Europium functionalized metal-organic framework-based dual-emission fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a europium-functionalized metal-organic framework-based dual-emission fluorescent probe and a preparation method and application thereof, and relates to the field of organic metal frameworks and fluorescence detection.The preparation method comprises the steps that ZrCl4, pyromellitic acid, 6-hydroxy-2-naphthoic acid and water are mixed, acetic acid is added, and a mixture is obtained; heating and refluxing the mixture, centrifuging to obtain residues, washing and drying to obtain UCHNA; the preparation method comprises the following steps: dispersing UCHNA in an aqueous solution of Eu (NO3) 3.6 H2O, heating to react, collecting white precipitate, washing and drying to obtain the Eu (NO3) 3.6 H2O. The invention has the beneficial effects that the UiO-66 type MOF is modified by using the luminous ligand 6-hydroxy-2-naphthoic acid, post-modification is performed by using Eu < 3 + >, the Eu < 3 + >-MOF with dual emission of blue and red wavebands is reasonably designed and synthesized, crystal violet (CV) and quinine (QN) are selectively and quickly responded through different response modes, the detection limit is low, and the Eu < 3 + >-MOF is superior to most reported sensors.

Description

Technical Field

[0001] The present invention relates to the field of metal organic frameworks and fluorescence detection, and in particular to a europium-functionalized metal-organic framework dual-emission fluorescence probe and a preparation method and application thereof. Background Art

[0002] As large-scale, intensive aquaculture continues to expand, frequent outbreaks of fungal, bacterial, and parasitic infections have highlighted the need for effective disease management. In this regard, crystal violet (CV) and quinine (QN) are widely used to protect the health of fish and shrimp, especially to treat and prevent these infections, providing an essential solution in an environment where disease outbreaks can cause significant economic losses. CV is a synthetic triphenylmethane dye that has been widely used to prevent and treat diseases in aquaculture due to its effective anthelmintic, fungicide, and bactericide effects. However, CV has high toxicity and may cause mutagenic and carcinogenic effects, and therefore, many countries have banned its use in aquaculture. QN is a cinchona alkaloid known as an important antimalarial drug, commonly used to prevent and treat malaria in fish, or as a preservative in aquatic products to extend their shelf life. Unfortunately, QN has high neurotoxic and cardiovascular toxicity. Excessive use of QN can lead to carcinomatosis, blurred vision, and even neurological and cardiovascular damage. Therefore, it is essential to monitor CV and QN values ​​in fish meat and aquaculture water.

[0003] In the past, several analytical techniques have been used to determine CV and QN, including high performance liquid chromatography, UV-visible spectrophotometry, flow injection chemiluminescence, surface enhanced Raman scattering, and electrochemical analysis. Nevertheless, these techniques have some problems, such as requiring expensive instruments or professional technicians, or insufficient sensitivity, or involving complex pretreatment procedures. At the same time, due to the complex contents in real samples, the realization of multi-target detection in one system is in great demand, but also a major challenge. So far, there has been no report on the simultaneous detection of CV and QN. Therefore, it is necessary to develop a simple, sensitive, rapid, and low-cost method for the simultaneous detection of CV and QN in aquatic products.

[0004] Fluorescence analysis is a good choice because of its simplicity, intuitiveness, and rapidity. In addition, multi-emission materials can not only reduce environmental interference through self-reference, but also achieve multi-target detection through different response modes, so they have attracted widespread attention. Lanthanide metal-organic frameworks (Ln-MOFs) combine the modifiability and porosity of MOFs with Ln 3+ The unique fluorescence properties can be combined with the luminescent ligand, Ln 3+Multiple emission fluorescence sensors can be easily designed and constructed for the detection of target substances. Many multi-emission Ln-MOFs have been synthesized and developed as fluorescence sensors for biomarkers, explosives, food or pollutants in the environment. However, there are certain limitations for the detection of multiple targets with a single fluorescence sensor. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide a dual-emission fluorescent probe based on a europium functionalized metal-organic framework and a preparation method and application thereof.

[0006] The present invention uses luminescent ligand 6-hydroxy-2-naphthoic acid to pre-modify UiO-66 type MOF, and uses Eu 3+ Post-modification (e.g. Fig.16 a), rationally designed and synthesized Eu with dual emission in blue and red bands 3+ -MOF (EuUCHNA). The fluorescence spectrum shows that the fluorescence spectrum of EuUCHNA exhibits different response patterns to CV and QN (such as Fig.16 b). QN effectively quenched both emission bands of EuUCHNA, while QN significantly enhanced the blue emission band of EuUCHNA and slightly weakened the red emission band of EuUCHNA. By combining visible fluorescence color changes with smartphones, instrument-free monitoring of CV and QN can be achieved in real samples. In addition, different response modes enable simultaneous quantification of CV and QN. This work will promote the development of fluorescent sensors for multi-target detection.

[0007] The technical solution of the present invention is as follows: The first aspect of the present invention provides a method for preparing a dual-emission fluorescent probe based on a europium functionalized metal-organic framework, comprising the following steps: ZrCl 4 , pyromellitic acid, 6-hydroxy-2-naphthoic acid and water are mixed, and acetic acid is added to obtain a mixture; the mixture is heated to reflux, centrifuged to obtain a residue, and the residue is washed and dried to obtain UCHNA; The UCHNA is dispersed in Eu(NO 3 ) 3 6H 2 O aqueous solution, heated to react, and the white precipitate was collected, washed, and dried to obtain a dual-emission fluorescent probe based on a europium-functionalized metal-organic framework.

