Eu3+ functionalized metal-organic framework dual-emission fluorescent probe, preparation method and application thereof

Abstract

The application discloses a europium functionalized metal-organic framework double-emission fluorescent probe and a preparation method and application thereof, relates to the field of organic metal frameworks and fluorescent detection, and the preparation method comprises the following steps: mixing ZrCl4, pyromellitic acid, 6-hydroxy-2-naphthoic acid and water, adding acetic acid to obtain a mixture; the mixture is heated to reflux, centrifuged to obtain a residue, washed and dried to obtain UCHNA; the UCHNA is dispersed in an aqueous solution of Eu(NO3)3.6H2O, heated to react, the white precipitate is collected, washed and dried, and the Eu-MOF is obtained. 3+ The application has the beneficial effect that a luminescent ligand 6-hydroxy-2-naphthoic acid is used to modify a UiO-66 type MOF, and Eu 3+ -MOF is synthesized through post-modification and reasonable design, and the Eu-MOF can selectively and rapidly respond to crystal violet (CV) and quinine (QN) through different response modes, and has a low detection limit, which is superior to most reported sensors.

Description

Technical Field

[0001] This invention relates to the field of organometallic frameworks and fluorescence detection, specifically to europium-functionalized metal-organic framework dual-emission fluorescent probes, their preparation methods, and applications. Background Technology

[0002] With the continued expansion of large-scale, intensive aquaculture, frequent outbreaks of fungal, bacterial, and parasitic infections underscore the necessity of effective disease management. In this regard, crystal violet (CV) and quinine (QN) are widely used to protect the health of fish and shrimp, particularly for the treatment and prevention of these infections, providing essential solutions in environments where disease outbreaks can cause significant economic losses. CV, a synthetic triphenylmethane dye, has been widely used for the prevention and treatment of aquatic diseases due to its effective anthelmintic, bactericidal, and fungicidal effects. However, CV is highly toxic and can cause mutagenic and carcinogenic effects; therefore, its use in aquaculture has been banned in many countries. QN, a cinchona alkaloid, is known as an important antimalarial drug and is commonly used to prevent and treat malaria in fish or as a preservative for seafood to extend its shelf life. Unfortunately, QN has high neurotoxicity and cardiovascular toxicity. Excessive use of QN can lead to osteomalacia, blurred vision, and even damage to the nervous and cardiovascular systems. Therefore, monitoring CV and QN values ​​in fish and aquaculture water is crucial.

[0003] In the past, several analytical techniques have been used to determine CV and QN, including high-performance liquid chromatography (HPLC), UV-Vis spectrophotometry, flow-injection chemiluminescence, surface-enhanced Raman scattering (SERS), and electrochemical analysis. However, these techniques have several limitations, such as requiring expensive instruments or highly skilled technicians, insufficient sensitivity, or complex pretreatment procedures. Furthermore, the complexity of real-world samples makes multi-target detection within a single system both desirable and challenging. To date, there are no reports of simultaneous detection of CV and QN. Therefore, there is a need to develop a simple, sensitive, rapid, and low-cost method for the simultaneous detection of CV and QN in aquatic products.

[0004] Fluorescence analysis offers advantages such as simplicity, intuitiveness, and speed, making it an excellent choice. Furthermore, multi-emission materials not only reduce environmental interference through self-reference but also enable multi-target detection through different response modes, thus attracting widespread attention. Lanthanide metal-organic frameworks (Ln-MOFs) combine the modifiability and porosity of MOFs with the properties of Ln... 3+ The unique fluorescence properties are combined, and can be utilized through luminescent ligands, Ln 3+Multiple emission fluorescence sensors can be easily designed and constructed with target materials. Many multi-emission Ln-MOFs have been synthesized and developed as fluorescent sensors for biomarkers, explosives, food, or environmental contaminants. However, multi-target detection by a single fluorescence sensor has certain limitations. Summary of the Invention

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

[0006] This invention employs the luminescent ligand 6-hydroxy-2-naphthoic acid to pre-modify UiO-66 type MOF, and uses Eu 3+ Post-modification (e.g.) Figure 16 As shown in a), a dual-emission Eu signal with blue and red bands was rationally designed and synthesized. 3+ -MOF (EuUCHNA). Fluorescence spectroscopy shows that EuUCHNA exhibits different response modes to CV and QN (e.g., Figure 16 (As shown in b). QN effectively quenched both emission bands of EuUCHNA, while significantly enhancing the blue emission band of EuUCHNA and slightly weakening the red emission band. By combining visible fluorescence color changes with a smartphone, instrument-free monitoring of CV and QN can be achieved in real samples. Furthermore, different response modes enable simultaneous quantification of CV and QN. This work will promote the development of fluorescence sensors for multi-target detection.

