Water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide as well as preparation method and application of water-soluble near-infrared fluorescent probe

Abstract

The invention discloses a water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide and a preparation method and application of the water-soluble near-infrared fluorescent probe, and belongs to the technical field of fluorescent probes, indole salt and cyclohexanone are used as raw materials, probes TNO-1 and TNO-2 are synthesized through a four-step reaction, the structural formula of the probes TNO-1 and TNO-2 is as follows: # imgabs0 #, after H2O2 is added into the probe TNO-1, the probe TNO-1 and the probe TNO-2 are subjected to a four-step reaction, the probe TNO-1 and the probe TNO-2 are subjected to a four-step reaction, and the probe The color of the probe changes from dark blue to light green visible to naked eyes, the maximum ultraviolet absorption peak has red shift (593 nm to 693 nm), the maximum fluorescence emission wavelength is shifted from 670 nm to 720 nm, the fluorescence intensity is enhanced by 14 times, the intensity and the concentration of hydrogen peroxide have a good linear relation, the sensitivity is high, the detection limit is 0.10646 mu mol / L, selected interference ions have no response to the probe TNO-1, the selectivity is good, and the detection sensitivity is high. Meanwhile, the higher the viscosity is, the higher the fluorescence intensity of the probe is, the fluorescence is enhanced by 11 times at the position of 675 nm, double-function detection of hydrogen peroxide and viscosity is achieved, and the probe has good water solubility, high biocompatibility and low toxicity, facilitates in-vivo imaging and is expected to be applied to the fields of chemical analysis and biomedicine.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis, and particularly relates to a water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide, a preparation method thereof, and an application thereof. Background Art

[0002] H2O2 is a reactive oxygen species that participates in various physiological and pathological processes in organisms and plays an indispensable role in cell differentiation and proliferation, toxicity and senescence, antibacterial, signal transduction, etc. For example, changes in the content of H2O2 may cause cell damage and cause diseases to the human body, such as cardiovascular diseases, skin diseases, etc., and may even cause cancer. Therefore, the detection of hydrogen peroxide has important research significance.

[0003] In recent years, the fluorescence probe method has been widely used for the detection of hydrogen peroxide due to its advantages such as high sensitivity, good selectivity, non-invasive detection, and real-time imaging. Currently, the fluorescence probes for detecting hydrogen peroxide have poor water solubility and biocompatibility; the emission wavelength is short, which will generate background interference on the fluorescence signal; it is difficult to detect viscosity, which is not conducive to deep tissue and in vivo imaging detection. Therefore, it is particularly important to develop and design a water-soluble near-infrared fluorescent probe that can achieve dual detection of hydrogen peroxide content and viscosity. Summary of the Invention

[0004] The purpose of the present invention is to provide a water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide. Through the performance comparison of the synthesis of two probes TNO-1 and TNO-2, the performance of TNO-1 is better, realizing the quantitative detection of hydrogen peroxide content. The probe structure also introduces a group with good biocompatibility, making the probe have good water solubility, which can increase the stability of the probe in organisms, improve the biocompatibility with organisms, reduce the interference and toxicity of probe molecules to organisms. At the same time, the probe has a carbon-carbon double bond structure, and the probe can perform molecular torsion, which can be used for the determination of viscosity. The change in the viscosity of the system increases the inhibition of the intramolecular charge transfer process of the probe molecule. It is not easy for the molecule to rotate at high viscosity, showing strong fluorescence intensity. This probe has good selectivity, high sensitivity, and low biological toxicity for the recognition of hydrogen peroxide, and realizes the dual-functional detection of hydrogen peroxide content and viscosity.

[0005] The present invention also provides a preparation method and an application of the above-mentioned water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A water-soluble near-infrared fluorescent probe TNO-1 and TNO-2 for detecting hydrogen peroxide, and the structural formulas of the probes TNO-1 and TNO-2 are as follows:

[0008]

[0009] The preparation methods of the above water-soluble near-infrared fluorescent probes TNO-1 and TNO-2 for detecting hydrogen peroxide, and the synthetic routes of the fluorescent probes TNO-1 and TNO-2 are as follows:

[0010]

[0011] Specifically, it includes the following steps:

[0012] 1) Compound 3 reacts with 4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate inner salt to obtain probe TNO-1;

[0013] 2) Compound 3 reacts with 2,3,3-trimethyl-1-propyl-3H-indolium iodide to obtain probe TNO-2.

