Application of aromatic ring compound in biological thiol glutathione detection

Abstract

The invention discloses application of an aromatic ring compound as a fluorescent probe in biological thiol glutathione detection, experiments find that the compound can react with glutathione (GSH), fluorescence is obviously enhanced after the reaction, Cys and Hcy which are highly similar to the GSH in structure do not have the phenomenon, and the compound can be applied to biological thiol glutathione detection. Therefore, the aromatic ring compound can be used as a fluorescent probe for detecting glutathione (GSH), meanwhile, experiments find that a detection system is not interfered by zwitterions, amino acids, different pH conditions and the like, and the aromatic ring compound has the characteristics of high sensitivity, low detection cost, convenience in operation, rapidness in determination, real-time detection and the like. The method has application prospects in the fields of preparation of detection test paper, fluorescent developing anti-counterfeiting optical materials and the like.

Description

TECHNICAL FIELD

[0001] The application relates to application of an aromatic ring compound in detection of biothiol glutathione and belongs to the technical field of analytical chemistry. BACKGROUND

[0002] Biothiols are important functional biomolecules, including glutathione (GSH), homocysteine (Hcy) and cysteine (Cys), which play a crucial role in maintaining the stability of the intracellular environment. Abnormal levels of biothiols in cells are closely related to various diseases: abnormal Cys levels can affect liver damage, skin damage, hair pigment loss, slow growth and cancer; Hcy is considered a risk factor for cardiovascular disease and Alzheimer's disease; GSH, as the most abundant biothiol in cells (1-10 mM), plays a key role in human health and disease due to its unique redox properties and nucleophilicity. Low levels of biothiols in the body can lead to slow growth, liver damage, weight loss and other adverse effects, while high levels of biothiols are closely related to Parkinson's disease, Alzheimer's disease and cardiovascular disease. Therefore, detecting and monitoring the levels of biothiols is crucial for understanding the functions of biothiols in physiological and pathological processes and for diagnosis.

[0003] In order to quantify biothiols in biological systems, several biothiol detection methods based on chromatography, electrochemistry and spectroscopy, and biosensor methods have been reported. A large number of low-toxicity probes have been reported and applied to real-time detection, such as fluorescent quantum dots, luminescent organic probes, and composite nanomaterials. However, there is little work on the detection of cell lysates including high molecular weight biothiols.

[0004] Current detection methods have certain limitations, such as insufficient selectivity: it is difficult to accurately distinguish between Cys, Hcy and GSH, which are highly structurally similar, especially in complex biological matrices; weak anti-interference ability: easily interfered by metal ions, active oxygen and electroactive substances in biological samples; complexity of operation and cost: chromatography and mass spectrometry rely on large instruments and are complex to operate; high preparation cost of fluorescent probes and biosensors; lack of real-time / in situ detection capability: most methods require post-processing after sampling, which cannot achieve dynamic real-time monitoring of biothiols in living cells and tissues; limited biocompatibility: probes or sensors used for in vivo detection are easily removed by the immune system or have toxicity, making it difficult to apply to living models.

[0005] GSH has similar structural units and reaction characteristics to Cys and Hcy, and is interfered by Hcy and Cys. Therefore, it is a great challenge to require the probe to specifically detect GSH in this complex environment. SUMMARY

[0006] The application provides a new use of an aromatic ring compound, namely, application of the aromatic ring compound as a fluorescent probe in detection of biological thiol glutathione.

[0007] The aromatic ring compound is as follows: .

[0008] Another object of the application is to provide a detection test paper containing the aromatic ring compound.