[0007] The technical solution of the present invention is as follows:

[0008] The first aspect of this invention provides a method for preparing a dual-emission fluorescent probe based on a europium-functionalized metal-organic framework, comprising the following steps:

[0009] ZrCl4, pyromellitic acid, 6-hydroxy-2-naphthoic acid and water were mixed, and acetic acid was added to obtain a mixture; the mixture was heated under reflux and centrifuged to obtain a residue, which was then washed and dried to obtain UCHNA;

[0010] The UCHNA was dispersed in an aqueous solution of Eu(NO3)3·6H2O, heated to react, and the white precipitate was collected, washed, and dried to obtain a europium-functionalized metal-organic framework dual-emission fluorescent probe.

[0011] Optionally, 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.

[0012] Optionally, the ratio of ZrCl4 to water and acetic acid is 1.5 mol to 2 mol: 15 mL to 25 mL: 4 mL to 6 mL.

[0013] Optionally, the temperature of the heating reflux is 80℃~120℃, and the heating reflux time is 20 h~28 h.

[0014] Optionally, the mass ratio of UCHNA to Eu(NO3)3·6H2O is 0.4~0.6:0.8~1.2.

[0015] Optionally, the temperature of the heating reaction is 70℃~90℃, and the heating reaction time is 20 h~28 h.

[0016] A second aspect of the present invention provides a europium-functionalized metal-organic framework dual-emission fluorescent probe, which is obtained by the preparation method described above.

[0017] A third aspect of the present invention provides an application of the europium-functionalized metal-organic framework dual-emission fluorescent probe obtained by the above preparation method or the above europium-functionalized metal-organic framework dual-emission fluorescent probe in the simultaneous detection of crystal violet and quinine in water or aquatic products.

[0018] A fourth aspect of the present invention provides a fluorescence sensor comprising a europium-functionalized metal-organic framework dual-emission fluorescent probe obtained by the above-described preparation method or the europium-functionalized metal-organic framework dual-emission fluorescent probe described above.

[0019] A fifth aspect of the present invention provides a method for simultaneously detecting crystal violet and quinine in water or aquatic products, comprising the detection using a europium-functionalized metal-organic framework dual-emission fluorescent probe obtained by the above-described preparation method or the above-described europium-functionalized metal-organic framework dual-emission fluorescent probe, comprising the following steps:

[0020] The meat of aquatic products is added to a mixture of CH3CN and PBS, sonicated, centrifuged, and the 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.

[0021] EuUCHNA was added to water to prepare an EuUCHNA aqueous suspension;

[0022] The pretreated sample or the pretreated water was added to the EuUCHNA aqueous suspension, and the fluorescence spectrum was measured.

[0023] This invention has at least one of the following beneficial effects:

[0024] This invention develops a dual-emission sensor (EuUCHNA) that selectively and rapidly responds to crystal violet (CV) and quinine (QN) through different response modes; these are two commonly used anti-infective drugs for treating fungi, bacteria, and parasites. The detection limits for CV and QN are 2.4 nM and 32.0 nM, respectively, superior to most reported sensors. The determination of CV and QN can be applied to aquatic products such as fish meat and aquaculture water such as fishpond water. Furthermore, EuUCHNA can simultaneously quantify CV and QN in actual fishpond water. This invention provides a promising fluorescent sensor and a novel analytical method for the simultaneous quantification of two targets in real-world samples using a single sensor. Attached Figure Description

[0025] Figure 1 Characterization diagrams of UCHNA and EuUCHNA synthesized in Example 1 are shown, where (a) PXRD spectra of UiO-66 (simulated), UCHNA, EuUCHNA, and EuUCHNA after soaking in water for 10 days; (b) FT-IR spectra of UCHNA and EuUCHNA; and (c) XPS spectra of UCHNA and EuUCHNA.

[0026] Figure 2 The TGA curves are for UCHNA and EuUCHNA synthesized in Example 1.

[0027] Figure 3 The fluorescence spectra of UCHNA synthesized in Example 1 are shown, where (a) is the solid-state fluorescence spectrum of UCHNA; and (b) is the liquid-state fluorescence spectrum of UCHNA.

[0028] Figure 4 The fluorescence spectra of EuUCHNA synthesized in Example 1 are shown below, including (a) excitation and emission spectra in the solid state; (b) aqueous suspension; (c) fluorescence spectrum of EuUCHNA in aqueous solution after 7 days; (d) effect of pH value on the fluorescence spectrum of EuUCHNA; and (e) and (f) fluorescence spectra of EuUCHNA at different concentrations (0.1-1.5 mg / mL).

