Stable R6G / Eu-MOF dual-emission fluorescence sensor and application thereof in orientin detection

Abstract

The invention relates to a stable R6G / Eu-MOF dual-emission fluorescence sensor and application thereof in orientin detection, and a preparation method of the R6G / Eu-MOF dual-emission fluorescence sensor comprises the following steps: synthesizing Eu-MOF by using europium chloride hexahydrate, 2-aminoterephthalic acid and 1, 10-phenanthroline, and then mixing the synthesized Eu-MOF with R6G to prepare the R6G / Eu-MOF dual-emission fluorescence sensor. The R6G / Eu-MOF dual-emission fluorescence sensor can be used for establishing a simple and rapid fluorescence sensing method to measure orientin, and orientin and other various traditional Chinese medicine monomers can be rapidly distinguished by using the difference of the intensity ratio of two emission peaks at 430 nm and 555 nm. The invention also utilizes the R6G / Eu-MOF dual-emission fluorescence sensor to detect orientin in a human serum sample, and the result accords with expectation.

Description

Technical Field

[0001] The invention relates to a stable R6G / Eu-MOF dual-emission fluorescence sensor and application thereof in orientin detection. Background Art

[0002] Orientin is a C-glycosyl flavonoid isolated from Polygonum orientale L., a traditional medicinal plant with a long history of treating rheumatic arthritis and rheumatoid arthritis. It has a variety of pharmacological activities, such as antioxidant, anti-inflammatory and anti-tumor, and is a natural compound that can benefit human health in many ways. Quantitative analysis of orientin can evaluate and select medicinal materials rich in this substance, and can help to gain a deeper understanding of the distribution and metabolic pathways of this drug in the body, thereby ensuring the dosage safety and effectiveness of orientin. Currently, the commonly used methods for the determination of orientin include thin layer chromatography and large instrumental analysis methods such as high performance liquid chromatography (HPLC), high performance liquid chromatography-electrochemical detection (HPLC-EC) or ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS). There is still a lack of a relatively fast and sensitive simple method for the fluorescence determination of orientin.

[0003] Luminescent metal organic frameworks (LMOFs) are an important subclass of MOFs materials, and their fluorescent light sources can be obtained by selecting ligands with fluorescent properties, photoactive metal ions, and fluorescent functionalized modification groups. Most current LMOF-based detection systems are single emission source types, and their detection signals are more susceptible to external influences, resulting in inaccurate results. This problem can be solved through dual fluorescence emission mode. Dual fluorescence emission systems are usually designed to emit dual fluorescence under single wavelength excitation. This internally calibrated dual emission can eliminate the dependence on specific measurement conditions, thereby improving the accuracy and reproducibility of the analysis. At present, encapsulating fluorescent dyes into MOFs to form dual emission centers is a common strategy. This strategy can provide more diverse functional sites and chemical interaction possibilities for the detection system, but the MOFs-dye composite materials based on this also have shortcomings such as relatively complex preparation process and poor material stability, which limits its application range. Therefore, it is of great significance to find simpler and more efficient ways to combine MOFs with dyes. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing a stable R6G / Eu-MOF dual-emission fluorescence sensor, the R6G / Eu-MOF dual-emission fluorescence sensor obtained by the method, and application of the sensor in orientin detection.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for preparing a stable R6G / Eu-MOF dual-emission fluorescence sensor comprises the following steps:

[0007] (1) Synthesis of Eu-MOF: Europium chloride hexahydrate, 2-aminoterephthalic acid and 1,10-phenanthroline were added to ultrapure water and ultrasonically mixed; then, an aqueous NaOH solution was added, and the resulting mixture was transferred to a polytetrafluoroethylene-lined hydrothermal reactor and heated at 155-170°C for 12-14 hours; after centrifugation and repeated washing with water and ethanol, it was dried at 60-65°C under vacuum for 5-8 hours to finally obtain yellow-brown powder Eu-MOF;

[0008] (2) Preparation of R6G / Eu-MOF: Eu-MOF powder and an equal mass of rhodamine 6G (R6G) were dispersed in ultrapure water at a concentration of 1 mg / mL, respectively. The two dispersed solutions were then mixed in an equal volume ratio and ultrasonically treated for 1 hour to finally obtain a R6G / Eu-MOF suspension, i.e., the R6G / Eu-MOF dual-emission fluorescence sensor.