[0009] Advantages and beneficial effects of the application: 1. The aromatic ring compound can interact with glutathione GSH in a solution environment at room temperature, and the fluorescence is obviously enhanced, so that the aromatic ring compound can be used as a fluorescent probe for specific detection of glutathione GSH; the aromatic ring compound has the characteristics of high sensitivity, low detection cost, convenient operation, rapid determination, real-time detection and the like; in addition, the test paper with the aromatic ring compound can be used for detection of GSH, and the existence of biological thiol glutathione can be directly judged by naked eyes, so that the aromatic ring compound has application prospects in the fields of preparation of detection test paper and fluorescent color developing anti-counterfeiting materials; 2. The aromatic ring compound can distinguish Cys, Hcy and GSH with high structural similarity, has strong specificity for GSH, and can well distinguish Cys, Hcy and GSH according to different light emission conditions after addition of biological thiol, and has low operation complexity, unlike chromatography and mass spectrometry which depend on large instruments and are simple to operate; 3. The aromatic ring compound has certain anti-interference ability, and can detect interference of anions and cations, part of common amino acids and different pH conditions in biological samples; considering the biological compatibility limitation, the aromatic ring compound provided by the application has low toxicity and suitable molecular weight, and can be applied to a living model; 4. The aromatic ring compound can be used as a ligand of platinum or palladium drugs for synthesis of a complex, and improves the light emission performance; when the platinum or palladium drugs are combined with GSH to play an anti-tumor role, real-time positioning and detection, biological imaging and the like can be realized through changes in fluorescence spectrum, and the aromatic ring compound can be used for cell and biological living body imaging research. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 Fig. 1 is a detection result of ligand 1a on thiols GSH, Cys and Hcy, wherein a graph is a fluorescence intensity graph of ligand 1a+GSH, and b graph is a fluorescence enhancement multiple statistical graph of ligand 1a after GSH, Cys and Hcy are respectively added; Figure 2 Fig. 2 is a detection result of ligand 1b on thiols GSH, Cys and Hcy, wherein a graph is a fluorescence intensity graph of ligand 1b+GSH, and b graph is a fluorescence enhancement multiple statistical graph of ligand 1b after GSH, Cys and Hcy are respectively added; Figure 3 Detection results of ligand 1c on thiols GSH, Cys, Hcy, wherein a is the fluorescence intensity chart of ligand 1c+GSH, and b is the fluorescence enhancement fold chart after adding GSH, Cys, Hcy to 1c respectively; Figure 4 Detection results of ligand 2a on thiols GSH, Cys, Hcy, wherein a is the fluorescence intensity chart of ligand 2a+GSH, and b is the fluorescence enhancement fold chart after adding GSH, Cys, Hcy to 2a respectively; Figure 5 Detection results of ligand 3a on thiols GSH, Cys, Hcy, wherein a is the fluorescence intensity chart of ligand 3a+GSH, and b is the fluorescence enhancement fold chart after adding GSH, Cys, Hcy to 3a respectively; Figure 6 Detection results of ligand 3b on thiols GSH, Cys, Hcy, wherein a is the fluorescence intensity chart of ligand 3b+GSH, and b is the fluorescence enhancement fold chart after adding GSH, Cys, Hcy to 3b respectively; Figure 7 Detection results of ligand 3c on thiols GSH, Cys, Hcy, wherein a is the fluorescence intensity chart of ligand 3c+GSH, and b is the fluorescence enhancement fold chart after adding GSH, Cys, Hcy to 3c respectively; Figure 8 Detection results of ligand 4a on thiols GSH, Cys, Hcy, wherein a is the fluorescence intensity chart of ligand 4a+GSH, and b is the fluorescence enhancement fold chart after adding GSH, Cys, Hcy to 4a respectively; Figure 9 Detection results of ligand 4b on thiols GSH, Cys, Hcy, wherein a is the fluorescence intensity chart of ligand 4b+GSH, and b is the fluorescence enhancement fold chart after adding GSH, Cys, Hcy to 4b respectively; Figure 10 Detection results of ligand 4c on thiols GSH, Cys, Hcy, wherein a is the fluorescence intensity chart of ligand 4c+GSH, and b is the fluorescence enhancement fold chart after adding GSH, Cys, Hcy to 4c respectively; Figure 11 Influence results of amino acids on detection of GSH by 10 compounds; Figure 12 Influence results of anions on detection of GSH by 10 compounds; Figure 13 Influence results of cations on detection of GSH by 10 compounds; Figure 14Results of detection of the effect of pH on GSH for 10 compounds Figure 15 Results of detection of the effect of pH on GSH for 10 compounds Figure 16 Results of detection of the effect of pH on GSH for 10 compounds Figure 17 Results of detection of the effect of pH on GSH for 10 compounds Figure 18 Results of detection of the effect of pH on GSH for 10 compounds DETAILED DESCRIPTION

[0011] The application will be further described in detail by the following examples, but the scope of the application is not limited to the content described, the method in the examples is a routine method unless otherwise specified, and the reagent used is a routine commercially available reagent or a reagent prepared according to a routine method unless otherwise specified. In the following examples, the fluorescence enhancement multiple is calculated by dividing the fluorescence intensity of the GSH added by the fluorescence intensity of the control at the maximum fluorescence emission peak, taking the fluorescence intensity of the control of water as the basis.

[0012] Example 1: Detection of N-N fluorescent ligand on glutathione (GSH), cysteine (Cys), homocysteine (Hcy) The structure of the N-N fluorescent ligand is as follows: ; Respectively, ligand 1a, 1b, 1c was weighed and added to dimethyl sulfoxide to prepare a 20 mmol / L mother liquor, 3 μL of ligand mother liquor was taken into a 5 mL centrifuge tube, 27 μL of dimethyl sulfoxide was added, and then 3 mL of water was added to dilute the ligand solution to a concentration of 20 μmol / L. GSH, Cys, Hcy aqueous solution was added to the ligand solution, and the final concentration of thiol was 5 mmol / L. The above solution was measured for fluorescence under 320 nm excitation light, and the fluorescence spectrum was obtained. The results are shown in Figures 1-3 : As can be seen from the figure: before the addition of thiol, the ligand 1a solution exhibits blue fluorescence, with the blue fluorescence emission peak having the maximum fluorescence intensity at 360±20 nm. After adding GSH to the ligand 1a solution, the solution changes from blue fluorescence to green fluorescence after 30 min, with the green fluorescence emission peak having the strongest fluorescence intensity at 450±20 nm, an increase of about 2.6 times; however, when Cys or Hcy is added to the 1a ligand solution, no green fluorescence is produced in the solution after 30 min. The ligand 1b solution was colorless before the addition of thiol, with the fluorescence emission peak having the maximum fluorescence intensity at 360±20 nm. After adding GSH to the 1b ligand solution, the solution changed from colorless to green fluorescence in about 30 minutes, and the fluorescence intensity of the green fluorescence emission peak at 500±20 nm increased by about 6.4 times. However, no green fluorescence was produced after 30 minutes when Cys or Hcy were added to the 1b ligand solution.

[0013] Before the addition of thiol, the ligand 1c solution was colorless, and the fluorescence emission peak had the maximum fluorescence intensity at 360±20 nm. After adding GSH to the ligand 1c solution, the solution changed from colorless to reddish-brown fluorescence after 30 min, and the fluorescence intensity of the reddish-brown fluorescence emission peak at 570±20 nm increased by about 25.7 times. However, when Cys or Hcy were added to the 1c ligand solution, no reddish-brown fluorescence was produced after 30 min.

[0014] Example 2: Selective detection of thiols using S-containing CN fluorescent ligands The structural formula of the S-containing CN fluorescent ligand is as follows: ; The ligand configuration and reaction system in this embodiment are the same as in Example 1. Fluorescence was measured under 320 nm excitation light to obtain the fluorescence spectrum, and the results are shown below. Figure 4 : As can be seen from the figure, the ligand 2a solution was colorless before the addition of thiol, and the fluorescence emission peak had the maximum fluorescence intensity at 380±20 nm. After adding GSH to the ligand 2a solution, the solution changed from no fluorescence to blue-green fluorescence after 30 min. The fluorescence intensity of the blue-green fluorescence emission peak was the strongest at 370±20 nm, and the fluorescence intensity of the blue-green fluorescence emission peak at 510±20 nm was increased by about 6.7 times. However, when Cys or Hcy were added to the 2a ligand solution, no blue-green fluorescence was produced after 30 min.