[0008] Optionally, the ZrCl 4 The molar ratio of 1,2-dimethylbenzene tetracarboxylic acid and 6-hydroxy-2-naphthoic acid is 1.5~2:2.5~3.5:0.2~0.5.

[0009] Optionally, the ZrCl 4 The usage ratio of water and acetic acid is 1.5mol~2mol:15mL~25mL:4mL~6mL.

[0010] Optionally, the heating reflux temperature is 80° C. to 120° C., and the heating reflux time is 20 h to 28 h.

[0011] Optionally, the UCHNA and Eu(NO 3 ) 3 6H 2 The mass ratio of O is 0.4~0.6:0.8~1.2.

[0012] Optionally, the temperature of the heating reaction is 70° C. to 90° C., and the time of the heating reaction is 20 h to 28 h.

[0013] The second aspect of the present invention provides a dual-emission fluorescent probe based on a europium-functionalized metal-organic framework, which is obtained by the above-mentioned preparation method.

[0014] The third aspect of the present invention provides a dual-emission fluorescent probe based on europium functionalized metal-organic framework obtained by the above-mentioned preparation method or the application of the above-mentioned dual-emission fluorescent probe based on europium functionalized metal-organic framework in the simultaneous detection of crystal violet and quinine in water or aquatic products.

[0015] A fourth aspect of the present invention provides a fluorescence sensor, comprising the europium-functionalized metal-organic framework dual-emission fluorescence probe obtained by the above preparation method or the europium-functionalized metal-organic framework dual-emission fluorescence probe.

[0016] A fifth aspect of the present invention provides a method for simultaneously detecting crystal violet and quinine in water or aquatic products, comprising the europium-functionalized metal-organic framework dual-emission fluorescent probe obtained by the above-mentioned preparation method or the above-mentioned europium-functionalized metal-organic framework dual-emission fluorescent probe for detection, comprising the following steps: Add the meat of seafood to CH 3 In a mixture of CN and PBS, ultrasonic treatment is performed, centrifugation is performed, and a supernatant is collected; the supernatant is filtered through a filter membrane to obtain a pretreated sample; or the collected aquaculture water sample is centrifuged and filtered to obtain pretreated water; Adding EuUCHNA into water to prepare EuUCHNA water suspension; The pretreated sample or the pretreated water is added to the EuUCHNA water suspension, and the fluorescence spectrum is measured.

[0017] The present invention has at least one of the following beneficial effects: A dual-emission sensor (EuUCHNA) was developed to selectively and rapidly respond to crystal violet (CV) and quinine (QN), two commonly used anti-infective drugs for the treatment of fungi, bacteria, and parasites, through different response modes. The detection limits of CV and QN were 2.4 nM and 32.0 nM, respectively, which are better than most reported sensors. The determination of CV and QN can be used for aquatic products such as fish meat and aquaculture water such as fish pond water. In addition, EuUCHNA can simultaneously quantify CV and QN in actual fish pond water. The present invention provides a promising fluorescent sensor and a new analytical method for the simultaneous quantification of two targets in real samples using a single sensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Characterization diagrams of UCHNA and EuUCHNA synthesized in Example 1, including: (a) PXRD spectra of UiO-66 (simulation), UCHNA, EuUCHNA, and EuUCHNA after immersion in water for 10 days; (b) FT-IR spectra of UCHNA and EuUCHNA; (c) XPS spectra of UCHNA and EuUCHNA.

[0019] Figure 2 These are the TGA curves of UCHNA and EuUCHNA synthesized in Example 1.

[0020] Figure 3 The fluorescence spectrum of UCHNA synthesized in Example 1, where (a) the fluorescence spectrum of UCHNA solid; (b) the fluorescence spectrum of UCHNA liquid Figure 4 The fluorescence spectra of EuUCHNA synthesized in Example 1, including (a) excitation and emission spectra in the solid state; (b) aqueous suspension. (c) fluorescence spectra of EuUCHNA in aqueous solution for 7 days; (d) the effect of pH on the fluorescence spectrum of EuUCHNA; (e) and (f) fluorescence spectra of different concentrations of EuUCHNA (0.1-1.5 mg / ml).

[0021] Figure 5 The fluorescence spectra of EuUCHNA synthesized in Example 1, including: (a) fluorescence images of EuUCHNA in the presence of different substances; (b) fluorescence images of EuUCHNA and different substances in the presence of CV; (c) fluorescence images of EuUCHNA and different substances in the presence of QN; (d) fluorescence spectrum comparison of EuUCHNA, CV and different substances; (e) fluorescence spectrum comparison of EuUCHNA, QN and different substances; (f) fluorescence spectrum of EuUCHNA and different substances in the presence of CV; (g) fluorescence spectrum of EuUCHNA and different substances in the presence of QN.

[0022] Figure 6 : The fluorescence spectrum of EuUCHNA synthesized in Example 1, including: (a) fluorescence spectrum of EuUCHNA at CV (50 μM); (b) QN (40 μM).

