Dual-emission ratiometric fluorescent probe rb@zr-fcu-sti, its preparation and application in detection of malachite green

Abstract

The application discloses a double-emission ratio fluorescent probe RB@Zr-fcu-sti, a preparation method thereof and application thereof in detection of malachite green (MG). The preparation method comprises the following steps: adding Zr-fcu-sti and rhodamine B into anhydrous ethanol, ultrasonic dispersion, stirring reaction at room temperature in dark, centrifugation, washing, and vacuum drying. The double-emission ratio fluorescent probe RB@Zr-fcu-sti is applied to the fluorescence detection of malachite green, and has high sensitivity and fast response. With the increase of the concentration of the sensing system malachite green (MG), the red fluorescence emission of the probe is significantly quenched, while the intensity of the blue fluorescence emission remains basically unchanged, so that the fluorescence color of the sensing system gradually changes from purple to blue. The detection limit of the application to malachite green (MG) is as low as 2.69nM, and thus the application can be applied to high-sensitivity ratio fluorescence sensing of malachite green (MG).

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection and analysis technology, and specifically relates to a dual emission ratio fluorescent probe RB@Zr-fcu-sti and its preparation and application in malachite green detection. Background Technology

[0002] Malachite green (MG) possesses broad-spectrum antibacterial activity and has been widely used in aquaculture as an antibacterial, antifungal, and antiparasitic agent. However, due to its acute toxicity and potential carcinogenicity and teratogenicity in animals and humans, the addition of malachite green to aquaculture ponds is not only toxic to organisms but also has the potential to bioaccumulate through the food chain, ultimately affecting human health. Therefore, many countries have banned the use of malachite green in aquaculture. Nevertheless, due to its unique antibiotic properties and low cost, the illegal use of malachite green in aquaculture remains widespread. Therefore, finding a simple, rapid, and accurate method to monitor and quantify residual malachite green levels in the environment is of great importance.

[0003] Due to their high sensitivity, simple equipment, and ease of operation, fluorescence-based analytical methods provide a powerful tool for the detection of residual malachite green in the environment. In recent years, researchers have focused on developing various fluorescent probes for detecting malachite green, including "off" probes with only a single-channel fluorescence signal and "ratio-modulation" fluorescent probes with dual-channel fluorescence signals. In contrast, ratio-modulation methods based on dual-channel fluorescence signals incorporate correction for external interference, thereby minimizing the influence of experimental factors such as instrument bias and solvent effects, achieving higher analytical accuracy. However, there are currently few reports on probes for ratio-modulation fluorescence sensing of malachite green. Therefore, there is a need to develop novel "ratio-modulation" fluorescent probes for highly sensitive detection of malachite green.

[0004] Metal-organic frameworks (MOFs) are a class of porous crystalline materials with well-defined structures, designable topologies, and high porosity, and have been widely used in separation, catalysis, fluorescence sensing, and drug delivery. However, for fluorescence sensing, most MOF materials are not stable enough in water, limiting their application in aqueous media. Due to the strong interaction between the high-valence Zr(Ⅳ) group and the carboxyl group, UiO series MOF materials typically exhibit significant water stability. Importantly, certain UiO series materials, such as MOF-801, UiO-66-N3, UiO-67, and Zr-fcu-sti, can serve as host materials for loading fluorescent dye molecules to construct dual-emission ratiometric fluorescent probes. For example, It can achieve the removal of Cr2O7 from water. 2- High sensitivity ratio fluorescence detection[1] ;fluorescin@UiO-67 can achieve Al in water 3+ High sensitivity sensor [2] RhB / UiO-66-N3 can achieve the removal of sulfur in water. 2- sensitive sensing [3] However, there are currently no examples of constructing dual-emission ratiometric fluorescent probes based on Zr-fcu-sti host loaded with Rhodamine B dye. Summary of the Invention

[0005] Based on the aforementioned technical background, this invention, for the first time, constructs a dual-emission fluorescent probe RB@Zr-fcu-sti by loading Rhodamine B (RB) dye onto a Zr-fcu-sti substrate, and applies it to ratiometric fluorescence sensing of malachite green (MG), achieving highly sensitive detection of malachite green (MG). This invention is simple to operate and offers advantages such as fast response speed and high sensitivity during detection.

[0006] Specifically, the present invention is implemented using the following technical solution:

[0007] Firstly, the present invention provides a method for preparing a dual emission ratio fluorescent probe RB@Zr-fcu-sti, which involves reacting the compound Zr-fcu-sti with Rhodamine B in anhydrous ethanol at room temperature in the dark, followed by post-treatment.