[0015] Example 3: Detection of thiols GSH, Cys, and Hcy using CN ligands The structural formula of CN ligand is as follows: ; The configuration and reaction system of the ligand in this example are the same as those in Example 1. The fluorescence spectrum is obtained by measuring the fluorescence under excitation light at 320 nm. The results are shown in Figures 5-7 : As can be seen from the figure, the ligand 3a solution is colorless before adding the thiol, and the colorless fluorescence emission peak has the maximum fluorescence intensity at 370 ± 20 nm. After adding GSH to the ligand 3a solution, the solution changes from no fluorescence to dark blue fluorescence after 30 min, and the dark blue fluorescence emission peak has the strongest fluorescence intensity at 410 ± 20 nm, which is about 5.0 times stronger. However, no dark blue fluorescence is produced after adding Cys or Hcy to the ligand 3a solution for 30 min.

[0016] The ligand 3b solution is colorless before adding the thiol, and the fluorescence emission peak has the maximum fluorescence intensity at 360 ± 20 nm. After adding GSH to the ligand 3b solution, the solution changes from no fluorescence to dark blue fluorescence after 30 min, and the dark blue fluorescence emission peak has the strongest fluorescence intensity at 420 ± 20 nm, which is about 10.8 times stronger. However, no dark blue fluorescence is produced after adding Cys or Hcy to the ligand 3b solution for 30 min.

[0017] The ligand 3c solution presents weak dark blue fluorescence before adding the thiol, and the light blue fluorescence emission peak has the maximum fluorescence intensity at 360 ± 20 nm. After adding GSH to the ligand 3c solution, the solution changes from dark blue fluorescence to bright blue fluorescence after 30 min, and the bright blue fluorescence emission peak has the strongest fluorescence intensity at 450 ± 20 nm, which is about 19.8 times stronger. However, bright blue fluorescence is produced after adding Cys or Hcy to the ligand 3c solution for 30 min.

[0018] Example 4: Selective detection of thiol by cyclometalated palladium complex The structure of the cyclometalated palladium complex is as follows: ; The configuration and reaction system of the complex in this example are the same as those in Example 1. The fluorescence spectrum is obtained by measuring the fluorescence under excitation light at 320 nm. The results are shown in Figures 8-10 : As can be seen from the figure, the complex 4a solution presents a relatively weak light blue color before adding the thiol, and the fluorescence emission peak has the maximum fluorescence intensity at 360 ± 20 nm. After adding GSH to the complex 4a solution, the fluorescence intensity of the light blue fluorescence emission peak at 360 ± 20 nm is enhanced by about 60.4 times after 30 min. However, the fluorescence intensity is not enhanced after adding Cys or Hcy to the complex 4a solution for 30 min.

[0019] The complex 4b solution presents a dim blue color before adding the thiol, and the fluorescence emission peak has the maximum fluorescence intensity at 360±20 nm. After adding GSH to the complex 4b solution, the fluorescence intensity of the fluorescence emission peak at 360±20 nm is enhanced by about 90.6 times after 30 min, and the solution presents a dark blue color. However, after adding Cys or Hcy to the complex 4b solution, the fluorescence intensity is not obviously enhanced after 30 min.

[0020] The complex 4c solution presents a relatively weak dark blue color before adding the thiol, and the fluorescence emission peak has the maximum fluorescence intensity at 360±20 nm. After adding GSH to the complex 4c solution, the solution is changed from dark blue fluorescence to bright blue fluorescence after 30 min, and the fluorescence intensity of the bright blue fluorescence emission peak at 430±20 nm is the strongest. The fluorescence intensity of the bright blue fluorescence emission peak at 430±20 nm is enhanced by about 314.6 times. However, after adding Cys or Hcy to the complex 4c solution, the fluorescence intensity is not enhanced after 30 min, and there is no bright blue fluorescence.

[0021] Example 5: In order to further detect the detection specificity of the above-mentioned compound to GSH, whether the ligand luminescence in the detection system of the application is affected by the amino acid, pH, anion and cation existing in the body is detected The amino acid is proline (Pro), phenylalanine (Phe), methionine (Met) and glycine (Gly); the anion is NO3 - , HPO4 2- , Cl - , ClO - , ClO4 - , F - , I - , CH3COO - ; and the cation is K + , Ca 2+ , Na + , Mg 2+ , Li + , NH4 + , Co 2+ , Mn 2+ , Ba 2+ , Cs + , Cd 2+ , Rb + , Ni + .

[0022] In order to better simulate the tumor microenvironment, the corresponding tests on pH and viscosity are also carried out. The pH gradient is pH=5.8, pH=6.0, pH=6.2, pH=6.6, pH=7.0, pH=7.4, pH=7.8, pH=8.0 and pH=9.0.

[0023] 1. Effect of amino acids on fluorescence of the compound The above 10 compounds were configured into a ligand solution with a concentration of 20 pmol / L according to the method in Example 1. Proline (Pro), phenylalanine (Phe), methionine (Met), and glycine (Gly) aqueous solutions were added to the ligand solution, respectively, and the final concentration of the amino acid solution was 5 mmol / L. Meanwhile, water and GSH (5 mmol / L) were set as controls. The fluorescence of the above solutions was measured under excitation light at 320 nm, the fluorescence spectrum was obtained, and the fluorescence enhancement multiple was calculated. The results are shown in Table 1. Figure 11 As can be seen from the figure, after the addition of amino acids in the ligand 1a solution, the fluorescence intensity slightly decreased at 360±20 nm, there was no phenomenon of fluorescence intensity enhancement after the addition of GSH, and there was no red shift phenomenon. After the addition of amino acids, the fluorescence color of the solution was basically consistent with that before the addition, showing a relatively light blue fluorescence, and there was no case of red shift leading to the change of the solution from blue fluorescence to green fluorescence.

