Thionitrous acid near-infrared light activated probe as well as preparation method and application thereof

Abstract

The invention provides a thionitrous acid near-infrared light activated probe as well as a preparation method and application thereof. The probe is stable in property, good in selectivity, high in sensitivity and low in detection limit (142 nM). The probe can detect the physiological level HSNO in HCT116 cells, and is applied to fluorescence imaging of HSNO in HCT116 colon cancer nude mouse transplanted tumor, so that a visual detection tool is provided for revealing the physiological and pathological mechanism of HSNO.

Description

Technical Field

[0001] The invention belongs to the field of chemistry and analytical detection, and specifically relates to a thionitrite near-infrared light-activated probe and a preparation method and application thereof. Background Art

[0002] Thionitrious acid (HSNO), as the smallest molecular weight S-nitrosothiol, is a key signal transduction molecule connecting the H2S and NO signaling pathways. HSNO can not only regulate protein function through nitrosylation, but also convert NO and H2S into n , HNO and other important signal transduction molecules in the body are "transported" into the cells, thereby playing an important role in the cardiovascular, nervous and immune systems.

[0003] In recent years, more and more studies have shown that hydrogen sulfide can inhibit or activate the activity of NOS. NO affects the production of endogenous H2S by inhibiting or inducing the activity or expression of cystathionine-β-synthase and cystathionine-γ-lyase. There is a biphasic effect on cell proliferation and apoptosis, which is related to factors such as the level of H2S / NO, the duration of action, the type of tumor cells and sensitivity. In colon cancer, H2S and NO regulate cell proliferation through a bell-shaped concentration-response curve. Lower levels of endogenous H2S / NO promote tumor cell proliferation, while exposure to higher levels of exogenous H2S / NO shows anti-tumor activity. In colon cancer, H2S participates in the anti-tumor migration and invasion activity mediated by exogenous NO, suggesting that there is a cross-talk between H2S and NO mediated by the cGMP / VASP signaling pathway. It can be seen that the expression of H2S and NO and the crosstalk between them affect the development of colon cancer. HSNO is the source of NO and the product of the crosstalk between H2S and NO. Its level changes are closely related to the occurrence and development of colon cancer, but the specific mechanism is still unclear. It is urgent to develop an accurate HSNO detection method, which will help to better understand its physiological role and help study the relationship between colon cancer and HSNO level changes.

[0004] Existing HSNO detection methods include Fourier transform infrared spectroscopy and high-resolution mass spectrometry, which require the separation and extraction of cells or tissues and cannot be imaged in real time. The fluorescent probe method has high selectivity and high sensitivity, and can achieve real-time in situ monitoring of HSNO. At present, there are only two reports on HSNO fluorescent probes, but they cannot achieve non-invasive, real-time, and continuous in vivo detection of HSNO. Therefore, it is necessary to develop a molecular probe with strong tissue penetration, high spatiotemporal resolution, high sensitivity, and suitable for in vivo detection of HSNO.

[0005] The sensing and imaging of photoactivated fluorescent probes are regulated by light, thus providing high temporal and spatial resolution imaging. It is well known that most photoactivated protecting groups require UV / visible light for excitation, resulting in strong autofluorescence interference, accompanied by phototoxicity and photodamage, which limits their imaging applications at the deep tissue, organ and in vivo levels. In order to overcome these limitations, near-infrared light-activated probes have been developed, which can increase the tissue imaging depth of the probes and reduce phototoxicity and photodamage. Rare earth upconversion nanoparticles are important carriers for near-infrared light activation / regulation, which can absorb long-wavelength near-infrared excitation light and upconvert it into visible / ultraviolet light or short-wavelength near-infrared emission light. Therefore, the development of near-infrared light-activated fluorescent probes can achieve deep tissue and in vivo imaging while reducing phototoxicity and photodamage. Summary of the invention

[0006] The purpose of the present invention is to provide a thionitrite near-infrared light-activated probe based on the prior art, which not only has good selectivity and high sensitivity (detection limit of 142nM), but also successfully realizes fluorescence imaging detection of HSNO at the HCT116 cell level and in vivo level, providing a visual detection tool for revealing the physiological and pathological mechanism of HSNO, and is of great significance for studying diseases related to HSNO, such as cancer, cardiovascular disease and neurodegenerative diseases.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned photoactivated probe.

[0008] The third object of the present invention is to provide the use of the above-mentioned photoactivatable probe in detecting thionitrite.

[0009] The technical solution of the present invention is as follows:

[0010] The design of the probe UCNPs-R-PSN mainly includes the following three points: (1) The amino-functionalized upconversion nanoparticles UCNPs-SiO2-NH2 (NaYF4:Yb / Tm@NaYF4@SiO2-NH2) are used as carriers for near-infrared light activation and coupling of photoactivated fluorescent probes. (2) Ortho-nitrobenzyl ester derivatives are selected as photoactivation protecting groups, rhodamine derivatives are used as near-infrared fluorescent groups, and o-thiobenzoate and o-phenylenediamine are used as HSNO recognition groups to construct the photoactivated fluorescent probe R-PSN. (3) The photoactivated fluorescent probe is coupled to the upconversion nanoparticles through an amide bond to construct the UCNPs-based near-infrared light-activated fluorescent probe UCNPs-R-PSN.

[0011] A thionitrite near-infrared light-activated probe (UCNPs-R-PSN), the synthesis route of the light-activated probe is as follows:

[0012]

[0013]

[0014] The recognition mechanism is as follows: (1) UCNPs-R-PSN is irradiated with 980nm near-infrared light, and the upconversion nanoparticles generate upconversion 365nm ultraviolet light, which triggers the photolysis reaction of the photoactivated protective group, and the o-nitrobenzyl ester group breaks, releasing the compound PSN; (2) The S of HSNO is electrophilic, and undergoes a nucleophilic reaction and an intramolecular cyclization reaction with the exposed thiol group of the compound PSN, releasing benzodithiophene; (3) N=O in HSNO reacts with o-phenylenediamine in the structure of PSN-1 to generate benzotriazole and hydrolyze it to release the fluorescent mother core R-2, turning on the near-infrared fluorescence of the rhodamine derivative. The product after the reaction of UCNPs-R-PSN and HSNO was verified by high-resolution mass spectrometry. After the probe reacted with HSNO, compound R-2 was generated, which proved the reaction process of the probe and HSNO.

[0015] The present invention provides a thionitrite near-infrared light-activated probe, and the preparation method of the light-activated probe comprises the following steps:

[0016] (1) Synthesis of NaYF4:Yb / Tm core nanoparticles: YCl3 . 6H2O, YbCl3 . 6H2O、TmCl3 . 6H2O, oleic acid and octadecene are uniformly mixed, heated to 140-160°C to remove the low boiling point solvent, cooled to 20-30°C, added with an anhydrous methanol solution containing NaOH and NH4F to react by stirring, then heated to 80-100°C to remove the anhydrous methanol, the obtained mixture is rapidly heated to 280-320°C at a rate of 10-20°C / min to react by stirring, cooled to 20-30°C again, centrifuged and separated to obtain NaYF4:Yb / Tm core nanoparticles;

[0017] (2) Synthesis of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles: YCl3 .6H2O oleic acid and octadecene are uniformly mixed, heated to 140-160°C to remove the low boiling point solvent, cooled to 20-30°C, added with the mixed solution prepared by dispersing the NaYF4:Yb / Tm core nanoparticles obtained in step (1) in cyclohexane, heated to 80-100°C to remove the cyclohexane, cooled to 20-30°C again, added with anhydrous methanol solution containing NaOH and NH4F to react with stirring, heated to 80-100°C to remove the anhydrous methanol, and the obtained mixture is rapidly heated to 280-320°C at a rate of 10-20°C / min to react with stirring, cooled to 20-30°C, centrifuged, and separated to obtain NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles;

[0018] (3) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2: CTAB is dispersed in deionized water, and a mixed solution prepared by dispersing the NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles obtained in step (2) in cyclohexane is added thereto for stirring reaction to form a NaYF4:Yb / Tm@NaYF4-CTAB solution, to which ethanol and sodium hydroxide solution are added, stirred evenly, heated to 60-80°C, tetraethyl silicate is added dropwise for stirring reaction, the reaction solution is collected, centrifuged, and separated, the surfactant CTAB is removed from the obtained product by ion exchange, the obtained precipitate is resuspended in anhydrous ethanol and NaCl is added, the temperature is raised to 50-70°C for stirring reaction, centrifuged again, and separated to obtain SiO2-coated upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2;

[0019] (4) Synthesis of UCNPs-SiO2-NH2: Disperse the NaYF4:Yb / Tm@NaYF4@SiO2 obtained in step (3) in anhydrous ethanol, add (3-aminopropyl)triethoxysilane dropwise, stir and react at 60-80°C, centrifuge and separate to obtain amino-functionalized upconversion nanoparticles UCNPs-SiO2-NH2;

[0020] (5) Synthesis of compound T-1: Concentrated sulfuric acid, dichloromethane and anhydrous magnesium sulfate were uniformly mixed, 4-bromomethyl-3-nitrobenzoic acid and tert-butyl alcohol were added thereto, and the mixture was stirred and reacted at 25-35° C. to prepare compound T-1;

[0021] (6) Synthesis of compound T-2: Compound T-1 and o-mercaptobenzoic acid were mixed, anhydrous potassium carbonate and DMF were added, and the mixture was stirred at 25-35° C. to react to prepare compound T-1;

[0022] (7) Synthesis of compound R-2: Compound R-1 and 1,6-dihydroxynaphthalene were dissolved in methanesulfonic acid, and stirred at 80-90° C. to prepare compound R-2;

[0023] (8) Synthesis of compound R-3: Compound T-2, 4-dimethylaminopyridine and EDCI were dissolved in dichloromethane, and compound R-2 was added to react by stirring to prepare compound R-3;

[0024] (9) Synthesis of compound R-4: Compound R-3 was dissolved in dichloromethane, and DMF and oxalyl chloride were added to react with stirring to prepare an intermediate product; the intermediate product was dissolved in dichloromethane, and added to an anhydrous dichloromethane solution containing o-phenylenediamine and triethylamine, and reacted with stirring to prepare compound R-4;

[0025] (10) Synthesis of compound R-PSN: Compound R-4 was dissolved in dichloromethane, trifluoroacetic acid was added dropwise, and the mixture was stirred to react to prepare compound R-PSN;

[0026] (11) Synthesis of probe UCNPs-R-PSN: Compound R-PSN, UCNPs@SiO2-NH2, HBTU and HOBt were mixed and stirred for reaction. DIPEA was then added and the stirring reaction was continued to obtain the probe UCNPs-R-PSN.

[0027] In a preferred embodiment, in step (1), YCl3 . 6H2O, YbCl3 . 6H2O and TmCl3 . The molar ratio of 6H2O is 0.7-0.9:0.1-0.3:0.003-0.007, preferably 0.79:0.2:0.005,

[0028] In step (1), YCl3 . The mass volume ratio of 6H2O to oleic acid is 235-245:7 mg / mL, preferably 241:7 mg / mL.

[0029] In step (1), YCl3 . The mass volume ratio of 6H2O to octadecene is 235-245:15 mg / mL, preferably 235-245:15 mg / mL.

[0030] In step (1), YCl3 . The molar ratio of 6H2O to NaOH is 235-245:100, preferably 241:100; the YCl3 . The molar ratio of 6H2O to NH4F is 235-245:148, preferably 241:148.

[0031] In step (1), the temperature for removing the low boiling point solvent is 145-155°C, preferably 150°C; the temperature for removing anhydrous methanol is 85-95°C, preferably 90°C; the temperature is rapidly increased to 290-310°C, preferably 300°C, at a rate of 15°C / min.

[0032] In a preferred embodiment, in step (2), YCl3 . The mass volume ratio of 6H2O to oleic acid is 300-310:7 mg / mL, preferably 303:7 mg / mL.

[0033] In step (2), YCl3 . The mass volume ratio of 6H2O to octadecene is 300-310:15 mg / mL, preferably 303:15 mg / mL.

[0034] In step (2), YCl3 . The molar ratio of 6H2O to NaOH is 300-310:100, preferably 303:100. . The molar ratio of 6H2O to NH4F is 300-310:148, preferably 303:148.

[0035] In step (2), the temperature for removing the low boiling point solvent is 145-155°C, preferably 150°C; the temperature for removing cyclohexane is 85-95°C, preferably 90°C; the temperature for removing anhydrous methanol is 85-95°C, preferably 90°C; the temperature is rapidly increased to 290-310°C, preferably 300°C, at a rate of 15°C / min.