[0008] A preferred preparation method described above involves adding Zr-fcu-sti and Rhodamine B to anhydrous ethanol, ultrasonically dispersing, stirring and reacting at room temperature in the dark for 20-30 hours, centrifuging at 6000-10000 rpm for 2-8 minutes, washing the precipitate 1-6 times with anhydrous ethanol, and then vacuum drying. The mass ratio of Zr-fcu-sti to Rhodamine B is 100:1-5, and the mass ratio of Zr-fcu-sti to anhydrous ethanol is 100:30-80, mg / mL. Zr-fcu-sti is prepared by a known method; the specific preparation process can be found in J. Am. Chem. Soc. 2018, 140, 15022-15030.

[0009] In the preparation method described above, preferably, the mass ratio of compound Zr-fcu-sti to Rhodamine B is 100:2-3, and the mass ratio of compound Zr-fcu-sti to the volume ratio of anhydrous ethanol is 100:40-60, mg / mL.

[0010] In the preparation method described above, preferably, the washing involves placing the precipitate in anhydrous ethanol and sonicating it for 6-15 minutes, followed by centrifugation at 6000-10000 rpm for 2-8 minutes.

[0011] In the preparation method described above, preferably, the vacuum drying oven is used to treat the product at 50-70°C for 8-15 hours.

[0012] A more preferred preparation method described above is to add 100 mg Zr-fcu-sti and 2.75 mg Rhodamine B to anhydrous ethanol, disperse by ultrasonication, stir and react at room temperature in the dark, centrifuge, and wash the precipitate four times with anhydrous ethanol. The washing process is as follows: ultrasonication for 10 min, centrifugation at 8000 rpm for 5 min; place the washed solid product in a vacuum drying oven and treat it at 60°C for 12 h.

[0013] Secondly, this invention provides a dual-emission ratiometric fluorescent probe RB@Zr-fcu-sti obtained by the preparation method described above. The ratiometric fluorescent probe RB@Zr-fcu-sti provided by this invention has dual emission characteristics, that is, it simultaneously exhibits red fluorescence emission from rhodamine B dye and blue fluorescence emission from Zr-fcu-sti.

[0014] Thirdly, this invention also provides the application of the dual-emission ratio fluorescent probe RB@Zr-fcu-sti in the detection of malachite green. Specifically, the application can be carried out using the following method: Take 1 mL of RB@Zr-fcu-sti suspension with a concentration of 0.2 mg / mL, add different volumes of malachite green solution with a concentration of 2.5 μM, dilute to 2 mL, incubate in the dark for 10-20 min, measure the fluorescence emission spectrum of the RB@Zr-fcu-sti-malachite green system, establish the relationship between the change in fluorescence peak intensity ratio and the concentration of malachite green, and realize the quantitative detection of malachite green (MG).

[0015] In the aforementioned applications, preferably, the solvent used for the RB@Zr-fcu-sti suspension and the malachite green solution is a 10mM HEPES buffer solution with a pH of 7.3. Of course, the solvent used for volume adjustment can also be a HEPES buffer solution.

[0016] The malachite green (MG) ratiometric fluorescence sensing method provided by this invention features high sensitivity and fast response. As the concentration of malachite green (MG) in the sensing system increases (0-200 nM), the red fluorescence emission of the probe is significantly quenched, while the intensity of the blue fluorescence emission remains essentially unchanged, causing the fluorescence color of the sensing system to gradually evolve from purple to blue. Its detection limit for malachite green (MG) is as low as 2.69 nM. Furthermore, preferably, the optimal excitation wavelength for measuring the fluorescence emission spectrum of the RB@Zr-fcu-sti-malachite green system in the above method is 359 nm.

[0017] The key advantages of this invention are:

[0018] (1) The malachite green (MG) ratio fluorescence sensing method based on dual emission probe RB@Zr-fcu-sti has advantages such as a low detection limit (detection limit as low as 2.69 nM) and a short response time (response time is 5 min);

[0019] (2) Compared with existing technologies, this invention is the first to employ an adsorption-based pre-concentration strategy to improve the sensitivity of the probe to malachite green (MG) fluorescence sensing. Specifically, RB@Zr-fcu-sti with appropriate pore size can effectively adsorb and pre-concentrate malachite green (MG), promoting the donor-acceptor interaction between Rhodamine B (RB) molecules and malachite green (MG) molecules within the Zr-fcu-sti pores (i.e., shortening the spatial distance between RB and MG molecules), thereby significantly improving the sensitivity of the probe to malachite green (MG) fluorescence sensing. Resonant Energy Transfer (FRET) efficiency. Based on high efficiency Resonant energy transfer (FRET) shows that the red fluorescence emission of rhodamine B (RB) can be significantly quenched by malachite green (MG), while the intensity of the blue fluorescence of Zr-fcu-sti is almost unaffected. This phenomenon can be applied to high-sensitivity ratio fluorescence sensing of malachite green (MG). Attached Figure Description

