Misaligned 10-membered cucurbit ring assembly and its detection of ClO in kidney HK2 cells - Applications

Abstract

The present invention discloses a misaligned decacyclodextrin-based assembly and its application for detecting ClO⁻ in kidney HK2 cells. The supramolecular assembly is prepared by mixing a misaligned decacyclodextrin solution and a cationic rose bengal solution. The supramolecular assembly of the present invention can detect ClO⁻ in aqueous solutions and HK-2 cells, and has the characteristics of high selectivity, good sensitivity, wide detection range, fast response time, and minimal damage to samples; in addition, it also has the characteristics of simple preparation method of the fluorescent probe and good biocompatibility.

Description

Technical Field

[0001] The present invention relates to a supramolecular assembly and its application, in particular to a dislocated ten-membered cucurbitacin ring-based assembly and its application for detecting ClO in kidney HK2 cells - Applications Background Art

[0002] In some disinfection scenarios, hypochlorite anion (ClO - ) solution is widely used as a disinfectant because of its strong oxidizing property. However, long-term and large-scale use will cause damage to the environment, especially in sewage, where high content of ClO - It will cause great damage to the environment and ecology.

[0003] In addition, ClO - It is also an important reactive oxygen species and has been widely used in clinic for nearly a century as an efficient and rapid broad-spectrum antibacterial drug. In addition, endogenous ClO is also produced in organisms. - It is mainly catalyzed by myeloperoxidase in phagocytes to react with H2O2 and chloride ions (Cl - ) reaction. Although ClO - It can effectively eliminate bacteria in organisms, but excessive or incorrect ClO production during inflammation may occur. - It can cause or aggravate the onset of various diseases, such as Alzheimer's disease, inflammatory diseases, atherosclerosis, cardiovascular disease, cancer and kidney disease.

[0004] Given that ClO - Due to its importance in disinfection and biological clinical practice, people have to continue to use it extensively. However, in order to protect the environment and protect life and health, the ClO in the environment and biological systems - Monitoring is very important.

[0005] Currently used to detect ClO in environmental aqueous solutions or organisms - There are many methods for detecting environmental pollution, including iodine titration, colorimetry, chemiluminescence, coulometric method, polarography and radiolysis. However, these methods usually have complex equipment, high professional requirements and high costs, which greatly limits their popularization and application. In contrast, fluorescence method is considered to be an ideal method for environmental monitoring and biological research because of its advantages such as simple detection instrument, high selectivity, good sensitivity, wide detection range, fast response time and minimal damage to samples.

[0006] Therefore, how to design a ClO - The fluorescent probe for the detection of ClO in aqueous solution and in vivo is a solution to the fluorescence detection of ClO - The key. Summary of the invention

[0007] The object of the present invention is to provide a dislocated ten-membered cucurbitacinyl assembly and its application in detecting ClO- in kidney HK2 cells. The supramolecular assembly of the present invention can detect ClO- in aqueous solution and HK-2 cells. - , and has the characteristics of high selectivity, good sensitivity, wide detection range, fast response time, and minimal damage to the sample; in addition, it also has the characteristics of simple fluorescent probe preparation method and good biocompatibility.

[0008] The technical solution of the present invention is a dislocated ten-membered cucurbit ring-based assembly, the molecular formula of which is 2C 22 H 26 Cl2N2@C 54 H 60 N 40 O 20 , the structural formula is as follows:

[0009]

[0010] in, It is a dislocated ten-yuan melon ring.

[0011] The above-mentioned dislocated ten-membered cucurbitacin ring-based assembly is prepared by mixing a dislocated ten-membered cucurbitacin ring solution and a cationic pink solution.

[0012] The above-mentioned dislocated ten-membered cucurbitacinyl ring-based assembly, the preparation method of the supramolecular assembly is as follows:

[0013] (1) Take a dislocated ten-membered melon ring, add deionized water to dissolve it, and obtain solution A;

[0014] (2) Take cationic pink, add deionized water to dissolve, and obtain solution B;

[0015] (3) Solution A and solution B are mixed and reacted to obtain the misaligned ten-membered cucurbitacin ring-based assembly.

[0016] The above-mentioned dislocated ten-membered cucurbit ring-based assembly, the concentration of the dislocated ten-membered cucurbit ring in the solution A is 1.00×10 - 3 mol / L; the concentration of cationic pink in solution B is 1.00×10 -3 mol / L; when the solution A and the solution B are mixed, the molar ratio of the dislocated ten-membered cucurbitacin ring to the cationic pink is 1:2.