[0009] The specific operation method of step (1) is:

[0010] 0.511 g of europium chloride hexahydrate, 0.385 g of 2-aminoterephthalic acid and 0.280 g of 1,10-phenanthroline were added to 35 mL of ultrapure water and mixed by ultrasonication; after adding 4.2 mL of 0.65 M NaOH aqueous solution, the resulting mixture was transferred to a polytetrafluoroethylene-lined hydrothermal reactor, heated at 160° C. for 12 hours, centrifuged and repeatedly washed with water and ethanol, and dried at 60° C. under vacuum for 6 hours to finally obtain yellow-brown powder Eu-MOF, wherein the speed of the centrifuge was controlled to 11000 rpm and the centrifugation time was 5 min.

[0011] The specific operation method of step (2) is:

[0012] 1 mg Eu-MOF powder and 1 mg R6G were dispersed in 1 mL ultrapure water respectively, and then the two solutions were mixed in equal volumes and treated with ultrasound for 1 hour to finally obtain a R6G / Eu-MOF suspension, namely the R6G / Eu-MOF dual-emission fluorescence sensor.

[0013] A stable R6G / Eu-MOF dual-emission fluorescence sensor prepared by the preparation method.

[0014] The application of the R6G / Eu-MOF dual-emission fluorescence sensor in the detection of orientin.

[0015] A method for detecting orientin using the R6G / Eu-MOF dual-emission fluorescence sensor comprises the following steps: adding a R6G / Eu-MOF suspension and an orientin standard solution or a solution to be tested into an EP tube, then adding a 0.1M Tris-HCl solution with a pH of 7.4, vortexing the solution to ensure uniform mixing, then transferring it to a quartz cell, and performing fluorescence detection at room temperature. The fluorescence experiment is performed at an excitation wavelength of 330 nm, and recording the fluorescence emission spectrum within a range of 400 to 600 nm.

[0016] The method for detecting orientin using the R6G / Eu-MOF dual-emission fluorescence sensor has the following specific steps:

[0017] (1) Draw the orientin response standard curve and obtain the linear regression equation:

[0018] 10 μL of R6G / Eu-MOF suspension and 10 μL of orientin standard solution of different concentrations were added to 0.5 mL EP tubes, and then 180 μL of 0.1 M Tris-HCl solution with a pH of 7.4 was added; the solution was vortexed to ensure uniform mixing, and then transferred to a quartz cell for fluorescence detection at room temperature; the fluorescence experiment was performed at an excitation wavelength of 330 nm, and the fluorescence emission spectrum in the range of 400 to 600 nm was recorded; based on the quenching amount of the sample on the fluorescence emission signal of R6G / Eu-MOF at 555 nm and 430 nm, a standard curve of fluorescence quenching amount and orientin concentration was obtained; based on the obtained standard curve, a linear regression equation of relative fluorescence quenching amount and orientin concentration was obtained;

[0019] (2) Detection: 10 μL of R6G / Eu-MOF suspension and 10 μL of the test solution were added to 0.5 mL EP tubes respectively, and then 180 μL of 0.1 M Tris-HCl solution with a pH of 7.4 was added; the solution was vortexed to ensure uniform mixing, and then transferred to a quartz cell for fluorescence detection at room temperature; at an excitation wavelength of 330 nm, the quenching amount of the solution fluorescence signal at 555 nm and 430 nm was recorded; the fluorescence quenching amount was substituted into the linear regression equation obtained in step (1), and the orientin concentration of the test solution was calculated.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] The present invention successfully prepares a dual-emission fluorescent MOF (R6G / Eu-MOF) by simply mixing Eu-MOF and R6G. With the assistance of the cationic fluorescent dye R6G, the stability of Eu-MOF in aqueous solution is greatly improved. The obtained R6G / Eu-MOF exhibits dual-emission fluorescence at wavelengths of 430nm and 555nm respectively under 330nm excitation, and has good structure and fluorescence stability in aqueous solution. Due to the competitive absorption and dynamic quenching effect of orientin on the excitation energy of R6G / Eu-MOF, the addition of orientin can quench the fluorescence at two emission peaks at the same time. Therefore, the inventors established a simple and rapid fluorescence sensing method to determine orientin, with a linear range of 1.1-220μM and a detection limit of 0.26μM at 430nm and 0.16μM at 555nm (3σ / N).

[0022] In addition, the difference in the intensity ratio of the two emission peaks at 430nm and 555nm can be used to quickly distinguish orientin from many other Chinese medicine monomers.