[0020] Figure 1 Powder X-ray diffraction (PXRD) patterns of Zr-fcu-sti and RB@Zr-fcu-sti, and simulated diffraction pattern of Zr-fcu-sti crystal (CCDC No.: 1451622);

[0021] Figure 2 N2 adsorption-desorption curves of Zr-fcu-sti and RB@Zr-fcu-sti at 77 K;

[0022] Figure 3 : Investigation into the optimal incubation time;

[0023] Figure 4Fluorescence spectra of RB@Zr-fcu-sti in response to different concentrations (0-200 nM) of malachite green (MG);

[0024] Figure 5 Linear relationship of RB@Zr-fcu-sti response to different concentrations (0-200 nM) of malachite green (MG);

[0025] Figure 6 Fluorescence images of RB@Zr-fcu-sti responding to different concentrations (0-200 nM) of malachite green (MG) (excitation source: 365 nm);

[0026] Figure 7 : Selectivity of probe RB@Zr-fcu-sti for malachite green (MG) sensing;

[0027] Figure 8 : The interference immunity of probe RB@Zr-fcu-sti to malachite green (MG) sensing. Detailed Implementation

[0028] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the reagents used can be purchased from chemical or biological reagent companies.

[0029] Example 1

[0030] Preparation and characterization of dual-emission fluorescent probe RB@Zr-fcu-st i

[0031] The compound Zr-fcu-sti used in this invention was prepared by a conventional method, the preparation process of which can be found in reference J. Am. Chem. Soc. 2018, 140, 15022-15030. The preparation process is briefly described as follows: In a 50 mL polytetrafluoroethylene-lined reactor, 15 mL of DMF solvent was added to trans-4,4'-stilbene dicarboxylic acid (0.45 mmol), ZrCl4 (0.45 mmol), L-proline (2 mmol), and 45 μL of concentrated HCl. The mixture was heated at 120 °C for 16 h, allowed to cool naturally, and the precipitate was collected by centrifugation. The precipitate was then soaked successively in DMF for 72 h (with fresh DMF replaced every 12 h) and in methanol for 24 h (with fresh methanol replaced every 6 h). The mixture was then vacuum dried at 85 °C and a pressure <50 Pa for 12 h to obtain Zr-fcu-sti.

[0032] Preparation steps of RB@Zr-fcu-sti: Zr-fcu-sti (100 mg) and Rhodamine B (2.75 mg) were added to 50 mL of ethanol, ultrasonically dispersed for 5 min, and then stirred at room temperature in the dark for 24 h. The mixture was then centrifuged (8000 rpm, 5 min). The precipitate was washed four times with anhydrous ethanol (50 mL of anhydrous ethanol each time, the washing process being: ultrasonication for 10 min, centrifugation for 5 min [8000 rpm]). The washed solid product was placed in a vacuum drying oven and treated at 60 °C for 12 h to obtain 92.3 mg of pink solid, with a yield of 92.3% (based on the mass of Zr-fcu-sti).

[0033] The prepared Zr-fcu-sti and RB@Zr-fcu-sti samples were subjected to powder X-ray diffraction (PXRD) and SEM tests, respectively, as shown in the figures. Figure 1 and Figure 2 As shown in the PXRD diagram ( Figure 1 As shown in the image, the powder X-ray diffraction pattern of the Zr-fcu-sti sample exhibits two main peaks located at 5.2° and 6.0°, respectively, which match the simulated Zr-fcu-sti crystal diffraction pattern, confirming the successful synthesis of Zr-fcu-sti. Furthermore, RB@Zr-fcu-sti shows a nearly unchanged diffraction pattern, demonstrating that the material maintains good crystallinity. (BET test...) Figure 2 The results show that the BET specific surface area of ​​RB@Zr-fcu-sti is slightly reduced compared to Zr-fcu-sti (from 1216.5 m²). 2 / g decreased to 1171.2m 2 / g), indicating that the Rhodamine B molecule occupies part of the pores of Zr-fcu-sti.