[0029] Figure 5 The fluorescence spectra of EuUCHNA synthesized in Example 1 are shown below: (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) comparison of fluorescence spectra of EuUCHNA with CV and different substances; (e) comparison of fluorescence spectra of EuUCHNA with QN and different substances; (f) fluorescence spectra of EuUCHNA and different substances in the presence of CV; and (g) fluorescence spectra of EuUCHNA and different substances in the presence of QN.

[0030] Figure 6 The fluorescence spectra of EuUCHNA synthesized in Example 1 are shown, where (a) is the fluorescence spectrum of EuUCHNA at a concentration of CV (50 μM); and (b) is the fluorescence spectrum at a concentration of QN (40 μM).

[0031] Figure 7 The following figures are shown: (a) fluorescence color of EuUCHNA at different crystal violet concentrations; (b) fluorescence color of EuUCHNA at different quinine concentrations; (c) fluorescence spectrum of EuUCHNA in crystal violet (0 ~ 50 μM); (d) I0 / I at 613 nm. 613 Linear relationship with crystal violet concentration; (e) I0 / I at 450 nm 450 Linear relationship with CV concentration; (f) Fluorescence spectrum of EuUCHNA at quinine (0 ~ 40 μM); (g) I at 450 nm 450 Linear relationship between I0 and 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).

[0032] Figure 8 QN@EuUCHNA and CV@EuUCHNA in the figure represent the CIE colorimetric changes of EuUCHNA synthesized in Example 1 after the addition of quinine (40 μM) and crystal violet (50 μM), respectively.

[0033] Figure 9 The image shows (a) the fish meat sample at 613 nm. I 0 / I 613 (a) Linearity between CV concentration and concentration; (b) Fish meat sample I 450 / I 613 The linear relationship between QN concentration and the concentration of QN.

[0034] Figure 10 The figures show (a) verification of the additivity of CV and QN; (b) CV concentration of 2 μM; (c) CV concentration of 4 μM; and (d) CV concentration of 6 μM.

[0035] Figure 11The following figures show the effect of different concentrations of QN (2, 4, 6 μM) on the fluorescence spectrum of EuUCHNA in the presence of 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 QN concentrations (2, 4, 6 μM) in the presence of CV (2 μM); (d) CV (4 μM); (f) CV (6 μM).

[0036] Figure 12 The fluorescence spectra of EuUCHNA at different concentrations are shown as (a) CV (2, 4, 6 μM); (b) QN (4, 8, 12 μM).

[0037] Figure 13 The following are the (a) PXRD spectra of CV@EuUCHNA and QN@EuUCHNA; (b) XPS spectra of CV, QN, CV@EuUCHNA and QN@EuUCHNA; (c) UV-Vis spectrum of CV, and excitation and emission spectra of EuUCHNA.

[0038] Figure 14 The fluorescence lifetimes of EuUCHNA, CV@EuUCHNA and QN@EuUCHNA at 450 nm are shown in (a); and the fluorescence lifetimes of EuUCHNA, CV@EuUCHNA and QN@EuUCHNA at 613 nm are shown in (b).

[0039] Figure 15 The diagram shows (a) the liquid color recognition process; (b) the fluorescence color and RGB values ​​of EuUCHNA containing CV or QN; (c) the linear relationship between B / R and CV concentration; and (d) the linear relationship between B / R and QN concentration.

[0040] Figure 16 The diagram shows (a) the synthesis route of EuUCHNA; and (b) a schematic diagram of different response modes of EuUCHNA to CV and QN. Detailed Implementation

[0041] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0042] ZrCl4, 6-hydroxy-2-naphtholic 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 Co., Ltd. (China). Sulfaacetamide (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. CaCl2, MgCl2, Zn(NO3)2, KCl, K2CO3, K2SO4, and AgNO3 were all from Tianjin Fuchen Chemical Reagents.

[0043] Powder X-ray diffraction (PXRD) was performed on the Bruker-D8 Advanced using CuKα radiation. X-ray photoelectron spectroscopy (XPS) results were obtained from an ESCALAB 250Xi. Recordings from 4000 to 400 cm⁻¹ were performed using a Thermo Fisher Scientific Nicolet iS10. -1 Fourier transform infrared spectroscopy results of KBr particles within the range. Fluorescence spectra were recorded on a HORIBA FluoroMax-4. Ultraviolet-visible absorption spectroscopy (UV-vis) data were acquired on an INESA N4S spectrophotometer.

[0044] Example 1

[0045] The preparation method of europium-functionalized metal-organic framework dual-emission fluorescent probe includes the following steps:

[0046] Step 1: UCHNA Synthesis

[0047] ZrCl4 (0.47 g, 2.0 mmol), pyromellitic acid (H4betc, 0.78 g, 3.05 mmol), and 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, and then washed repeatedly with water and methanol. Finally, the residue was dried under vacuum at 80 °C to obtain 0.74 g of white powder UCHNA.