[0036] In a preferred embodiment, in step (3), the weight ratio of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles to CTAB is 1:3-7, preferably 1:5.

[0037] In step (3), the mass volume ratio of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles to tetraethyl silicate is 4-6:3 mg / μL, preferably 5:3 mg / μL.

[0038] In a preferred embodiment, in step (4), the mass volume ratio of NaYF4:Yb / Tm@NaYF4@SiO2 to (3-aminopropyl)triethoxysilane is 1-1.5:2.5 mg / μL, preferably 1:2 mg / μL.

[0039] In a preferred embodiment, in step (5), the molar ratio of 4-bromomethyl-3-nitrobenzoic acid to tert-butanol is 1:3-7, preferably 1:5.

[0040] In a preferred embodiment, in step (6), the molar ratio of compound T-1 to o-mercaptobenzoic acid is 1:0.8-1.5, preferably 1:1.

[0041] In a preferred embodiment, in step (7), the molar ratio of compound R-1 to 1,6-dihydroxynaphthalene is 1:0.8-1.5, preferably 1:1.

[0042] In a preferred embodiment, in step (8), the molar ratio of compound T-2 to 4-dimethylaminopyridine is 230-240:54, preferably 233:54. The molar ratio of compound T-2 to EDCI is 230-240:349, preferably 233:349. The molar ratio of compound T-2 to compound R-2 is 230-240:275, preferably 233:275.

[0043] In a preferred embodiment, in step (9), the molar ratio of compound R-3 to oxalyl chloride is 1:2-4, preferably 1:3. The molar ratio of compound R-3 to o-phenylenediamine is 1:2-4, preferably 1:3. The molar ratio of compound R-3 to triethylamine is 1:2-4, preferably 1:3.

[0044] In a preferred embodiment, in step (10), the mass volume ratio of compound R-4 to trifluoroacetic acid is 425-435:1 mg / mL, preferably 430:1 mg / mL.

[0045] In a preferred embodiment, in step (11), the mass ratio of compound R-PSN to UCNPs@SiO2-NH2 is 3-7:1, preferably 5:1; the molar ratio of compound R-PSN to HBTU is 1:0.8-1.5, preferably 1:1; the molar ratio of compound R-PSN to HOBt is 1:0.8-1.5, preferably 1:1; the molar ratio of compound R-PSN to DIPEA is 1:0.8-1.5, preferably 1:1.

[0046] Adopt the technical scheme of the present invention, the advantages are as follows:

[0047] The present invention provides a thionitrite near-infrared light-activated probe, which not only has good selectivity and high sensitivity (detection limit of 142nM), but also successfully realizes fluorescence imaging detection of HSNO at the HCT116 cell level and in vivo level. By intraperitoneal injection of exogenous HSNO, it is found that it has anti-tumor activity against HCT116 transplanted tumors. The probe is used to detect the HSNO level in HCT116 tumor slices. The results show that compared with the saline group, after HSNO treatment, the HSNO level in nude mouse HCT116 tumor slices is significantly increased. This finding preliminarily indicates that the change in HSNO level is associated with the occurrence and development of colon cancer, and provides a visual detection tool for revealing the physiological and pathological mechanism of HSNO. It is also of great significance for studying diseases related to HSNO, such as cancer, cardiovascular disease and neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the synthetic route of the probe UCNPs-R-PSN;

[0049] Figure 2 This is the recognition mechanism of the probe UCNPs-R-PSN to detect HSNO;

[0050] Figure 3 is the HRMS of the reaction product of probe UCNPs-R-PSN+HSNO. (calculated for C 28 H 23 NO4[M+H] + ,438.1700; found,438.1699);

[0051] Figure 4 The compound R-PSN 1 H NMR spectrum;

[0052] Figure 5 The compound R-PSN 13 CNMR spectrum;

[0053] Figure 6 This is the high-resolution mass spectrum of compound R-PSN: [MH] - calculated for C 49 H 38 N4O8S,841.2338; found,841.2333;

[0054] Figure 7 is a characterization of NaYF4:Yb / Tm; where, Figure 7 A is the transmission electron microscopy image of NaYF4:Yb / Tm; Figure 7B is the particle size distribution diagram of NaYF4:Yb / Tm; Figure 7 C is the fluorescence spectrum of NaYF4:Yb / Tm; Figure 7 D is the XRD pattern of NaYF4:Yb / Tm;

[0055] Figure 8 is the characterization of NaYF4:Yb / Tm@NaYF4; among them, Figure 8 A is the transmission electron microscopy image of NaYF4:Yb / Tm@NaYF4; Figure 8 B is the particle size distribution diagram of NaYF4:Yb / Tm@NaYF4;

[0056] Fig. 9 is the characterization of the probe UCNPs-R-PSN; among them, Fig. 9 A is the transmission electron microscopy image of UCNPs-R-PSN; Fig. 9 B is the particle size distribution diagram of UCNPs-R-PSN; Fig. 9 C is the Zeta potential value of UCNPs-SiO2, UCNPs-SiO2-NH2 and UCNPs-R-PSN;

[0057] Fig.10 is the UV absorption spectrum of the probe UCNPs-R-PSN before and after the reaction with HSNO; probe UCNPs-R-PSN (0.1 mg / mL, in DMSO), probe UCNPs-R-PSN (0.1 mg / mL, in DMSO) + 980 nm light (3 W / cm 2 , 10min), probe UCNPs-R-PSN (0.1mg / mL, in DMSO) + 980nm light (3W / cm 2 , 10min)+HSNO (100μM), compound R-2 (40μM) incubated in PBS buffer (50mM, pH=7.4, 20% DMSO) for 10min; Among them, at 450nm, the curves from high to low are compound R-2, probe UCNPs-R-PSN+light+HSNO, probe UCNPs-R-PSN and probe UCNPs-R-PSN+light, and the latter two curves are basically overlapping;

[0058] Fig.11 It is the loading amount determination of compound R-PSN in probe UCNPs-R-PSN; wherein, Fig.11The black line in A (at 275 nm, the higher curve): UV-visible absorption spectrum of compound R-PSN (65 μM), the red line (at 275 nm, the lower curve): UV-visible absorption spectrum of the unreacted compound R-PSN remaining in the reaction supernatant obtained by centrifugation (10000 rpm / min) after compound R-PSN (65 μM) was loaded onto the surface of upconversion nanoparticles; Fig.11 B is the linear fitting standard curve of the absorbance of different concentrations of R-PSN (5-80 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM) at 267 nm;

[0059] Fig.12 is the fluorescence spectrum of the probe UCNPs-R-PSN and HSNO response; among them, the probe UCNPs-R-PSN (0.1 mg / mL), the probe UCNPs-R-PSN (0.1 mg / mL) + HSNO (100 μM), the probe UCNPs-R-PSN (0.1 mg / mL) + 980 nm light (3 W / cm 2 , 10min), probe UCNPs-R-PSN (0.1mg / mL) + 980nm light (3W / cm 2 , 10 min) + HSNO (100 μM) in PBS buffer (50 mM, pH = 7.4, 20% DMSO), incubated at 37 ° C for 10 min (λ ex =575nm, slit: 10nm);

[0060] Fig.13 is the change in fluorescence response intensity of the probe UCNPs-R-PSN after being irradiated at 980nm and incubated with HSNO (0-80μM) in PBS buffer (50mM, pH=7.4, 20% DMSO) for 10min; Fig.13 A is UCNPs-R-PSN (0.1 mg / mL) after 980 nm (3 W / cm 2 ) after irradiation for 10 min, and then incubated with different concentrations of HSNO (0-80 μM, 0 μM, 1 μM, 1.2 μM, 1.4 μM, 1.6 μM, 1.8 μM, 2 μM, 2.2 μM, 2.4 μM, 2.6 μM, 2.8 μM, 3 μM, 4 μM, 6 μM, 8 μM, 10 μM, 20 μM, 40 μM, 80 μM) for 10 min; Fig.13 B is the change in fluorescence intensity of UCNPs-R-PSN (0.1 mg / mL) after irradiation at 980 nm and incubation with different concentrations of HSNO (0-80 μM) for 10 min; Fig.13 C is the linear relationship between fluorescence intensity and HSNO concentration (0-2 μM) (λ ex =575nm, slit: 10nm);

[0061] Fig.14 is the fluorescence spectra of the probe UCNPs-R-PSN and HSNO incubated for different time periods; Fig.14 A is the probe UCNPs-R-PSN (0.1 mg / mL) after 980nm (3W / cm 2 ) after irradiation for 10 min, and then incubated with HSNO (100 μM) in PBS buffer (50 mM, pH=7.4, 20% DMSO) at 37°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 and 90 min, respectively; Fig.14 B is the change in fluorescence intensity of the probe UCNPs-R-PSN with HSNO (100 μM) in PBS buffer (50 mM, pH = 7.4, 20% DMSO) at 37°C over time (λ ex =575nm, slit: 10nm);

[0062] Fig.15 is the effect of pH on the reaction between UCNPs-R-PSN and HSNO; Fig.15 A probe UCNPs-R-PSN (0.1 mg / mL) was subjected to 980 nm (3 W / cm 2 ) after irradiation for 10 min, and then incubated with HSNO (100 μM) in PBS buffer with different pH values ​​(50 mM, pH=4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.4; 8.0; 8.5; 9.0; 20% DMSO) at 37°C for 10 min; Fig.15 Probe B UCNPs-R-PSN (0.1 mg / mL) was subjected to 980 nm (3 W / cm 2 ) after 10 min of irradiation, and then incubated with HSNO (100 μM) at 37 °C for 10 min. The fluorescence intensity changes with different pH values ​​(λ ex =575nm, slit: 10nm);

[0063] Fig.16 is the selectivity of the probe UCNPs-R-PSN to HSNO; among them, Fig.16 A is UCNPs-R-PSN (0.1 mg / mL) after 980 nm (3 W / cm 2) was irradiated for 10 min, and then incubated with HSNO (100 μM) and amino acids (blank; 1 mM Gly; 1 mM D-Ala; 1 mM Val; 1 mM Ile; 1 mM L-Cys; 1 mM L-Thr; 1 mM Asp; 1 mM L-Glu; 1 mM L-Arg; 1 mM L-Lys; 1 mM L-His; 1 mM Hcy) in PBS buffer (50 mM, pH = 7.4, 20% DMSO) at 37°C for 10 min; Fig.16 B is UCNPs-R-PSN after 980nm irradiation, incubated with HSNO (100μM) and amino acids for 10min. Black represents the fluorescence intensity of UCNPs-R-PSN at 645nm after incubation with various amino acids, and red represents the fluorescence intensity of UCNPs-R-PSN at 645nm after incubation with a mixture of various amino acids and HSNO (100μM). 1. Blank + HSNO (100μM); 2. Gly (1mM) + HSNO (100μM); 3. D-Ala (1mM) + HSNO (100μM); 4. Val (1mM) + HSNO (100μM); 5. Ile (1mM) + HSNO (100μM); 6. L-Cys (1mM) + HSNO (100μM); 7. L-Thr (1mM) + HSNO ( 100μM); 8.Asp(1mM)+HSNO(100μM); 9.L-Glu(1mM)+HSNO(100μM); 10.L-Arg(1mM)+HSNO(100μM ); 11.L-Lys(1mM)+HSNO(100μM); 12.L-His(1mM)+HSNO(100μM); 13.Hcy(1mM)+HSNO(100μM)(λ ex =575nm, slit: 10nm);