[0023] Adding orientin can quench both emissions simultaneously, and the ratio of the two emissions can be used to quantitatively determine orientin with good selectivity. The present invention also uses the R6G / Eu-MOF dual-emission fluorescence sensor to detect orientin in human serum samples, and the results are in line with expectations. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a characteristic diagram of Eu-MOF of the present invention; wherein, Figure 1 a is the scanning electron microscope image of Eu-MOF; Figure 1 b is the N2 adsorption–desorption isotherm of Eu-MOF; Figure 1 The inset in b is the pore size distribution curve of the synthesized Eu-MOF; Figure 1 c is the experimental XRD pattern of Eu-MOF and the simulated XRD pattern according to the CIF file (CCDC number is 222296); Figure 1 d is the Zeta potential characterization diagram of Eu-MOF; Figure 1 e is the fluorescence spectrum of Eu-MOF after dispersion in DMF, where the inset is the luminescence photo of Eu-MOF solid powder; Figure 1 f is the UV-visible absorption spectrum and fluorescence spectrum of Eu-MOF after dispersion in water, and the inset is the fluorescence photograph of Eu-MOF dispersed in water.

[0025] Figure 2 is the fluorescence behavior diagram of R6G / Eu-MOF; Figure 2 a is the excitation spectrum and emission spectrum of R6G / Eu-MOF; Figure 2The inset of a is a photograph of Eu-MOF (①) and R6G / Eu-MOF (②) solutions under white light (left) and 365 nm ultraviolet irradiation (right); Figure 2 b is the change of fluorescence intensity at 430nm, 555nm and ΔF430 / ΔF555 at different pH values; Figure 2 c is the change of fluorescence intensity of Eu-MOF and R6G / Eu-MOF at 430 nm wavelength under different storage days; Figure 2 d is the fluorescence intensity ratio of R6G / Eu-MOF under different storage days.

[0026] Figure 3 This is the effect of Chinese medicine monomers on R6G / Eu-MOF fluorescence; Figure 3 a is the effect of different herbal monomers on the fluorescence intensity of R6G / Eu-MOF (F430 and F555); Figure 3 b is the heat map of the quenching rate of different herbal monomers on R6G / Eu-MOF (F430 and F555).

[0027] Figure 4 is the fluorescence quenching behavior of orientin on R6G / Eu-MOF; Figure 4 a is the fluorescence spectra of R6G / Eu-MOF suspension after adding orientin at low, medium and high concentrations; Figure 4 The inset in a is the image of R6G / Eu-MOF suspension without, with, orientin at 75 μM and 150 μM respectively, under 365 nm UV light irradiation; Figure 4 b is the graph showing the change of solution fluorescence intensity with orientin addition time; Figure 4 c is the UV absorption spectra of orientin, R6G / Eu-MOF and R6G / Eu-MOF+orientin;

[0028] Figure 4 d is the fluorescence lifetime decay curve of R6G / Eu-MOF and R6G / Eu-MOF+orientin under 430nm emission excited by 375nm-EPL light source.

[0029] Figure 5 is the fluorescence determination result of orientin; Figure 5 a is a schematic diagram of the preparation method and the detection method of erythrosin of the present invention, Figure 5 b is the fluorescence spectra of R6G / Eu-MOF at different orientin concentrations; Figure 5 c is the linear relationship between the fluorescence intensity change at 430 nm and 550 nm and the concentration of orientin.

[0030] Figure 6The effect of orientin and some common ions and amino acids on the fluorescence intensity of R6G / Eu-MOF, where the concentration of orientin is 45 μM and the concentration of other substances is 500 μM. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0032] 1. Experimental part:

[0033] 1.1 Materials and reagents

[0034] Europium chloride hexahydrate, rhodamine 6G, and N,N-dimethylformamide were purchased from Shanghai MacLean Biochemical Co., Ltd. (Shanghai, China), and 2-aminoterephthalic acid and 1,10-phenanthroline hydrate were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Phosphate buffer 1× (Tris-HCl) and normal human serum were purchased from Beijing Solebao Biotechnology Co., Ltd. (Beijing, China). Drug monomers such as orientin, paeoniflorin, hesperidin, and quercetin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Reagents such as sodium hydroxide, potassium chloride, sodium chloride, magnesium chloride, and calcium chloride were purchased from China National Pharmaceutical Group Corporation (Beijing, China). All reagents and organic ligands were obtained from commercial sources without further purification.