[0048] Step 2: EuUCHNA Synthesis

[0049] 0.50 g of UCHNA was dispersed in 40 mL of distilled aqueous solution containing 1.00 g of Eu(NO3)3·6H2O. The mixture was stirred at 80 °C for 24 h. After cooling, the white precipitate was collected and washed three times alternately with water and methanol. The solid product was dried at 80 °C for 8 h to obtain 0.50 g of white powder EuUCHNA.

[0050] The synthesized EuUCHNA was characterized, and the characterization results are as follows:

[0051] The structure of the synthesized MOF was determined by powder X-ray diffraction (PXRD). Figure 1 As shown in Figure a, the PXRD peaks of UCHNA and EuUCHNA are mainly distributed around 7.5°, 8.6°, 12.2°, 14.4°, 17.3°, 25.9°, and 30.0°. These peaks are consistent with the simulated PXRD pattern of UiO-66, indicating that the synthesized MOF material has the same basic framework structure as UiO-66. Furthermore, after immersion in water for 10 days, the PXRD pattern of EuUCHNA remained well-preserved, demonstrating its high structural stability in water. The FT-IR spectrum is shown below. Figure 1 As shown in b, UCHNA and EuUCHNA are at 1574 cm. -1 and 1426 cm -1 Both asymmetric and symmetric stretching vibrations of the carboxyl group were observed nearby. Notably, the 1715 cm⁻¹ region associated with the uncoordinated carboxyl group in UCHNA was observed. -1 The vibrations become weaker in EuUCHNA, indicating that most of the free carboxyl groups in EuUCHNA are associated with Eu. 3+ Coordination. X-ray photoelectron spectroscopy (XPS) analysis further verified Eu's coordination. 3+ and COO Coordination bonds between groups (Table 6).

[0052] like Figure 1 As shown in Figure c, compared to UCHNA, EuUCHNA exhibits a new peak at 1125.01 eV, attributed to the three-dimensional binding energy of Eu. The O1s binding energy shifts from 531.70 eV for UCHNA to 531.65 eV for EuUCHNA. The Eu binding energy was determined by ICP-MS. 3+ The doping amount in EuUCHNA. The Eu content was calculated based on ICP-MS results. 3+ With Zr 4+ The molar ratio was 1:4.7. Thermogravimetric analysis (TGA) showed that the synthesized MOF was thermally stable at around 300 °C. Before 300 °C, the weight loss of the synthesized MOF was approximately 24%, which is related to the loss of incorporated solvent molecules. Figure 2 ).

[0053] Example 2

[0054] The EuUCHNA obtained in Example 1 was subjected to fluorescence sensing experiments.

[0055] This study investigated the fluorescence response of EuUCHNA to several common fishery drugs and potential species in fish meat and pond water. The substances studied included: sulfadiazine (SG), florfenicol (FF), thiamine (TAP), sulfaacetamide (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), CaCl2, MgCl2, Zn(NO3)2, KCl, K2CO3, K2SO4, and AgNO3. Typically, 12.5 mg of EuUCHNA was sonicated in 50 mL of H2O for 20 minutes to form a homogeneous suspension (0.25 mg / mL). Then 20 μL (0.01 mol) of each analyte was added. L -1 Add to 2 mL or more of EuUCHNA aqueous suspension for fluorescence analysis.

[0056] The results of the fluorescence sensing experiment are as follows:

[0057] I. Fluorescence properties:

[0058] For UCHNA, a violet-blue emission was observed in the solid state, while a bright blue emission was observed with the naked eye under a UV lamp in an aqueous suspension. Figure 3 a and Figure 3 b). Its fluorescence spectrum exhibits a weak, broad band in the solid state, with a maximum emission wavelength of 400 nm, and a strong emission band in the aqueous suspension, located at 450 nm, which is related to the π-π* transition of the 6-HNA ligand. For EuUCHNA, bright red and magenta emission can be observed with the naked eye in both 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 showed emission bands of the 6-HNA ligand and Eu in both solid and aqueous suspensions. 3+ Characteristic emission bands of ions ( Figure 4 (a and 4b). The dominant peak is located at 613 nm, corresponding to Eu. 3+ Ionic5 D0→ 7 F2 transition indicates that the ligand interacts with Eu. 3+ There is a strong antenna effect between them.

[0059] It is worth noting that 6-HNA ligands and Eu 3+ The emission of ions is very stable in aqueous medium; after immersion in water for 7 days, the fluorescence intensity at 450 and 613 nm did not change significantly. Figure 4 c). Furthermore, pH-dependent fluorescence studies showed that emission at 450 nm and 613 nm was highly stable within a pH range of 4–8, indicating a wide range of practical applications. Figure 4 d).

