Near-infrared two-region fluorescent probe for detecting nitric oxide as well as preparation method and application of near-infrared two-region fluorescent probe

Abstract

The invention discloses a near-infrared two-region fluorescent probe for detecting nitric oxide as well as a preparation method and application of the near-infrared two-region fluorescent probe. The molecular formula of the fluorescent probe is C71H96N3O16 < + >. According to the fluorescent probe, 4-pentaethylene glycol monomethyl ether benzylamine is adopted as a nitric oxide response group, and a heptamethine cyanine dye containing two pentaethylene glycol monomethyl ether benzoindole derivatives is adopted as a fluorophore. The probe responds to nitric oxide and then emits near-infrared two-region fluorescence with the wavelength of 1000-1400 nm, and the probe has good selectivity on nitric oxide. The three pentaethylene glycol monomethyl ether in the probe endows the probe with good water solubility and biocompatibility, so that the probe can be used for detecting nitric oxide in various aqueous phase systems (including physiological fluids and plants).

Description

Technical Field

[0001] The present invention belongs to the field of analysis and detection of nitric oxide in animals and plants, and particularly relates to a near-infrared second near-infrared fluorescence probe for detecting nitric oxide, a preparation method thereof, and applications thereof. Background Art

[0002] Nitric oxide (NO), as a transferable free radical, plays a key role in physiological processes such as vascular tone regulation, neuronal signal transduction, tumor microenvironment regulation, and immune response. Under normoxic conditions, NO is biosynthesized by nitric oxide synthase (NOS), and NOS generates NO by oxidizing L-arginine to L-citrulline; while in a hypoxic environment, NO can also be produced through the enzymatic / non-enzymatic reduction pathway of nitrite, and this phenomenon has been confirmed in ischemic myocardial tissue and triggers NO-mediated pathological damage. Given the multi-dimensional regulatory role of NO in physiological and pathological processes of organisms, its accurate quantification and dynamic monitoring have important research value for disease diagnosis. Since in vitro samples (such as cultured cells, tissue sections, etc.) cannot fully simulate the complexity of in vivo samples (such as the interaction between cells and their surrounding environment), it is crucial to develop new methods for real-time detection of NO in organisms.

[0003] Currently, the methods for detecting NO have deficiencies in in vivo applications. For example, colorimetric detection methods can be used to analyze NO in cell lysates, but this method indirectly detects through nitrite and nitrate, and requires operation under acidic conditions, lacking biocompatibility. In contrast, techniques such as electron paramagnetic resonance (EPR) spectroscopy and magnetic resonance imaging (MRI) have been used for in vivo NO detection imaging, but these methods are respectively limited by low resolution and low sensitivity. Amperometric titration has high sensitivity (pM), but requires invasive operation and can only detect NO in direct contact with the probe. Chemiluminescence methods utilize the reaction of NO with ozone (O3) to generate excited NO2, and when the excited NO2 returns to the ground state, it emits light at a specific wavelength, and the light intensity is measured by a chemiluminescence detector to quantify NO, with high sensitivity and specificity, and can detect low concentrations of NO. However, the instrument equipment required for this method is relatively expensive, the operation is relatively complex, and special treatment of samples is required. Fluorescence imaging is a non-invasive method that can achieve high-resolution and high-contrast imaging. Especially near-infrared fluorescence probes with near-infrared second near-infrared wavelength emission show great potential due to the advantages of low autofluorescence interference level and high imaging signal-to-noise ratio. Therefore, it is very necessary to develop activatable near-infrared fluorescence probes with near-infrared second near-infrared wavelength emission.