[0014] Preferably, the specific preparation process of the fluorescent probe TNO-1 in step (1) is as follows: Under a protective atmosphere, compound 3, 4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate inner salt, L-proline, and ethanol are successively placed in a reaction flask, and reflux reaction is carried out. The reaction progress is monitored by TLC. After the reaction, the solution is rotary evaporated and purified by column chromatography to obtain probe TNO-1. The molar ratio of compound 3, 4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate inner salt, and L-proline is (1-2):1:(0.8-1.2), and the concentration of compound 3 in ethanol is 0.02-0.05 mol / L. When eluting by column chromatography, the eluent dichloromethane:methanol = 15-40:1 (volume ratio).

[0015] Preferably, the specific preparation process of the fluorescent probe TNO-2 for detecting reactive oxygen species in step (2) is as follows: Under a protective atmosphere, compound 3, 2,3,3-trimethyl-1-propyl-3H-indolium iodide, L-proline, and ethanol are successively placed in a reaction flask, and reflux reaction is carried out. The reaction progress is monitored by TLC. After the reaction, the solution is rotary evaporated and purified by column chromatography to obtain probe TNO-2. The molar ratio of compound 3, 2,3,3-trimethyl-1-propyl-3H-indolium iodide, and L-proline is (1-2):1:(0.8-1.2), and the concentration of compound 3 in ethanol is 0.02-0.05 mol / L. When eluting by column chromatography, the eluent dichloromethane:methanol = 20-50:1 (volume ratio).

[0016] Application of the above water-soluble near-infrared fluorescent probe in detecting hydrogen peroxide

[0017] The above fluorescent probe is used for dual-functional detection of hydrogen peroxide concentration and viscosity.

[0018] Furthermore, it is used for fluorescence detection and visual qualitative detection of H2O2. The wavelength during fluorescence detection is 720 nm.

[0019] When detecting hydrogen peroxide or the concentration of hydrogen peroxide by fluorescence detection and visual qualitative detection, it is carried out in a mixed solution of acetonitrile and PBS buffer solution with a volume ratio of 1:1, and the pH of the PBS buffer solution is 7.4.

[0020] When detecting the viscosity of hydrogen peroxide, it is carried out in a mixed system of glycerol and water, and the volume ratio of glycerol in the mixed system is 10% - 90%.

[0021] All kinds of raw materials used in the present invention are common commercially available products, or are obtained by methods known to those skilled in the art or methods disclosed in the prior art.

[0022] The present invention prepared a novel probe TNO-1 using cyclohexanone and 3,4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate inner salt as raw materials, and characterized it by nuclear magnetic resonance and fluorescence emission spectrometer; after adding H2O2 to the probe TNO-1, the dark blue color of the probe turns light green, the maximum ultraviolet absorption peak shows a red shift (from 593 nm to 693 nm), the maximum fluorescence emission wavelength shifts from 670 nm to 720 nm, the fluorescence intensity increases by 14 times, there is a good linear relationship between the intensity and the hydrogen peroxide concentration, the sensitivity is high, the detection limit is 0.10646 μmol / L, the selected interfering ions have no response to the probe TNO, the selectivity is good, it has a response to viscosity, the greater the viscosity, the greater the fluorescence intensity of the probe, realizing the dual-functional detection of hydrogen peroxide and viscosity. Description of the Drawings

[0023] Figure 1 、 Figure 2 are the ultraviolet absorption spectrum and fluorescence spectrum of the fluorescence probe TNO-1 for the response relationship of H2O2 with time;

[0024] Figure 3 、 Figure 4 are the ultraviolet absorption spectrum and fluorescence spectrum of the fluorescence probe TNO-2 for the response relationship of H2O2 with time;

[0025] Figure 5 、 Figure 6 are the ultraviolet absorption spectrum and fluorescence spectrum diagram of the fluorescence probe TNO-1 with different H2O2 concentrations;