[0060] II. Sensing performance:

[0061] First, the concentration of EuUCHNA was optimized by recording its fluorescence spectrum. For example... Figure 4 As shown in e and 4f, when the EuUCHNA concentration is 0.25 mg·mL -1 At that time, the fluorescence intensity at 450 and 613 nm was relatively strong; therefore, the subsequent sensing experiments used 0.25 mg·mL⁻¹. -1 EuUCHNA. To study the fluorescence response of EuUCHNA, 20 μL (0.01 mol·L⁻¹) of veterinary drugs, some metal ions, and amino acids were added. -1 The substances were added to 2 ml of EuUCHNA aqueous suspension. Under UV light, we could clearly see that most reagents had negligible effects on the fluorescence emission of EuUCHNA, but the fluorescence of EuUCHNA was significantly quenched after the addition of CV. Upon the addition of QN, the emission color changed from magenta to bright blue, which was visible to the naked eye. Figure 5 a, Figure 5 d、 Figure 5 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, while 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 EuUCHNA fluorescence selectively responds to CV and QN. The anti-interference ability of common veterinary drugs, metal ions, and amino acids on EuUCHNA detection of CV and QN was investigated (CV: 20 μM; QN: 100 μM; potential interfering substances: 200 μM). CV and QN were added to a mixture of EuUCHNA and interferon, respectively, and fluorescence spectra were measured. In the presence of interference, CV effectively quenched fluorescence at 450 nm and 613 nm. Figure 5f), QN can significantly enhance fluorescence at 450 nm (f), Figure 5 (g), indicating that EuUCHNA exhibits good anti-interference ability for both CV and QN fluorescence responses.

[0062] Example 3

[0063] The EuUCHNA obtained in Example 1 was subjected to quantitative determination of CV and QN, as follows:

[0064] Generally, standard high-concentration CVs (1 mM and 10 mM) are first prepared in H2O, and QNs are prepared in DMF. Then, different volumes of high-concentration CVs and QNs are added to 1980 μL of EuUCHNA (0.25 mg). mL -1 Add H2O and DMF to the aqueous suspension to adjust the total volume to 2000 μL. Add 0.2, 0.5, 1.0, and 1.5 μL of 1mM CV solution and 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 μL of 10mM CV solution to EuUCHNA, respectively. Add 0.2, 0.5, 1.0, 1.5, 1.5, 2.0, 3.0, 1.0, and 1.5 μL of 1mM QN solution and 0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 μL of 10mM QN solution 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.

[0065] The results are as follows:

[0066] To quantitatively determine CV and QN, standard solutions of CV and QN were added, and the fluorescence spectra of EuUCHNA were recorded. As the concentration of CV increased, the fluorescence color gradually darkened. Figure 7 a), 450 nm ( I 450 ) and 613 nm ( I 613 The fluorescence intensity at point () gradually decreases. Clearly, I 613 The quenching efficiency is much greater than I 450 ( Figure 7 c).

[0067] Within the range of 0.1 ~ 15 μM, I 450 and I 613 It exhibits 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 (in μM) was used to calculate the limits of detection (LODs) as 2.4 nM and 61.1 nM, respectively, based on a signal-to-noise ratio of 3. As the QN concentration increased, the fluorescence color gradually changed from magenta to bright blue. Figure 7 (b) When the QN concentration reached 40 μM, the fluorescence intensity at 613 nm decreased by only about 13.8%, while the fluorescence intensity at 450 nm increased by about 10.4 times. Interestingly, in the range of 0.1 ~ 40 μM, I 450 It exhibits 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) applies to high QN concentrations (2.5 ~ 40.0 μM). The LOD value of QN is calculated to be 32.0 nM. Furthermore, since I 450 and I 613 They exhibit significantly different sensitivities, therefore the intensity ratio (I 450 / I 613 The ratio signal is used to quantize QN. For lower QN concentrations (0.1 ~ 1.0 μM), the equation is as follows: I 450 / I 613 = 0.244· C QN + 0.2096 ( R 2 = 0.9966, Equation (5); For high QN concentrations (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 calculated corresponding LOD value is 76.2 nM. The LOD values ​​of CV and QN are superior to most reported fluorescence sensors (Table 1, Table 2). The low LOD values ​​of CV and QN allow for the detection of trace levels of CV and QN in real samples.

[0068] like Figure 7 i and Figure 7 As shown in j, time-resolved studies indicate that... I 450 and I 613 The fluorescence intensity can be quenched by CV, while I 450 It can be enhanced by QN and reach stability within 1 minute, demonstrating EuUCHNA's rapid response to CV and QN.

[0069]

[0070] Table 1. Changes in detection CV between this embodiment and previously reported fluorescence detection methods.