[0017] In the aforementioned dislocated ten-membered cucurbitacinyl ring assembly, the reaction in step (3) is carried out at room temperature and pressure.

[0018] A dislocated ten-membered cucurbit ring-based assembly is used to detect ClO in aqueous solution. -application.

[0019] The above-mentioned misaligned ten-membered cucurbit ring-based assembly was used to detect ClO in aqueous solution. - The specific detection methods are as follows:

[0020] (1) Take the dislocated ten-membered cucurbitacinyl ring assembly and dilute it with water until the concentration of the dislocated ten-membered cucurbitacinyl ring assembly is 3.00×10 -5 mol / L, and the probe standard solution was obtained;

[0021] (2) adding the aqueous solution to be tested to the prepared probe standard solution, leaving it for 5 min, and then measuring the fluorescence emission spectrum at a fixed excitation wavelength of 523 nm, and plotting the change curve of the fluorescence intensity at the excited laser wavelength;

[0022] (3) According to the curve of step (2), the change value ΔI of the fluorescence emission spectrum intensity at 586 nm corresponding to the addition of the water to be tested to the probe standard solution can be calculated to obtain the ClO in the aqueous solution. - ions are detected.

[0023] A misaligned ten-membered cucurbit ring-based assembly was used to detect ClO in kidney HK2 cells. - application.

[0024] The above-mentioned misaligned ten-membered cucurbit ring assembly was used to detect ClO in kidney HK2 cells. - The specific detection methods are as follows:

[0025] (1) Take the dislocated ten-membered cucurbitacinyl ring assembly and dilute it with water until the concentration of the dislocated ten-membered cucurbitacinyl ring assembly is 3.00×10 -5 mol / L, and the probe standard solution was obtained;

[0026] (2) Under serum-free DMEM culture conditions, cells were first treated with 20 μM probe standard solution for 30 min, and then 20 μM ClO - After HK-2 cells were treated with ions for 2 h, the fluorescence of the intracellular probe was observed after the cells were fixed. No obvious changes were found. If the fluorescence decreased or quenched, it indicated that HK-2 contained ClO - ions; otherwise, it means that it does not contain ClO - ion.

[0027] Beneficial effects of the present invention

[0028] 1. The supramolecular assembly of the present invention is formed by a dislocated ten-membered cucurbitacin ring and a cationic rutin, and can be used as a fluorescent probe to detect ClO in aqueous solution and HK-2 cells. - The detection process has the advantages of high selectivity, good sensitivity, wide detection range, fast response time and minimal damage to the sample.

[0029] 2. The supramolecular assembly of the present invention is prepared by mixing an aqueous solution of a dislocated ten-membered cucurbitacin ring and a cationic rosin, and reacting the mixture at room temperature and pressure. The preparation process is simple, the detection process is easy to operate, and the supramolecular assembly is conducive to wide promotion.

[0030] 3. The supramolecular assembly of the present invention also has good biocompatibility and is suitable for ClO in the body. - Detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Attached Figure 1 The structural formulas are the misaligned ten-membered cucurbitacin (a) (abbreviated as ns-Q

[10] ) and cationic phenoxyethanol (b) (abbreviated as APFG).

[0032] Attached Figure 2 is the molecular structure formula of the supramolecular fluorescent probe (APFG@ns-Q

[10] ) of the present invention.

[0033] Attached Figure 3 The fluorescence spectra of the misaligned ten-membered cucurbitacin (ns-Q

[10] ) and cationic fluorescein (APFG) were obtained by data processing using the origin software. (a) The fluorescence spectra of APFG when ns-Q

[10] was gradually added to 0, 0.1... to 1 times; (b) The linear relationship between the concentration ratio of ns-Q

[10] / APFG and the fluorescence intensity at the emission wavelength of 586nm; (c) The Job'plot working curve of APFG and ns-Q

[10] .

[0034] Attached Figure 4 This is the NMR titration and inclusion pattern diagram of APFG and ns-Q

[10] .

[0035] Attached Figure 5 The fluorescence recognition response diagram of the probe to 21 sodium salt anions (HPO4 2- , HSO3 - , I - ,Br - ,CO3 2- , HSO4 - ,PO4 3- , SO3 2- , NO3 - , SCN - , SO4 2- , IO3 - , CH3COO - , H2PO4 2- , SiO4 2- , HCO3 2- , S 2- , OH - , Cl- , NO2 - , ClO - ). This indicates that the fluorescent probe of the present invention has strong selective recognition.