[0023] Figure 7 Shown are (a) the fluorescence color of EuUCHNA at different crystal violet concentrations; (b) the fluorescence color of EuUCHNA at different quinine concentrations; (c) the fluorescence spectrum of EuUCHNA under crystal violet (0 ~ 50 μM); (d) I 0 / I 613 Linear relationship with crystal violet concentration; (e) I at 450 nm 0 / I 450 Linear relationship with CV concentration; (f) Fluorescence spectrum of EuUCHNA under quinine (0 ~ 40 μM); (g) I 450 / I 0 Linear relationship with quinine concentration; (h) I 450 / I 613 Linear relationship with quinine concentration; (i) Time-resolved fluorescence response of EuUCHNA (0.25 mg / mL) to crystal violet (15 μM); (j) Time-resolved fluorescence response of EuUCHNA (0.25 mg / mL) to quinine (40 μM).

[0024] Figure 8 QN@EuUCHNA and CV@EuUCHNA in FIG. 1 are the CIE chromaticity changes of EuUCHNA synthesized in Example 1 when quinine (40 μM) and crystal violet (50 μM) are added respectively.

[0025] Fig. 9 (a) The fish sample at 613 nm I 0 / I 613 Linearity between CV concentration and (b) fish samples I 450 / I 613 The linearity between the concentration of QN and the

[0026] Fig.10 Shown are (a) verification of the additivity of CV and QN; (b) CV concentration of 2 μM; (c) CV concentration of 4 μM; (d) CV concentration of 6 μM.

[0027] Fig.11Shown are the effects of different concentrations of QN (2, 4, 6 μM) on the fluorescence spectrum of EuUCHNA in the presence of (a) CV (2 μM); (c) CV (4 μM); (e) CV (6 μM); (b) CV (2 μM); (b) Fluorescence intensity of EuUCHNA at 613 nm with different concentrations of QN (2, 4, 6 μM) in the presence of (d) CV (4 μM); (f) CV (6 μM) Fig.12 Shown are the fluorescence spectra of EuUCHNA at different concentrations of (a) CV (2, 4, 6 μM); (b) QN (4, 8, 12 μM).

[0028] Fig.13 Shown are (a) PXRD spectra of CV@EuUCHNA and QN@EuUCHNA; (b) XPS spectra of CV, QN, CV@EuUCHNA and QN@EuUCHNA; (c) UV-visible spectrum of CV, excitation and emission spectra of EuUCHNA.

[0029] Fig.14 Shown are (a) the fluorescence lifetime of EuUCHNA, CV@EuUCHNA and QN@EuUCHNA at 450 nm; (b) the fluorescence lifetime of EuUCHNA, CV@EuUCHNA and QN@EuUCHNA at 613 nm.

[0030] Fig.15 Shown are (a) liquid color recognition process diagram; (b) fluorescence color and RGB value of EuUCHNA containing CV or QN; (c) linear relationship between B / R and CV concentration; (d) linear relationship between B / R and QN concentration.

[0031] Fig.16 Shown are (a) the synthesis route of EuUCHNA; (b) schematic diagram of the different response modes of EuUCHNA to CV and QN. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] ZrCl 4, 6-hydroxy-2-naphthoic acid (6-HNA), acetic acid, 5-fluorocytosine (5-FC), gentamicin sulfate (USP), amikacin sulfate (AS), and kanamycin sulfate (KAM) were obtained from Macklin Reagent (China). Phthalic acid and Eu(NO3)3·6H2O were provided by Shanghai Xushuo Biotechnology Co., Ltd. Quinine (QN) was purchased from Leyan Reagent Company, China. Sulfacetamide (SA), DMF, neomycin sulfate (NMA), sulfaguanidine (SG), florfenicol (FF), and thiamine (TAP) were purchased from Aladdin Reagent. L-phenylalanine (Phe), L-glycine (Gly), L-alanine (Ala), L-glutamic acid (Glu), L-leucine (Leu), L-threonine (Thr), and L-aspartic acid (Asp) were all from Shanghai Lanji Biotechnology Co., Ltd. CaCl 2 MgCl 2 、Zn(NO 3 ) 2 , KCl, K 2 CO 3 , K 2 SO 4 AgNO 3 All are from Tianjin Fuchen Chemical Reagent.

[0034] Powder X-ray diffraction (PXRD) was performed on a Bruker-D8 Advanced using CuKα radiation. X-ray photoelectron spectroscopy (XPS) results were obtained from an ESCALAB 250Xi. The spectral range from 4000 to 400 cm was recorded using a Nicolet iS10 from Thermo Fisher Scientific. -1 Fourier transform infrared spectroscopy results of KBr particles in the range. Fluorescence spectra were recorded on a HORIBA FluoroMax-4. UV-visible absorption spectroscopy data were collected on an INESA N4S spectrophotometer.

[0035] Example 1 The preparation method of the europium-functionalized metal-organic framework dual-emission fluorescent probe comprises the following steps: Step 1: UCHNA synthesis ZrCl 4 (0.47 g, 2.0 mmol), pyromellitic acid(H 4betc, 0.78 g, 3.05 mmol), 6-hydroxy-2-naphthoic acid (6-HNA, 0.06 g, 0.34 mmol) were mixed with 20 mL of water and 5 mL of acetic acid was added. The mixture was then refluxed at 100 °C for 24 h, centrifuged to obtain the residue, which was then repeatedly washed with water and methanol. Finally, 0.74 g of white powder UCHNA was obtained by vacuum drying at 80 °C.