[0071] The cited references are as follows:

[0072] [1]Y. Bai, M. Liu, Y. He, G. Song, 2023. Portable smartphone platformbased on Ti3C2MQDs / CDs assembly for ratiometric fluorescence quantitativemonitoring of crystal violet. Anal Methods 15, 5510-5517.

[0073] [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 ItsApplication in the Detection of Crystal Violet. Nanomaterials 9, 1556.

[0074] [3]J. Zhou, M. Qing, Y. Ling, L. Wang, N.B. Li, H.Q. 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.

[0075] [4]K. Yi, 2017. A novel method for the quantitative determination ofcrystal violet utilizing YVO4: Eu 3+ nanoparticles. Chem Lett 46, 520-523.

[0076] [5]Y. Hu, Z. Gao, 2020. Yellow emissive Se, N-codoped carbon dotstoward sensitive fluorescence assay of crystal violet. J Hazard Mater 388,122073.

[0077] [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.

[0078] Table 2. Changes in the detection QN of this embodiment and previously reported fluorescence detection methods.

[0079]

[0080] The cited references are as follows:

[0081] [7]VC Tsaftari, M. Tarara, PD Tzanavaras, GZ Tsogas, 2023. Anovel equipment-free paper-based fluorometric method for the analytical determination of quinine in soft drink samples. Sensors 23, 5153.

[0082] [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.

[0083] [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.

[0084] Example 4

[0085] The EuUCHNA obtained in Example 1 was used to detect CV and QN in real samples. The specific method is as follows:

[0086] I. Detection of CV and QN in aquaculture water

[0087] The aquaculture water was taken from Yezhi Lake in Wuhan, Hubei Province. The water was centrifuged at 9000 rpm for 10 min and then filtered through a 0.22µm aqueous membrane to obtain pretreated water.

[0088] For CV determination, standard high-concentration CV (1 and 10 mM) were prepared using the above filtrate. 0.6 μL of 1 mM CV, 0.6 μL of 1 mM CV, and 2.6 μL of 10 mM CV were added to 1980 μL of EuUCHNA aqueous suspension (0.25 mg / mL), respectively. Pretreated water was added to the mixture to adjust the total volume to 2000 μL. The final CV concentrations were 0.3, 3.0, and 13.0 μM, respectively, for recovery tests. The fluorescence spectra of each sample were measured under the conditions of an excitation wavelength of 312 nm and a slit width of 3.3 / 3.3 nm.

[0089] 20 μL of pretreated water was added to 1980 μL of EuUCHNA aqueous suspension (0.25 mg / mL). Then, 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 mixture, and QN was determined. The final concentrations of QN were 0.3, 3.0, 13.0, and 23.0 μM, respectively, for recovery tests.

[0090] II. Simultaneous determination of CV and QN in aquatic water

[0091] Grass carp purchased from a local supermarket was minced from its back muscle. 8.00 g of the minced fish was weighed and added to 4 mL of CH3CN and 20 mL of PBS (pH = 6.86). The mixture was sonicated for 10 minutes and centrifuged at 9000 r / min for 10 minutes, and the supernatant was collected. The supernatant was filtered through a 0.45 μm mixed-system filter membrane to obtain the pretreated sample. Generally, 20 µL of the pretreated sample was added to a 1980 μL LEuUCHNA (0.25 mg / mL) aqueous suspension, and different volumes of standard concentrations of CV (1 and 10 mM in H2O) and QN (1 and 10 mM in DMF) were added, and their fluorescence spectra were measured. The final concentrations of CV were 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0, and 15.0 μM, 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, showing a linear relationship. In the recovery experiment, the final concentrations of CV were 0.3, 3.0, and 13.0 μM, and the final concentrations of QN were 3.0, 13.0, and 23.0 μM.

[0092] The measurement results are as follows:

[0093] I. Using fishpond water and fish meat as research subjects, the feasibility of the established method for determining CV and QN was explored through standard scrambling and recovery experiments. The linear relationship between fluorescence intensity and concentration of CV and QN is as follows: Figure 9 As shown in Table 3, the recovery rates of CV and QN are within the range of 90-112.33%, and the RSD values ​​meet the requirement of <5%. These results indicate that EuUCHNA can be used to determine the content of CV and QN in actual samples.

[0094] Table 3. Recovery rates of CV and QN in actual fishpond water and fish meat samples.

[0095]

[0096] II. Simultaneous Quantification of CV and QN

[0097] Because QN at low concentrations I 613 The impact is negligible, and we believe that simultaneous quantification of CV and QN can be achieved. Firstly, to investigate the effect of QN on CV detection systems... I 613 To investigate the effects of different concentrations of QN (4, 8, 12 μM), different concentrations were added to EuUCHNA suspensions containing standard CV concentrations (2, 4, 6 μM), and the fluorescence spectra of QN at different concentrations (4, 8, 12 μM) were measured. Figure 10 a and Figure 11 As shown, after adding QN I 613 The lack of significant change indicates that QN does not affect the CV inspection system. I 613 .