[0036] Attached Figure 6 The probe contains 20 sodium salt anions (HPO4 2- , HSO3 - , I - ,Br - ,CO3 2- , HSO4 - ,PO4 3- , SO3 2- , NO3 - , SCN - , SO4 2- , IO3 - , CH3COO - , H2PO4 2- , SiO4 2- , HCO3 2- , S 2- , OH - , Cl - , NO2 - ) for ClO - The anti-interference diagram of ions shows that the fluorescent probe of the present invention has strong anti-interference performance.

[0037] Attached Figure 7 For the probe pair ClO - Mechanism of specific recognition of ions.

[0038] Attached Figure 8 The probe standard solution was added with different concentrations of ClO - Fluorescence titration spectrum curve of ion solution.

[0039] Attached Fig. 9 Add ClO to the probe standard solution - Detection limit of fluorescence titration when the ion is in solution.

[0040] Attached Fig.10 Fluorescence comparison of cuvettes selected for probe specificity to 21 sodium salt anions.

[0041] Attached Fig.11 The bar graph shows the biocompatibility of the probe in HK-2 cells.

[0042] The biocompatibility test method is as follows: HK-2 cells were seeded in a 96-well U-bottom plate, cultured at 5% CO2 and 37°C for 24 hours, and then treated with different concentrations (0-30μM) of APFG@ns-Q

[10] for 48 hours. Subsequently, after removing the supernatant, MTT (5 mg / mL culture medium, 20μL / well) was added to the wells and then incubated at 37°C for 4 hours. The supernatant was removed and 150μL of deionized water was added to each well for dissolution. The plate was shaken for 4 minutes and the absorbance value was tested.

[0043] Attached Fig.12 The cell imaging images of HK-2 cells by the probe. (a) is the staining image of HK-2 cells by the probe; (b) is the staining image of HK-2 cells by the probe containing 20 μmol / L ClO - Cell staining of ions. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0045] Embodiments of the present invention

[0046] Example 1

[0047] Preparation method of each reagent:

[0048] (1) 163.66 mg of ns-Q

[10] was placed in a 100 mL volumetric flask, and then deionized water was added to the scale line and mixed to obtain a concentration of 1.00 × 10 -3 mol / L dislocated ten-membered cucurbit ring solution;

[0049] (2) Take 38.94 mg of APFG and place it in a 100 mL volumetric flask. Then add deionized water to the mark and mix well to obtain a concentration of 1.00 × 10 -3 mol / L cationic pink solution;

[0050] (3) The concentration is 1.00×10 -3 mol / L dislocation ten-membered cucurbit ring solution and cationic peach red solution were uniformly mixed in a volume ratio of 1:2, that is, 15 μL of a concentration of 1.00×10 -3 mol / L ns-Q

[10] solution and 30 μL of 1.00×10 -3 mol / L APFG solution was placed in a 100 mL volumetric flask, and then deionized water was added to the scale and mixed to obtain the fluorescent probe standard solution (APFG@ns-Q

[10] , with a concentration of 3.00×10 -5 mol / L).

[0051] (4) Preparation of other ion solutions:

[0052] Accurately weigh the analytically pure standard products of various potassium salt anions required, dissolve them in deionized water, and obtain a concentration of 1.00×10 -1 mol / L standard solutions of other anions.

[0053] Example 2

[0054] Detection of ClO in water using fluorescent probes - The method steps are as follows:

[0055] 1) Take 3000 μL of the fluorescent probe standard solution of Example 1, whose concentration is 3.00×10 -5 mol / L;

[0056] 2) adding the solution to be tested to the probe standard solution of step 1), leaving it for 5 minutes, then measuring the fluorescence emission spectrum at a fixed excitation wavelength of 523 nm, and plotting a curve of the change of the fluorescence intensity at an emission wavelength of 586 nm;

[0057] 3) According to the curve of step 2), the change value ΔI of the fluorescence emission spectrum intensity at 586 nm before and after the addition of the water to be tested is calculated. If the intensity of the fluorescence emission spectrum at 586 nm before and after the addition of the water to be tested is significantly weakened, it indicates that the water to be tested contains ClO - ions; if the fluorescence emission spectrum at 586nm does not change significantly before and after adding the water to be tested, it indicates that the water does not contain ClO - ion.