[0034] Example 2

[0035] Application of dual-emission fluorescent probe RB@Zr-fcu-st i in the detection of malachite green (MG)

[0036] Step (1): Optimal incubation time investigation: Weigh 10 mg of RB@Zr-fcu-sti solid, add 50 mL of 10 mM HEPES buffer solution (pH = 7.3), and sonicate for 30 min to obtain a uniformly dispersed RB@Zr-fcu-sti suspension with a concentration of 0.2 mg / mL. Add 1.0 mL of the prepared RB@Zr-fcu-sti suspension (0.2 mg / mL) to the system, then add 920 μL of HEPES buffer, followed by 80 μL of 2.5 μM malachite green (MG) HEPES buffer. Measure the fluorescence spectrum using a fluorescence spectrophotometer at 0 min, 1 min, 2 min, 3 min, 5 min, 10 min, 15 min, and 30 min (mix thoroughly before measurement; excitation wavelength is 359 nm).

[0037] Results of the study on optimal incubation time are as follows Figure 3 As shown. After the addition of malachite green (MG), the fluorescence intensity of RB@Zr-fcu-sti increased rapidly and reached a stable level after 5 minutes. Figure 3 The results show that the malachite green (MG) ratio fluorescence sensing method based on RB@Zr-fcu-sti has a shorter response time.

[0038] Step (2): Fluorescence titration experiment: Add 1.0 mL of RB@Zr-fcu-sti suspension (0.2 mg / mL) prepared in step (1) to the system, and then add different volumes (0, 20, 40, 60, 80, 100, 120, 140 and 160 μL) of HEPES buffer with a concentration of 2.5 μM malachite green (MG) to the system. Make up the volume to 2 mL with HEPES buffer (the concentrations of malachite green in the system are 0 nM, 25 nM, 50 nM, 75 nM, 100 nM, 125 nM, 150 nM, 175 nM and 200 nM, respectively). Incubate in the dark for 5 min, mix thoroughly, and immediately measure the fluorescence spectrum (excitation wavelength 359 nm) using a fluorescence spectrophotometer. By establishing the relationship between the change in the fluorescence peak intensity ratio and the concentration of malachite green (MG), the quantitative detection of malachite green (MG) can be achieved.

[0039] The results of the fluorescence titration experiment are as follows Figure 4 and Figure 5 As shown, with increasing malachite green concentration, the red emission of Rhodamine B dye was significantly quenched, while the blue emission remained almost unchanged. Figure 4 The arrow indicates that the red fluorescence emission gradually weakens as the concentration gradually increases. Simultaneously, the fluorescence intensity ratio (F...) B / F R ) / (F B,0 / F R,0A good linear relationship was established between the concentration of malachite green and the concentration of malachite green (R). 2 =0.996). The linear calibration relationship is expressed as (F B / F R ) / (F B,0 / F R,0 ) = 0.00603 × [MG] + 0.968, where (F B / F R ) and (F B,0 / F R,0 The values ​​represent the ratios of blue fluorescence intensity to red fluorescence intensity in the presence and absence of malachite green, respectively. Figure 5 Based on the data from the standard curve, the limit of detection (LOD) was calculated to be 2.69 nM. Figure 4 As can be seen, RB@Zr-fcu-sti exhibits good sensitivity and a low detection limit for malachite green. Furthermore, as... Figure 6 As shown, the system appears purple without the addition of malachite green, but with the addition of malachite green, the red fluorescence emission gradually quenches, causing the fluorescence color of the system to gradually change to blue.

[0040] Step (3): Fluorescence selectivity experiment: Add 1.0 mL of the RB@Zr-fcu-sti suspension (0.2 mg / mL) prepared in step (1) to the system, and then add 120 μL of 2.5 μM malachite green HEPES buffer or 30 μL of 0.1 mM of 11 interfering substances (K) to the system. + Na + NH4 + CO3 2- SO4 2- H2PO4 - HPO4 2- Ac - The HEPES buffer (containing nitrofurantoin, secnidazole, and enrofloxacin) was adjusted to 2 mL to make the concentration of malachite green 150 nM and the concentration of interferon 1.5 μM. The mixture was incubated in the dark for 5 min, and after mixing well, its fluorescence emission spectrum was immediately scanned with a fluorescence spectrophotometer.

[0041] Fluorescence selectivity experiment see Figure 7 .like Figure 7 As shown, the addition of 1.50 μM of interfering agent did not significantly affect the fluorescence of RB@Zr-fcu-sti, demonstrating good selectivity for malachite green (MG).