[0004] At present, some fluorescent probes for detecting NO have been developed. For example, Patent CN109574980 A, "Fluorescent Probe Molecule for Detecting Nitric Oxide Based on Rhodamine Derivative, Preparation Thereof and Use Thereof", designs a fluorescent probe molecule based on rhodamine derivative, and this fluorescent probe molecule has an imine group capable of reacting with nitric oxide. As the concentration of nitric oxide increases, the fluorescence emission intensity of this probe at 590 nm gradually increases. However, since its emission wavelength is in the visible light region, and the probe only has an enhancement of about 5 times at 590 nm after reacting with nitric oxide, this is not conducive to its effective detection and monitoring of peroxynitrite in vivo. Another patent, CN118126073A, "Fluorescent Probe Compound for Detecting Nitric Oxide, Preparation Method Thereof and Application Thereof", designs a novel fluorescent probe. This probe uses a fluoroboron dipyrromethene derivative as the fluorescent chromophore, and the secondary amine group on the probe molecule serves as the nitric oxide response site. This fluorescent probe compound itself does not emit light due to the effect of PET (photoinduced electron transfer); when it encounters nitric oxide in an aerobic environment, a nitrosation reaction occurs with the secondary amine group of the probe molecule, blocking the original PET effect, and thus emitting fluorescence at 588 nm. Although this probe has certain response characteristics, its visible light emission wavelength limits its application in in vivo imaging, and the low water solubility caused by its molecular structure further restricts its feasibility for biological detection. Therefore, developing a fluorescent probe with both a second near-infrared emission wavelength and good water solubility is of great significance for simply, conveniently, quickly, and accurately detecting nitric oxide in vivo. Such probes can not only effectively avoid the interference problem caused by autofluorescence in vivo, but also provide higher detection sensitivity and specificity, meeting the requirements of practical applications. Summary of the Invention

[0005] Aiming at the deficiencies existing in the prior art, the purpose of the present invention is to provide a second near-infrared fluorescent probe for detecting nitric oxide, its preparation method and application. Specifically, the present invention provides a second near-infrared fluorescent probe capable of detecting nitric oxide, and details the preparation method of this probe and its application in the detection of nitric oxide in an aqueous phase. The fluorescent probe of the present invention uses a heptamethine cyanine dye containing two pentaethylene glycol monomethyl ether benzindole derivatives as the fluorophore. This structural design with a long conjugation degree enables the probe to emit second near-infrared fluorescence of 1000 - 1400 nm after reacting with nitric oxide, thus effectively avoiding the interference problem caused by autofluorescence of short-wavelength probes in vivo. In addition, the pentaethylene glycol monomethyl ether on both sides of the probe and the pentaethylene glycol monomethyl ether in the recognition site structure significantly improve its water solubility, solving the problem that some other probes are difficult to achieve real-time and in-situ detection of nitric oxide in vivo due to poor water solubility. Therefore, the present invention not only overcomes the limitations in the prior art, but also provides a new tool with high sensitivity, strong specificity and suitable for real-time monitoring of nitric oxide in vivo, having important practical application value.

[0006] The object of the present invention is achieved by the following technical solutions.

[0007] A near-infrared second-region fluorescent probe for detecting nitric oxide, wherein the fluorescent probe is 2-[(1E)-2-{3-[(1Z)-2-[(3Z)-2-(2,5,8,11,14-pentaoxahexadec-16-yl)-2-azatricyclo[6.3.1.04,12]dodeca-1(11),4(5),6,8(12),9-pentaen-3-ylidene]ethylidene]-2-({[4-(2,5,8,11,14-pentaoxahexadec-16-yloxy)phenyl]methyl}amino)cyclopent-1-enyl}vinyl]-3-(2,5,8,11,14-pentaoxahexadec-16-yl)-3-azatricyclo[6.3.1.04,12]dodeca-1(12),2,4(5),6,8(9),10-hexaen-3-ium salt; with the molecular formula C 71 H 96 N3O 16 + , and the specific structure is as follows: .

[0008] The present invention provides a preparation method of a near-infrared second-region fluorescent probe for detecting nitric oxide, comprising the following steps: Dissolve the compound FD1080-Cl shown by the following formula in N,N-dimethylformamide, add triethylamine, introduce an inert gas to make the reaction system in an inert atmosphere, stir and react under an ice-water bath, then add 4-pentaethylene glycol monomethyl ether benzylamine, and then raise the temperature for reaction; after the reaction is completed, separate and purify to obtain the near-infrared second-region fluorescent probe.

[0009] .

[0010] Preferably, the molar ratio of the compound FD1080-Cl to triethylamine is 1:(2 - 4).

[0011] Preferably, the molar ratio of the compound FD1080-Cl to 4-pentaethylene glycol monomethyl ether benzylamine is 1:(1 - 2). ]>

[0012] Preferably, the amount of N,N-dimethylformamide added per mmol of the compound FD1080-Cl is 15 - 20 mL.

