Supramolecular fluorescent probe and application thereof

Abstract

The application discloses a supramolecular fluorescent probe which is prepared by a clathration reaction of a ring-opening cucurbituril and a cationic rose, wherein the molar ratio of the ring-opening cucurbituril and the cationic rose is 1:1; the supramolecular fluorescent probe is prepared by supramolecular assembly of the ring-opening cucurbituril and the cationic rose, and the flexible cavity of the ring-opening cucurbituril twists and restrains the cationic rose based on the principle of size adaptation, so that the intermolecular charge transfer emission state of the cationic rose is enhanced, the supramolecular fluorescent probe produces extremely strong orange-red fluorescence, the supramolecular fluorescent probe can realize dual-mode detection of hypochlorite anions in ultraviolet-visible light and fluorescence, has high sensitivity and extremely strong anti-interference ability, and can be widely applied in the fields of biomedicine, environmental monitoring, food safety and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of new materials, and particularly relates to a supramolecular fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Hypochlorous acid (HClO) is a highly efficient and powerful oxidant that can eliminate bacteria, viruses and other harmful substances. As a mild disinfectant, HClO is widely used in drinking water treatment, food processing, medical and environmental health, especially in various public places. However, excessive HClO can harm the human body, leading to various diseases, including coronary artery disease, Alzheimer's disease and kidney disease (Sensors and Actuators B: Chemical 284 (2019) 23-29). Therefore, it is necessary to develop a highly selective method for detecting HClO. Traditional methods for detecting HClO include titration, spectroscopic analysis, electrochemical analysis, chromatography and colorimetric method. However, these methods have the disadvantages of limited sensitivity and selectivity, time-consuming operation, high equipment requirements, etc. (ACS Applied Materials & Interfaces 11 (17) (2019) 15298-15305), and there is an urgent need to develop a supramolecular fluorescent probe with higher sensitivity for detection.

[0003] Cationic rose is an important organic dye. Because of its bright color and excellent dyeing performance, it has been widely used in the textile printing and dyeing industry and biomedical field and has been deeply researched. However, the molecular structure of cationic rose in aqueous solution tends to be more stable local excited state (LE state), and the twisting of its electron donor and acceptor parts causes the separation of HOMO-LUMO orbitals, resulting in the decoupling of electrons, which shows weak red fluorescence in aqueous solution. Current research on the regulation of twisted intermolecular charge transfer includes changing the type of solvent and increasing the solution viscosity, but all lack good water dispersibility and the potential for cell imaging.

[0004] Acyclic cucurbituril (ACB) is a new type of supramolecular host designed and synthesized by the Isaacs group in 2012 by combining the features of cucurbituril and pillararene. Unlike traditional cucurbituril, it has a flexible and variable c-shaped cavity, and is easy to perform chemical modification on the terminal aromatic wall. In addition, the negatively charged carbonyl port of acyclic cucurbituril has strong affinity for cationic compounds, which is conducive to better recognition and assembly of guest molecules (Chemical Science 15(14) (2024) 5163-5173). The hydrophobic cavity of acyclic cucurbituril has a orthogonal configuration that limits the torsion of the electron donor and acceptor of the guest molecule to 90°. This spatial restriction effectively hinders the transition of some dye molecules in aqueous solution from the intramolecular charge transfer (ICT) emission state to the non-radiative twisted intramolecular charge transfer (TICT) state, thereby promoting the enhancement of fluorescence (Analytica Chimica Acta 1254 (2023) 341095).

[0005] The supramolecular fluorescent probe is prepared by supramolecular assembly of acyclic cucurbituril and cationic rose, realizes dual-mode detection of hypochlorite, and has the ability of cell imaging and real water sample detection. The method has not been reported. SUMMARY

[0006] The application provides a supramolecular fluorescent probe based on acyclic cucurbituril and cationic rose, which has the advantages of good water dispersibility, low toxicity and high sensitivity, can realize dual-mode detection of hypochlorite, and also has the function of dyeing and imaging cells.