[0036] Step 2: EuUCHNA synthesis 0.50 g UCHNA was dispersed in a solution containing Eu(NO 3 ) 3 6H 2 O (1.00 g) in 40 mL of distilled water was stirred at 80 °C for 24 h, and the white precipitate was collected after cooling and washed alternately with water and methanol for 3 times. The solid product was dried at 80 °C for 8 h to obtain 0.50 g of white powder EuUCHNA.

[0037] The synthesized EuUCHNA was characterized and the results are as follows: The structure of the synthesized MOF was analyzed by powder X-ray diffraction (PXRD). Figure 1 As shown in a, the PXRD peaks of UCHNA and EuUCHNA are mainly distributed near 7.5°, 8.6°, 12.2°, 14.4°, 17.3°, 25.9° and 30.0°. They are consistent with the simulated PXRD spectrum of UiO-66, indicating that the synthesized MOF material has the same basic framework structure as UiO-66. In addition, after EuUCHNA was immersed in water for 10 days, its PXRD spectrum remained well, indicating that its structure was highly stable in water. FT-IR spectrum is shown in Figure 1 As shown in b, UCHNA and EuUCHNA have a peak at 1574 cm -1 and 1426 cm -1 The asymmetric stretching vibration and symmetric stretching vibration of the carboxyl group were observed nearby. It is worth noting that the 1715 cm -1 The vibration becomes weaker in EuUCHNA, which indicates that most of the free carboxyl groups in EuUCHNA are bound to Eu 3+ X-ray photoelectron spectroscopy (XPS) analysis further confirmed that Eu 3+ Coordination bonds with −COO− groups (Table 6).

[0038] like Figure 1As shown in c, compared with UCHNA, EuUCHNA has a new peak at 1125.01 eV, which is attributed to the three-dimensional binding energy of Eu. The binding energy of O1s shifts from 531.70 eV of UCHNA to 531.65 eV of EuUCHNA. 3+ The amount of EuUCHNA doped in the EuUCHNA was calculated based on the ICP-MS results. 3+ With Zr 4+ The molar ratio of is 1:4.7. Thermogravimetric analysis (TGA) shows that the synthesized MOF is thermally stable at a temperature of about 300 °C. Before 300 °C, the weight loss rate of the synthesized MOF is about 24%, which is related to the loss of the incorporated solvent molecules ( Figure 2 ).

[0039] Example 2 The EuUCHNA obtained in Example 1 was subjected to fluorescence sensing experiments.

[0040] This example studies the fluorescence response of EuUCHNA to some common fishery drugs and some potential species in fish meat and fish pond water. The substances studied include: sulfadiazine (SG), florfenicol (FF), thiamin (TAP), sulfacetamide (SA), neomycin sulfate (NMS), 5-fluorocytosine (5-FC), gentamicin (USP), amikacin sulfate (AS), kanamycin sulfate (KAM), L-phenylalanine (Phe), L-glycine (Gly), L-alanine (Ala), L-glutamic acid (Glu), L-leucine (Leu), L-threonine (Thr), L-aspartic acid (Asp) CaCl 2 MgCl 2 、Zn(NO 3 ) 2 , KCl, K 2 CO 3 , K 2 SO 4 AgNO 3 In general, 12.5 mg EuUCHNA in 50 mL H 2 O for 20 min to form a homogeneous suspension (0.25 mg / mL). Then, 20 μL of each analyte (0.01 mol∙L -1 ) was added to more than 2 mL of EuUCHNA aqueous suspension for fluorescence analysis.

[0041] The results of the fluorescence sensing experiment are as follows: 1. Fluorescence characteristics: For UCHNA, violet-blue emission was observed in the solid state, while bright blue emission was observed by naked eyes in aqueous suspension under UV light ( Figure 3 a and Figure 3 b). Its fluorescence spectrum shows a weak broadband in the solid state with a maximum emission wavelength of 400 nm, and a strong emission band at 450 nm in the aqueous suspension, which is related to the π-π* transition of the 6-HNA ligand. For EuUCHNA, bright red and magenta emissions can be observed by the naked eye in solid and aqueous media ( Figure 4 a and Figure 4 b), which indicates that Eu 3+ It was successfully functionalized onto UCHNA. Its fluorescence spectrum observed the emission band of 6-HNA ligand and Eu in both solid and aqueous suspension. 3+ Characteristic emission bands of ions ( Figure 4 a and 4b). The dominant peak is located at 613 nm, corresponding to Eu 3+ Ionic 5 D 0 → 7 F 2 transition, indicating that the ligand and Eu 3+ There is a strong antenna effect between them.

[0042] It is worth noting that 6-HNA ligand and Eu 3+ The emission of the ions is very stable in water medium, and after immersion in water for 7 days, there is no significant change in the fluorescence intensity at 450 and 613 nm ( Figure 4 c). In addition, pH-dependent fluorescence studies showed that both 450 nm and 613 nm emissions were highly stable in the pH range of 4-8, indicating a wide range of practical applications ( Figure 4 d).