[0013] Preferably, the inert atmosphere is to evacuate the system and fill it with nitrogen, and repeat at least three times, and the inert gas is nitrogen.

[0014] Preferably, the stirring reaction time under the ice-water bath is 20 - 30 minutes.

[0015] Preferably, the stirring reaction temperature is 60-80° C. and the reaction time is 4-6 h.

[0016] Preferably, the purification method is silica gel chromatography.

[0017] Preferably, the eluent used in the silica gel chromatography is dichloromethane / methanol.

[0018] The present invention also provides application of the near-infrared second-region fluorescent probe for detecting nitric oxide in detecting nitric oxide in a living body.

[0019] Furthermore, the near-infrared second-region fluorescent probe for detecting nitric oxide is used to detect nitric oxide in organisms.

[0020] Compared with the prior art, the outstanding advantages of the fluorescent probe provided by the present invention include: (1) Specific detection in the near-infrared region II: The probe of the present invention uses 4-pentaethylene glycol monomethyl ether benzylamine as a specific response group and is combined with the heptamethine cyanine fluorophore of a benzindole derivative. In addition, a five-membered ring is introduced in the middle position of the heptamethine to improve the overall stability of the molecule and promote the delocalization of the π electrons of the heptamethine chain, making the conjugated system more continuous, thereby broadening the absorption spectrum or red-shifting the emission wavelength. Compared with the six-membered ring, the five-membered ring is more favorable in subsequent reaction kinetics and has a higher yield. The smaller ring size can reduce steric hindrance and improve biocompatibility or labeling efficiency. After reacting with NO, the excitation wavelength of the probe is red-shifted to the near-infrared region II of 1000-1400 nm, significantly reducing the autofluorescence interference in the test system (such as chlorophyll in plants). At the same time, near-infrared photons have stronger tissue penetration ability, effectively expanding the applicable scenarios of NO detection in organisms.

[0021] (2) Optimization of water solubility and stability: The probe molecule of the present invention introduces pentaethylene glycol monomethyl ether modification at both the onium salt site of the benzindole derivative and the benzylamine response group. Compared with probes without hydrophilic groups or triethylene glycol monomethyl ether derivatives, its water solubility is significantly improved. Its excellent water solubility and biocompatibility enable it to avoid fluorescence quenching caused by aggregation when used for NO detection in vivo. Therefore, the fluorescent probe of the present invention is particularly suitable for the detection of nitric oxide in vivo.

[0022] (3) The fluorescent probe of the present invention has strong anti-interference ability, does not respond to some ions and amino acids that may be present in the body, and has good selectivity for nitric oxide. Therefore, the probe is simple to operate, fast, accurate, and highly reliable for NO detection in vivo.

[0023] (4) When the fluorescent probe of the present invention reacts with nitric oxide, the fluorescence intensity at the peak of 1104 nm has a good linear relationship with the concentration of NO. The linear regression equation is Y = 23.91812*X + 159.50717, R 2 = 0.998. Therefore, this probe can quantitatively detect nitric oxide in organisms. In addition, the synthetic route of the near-infrared second-region fluorescent probe of the present invention is simple, efficient, and low-cost, with broad application prospects. Brief Description of the Drawings

[0024] Figure 1 is the synthetic route diagram of the fluorescent probe of the present invention.

[0025] Figure 2 is the nuclear magnetic resonance hydrogen spectrum diagram of the fluorescent probe in Example 1.

[0026] Figure 3 is the high-resolution mass spectrum diagram of the fluorescent probe in Example 1.

[0027] Figure 4 is the fluorescence emission spectrum diagram measured by adding different concentrations of nitric oxide to the fluorescent probe in Application Example 1.

[0028] Figure 5 is the linear relationship between the fluorescence intensity of the fluorescent probe in Application Example 1 at 1104 nm and the concentration of nitric oxide.

[0029] Figure 6 is the spectrum diagram of the change in fluorescence intensity with time measured by adding nitric oxide to the fluorescent probe in Application Example 2.

[0030] Figure 7 is the relationship diagram between the fluorescence intensity at 1104 nm and time measured by adding nitric oxide to the fluorescent probe in Application Example 2.

[0031] Figure 8 is the selectivity test diagram of the fluorescent probe in Application Example 3.