[0064] Fig.17 is the selectivity of the probe UCNPs-R-PSN to HSNO; among them, Fig.17 A is UCNPs-R-PSN (0.1 mg / mL) after 980 nm (3 W / cm 2 ) for 10 min, and then with HSNO (100 μM) and inorganic salt ions (blank; 1 mM Sn 2+ ; 1mM Cd 2+ ; 1mM Mn 2+ ; 1 mM Co 2+ ; 1mM Cu 2+ ; 1mM Hg 2+ ; 1mM Zn 2+ ; 1mM Fe2+ ; 1mM Fe 3+ ; 1 mM Ca 2+ ; 1mM Al 3+ ; 1mM Ni 2+ ; 1 mM Mg 2+ ; 1mM Li + ; 1mM Na + ; 1mM K + ; 1mM Ag + ) Fluorescence spectra of cells incubated in PBS buffer (50 mM, pH = 7.4, 20% DMSO) at 37 °C for 10 min; Fig.17 B is the fluorescence intensity of UCNPs-R-PSN after irradiation at 980nm and incubation with HSNO (100μM) and inorganic salt ions for 10min. The black color indicates the fluorescence intensity of UCNPs-R-PSN at 645nm after incubation with various inorganic salt ions, and the red color indicates the fluorescence intensity of UCNPs-R-PSN at 645nm after incubation with a mixture of various inorganic salt ions and HSNO (100μM); 1. Blank + HSNO (100μM); 2. Sn 2+ (1mM)+HSNO(100μM);3.Cd 2+ (1mM)+HSNO(100μM);4.Mn 2+ (1mM)+HSNO(100μM);5.Co 2+ (1mM)+HSNO(100μM);6.Cu 2+ (1mM)+HSNO(100μM);7.Hg 2+ (1mM)+HSNO(100μM);8.Zn 2+ (1mM)+HSNO(100μM);9.Fe 2+ (1mM)+HSNO(100μM);10.Fe 3+ (1mM)+HSNO(100μM);11.Ca 2+ (1mM)+HSNO(100μM);12.Al 3+ (1mM)+HSNO(100μM);13.Ni 2+ (1mM)+HSNO(100μM);14.Mg 2+ (1mM)+HSNO(100μM);15.Li + (1mM)+HSNO(100μM);16.Na + (1mM)+HSNO(100μM);17.K + (1mM)+HSNO(100μM);18.Ag +(1mM)+HSNO(100μM)(λ ex =575nm, slit: 10nm);

[0065] Fig.18 is the selectivity of probe UCNPs-R-PSN to HSNO; among them, Fig.18 A is UCNPs-R-PSN (0.1 mg / mL) after 980 nm (3 W / cm 2 ) after irradiation for 10 min, and then incubated with HSNO (100 μM) and RSS (blank; 100 μM Na2S; 100 μM Na2S+100 μM DEA·NONOate; 100 μM Na2S2; 100 μM Na2S2+100 μM DEA·NONOate; 500 μM NaHS; 500 μM Na2SO3; 500 μM NaHSO3; 100 μM CH3SSSCH3; 500 μM CysSSCys; 1 mM GSSG, 500 μM S8; 10 mM GSH) in PBS buffer (50 mM, pH=7.4, 20% DMSO) at 37°C for 10 min; Fig.18 After irradiation at 980 nm, BUCNPs-R-PSN was incubated with HSNO (100 μM) and active sulfur for 10 min; black represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after incubation with various active sulfurs, and red represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after incubation with a mixture of various active sulfurs and HSNO (100 μM); 1. Blank + HSNO (100 μM); 2. Na2S (100 μM) + HSNO (100 μM); 3. Na2S (100 μM) + DEA·NONOate (100 μM) + HSNO (100 μM); 4. Na2S2 (100 μM) + HSNO (100 μM); 5. Na 2S2(100μM)+DEA·NONOate(100μM)+HSNO(100μM); 6.NaHS(500μM)+HSNO(100μM); 7.Na2SO3(500μM)+HSNO(100μM); 8.NaHSO3(500μM)+HSNO(100μM); 9.CH3SS SCH3 (100μM) + HSNO (100μM); 10. CysSSCys (500μM) + HSNO (100μM); 11. GSSG (1mM) + HSNO (100μM); 12. S8 (500μM) + HSNO (100μM); 13. GSH (10mM) + HSNO (100μM) (λ ex=575nm, slit: 10nm);

[0066] Fig.19 is the selectivity of the probe UCNPs-R-PSN to HSNO; among them, Fig.19 A is UCNPs-R-PSN (0.1 mg / mL) after 980 nm (3 W / cm 2 ) for 10 min, and then HSNO (100 μM) and ROS (blank; 100 μM H2O2; 100 μM ClO - ; 100μM t BuOOH; 100 μM · OH; 100 μM 1 O2; 100μM O2 ·- ; 100μM NO3 - ; 100μM NO2 - ; 100 μM ONOO - ; 1mM L-AA; 1mM DHA; 1mM MGO) in PBS buffer (50mM, pH=7.4, 20% DMSO) at 37°C for 10min; Fig.19 B is UCNPs-R-PSN after 980nm irradiation, incubated with HSNO (100μM) and reactive oxygen species for 10min. Black represents the fluorescence intensity of UCNPs-R-PSN at 645nm after incubation with various reactive oxygen species, and red represents the fluorescence intensity of UCNPs-R-PSN at 645nm after incubation with a mixture of various reactive oxygen species and HSNO (100μM). 1. Blank + HSNO (100μM); 2. H2O2 (100μM) + HSNO (100μM); 3. ClO - (100μM)+HSNO(100μM);4. t BuOOH(100μM)+HSNO(100μM); 5. · OH(100μM)+HSNO(100μM);6. 1 O2(100μM)+HSNO(100μM); 7.O2 ·- (100μM)+HSNO(100μM);8.NO3 - (100μM)+HSNO(100μM); 9.NO2 - (100μM)+HSNO(100μM); 10.ONOO -(100μM)+HSNO(100μM); 11.L-AA(1mM)+HSNO(100μM); 12.DHA(1mM)+HSNO(100μM); 13.MGO(1mM)+HSNO(100μM)(λ ex =575nm, slit: 10nm);

[0067] Fig. 20 The effect of probe UCNPs-R-PSN on cell survival rate. The effect of HCT116 cells incubated with different concentrations of probe UCNPs-R-PSN (0, 50, 100, 200, 400, 500 μg / mL) on cell survival rate after 48 h; data are expressed as mean ± standard deviation (n = 3);

[0068] Fig.21 The effect of probe UCNPs-R-PSN on cell survival rate; the effect of HCT116 cells co-incubated with the probe (final concentration 100 μg / mL) for different time periods (0, 6, 12, 24, 48 h) on cell survival rate. Data are expressed as mean ± standard deviation (n = 3);

[0069] Fig. 22 It is the fluorescence imaging of HSNO detected by probe UCNPs-R-PSN; among them, Fig. 22 A: probe (final concentration 0.1 mg / mL, 5 μL DMSO) incubated with HCT116 cells for 30 min; Fig. 22 B: The probe (final concentration 0.1 mg / mL, 5 μL DMSO) was incubated with HCT116 cells for 30 min, and then 100 μM HSNO was added and incubated for 10 min; Fig. 22 C) probe (final concentration 0.1 mg / mL, 5 μL DMSO) was incubated with HCT116 cells for 30 min, and then irradiated with 980 nm (1 W / cm 2 ) Continuous irradiation for 10 min; Fig. 22 D is a probe (final concentration 0.1 mg / mL, 5 μL DMSO) incubated with HCT116 cells for 30 min, then 100 μM HSNO was added for 10 min, and then 980 nm (1 W / cm 2 ) Continuous irradiation for 10 min; Fig. 22 E is the probe (final concentration 0.1 mg / mL, 5 μL DMSO) incubated with HCT116 cells for 30 min, then added with 150 μM Na2S for 10 min, and then irradiated with 980 nm (1 W / cm 2 ) Continuous irradiation for 10 min; Fig. 22F is a probe (final concentration 0.1 mg / mL, 5 μL DMSO) incubated with HCT116 cells for 30 min, then 1 mM GSNO was added for 10 min, and then 980 nm (1 W / cm 2 ) Continuous irradiation for 10 min; Fig. 22 G is Fig. 22 Mean fluorescence intensity of cells in A, 22B, 22C, 22D, 22E, and 22F;

[0070] Fig.23 yes Fig. 22 Corresponding bright field images of cells;

[0071] Fig.24 It is the fluorescence imaging of HSNO detected by probe UCNPs-R-PSN; among them, Fig.24 A: probe (final concentration 0.1 mg / mL, 5 μL DMSO) incubated with HCT116 cells for 30 min; Fig.24 B is the probe (final concentration 0.1 mg / mL, 5 μL DMSO) co-incubated with HCT116 cells for 30 min, and then irradiated with 980 nm (1 W / cm 2 ) Continuous irradiation for 10 min; Fig.24 C The cells were incubated with AOAA (20 μM) for 45 min, and then with the probe (final concentration 0.1 mg / mL, 5 μL DMSO) for 30 min. Then, 980 nm (1 W / cm 2 ) Continuous irradiation for 10 min; Fig.24 D probe (final concentration 0.1 mg / mL, 5 μL DMSO) was incubated with CBS siRNA-transfected HCT116 cells for 30 min; Fig.24 The E probe (final concentration 0.1 mg / mL, 5 μL DMSO) was co-incubated with CBS siRNA-transfected HCT116 cells for 30 min, and then irradiated with 980 nm (1 W / cm 2 ) Continuous irradiation for 10 min; Fig.24 F is Fig.24 Mean fluorescence intensity of cells in A, 24B, 24C, and 22D; Fig.24 G is the expression level of CBS protein in HCT116 cells and HCT116 cells transfected with CBS siRNA; data are expressed as mean ± SD (n = 3), compared with the control group cells, ****P < 0.0001;

[0072] Fig.25 yes Fig.24 Corresponding bright field images of cells;

[0073] Fig.26The probe UCNPs-R-PSN was used to detect HSNO in the nude mouse model of colon cancer transplantation; Fig.26 A: The probe UCNPs-R-PSN (50 mg / mL, 10 μL, saline: dimethyl sulfoxide = 9:1 v / v) was injected into the mouse tumor; Fig.26 B shows the mouse intratumoral injection of probe UCNPs-R-PSN (50 mg / mL, 10 μL, saline: dimethyl sulfoxide = 9:1 v / v), followed by 980 nm (2 W / cm 2 ) Continuous irradiation for 5 minutes, followed by a 5-minute interval, for a total of 4 cycles, for a total of 20 minutes of irradiation; Fig.26 C shows that the probe UCNPs-R-PSN (50 mg / mL, 10 μL, saline: dimethyl sulfoxide = 9:1 v / v) was injected into the mouse tumor, and then exogenous HSNO (100 μM, 100 μL) in PBS solution (50 mM, pH = 7.4) was incubated for 30 min, followed by 980 nm (2 W / cm 2 ) Continuous irradiation for 5 minutes, followed by a 5-minute interval, for a total of 4 cycles, for a total of 20 minutes of irradiation; Fig.26 D is a quantitative representation of the fluorescence intensity of the tumor site of each group of mice. The data are expressed as mean ± standard deviation (n = 3);

[0074] Fig. 27 The fluorescence intensity of HSNO in nude mice with colon cancer was detected by using the probe UCNPs-R-PSN. The mice in the control group were injected with the probe UCNPs-R-PSN (50 mg / mL, 10 μL, saline: dimethyl sulfoxide = 9:1 v / v) into the tumor, and then given 980nm (2W / cm 2 ) for 5 min, followed by a 5-min interval, for a total of 4 cycles, for a total of 20 min; the mice in the HSNO group were injected intratumorally with probe UCNPs-R-PSN (50 mg / mL, 10 μL, saline: dimethyl sulfoxide = 9:1 v / v), and then given exogenous HSNO (100 μM, 100 μL) in PBS solution (50 mM, pH = 7.4) for 30 min, followed by 980 nm (2 W / cm 2 ) Continuous irradiation for 5 minutes, followed by a 5-minute interval, for a total of 4 cycles, for a total of 20 minutes of irradiation;

[0075] Fig.28 It is a quantitative expression Fig. 27 Fluorescence signal intensity of the two groups of mice in ; data are expressed as mean ± standard deviation (n = 3);

[0076] Fig.29 It is the effect of exogenous HSNO on tumor growth; among them, Fig.29A is the weight change of nude mice transplanted with colon cancer within 12 days; Fig.29 B shows the changes in tumor volume of nude mice transplanted with colon cancer within 12 days; Fig.29 C is a photo of the tumor tissue in the right armpit of a nude mouse transplanted with colon cancer 12 days after treatment; Fig.29 D: Nude mice with colon cancer transplanted tumors were killed after 12 days of treatment, and the tumor tissues were dissected and stained with H&E, respectively, Saline group and HSNO group (Scalebar: 50 μm); data are expressed as mean ± standard deviation (n = 6);

[0077] Fig.30 This is the confocal imaging of HSNO in tumor slices of nude mice with colon cancer transplants; Fig.30 A is the Saline group: nude mice with colon cancer xenografts were given normal saline (100 μL) through the tail vein on days 0, 3, 6, 9, and 12, and the probe UCNPs-R-PSN (50 mg / mL, 10 μL, normal saline: dimethyl sulfoxide = 9:1 v / v) was injected into the tumor on day 12. Then, 980 nm (2 W / cm 2 ) Continuous irradiation for 5 minutes, followed by an interval of 5 minutes, for a total of 20 minutes, after which the tumor tissue was removed and frozen sections were performed; Fig.30 B is the HSNO group: nude mice with colon cancer transplanted tumors were given HSNO (1.6 mg / kg, saline: DMSO = 99:1) through the tail vein on days 0, 3, 6, 9, and 12, respectively. On day 12, the probe UCNPs-R-PSN (50 mg / mL, 10 μL, saline: dimethyl sulfoxide = 9:1 v / v) was injected into the tumor. Then, 980 nm (2 W / cm 2 ) Continuous irradiation for 5 minutes, followed by a 5-minute interval, for a total of 20 minutes, after which the tumor tissue was removed and frozen sections were made. Laser confocal microscopy was used for imaging; Fig.30 C is Fig.30 A partial enlarged view; Fig.30 D is Fig.30 A partial enlarged view of B; Fig.30 E is the fluorescence intensity of tumor sections in the Saline group and the HSNO group; the excitation light was 561 nm, and the emission wavelength was 600 nm to 700 nm; the data are expressed as mean ± standard deviation (n = 3), ***P < 0.001. DETAILED DESCRIPTION

[0078] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0079] 1. Implementation Methods

[0080] 1 Materials and Instruments

[0081] 1.1 Solution preparation

[0082] (1) Preparation of probe UCNPs-R-PSN solution: UCNPs-R-PSN (20.0 mg) was dissolved in 1.0 mL of anhydrous DMSO solution to prepare a 20 mg / mL probe solution, which was stored in a -20°C refrigerator away from light.