[0058] Example 3

[0059] The above fluorescent probes detect ClO in HK-2 cells - The method is as follows:

[0060] 1) Take 3000 μL of the fluorescent probe standard solution of Example 1, whose concentration is 3.00×10 -5 mol / L;

[0061] 2) In serum-free DMEM culture conditions, cells were first treated with 20 μM probe for 30 min, and then 20 μmol / L ClO - After HK-2 cells were treated with ions and co-cultured for 2 h, the fluorescence of the intracellular probe was observed after the cells were fixed. No obvious change was found. If the fluorescence decreased or quenched, it indicated that ClO was present. - ions; if not, it means it does not contain ClO - ion.

[0062] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A dislocated ten-membered cucurbit ring-based assembly, characterized in that: Its molecular formula is 2C 22 H 26 Cl2N2@C 54 H 60 N 40 O 20， The structural formula is as follows: in, It is a dislocated ten-yuan melon ring.

2. The dislocated ten-membered cucurbitacinyl ring assembly according to claim 1, characterized in that: The assembly is prepared by mixing a dislocated ten-membered cucurbitacin solution and a cationic rosin solution.

3. The dislocated ten-membered cucurbitacinyl ring assembly according to claim 2, characterized in that: The preparation method of the assembly is as follows: (1) Take a dislocated ten-membered melon ring, add deionized water to dissolve it, and obtain solution A; (2) Take cationic pink, add deionized water to dissolve, and obtain solution B; (3) The dislocated ten-membered cucurbitacin ring-based assembly can be obtained by mixing solution A and solution B.

4. The dislocated ten-membered cucurbitacinyl ring assembly according to claim 3, characterized in that: The concentration of the dislocated ten-membered cucurbit ring in the solution A is 1.00×10 -3 mol / L; the concentration of cationic pink in solution B is 1.00×10 -3 mol / L; when the solution A and the solution B are mixed, the molar ratio of the dislocated ten-membered cucurbitacin ring to the cationic pink is 1:

2.

5. The dislocated ten-membered cucurbitacinyl ring assembly according to claim 3, characterized in that: The reaction in step (3) is carried out at normal temperature and pressure.

6. A method for detecting ClO in aqueous solution by using the dislocated ten-membered cucurbitacin ring assembly according to any one of claims 1 to 5 - application.

7. The dislocated ten-membered cucurbit ring-based assembly according to claim 6 is used to detect ClO in aqueous solution. - The application is characterized in that The specific detection methods are as follows: (1) Take the dislocated ten-membered cucurbitacinyl ring assembly and dilute it with water until the concentration of the dislocated ten-membered cucurbitacinyl ring assembly is 3.00×10 -5 mol / L, and the probe standard solution was obtained; (2) adding the aqueous solution to be tested to the prepared probe standard solution, leaving it for 5 min, and then measuring the fluorescence emission spectrum at a fixed excitation wavelength of 523 nm, and plotting the change curve of the fluorescence intensity at the excited laser wavelength; (3) According to the curve of step (2), the change value ΔI of the fluorescence emission spectrum intensity at 586 nm corresponding to the addition of the water to be tested to the probe standard solution can be calculated to obtain the ClO in the aqueous solution. - ions are detected.

8. A method for detecting ClO in kidney HK2 cells for non-disease diagnosis or treatment purposes using the misplaced ten-membered cucurbit ring-based assembly according to any one of claims 1 to 5. - application.

9. The misplaced ten-membered cucurbit ring-based assembly according to claim 8 is used to detect ClO in kidney HK2 cells for non-disease diagnosis or treatment purposes. - The application is characterized in that The specific detection methods are as follows: (1) Take the dislocated ten-membered cucurbitacinyl ring assembly and dilute it with water until the concentration of the dislocated ten-membered cucurbitacinyl ring assembly is 3.00×10 -5 mol / L, and the probe standard solution was obtained; (2) Under serum-free DMEM culture conditions, cells were first treated with 20 μM probe standard solution for 30 min, and then 20 μM ClO - After HK-2 cells were treated with ions for 2 h, the fluorescence of the intracellular probe was observed after the cells were fixed. No obvious change was found. If the fluorescence decreased or quenched, it indicated that HK-2 contained ClO - ion; On the contrary, it means that ClO is not contained. - ion.

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