[0007] To achieve the above-mentioned purpose, the application places equimolar ratio of acyclic cucurbituril and cationic rose in pure water, stirs and dissolves at room temperature, and then avoids light and stirs to carry out inclusion reaction for 1-48h. The reaction product is purified and freeze-dried to prepare a supramolecular fluorescent probe (structural formula as follows);

[0008]

[0009] The structural formula of the acyclic cucurbituril is selected from the following:

[0010]

[0011] In which R is selected from -(CH2)3SO3Na, -O(CH2)2PO3Na2.

[0012] The structural formula of the cationic rose is as follows:

[0013]

[0014] The purification is by dialysis or membrane filtration.

[0015] The drying is freeze drying, vacuum drying or spray drying.

[0016] Another object of the present application is to apply the above-mentioned supramolecular fluorescent probe in the ultraviolet or fluorescent dual-mode detection of hypochlorite anions.

[0017] The absorbance of the ultraviolet-visible absorption spectrum is taken at a maximum absorption wavelength of 535 nm; the excitation wavelength of the fluorescence spectrum is 536 nm, and the emission wavelength is 586 nm.

[0018] The detection limit of the supramolecular fluorescent probe of formula I is 0.08 mg / L under the ultraviolet absorption spectrum and 0.012 mg / L under the fluorescence spectrum; the detection limit of the supramolecular fluorescent probe of formula II is 0.025 mg / L under the ultraviolet absorption spectrum and 0.015 mg / L under the fluorescence spectrum.

[0019] The supramolecular fluorescent probe of the present application has detection capability for hypochlorite ions in cells and real water samples.

[0020] The present application also provides a portable test paper containing the above-mentioned supramolecular fluorescent probe.

[0021] The test paper material for preparing the test paper is a strip-shaped qualitative filter paper, the concentration of the dyeing solution (supramolecular fluorescent probe) is 1 mg / mL, the dyeing time is 24 h, and the detection limit is 0.25 mg / L.

[0022] The present application focuses on acyclic cucurbiturils (ACBs), which have a unique structure constructed by one glycoluril tetramer unit and two aryl end groups carrying anionic sulfonate groups. This special structure has remarkable characteristics. On the one hand, it creates a C-shaped cavity with adjustable size, which can be fine-tuned according to the size difference of guest molecules. On the other hand, it greatly improves the water solubility. In terms of interaction with guest molecules, the aryl end groups can effectively carry out π-π interaction, and the multiple carbonyl structure can form multiple hydrogen bond interactions with the guest molecules. Based on the above characteristics, acyclic cucurbiturils exhibit excellent inclusion ability for neutral molecules or cations, which is of great significance in the technical solutions and application scenarios of the present application, and can be used as a core element for constructing various functional materials or in the fields of molecular recognition, separation and the like.

[0023] The acyclic cucurbiturils in the present application are prepared according to the method described in the existing literature (Nat. Chem., 2012, 4: 503.).

[0024] The present application has the following beneficial technical effects:

[0025] The supramolecular fluorescent probe based on the self-assembly of the open-loop cucurbituril and the cationic pink provided by the application is simple, safe and efficient in preparation, the supramolecular fluorescent probe applied to the detection of hypochlorite can not only realize dual-mode detection of ultraviolet and fluorescence, but also has the characteristics of high sensitivity, fast response speed, strong anti-interference ability and good biocompatibility, and the prepared test paper can also realize visual detection, and the supramolecular fluorescent probe can be applied to the fields of biomedical science, environmental science, chemical analysis and material science. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a nuclear magnetic resonance hydrogen spectrum comparison analysis diagram of the cationic pink, the inclusion compound and the open-loop cucurbituril M1 in Example 1, and the spectrum lines from top to bottom are the cationic pink, the inclusion compound and the open-loop cucurbituril M1 respectively;

[0027] Figure 2 It is a 2D-ROESY spectrum analysis diagram of the inclusion compound in Example 1;

[0028] Figure 3 It is a nuclear magnetic resonance hydrogen spectrum comparison analysis diagram of the cationic pink, the inclusion compound and the open-loop cucurbituril M2 in Example 2, and the spectrum lines from top to bottom are the open-loop cucurbituril M2, the inclusion compound and the cationic pink respectively;

[0029] Figure 4 It is a 2D-ROESY spectrum analysis diagram of the inclusion compound in Example 2;

[0030] Figure 5 It is an infrared spectrum comparison analysis diagram of the inclusion compound in Example 1; (a) the inclusion compound; (b) the physical mixture; (c) the cationic pink; (d) the open-loop cucurbituril M1;