[0035] 1.2 Instruments

[0036] The specific surface area and pore size were measured by ASAP 2020 specific surface area and porosity analyzer (Micromeritics, USA). Scanning electron microscope (SEM) images were obtained by TESCAN MIRALMS scanning electron microscope (TESCAN, Czech Republic). Powder X-ray diffraction (PXRD) patterns were recorded by Rigaku Smartlab SE X-ray powder diffractometer (Rigaku, Japan). X-ray photoelectron spectra were measured by K-Alpha X-ray photoelectron spectrometer (Thermo Fisher Scientific, USA) to analyze the elemental composition, chemical state and molecular structure of the materials. Fluorescence lifetime decay curves were obtained by photoluminescence spectrometer FLS1000 (Edinburgh, UK). Fluorescence spectra were recorded by Cary Eclipse fluorescence spectrophotometer. (VARIAN, USA) records fluorescence spectra. UV-visible absorption spectra were obtained by Shimadzu UV-1800 spectrophotometer (Shimadzu, Japan).

[0037] 1.3 Synthesis of Eu-MOF

[0038] The synthesis of the Eu-MOF material used in the present invention can refer to the prior art (Anal.Chem.2019,91,7,4845), specifically: europium chloride hexahydrate (0.511g), 2-aminoterephthalic acid (0.385g) and 1,10-phenanthroline (0.280g) are added to ultrapure water (35mL) and ultrasonically mixed. After adding 4.2mL of NaOH aqueous solution (0.65M), the resulting mixture is transferred to a polytetrafluoroethylene-lined hydrothermal reactor and heated at 160°C for 12 hours. After centrifugation (11000rpm / 5min) and repeated washing with water and ethanol, it is dried under vacuum at 60°C for 6 hours to finally obtain a yellow-brown powder Eu-MOF for subsequent use.

[0039] Characterization of Eu-MOF:

[0040] Scanning electron microscope images of the prepared Eu-MOF were taken, the N2 adsorption-desorption isotherms of Eu-MOF were measured, the crystal structure of Eu-MOF was studied using XRD diffractometer, and the fluorescence characteristics of Eu-MOF were studied, and its absorption wavelength, maximum fluorescence excitation wavelength and emission wavelength were tested. The specific results are as follows: Figure 1 As shown, the above test methods are all routine operations, so each method will not be described in detail.

[0041] From the scanning electron microscope images ( Figure 1 a) It can be seen that the prepared Eu-MOF is a uniform rhombus crystal with a size of about 7.3 μm. The N2 adsorption-desorption isotherm of Eu-MOF is a typical type I isotherm, with a large adsorption amount in the P / P0≈0 region ( Figure 1 b), confirming the existence of microporous structure. The surface area and average pore size of the material calculated by BET specific surface area test method are 6.9122m2 / g and 7.8473nm ( Figure 1 b illustration). In addition, the XRD patterns of the synthesized Eu-MOFs are consistent with the simulated XRD patterns in the crystallographic information file (CIF) (CCDC number 222296), proving that the Eu-MOFs were successfully prepared ( Figure 1 c). The zeta potential of the Eu-MOF prepared in the present invention is about -5.3 mV, indicating that its surface has a slight negative charge ( Figure 1 d). This negative charge allows it to interact with positively charged species through electrostatic forces and also helps maintain the dispersion stability of Eu-MOF in an aqueous environment. However, such a weak charge may not be sufficient to prevent it from aggregating over time or under certain conditions (such as changes in pH or ionic strength).

[0042] The present invention also studies the ultraviolet absorption spectrum and fluorescence spectrum characteristics of Eu-MOF. Under 365-nanometer ultraviolet light, Eu-MOF powder emits bright red fluorescence, which is derived from the fluorescence of the central Eu3+ ion ( Figure 1 e inset). When dissolved in DMF solution and excited at 330 nm, Eu-MOF exhibited five emission peaks in the range of 400–700 nm, which are 430, 593, 618, 651, and 696 nm, respectively, representing the characteristic emission peaks of Eu3+ ( Figure 1 e). However, when this material is dispersed in aqueous solution, it exhibits bright blue fluorescence ( Figure 1 f). The results confirmed the solvent-dependent fluorescence behavior of Eu-MOF. In addition, Eu-MOF has a wide absorption wavelength range from 330 nm to about 400 nm, with a maximum absorption wavelength of about 350 nm ( Figure 1 f).