[0098] To investigate whether the quenching effect of CV and the enhancement effect of QN are independent, standard concentrations of CV (2, 4, 6 μM) and QN (4, 8, 12 μM) were first added, and the fluorescence spectra of EuUCHNA were measured. Figure 12 The fluorescence intensity (IF) at 450 nm was calculated by increasing QN and decreasing CV. 450, cal ), can be I 450，cal = I 450,QN - ( I 450,0 - I 450，CV The formula is determined by equation (7). Where... I 450,0 , I 450,QN , I 450,CV The fluorescence intensities at 450 nm for EuUCHNA and EuUCHNA with added CV and QN, respectively, are shown. Then, the fluorescence spectra of EuUCHNA with mixtures of the same concentrations of CV and QN were recorded, and the fluorescence intensity at 450 nm for each mixture is expressed as follows: I 450，exp .like Figure 10 As shown in b, 10c, and 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.

[0099] Therefore, in the case of a hybrid CV and QN approach, CV can be prioritized. I 613The concentration of CV is determined by formula (1), and then the fluorescence intensity at 450 nm can be calculated by formula (2). I 450，CV ). Calculated I 450、CV It can be used as the starting point for the QN enhancement process. Then the concentration of QN can be calculated by formula (4).

[0100] To validate the simultaneous quantification method described above, a series of nine mixed standard concentrations of CV and QN were added to the EuUCHNA suspension, and fluorescence spectra were recorded. As shown in Table 4, the measured CV and QN concentrations were consistent with the added spiking amounts, indicating that the proposed calculation method is reliable for the simultaneous quantification of CV and QN. The established method was then applied to river water for the simultaneous quantification of CV and QN. The recoveries of CV and QN ranged from 92.50% to 108.67%, and the RSD values ​​were acceptable (Table 5).

[0101] Table 4 shows the analytical results of CV and QN in the mixture.

[0102]

[0103] Table 5 shows the results of simultaneous detection of CV and QN in fishpond water.

[0104]

[0105] III. Fluorescence Response Mechanism

[0106] The fluorescence response mechanism of EuUCHNA was investigated using PXRD, UV-vis, and XPS analyses. The PXRD patterns of EuUCHNA after sensing CV and QN were consistent with the original patterns, indicating that the fluorescence response is independent of the structural framework collapse. Figure 13a) XPS data show that the binding energy of Eu 3d in EuUCHNA changed from 1125.01 and 1125.2 eV to 1125.25 eV after detecting CV and QN, respectively. Furthermore, the N1s binding energy also changed significantly after incorporating the EuUCHNA sensor compared to the original CV and QN (Table 6). These results indicate a weak interaction between the EuUCHNA sensor and the N atoms of CV or QN. The UV-Vis absorption spectrum of CV largely overlaps with the emission and excitation spectra of EuUCHNA, which may be related to fluorescence resonance energy transfer (FRET) or internal filtering 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 7), indicating that EuUCHNA underwent a dynamic quenching process. Therefore, the quenching effect of CV on EuUCHNA is dynamic, and the IFE and FRET effects also participate in this process.

[0107] The significant enhancement of EuUCHNA fluorescence at 450 nm in response to QN is mainly attributed to two factors. First, the incorporation of QN induces a change in the electronic structure of the 6-HNA ligand, leading to enhanced fluorescence at 450 nm. Specifically, the -OH group on the 6-HNA ligand forms a strong hydrogen bond with the N or O atom of QN. Furthermore, since QN is an alkaloid, the -OH group of the 6-HNA ligand may undergo deprotonation. This deprotonation process, combined 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, ultimately increasing the fluorescence intensity at 450 nm.

[0108] Secondly, due to the strong hydrogen bond interaction with the -OH group of the 6-HNA ligand, and even partial protonation, the emission wavelength of the QN molecule itself undergoes a red shift, moving to approximately 450 nm. Therefore, the combined effect of the fluorescence of the 6-HNA ligand and the emission of the protonated QN results in an overall enhancement of the fluorescence intensity at 450 nm.