[0042] Step (4): Fluorescence interference experiment: Add 1.0 mL of the RB@Zr-fcu-sti suspension (0.2 mg / mL) prepared in step (1) to the system, and then add 30 μL of 11 interfering substances (K) with a concentration of 0.1 mM to the system in sequence. + Na + NH4 + CO3 2- SO4 2- H2PO4 - HPO4 2- Ac - (nitrofurantoin, secnidazole and enrofloxacin), then 120 μL of 2.5 μM malachite green HEPES buffer was added to each, and the volume was adjusted to 2 mL with HEPES buffer so that the concentrations of malachite green and the interfering substances in the system were 150 nM and 1.5 μM, respectively. The mixture was incubated in the dark for 5 min, and after mixing well, its fluorescence emission spectrum was immediately scanned with a fluorescence spectrophotometer.

[0043] Fluorescence interference experiment see Figure 8 .like Figure 8 As shown, the presence of 1.50 μM of interfering material did not significantly interfere with the response of RB@Zr-fcu-sti to malachite green. The results indicate that the ratiometric fluorescent probe RB@Zr-fcu-sti exhibits good anti-interference properties in its response to malachite green.

[0044] References:

[0045] [1]Yoo Jounghyun, Ryu UnJin, Kwon Woosung, Choi Kyung Min. A multi-dyecontaining MOF for the ratiometric detection and simultaneous removal ofCr2O7 2- in the presence of interfering ions. Sensors and Actuators B: Chemical, 2019, 283, 426-433.

[0046] [2]Yang Li,Liu Yao,Chen Lili,Guo Lulu,Lei You,Wang Li.Stabledual-emissive fluorescin@UiO-67metal-organic frameworks for visual and ratiometricsensing of Al 3+and ascorbic acid Spectrochimica Acta Part A:Molecular andBiomolecular Spectroscopy,2021,261,15,120068.

[0047] [3]Gao Xia,Sun Guangming,Wang Xinke,Lin Xiaodong,Wang Shuo,LiuYaqing.RhB / UiO-66-N3MOF-based ratiometric fluorescent detection andintracellular imaging of hydrogen sulfide.Sensors and Actuators B:Chemical,2021,331,129448。

Claims

1. The application of a dual emission ratio fluorescent probe RB@Zr-fcu-sti in the detection of malachite green, wherein the dual emission ratio fluorescent probe RB@Zr-fcu-sti is obtained by reacting the compound Zr-fcu-sti with Rhodamine B in anhydrous ethanol at room temperature in the dark and followed by post-treatment.

2. Use according to claim 1, wherein Zr-fcu-sti and Rhodamine B were added to anhydrous ethanol, ultrasonically dispersed, and stirred at room temperature in the dark for 20-30 h. The mixture was then centrifuged at 6000-10000 rpm for 2-8 min, and the precipitate was washed 1-6 times with anhydrous ethanol and then vacuum dried. The mass ratio of Zr-fcu-sti to Rhodamine B was 100:1-5, and the mass ratio of Zr-fcu-sti to the volume ratio of anhydrous ethanol was 100:30-80, mg / mL.

3. The application as described in claim 2, characterized in that, The mass ratio of compound Zr-fcu-sti to Rhodamine B is 100:2-3, and the mass ratio of compound Zr-fcu-sti to the volume ratio of anhydrous ethanol is 100:40-60, mg / mL.

4. The application as described in claim 2, characterized in that, The washing process involves placing the precipitate in anhydrous ethanol and sonicating it for 6-15 minutes, followed by centrifugation at 6000-10000 rpm for 2-8 minutes.

5. The application as described in claim 2, characterized in that, The vacuum drying process involves treating the sample at 50-70°C for 8-15 hours.

6. The application as described in any one of claims 1-5, characterized in that, 100 mg Zr-fcu-sti and 2.75 mg Rhodamine B were added to anhydrous ethanol, ultrasonically dispersed, stirred at room temperature in the dark, centrifuged, and the precipitate was washed four times with anhydrous ethanol. The washing process was: ultrasonic for 10 min, centrifuged at 8000 rpm for 5 min. The washed solid product was placed in a vacuum drying oven and treated at 60 ℃ for 12 h.

7. The application as described in claim 1, characterized in that, The following steps were used: 1 mL of RB@Zr-fcu-sti suspension with a concentration of 0.2 mg / mL was taken, and different volumes of malachite green solution with a concentration of 2.5 μM were added. The volume was adjusted to 2 mL, and the mixture was incubated in the dark for 10-20 min. The fluorescence emission spectrum of the RB@Zr-fcu-sti-malachite green system was measured, and the relationship between the change in the fluorescence peak intensity ratio and the concentration of malachite green was established to achieve quantitative detection of malachite green.

8. The application as described in claim 7, characterized in that, The solvent used for the RB@Zr-fcu-sti suspension and the malachite green solution was HEPES buffer solution with a concentration of 10 mM and a pH of 7.3.

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