[0032] Figure 9 is the imaging diagram of the fluorescent probe in Application Example 4. Detailed Description of the Embodiments

[0033] The following further illustrates the specific implementation of the present invention in conjunction with the drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that for the processes not specifically described in detail below, those skilled in the art can implement or understand them with reference to the prior art. The reagents or instruments used without indicating the manufacturer are regarded as conventional products that can be purchased commercially.

[0034] The synthetic route of the near-infrared second-region fluorescent probe for detecting nitric oxide of the present invention is as Figure 1 shown.

[0035] Example 1 Take 109.65 mg (0.10 mmol) of compound FD1080-Cl p-toluenesulfonate in a 25 mL two-necked flask, and add 1.5 mL of N,N-dimethylformamide to dissolve it. Subsequently, add 20.24 mg (0.2 mmol) of triethylamine, and introduce nitrogen to make the reaction system under nitrogen protection. After stirring in an ice-water bath for 20 minutes, add 35.72 mg (0.10 mmol) of 4-pentaethylene glycol monomethyl ether benzylamine, and then heat the reaction system to 60 °C and react for 4 h. After the reaction is completed, add 5 mL of water, and extract the reaction system with 5 mL of DCM (dichloromethane). The organic phase is washed with water 2-3 times, the organic phase is collected and dried with anhydrous magnesium sulfate. After removing DCM by rotary evaporation, the obtained oily liquid is purified by silica gel column chromatography (the eluent used is dichloromethane / methanol, V / V = 30:1), and finally 72.77 mg of the fluorescent probe is obtained (yield: 51.33%).

[0036] Characterize it by 1H NMR: 1 H NMR (400 MHz, DMSO) δ 9.86 (s, 1H), 8.14 (d, J J = 10.4 Hz, 2H), 7.98 (d, J J = 8.3 Hz, 2H), 7.50 (d, J J = 6.5 Hz, 8H), 7.44 (d, J J = 8.8 Hz, 2H), 7.24 – 7.12 (m, 4H), 6.36 (d, J J = 13.1 Hz, 2H), 5.11 (s, 2H), 4.44 – 4.29 (m, 4H), 4.13 (t, J J = 4.6 Hz, 2H), 3.81 (t, J J = 5.4 Hz, 4H), 3.75 (t, J J = 4.6 Hz, 2H), 3.64 – 3.36 (m, 48H), 3.20 (s, 3H), 3.18 (s, 6H), 2.96 (d, J J = 12.0 Hz, 4H). The 1H NMR spectrum of the probe is as Figure 2 shown.0]

[0037] In addition, further verify it by high-resolution mass spectrometry: HR-MS (ESI, m / z): The theoretically calculated molecular mass-to-charge ratio C 71 H 94BN2O 13 + [M] + : 1246.6785, the actual molecular mass-to-charge ratio is: 1246.6738. The high-resolution mass spectrum of this probe is as Figure 3 shown.

[0038] Example 2 Take 548.23 mg (0.50 mmol) of compound FD1080-Cl p-toluenesulfonate in a 50 mL two-necked flask, add 8.5 mL of N,N-dimethylformamide to dissolve it. Subsequently, add 151.79 mg (1.5 mmol) of triethylamine, and introduce nitrogen to make the reaction system under nitrogen protection. After stirring in an ice-water bath for 25 minutes, add 267.91 mg (0.75 mmol) of 4-pentaethylene glycol monomethyl ether benzylamine, then heat the reaction system to 70 °C and react for 5 h. After the reaction is completed, add 25 mL of water, and extract the reaction system with 25 mL of DCM. The organic phase is washed with water 2-3 times, the organic phase is collected and dried with anhydrous magnesium sulfate. After rotary evaporation to remove DCM, the obtained oily liquid is purified by silica gel column chromatography (the eluent used is dichloromethane / methanol, V / V = 30:1), and finally 377.39 mg of the fluorescent probe is obtained (yield: 53.24%).

[0039] The characterization results of the fluorescent probe obtained in this example are the same as those in Example 1.