[0043] 2. Sensing performance: First, the concentration of EuUCHNA was optimized by recording its fluorescence spectrum. Figure 4 e and 4f, when the EuUCHNA concentration was 0.25 mg mL -1 When the fluorescence intensity at 450 and 613 nm was relatively strong, the following sensing experiment used 0.25 mg mL -1 To study the fluorescence response of EuUCHNA, 20 μL (0.01 mol·L -1) were added to 2 ml of EuUCHNA aqueous suspension. Under UV light, we can clearly see that most reagents have negligible effects on the fluorescence emission of EuUCHNA, while after adding CV, the fluorescence of EuUCHNA is significantly quenched. After adding QN, the emission color changes from magenta to bright blue, which is visible to the naked eye ( Figure 5 a. Figure 5 d. Figure 5 e). Therefore, after adding CV, the fluorescence intensity at 450 nm and 613 nm decreased significantly; after adding QN, the fluorescence intensity at 450 nm increased significantly, and the change at 613 nm was negligible; the fluorescence spectrum did not change significantly with the addition of other substances ( Figure 6 ). These results indicate that the fluorescence of EuUCHNA responds selectively to CV and QN. The anti-interference ability of potential interfering factors such as common veterinary drugs, metal ions and amino acids on the detection of CV and QN by EuUCHNA was studied (CV: 20 μM; QN: 100 μM; potential interfering substances: 200 μM). CV and QN were added to the mixture of EuUCHNA and interferon, respectively, and the fluorescence spectra were measured. In the presence of interference, CV can effectively quench the fluorescence at 450 nm and 613 nm ( Figure 5 f), QN can significantly enhance the fluorescence at 450 nm ( Figure 5 g), indicating that EuUCHNA exhibits good anti-interference ability in the fluorescence responses to both CV and QN.

[0044] Example 3 The EuUCHNA obtained in Example 1 was subjected to quantitative determination of CV and QN as follows: Generally speaking, first in H 2 Standard high concentrations (1 mM and 10 mM) of CV were prepared in 4% CO, and QN was prepared in DMF. Then, different volumes of high concentrations of CV and QN were added to 1980 μL EuUCHNA (0.25 mg∙mL -1 ) water suspension, and then add H 2O and DMF to adjust the total volume to 2000 μL. 0.2, 0.5, 1.0, 1.5 μL of 1 mM CV solution and 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 μL of 10 mM CV solution were added to EuUCHNA, and 0.2, 0.5, 1.0, 1.5, 1.5, 2.0, 3.0, 1.0, 1.5 μL of 1 mM QN solution and 0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 μL of 10 mM QN solution were added to EuUCHNA, respectively. The final concentrations of CV were 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, 10.0, 12.5, and 15 μM, and the final concentrations of QN were 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 μM.

[0045] The results are as follows: In order to quantitatively determine CV and QN, CV and QN standard solutions were added respectively, and the fluorescence spectrum of EuUCHNA was recorded. As the concentration of CV increased, the fluorescence color gradually darkened ( Figure 7 a), 450 nm ( I 450 ) and 613 nm ( I 613 ) gradually decreases. Obviously, I 613 The quenching efficiency is much greater than I 450 ( Figure 7 c).

[0046] In the range of 0.1 ~ 15 μM, I 450 and I 613 It has a good linear relationship with CV concentration. The linear equation can be expressed as ( I 0 / I ) 613 = 0.23 C CV + 0.9701 ( R 2 = 0.9985, formula (1)) and ( I 0 / I ) 450 = 0.0329 C CV+0.9866 ( R 2 = 0.9975, formula (2)) ( C CV The concentration of CV is expressed as μM. The detection limits (LOD) were calculated to be 2.4 nM and 61.1 nM, respectively, based on a signal-to-noise ratio of 3. As the concentration of QN increases, the fluorescence color gradually changes from magenta to bright blue ( Figure 7 b). When the QN concentration reached 40 μM, the fluorescence intensity at 613 nm was only reduced by about 13.8%, while the fluorescence intensity at 450 nm was enhanced by about 10.4 times. Interestingly, in the range of 0.1 ~ 40 μM, I 450 There are two good linear relationships with QN concentration. I 0 / I ) 450 = 1.0698 C QN + 1.0493 ( R 2 = 0.9982, formula (3)) is applicable to lower QN concentrations (0.1 ~ 2.5 μM), ( I 0 / I ) 450 = 0.3372 C QN + 3.3737 ( R 2 = 0.9948, formula (4)) is applicable to high QN concentrations (2.5 ~ 40.0 μM). The LOD value of QN was calculated to be 32.0 nM. In addition, due to I 450 and I 613 showed significantly different sensitivities, so the intensity ratio ( I 450 / I 613 ) is used as a ratiometric signal to quantify QN. For lower QN concentrations (0.1 ~ 1.0 μM), the equation is I 450 / I 613 = 0.244 C QN + 0.2096 ( R 2 = 0.9966, formula (5)); for high QN concentration (1.0 ~ 40.0 μM), the equation is I 450 / I 613 = 0.0996 C QN + 0.4545 ( R 2 = 0.9981, formula (6). The corresponding LOD value was calculated to be 76.2 nM. The LOD values ​​of CV and QN are better than most reported fluorescent sensors (Table 1, Table 2). The low LOD values ​​of CV and QN allow the detection of trace levels of CV and QN in real samples.

[0047] like Figure 7 i and Figure 7 As shown in j, time-resolved studies have shown that I 450 and I 613 The fluorescence intensity of I 450 It can be enhanced by QN and reach stability within 1 min, indicating the rapid response of EuUCHNA to CV and QN.