[0031] Figure 6 It is an infrared spectrum comparison analysis diagram of the inclusion compound in Example 2; (a) the inclusion compound; (b) the physical mixture; (c) the cationic pink; (d) the open-loop cucurbituril M2;

[0032] Figure 7 It is an X-ray diffraction spectrum comparison analysis diagram of the inclusion compound in Example 1; (a) the inclusion compound; (b) the physical mixture; (c) the cationic pink; (d) the open-loop cucurbituril M1;

[0033] Figure 8 It is an X-ray diffraction spectrum comparison analysis diagram of the inclusion compound in Example 2; (a) the inclusion compound; (b) the physical mixture; (c) the cationic pink; (d) the open-loop cucurbituril M2;

[0034] Figure 9The UV-visible spectrophotometry and fluorescence detection results of the open-loop cucurbituril M1 and cationic pink inclusion compound in Example 3, wherein Figure A is the UV-visible absorption spectrum, Figure B is the linear fitting graph of the hypochlorite concentration (0-17.5 mg / L) and the absorbance value; Figure C is the fluorescence spectrum, and Figure D is the linear fitting graph of the hypochlorite concentration (0-4 mg / L) and the fluorescence intensity;

[0035] Figure 10 The UV-visible spectrophotometry and fluorescence detection results of the open-loop cucurbituril M2 and cationic pink inclusion compound in Example 3, wherein Figure A is the UV-visible absorption spectrum, Figure B is the linear fitting graph of the hypochlorite concentration (0.08-10 mg / L) and the absorbance value; Figure C is the fluorescence spectrum, and Figure D is the linear fitting graph of the hypochlorite concentration (0-10 mg / L) and the fluorescence intensity;

[0036] Figure 11 The test results of the anti-interference ability of the open-loop cucurbituril M1 and cationic pink inclusion compound in Example 4 in the detection application of hypochlorite after adding different interference substances;

[0037] Figure 12 The imaging schematic diagram of the open-loop cucurbituril M1 and cationic pink inclusion compound in cells in Example 5, wherein APFG is cationic pink + cells, ACBs@APFG is open-loop cucurbituril and cationic pink inclusion compound + cells, ACBs@APFG CIO - (5 μM) is open-loop cucurbituril and cationic pink inclusion compound + cells + 5 μmol / L sodium hypochlorite, ACBs@APFG CIO - (10 μM) is open-loop cucurbituril and cationic pink inclusion compound + cells + 10 μmol / L sodium hypochlorite

[0038] Figure 13 The color development schematic diagram of the test paper under visible light (right graph) and under fluorescence (left graph) in Example 6;

[0039] Figure 14 The linear relationship graph of the RGB value under visible light and the concentration of sodium hypochlorite solution;

[0040] Figure 15 The mass spectrometry mechanism graph of the supramolecular fluorescent probe for detecting hypochlorite in Example 7. DETAILED DESCRIPTION

[0041] The technical solutions of the present application are further described below by examples, but the content of the present application is not limited thereto. The methods in the present examples are all conventional methods unless otherwise specified, and the materials, reagents and the like are all obtained from commercial channels or prepared according to conventional methods unless otherwise specified;

[0042] Example 1: Preparation of the inclusion complex of the ring-opened cucurbituril Ml and cationic pink

[0043] Take 1 mmol of ring-opened cucurbituril Ml in 8 mL of ultrapure water, after stirring and dissolving at room temperature, 1 mmol of cationic pink is slowly added to the above aqueous solution, and after the addition of cationic pink is completed, it is immediately stirred for 24 h under light-protected conditions, after the reaction is completed, the reaction product is placed in a dialysis bag with a molecular weight cut-off of 500 Da for dialysis for 48 h in order to effectively remove free cationic pink, and finally the dialysis product is freeze-dried to obtain the powder of the inclusion complex of ring-opened cucurbituril Ml and cationic pink;

[0044] The structure of the ring-opened cucurbituril Ml is as follows:

[0045]