[0024] After the addition of amino acids in the ligand 1b solution, only the sample with the addition of glycine (Gly) had a slight increase in fluorescence intensity at 500±20 nm, and the fluorescence change multiple could reach 2.5 times at most. The fluorescence intensity of the remaining three samples was basically the same before and after the addition of the three kinds of amino acids, and the fluorescence intensity was not as high as that after the addition of GSH (6.4 times), and there was no red shift phenomenon. After the addition of amino acids, the fluorescence color of the solution was basically consistent with that before the addition, showing a relatively light colorless fluorescence, and there was no case of red shift leading to the change of the solution from no fluorescence to dark green fluorescence.

[0025] After the addition of amino acids in the ligand 1c solution, the fluorescence intensity of the sample basically showed a downward trend, and the fluorescence intensity did not have a very high phenomenon of enhancement multiple (25.7 times) after the addition of GSH, and there was no red shift phenomenon. After the addition of amino acids, the fluorescence color of the solution was basically consistent with that before the addition, showing a relatively light colorless fluorescence, and there was no case of red shift leading to the change of the solution from no fluorescence to red-brown fluorescence.

[0026] After the addition of amino acids in the ligand 2a solution, the fluorescence intensity did not have a very high phenomenon of enhancement multiple (4.2 times) after the addition of GSH, and the fluorescence intensity did not have a red shift phenomenon after the addition of GSH. After the addition of amino acids, the fluorescence color of the solution was basically consistent with that before the addition, showing a relatively light colorless fluorescence, and there was no case of red shift leading to the change of the solution from no fluorescence to blue-green fluorescence.

[0027] ​After adding amino acids into ligand 3a solution, the fluorescence intensity was higher (5.0 times) than that without adding GSH, and there was no red shift phenomenon. After adding amino acids, the fluorescence color of the solution was basically consistent with that before adding, showing a relatively light colorless fluorescence, and there was no red shift to change the solution from no fluorescence to dark blue fluorescence.

[0028] After adding amino acids into ligand 3b solution, the fluorescence intensity slightly decreased, and the fluorescence intensity did not appear the high enhancement multiple (10.8 times) after adding GSH, and there was no red shift phenomenon. After adding amino acids, the fluorescence color of the solution was basically consistent with that before adding, showing a relatively light colorless fluorescence, and there was no red shift to change the solution from no fluorescence to dark blue fluorescence.

[0029] After adding amino acids into ligand 3c solution, the fluorescence intensity slightly decreased, and the fluorescence intensity did not appear the high enhancement multiple (19.8 times) after adding GSH, and there was no red shift phenomenon. After adding amino acids, the fluorescence color of the solution was basically consistent with that before adding, showing a relatively light colorless fluorescence, and there was no red shift to change the solution from no fluorescence to dark blue fluorescence.

[0030] After adding amino acids into complex 4a solution, the fluorescence intensity of the sample at 360±20 nm basically showed a weak enhancement trend, and the fluorescence change multiple was 1.2-1.8 times. Only the sample with added glycine (Gly) showed a decrease in fluorescence intensity, but the overall fluorescence intensity was not as high (60.4 times) as that after adding GSH. After adding amino acids, the fluorescence color of the solution was basically consistent with that before adding, showing a relatively light blue fluorescence.

[0031] After adding amino acids into complex 4b solution, the fluorescence intensity of the sample at 360±20 nm basically showed an enhancement trend, and the fluorescence change multiple was 1.2-1.4 times. However, the overall fluorescence intensity was not as high (90.6 times) as that after adding GSH. After adding amino acids, the fluorescence color of the solution was basically consistent with that before adding, showing a relatively light blue fluorescence.

[0032] After adding amino acids into complex 4c solution, the fluorescence intensity of the sample at 430±20 nm basically showed an enhancement trend, and the fluorescence change multiple was 1.4-1.7 times. The enhancement multiple (314.6 times) after adding GSH was not as high as that without adding GSH, and there was no red shift phenomenon. After adding amino acids, the fluorescence color of the solution was basically consistent with that before adding, showing a relatively weak light blue fluorescence, and there was no red shift to change the solution from light blue fluorescence to bright blue fluorescence.

[0033] 2. Effect of anions on the fluorescence of the compound The detection system was synchronized with Step 1, except that an anion NO3 - , HPO4 2- , Cl - , ClO - , ClO4 - , F - , I - , CH3COO - ion solution was added, and the final concentration of the solution was 5 mmol / L. The fluorescence spectrum was obtained by measuring the fluorescence under 320 nm excitation light, and the results are shown in Figure 12 ; As can be seen from the figure, after adding the above anions to the ligand 1a solution, the fluorescence intensity mostly fluctuates up and down under the condition of not adding anions, but the overall trend does not change significantly. The fluorescence intensity is enhanced after adding some anions such as F - , and the maximum fluorescence enhancement change multiple at 450±20 nm is 1.1-1.5 times, which is lower than the fluorescence enhancement multiple (2.6 times) after adding GSH. The fluorescence intensity is weakened after adding some anions, but there is no red shift phenomenon after adding GSH. After adding anions NO3 - , HPO4 2- , Cl - , ClO - , ClO4 - , F - , I - , CH3COO - , the fluorescence color of the solution basically remains the same as before adding, showing a relatively light blue fluorescence, and there is no red shift to change the solution from blue fluorescence to green fluorescence.

[0034] After adding the above anions to the ligand 1b solution, only the samples with NO3 - , ClO4 - , F - have a decreased fluorescence intensity, and the remaining samples have a certain enhancement of fluorescence intensity after adding the corresponding anions, compared with before adding. The fluorescence enhancement change multiple at 500±20 nm is 1.1-2.7 times, which is lower than the fluorescence enhancement multiple (6.4 times) after adding GSH. The fluorescence intensity does not have a red shift phenomenon after adding GSH in general. After adding anions NO3 - , HPO4 2- , Cl - , ClO - , ClO4 - , F - , I - , CH3COO -Afterwards, the fluorescence color of the solution remained basically the same as before the addition, showing a relatively light colorless fluorescence, and there was no red shift that caused the solution to change from no fluorescence to green fluorescence.