[0083] (2) Preparation of GSNO stock solution: Weigh GSNO (11.37 mg, 0.034 mmol), dissolve it in PBS buffer solution (50 mM, pH = 7.4, 33.8 mL) to prepare a 1 mM stock solution, and store it at -20°C.

[0084] (3) Preparation of Na2S stock solution: Weigh Na2S (3.21 mg, 0.041 mmol) and dissolve it in PBS buffer solution (50 mM, pH = 7.4, 13.7 mL) to prepare a 3 mM stock solution, and then dilute the stock solution to a 300 μM solution for later use.

[0085] (4) Preparation of HSNO stock solution: In a dark environment, at room temperature and under nitrogen protection, freshly prepared 1 mM GSNO solution and 0.3 mM Na2S solution were placed in a PBS buffer solution (50 mM, pH = 7.4) to obtain a 300 μM HSNO stock solution. Next, the stock solution was diluted to a 100 μM solution and used immediately.

[0086] (5) Preparation of glutathione (GSH) stock solution: Add GSH (15.36 mg, 0.05 mmol) to deionized water (10.0 mL) to prepare a 5.0 mM stock solution. Then dilute the stock solution into 1.0 mM and 10.0 μM solutions for later use.

[0087] (6) Preparation of L-cysteine ​​(L-Cys) stock solution: Cys (6.05 mg, 0.05 mmol) was added to deionized water (10.0 mL) to prepare a 5.0 mM stock solution. The stock solution was then diluted to 1.0 mM and 10.0 μM solutions for later use.

[0088] (7) Preparation of DEA·NONOate stock solution (as a source of NO): DEA·NONOate (15.5 mg, 0.1 mmol) was added to 0.01 M sodium hydroxide solution (10 mL) to prepare a 10.0 mM stock solution. The stock solution was then diluted to 1.0 mM and 10.0 μM solutions for later use.

[0089] (8) Preparation of H2O2 stock solution: Add 1 mL of H2O2 (30%) to deionized water (9.0 mL). Determine the concentration of the H2O2 stock solution by measuring the absorbance at 240 nm. The molar extinction coefficient is 43.6 M -1 cm -1 , the calculation formula is c=A / (bε).

[0090] (9)ClO - Preparation of stock solution: Add NaClO (10%) to deionized water and then measure the absorbance at 209 nm. The molar extinction coefficient is 350 M -1 cm -1 , determination of ClO - The concentration of the stock solution is calculated using the formula c = A / (bε).

[0091] (10) t Preparation of BuOOH stock solution: Commercial products t BuOOH (5 mM).

[0092] (11) · Preparation of OH solution: Add ferrous sulfate (15.20 mg, 0.10 mmol) to hydrogen peroxide solution (10 mL) and mix to prepare 10 mM · OH stock solution.

[0093] (12) 1 Preparation of O2 stock solution: methylene blue (31.98 mg, 0.10 mmol) was added with 5 mL of NaClO4 (1 mM) solution and 5 mL of H2O2 (30%) solution under LED light to prepare a 10 mM stock solution for immediate use.

[0094] (13)O2 ·- Preparation of stock solution: Under nitrogen protection, add KO2 (7.10 mg, 0.10 mmol) to anhydrous DMSO (10 mL) to prepare a 10 mM stock solution for immediate use.

[0095] (14) NO3 - Preparation of stock solution: Add NaNO3 (8.50 mg, 0.1 mmol) to deionized water (10 mL) to prepare a NO stock solution with a concentration of 10 mM. 3- Stock solution.

[0096] (15) NO2 - Preparation of stock solution: Add NaNO2 (6.90 mg, 0.1 mmol) to deionized water (10 mL) to prepare a NO solution with a concentration of 10 mM. 2- Stock solution.

[0097] (16)ONOO - Preparation of stock solution: NaNO2 (0.6 M, 10 mL) and H2O2 (0.7 M, 10 mL) were added to deionized water and stirred vigorously. HCl (0.6 M, 10 mL) was added at 0 °C, and then NaOH solution (1.5 M, 20 mL) was quickly added. ONOO was determined by UV analysis. - The molar extinction coefficient is 1670M -1 cm -1 , the calculation formula is c=A / (bε).

[0098] In addition to the above substances, other stock solutions are prepared, such as amino acids (Gly, D-Ala, Val, Ile, L-Thr, Asp, L-Glu, L-Arg, L-Lys, L-His, Hcy); inorganic salts (Sn 2+ 、Cd 2+ , Mn 2+ 、Co 2+ , Cu 2+ , Hg 2+ 、Zn 2+ , Fe 2+ , Fe 3 + , Ca 2+ 、Al 3+ 、Ni 2+ Mg 2+ , Li + 、Na + , K + 、Ag + ); active sulfur (Na2S2, NaHS, Na2SO3, NaHSO3, CH3SSSCH3, CysSSCys, GSSG, S8) were directly dissolved in deionized water for preparation.

[0099] 1.2 Cells

[0100] Species and strain: HCT116 (human colon cancer cell line) Source: Wuhan Savier Biotechnology Co., Ltd.

[0101] 1.3 Experimental animals

[0102] The species and strain were healthy male Balb / C nude mice, weighing between 20 and 25 g, which were obtained from Changzhou Cavens Experimental Animal Co., Ltd.

[0103] 2. Methods

[0104] 2.1 Synthesis route of probe UCNPs-R-PSN

[0105] NaYF4:Yb / Tm@NaYF4 core-shell nanomaterials were synthesized by high-temperature organic thermal decomposition method, and then the surface of NaYF4:Yb / Tm@NaYF4 was coated with silica and (3-aminopropyl)triethoxysilane for surface modification to obtain amino-functionalized upconversion nanoparticles UCNPs@SiO2-NH2, which were used as carriers for near-infrared light activation and coupling of fluorescent probes. (2) Ortho-nitrobenzyl derivatives were selected as photoactivation protective groups, and rhodamine derivatives were used as near-infrared fluorescent groups; o-mercaptobenzoic acid and o-phenylenediamine were used as recognition groups of HSNO, and the photoactivated fluorescent probe R-PSN was synthesized through esterification reaction, nucleophilic addition elimination, nucleophilic substitution and other reactions. (3) The photoactivated fluorescent probe R-PSN was coupled to the upconversion nanoparticles UCNPs through amide condensation reaction, and the near-infrared light-activated fluorescent probe UCNPs-R-PSN based on UCNPs was constructed.

[0106] 2.1.1 Synthesis of upconversion nanoparticles UCNPs@SiO2-NH2

[0107] (1) Synthesis of NaYF4:Yb / Tm core nanoparticles: ① Weigh YCl3 . 6H2O (241.00mg, 0.79mmol), YbCl3 . 6H2O (77.00mg, 0.20mmol), TmCl3 . 6H2O (1.910 mg, 0.005 mmol) was added to a 100 mL three-necked flask, and after slowly stirring, 7 mL of oleic acid (OA) and 15 mL of octadecene (1-ODE) were added. Under nitrogen purge, the resulting mixed solution was heated to 150 ° C and kept warm for 1 h to remove the low boiling point solvent to obtain a light yellow clear solution. ② After the light yellow clear solution was naturally cooled to 20-30 ° C, an anhydrous methanol solution (10 mL) containing NaOH (100.00 mg, 2.50 mmol) and NH4F (148.00 mg, 4.00 mmol) was quickly added and stirred at 30 ° C for 1 h. ③ Under nitrogen purge, the temperature was continued to rise to 90 ° C and kept warm for 1 h to remove anhydrous methanol. ④ Under the device of circulating cooling water, the obtained mixed solution was rapidly heated to 300°C at a rate of 15°C / min, and maintained at 300°C for 1h to obtain a yellow solution. ⑤ After the yellow clear solution was naturally cooled to 20-30°C, anhydrous ethanol was added, and centrifuged at 10000rpm / min for 5min to obtain the product after centrifugation. ⑥ A mixed solution of cyclohexane and anhydrous ethanol with a volume ratio of 1:1 was added to the obtained product, ultrasonically washed for 5min, and centrifuged at 10000rpm / min for 5min to obtain a product precipitate, and the washing and centrifugation steps were repeated three times to obtain a white solid, and the white solid product was dispersed in 10mL cyclohexane.

[0108] (2) Synthesis of NaYF4:Yb / Tm@NaYF4 core-shell double-layer rare earth nanoparticles: ① Weigh YCl3 . 6H2O (303.00 mg, 1.00 mmol) was added to a 100 mL three-necked flask, and after slowly stirring, 7 mL of oleic acid (OA) and 15 mL of octadecene (1-ODE) were added. Under nitrogen purge, the resulting mixed solution was heated to 150°C and kept warm for 1 h to remove the low boiling point solvent, thereby obtaining a light yellow clear solution. ② After the light yellow clear solution was naturally cooled to 20-30°C, the cyclohexane solution (10 mL) of NaYF4:Yb / Tm nanoparticles obtained in step (1) was added to the above light yellow clear solution, stirred at 30°C for 30 min, and then heated to 90°C and kept at this temperature for 1 h to remove cyclohexane. ③ After the cyclohexane is completely removed, the mixed solution is cooled naturally to 20-30°C, and anhydrous methanol solution (10 mL) containing NaOH (100.00 mg, 2.50 mmol) and NH4F (148.00 mg, 4.00 mmol) is quickly added, and stirred at 30°C for 1 hour. ④ Under nitrogen purge, continue to heat to 90°C and keep warm for 1 hour to remove anhydrous methanol. ⑤ Under the circulating cooling water device, the obtained mixed solution is quickly heated to 300°C at a rate of 15°C / min, and maintained at 300°C for 1 hour to obtain a yellow solution. ⑥ After the yellow clear solution is cooled naturally to 20-30°C, anhydrous ethanol is added, and centrifuged at 10000rpm / min for 5 minutes to obtain the product after centrifugation. ⑦ Add a mixed solution of cyclohexane and anhydrous ethanol in a volume ratio of 1:1 to the obtained product, ultrasonically wash for 5 minutes, centrifuge at 10000 rpm / min for 5 minutes to obtain a product precipitate, wash, and repeat the centrifugation steps three times to obtain NaYF4:Yb / Tm@NaYF4, and then disperse the product in 10 mL of cyclohexane.