[0043] 1.4 Preparation of R6G / Eu-MOF

[0044] To prepare the R6G / Eu-MOF mixed solution, 1 mg of Eu-MOF powder and 1 mg of R6G were dispersed in 1 mL of ultrapure water, respectively. Then the two solutions were mixed in equal volumes and treated with ultrasound for 1 h to finally obtain the R6G / Eu-MOF suspension.

[0045] Fluorescence behavior of R6G / Eu-MOF:

[0046] The fluorescence behavior of the obtained R6G / Eu-MOF suspension was tested, and the test results are as follows: Figure 2 The main tests were the solution colors of R6G / Eu-MOF and R6G / Eu-MOF suspension under white light and 365nm UV light, the fluorescence spectrum of R6G / Eu-MOF, the effect of different pH values ​​on the fluorescence of R6G / Eu-MOF, and the stability of R6G / Eu-MOF and pure Eu-MOF materials in aqueous solution. The specific results are as follows:

[0047] Under white light irradiation, the R6G / Eu-MOF suspension exhibits an orange-red color ( Figure 2 a Left illustration②), while the Eu-MOF solution is a transparent solution ( Figure 2 a Left illustration ①). When excited by 365nm ultraviolet light, the R6G / Eu-MOF suspension emits bright yellow fluorescence ( Figure 2 a right illustration②). Under the same conditions, the Eu-MOF suspension showed strong blue fluorescence ( Figure 2 a right illustration ①). Figure 2As shown in (a), the fluorescence spectrum of R6G / Eu-MOF shows dual emission characteristics, corresponding to the emission peak of Eu-MOF (430 nm) and the emission peak of R6G (555 nm), respectively.

[0048] In the pH range of 4.5-7.5, the fluorescence intensities of the two emission peaks and their ratio (F 430 / F 555 ) Almost no change( Figure 2 b), which indicates that R6G / Eu-MOF can remain stable in acidic to neutral environments.

[0049] In addition, the present invention investigates the fluorescence stability of the material in aqueous solution, that is, the fluorescence data of the R6G / Eu-MOF suspension and the pure Eu-MOF suspension are tested every 10 days, and the fluorescence intensity curve is recorded and plotted. Figure 2 As shown in Figure c, the fluorescence intensity of Eu-MOF at 430nm wavelength decreased significantly over time, and only 21% of the original intensity remained at 45 days. In contrast, the signal attenuation problem of R6G / Eu-MOF at 430nm wavelength was significantly improved, and its fluorescence intensity still maintained 85% of the original intensity at 45 days. In addition, the intensity ratio of the dual fluorescence signal F430 / F555 of R6G / Eu-MOF remained basically stable over time ( Figure 2 d). This fully demonstrates that with the simple mixing assistance of R6G, the fluorescence stability of Eu-MOF has been significantly improved, and at the same time, the hybrid probe system has been endowed with more stable and diverse fluorescence properties.

[0050] In order to explore the reasons for the enhanced stability of the hybrid probe, the present invention also investigated the zeta potential of the R6G / Eu-MOF hybrid probe system. The results show that the charge of R6G is about 28mV, the surface charge of pure Eu-MOF is about -5.3mV, and when Eu-MOF is mixed with R6G, the zeta potential of R6G / Eu-MOF is converted to a positive charge of 10.3mV. Since R6G is a cationic dye, it can interact with the negatively charged Eu-MOF surface through electrostatic forces. This electrostatic force promotes the adsorption of R6G molecules on the surface of Eu-MOF, which may improve the surface charge characteristics of Eu-MOF and enhance its stability in water. In addition, the R6G molecule has a rigid planar structure, and the ligand 1,10-phenanthroline in Eu-MOF also contains a π electron system. Therefore, the π system of the R6G molecule can interact with the ligand system in Eu-MOF through π-π stacking. This stacking effect can enhance the binding strength between R6G and Eu-MOF, and help improve the structural stability of Eu-MOF. These interaction mechanisms work together to significantly enhance the stability of Eu-MOF and give Eu-MOF enhanced fluorescence properties or improved sensing properties. This is also the reason why the R6G / Eu-MOF hybrid probe system proposed in the present invention is simple and effective.

[0051] 1.5 Effect of Chinese medicine monomers on R6G / Eu-MOF fluorescence

[0052] As we all know, due to the similar structures and properties of TCM monomers, it is difficult to distinguish them using simple spectral methods. Here, the inventors tried to use the dual fluorescence emission R6G / Eu-MOF as a signal probe to explore the possibility of simply distinguishing some TCM monomers.