[0035] After adding the aforementioned anions to the ligand 1c solution, the fluorescence intensity of the samples generally showed an increasing trend, with a fluorescence enhancement factor of 1.3-3.5 times at 570±20 nm. However, the fluorescence intensity did not exhibit the extremely high enhancement factor (25.7 times) seen after adding GSH, and no redshift was observed. The addition of the anion NO3... - HPO4 2- Cl - ,ClO - ClO4 - F - I - CH3COO - Afterwards, the fluorescence color of the solution remained basically the same as before the addition, showing a relatively light colorless fluorescence, and there was no red shift that caused the solution to change from no fluorescence to reddish-brown fluorescence.

[0036] After adding the above anions to the ligand 2a solution, the fluorescence intensity of the samples generally showed a decreasing trend. Adding F at 500±20 nm further reduced the fluorescence intensity. - CH3COO - The maximum fluorescence enhancement fold of the samples was 1.6-2.8 times, and the fluorescence intensity did not exhibit the extremely high enhancement fold (4.2 times) seen after the addition of GSH, nor did it show a red shift after the addition of GSH; the addition of anion NO3... - HPO4 2- Cl - ,ClO - ClO4 - F - I - CH3COO - Afterwards, the fluorescence color of the solution remained basically the same as before the addition, showing a relatively light colorless fluorescence, and there was no red shift that caused the solution to change from no fluorescence to blue-green fluorescence.

[0037] After adding the aforementioned anions to the ligand 3a solution, the fluorescence intensity fluctuated, but the overall trend remained largely unchanged. The addition of some anions, such as Cl... - ClO4 - F - The fluorescence intensity increased, with a maximum enhancement factor of 1.2-1.8 times at 410±20 nm, which is lower than the fluorescence enhancement factor after adding GSH (5.0 times). The addition of some anions, such as NO3-, further contributed to the increase. - HPO4 2-, ClO - , I - , CH3COO - The fluorescence intensity is weakened, and the red shift phenomenon after adding GSH does not occur. After adding anion NO3 - , HPO4 2- , Cl - , ClO - , ClO4 - , F - , I - , CH3COO - , the fluorescence color of the solution basically remains consistent with that before adding, showing relatively light colorless fluorescence, and there is no red shift to cause the solution to change from no fluorescence to dark blue fluorescence.

[0038] After adding the above anions in the ligand 3b solution, the fluorescence intensity is increased or decreased. After adding some anions such as HPO4 2- , F - , CH3COO - The fluorescence intensity is enhanced, and the maximum change multiple of the fluorescence enhancement at 420±20 nm is 1.1-3.6 times, which is lower than the fluorescence enhancement multiple (10.8 times) after adding GSH. After adding some anions such as NO3 - , Cl - , ClO4 - , ClO - , I - The fluorescence intensity is weakened, but the overall trend does not change significantly. The red shift phenomenon after adding GSH does not occur. After adding anion NO3 - , HPO4 2- , Cl - , ClO - , ClO4 - , F - , I - , CH3COO - , the fluorescence color of the solution basically remains consistent with that before adding, showing relatively light colorless fluorescence, and there is no red shift to cause the solution to change from no fluorescence to dark blue fluorescence.

[0039] After adding the above anions in the ligand 3c solution, the fluorescence intensity of the sample basically presents a weakening trend. After adding NO3 - The maximum change multiple of the fluorescence enhancement of the sample is 1.1 times. The fluorescence intensity does not have the extremely high phenomenon of the multiple of the fluorescence enhancement (19.8 times) after adding GSH. The red shift phenomenon after adding GSH does not occur. After adding anion NO3 - , HPO4 2- , Cl -, CI O - , CI O4 - , F - , I - , CH3COO - Afterwards, the fluorescence color of the solution basically remained consistent with that before the addition, showing a relatively light blue fluorescence.

[0040] After adding the above-mentioned anions into the solution of complex 4a respectively, the fluorescence intensity of the samples after adding NO3 - , CI - , CI O - , I - was slightly enhanced, and the maximum fluorescence enhancement change multiple at 360±20 nm was 1.1-2.3. The fluorescence intensity of the samples after adding other anions was weakened, and generally, there was no phenomenon of extremely high enhancement multiple (60.4 times) after adding GSH. After adding anions NO3 - , HPO4 2- , CI - , CI O - , CI O4 - , F - , I - , CH3COO - , the fluorescence color of the solution basically remained consistent with that before the addition, showing a relatively light blue fluorescence.

[0041] After adding the above-mentioned anions into the solution of complex 4b respectively, the fluorescence intensity of the samples after adding NO3 - , CI O4 - was slightly weakened, and the fluorescence intensity of the samples after adding other anions was highly weakened. Generally, there was no phenomenon of enhancement after adding GSH. After adding anions NO3 - , HPO4 2- , CI - , CI O - , CI O4 - , F - , I - , CH3COO - , the fluorescence color of the solution basically remained consistent with that before the addition, showing a relatively light blue fluorescence.

[0042] After adding the above-mentioned anions into the solution of complex 4c respectively, the fluorescence intensity of the samples was basically weakened, and generally, there was no phenomenon of enhancement after adding GSH, and there was no red shift phenomenon. After adding anions NO3 - , HPO4 2- , CI -, ClO - , ClO4 - , F - , I - , CH3COO - Afterwards, the fluorescence color of the solution remained basically consistent with that before the addition, showing a relatively weak light blue fluorescence, and there was no red shift leading to the solution changing from light blue fluorescence to bright blue fluorescence.