[0048] Table 1. Changes in detection CVs of this example and reported fluorescence detection methods The references cited are as follows: [1]Y. Bai, M. Liu, Y. He, G. Song, 2023. Portable smartphone platformbased on Ti 3 C 2 MQDs / CDs assembly for ratiometric fluorescence quantitative monitoring of crystal violet. Anal Methods 15, 5510-5517. [2]D. Zhao, X. Liu, Z. Zhang, R. Zhang, L. Liao, X. Xiao, et al., 2019. Synthesis of Multicolor Carbon Dots Based on Solvent Control and Its Application in the Detection of Crystal Violet. Nanomaterials 9, 1556. [3]J. Zhou, M. Qing, Y. Ling, L. Wang, NB Li, HQ Luo, 2021. Double-stranded DNA nanobridge enhanced fluorescence of crystal violet / G-quadruplex complex for detection of lead ions and crystal violet. SensActuators B chem 340, 129968. [4]K. Yi, 2017. A novel method for the quantitative determination ofcrystal violet utilizing YVO 4 : Eu 3+ nanoparticles. Chem Lett 46, 520-523. [5]Y. Hu, Z. Gao, 2020. Yellow emissive Se, N-codoped carbon dotstoward sensitive fluorescence assay of crystal violet. J Hazard Mater 388,122073. [6]Y. Han, Y. Chen, J. Liu, X. Niu, Y. Ma, S. Ma, et al., 2018. Room-temperature synthesis of yellow-emitting fluorescent silicon nanoparticles for sensitive and selective determination of crystal violet in fish tissues.Sens. Actuators B chem 263, 508-516. Table 2. Changes in QN detected by this example and reported fluorescence detection methods The references cited are as follows: [7]VC Tsaftari, M. Tarara, PDTzanavaras, GZ Tsogas, 2023. Anovel equipment-free paper-based fluorometric method for the analytical determination of quinine in soft drink samples. Sensors 23, 5153. [8]MV Gorbunova, TA Terentev, VV Apyari, SG Dmitrienko, YAZolotov, 2023. Monitor calibrator as an alternative to spectrofluorimeter:determination of quinine in beverages and medicinal preparations. J Anal Chem78, 337-343. [9]TY Liu, XL Qu, B. Yan, 2020. A sensitive metal-organicframework nanosensor with cation-introduced chirality for enantioselectiverecognition and determination of quinine and quinidine in human urine. JMater Chem 8, 14579-14586. Example 4 The EuUCHNA obtained in Example 1 was used to detect CV and QN in real samples. The specific method is as follows: 1. Detection of CV and QN in aquaculture water The aquaculture water was obtained from Yezhi Lake in Wuhan City, Hubei Province. It was centrifuged at 9000 rpm for 10 min and then filtered through a 0.22µm water membrane to obtain pretreated water.

[0049] When measuring CV, the above filtrate was used to prepare standard high concentration CV (1 and 10 mM), and 0.6 μL of 1 mM CV, 0.6 μL and 2.6 μL of 10 mM CV were added to 1980 μL of EuUCHNA aqueous suspension (0.25 mg / mL), and pretreated water was added to the mixture to adjust the total amount to 2000 μL. The final concentrations of CV were 0.3, 3.0 and 13.0 μM, respectively, for recovery test. The fluorescence spectrum of each sample was measured under the conditions of excitation wavelength of 312 nm and slit of 3.3 / 3.3 nm.

[0050] 20 μL of pretreated water was added to 1980 μL of EuUCHNA aqueous suspension (0.25 mg / mL), and 0.6 μL of 1 mM QN, ​​0.6, 2.6, and 4.6 μL of 10 mM QN (DMF solution) were added to the above mixture to determine QN. The final concentrations of QN were 0.3, 3.0, 13.0, and 23.0 μM, respectively, for recovery experiments.

[0051] 2. Simultaneous determination of CV and QN in aquatic water Grass carp was purchased from a local supermarket and the back muscles of the grass carp were minced. 8.00 g of fish meat was weighed and 4 mL of CH 3 CN and 20 mL PBS (pH = 6.86). The mixture was sonicated for 10 minutes, centrifuged at 9000 r / min for 10 minutes, and the supernatant was collected. The obtained supernatant was filtered with a 0.45 μm mixed filter to obtain the pretreated sample. Generally, 20 μL of the pretreated sample was added to 1980 μL of EuUCHNA (0.25 mg / mL) aqueous suspension, and different volumes of standard concentrations of CV (H 2 The fluorescence spectra of CV and QN were measured in 1% ethanol (1, 10 mM in DMF) and 1% ethanol (1, 10 mM in DMF). The final concentrations of CV and QN were 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0 and 15.0 μM, respectively, and the final concentrations of QN were 0.1, 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 30.0 and 40.0 μM, respectively, showing a linear relationship. In the recovery test, the final concentrations of CV and QN were 0.3, 3.0 and 13.0 μM, respectively, and the final concentrations of QN were 3.0, 13.0 and 23.0 μM, respectively.

[0052] The results are as follows: First, fish pond water and fish meat were used as research objects. The feasibility of the established method for determining CV and QN was explored through standard addition and recovery tests. The linear relationship between the fluorescence intensity and concentration of CV and QN is shown in Fig. 9The recovery results are shown in Table 3, from which it can be clearly seen that the recovery of CV and QN is in the range of 90-112.33%, and the RSD value meets (<5%). The results show that EuUCHNA can be used to determine the content of CV and QN in actual samples.

