 nm ultraviolet light, it can be clearly seen by the naked eye that as the content of arginine or histidine increases, the solution changes from red to yellow-green, accompanied by the appearance of multiple distinguishable colors such as red-orange, yellow-orange, orange, yellow-green, and green, as shown in FIG. Figure 4(a) Figure 5 (a) shows that color recognition visualization detection is realized, which not only meets the physiological mechanism of naked eye visualization recognition, but also meets the quantitative requirements of color recognition visualization of smartphone terminals. Figure 4 (b) Figure 5 (b) Relationship between arginine and histidine concentrations and G / R values. In the concentration range of 0-600 μM, the linear relationship between arginine concentration and G / R value is Y=0.0011X+0.5775, R 2 The calculated value is 0.9911. The linear relationship between histidine concentration and G / R value is Y=0.001X+0.5954, R 2 The calculated value is 0.9922, and there is a good linear relationship between the G / R value and the concentration of arginine or histidine, which successfully realizes the rapid diagnosis of amino acids on site without instrumentation. This visual detection method using a smartphone provides more convenience and practicality.

[0028] The present invention is described in detail above in combination with the implementation modes and exemplary examples. The contents described in this specification are merely an enumeration of the implementation forms of the inventive concept, and the protection scope of the present invention should not be considered as being limited to the specific forms described in the embodiments.

Claims

1. A ratiometric fluorescent probe for visual detection of arginine and histidine, characterized in that: The preparation method of the ratiometric fluorescent probe comprises the following steps: S1 uses europium (III) as the central ion, pyridine 3,5-dicarboxylic acid and pyromellitic acid as ligands, and water and N,N-dimethylacetamide as solvents. The mixture is uniformly mixed by ultrasonication, and the resulting solution is transferred to a reaction kettle, and then placed in an oven for reaction, and then cooled to room temperature, centrifuged, washed, and dried to obtain; S2 is the same as the above steps, except that sodium fluorescein is added during the synthesis to obtain a ratiometric fluorescent probe.

2. The fluorescent probe according to claim 1, characterized in that The structural formula of the fluorescent probe is expressed as: FS@Eu-MOF.

3. The method for preparing a ratiometric fluorescent probe according to claim 1, characterized in that: Pyridine 3,5-dicarboxylic acid, pyromellitic acid and sodium fluorescein were added in the same ratio, and the volume of water added was four times the volume of N,N-dimethylacetamide added.

4. The method for preparing a ratiometric fluorescent probe according to claim 1, characterized in that: The temperature of the oven is 90-120°C and the reaction time is 48-96 h.

5. The use of the ratiometric fluorescent probe for color recognition and visualization rapid detection of arginine and histidine according to claim 1, characterized in that The application method comprises the following steps: S1 Prepare a ratiometric fluorescent probe suspension to a concentration of 0.5-1 mg / mL. Detect the fluorescence intensity of the fluorescence emission spectrum of the solution at 514 nm and 619 nm excitation wavelengths. 514 / I 619 As the ordinate, and with different concentrations of arginine and histidine as the abscissa, a standard curve was drawn to obtain the corresponding curve equation; S2 After the ratio fluorescent probe suspension and the added arginine and histidine solutions of different concentrations are fully mixed, let it stand for 10-30 min; take photos under fluorescence, and use a smartphone to analyze the sample through image acquisition and color output functions. The color of the image is converted into data, and a standard curve is drawn with the converted G / R ratio as the vertical coordinate and solutions containing different concentrations of arginine and histidine as the horizontal coordinate to obtain the corresponding curve equation.

Images