[0043] 3. Effect of cations on the fluorescence of the compound The detection system was synchronized with Step 1, except that cations K + , Ca 2+ , Na + , Mg 2+ , Li + , NH4 + , Co 2+ , Mn 2+ , Ba 2 + , Cs + , Cd 2+ , Rb + , Ni + solution, the final concentration of the solution was 5 mmol / L, the fluorescence of the above solution was measured under 320 nm excitation light, and the fluorescence spectrum was obtained, the results are shown in Figure 13 : As can be seen from the figure: after adding the above cations to the ligand 1a solution, the fluorescence intensity was lower than that after adding GSH, and there was no red shift phenomenon after adding GSH; after adding cations K + , Ca 2+ , Na + , Mg 2+ , Li + , NH4 + , Co 2+ , Mn 2+ , Ba 2+ , Cs + , Cd 2+ , Rb + , Ni + , the fluorescence color of the solution remained basically consistent with that before the addition, showing a relatively light light blue fluorescence, and there was no red shift leading to the solution changing from blue fluorescence to green fluorescence.

[0044] After adding the above cations to the ligand 1b solution, the fluorescence intensity of the sample decreased, which was lower than that after adding GSH, and the red shift phenomenon after adding GSH did not occur in general; after adding cations K + , Ca 2+ , Na + , Mg2+ Li + NH4 + Co 2+ Mn 2+ Ba 2+ Cs + Cd 2+ Rb + Ni + Afterwards, the fluorescence color of the solution remained basically consistent with that before the addition, showing a relatively light colorless fluorescence, and there was no red shift leading to the solution changing from no fluorescence to green fluorescence.

[0045] After adding the above-mentioned cations into the ligand 1c solution respectively, the fluorescence intensity increased or decreased, the fluorescence enhancement change multiple at 570±20 nm was 1.1-1.3 times, but the fluorescence intensity was not as high as that after adding GSH (25.7 times), and there was no red shift phenomenon; after adding the cation K + Ca 2+ Na + Mg 2+ Li + NH4 + Co 2+ Mn 2+ Ba 2+ Cs + Cd 2+ Rb + Ni + Afterwards, the fluorescence color of the solution remained basically consistent with that before the addition, showing a relatively light colorless fluorescence, and there was no red shift leading to the solution changing from no fluorescence to green fluorescence.

[0046] After adding the above-mentioned cations into the ligand 2a solution respectively, the fluorescence intensity increased or decreased, the maximum fluorescence enhancement change multiple at 500±20 nm was 1.1 times, which was lower than that after adding GSH (4.2 times), and there was no red shift phenomenon of the fluorescence intensity after adding GSH; after adding the cation K + Ca 2+ Na + Mg 2+ Li + NH4 + Co 2+ Mn 2+ Ba 2+ Cs + Cd 2+ Rb + Ni +Afterwards, the fluorescence color of the solution basically keeps consistent with that before adding, showing as relatively light colorless fluorescence, and there is no red shift to cause the solution to change from no fluorescence to blue-green fluorescence.

[0047] Afterwards, the fluorescence color of the solution basically keeps consistent with that before adding, showing as relatively light colorless fluorescence, and there is no red shift to cause the solution to change from no fluorescence to blue-green fluorescence. + 、Co 2+ 、Ba 2+ 、Cs + 、Rb + The fluorescence intensity is weakened, and there is no red shift phenomenon of fluorescence intensity after adding GSH; after adding cations K + 、Ca 2+ 、Na + 、Mg 2+ 、Li + 、NH4 + 、Co 2+ 、Mn 2+ 、Ba 2+ 、Cs + 、Cd 2+ 、Rb + 、Ni + , the fluorescence color of the solution basically keeps consistent with that before adding, showing as relatively light colorless fluorescence, and there is no red shift to cause the solution to change from no fluorescence to dark blue fluorescence.

[0048] Afterwards, the fluorescence color of the solution basically keeps consistent with that before adding, showing as relatively light colorless fluorescence, and there is no red shift to cause the solution to change from no fluorescence to blue-green fluorescence. 2+ 、NH4 + 、Mn 2+ 、Cd 2+ The fluorescence intensity is enhanced, and the maximum value of fluorescence enhancement change multiple at 420±20 nm is 1.1-1.6 times, which is lower than the fluorescence enhancement multiple (10.8 times) after adding GSH; after adding some cations such as K + 、Co 2+ 、Ba 2+ 、Cs + 、Rb + The fluorescence intensity is weakened, but the overall trend does not change significantly, and there is no red shift phenomenon of fluorescence intensity after adding GSH; after adding cations K + 、Ca 2+ 、Na + 、Mg 2+ 、Li + 、NH4 + 、Co 2+ 、Mn2+ , Ba 2+ , Cs + , Cd 2+ , Rb + , Ni + Afterwards, the fluorescence color of the solution remained basically consistent with that before the addition, showing a relatively light colorless fluorescence, and there was no red shift leading to the solution changing from no fluorescence to dark blue fluorescence.

[0049] After adding the above-mentioned cations into the ligand 3c solution, the fluorescence intensity increased or decreased, the fluorescence intensity of the samples added with some cations such as Li + , NH4 + , Ni + increased, the maximum value of the fluorescence enhancement change multiple at 450±20 nm was 1.1-1.9 times, which was lower than the fluorescence enhancement multiple (19.8 times) after adding GSH, the fluorescence intensity of the samples added with other cations decreased, but the overall trend did not change significantly, and there was no red shift phenomenon of the fluorescence intensity after adding GSH; after adding the cations K + , Ca 2+ , Na + , Mg 2+ , Li + , NH4 + , Co 2+ , Mn 2+ , Ba 2+ , Cs + , Cd 2+ , Rb + , Ni + , the fluorescence color of the solution remained basically consistent with that before the addition, showing a relatively light blue fluorescence, and there was no red shift leading to the solution changing from no fluorescence to dark blue fluorescence.