[0109] (3) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2: ① Weigh CTAB (100.00 mg, 0.27 mmol) and dissolve it in 20 mL of deionized water, and heat it to 60°C to make the CTAB solution clear and transparent. ② Add cyclohexane solution (1 mL) containing 20.00 mg of NaYF4:Yb / Tm@NaYF4 to the above CTAB solution, and stir the resulting mixed solution at 30°C and 1000 rpm / min for 12 h to completely evaporate the cyclohexane to form a clear and transparent NaYF4:Yb / Tm@NaYF4-CTAB solution. ③ Add ethanol (3 mL) and 2M sodium hydroxide solution (150 μL) to the resulting clear and transparent solution, stir evenly at 1000 rpm / min, and heat it to 70°C. After the temperature stabilizes, add 60 μL of tetraethyl silicate (TEOS) dropwise at a rate of 5 μL each time, and continue stirring and reacting for 2 h. ④After the reaction is completed, the reaction solution is quickly collected and washed with anhydrous ethanol at 10000rpm / min for 5min, and the above operation is repeated 5 times. ⑤The surfactant CTAB is removed by a fast and efficient ion exchange method, the above precipitate is resuspended in 30mL of anhydrous ethanol and NaCl (6.00g, 0.10mol) is added, and the reaction is stirred at 60℃ and 1000rpm / min for 6h. After the reaction is completed, the reaction solution is allowed to stand for 10min, the supernatant is collected, and washed with anhydrous ethanol at 10000rpm / min for 5min, and the above operation is repeated 5 times. Finally, SiO2-encapsulated upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2 (UCNPs-SiO2) are obtained.

[0110] (4) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2-NH2: Weigh 10.00 mg UCNPs-SiO2 (NaYF4:Yb / Tm@NaYF4@SiO2) and dissolve it in 10 mL of anhydrous ethanol. Add (3-aminopropyl)triethoxysilane (20 μL) dropwise, and stir the resulting mixed solution rapidly at 70 °C for 3 h. After the reaction, wash with anhydrous ethanol at 10,000 rpm / min for 5 min, and repeat the above operation 5 times. Finally, amino-functionalized upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2-NH2 (UCNPs-SiO2-NH2) were obtained.

[0111] 2.1.2 Synthesis route of compound R-PSN

[0112] (1) 4-bromomethyl-3-nitrobenzoic acid is selected as the starting material, and compound a is reacted with tert-butyl alcohol to obtain compound T-1 through an esterification reaction, and compound T-1 and o-mercaptobenzoic acid are reacted through a nucleophilic substitution reaction to obtain compound T-2. (2) 4-diethylaminoketo acid and 1,6-dihydroxynaphthalene are selected as the starting materials, and compound R-2 is obtained through a condensation reaction, and compound R-2 and compound T-1 are reacted through an amide condensation reaction to obtain compound R-3, and compound R-3 is condensed with oxalyl chloride and o-phenylenediamine to obtain compound R-4, and compound R-4 is deprotected from the tert-butyl protecting group by trifluoroacetic acid to obtain the final product R-PSN.

[0113] Synthesis of compound T-1: Concentrated sulfuric acid (210.00 μL, 3.75 mmol) was added to 30 mL of dichloromethane, and anhydrous magnesium sulfate (1.81 g, 15.00 mmol) was added, and stirred at 30 ° C for 15 min, and 4-bromomethyl-3-nitrobenzoic acid (1.00 g, 3.75 mmol) and tert-butyl alcohol (2.00 mL, 18.75 mmol) were added, and stirred at 30 ° C in the dark for 18 h. The resulting reaction solution was transferred to 200 mL of saturated NaCl solution for extraction, and then washed with dichloromethane (3×50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a brown oil. Purification by silica gel column chromatography (silica, PE: EtOAc, 3: 1 v / v) gave 850.00 mg of white solid compound T-1 with a yield of 71.95%. R f =0.6, TLC (silica, PE:EtOAc, 1:1v / v). 1 H NMR (400MHz, CDCl3) δ8.58-8.57(d,J=1.8Hz,1H),8.20-8.18(m,1H),7.65-7.63(d,J=8.0Hz,1H),4.84(s,2H),1.61(s,9H).

[0114] Synthesis of compound T-2: Compound T-1 (850.00 mg, 2.69 mmol) and o-mercaptobenzoic acid (416.50 mg, 2.69 mmol) were added to 20 mL of ultrapure water, anhydrous potassium carbonate (439.20 mg, 3.17 mmol) was added, and a catalytic amount of DMF (2 mL) was added and stirred at 30 ° C in the dark for 12 h. The resulting reaction solution was transferred to 200 mL of saturated NaCl solution and extracted with dichloromethane (3×50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a light yellow solid. Purification by silica gel column chromatography (silica, DCM: MeOH, 20: 1 v / v) gave 905.00 mg of white solid compound T-2 with a yield of 86.49%. Rf =0.3, TLC (silica, DCM:MeOH, 10:1v / v). 1 H NMR (400MHz, CDCl3) δ8.56 (s, 1H), 8.12-8.10 (d, J = 8.0Hz, 2H), 7.69-7.67 (d, J = 8. 0Hz,1H),7.46-7.42(t,J=7.8Hz,1H),7.27-7.19(m,3H),4.56(s,2H),1.60(s,9H).

[0115] Synthesis of compound R-2: Compound R-1 (2.00 g, 6.39 mmol) and 1,6-dihydroxynaphthalene (1.02 g, 6.39 mmol) were dissolved in 20 mL of methanesulfonic acid, and the resulting mixed solution was stirred at 90 ° C for 8 h. The resulting reaction solution was naturally cooled to 20-30 ° C, and then transferred to ice water and stirred thoroughly. Finally, the filter residue was collected by suction to obtain a dark red solid, which was purified by silica gel column chromatography (silica, DCM: MeOH, 30: 1 v / v) to obtain 2.40 g of red solid compound R-2, with a yield of 85.95%. R f =0.5, TLC (silica, DCM:MeOH, 30:1v / v). 1 H NMR (400MHz, DMSO-D6) δ10.12(s,1H),8.45-8.43(d,J=9.0Hz,1H),8.04-8.02(d,J=7.4Hz,1H),7.79 -7.70(m,2H),7.36-7.34(d,J=8.8Hz,1H),7.28-7.24(m,2H),7.15-7.14(d,J=2.2Hz,1H),6. 73-6.72(d,J=2.4Hz,1H),6.59-6.50(m,3H),3.42-3.36(m,4H),1.14-1.11(t,J=7.0Hz,6H).

[0116] Synthesis of compound R-3: Compound T-2 (905.00 mg, 2.33 mmol), 4-dimethylaminopyridine (65.36 mg, 0.54 mmol), and EDCI (668.69 mg, 3.49 mmol) were dissolved in 20 mL of dichloromethane and stirred in an ice-water bath for 5 min. Compound R-2 (1.20 g, 2.75 mmol) was then added and stirred overnight at 30 ° C in the dark. The resulting reaction solution was concentrated under reduced pressure to obtain a dark reddish brown oil, which was purified by silica gel column chromatography (silica, DCM: MeOH, 50: 1 v / v) to obtain 1.47 g of dark red solid compound R-3, with a yield of 66.16%. Rf =0.4, TLC (silica, DCM:MeOH, 50:1v / v). 1 HNMR(400MHz,DMSO-D6)δ8.71-8.69(d,J=9.0Hz,1H),8.40-8.38(m,1H),8.22-8.20(d,J=7 .8Hz,1H),8.13-8.11(d,J=8.0Hz,1H),8.07-8.04(d,J=8.6Hz,1H),7.88-7.72(m,4H),7.66 -7.60(m,4H),7.44-7.40(t,J=7.4Hz,1H),7.32-7.30(d,J=7.0Hz,1H),6.82-6.79(m,2H),6 .60-6.53(m,2H),4.69(s,2H),3.44-3.39(m,4H),1.53(s,9H),1.16-1.12(t,J=7.0Hz,6H).

[0117] Synthesis of compound R-4: Compound R-3 (1.47 g, 1.82 mmol) was dissolved in 15 mL of anhydrous dichloromethane solution and a catalytic amount of anhydrous DMF (50 μL) was added, and then oxalyl chloride (695.00 mg, 5.48 mmol) was added dropwise and stirred in an ice-water bath for 20 min. The resulting reaction solution was concentrated under reduced pressure to obtain a crude intermediate. The crude intermediate was redissolved in 10 mL of anhydrous dichloromethane solution and slowly added dropwise to 15 mL of anhydrous dichloromethane solution containing o-phenylenediamine (590.00 mg, 5.46 mmol) and triethylamine (550.00 mg, 5.46 mmol), and reacted at 30 ° C in the dark for 4 h. The reaction solution was transferred to 200 mL of saturated NaCl solution for quenching, and then extracted with dichloromethane (3×50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a dark green oil, which was purified by silica gel column chromatography (silica, DCM:MeOH, 80:1 v / v) to obtain 860.00 mg of a light yellow solid compound R-4, with a yield of 52.62%. f =0.6, TLC (silica, DCM:MeOH, 40:1v / v).

[0118] Synthesis of compound R-PSN: Compound R-4 (860.00 mg, 0.86 mmol) was dissolved in 10 mL of anhydrous dichloromethane, trifluoroacetic acid (2 mL) was added dropwise, and stirred for 3 h in an ice-water bath in the dark. The reaction solution was concentrated under reduced pressure to obtain a dark red solid, which was purified by silica gel column chromatography (silica, DCM: MeOH, 30: 1 v / v) to obtain 340.00 mg of light yellow solid compound R-PSN, with a yield of 46.96%. f =0.25, TLC (silica, DCM:MeOH, 30:1v / v). 1 H NMR (400MHz, DMSO-D6) δ8.49-8.44(m,2H),8.21-8.16(m,2H),7.97-7.95(d,J=4.8Hz,1H),7 .84-7.79(m,2H),7.65-7.59(m,4H),7.55-7.52(d,J=9.0Hz,2H),7.42-7.38(t,J=7.4Hz,1H) ,7.19-7.18(d,J=4.8Hz,1H),7.00(s,1H),6.80-6.76(t,J=7.6Hz,2H),6.60(s,1H),6.49(s ,2H),6.11(s,1H),5.89(s,1H),4.69(s,2H),3.50-3.42(m,4H),1.13-1.09(t,J=7.0Hz,6H). 13 C NMR (400MHz, DMSO-D6) δ166.3,165.8,164.8,153.2,149.9,149.2,149.2,147.8,146 .4,140.4,136.5,134.7,134.2,134.2,133.7,133.4,132.1,132.1,129.4,128.9,12 8.4,128.0,127.4,126.3,126.0,124.8,124.2,123.6,122.3,122.3,121.8,120.9,120.0,119.5,119.5,116.5,116.0,111.1,106.4,98.1,67.5,44.2,33.9,13.0. For related spectra, see Figure 4-6 .

[0119] 2.1.3 Synthesis of probe UCNPs-R-PSN

[0120] Synthesis of probe UCNPs-R-PSN: Compound R-PSN (50.00 mg, 0.059 mmol), NaYF4:Yb / Tm@NaYF4@SiO2-NH2 (10.00 mg), HBTU (23.00 mg, 0.059 mmol), HOBt (8.00 mg, 0.059 mmol) were dissolved in 8 mL of anhydrous DMF and stirred for 5 min at 20-30 ° C and protected from light under nitrogen protection. DIPEA (11.00 μL, 0.06 mmol) was added to the reaction solution and stirred for 12 h at 20-30 ° C and protected from light. After the reaction was complete, the reaction was quenched with deionized water and centrifuged at 10000 rpm / min for 10 min. The obtained precipitate was washed with anhydrous ethanol at 10000 rpm / min for 10 min, and the above operation was repeated 5 times. Finally, a light yellow solid UCNPs-R-PSN was obtained.

[0121] 2.2 Mechanism verification of the recognition of HSNO by probe UCNPs-R-PSN

[0122] In the dark, the probe UCNPs-R-PSN (final concentration 0.1 mg / mL) was firstly irradiated at 980 nm (3 W / cm 2 ) for 10 min, and then incubated with HSNO (final concentration 300 μM) in PBS (pH = 7.4, 50 mM, 1 mL) buffer solution at 37 ° C for 10 min. The organic phase was extracted with ethyl acetate (3×1 mL), and the product was separated and confirmed by high-resolution mass spectrometry to verify the mechanism of probe UCNPs-R-PSN recognizing HSNO.

[0123] 2.3 Determination of the loading amount of R-PSN in probe UCNPs-R-PSN

[0124] The loading amount of the compound R-PSN on the upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2-NH2 was determined according to the Lambert-Beer law. First, the UV absorption spectra of R-PSN at different concentrations were detected using a UV-2401PC UV-visible spectrophotometer, and then the standard curve of R-PSN at a wavelength of 267nm was fitted.

[0125] R-PSN was coupled to the upconversion nanoparticle NaYF4:Yb / Tm@NaYF4@SiO2-NH2 through an amide condensation reaction. First, the absorbance of the initial concentration of R-PSN before the reaction was measured at 267nm, and then the upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2-NH2, HBTU, and HOBt were added and stirred at room temperature in the dark for 12h. After the reaction was completed, the product was collected by centrifugation (10000rpm, 10min), and then the absorbance of the supernatant after the reaction was measured at 267nm. Finally, the loading amount of R-PSN was calculated by the following formula.