[0053] Table 3. Recovery results of actual fish pond water and fish samples for CV and QN detection.

[0054] 2. Simultaneous quantification of CV and QN Since QN has a I 613 The influence of is negligible, and we believe that simultaneous quantification of CV and QN can be achieved. First, in order to study the effect of QN in the CV detection system I 613 To investigate the effect of QN on the fluorescence of EuUCHNA, different concentrations of QN (4, 8, 12 μM) were added to the EuUCHNA suspension containing the standard CV concentration (2, 4, 6 μM), and the fluorescence spectra of different concentrations of QN (4, 8, 12 μM) were measured. Fig.10 a and Fig.11 As shown, after adding QN I 613 There is no obvious change, indicating that QN does not affect the CV detection system I 613 .

[0055] In order to investigate whether the quenching effect of CV and the enhancement effect of QN are independent of each other, firstly, some standard concentrations of CV (2, 4, 6 μM) and QN (4, 8, 12 μM) were added, and the fluorescence spectra of EuUCHNA were measured ( Fig.12 ). The fluorescence intensity at 450 nm (I 450, cal ), can be I 450，cal = I 450,QN - ( I 450,0 - I 450，CV ) formula (Formula (7)). I 450,0 , I 450,QN , I 450,CV The fluorescence intensities of EuUCHNA and EuUCHNA after adding CV and QN at 450 nm are respectively represented. Then, the fluorescence spectra of the mixtures of CV and QN with the same concentrations to EuUCHNA are recorded, and the fluorescence intensity of each mixture at 450 nm is represented asI 450，exp .like Fig.10 b, 10c, 10d, I 450，cal Value and I 450，exp The values ​​are consistent, verifying that the enhancement of QN and the quenching of CV are indeed independent of each other.

[0056] Therefore, in the case of a mixture of CV and QN, CV can be used first. I 613 The concentration of CV is determined by formula (1), and then we can calculate the fluorescence intensity at 450 nm by formula (2) I 450，CV ). I 450、CV It can be used as the starting point of the QN enhancement process. Then the concentration of QN can be calculated by formula (4).

[0057] To verify the above simultaneous quantitative method, a series of 9 mixed standard concentrations of CV and QN were added to the EuUCHNA suspension, and the fluorescence spectra were recorded. As shown in Table 4, the measured CV and QN concentrations were consistent with their spiked amounts, indicating that the proposed calculation method is reliable for the simultaneous quantification of CV and QN. The established method was applied to river water for the simultaneous quantitative determination of CV and QN. The recoveries of CV and QN were between 92.50 and 108.67%, and the RSD values ​​were acceptable (Table 5).

[0058] Table 4 Analysis results of CV and QN in its mixture.

[0059] Table 5 Results of simultaneous detection of CV and QN in fish pond water.

[0060] 3. Fluorescence response mechanism The fluorescence response mechanism of EuUCHNA was explored by PXRD spectrum, UV-vis spectrum and XPS analysis. The PXRD spectrum of EuUCHNA after CV and QN induction was consistent with the original spectrum, indicating that the fluorescence response was not related to the collapse of the structural framework ( Fig.13a). XPS data showed that the binding energy of Eu 3d in EuUCHNA shifted from 1125.01 and 1125.2 eV to 1125.25 eV after the detection of CV and QN, respectively. In addition, the binding energy of N1s also changed significantly after the incorporation of EuUCHNA sensor compared with the original CV and QN (Table 6). These results indicate that there is a weak interaction between the EuUCHNA sensor and the N atoms of CV or QN. The UV-visible absorption spectrum of CV has a large overlap with the emission and excitation spectra of EuUCHNA, which may be related to fluorescence resonance energy transfer (FRET) or internal filter effect (IFE). The fluorescence lifetime of EuUCHNA decreased from 180.4 μs to 136.8 μs at 613 nm and from 5.0 ns to 4.5 ns at 450 nm (see Table S4), indicating that a dynamic quenching process occurred in EuUCHNA. Therefore, the quenching effect of CV on EuUCHNA is dynamic, and IFE and FRET effects are also involved in this process.

[0061] As for the induction of QN, the fluorescence of EuUCHNA at 450 nm was significantly enhanced, which was mainly attributed to two aspects. First, the incorporation of QN triggered the change of the electronic structure of the 6-HNA ligand, resulting in its enhanced fluorescence at 450 nm. Specifically, the -OH group on the 6-HNA ligand formed a strong hydrogen bond interaction with the N or O atom of QN. In addition, since QN is an alkaloid, the -OH group of the 6-HNA ligand may be deprotonated. This deprotonation process, coupled with the hydrogen bond interaction, increases the electron density around the O atom of the -OH group on the 6-HNA ligand, thereby optimizing the intramolecular electron transfer efficiency within the naphthalene ring of the 6-HNA ligand, and ultimately increases the fluorescence intensity at 450 nm.