[0046] The results of the comparative analysis of the nuclear magnetic resonance hydrogen spectrum of the prepared inclusion complex are shown in Figure 1 The reason for the high-field displacement of the proton (H-8-16) peak of the N-(2-chloroethyl)-N-methyl aniline unit of cationic pink is the shielding effect of the ring-opened cucurbituril cavity. On the other hand, the deshielding effect of the ring-opened cucurbituril leads to the protons H-1, 2 to be displaced in the front field, while the rest of the proton peaks remain relatively unchanged. The 2D-ROESY spectrum analysis chart of the inclusion complex is shown in Figure 2 From the figure, it can be seen that H-4, H-5, H-6, H-8 on cationic pink all have significant correlation signals with H-a', H-b', H-c', H-g hydrogen protons inside the cavity of the ring-opened cucurbituril Ml, which all indicate the formation of the inclusion complex.

[0047] The comparative chart of the infrared spectra of the inclusion complex prepared in this example, the ring-opened cucurbituril Ml, the cationic pink, and the physical mixture of the ring-opened cucurbituril Ml and the cationic pink (1:1 mixed and ground) is shown in Figure 5 The comparative chart of the powder X-ray diffraction is shown in Figure 8 From the figure, it can be seen that the X-ray diffraction spectrum and the infrared spectrum of the prepared inclusion complex are obviously different from those of cationic pink itself, and different from those of the physical mixture of cationic pink and ring-opened cucurbituril Ml, thus it can be confirmed that the inclusion complex has been successfully prepared.

[0048] Example 2: Preparation of the inclusion complex of the ring-opened cucurbituril M2 and cationic pink

[0049] 1 mmol of open-ring cucurbituril M2 was placed in 8 mL of ultrapure water, stirred and dissolved at room temperature, and 1 mmol of cationic rutin was slowly added to the above aqueous solution. After the addition of cationic rutin was completed, stirring was continued for 24 hours under light-proof conditions. After the reaction was completed, the reaction product was placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed for 48 hours to effectively remove the free cationic rutin. Finally, the dialyzed product was freeze-dried to obtain a powdery inclusion complex of open-ring cucurbituril M2 and cationic rutin.

[0050] The structural formula of open-ring cucurbituril M2 is as follows:

[0051]

[0052] The results of the comparative analysis of the H NMR spectrum of the above-prepared inclusion compound are as follows: Figure 3 As shown in Figure 2, the reason why the proton (H-8~16) peak of the N-(2-chloroethyl)-N-methylaniline unit of cationic pink undergoes high-field shift is the shielding effect of the open-ring cucurbituril cavity, while the proton peaks of the remaining parts remain relatively unchanged, indicating the formation of inclusion complex; the 2D-ROESY spectrum analysis of the inclusion complex is shown in Figure 2. Figure 4 As shown, H-8, H-10, and H-12 on the cationic cucurbituril all have significant correlation signals with the H-a', H-b', H-c', and Hg hydrogen protons inside the open-ring cucurbituril M2 cavity, so the formation of the inclusion complex was further determined based on 2D-ROESY.

[0053] The infrared spectra comparison of the inclusion complex, open-ring cucurbituril M2, cationic pink, and the physical mixture of open-ring cucurbituril M2 and cationic pink prepared in this example are shown in FIG. Figure 6 , powder X-ray diffraction comparison diagram is shown in Figure 8 As can be seen from the figure, the X-ray diffraction pattern and infrared spectrum of the prepared inclusion complex are significantly different from those of cationic pink itself, and are different from the physical mixture of cationic pink and open-ring cucurbituril M2, so it can be confirmed that the inclusion complex has been successfully prepared.

[0054] Example 3: Determination of detection limit of supramolecular fluorescent probe

[0055] 1. Transfer 700 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 4.0) and 50 μL of the inclusion complex of open-ring cucurbituril M1 and cationic pink (1 mg / mL) into a 15 mL centrifuge tube and incubate at room temperature for 30 min. Then, sodium hypochlorite solution of different concentrations was added to the incubated solution, and the mixed solution was incubated at room temperature for 10 min.