[0126]

[0127] 2.4 Fluorescence spectroscopy

[0128] The probe UCNPs-R-PSN was dissolved in DMSO solution under light-protected conditions and then illuminated at 980 nm (3 W / cm 2 ) was irradiated under 40°C, and a freshly prepared mixture of 1 mM nitrosoglutathione (GSNO) and 0.3 mM Na2S was dissolved in PBS buffer (pH 7.4, 50 mM) as a source of HSNO (300 μM) (hereinafter referred to as "HSNO solution"). The probe after near-infrared light irradiation was then mixed with 300 μM HSNO solution, and after incubation at 37°C for a period of time, the sample was poured into a quartz cuvette, and its fluorescence intensity was measured using a fluorescence spectrophotometer. During the experiment, PBS buffer (pH 7.4, 50 mM) was used as a blank control. Each set of data was measured at least three times in parallel, and the final results were expressed as mean ± SD.

[0129] Fluorescence spectrophotometer detection conditions: For the reaction between probe UCNPs-R-PSN and HSNO, the excitation wavelength was set to 575 nm, the excitation slit width and emission slit width were both set to 10 nm, the instrument scanning speed was set to 1200 nm / min, the emission spectrum range was set to 600-750 nm, and the voltage of the photomultiplier tube was set to 650 V.

[0130] 2.5 Determination of the detection limit of HSNO by probe UCNPs-R-PSN

[0131] First, the probe UCNPs-R-PSN was passed through a 980nm (3W / cm 2) irradiated, and then co-incubated with different concentrations of HSNO solution (0, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 4, 6, 8, 10, 20, 40, 80 μM) in PBS (pH 7.4, 50 mM, DMSO: PBS = 1: 5) buffer for 10 min, and then the fluorescence intensity was measured. The linear equation between the calculated fluorescence intensity and HSNO concentration. The detection probe UCNPs-R-PSN was only detected by 980nm (3W / cm 2 ) and the fluorescence intensity in PBS buffer without HSNO was measured 10 times in total, and the standard deviation of the 10 detection results was calculated. The calculation formula of the detection limit is: LOD = 3σ / k; where σ is the standard deviation of the blank sample, and k is the slope of the linear equation of fluorescence intensity and HSNO concentration.

[0132] 2.6 Cell culture methods

[0133] HCT116 cell culture: McCoy's 5A medium containing 10% fetal bovine serum and 100 μg / mL penicillin and streptomycin was used, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2. Cells were passaged every two days and transplanted into new culture dishes at a ratio of 1:3.

[0134] 2.7 Determination of cytotoxicity of probe UCNPs-R-PSN by CCK8 method

[0135] Prepare cell suspension: Collect cells from T25 cell culture flasks, pipette appropriate amount of cells and dilute to appropriate concentration (1×10 5 cells / mL).

[0136] The prepared cell suspension was placed at a volume of approximately 1×10 4 The cells were inoculated into a 96-well plate, with a volume of 100 μL per well, and 3 parallel replicate wells were set up for each group. The 96-well plate was then placed in a cell culture incubator for 24 hours to allow the cells to fully adhere to the wall and proliferate.

[0137] Different concentrations of probes (0, 50, 100, 200, 400, 500 μg / mL) were added to each well and incubated in a cell culture incubator for 48 h.

[0138] After 48 hours of co-incubation, discard the original culture medium in each well. Gently add serum-free culture medium (McCOY's 5A culture medium) containing 10% CCK-8 enhanced solution to each well under light-proof conditions. Be careful during the addition process to prevent the generation of bubbles. Subsequently, wrap the culture plate with aluminum foil and place it in the incubator for another 30 minutes. Finally, use an ELISA reader to measure the absorbance value at 450nm.

[0139] Cell survival rate calculation formula: Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0140] As: absorbance of the experimental well (containing probe, CCK8, and cell culture medium);

[0141] Ac: absorbance of control well (containing CCK8, cell culture medium, and no probe);

[0142] Ab: blank well (does not contain any cells, only the absorbance of CCK8 and cell culture medium).

[0143] 2.8 Cellular fluorescence imaging

[0144] HCT116 cells were collected from T25 cell culture flasks, and appropriate amount of cells were taken and diluted to a suitable concentration to prepare a cell suspension. The prepared cell suspension was diluted to about 1×10 5 The cells were inoculated into confocal dishes and continued to be cultured.

[0145] 2.8.1 Fluorescence imaging of physiological and exogenous HSNO in cells

[0146] The cells were divided into the following 6 groups: ①probe+cell group: the probe UCNPs-R-PSN (final concentration 0.1 mg / mL, 5 μL DMSO) was co-incubated with HCT116 cells for 30 min. ②probe+cell+HSNO group: the probe UCNPs-R-PSN (final concentration 0.1 mg / mL, 5 μL DMSO) was first co-incubated with HCT116 cells for 30 min, and then 100 μM HSNO was added and co-incubated for 10 min. ③probe+cell+980nm light: the probe UCNPs-R-PSN (final concentration 0.1 mg / mL, 5 μL DMSO) was first co-incubated with HCT116 cells for 30 min, and then 980nm (1 W / cm 2) for 10 minutes. ④AOAA+probe+cell+980nmlight: First, cells were incubated with AOAA (20μM) for 45 minutes, then with probe UCNPs-R-PSN (final concentration 0.1mg / mL, 5μL DMSO) for 30 minutes, and then irradiated with 980nm (1W / cm 2 ) for 10 minutes. ⑤cell+probe+Na2S+980nmlight: First, the probe UCNPs-R-PSN (final concentration 0.1mg / mL, 5μL DMSO) was incubated with HCT116 cells for 30 minutes, and then 150μM Na2S was added for 10 minutes. Finally, 980nm (1W / cm 2 ) for 10 minutes. ⑥cell+probe+GSNO+980nm light: First, the probe UCNPs-R-PSN (final concentration 0.1mg / mL, 5μL DMSO) was co-incubated with HCT116 cells for 30 minutes, and then 1mM GSNO was added for co-incubation for 10 minutes. Finally, 980nm (1W / cm 2 ) for 10 minutes. ⑦cell+probe+HSNO+980nm light: First, the probe UCNPs-R-PSN (final concentration 0.1mg / mL, 5μLDMSO) was co-incubated with HCT116 cells for 30 minutes, and then 100μM HSNO was added for 10 minutes. Finally, 980nm (1W / cm 2 ) for 10 min. Before imaging, the confocal dish was rinsed three times with PBS buffer. The images were taken using a Leica STELLARIS 5 laser confocal microscope (63× oil objective).

[0147] The excitation wavelength of channel 1 was 561 nm, and the emission wavelength range was set to 600-750 nm. Leica software (Lecia) was used for analysis. All data are expressed as mean±SD (n=3).

[0148] 2.9 Animal husbandry

[0149] This study used healthy male Balb / C nude mice weighing 20-25 g and provided by Changzhou Cavens Laboratory Animal Co., Ltd. The animal experiment protocol has been approved by the Animal Care and Use Committee of Xuzhou Medical University and complies with relevant provisions of Chinese law. Before the start of the experiment, the nude mice need to acclimate to the environment one week in advance. Under the lighting conditions of natural circadian rhythm, the cage group was used, the ambient temperature was maintained at 22±2℃, the humidity was 50±10%, and food and water were freely available.

[0150] 2.9.1 Construction of HCT116 colon cancer subcutaneous transplant tumor model in nude mice

[0151] HCT116 cells were cultured in T75 flasks until they reached the logarithmic growth phase, then trypsinized and transferred to serum-free McCoy's 5A medium to obtain a cell suspension concentration of approximately 4 × 10 7 Then, 4×10 cells / mL were selected from 4-6 week old Balb / C nude mice weighing 20-25 g. 6 The cells (100 μL cell suspension) were inoculated subcutaneously in the middle and posterior part of the axilla of the left upper limb of nude mice.

[0152] 2.10 Fluorescence imaging of nude mouse transplanted tumor model

[0153] Select patients with tumor volume greater than 200 mm 3 , the nude mice in good condition were randomly divided into three groups, six in each group. The three groups were Probe group, Probe+980nm light group and Probe+HSNO+980nm light group. After each group of mice was anesthetized with isoflurane gas, (1) Probe group: mice were injected with probe UCNPs-R-PSN (50mg / mL, 10μL, saline: dimethyl sulfoxide = 9:1v / v) intratumorally; (2) Probe+980nm light group: mice were injected with probe UCNPs-R-PSN (50mg / mL, 10μL, saline: dimethyl sulfoxide = 9:1v / v) intratumorally, and then given 980nm (2W / cm 2 ) The tumor was irradiated continuously for 5 min, followed by an interval of 5 min, for a total of 20 min; (3) Probe+HSNO+980 nm light group: mice were injected intratumorally with probe UCNPs-R-PSN (50 mg / mL, 10 μL, saline: dimethyl sulfoxide = 9:1 v / v), followed by intratumoral injection of exogenous HSNO (100 μM, 100 μL) in PBS solution (50 mM, pH = 7.4) for 30 min, followed by 980 nm (2 W / cm 2 ) The tumor was irradiated continuously for 5 minutes, followed by a 5-minute interval, for a total of 20 minutes.

[0154] The images were taken using the LB983 NightOWL IILB983 small animal living imager. Imaging conditions: excitation wavelength 580nm, emission wavelength 610nm. Images and data were analyzed using Living Image software.

[0155] 2.11 Data Processing

[0156] Each group of experimental data was repeated at least 3 times, and the final results were expressed as mean ± SD, and statistical analysis was performed using SPSS16.0 software. One-way ANOVA with a completely randomized design was used to compare the differences between multiple groups of data. P < 0.05 indicated that the difference was statistically significant.

[0157] 3 Preparation and characterization of probe UCNPs-R-PSN

[0158] 3.1 Synthesis and characterization of NaYF4:Yb / Tm

[0159] Firstly, a high temperature organic thermal decomposition method was used to synthesize Tm 3+ The rare earth upconversion nanoparticles NaYF4:Yb / Tm were prepared as the luminescence center, and the synthesized NaYF4:Yb / Tm nanoparticles were characterized by transmission electron microscopy (TEM), fluorescence spectroscopy and X-ray diffraction (XRD). Figure 7 A and Figure 7 As shown in B, the transmission electron microscope image shows that NaYF4:Yb / Tm is a monodisperse, uniformly sized, and regularly shaped spherical nanoparticle with an average particle size of 30.24±0.22nm. The fluorescence spectrum of NaYF4:Yb / Tm was measured under 980nm near-infrared light excitation. Figure 7 C shows that the synthesized nanoparticles have strong fluorescence emission peaks at 365nm, 450nm, and 475nm. X-ray diffraction (XRD) was used to characterize NaYF4:Yb / Tm. The results showed that ( Figure 7 D), the spectrum of the nanoparticles has a flat baseline and a narrow half-peak width, which is completely consistent with the standard PDF card (PDF#28-1192) of NaYF4:Yb / Tm and has no impurity peaks, indicating that the synthesized nanoparticles have a high degree of crystallinity. Combined with the above results, the present invention successfully constructed NaYF4:Yb / Tm core nanoparticles.

[0160] 3.2 Synthesis and characterization of NaYF4:Yb / Tm@NaYF4

[0161] First, NaYF4 was wrapped on the outside by epitaxial growth to obtain nanoparticles NaYF4:Yb / Tm@NaYF4 with a core-shell double-layer structure. Then the synthesized NaYF4:Yb / Tm@NaYF4 nanoparticles were characterized by transmission electron microscopy (TEM) and particle size distribution. Figure 8 A and Figure 8As shown in Figure B, transmission electron microscopy results show that the synthesized NaYF4:Yb / Tm@NaYF4 is a monodisperse, uniformly sized, and regularly shaped short rod-shaped nanoparticle with an average particle size of 39.89±0.31nm. The diameter of NaYF4:Yb / Tm@NaYF4 nanoparticles has increased significantly compared to NaYF4:Yb / Tm, indicating that NaYF4:Yb / Tm@NaYF4 nanoparticles with a core-shell double-layer structure have been constructed.