[0050] After adding the above-mentioned cations into the complex 4a solution, the fluorescence intensity increased or decreased, the fluorescence intensity of the samples did not increase after adding GSH (60.4 times) in general, except that the fluorescence intensity of the samples added with K + , Na + , Li + , Cs + , Rb + decreased, the fluorescence intensity of the samples added with other cations increased, the maximum value of the fluorescence enhancement change multiple at 360±20 nm was 1.1-1.3 times; after adding the cations K + , Ca 2+ , Na + , Mg 2+ , Li + , NH4 + , Co 2+ , Mn2+ , Ba 2 + , Cs + , Cd 2+ , Rb + , Ni + Afterwards, the fluorescence color of the solution basically remained consistent with that before the addition, showing a relatively weak light blue fluorescence.

[0051] After adding the above-mentioned cations into the solution of complex 4b respectively, the fluorescence intensity of the sample basically showed a weakening trend, and only the sample added with Ni + had a certain enhancement of fluorescence intensity, and the maximum fluorescence enhancement change multiple at 465 ± 20 nm was 2.6 times. The fluorescence intensity of the samples added with other cations weakened to a greater extent, and generally, there was no phenomenon of extremely high enhancement multiple (90.6 times) after the addition of GSH. After adding the cations K + , Ca 2+ , Na + , Mg 2+ , Li + , NH4 + , Co 2+ , Mn 2+ , Ba 2+ , Cs + , Cd 2+ , Rb + , Ni + , the fluorescence color of the solution basically remained consistent with that before the addition, showing a relatively light blue fluorescence.

[0052] After adding the above-mentioned cations into the solution of complex 4c respectively, the fluorescence intensity of the sample basically showed a weakening trend, and generally, there was no phenomenon of enhancement after the addition of GSH, and there was no red shift phenomenon. Among them, except that the fluorescence intensity of the samples added with K + , Na + had a higher degree of weakening, the fluorescence intensity of the samples added with other cations had a smaller degree of weakening. After adding the cations K + , Ca 2+ , Na + , Mg 2+ , Li + , NH4 + , Co 2+ , Mn 2+ , Ba 2+ , Cs + , Cd 2+ , Rb + , Ni +The fluorescence color of the solution remained basically consistent with that before adding, showing weak light blue fluorescence, and there was no red shift to cause the solution to change from light blue fluorescence to bright blue fluorescence.

[0053] 4. Effect of pH on fluorescence of the compounds The above 10 compounds were weighed separately, dissolved in dimethyl sulfoxide to prepare a mother liquor of 20 mmol / L, 3 μL of the mother liquor was taken into a 5 mL centrifuge tube, 27 μL of dimethyl sulfoxide was added; 3 mL of citric acid-sodium phosphate buffer solution with pH of 5.8, 6.0, 6.2, 6.6, 7.0, 7.4, 7.8, 8.0, and 9.0 was added respectively to prepare a ligand solution with a concentration of 20 μmol, the fluorescence was measured under excitation light at 320 nm using the above solution, and the fluorescence spectrum was obtained, the results are shown in Figure 14 : As can be seen from the figure, the fluorescence intensity of ligand 1a in the above solutions with different pH values was comparable to that of the control sample with added water at pH=5.8, pH=6.0, pH=6.6, pH=7.0, pH=7.4, pH=8.0, and pH=9.0, the fluorescence intensity was weakened to a greater extent at pH=6.2, and the fluorescence intensity was enhanced to a higher extent at pH=7.8, the maximum change multiple of fluorescence enhancement at 450±20 nm was 1.1-1.5 times, which was lower than the fluorescence enhancement multiple (2.6 times) after adding GSH, but there was no red shift phenomenon after adding GSH; the fluorescence color of the solution remained basically consistent with that before adding, showing relatively weak light blue fluorescence, and there was no red shift to cause the solution to change from light blue fluorescence to green fluorescence.

[0054] The fluorescence intensity of ligand 1b in the above solutions with different pH values was all enhanced, the maximum change multiple of fluorescence enhancement at 500±20 nm was 1.3-4.8 times, which was lower than the fluorescence enhancement multiple (6.4 times) after adding GSH, among which the sample fluorescence intensity was the highest at pH=7.4, but the fluorescence intensity did not have the red shift phenomenon after adding GSH in general; the fluorescence color of the solution remained basically consistent with that before adding, showing relatively weak colorless fluorescence, and there was no red shift to cause the solution to change from no fluorescence to green fluorescence.

[0055] The ligand 1c in the above-mentioned solutions with different pH values has a fluorescence intensity comparable to that of the control sample with added water, the maximum fluorescence enhancement change multiple at 570±20nm is 1.1-1.8 times, which is lower than the fluorescence enhancement multiple (25.7 times) after adding GSH, but the fluorescence intensity does not generally show the extremely high enhancement multiple phenomenon after adding GSH, and there is no red shift phenomenon; in the above-mentioned pH solutions, the solution fluorescence color basically remains consistent with that before adding, showing relatively shallow colorless fluorescence, and there is no red shift leading to the solution changing from no fluorescence to red-brown fluorescence.

[0056] The ligand 2a in the above-mentioned solutions with different pH values basically shows an enhancement trend in sample fluorescence intensity, but the overall trend does not change significantly, the maximum fluorescence enhancement change multiple at 500±20nm is 1.1-2.8 times, which is lower than the fluorescence enhancement multiple (4.2 times) after adding GSH, and there is no red shift phenomenon of fluorescence intensity after adding GSH; in the above-mentioned pH solutions, the solution fluorescence color basically remains consistent with that before adding, showing relatively shallow colorless fluorescence, and there is no red shift leading to the solution changing from no fluorescence to blue-green fluorescence.

[0057] The ligand 3a in the above-mentioned solutions with different pH values has an increase and a decrease in fluorescence intensity, the maximum fluorescence enhancement change multiple at 410±20nm is 1.1-2.4 times, which is lower than the fluorescence enhancement multiple (5.0 times) after adding GSH, the fluorescence intensity is enhanced more obviously at pH=5.8 and pH=6.0, the sample fluorescence intensity under the conditions of pH=6.2 and pH=6.6 fluctuates up and down in the aqueous solution, and the fluorescence intensity under other conditions shows a certain weakening, but there is no red shift phenomenon of fluorescence intensity after adding GSH; in the above-mentioned pH solutions, the solution fluorescence color basically remains consistent with that before adding, showing relatively shallow colorless fluorescence, and there is no red shift leading to the solution changing from no fluorescence to dark blue fluorescence.