[0062] Secondly, the QN molecule itself has a strong hydrogen bond interaction with the -OH group of the 6-HNA ligand, and even partially protonated, and its emission wavelength is red-shifted to about 450 nm. Therefore, the fluorescence of the 6-HNA ligand and the emission of the protonated QN work together to enhance the overall fluorescence intensity at 450 nm. Table 6. XPS data Table 7. Fluorescence lifetime data Example 5 The EuUCHNA obtained in Example 1 was combined with a smartphone to detect CV and QN as follows: A series of standard solutions of CV and QN were added to a solution of EuUCHNA aqueous suspension (0.25 mg mL -1) in a 96-well plate and mixed well. The final concentrations of CV were 1, 2, 3, 4, 5, 10, 15, 20, 25 μM, and the final concentrations of QN were 0.5, 1, 1.5, 2, 5, 10, 15 μM. The fluorescence image of each mixture was analyzed by RGB under UV light by a smartphone camera.

[0063] The results are as follows: Smartphones are used in the scientific field because of their portability, powerful computing power, and high image resolution. This example uses smartphones to develop a low-cost, instrument-free CV and QN detection method because the fluorescent colors can be distinguished by the naked eye.

[0064] Fluorescence photos were taken in a dark box using a 254 nm UV lamp (PHILIPS, 6 W). The camera was taken with a vivoS9e smartphone, whose rear camera configuration is 64 million + 8 million + 2 million pixels. RGB values ​​were recorded using a free color recognition software called "Color Recognizer". Fig.15 As shown in the figure, when detecting CV, the G and B values ​​of the fluorescent color are lower than those of EuUCHNA, while when detecting QN, the G and B values ​​are higher than those of EuUCHNA. Therefore, the difference in G and B values ​​provides a method for effectively distinguishing CV from QN. The B / R values ​​are respectively related to CV (1 ~ 25 μM, R 2 = 0.9994) and QN (0.5 ~ 15 μM, R 2 = 0.9909) showed a good linear relationship ( Fig.15 ). The LOD values ​​of CV and QN calculated by smartphone images were 0.33 μM and 0.17 μM, respectively.

[0065] The practicality and reliability of smartphone imaging detection of CV and QN were evaluated by the spike recovery test of distilled water and fish pond water. The results are shown in Table 8. The CV spike recovery rate was 93.73~108.16%, and the QN spike recovery rate was 91.07%~113.20%. The RSD value was acceptable, with good spike recovery and RSD value.

[0066] Table 8 Recovery results of CV and QN detection A novel dual-emission europium MOF (EuUCHNA) was rationally designed and constructed. The fluorescence of EuUCHNA is quite stable in water, very stable in the pH range of 4-8, and sensitive to CV and QN with different response modes. The detection limits of CV and QN are both low at 2.4 nM and 32.0 nM. More importantly, it achieves the simultaneous quantification of CV and QN in fish pond water. In addition, the combination of a smartphone realizes the instrument-free determination of CV and QN in real water. This work provides a new method for the simultaneous quantification of two analytes with a single fluorescence sensor.

[0067] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a dual-emission fluorescent probe based on a europium-functionalized metal-organic framework, characterized in that: The following steps are involved: Mix ZrCl4, pyromellitic acid, 6-hydroxy-2-naphthoic acid and water, and add acetic acid to obtain a mixture; The mixture is heated to reflux, centrifuged to obtain a residue, and the residue is washed and dried to obtain UCHNA; The UCHNA is dispersed in a Eu(NO3)3·6H2O aqueous solution, heated for reaction, and a white precipitate is collected, washed, and dried to obtain a dual-emission fluorescent probe based on a europium-functionalized metal-organic framework.

2. The preparation method according to claim 1, characterized in that: The molar ratio of ZrCl4, pyromellitic acid and 6-hydroxy-2-naphthoic acid is 1.5-2: 2.5-3.5: 0.2-0.

5.

3. The preparation method according to claim 1, characterized in that: The usage ratio of ZrCl4 to water and acetic acid is 1.5 mol~2 mol: 15 mL~25 mL: 4 mL~6 mL.

4. The preparation method according to claim 1, characterized in that: The heating reflux temperature is 80° C. to 120° C., and the heating reflux time is 20 h to 28 h.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the UCHNA to Eu(NO3)3·6H2O is 0.4-0.6:0.8-1.

2.

6. The preparation method according to claim 1, characterized in that: The temperature of the heating reaction is 70° C. to 90° C., and the time of the heating reaction is 20 h to 28 h.

7. Dual-emission fluorescent probe based on europium functionalized metal-organic framework, characterized in that: Obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the europium-functionalized metal-organic framework dual-emission fluorescent probe according to claim 7 in the simultaneous detection of crystal violet and quinine in water or aquatic products.

9. A fluorescence sensor, characterized in that: It includes the dual-emission fluorescent probe based on europium functionalized metal-organic framework as described in claim 7.

10. A method for simultaneously detecting crystal violet and quinine in water or aquatic products, characterized in that: The detection is performed using the europium-functionalized metal-organic framework dual-emission fluorescent probe according to claim 7, comprising the following steps: The meat of the aquatic product is added to a mixture of acetonitrile and PBS buffer solution, subjected to ultrasonic treatment, centrifuged, the supernatant is collected, and the supernatant is filtered through a filter membrane to obtain a pre-treated sample; or the collected aquaculture water sample is subjected to centrifugation and filtration treatment to obtain pre-treated water; Adding EuUCHNA into water to prepare EuUCHNA water suspension; The pretreated sample or the pretreated water is added to the EuUCHNA water suspension, and the fluorescence spectrum is measured.

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