[0162] 3.3 Synthesis and characterization of UCNPs-R-PSN

[0163] First, NaYF4:Yb / Tm@NaYF4 coated with silica was prepared by organic template method to obtain UCNPs@SiO2 (NaYF4:Yb / Tm@NaYF4@SiO2) nanoparticles. Then, the surface was amino-modified by aminosilane coupling agent to obtain NaYF4:Yb / Tm@NaYF4@SiO2-NH2 (UCNPs-SiO2-NH2). Finally, the photoactivated fluorescent probe R-PSN was loaded onto the surface of UCNPs by amide condensation reaction to obtain the final product UCNPs-R-PSN nanoparticles. Fig. 9 A is the TEM characterization result of UCNPs-R-PSN nanoparticles, which shows an obvious core-shell layered structure and an average particle size of 53.90±0.54nm ( Fig. 9 B). According to the Zeta potential results, the surface of UCNPs modified with SiO2 exhibits negative charge (-28.32mV), which is due to the presence of surface hydroxyl groups. After APTES modification, the number of amino groups on the surface of UCNPs-SiO2-NH2 is much greater than the number of hydroxyl groups, making the surface positively charged (+14.93mV). After further coupling with the compound R-PSN, UCNPs-R-PSN was obtained, and its Zeta potential value increased significantly ( Fig. 9 C). The above experimental results prove that the compound R-PSN was successfully coupled to the NaYF4:Yb / Tm@NaYF4@SiO2-NH2 surface.

[0164] Study on the detection performance of 4-probe UCNPs-R-PSN for HSNO

[0165] 4.1 UV absorption spectrum of the reaction between probe UCNPs-R-PSN and HSNO

[0166] First, the changes in the UV absorption spectrum of the probe UCNPs-R-PSN before and after the reaction with HSNO were studied. Fig.10As shown in the figure, the probe UCNPs-R-PSN itself has no absorption peak between 450-550nm; while the probe UCNPs-R-PSN first passes through 980nm (3W / cm 2 ) for 10 min, and then after reacting with HSNO, a maximum ultraviolet absorption peak appeared at 545 nm, which is basically consistent with the ultraviolet absorption peak of compound R-2, further indicating that compound R-PSN is loaded onto the surface of upconversion nanomaterials, and the mechanism by which the probe UCNPs-R-PSN reacts with HSNO to produce fluorescence is consistent with expectations.

[0167] 4.2 Loading amount of compound R-PSN in probe UCNPs-R-PSN

[0168] After successfully preparing the probe UCNPs-R-PSN and verifying the coupling of the compound R-PSN on the surface of the upconversion nanoparticles, the loading performance of the upconversion nanoparticles was studied. Fig.11 In A, the black line represents the UV-visible absorption spectrum of the compound R-PSN (65 μM), and the red line represents the reaction supernatant obtained by centrifugation (10000 rpm / min, 5 min) after the compound R-PSN (65 μM) is loaded onto the surface of the upconversion nanoparticles through an amide condensation reaction. The supernatant contains the remaining unreacted R-PSN, and its absorbance value is measured by UV-visible absorption spectroscopy. Compared with before the amide condensation reaction, the absorbance of R-PSN in the reaction supernatant at 267 nm decreased significantly. Fig.11 As shown in Figure B, the ultraviolet absorbance value of the compound R-PSN in the reaction supernatant after centrifugation is 0.967. After substituting into the standard curve of R-PSN at 267nm, the concentration of R-PSN in the solution after loading is calculated to be 29.02μM; the initial concentration of R-PSN in the reaction solution is 65μM. Therefore, according to the change in the concentration of R-PSN in the supernatant before and after the reaction, the loading amount of R-PSN on the upconversion nanoparticles is 55.35%.

[0169] 4.3 Fluorescence spectra of the reaction between probe UCNPs-R-PSN and HSNO

[0170] In order to study the response performance of the probe UCNPs-R-PSN to HSNO, Fig.12As shown in the figure, the probe UCNPs-R-PSN itself has almost no fluorescence; when only irradiated with 980nm and incubated without HSNO, the probe UCNPs-R-PSN has almost no fluorescence; when not irradiated with 980nm and incubated with HSNO, the probe UCNPs-R-PSN has almost no fluorescence; after the probe is irradiated with 980nm and then incubated with HSNO, the probe UCNPs-R-PSN will show a significantly enhanced fluorescence signal at 645nm. The results show that after irradiation with 980nm, the probe UCNPs-R-PSN incubated with HSNO can produce a significantly enhanced fluorescence signal.

[0171] 4.4 Linear relationship and detection limit of the reaction between probe UCNPs-R-PSN and HSNO

[0172] The response of the probe UCNPs-R-PSN to HSNO was further studied. Fig.13 As shown in the figure, the probe UCNPs-R-PSN itself has no fluorescence signal. When the probe UCNPs-R-PSN was irradiated at 980nm and incubated with different concentrations of HSNO (0-80μM), the fluorescence intensity at 645nm gradually increased. In the HSNO concentration range of 0-2μM, the fluorescence intensity showed a good linear relationship with the HSNO concentration. In addition, in PBS buffer, the probe detection limit was 142nM, indicating that the probe UCNPs-R-PSN has a high detection sensitivity.

[0173] 4.5 Reaction time of probe UCNPs-R-PSN and HSNO

[0174] The present invention further explores the reaction time of the incubation of the probe UCNPs-R-PSN and HSNO. Fig.14 As shown in the figure, after irradiation at 980nm, the probe UCNPs-R-PSN was incubated with HSNO (100μM) for about 10min, and the fluorescence intensity reached a peak. In addition, after incubation for 90min, the fluorescence intensity did not significantly weaken, indicating that the generated fluorescence response was stable. The above results show that the fluorescence response generated by the probe UCNPs-R-PSN and HSNO has good stability and is suitable for long-term imaging.

[0175] 4.6 Effect of reaction system pH on the reaction between probe UCNPs-R-PSN and HSNO

[0176] In order to study the effect of different pH environments on the reaction between UCNPs-R-PSN and HSNO, the probe was firstly passed through 980nm (3W / cm 2) for 10 min, and then incubated with HSNO (100 μM) in PBS buffer with different pH values ​​(50 mM, pH = 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.4; 8.0; 8.5; 9.0; 20% DMSO). Fig.15 As shown in the figure, in the absence of HSNO, the probe is relatively stable at pH 4.0-9.0 and has no fluorescence emission. When the probe reacts with HSNO, the fluorescence signal decreases slightly with the increase of pH. It is speculated that this may be because the SN single bond in HSNO is relatively stable under acidic conditions, but is more easily decomposed into nitrous acid and hydrogen sulfide under neutral and alkaline conditions. The above experimental results suggest that the probe UCNPs-R-PSN is suitable for the detection of HSNO under physiological conditions (pH = 7.4).

[0177] 4.7 Selectivity of probe UCNPs-R-PSN in detecting HSNO

[0178] Considering the complex environment in the body, the high selectivity of the probe to the detected object is the key to achieve accurate detection. In order to verify the selectivity of the probe UCNPs-R-PSN to HSNO, the probe UCNPs-R-PSN was firstly subjected to 980nm (3W / cm 2 ) for 10 min, and then mixed with the following interfering substances: amino acids (Gly, D-Ala, Val, Ile, L-Cys, L-Thr, Asp, L-Glu, L-Arg, L-Lys, L-His, Hcy), inorganic salts (Sn 2+ 、Cd 2+ , Mn 2+ 、Co 2+ , Cu 2+ , Hg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Ca 2+ 、Al 3+ 、Ni 2+ Mg 2+ , Li + 、Na + , K + 、Ag + ), active sulfur (Na2S, Na2S2, NaHS, Na2SO3, NaHSO3, CH3SSSCH3, CysSSCys, GSSG, S8, GSH), active oxygen (H2O2, ClO - , t BuOOH, · OH, 1 O2, O2 ·- ), active nitrogen (NO3- 、NO2 - 、ONOO - , DEA·NONOate) and other interfering substances (L-AA, DHA, MGO), and then the fluorescence intensity was measured after incubation for 10 min. After irradiation at 980 nm, the probe UCNPs-R-PSN incubated with HSNO produced a significantly enhanced fluorescence signal, while there was almost no response to other substances ( Figure 16-Figure 19 ). The above experimental results show that the probe UCNPs-R-PSN has good selectivity for HSNO and is not interfered by other substances.

[0179] 5. Cell-level fluorescence imaging experiment

[0180] 5.1 Cytotoxicity assay

[0181] The safety of the probe UCNPs-R-PSN in cells was investigated using the CCK8 method. Human colon cancer cells HCT116 were selected and incubated with different concentrations of probe UCNPs-R-PSN (0, 50, 100, 200, 400, 500 μg / mL) for 48 hours. The results are shown in Fig. 20 As shown, the cell viability was always maintained above 85%, indicating that the probe had low cytotoxicity.

[0182] In order to observe the changes in cell activity over time after the cells were incubated with the probe, the cell activity was further detected after the probe (final concentration 100 μg / mL) was incubated with cells for different time periods (0, 6, 12, 24, and 48 h). Fig.21 As shown, after incubation with the probe for 48 h, the cell viability was still above 90%, indicating that the probe had low toxicity to cells and had little effect on cell viability.

[0183] 5.2 Fluorescence imaging of exogenous HSNO in cells

[0184] The high sensitivity and good selectivity of the probe UCNPs-R-PSN to HSNO were successfully verified by in vitro experiments. This paper further explored the fluorescence imaging performance of the probe to HSNO in cells. First, the probe UCNPs-R-PSN was incubated with cells for only 10 minutes, and no fluorescence signal was observed ( Fig. 22 A). Subsequently, the probe was incubated with cells for 10 min before adding HSNO (100 μM), and no fluorescence signal was observed ( Fig. 22 B). When the probe is co-incubated with cells and then irradiated with 980nm light, only a weak red fluorescence appears in the cells ( Fig. 22C). However, when exogenous 100 μM HSNO (a mixture of freshly prepared 0.3 mM GSNO and 0.1 mM Na2S dissolved in PBS buffer was introduced into cells co-incubated with the probe, followed by 980 nm illumination, the intracellular red fluorescence signal was significantly enhanced (100-fold) ( Fig. 22 D). In contrast, when the probe was first incubated with cells and then Na2S (150 μM) or GSNO (1 mM) was added and irradiated with 980 nm light, only weak fluorescence signals were observed in both groups ( Fig. 22 E and Fig. 22 F). This indicates that the probe can effectively detect exogenous HSNO without interference from Na2S and GSNO. Fig.23 yes Fig. 22 The corresponding cell bright field images show that the cell morphology remains normal during confocal imaging, which proves that the probe can detect exogenous HSNO in living cells.

[0185] 5.3 Fluorescence imaging of HSNO in HCT116 cells transfected with CBS-siRNA

[0186] Thionitrite is an important crosstalk product between H2S and NO, and its level changes are usually regulated by H2S and NO. Studies have shown that endogenous H2S in cells can react with exogenous NO donors to generate HSNO in situ. This indicates that H2S plays a key role in the generation of endogenous HSNO in cells. When the CBS gene is knocked out, the intracellular H2S level is significantly reduced, which in turn leads to a decrease in the endogenous HSNO level. The present invention uses CBS-siRNA to transfect HCT116 cells to reduce the expression of CBS protein, thereby reducing the content of endogenous HSNO. Fig.24 As shown in G, the expression of CBS protein in HCT116 cells transfected with CBS-siRNA was significantly reduced compared with the control group. Fig.24 A and Fig.24 As shown in D, no fluorescence signal was observed when only the probe UCNPs-R-PSN was co-incubated with HCT116 cells or HCT116 cells transfected with CBS-siRNA. Fig.24 B and Fig.24 As shown in E, compared with HCT116 cells irradiated at 980 nm, the fluorescence intensity of CBS-siRNA transfected cells irradiated at 980 nm after incubation with the probe was significantly reduced, indicating that the intracellular HSNO level was decreased.

[0187] Studies have shown that aminooxyacetic acid (AOAA) is an inhibitor of the H2S synthase cystathionine β-synthase (CBS), which can reduce endogenous H2S levels. In this experiment, AOAA (20μM) was used to pretreat cells for 45 minutes to inhibit the generation of endogenous H2S, thereby inhibiting the reaction of endogenous H2S with NO and reducing the level of HSNO. The cells were then incubated with the probe and then irradiated with 980nm light. The results showed that the red fluorescence signal was also significantly reduced ( Fig.24 C) Description Fig.24 The red fluorescence signal in B is caused by the physiological level of HSNO in cells. These results indicate that the probe can detect endogenous as well as physiological levels of HSNO with high sensitivity.