[0026] Figure 7 is the linear fitting diagram of the fluorescence emission intensity of the fluorescence probe TNO-1 at 720 nm with the change of H2O2 concentration;

[0027] Figure 8 、 Figure 9are the ultraviolet absorption spectra and fluorescence spectra of the fluorescent probe TNO-2 at different H2O2 concentrations;

[0028] Figure 10 is the linear fitting graph of the fluorescence emission intensity of the fluorescent probe TNO-2 at 720 nm with the change of H2O2 concentration;

[0029] Figure 11 、 Figure 12 are the ultraviolet absorption spectra and fluorescence spectra of the fluorescent probe TNO-1 for different ion selectivities;

[0030] Figure 13 are the solution color photos taken under a fluorescent lamp after adding 16 different ions to the fluorescent probe TNO-1;

[0031] Figure 14 are the solution color photos taken under a fluorescent lamp after adding 16 different ions to the fluorescent probe TNO-2;

[0032] Figure 15 、 Figure 16 are the ultraviolet absorption spectra and fluorescence spectra of the fluorescent probe TNO-2 for different ion selectivities;

[0033] Figure 17 is the fluorescence intensity comparison of the probes TNO-1 and TNO-2 at 720 nm after adding 16 different ions;

[0034] Figure 18 、 Figure 19 are the fluorescence intensity graphs of the probes TNO-1 and TNO-2 in different solvents;

[0035] Figure 20 is the fluorescence response of the probe TNO-1 to different ratios of glycerol and water;

[0036] Figure 21 is the linear fitting graph of the fluorescence emission intensity of the probe TNO-1 at 675 nm in different ratios of glycerol and water;

[0037] Figure 22 is the fluorescence response of the probe TNO-2 to different ratios of glycerol and water;

[0038] Figure 23 is the linear fitting graph of the fluorescence emission intensity of the probe TNO-2 at 675 nm in different ratios of glycerol and water;

[0039] Figure 24 is the influence of pH on the response of the probe TNO-1 to hydrogen peroxide;

[0040] Figure 25 is the influence of pH on the response of the probe TNO-2 to hydrogen peroxide;

[0041] Figure 26 It is the application simulation of probe TNO-1 on filter paper with hydrogen peroxide at different concentrations;

[0042] Figure 27 It is the application simulation of probe TNO-1 on filter paper with different ions. Specific implementation mode

[0043] The present invention will be further described in detail below through preferred embodiments, but the protection scope of the present invention is not limited thereto.

[0044] Example 1

[0045] (1) Preparation of Compound 1

[0046] The synthesis route is as follows:

[0047]

[0048] 150 mmol of N,N-dimethylformamide, 50.0 mL of chloroform and 1 magnetic stir bar were successively added to a 250 mL three-necked flask. 150 mmol of phosphorus tribromide was added to a constant pressure dropping funnel and installed on the three-necked flask. Under nitrogen protection and in an ice bath, phosphorus tribromide was slowly added and stirred for 1 h. After the reaction was completed, 50 mmol of cyclohexanone was added to the constant pressure dropping funnel. After slowly dropping the mixed solution, it was stirred at 25 °C for 16 h. The product was poured into a beaker containing 200 mL of water. After adding solid sodium bicarbonate to adjust the reaction solution to be neutral, the solution was poured into a separatory funnel, extracted twice with dichloromethane, washed three times with saturated sodium chloride solution, dried with anhydrous magnesium sulfate for half an hour, and the dried solution was poured into a 50 mL distillation flask and concentrated by rotary evaporation to obtain a yellow oily liquid, and 2.00 g of Compound 1 was obtained.

[0049] (2) Preparation of Compound 2

[0050] The synthesis route is as follows:

[0051]

[0052] 600 mmol of N,N-dimethylformamide, 12 mmol of 4-bromo-2-hydroxybenzaldehyde, 30 mmol of cesium carbonate, 80 mmol of Compound 1 and 1 magnetic stir bar were successively added to a 100 mL round-bottomed flask. Under nitrogen protection, it was stirred at 25 °C for 50 h. After the reaction was completed, the product was transferred to a separatory funnel and washed successively with water, ethyl acetate and saturated brine, dried with anhydrous magnesium sulfate for 1 h, and the crude product of Compound 2 was obtained after rotary evaporation. The crude product of Compound 2 was further separated by column chromatography, and the eluent was petroleum ether:ethyl acetate = 15:1 (volume ratio) to obtain 1.40 g of yellow granular solid Compound 2 with a yield of 39%.