[0053] The inventors studied the fluorescence response of R6G / Eu-MOFs to different Chinese medicine monomers in Tris-HCl (0.1M, pH 7.4) at room temperature. The specific method is: a certain amount of standard solutions of various Chinese medicine monomers (including flavonoids: orientin, vitexin, isovitexin, baicalin, hesperidin, naringin, scutellaria baicalensis flavonoid II and quercetin; phenylpropanoids: verbascoside; terpenoids: tanshinone IIA, solanesol, oleanolic acid, ursolic acid and paeoniflorin; polyphenols: gallic acid and echinacoside) of equal concentration were added to the corresponding 0.5mL EP tubes, and then R6G / Eu-MOF suspension (10μL) and 180μL Tris-HCl (0.1M, pH 7.4) were added respectively, vortexed to ensure that the solution was evenly mixed, and then transferred to quartz cells respectively, and fluorescence detection was performed at room temperature. The fluorescence experiment was conducted under an excitation wavelength of 330 nm, and the changes in fluorescence signals at the two emission peaks of 430 nm and 555 nm were recorded. Figure 3a). Finally, the fluorescence intensity obtained from the test was used to calculate the fluorescence quenching rate of each monomer on the material at 430nm and 555nm, and a heat map of the fluorescence quenching rate of different monomers at 430nm and 555nm was drawn ( Figure 3 b).

[0054] from Figure 3 It can be seen intuitively that different substances have different effects on the signal intensities of the two emission peaks of MOF. Among them, polyphenols and terpenoids both enhance the fluorescence intensity at 555nm to varying degrees (especially ursolic acid), but have little effect on the fluorescence intensity at 430nm. Among flavonoids, quercetin and orientin both have a significant quenching effect on the fluorescence at 555nm, but the fluorescence quenching effect of quercetin on 430nm is not as obvious as orientin. This shows that among these monomers, orientin has the most obvious effect on the dual fluorescence signal of R6G / Eu-MOF, and there is a significantly differentiated fluorescence quenching effect between the two fluorescence signals. Therefore, these substances can be simply and quickly distinguished according to the degree of signal influence of different drug monomers on the dual fluorescence signal of R6G / Eu-MOF.

[0055] 1.6 Fluorescence quenching behavior of orientin on R6G / Eu-MOF

[0056] 10 μL of control buffer and 75 μM and 150 μM orientin standard solutions were added to the R6G / Eu-MOF suspension and mixed thoroughly to observe the fluorescence quenching behavior of orientin on R6G / Eu-MOF ( Figure 4 a). Under ultraviolet light ( Figure 4 a illustration), the fluorescence intensity of R6G / Eu-MOF can be observed with the naked eye to gradually decrease with the increase of orientin concentration. The fluorescence spectrum ( Figure 4 a) further confirms this.

[0057] The present invention investigates the effect of the reaction time of orientin and R6G / Eu-MOF on the fluorescence intensity ( Figure 4 b) The fluorescence intensity at 430 nm and 555 nm decreased rapidly within 1 minute after the addition of orientin and then remained almost constant, indicating that the quenching process was quite rapid.

[0058] The present invention also explores the possible quenching mechanism of orientin on R6G / Eu-MOF. First, the UV-visible absorption spectra of R6G / Eu-MOF and orientin were studied. Figure 4As shown in Figure c, the absorption wavelength range of orientin at around 350nm highly overlaps with the absorption and excitation wavelength range of R6G / Eu-MOF. This indicates that there is a competitive absorption of light source energy between them, that is, an internal filtering effect. In addition, after adding R6G / Eu-MOF, the absorbance of orientin decreased, indicating that there may be functional group interactions between orientin and R6G / Eu-MOF. In addition, as Figure 4 As shown in Figure d, after adding orientin, the fluorescence lifetime of R6G / Eu-MOF decreased from the original 15.3ns to 13.6ns, indicating that orientin also participated in the dynamic quenching effect. It can be seen that orientin has a significant fluorescence quenching effect on R6G / Eu-MOF, which is the result of the combined action of multiple effects.

[0059] 1.7 Fluorescence determination of orientin

[0060] The material synthesis of the present invention and the determination principle of red cyperus glycoside are Figure 5 The inventors further evaluated the application of R6G / Eu-MOF in the detection of orientin.