[0040] Example 3 Take 1096.45 mg (1 mmol) of compound FD1080-Cl p-toluenesulfonate in a 100 mL two-necked flask, add 20 mL of N,N-dimethylformamide to dissolve it. Subsequently, add 404.76 mg (4 mmol) of triethylamine, and introduce nitrogen to make the reaction system in a nitrogen atmosphere. After stirring in an ice-water bath for 30 minutes, add 714.43 mg (2 mmol) of 4-pentaethylene glycol monomethyl ether benzylamine, then heat the reaction system to 80 °C and react for 6 h. After the reaction is completed, add 50 mL of water, and extract the reaction system with 50 mL of DCM. The organic phase is washed with water 2-3 times, the organic phase is collected and dried with anhydrous magnesium sulfate. After rotary evaporation to remove DCM, the obtained oily liquid is purified by silica gel column chromatography (the eluent used is dichloromethane / methanol, V / V = 30:1), and finally 795.61 mg of the fluorescent probe is obtained (yield: 56.12%).

[0041] The characterization results of the fluorescent probe obtained in this example are the same as those in Example 1.

[0042] Application Example 1 Add hydrogen peroxide with different concentrations to the fluorescent probe to test the fluorescence emission spectrum: The fluorescent probe prepared in Example 1 was dissolved in DMSO to prepare a test mother liquor with a probe concentration of 1 mM. 30 μL was taken from the probe mother liquor, and then added to PBS buffer (pH = 7.4), and different amounts of NO solution (MAHMANONOate as the NO donor) were added respectively (the final concentrations of nitric oxide were 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 80 μM, 100 μM), and the total test volume was kept at 3.0 mL. The total volume of the test system was 3 mL, the probe concentration was 10 μM, and the volume percentage of DMSO was 1%. Using a wavelength of 980 nm as the excitation wavelength, the test results are as Figure 4 shown. As can be seen from Figure 4 , when no nitric oxide was added, the fluorescence of the probe was relatively weak. As the concentration of nitric oxide increased, the fluorescence intensity of the test system in the range of 1000 - 1400 nm (peak at 1104 nm) gradually increased. The fluorescence intensity at 1104 nm was linearly fitted with the nitric oxide concentration to obtain a fitting curve Y = 23.91812*X + 159.50717, R 2 = 0.998, as shown Figure 5 below. According to the lowest detection limit calculation formula LOD = 3σ / K (LOD is the lowest detection limit, σ is the standard deviation of multiple measurements (≥20 times) of the blank sample (i.e., the probe solution with a nitric oxide concentration of 0 μM), and K is the slope of the fitting curve), the lowest detection limit of this probe was calculated to be 0.45 μM. The above test results show that the fluorescent probe of the present invention has a good response effect on nitric oxide, and can realize the characterization of the change in nitric oxide concentration through the change in fluorescence intensity, and is suitable for the detection of nitric oxide in animals and plants.

[0043] Application Example 2 Response time test of the fluorescent probe to hydrogen peroxide: 30 μL of the probe mother liquor of Application Example 1 was added to a centrifuge tube, and then PBS buffer (pH = 7.4) was added, and a nitric oxide solution (the final concentration of the nitric oxide solution was 100 μM) was added. The total volume of the test system was 3 mL. The test was carried out after incubation at room temperature for 5 min, 10 min, 15 min, 25 min, 35 min, 45 min, 55 min, and 65 min respectively. The test results are as Figure 6 , Figure 7 shown. As can be seen from Figure 6 , the fluorescence intensity of the test system in the range of 1000 - 1400 nm (peak at 1104 nm) gradually increased with the increase of time and tended to be stable after 45 min. As can be seen from Figure 7It can be seen that the fluorescence intensity at 1104 nm gradually increases with time and basically no longer changes after 45 min, indicating that the response time of the probe to nitric oxide is 45 min.

[0044] Application Example 3 Selectivity test of the fluorescent probe for hydrogen peroxide: Add 30 μL of the probe mother liquor in Application Example 1 to a centrifuge tube, then add PBS buffer (pH = 7.4), and add the following analytes respectively: a. Blank (i.e., the probe solution without adding any analytes), b. Al 3+ (1 mM), c. Fe 3 + (1 mM), d. K + (1 mM), e. Cu 2+ (1 mM), f. Glutathione (1 mM), g. Cysteine (1 mM), h. Leucine (1 mM), i. Glutamic acid (1 mM), j. Tryptophan (1 mM), k. Alanine (1 mM), l. ClO - (100 μM), m. NaNO2 (100 μM), n. ONOO - (100 μM), o. H2O2 (100 μM), p. NO (100 μM). The total volume of each system is 3 mL, the concentration of the fluorescent probe in each test sample is 10 μM, and the final concentration of each analyte in the test system is the concentration in the parentheses of each analyte. During the selectivity test, the samples are incubated at room temperature for 45 min and then tested. The test results are as Figure 8 (the fluorescence intensity is the fluorescence intensity at the peak of 1104 nm) shown. It can be seen from Figure 8 that when other analytes are added, the fluorescence intensity of the test group does not change significantly; only when nitric oxide is added, the fluorescence intensity of the test group increases significantly. The experimental results show that the probe has good selectivity for nitric oxide, which will be beneficial to its application in the detection of nitric oxide in animals and plants.