[0053] (3) Synthesis of Compound 3

[0054] The synthesis route is as follows:

[0055]

[0056] In a dry two-necked flask, 1.1 mmol of Compound 2, 3.4 mmol of potassium acetate, 2.2 mmol of bis(pinacolato)diboron, 0.08 mmol of palladium dichloride, 15 mL of 1,4-dioxane and 1 magnetic stir bar were added successively. Under nitrogen protection, the mixture was heated to reflux for 6 h. The solvent was removed by rotary evaporation to obtain the crude product of Compound 3. The crude product of Compound 3 was further separated by column chromatography using dichloromethane as the eluent to obtain 0.17 g of yellow viscous solid Compound 3 with a yield of 48%.

[0057] (4) Preparation of Fluorescent Probe TNO

[0058] The synthesis route is as follows:

[0059]

[0060] Synthesis of TNO-1: 0.16 mmol of Compound 3, 0.16 mmol of 4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate, 0.15 mmol of proline and 5 mL of ethanol were successively placed in a 50 mL two-necked flask. A reflux condenser was installed, and the mixture was heated and stirred under nitrogen protection for 5 h. The crude product was further separated by column chromatography using methanol:dichloromethane = 1:30 as the eluent to obtain 0.06 g of pure probe TNO-1 with a yield of 64%. 10 mg of probe TNO-1 was taken and dissolved in deuterated chloroform for NMR scanning. The 1H NMR and 13C NMR data are as follows:

[0061] 1 H NMR(400MHz,DMSO)δ8.63(d,J=15.2Hz,1H),7.84(dd,J=14.3,7.7Hz,2H),7.49-7.59(m,,5H),7.40(s,1H),6.94(d,J=15.2Hz,1H),4.66(t,J=7.4Hz,2H),2.73(d,J=5.1Hz,4H),2.61(t,J=6.3Hz,2H),2.11(m,J=6.6Hz,2H),1.83(m,J=6.5Hz,2H),1.82(s,6H),1.34(s,12H).

[0062] 13C NMR (101 MHz, DMSO) δ 179.16, 159.22, 152.25, 146.29, 143.14, 141.71, 132.17, 131.29, 131.06, 129.44, 128.15, 127.52, 124.59, 123.29, 121.10, 115.42, 114.32, 107.44, 73.98, 55.39, 51.30, 47.91, 44.74, 29.40, 27.75, 25.17.

[0063] Synthesis of TNO-2: 0.16 mmol of compound 3, 0.16 mmol of 2,3,3-trimethyl-1-propyl-3H-indolium iodide, 0.15 mmol of proline, and 5 mL of ethanol were successively placed in a 50 mL two-necked flask. A reflux condenser was installed, and nitrogen protection was added. The mixture was heated and stirred under reflux for 5 h. The crude product was further separated by column chromatography, and the eluent was methanol:dichloromethane = 1:50 to obtain 0.04 g of pure probe TNO-1 with a yield of 64%. Take 1 mg of probe TNO-2, dissolve it in deuterated chloroform, and perform NMR scanning. The 1H NMR and 13C NMR data are as follows:

[0064] 1 H NMR (400 MHz, DMSO) δ 8.64 (d, J = 15.2 Hz, 1H), 7.82 (dd, J = 17.0, 7.6 Hz, 2H), 7.70–7.48 (m, 5H), 7.44 (s, 1H), 6.72 (d, J = 15.2 Hz, 1H), 4.48 (t, J = 7.1 Hz, 2H), 2.83–2.62 (m, 4H), 1.91–1.83 (m, 4H), 1.81 (s, 6H), 1.35 (s, 12H), 1.00 (t, J = 7.4 Hz, 3H).