[0056] The solution after incubation was detected by UV-visible spectrophotometry, the absorbance was measured at 535 nm wavelength, and the linear relationship between the concentration of sodium hypochlorite and the absorbance was obtained with the concentration of sodium hypochlorite as the abscissa and the absorbance as the ordinate, and the results are shown in Table 1. Figure 9 A, 9B;

[0057] The fluorescence intensity of the solution after incubation was measured at an excitation wavelength of 536 nm and an emission wavelength of 586 nm, the concentration of sodium hypochlorite was taken as the abscissa, and the fluorescence intensity was taken as the ordinate, to obtain the linear relationship between the concentration of sodium hypochlorite and the absorbance, and the results are shown in Table 2. Figure 9 C, 9D;

[0058] Figure 9 The results show that the detection limit of the open-ring cucurbituril M1 and cationic rose package compound by UV-visible spectrophotometry is 0.08 mg / L, and the detection limit under fluorescence spectrum is 0.012 mg / L.

[0059] 2. The detection limit determination method of open-ring cucurbituril M2 and cationic rose package compound is the same as step 1, and the results are shown in Table 3. Figure 10 The results show that the detection limit of open-ring cucurbituril M2 and cationic rose package compound by UV-visible spectrophotometry is 0.025 mg / L, and the detection limit under fluorescence spectrum is 0.015 mg / L.

[0060] Example 4: Specific detection experiment of supramolecular fluorescent probe

[0061] 1. 0.02 mg / L of metal ions (Na+, Mg, Ca 2+ , Zn 2+ , Co 2+ , Al 3+ , Na + or K + ), anions (SO4 2- , PO4 2- , CO3 2- , SO3 2- , Cl - , Br - , I - , NO3 - , NO2 - , CO3 2- , NO3 - , HCO3 - , SO3 2- , HPO4 2- , Cl - , I - , Br - , IO3 - , BrO3 -H2PO4 - or HPO4 - ), other possible interferents GSH and H2O2, incubate for 10 minutes at room temperature, measure the fluorescence intensity at 536 nm excitation wavelength, 586 nm emission wavelength, then add 0.02 mg / L sodium hypochlorite to it, detect the fluorescence intensity again, the results are shown in Figure 11 , the results show that the inclusion complex of open-loop cucurbituril M1 and cationic pink has high selectivity and anti-interference ability for the detection of hypochlorite.

[0062] 2, the detection method of the inclusion complex of open-loop cucurbituril M2 and cationic pink is the same as above, the results show that the inclusion complex of open-loop cucurbituril M2 and cationic pink has high selectivity and anti-interference ability for the detection of hypochlorite.

[0063] Example 5: Cell imaging detection experiment of the inclusion complex of open-loop cucurbituril and cationic pink

[0064] 20 μL of the inclusion complex of open-loop cucurbituril M1 and cationic pink with a concentration of 1 × 10 -4 mol / L dissolved in DMEM medium was incubated with HeLa cells or L02 cells at 37℃, 5% CO2 in a humidified incubator for 12 hours, then 5 μM, 10 μM of sodium hypochlorite was added to it respectively, incubated for 2h, then the cells were washed with PBS for 5 times to remove the non-internalized free molecules and eliminate their interference, the cell imaging ability of the inclusion complex of open-loop cucurbituril and cationic pink was studied by using laser confocal scanning microscope (CLSM, Eclips Ti, Nikon), and cationic pink + cells were set as control (APFG);

[0065] The cell imaging results are shown in Figure 12 , the experimental group added with the inclusion complex of open-loop cucurbituril M1 and cationic pink showed stronger red fluorescence than the group added with cationic pink, and the red fluorescence gradually weakened after the addition of hypochlorite, which indicated that hypochlorite could weaken the red fluorescence of the inclusion complex of open-loop cucurbituril M1 and cationic pink, so the inclusion complex had the ability to detect intracellular hypochlorite anions.