[0045] Application Example 4 Near-infrared second-region imaging test of the fluorescent probe: Add 20 μL of the probe mother liquor in Application Example 1 to a centrifuge tube, then add PBS buffer (pH = 7.4), and add nitric oxide solutions with different concentrations respectively (the final concentrations of the nitric oxide solutions are 0 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM). The total volume of the test system is 1 mL (the probe concentration is 10 μM). After incubating at room temperature for 45 min, place the sample in the imaging chamber of the NIR-II imaging system for imaging. In the imaging experiment, the selected laser excitation wavelength is 980 nm, the power is 60 mW / cm2, and the wavelength range of the emission filter is 900 - 1700 nm. The test results are as Figure 9 shown. It can be seen from Figure 9 that as the nitric oxide concentration in the sample increases, the fluorescence intensity gradually increases. The experimental results show that this fluorescent probe can effectively detect nitric oxide and reflect the nitric oxide level in animals and plants through the change of fluorescence intensity. This probe provides an effective solution for imaging and tracking the detection of nitric oxide in animals and plants, demonstrating its potential in the detection of nitric oxide in animals and plants.

[0046] The above examples are preferred embodiments of the present invention, but the embodiments of the invention are not limited by the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A near-infrared second near-infrared fluorescence probe for detecting nitric oxide, characterized in that, The molecular formula of the probe is C 71 H 96 N3O 16 + , and it has the following structural formula: 。 2. The preparation method of the near-infrared second-window fluorescence probe for detecting nitric oxide according to claim 1, characterized in that, It includes the following steps: Dissolve the compound FD1080-Cl shown below in N,N-dimethylformamide (DMF), add triethylamine, introduce an inert gas to make the reaction system in an inert atmosphere, stir the reaction under an ice-water bath, then add 4-pentaethylene glycol monomethyl ether benzylamine, and then raise the temperature to carry out the reaction; after the reaction is completed, separate and purify to obtain the near-infrared second-region fluorescence probe. 。 3. The preparation method of the near-infrared second-region fluorescent probe for detecting nitric oxide according to claim 2, characterized in that, The molar ratio of the compound FD1080-Cl to triethylamine is 1:(2 - 4).

4. The preparation method of the near-infrared second-window fluorescence probe for detecting nitric oxide according to claim 2, characterized in that, The molar ratio of the compound FD1080-Cl to 4-pentaethylene glycol monomethyl ether benzylamine is 1:(1 - 2).

5. The preparation method of the near-infrared second-region fluorescent probe for detecting nitric oxide according to claim 2, wherein, The amount of N,N-dimethylformamide added per mmol of the compound FD1080-Cl is (15 - 20) mL.

6. The preparation method of the near-infrared second near-infrared fluorescence probe for detecting nitric oxide according to claim 2, wherein, The inert gas is nitrogen.

7. The preparation method of the near-infrared second near-infrared fluorescence probe for detecting nitric oxide according to claim 2, characterized in that, The stirring reaction time under the ice-water bath is 20 - 30 minutes; the temperature for the stirring reaction after raising the temperature is 60 - 80 °C, and the reaction time is 4 - 6 h.

8. The preparation method of the near-infrared second-window fluorescence probe for detecting nitric oxide according to claim 2, wherein, The purification method is silica gel chromatography.

9. The preparation method of the near-infrared second near-infrared fluorescence probe for detecting nitric oxide according to claim 8, characterized in that, The eluent for the silica gel chromatography is dichloromethane / methanol.

10. Use of the near-infrared second-region fluorescence probe for detecting nitric oxide described in claim 1 in detecting nitric oxide in animals and plants.

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