[0065] 13 C NMR (101 MHz, DMSO) δ 179.24, 159.44, 152.23, 146.10, 143.01, 141.79, 132.05, 131.37, 129.44, 128.23, 127.58, 124.54, 123.38, 121.17, 114.95, 114.38, 106.91, 84.77, 73.98, 51.37, 46.98, 29.30, 27.75, 25.42, 25.17, 24.96, 24.12, 21.62, 20.21, 11.47.

[0066] (5) Application test

[0067] 1) Preparation of the detection stock solution

[0068] a. Preparation of the probe TNO-1 stock solution: Weigh 0.0030 g (0.005 mmol, M = 601.57 g / mol) of the probe TNO-1 using an electronic analytical balance and transfer it into a 10 mL small brown bottle. Then, use a pipette to add 5.00 mL of acetonitrile to prepare a probe TNO-1 stock solution with a concentration of 1.0×10 -3 mol·L -1 .

[0069] b. Preparation of the probe TNO-2 stock solution: Weigh 0.0033 g (0.005 mmol, M = 649.42 g / mol) of the probe TNO-2 using an electronic analytical balance and transfer it into a 10 mL small brown bottle. Then, use a pipette to add 5.00 mL of acetonitrile to prepare a probe TNO-2 stock solution with a concentration of 1.0×10 -3 mol·L -1 .

[0070] c. Preparation of the stock solutions of the ions to be measured: Potassium fluoride (F - ), sodium chloride (Cl - ), potassium bromide (Br - ), hydrogen peroxide (H2O2), potassium iodide (I - ), sodium hypochlorite (ClO - ), sodium nitrite (NO2 - ), sodium nitrate (NO3 - ), sodium bisulfate (HSO4 - ), sodium bisulfite (HSO3 - ), potassium dihydrogen phosphate (H2PO4 - ), sodium sulfide (S 2- ), sodium hydrosulfide (HS - ), cysteine (Cys) solution, glutathione (GSH) solution, and homocysteine (Hcy) solution are prepared into stock solutions of ions with a concentration of 1.0×10 -2 mol·L -1 .

[0071] d. Preparation of the PBS buffer solution (0.01 mol·L -1 , pH = 7.4):

[0072] Weigh 4.2500 g of sodium chloride, 1.1 g of disodium hydrogen phosphate, and 0.1003 g of sodium dihydrogen phosphate, add them to a 500 mL volumetric flask, dissolve with deionized water and make up the volume to the mark. Finally, prepare a PBS buffer solution with pH = 7.4.

[0073] e. Preparation of the system solution with acetonitrile:PBS = 1:1:

[0074] Measure 50 mL of anhydrous acetonitrile and 50 mL of PBS buffer solution, pour them into a conical flask, mix well and set aside.

[0075] 2) Detection and analysis

[0076] a. Spectral detection of the relationship between probe TNO and excessive H2O2 over time

[0077] Add 3 mL of the system solution (acetonitrile:PBS = 1:1) to the cuvette for baseline correction and zero adjustment, then add 30 μL of 1.00×10 -3 mol·L -1 probe TNO stock solution, scan an absorption curve of pure probe TNO, and then add 30 μL of 1.00×10 -2 mol·L -1 hydrogen peroxide ion solution, stir well, scan until the peak no longer changes, and record the time required for the reaction process. Set the scanning range of the ultraviolet-visible spectrophotometer to 200 - 800 nm, with an interval of 1 nm.

[0078] Add 3 mL of the system solution (acetonitrile:PBS = 1:1) and 30 μL of 1.00×10 -3 mol·L -1 probe TNO buffer solution to the cuvette, first scan a fluorescence curve of pure probe TNO, and then add 30 μL of 1.00×10 -2 mol·L -1 hydrogen peroxide ion solution, mix well, and detect the change of the fluorescence spectrum over time within 15 minutes. The excitation wavelength of the fluorescence is 600 nm, the excitation slit width is 5 nm, the emission slit width is 5 nm, the test voltage is 600 V, and perform scanning.

[0079] From Figure 1 the ultraviolet spectrum, it can be seen that: for probe TNO-1, the absorption redshift occurs, the absorption peak at 593 nm gradually decreases over time, and the absorption peak at 693 nm gradually increases over time. After the reaction proceeds for about 45 min, the absorption peak reaches the highest position. At the reaction equilibrium, it can be seen that the color of the solution changes from blue to light green.