[0061] 10 μL of orientin standard solution of different concentrations were added to the corresponding 0.5 mL EP tubes, and then R6G / Eu-MOF suspension (10 μL) and 180 μL Tris-HCl (0.1 M, pH 7.4) were added. Each solution was vortexed to ensure uniform mixing, and then transferred to a quartz cell for fluorescence detection at room temperature. The fluorescence experiment was performed at an excitation wavelength of 330 nm, and the fluorescence emission spectrum in the range of 400 to 600 nm was recorded.

[0062] like Figure 5 As shown in a, with the increase of orientin concentration, the fluorescence intensity of F430 and F555 gradually weakened. In the range of 1.1 μM to 220 μM, 430 nm ( Figure 5 b) and 555nm( Figure 5 The fluorescence intensity at c) shows a good linear relationship with the concentration of orientin. The detection limits of orientin in the invention are calculated to be 0.26 μM at 430 nm and 0.16 μM at 555 nm (3σ / N), respectively, proving that the method can detect orientin sensitively and quickly.

[0063] Since OT has a fluorescence quenching effect on both Eu-MOF and R6G, the present invention also evaluates the ability of Eu-MOF and R6G to detect OT separately. The results show that the fluorescence intensity of Eu-MOF and R6G also shows a linear correlation with the OT concentration. However, as shown in Table 1, when used alone, their sensing performance is not as good as that of the R6G / Eu-MOF hybrid probe system. Compared with using it alone, the R6G / Eu-MOF hybrid system has a wider linear range of detection of OT and higher sensitivity.

[0064] Table 1 Comparison of detection performance parameters of different probes for OT

[0065]

[0066] Detection selectivity of 1.8R6G / Eu-MOF dual-emission fluorescence sensor for orientin

[0067] To verify the selectivity of R6G / Eu-MOF material for orientin sensing, the fluorescence intensity of R6G / Eu-MOF in the presence of some amino acids and common ions was recorded. 2+ , Ca 2+ 、Na 2+ Mg 2+ , K + 、Zn 2+ , Cys, Met, Tyr), and then transferred to a quartz cell to measure the fluorescence spectrum. Figure 6 As shown in the figure, under the same conditions, in the presence of orientin (orientin concentration was 45 μM), the ΔF430 and ΔF555 of R6G / Eu-MOF changed significantly, while other substances had a weak effect on the fluorescence signal of R6G / Eu-MOF.

[0068] In order to investigate the stability and repeatability of this method, the intra-day and inter-day differences in the detection of orientin were further systematically investigated. The results are shown in Table 2.

[0069] Table 2 Stability and repeatability test results of R6G / Eu-MOF for detecting orientin

[0070]

[0071] As can be seen from the table, the intra-day relative standard deviation of the detection method is in the range of 0.7%-6.5%, and the inter-day average relative standard deviation is in the range of 1.2%-2.7%. The results show that the method has good stability and repeatability.

[0072] 1.9 Analytical Application in Human Serum Samples

[0073] In order to evaluate the applicability of this method, we used this method to detect orientin in human serum samples (Table 3). The purchased human serum reagent was diluted 10 times with Tris-HCl (0.1M, pH 7.4), and then orientin of known concentration was added to the diluted serum for fluorescence detection. The detection method was carried out according to 1.5. The results showed that the recovery rate of R6G / Eu-MOF in human serum was 97.5%-107.2%, and the relative standard deviation (RSD) was 1.8%-4.6%. In addition, the test results were basically consistent with the HPLC determination results. It shows that the invention has good precision and accuracy in the determination of orientin content in samples.

[0074] Table 3 Determination results of orientin in human serum samples

[0075]

[0076] 2. Conclusion

[0077] In summary, the dual-emission fluorescent R6G / MOF material proposed in the present invention not only inherits and maintains the excellent optical properties of R6G and Eu-MOF, but also has good structural stability and fluorescence stability in aqueous solution. The experimental results show that R6G / MOF can overcome the influence of pH value and the fluorescence attenuation of the initial Eu-MOF with the extension of storage time. More importantly, R6G / Eu-MOF has potential application value in the rapid analysis and identification of drugs, and successfully realizes the detection of orientin. As far as the inventor knows, this is the first fluorescent sensor for detecting orientin. The rapid fluorescence detection method with self-calibration function in this invention has potential application value in the rapid analysis and identification of small molecule drugs such as orientin, and is expected to provide an effective new method for the rapid detection of related drug components in complex matrices.