[0058] The ligand 3b in the above-mentioned solutions with different pH values has a fluorescence intensity comparable to that of the control sample with added water, and there is no red shift phenomenon of fluorescence intensity after adding GSH; in the above-mentioned pH solutions, the solution fluorescence color basically remains consistent with that before adding, showing relatively shallow colorless fluorescence, and there is no red shift leading to the solution changing from no fluorescence to dark blue fluorescence.

[0059] The sample fluorescence intensity of ligand 3c in the above solutions with different pH values basically showed a weakening trend. Only under the condition of pH = 5.8, the sample fluorescence intensity at 450 ± 20 nm showed enhancement, and the enhancement multiple was lower than the fluorescence enhancement multiple (19.8 times) after the addition of GSH. The fluorescence intensity did not show the red shift phenomenon after the addition of GSH. In the above pH solutions, the fluorescence color of the solution basically remained consistent with that before the addition, showing relatively shallow colorless fluorescence, and there was no red shift to cause the solution to change from colorless fluorescence to dark blue fluorescence.

[0060] The sample fluorescence intensity of complex 4a in the above solutions with different pH values basically showed a weakening trend. Only under the condition of pH = 8.0, the sample fluorescence intensity showed a certain degree of enhancement, and the maximum fluorescence enhancement multiple was 1.6 times. The fluorescence intensity of the samples under other pH conditions was reduced to a higher degree, and in general, there was no phenomenon of extremely high enhancement multiple (60.4 times) after the addition of GSH. In the above pH solutions, the fluorescence color of the solution basically remained consistent with that before the addition, showing relatively shallow light blue fluorescence.

[0061] The sample fluorescence intensity of complex 4b in the above solutions with different pH values basically showed an enhancement trend. The maximum fluorescence enhancement multiple at 360 ± 20 nm was 1.1 times. In general, there was no phenomenon of extremely high enhancement multiple (90.6 times) after the addition of GSH. In the above pH solutions, the fluorescence color of the solution basically remained consistent with that before the addition, showing relatively shallow light blue fluorescence.

[0062] The sample fluorescence intensity of complex 4c in the above solutions with different pH values basically showed an enhancement trend. The fluorescence intensity did not show the phenomenon of extremely high enhancement multiple after the addition of GSH. The maximum fluorescence enhancement multiple at 430 ± 20 nm was 1.5-6.5 times, which was lower than the fluorescence enhancement multiple (314.6 times) after the addition of GSH, and there was no red shift phenomenon. In the above pH solutions, the fluorescence color of the solution basically remained consistent with that before the addition, showing relatively weak light blue fluorescence, and there was no red shift to cause the solution to change from light blue fluorescence to bright blue fluorescence.

[0063] Example 6: Detection of the detection specificity of ligand 1c, ligand 2a, ligand 3a, ligand 3b, ligand 3c, and complex 4c for GSH by test paper experiment Take ligand 1c, ligand 2a, ligand 3a, ligand 3b, ligand 3c, complex 4c respectively, add dimethyl sulfoxide to dissolve to prepare 100 mmol / L mother liquor, take 2 µL mother liquor into 5 mL centrifuge tube, add 18 µL dimethyl sulfoxide; then add 2 mL ultrapure water to prepare ligand solution with a concentration of 100 µmol; prepare GSH solution with a final concentration of 2.5 mM, 5 mM and 10 mM; Take 10 µL of the above solution with a pipette and drop it on a clean long strip-shaped test paper, let it diffuse and dry, repeat the operation until the ligand solution has a relatively obvious trace under the ultraviolet lamp, then add GSH solution with a final concentration of 2.5 mM, 5 mM and 10 mM respectively, and set the one without adding GSH as a control; the results are as follows Figure 15 As can be seen from the figure, compared with the control, the test paper with GSH added shows obvious fluorescence under the ultraviolet lamp.

[0064] Example 7: Detection specificity of ligand 1c and complex 4c for GSH by confocal detection Hela and Hacat cells were incubated in confocal dishes for 24 h to adhere to the cells, and BSO solution with a final concentration of 0, 40, 60 and 80 µM (used to inhibit the generation of biological thiol molecules such as GSH in living cells) was added to each confocal dish, the cells were pretreated for 24 h, then ligand 1c or complex 4c (with a final concentration of 20 µM) was added, and the incubation was continued for 24 h, after which the cells were washed with PBS, and then the change in fluorescence intensity in the cells was observed by laser confocal microscopy, and the results are as follows Figure 16 As can be seen from the figure, with the increase of BSO concentration, the fluorescence intensity gradually weakens.

[0065] Example 8: Detection specificity of ligand 1c and complex 4c for GSH by confocal co-localization detection Hela cells were incubated in confocal dishes for 24 h, and after the cells adhered, ligand 1c and complex 4c with a final concentration of 20 µM were added to each confocal dish and incubated for 24 h, after which different commercial dyes (Mito-Green, Lyso-Blue, ER-Blue; Mito-Red, Lyso-Red, ER-Red) were added and incubated for 2 h, the culture medium was removed, PBS was added for washing, and then the change in fluorescence intensity in the cells was observed by laser confocal microscopy, The results are as follows Figures 17-18 As can be seen from the figure, ligand 1c or complex 4c can bind with GSH generated in the cells, and the fluorescence produced after aggregation in mitochondria, lysosomes and endoplasmic reticulum.

Claims

1. Application of a class of aromatic ring compounds as fluorescent probes in the detection of biological thiols glutathione, characterized in that, The aromatic ring compound is as follows: 。 2. A test paper containing the aromatic ring compound of claim 1.

Images