[0080] From Figure 2 the fluorescence spectrum, it can be seen that for probe TNO-1, the fluorescence intensity at 670 nm gradually decreases over time, the fluorescence intensity at 720 nm increases over time, and reaches the equilibrium state at 45 min.

[0081] From Figure 3As can be seen from the ultraviolet spectrum, after adding an excess of hydrogen peroxide to the probe TNO-2, the ultraviolet instrument shows that the absorption also redshifts. The absorption peak at 590 nm gradually decreases over time, and a new peak appears at 690 nm and its absorption peak gradually increases over time. After about 49 minutes, the absorption peak at the high wavelength reaches the highest point. At the reaction equilibrium, it can be seen that the color of the solution changes from blue to light green.

[0082] From Figure 4 As can be seen from the fluorescence spectrum, the fluorescence intensity of the original probe shows the highest peak at 660 nm and gradually decreases after adding hydrogen peroxide. The fluorescence intensity at 720 nm increases with time and reaches the equilibrium state at 49 minutes.

[0083] Both probes TNO-1 and TNO-2 show obvious and clear changes in the ultraviolet spectrum and fluorescence spectrum with the change of time for an excess of hydrogen peroxide. After the reaction, the wavelength shows an obvious redshift, the ultraviolet absorption peak changes significantly, the growth multiple of the fluorescence intensity is large, and the colors of the two reaction solutions also change significantly, both changing from the original blue to light green. However, the absorbance of the pure probe TNO-1 is significantly higher than that of TNO-2, indicating that TNO-1 has better water solubility. The obvious spectral changes of TNO-1 before and after the reaction indicate that it has a better response effect to hydrogen peroxide.

[0084] b. Spectral detection of the relationship between probe TNO and different H2O2 concentrations

[0085] Take 3 mL of the system solution, add 30 μL of 1.00×10 -3 mol·L -1 probe TNO, add different equivalents of H2O2 aqueous solution to prepare different concentrations, shake well, and detect the ultraviolet spectrum and fluorescence spectrum. The conditions are set as above.

[0086] From Figure 5 As can be seen from the ultraviolet spectrum, after adding hydrogen peroxide with different concentrations to the TNO-1 probe, the ultraviolet absorption peak at the low wavelength decreases in a gradient, and the peak at the high wavelength increases in a gradient. After adding 7 times the equivalent of hydrogen peroxide, the ultraviolet spectrum basically no longer changes. From Figure 6 As can be seen from the fluorescence spectrum, the fluorescence intensity continuously increases with the increase of concentration. By linearly fitting the fluorescence intensity at 720 nm in the fluorescence spectrum, we get Figure 7 , and it is found that there is a good linear relationship between the fluorescence intensity of the solution and the concentration of hydrogen peroxide. After linear fitting, the expression of the linear equation is: y = 34.48714 + 45.85869x, the linear correlation coefficient is R2 = 0.99157, and the detection limit is 0.10646 μM.

[0087] From Figure 8As can be seen from the ultraviolet spectrum, the response trend of the TNO-2 probe is the same as that of the TNO-1 probe. After adding 7 equivalents of hydrogen peroxide, the ultraviolet spectrum hardly changes any more. From Figure 9 As can be seen from the fluorescence spectrum, the fluorescence intensity increases continuously with the increase of concentration, and reaches a stable value at 7 equivalents. Similarly, linear fitting is performed on the fluorescence intensity at 720 nm, and Figure 10 is obtained. There is a good linear relationship between the fluorescence intensity of the solution before 7 equivalents of hydrogen peroxide. After linear fitting, the expression of the linear equation is: y = 7.57143 + 36.53571x, the linear correlation coefficient is R2 = 0.99925, and the detection limit is 0.11189 μM. The fitting degree of the regression line to the observed values of TNO-2 is slightly better than that of TNO-1.