Claims

1. A method for preparing a stable R6G / Eu-MOF dual-emission fluorescence sensor, characterized in that: It includes the following steps: (1) Synthesis of Eu-MOF: Europium chloride hexahydrate, 2-aminoterephthalic acid and 1,10-phenanthroline were added to ultrapure water and ultrasonically mixed; then, an aqueous solution of NaOH was added, and the resulting mixture was transferred to a polytetrafluoroethylene-lined hydrothermal reactor and heated at 155-170°C for 12-14 hours; After centrifugation and repeated washing with water and ethanol, it was dried under vacuum at 60-65°C for 5-8 hours to finally obtain yellow-brown powder Eu-MOF; Preparation of R6G / Eu-MOF: Eu-MOF powder and an equal mass of rhodamine 6G (R6G) were dispersed in ultrapure water at a concentration of 1 mg / ml, respectively, and the two dispersed solutions were then mixed in an equal volume ratio and ultrasonically treated for 1 hour to finally obtain a R6G / Eu-MOF suspension, namely the R6G / Eu-MOF dual-emission fluorescence sensor.

2. The preparation method according to claim 1, characterized in that: The specific operation method of step (1) is: 0.511 g of europium chloride hexahydrate, 0.385 g of 2-aminoterephthalic acid and 0.280 g of 1,10-phenanthroline were added to 35 mL of ultrapure water and mixed by ultrasonication; after adding 4.2 mL of 0.65 M NaOH aqueous solution, the resulting mixture was transferred to a polytetrafluoroethylene-lined hydrothermal reactor, heated at 160° C. for 12 hours, centrifuged and repeatedly washed with water and ethanol, and dried at 60° C. under vacuum for 6 hours to finally obtain yellow-brown powder Eu-MOF, wherein the speed of the centrifuge was controlled to 11000 rpm and the centrifugation time was 5 min.

3. The preparation method according to claim 1, characterized in that: The specific operation method of step (2) is: disperse 1 mg Eu-MOF powder and 1 mg Rhodamine 6G in 1 mL ultrapure water respectively, then mix the two solutions in equal volumes, and then treat with ultrasound for 1 hour to finally obtain a R6G / Eu-MOF suspension, namely the R6G / Eu-MOF dual-emission fluorescence sensor.

4. A stable R6G / Eu-MOF dual-emission fluorescence sensor prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the R6G / Eu-MOF dual-emission fluorescence sensor as claimed in claim 4 in the detection of orientin.

6. A method for detecting orientin using the R6G / Eu-MOF dual-emission fluorescence sensor according to claim 4, characterized in that: The method comprises the following steps: adding R6G / Eu-MOF suspension and orientin standard solution or test solution into an EP tube, then adding 0.1 mol / L Tris-HCl solution with a pH of 7.4, vortexing the solution to ensure uniform mixing, and then transferring the solution into a quartz cell to perform fluorescence detection at room temperature. The fluorescence experiment records the fluorescence emission spectrum within the range of 400 to 600 nm at an excitation wavelength of 330 nm.

7. The method according to claim 6, characterized in that: The specific steps are: (1) Draw the orientin response standard curve and obtain the linear regression equation: 10 μL of R6G / Eu-MOF suspension and 10 μL of orientin standard solution of different concentrations were added to 0.5 mL EP tubes, and then 180 μL of 0.1 mol / L Tris-HCl solution with a pH of 7.4 was added; the solution was vortexed to ensure uniform mixing, and then transferred to a quartz cell for fluorescence detection at room temperature; the fluorescence experiment was performed at an excitation wavelength of 330 nm, and the fluorescence emission spectrum in the range of 400 to 600 nm was recorded; based on the quenching amount of the sample on the fluorescence emission signal of R6G / Eu-MOF at 555 nm and 430 nm, a standard curve of fluorescence quenching amount and orientin concentration was obtained; based on the obtained standard curve, a linear regression equation of relative fluorescence quenching amount and orientin concentration was obtained; (2) Detection: 10 μL of R6G / Eu-MOF suspension and 10 μL of the solution to be tested were added to 0.5 mL EP tubes respectively, and then 180 μL of 0.1 mol / L Tris-HCl solution with a pH of 7.4 was added; the solution was vortexed to ensure uniform mixing, and then transferred to a quartz cell for fluorescence detection at room temperature; at an excitation wavelength of 330 nm, the quenching amount of the solution fluorescence signal at 555 nm and 430 nm was recorded; the fluorescence quenching amount was substituted into the linear regression equation obtained in step (1), and the orientin concentration of the solution to be tested was calculated.

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