[0066] Example 6: Detection experiment of the inclusion complex of open-loop cucurbituril and cationic pink on water samples

[0067] The real water samples tap water were taken from the laboratory of Kunming University of Science and Technology, and river water was taken from Luoyu River in Chenggong District of Kunming City, 3 different concentrations (0 mg / L, 1 mg / L, 3 mg / L) of sodium hypochlorite solution and 1 mg of the inclusion complex of open-loop cucurbituril M1 and cationic pink were added to the sample to be tested, and the fluorescence intensity was measured at 536 nm excitation wavelength, 586 nm emission wavelength after incubation for 10 minutes at room temperature, and the recovery rate was calculated; the results are shown in Table 1;

[0068] Table 1

[0069]

[0070] Example 7: Preparation and application of test paper

[0071] Place a strip of qualitative filter paper into a 1 mg / mL solution of the inclusion complex of open-ring cucurbituril and cationic pink, ensuring that the filter paper is completely immersed in the dye solution. Use tweezers to gently press the filter paper to fully soak it and expel air bubbles to ensure uniform dyeing. After soaking, remove the filter paper with tweezers, place it flat on a glass plate, and place it in a vacuum drying oven to dry for 24 hours. Remove it and place it in a light-proof, sealed bag for later use.

[0072] Remove the test strip from its sealed packaging and quickly use a dropper to draw up an appropriate amount of the sample to be tested (sodium hypochlorite solution, concentration range 0-20 mg / L). Add it dropwise to the reaction area of ​​the test strip, ensuring that the liquid evenly covers the reaction area and avoids local drying or accumulation of liquid. The amount of liquid added should just wet the reaction area without overflowing, usually 2-3 drops (about 0.1-0.2 mL). Allow the test strip to stand at room temperature (20-25°C), out of direct sunlight, and wait for the color reaction to stabilize, which generally takes 1-3 minutes. Do not shake or touch the test strip during this time to prevent uneven color from affecting the judgment.

[0073] Place the developed test paper under natural light and use a smartphone to collect color images of filter paper strips with different concentrations ( Figure 13 The RGB value of the captured image was read using the image processing software Fiji Image J. The linear relationship between the RGB value and the concentration of the sodium hypochlorite solution is shown in Figure 14 ,From the results, it can be seen that the RGB value of the test paper has a strong linear correlation with the hypochlorite concentration, and the detection limit is 0.25 mg / L according to the fitting curve;

[0074] The filter paper strips with different concentrations were colored under a handheld UV lamp. The results are shown in Figure 13 As can be seen in the left figure, the color of the test paper changes from red to blue as the concentration of hypochlorite increases.

[0075] Example 8: Fluorescence quenching mechanism of the open-ring cucurbituril and cationic rosin inclusion complex

[0076] Prepare 3×10 -5: mol / L of open-ring cucurbituril and cationic rosin inclusion complex in acetate buffer (0.1 M, pH = 4.0), to which 3×10 -4 mol / L hypochlorite aqueous solution was vortexed for 2 h, and the possible quenching mechanism of hypochlorite was explored by high-resolution mass spectrometry;

[0077] Results are shown in Figure 2 Figure 15 The experiment showed that the molecular ion peak of APFG (m / z = 388.1473) disappeared after the addition of hypochlorite, however, two new molecular ion peaks (m / z = 209.0971, [M+H] + = 210.0840), (m / z = 197.0607) appeared after the double bond cleavage. Since the inclusion complex reacted in the acetic acid buffer solution, the acetic acid ion attacked the aldehyde group, which was also confirmed by the molecular ion peak (m / z = 299.0880). Based on the above, we speculate that the structure of the intermolecular electron transfer is destroyed after the double bond of cationic rose b is oxidized and cleaved by hypochlorite, leading to fluorescence quenching.

Claims

1. A supramolecular fluorescent probe, characterized in that: It is prepared by inclusion reaction of open-ring cucurbituril and cationic rosin, wherein the molar ratio of open-ring cucurbituril to cationic rosin is 1:1; The open-ring cucurbituril structural formula is as follows: ； or ； wherein R is selected from -(CH2)3SO3Na, -O(CH2)2PO3Na2; The cationic pink structural formula is as follows: .

2. The supramolecular fluorescent probe according to claim 1, wherein: Equimolar ratios of open-ring cucurbituril and cationic rosin are placed in pure water, stirred and dissolved at room temperature, and then stirred in the dark for 1 to 48 hours. The reaction product is purified, concentrated and dried to prepare a supramolecular fluorescent probe.

3. Use of the supramolecular fluorescent probe according to claim 1 in detecting hypochlorite anions.

4. The use according to claim 3, characterized in that: Hypochlorite anion is detected by UV-visible spectrophotometry or fluorescence.

5. A test strip, characterized in that: The method comprises the supramolecular fluorescent probe according to claim 1.

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