[0088] c. Spectral detection of the selectivity of probe TNO to different substances

[0089] Take 16 portions of 3 mL of the system solution, add 30 μL of the probe TNO-1 or TNO-2 sample solution (1.00×10 -3 mol·L -1 ), and add 30 μL of 1.00×10 -2 mol·L -1 ionic solution respectively. (1) F - , (2) Cl - , (3) Br - , (4) H2O2, (5) I - , (6) ClO - , (7) NO2 - , (8) NO3 - , (9) HSO4 - , (10) HSO3 - , (11) H2PO4 - , (12) S 2- , (13) HS - , (14) cysteine (Cys) solution, (15) glutathione (GSH) solution, (16) homocysteine (Hcy) solution, and measure the ultraviolet absorption spectrum and fluorescence spectrum.

[0090] From Figure 11 As can be seen from the ultraviolet spectrum, after adding different ions to the probe TNO-1, compared with the original absorption peak of the probe, only when hydrogen peroxide is added, the ultraviolet absorption peak moves towards a higher wavelength and the highest absorbance is higher than that of the original probe. There may be reactions in other ionic solutions, but the changes are not obvious, and the wavelength hardly shifts, only the absorbance decreases slightly.

[0091] From Figure 12It can be seen from the fluorescence spectrum that only after the addition of hydrogen peroxide does the fluorescence spectrum shift significantly. The peak of the fluorescence intensity shifts towards higher wavelengths, and the fluorescence intensity increases by nearly 10 times. When other ions are added, small peaks appear at higher wavelengths, and the fluorescence intensity does not change significantly.

[0092] Figure 13 、 14 Figure 14 shows the color comparison after the solution reacts for the same time under a fluorescent lamp. Among them, only the solution with added H2O2 changes from blue to light green under the fluorescent lamp, and no obvious changes occur in others. The probes TNO-1 and TNO-2 have good selectivity for H2O2. From Figure 17 the comparison figure, it can be seen that the probe TNO-1 has a more obvious fluorescence response to hydrogen peroxide and good selectivity.

[0093] From Figure 15 the ultraviolet spectrum, it can be seen that the spectral changes of the probe TNO-2 and the probe TNO-1 are roughly the same. After the addition of hydrogen peroxide, the absorption peak of the ultraviolet spectrum moves towards higher wavelengths. When other ions are added, the absorbance of the solution changes insignificantly. For most ions, the wavelength of the response change is not obvious and basically does not shift, only the absorbance decreases slightly.

[0094] From Figure 16 the fluorescence spectrum, it can also be seen that only after the addition of hydrogen peroxide does the fluorescence spectrum shift significantly. The peak of the fluorescence intensity shifts towards higher wavelengths, and the fluorescence intensity also increases by nearly 10 times. When other ions are added, the fluorescence intensity does not change significantly.

[0095] d. Spectral detection of the fluorescence response of the probe TNO to different viscosities

[0096] Take eleven 5 mL EP tubes, and use a pipette to transfer 3 mL of solvents such as glycerol, methanol, acetonitrile, ethanol, dichloromethane, chloroform, N,N-dimethylformamide, dimethyl sulfoxide, water, Hepes, and PBS into them respectively. Then add 30 μL of 1×10 -3 mol·L -1 probe solution to each test tube, shake well, and transfer the solutions in the above EP tubes to a cuvette for fluorescence scanning to detect the fluorescence intensity.

[0097] Prepare aqueous solutions with different viscosity gradients. According to the following water and glycerol solution systems with different proportional coefficients, prepare mixed solution systems with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 in sequence. Transfer 3 mL of the above solutions to EP tubes in sequence, and then add 30 μL of 1×10 -3 mol·L -1 probe stock solution to each tube, shake well, and transfer the solutions in the above EP tubes to a cuvette for fluorescence scanning to detect the fluorescence intensity.

[0098] According toFigure 18 and Figure 19 In comparison with Figure 19 , the fluorescence solvent effects of probes TNO-1 and TNO-2 are basically similar. In different solvents, the fluorescence intensity of the probes changes. The fluorescence intensity of the probe in glycerol is very obvious, and it is the second in methanol.

[0099] According to Figure 20 and Figure 22 In a mixed solution of glycerol and water with different ratios, as the content of glycerol increases, that is, the viscosity increases, the peak height of the fluorescence intensity at about 675 nm gradually increases step by step. According to the molecular torsion property theory, when the system viscosity increases, the rotation of the probe itself will be inhibited, resulting in an increase in the fluorescence signal, which is verified in this experiment. The fluorescence intensity at 675 nm of the fluorescence spectrum is taken for fitting to obtain Figure 21 and Figure 23 , and it is found that there is a good linear relationship. Probe TNO has a good viscosity dependence. The change of the fluorescence response of TNO-1 to viscosity is obvious, and the effect of detecting viscosity is better than that of TNO-2.