[0109] Table 6. XPS Data

[0110]

[0111] Table 7. Fluorescence lifetime data

[0112]

[0113] Example 5

[0114] The EuUCHNA obtained in Example 1 is combined with a smartphone to detect CV and QN, as follows:

[0115] A series of standard solutions of CV and QN were added to an aqueous suspension containing EuUCHNA (0.25 mg·mL⁻¹). -1 In 96-well plates, the mixtures were homogeneously prepared, with final concentrations of CV (chemical chromatogram) of 1, 2, 3, 4, 5, 10, 15, 20, and 25 μM, and final concentrations of QN (quantitative chromatogram) of 0.5, 1, 1.5, 2, 5, 10, and 15 μM. Fluorescence images of each mixture were analyzed using a smartphone camera under UV light in an RGB configuration.

[0116] The results are as follows:

[0117] Smartphones are widely used in scientific fields due to their portability, powerful computing capabilities, and high image resolution. This embodiment utilizes a smartphone to develop a low-cost, instrument-free method for CV and QN detection, since fluorescence color can be distinguished with the naked eye.

[0118] The fluorescence photographs were taken in a darkroom using a 254 nm ultraviolet lamp (PHILIPS, 6 W). The camera was a vivo S9e smartphone with a triple-lens rear camera setup of 64MP + 8MP + 2MP. RGB values ​​were recorded using a free color recognition software called "Color Recognizer." Figure 15 As shown, when detecting CV, the G and B values ​​of the fluorescence 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 between CV and QN. The B / R value is related to CV (1 ~ 25 μM, ...). R 2 = 0.9994) and QN (0.5 ~ 15 μM, R 2 The concentration of ( = 0.9909) showed a good linear relationship. Figure 15 The LOD values ​​for CV and QN, calculated from smartphone images, are 0.33 μM and 0.17 μM, respectively.

[0119] The practicality and reliability of smartphone imaging detection for CV and QN were evaluated through spiked recovery tests on distilled water and fishpond water. The results are shown in Table 8. The CV recovery rate was 93.73%–108.16%, and the QN recovery rate was 91.07%–113.20%. The RSD values ​​were acceptable, indicating good recovery rates and RSD values.

[0120] Table 8 Recovery results of CV and QN detections

[0121]

[0122] This invention presents a novel dual-emission europium MOF (EuUCHNA). EuUCHNA exhibits remarkably stable fluorescence in water, is highly stable within a pH range of 4–8, and is sensitive to CV and QN responses with varying modalities. The detection limits for both CV and QN are low at 2.4 nM and 32.0 nM, respectively. More importantly, it enables simultaneous quantification of CV and QN in fishpond water. Furthermore, the integration with a smartphone facilitates instrument-free determination of CV and QN in actual water. This work provides a novel method for the simultaneous quantification of two analytes using a single fluorescence sensor.

[0123] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a europium-functionalized metal-organic framework dual-emission fluorescent probe, characterized in that, Includes the following steps: ZrCl4, pyromellitic acid, 6-hydroxy-2-naphthoic acid and water were mixed, and acetic acid was added to obtain a mixture; The mixture was heated under reflux, centrifuged to obtain a residue, and the residue was washed and dried to obtain UCHNA; The UCHNA was dispersed in an aqueous solution of Eu(NO3)3·6H2O, heated to react, and the white precipitate was collected, washed, and dried to obtain a dual-emission fluorescent probe based on europium-functionalized metal-organic frameworks. 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; The mass ratio of UCHNA to Eu(NO3)3·6H2O is 0.4~0.6:0.8~1.

2.

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

3. The preparation method according to claim 1, characterized in that, The temperature of the heating reflux is 80℃~120℃, and the heating reflux time is 20 h~28 h.

4. The preparation method according to claim 1, characterized in that, The heating reaction temperature is 70℃~90℃, and the heating reaction time is 20 h~28 h.

5. A dual-emission fluorescent probe based on europium-functionalized metal-organic frameworks, characterized in that, It is obtained by the preparation method described in any one of claims 1 to 4.

6. The application of the europium-functionalized metal-organic framework dual-emission fluorescent probe as described in claim 5 in the simultaneous detection of crystal violet and quinine in water or aquatic products.

7. A fluorescence sensor, characterized in that, Includes the europium-functionalized metal-organic framework-based dual-emission fluorescent probe as described in claim 5.

8. A method for simultaneously detecting crystal violet and quinine in water or aquatic products, characterized in that, The detection using the europium-functionalized metal-organic framework dual-emission fluorescent probe as described in claim 5 includes the following steps: The meat of aquatic products is added to a mixture of acetonitrile and PBS buffer solution, sonicated, centrifuged, and the supernatant is collected. The supernatant is then filtered through a filter membrane to obtain a pretreated sample; or, the collected aquaculture water sample is centrifuged and filtered to obtain pretreated water. The europium-functionalized metal-organic framework dual-emission fluorescent probe was added to water to prepare an EuUCHNA aqueous suspension. The pretreated sample or the pretreated water was added to the EuUCHNA aqueous suspension, and the fluorescence spectrum was measured.

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