[0100] e. Spectral detection of the fluorescence response of probe TNO to H2O2 at different pH values

[0101] Prepare PBS buffer solutions with pH values from 1 to 14. Use a pipette to transfer 1.5 mL of acetonitrile and 1.5 mL of each pH solution into an EP tube and mix well. Then add 30 μL of 1×10 -3 mol·L -1 probe solution to each EP tube. Transfer the above EP tubes to a cuvette for fluorescence scanning. Then add 30 μL of 1×10 -2 mol·L -1 hydrogen peroxide solution, and perform fluorescence scanning on the above solution again after reacting for 45 min.

[0102] Compare the fluorescence intensity of the probe solution at different pH values with the fluorescence intensity of the solution after the response to hydrogen peroxide. From Figure 24 and Figure 25 , it can be seen that the fluorescence spectrum shows that between pH = 7 - 10, the change in the comparison of the fluorescence intensities of probes TNO-1 and TNO-2 is obvious, indicating that the response effect of the probe to hydrogen peroxide is good in this range and has good stability, and the probe has a good effect in detecting hydrogen peroxide in the physiological environment.

[0103] f. Application of probe TNO

[0104] Conduct a simple application experiment on probe TNO-1. Cut filter paper of appropriate size to simulate the detection of hydrogen peroxide at different concentrations. Drop 20 μL of the probe system on the filter paper, and then drop 10 μL of 1×10 -3 mol·L -1TNO-1 solution, and then successively add hydrogen peroxide solutions with a volume gradient of 1×10 -2 mol·L -1 to filter papers 2, 3, 4, 5, and 6, so that the molar ratio is 2, 4, 6, 8, and 10. It can be clearly observed from Figure 26 that as the concentration of hydrogen peroxide increases, the color of the filter paper also changes and gradually fades, indicating that the probe can react with hydrogen peroxide under actual simulation.

[0105] For the detection simulation of different ions, 20 μL of the probe system is dropped on the filter paper, and then 10 μL of the probe TNO-1 solution of 1×10 -3 mol·L -1 is added. Then, hydrogen peroxide solutions with a concentration of 1×10 -2 mol·L -1 Hcy, Cys are successively added to the above filter papers 2, 3, and 4. It can be clearly observed from Figure 27 that no obvious changes occur in 2 and 3, indicating that the probe has a good reaction to hydrogen peroxide.

Claims

1. A water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide, characterized in that, The structural formula is as follows:

2. The preparation method of the water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide according to claim 1, characterized in that, It includes the following steps: (1) Compound 3 reacts with 4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate to obtain the probe TNO-1; (2) Compound 3 reacts with 2,3,3-trimethyl-1-propyl-3H-indolium iodide to obtain the probe TNO-2.

3. The preparation method of the water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide according to claim 2, wherein, The specific preparation process of step (1) or (2) is as follows: Under a protective atmosphere, compound 3, 4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate or 2,3,3-trimethyl-1-propyl-3H-indolium iodide, L-proline and ethanol are placed in a reaction flask, stirred and refluxed, monitored by TLC, and purified by column chromatography to obtain the probe.

4. The preparation method of the water-soluble near-infrared fluorescent probe for detecting hydrogen peroxide according to claim 3, characterized in that, The molar ratio of compound 3, 4-(2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate or 2,3,3-trimethyl-1-propyl-3H-indolium iodide to L-proline is (1-2):1:(0.8-1.2); The concentration of compound 3 in ethanol is 0.02-0.05 mol / L.

5. Use of the water-soluble near-infrared fluorescent probe according to claim 1 in the detection of hydrogen peroxide.

6. The application according to claim 5, wherein For the dual-functional detection of hydrogen peroxide concentration and viscosity.

7. The application according to claim 5, wherein For the fluorescence detection and visual qualitative detection of H2O2.

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