[0188] Fluorescence imaging of 6-probe UCNPs-R-PSN in animals

[0189] The present invention continues to investigate the imaging performance of probe UCNPs-R-PSN for HSNO detection at the in vivo level. The experimental results show that mice injected with probe UCNPs-R-PSN only have almost no fluorescence signal ( Fig.26 A), mice injected with the probe and then irradiated with 980nm also had almost no fluorescence signal ( Fig.26 B). Compared with the first two groups, the fluorescence intensity of mice irradiated at 980 nm after intratumoral injection of probe UCNPs-R-PSN and then HSNO (100 μM) increased significantly (19 times) ( Fig.26 C) Fig. 27 As shown in Figure 2, after the administration of exogenous HSNO, the fluorescence signal of the mouse gradually increased, reaching a peak at around 25 minutes, and there was still a strong signal at 120 minutes ( Fig.28 ). It can be seen that the probe UCNPs-R-PSN has good stability and is suitable for detecting HSNO in mice.

[0190] 7. Study the effect of exogenous HSNO on tumor growth

[0191] In order to evaluate the effect of HSNO on tumor growth, the present invention randomly divided HCT116 tumor-bearing mice into two groups: (1) saline group: saline (100 μL) was administered through the tail vein on days 0, 3, 6, 9, and 12; (2) HSNO group: HSNO (1.6 mg / kg, Saline:DMSO=99:1) was administered through the tail vein on days 0, 3, 6, 9, and 12. The tumor volume was recorded every 3 days for a 12-day treatment. The changes in body weight and tumor volume of each group of mice were recorded within 12 days to evaluate the therapeutic effect. The results are shown in Figure 2. Fig.29As shown in A, during the treatment, the tumor volume of mice in the saline group increased rapidly, while the HSNO group had a significant inhibitory effect on tumor growth. Fig.29 As shown in B, after 12 days of treatment, there was no significant difference in the body weight of mice in the HSNO treatment group compared with the mice in the normal saline group. Subsequently, the mice in the normal saline group and the HSNO group were euthanized, and the tumor tissues of the tumor-bearing mice were removed. The ex vivo tumor tissues were weighed and photographed for comparison ( Fig.29 C), H&E pathology staining and frozen section processing of the tumor were performed simultaneously. Fig.29 D The results showed that the tumor cells of mice in the normal saline group were dense, the nuclei were darkly stained and showed obvious atypia, the blood vessels in the tumor were relatively abundant, and no necrotic foci were observed. In the sections of the HSNO group, a large number of nuclear atypia were observed, the nuclear-cytoplasmic ratio was increased, and there was obvious inflammatory cell infiltration and fibrous connective tissue hyperplasia, and large areas of cell necrosis were present. The above results indicate that exogenous HSNO has anti-tumor activity. This paper further studies the performance of the probe in detecting HSNO in tumor sections. Fig.30 As shown in the figure, the fluorescence intensity of the tumor slices in the saline group was low, indicating that the HSNO level was low, while after exogenous HSNO treatment, the fluorescence intensity of the tumor slices increased significantly, indicating that the HSNO level was significantly increased. This preliminarily indicates that changes in HSNO levels are associated with the occurrence and development of colon cancer.

[0192] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A thionitrite near-infrared light-activated probe, characterized in that: The synthesis route of the photoactivated probe is as follows:

2. The thionitrite near-infrared light-activated probe according to claim 1, characterized in that The method for preparing the photoactivated probe comprises the following steps: (1) Synthesis of NaYF4:Yb / Tm core nanoparticles: YCl3 . 6H2O, YbCl3 . 6H2O、TmCl3 . 6H2O, oleic acid and octadecene are uniformly mixed, heated to 140-160°C to remove the low boiling point solvent, cooled to 20-30°C, added with an anhydrous methanol solution containing NaOH and NH4F to react by stirring, then heated to 80-100°C to remove the anhydrous methanol, the obtained mixture is rapidly heated to 280-320°C at a rate of 10-20°C / min to react by stirring, cooled to 20-30°C again, centrifuged and separated to obtain NaYF4:Yb / Tm core nanoparticles; (2) Synthesis of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles: YCl3 . 6H2O oleic acid and octadecene are uniformly mixed, heated to 140-160°C to remove the low boiling point solvent, cooled to 20-30°C, added with the mixed solution prepared by dispersing the NaYF4:Yb / Tm core nanoparticles obtained in step (1) in cyclohexane, heated to 80-100°C to remove the cyclohexane, cooled to 20-30°C again, added with anhydrous methanol solution containing NaOH and NH4F to react with stirring, heated to 80-100°C to remove the anhydrous methanol, and the obtained mixture is rapidly heated to 280-320°C at a rate of 10-20°C / min to react with stirring, cooled to 20-30°C, centrifuged, and separated to obtain NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles; (3) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2: CTAB is dispersed in deionized water, and a mixed solution prepared by dispersing the NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles obtained in step (2) in cyclohexane is added thereto for stirring reaction to form a NaYF4:Yb / Tm@NaYF4-CTAB solution, to which ethanol and sodium hydroxide solution are added, stirred evenly, heated to 60-80°C, tetraethyl silicate is added dropwise for stirring reaction, the reaction solution is collected, centrifuged, and separated, the surfactant CTAB is removed from the obtained product by ion exchange, the obtained precipitate is resuspended in anhydrous ethanol and NaCl is added, the temperature is raised to 50-70°C for stirring reaction, centrifuged again, and separated to obtain SiO2-coated upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2; (4) Synthesis of UCNPs-SiO2-NH2: Disperse the NaYF4:Yb / Tm@NaYF4@SiO2 obtained in step (3) in anhydrous ethanol, add (3-aminopropyl)triethoxysilane dropwise, stir and react at 60-80°C, centrifuge and separate to obtain amino-functionalized upconversion nanoparticles UCNPs-SiO2-NH2; (5) Synthesis of compound T-1: Concentrated sulfuric acid, dichloromethane and anhydrous magnesium sulfate were uniformly mixed, 4-bromomethyl-3-nitrobenzoic acid and tert-butyl alcohol were added thereto, and the mixture was stirred and reacted at 25-35° C. to prepare compound T-1; (6) Synthesis of compound T-2: Compound T-1 and o-mercaptobenzoic acid were mixed, anhydrous potassium carbonate and DMF were added, and the mixture was stirred at 25-35° C. to react to prepare compound T-1; (7) Synthesis of compound R-2: Compound R-1 and 1,6-dihydroxynaphthalene were dissolved in methanesulfonic acid, and stirred at 80-90° C. to prepare compound R-2; (8) Synthesis of compound R-3: Compound T-2, 4-dimethylaminopyridine and EDCI were dissolved in dichloromethane, and compound R-2 was added to react by stirring to prepare compound R-3; (9) Synthesis of compound R-4: Compound R-3 was dissolved in dichloromethane, and DMF and oxalyl chloride were added to react with stirring to prepare an intermediate product; the intermediate product was dissolved in dichloromethane, and added to an anhydrous dichloromethane solution containing o-phenylenediamine and triethylamine, and reacted with stirring to prepare compound R-4; (10) Synthesis of compound R-PSN: Compound R-4 was dissolved in dichloromethane, trifluoroacetic acid was added dropwise, and the mixture was stirred to react to prepare compound R-PSN; (11) Synthesis of probe UCNPs-R-PSN: Compound R-PSN, UCNPs@SiO2-NH2, HBTU and HOBt were mixed and stirred for reaction. DIPEA was then added and the stirring reaction was continued to obtain the probe UCNPs-R-PSN.

3. The thionitrite near-infrared light-activated probe according to claim 1, characterized in that: In step (1), YCl3 . 6H2O, YbCl3 . 6H2O and TmCl3 . The molar ratio of YCl3 . The mass volume ratio of 6H2O to oleic acid is 235-245:7 mg / mL, preferably 241:7 mg / mL; the YCl3 . The mass volume ratio of 6H2O to octadecene is 235-245:15 mg / mL, preferably 235-245:15 mg / mL; the YCl3 . The molar ratio of 6H2O to NaOH is 235-245:100, preferably 241:100; the YCl3 . The molar ratio of 6H2O to NH4F is 235-245:148, preferably 241:148; the temperature for removing low boiling point solvents is 145-155°C, preferably 150°C; the temperature for removing anhydrous methanol is 85-95°C, preferably 90°C; the temperature is rapidly raised to 290-310°C, preferably 300°C, at a rate of 15°C / min.

4. The thionitrite near-infrared light-activated probe according to claim 1, characterized in that: In step (2), the YCl3 . The mass volume ratio of 6H2O to oleic acid is 300-310:7 mg / mL, preferably 303:7 mg / mL; the YCl3 . The mass volume ratio of 6H2O to octadecene is 300-310:15 mg / mL, preferably 303:15 mg / mL; the YCl3 . The molar ratio of 6H2O to NaOH is 300-310:100, preferably 303:100; the YCl3 . The molar ratio of 6H2O to NH4F is 300-310:148, preferably 303:148; The temperature for removing low boiling point solvents is 145-155°C, preferably 150°C; the temperature for removing cyclohexane is 85-95°C, preferably 90°C; the temperature for removing anhydrous methanol is 85-95°C, preferably 90°C; the temperature is rapidly increased to 290-310°C, preferably 300°C, at a rate of 15°C / min.

5. The thionitrite near-infrared light-activated probe according to claim 1, characterized in that: In step (3), the weight ratio of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles to CTAB is 1:3-7, preferably 1:5; the mass volume ratio of the NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles to tetraethyl silicate is 4-6:3 mg / μL, preferably 5:3 mg / μL; in step (4), the mass volume ratio of the NaYF4:Yb / Tm@NaYF4@SiO2 to (3-aminopropyl)triethoxysilane is 1-1.5:2.5 mg / μL, preferably 1:2 mg / μL.

6. The thionitrite near-infrared light-activated probe according to claim 1, characterized in that: In step (5), the molar ratio of 4-bromomethyl-3-nitrobenzoic acid to tert-butyl alcohol is 1:3-7, preferably 1:5; in step (6), the molar ratio of compound T-1 to o-mercaptobenzoic acid is 1:0.8-1.5, preferably 1:1; in step (7), the molar ratio of compound R-1 to 1,6-dihydroxynaphthalene is 1:0.8-1.5, preferably 1:

1.

7. The thionitrite near-infrared light-activated probe according to claim 1, characterized in that: In step (8), the molar ratio of the compound T-2 to 4-dimethylaminopyridine is 230-240:54, preferably 233:54; the molar ratio of the compound T-2 to EDCI is 230-240:349, preferably 233:349; the molar ratio of the compound T-2 to the compound R-2 is 230-240:275, preferably 233:275; in step (9), the molar ratio of the compound R-3 to oxalyl chloride is 1:2-4, preferably 1:3; the molar ratio of the compound R-3 to o-phenylenediamine is 1:2-4, preferably 1:3; the molar ratio of the compound R-3 to triethylamine is 1:2-4, preferably 1:3; in step (10), the mass volume ratio of the compound R-4 to trifluoroacetic acid is 425-435:1 mg / mL, preferably 430:1 mg / mL.

8. The thionitrite near-infrared light-activated probe according to claim 1, characterized in that: In step (11), the mass ratio of the compound R-PSN to UCNPs@SiO2-NH2 is 3-7:1, preferably 5:1; the molar ratio of the compound R-PSN to HBTU is 1:0.8-1.5, preferably 1:1; the molar ratio of the compound R-PSN to HOBt is 1:0.8-1.5, preferably 1:1; the molar ratio of the compound R-PSN to DIPEA is 1:0.8-1.5, preferably 1:

1.

9. The method for preparing the thionitrite near-infrared light-activated probe according to claim 1, characterized in that: The steps include: (1) Synthesis of NaYF4:Yb / Tm core nanoparticles: YCl3 . 6H2O, YbCl3 . 6H2O、TmCl3 . 6H2O, oleic acid and octadecene are uniformly mixed, heated to 140-160°C to remove the low boiling point solvent, cooled to 20-30°C, added with an anhydrous methanol solution containing NaOH and NH4F to react by stirring, then heated to 80-100°C to remove the anhydrous methanol, the obtained mixture is rapidly heated to 280-320°C at a rate of 10-20°C / min to react by stirring, cooled to 20-30°C again, centrifuged and separated to obtain NaYF4:Yb / Tm core nanoparticles; (2) Synthesis of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles: YCl3 . 6H2O oleic acid and octadecene are uniformly mixed, heated to 140-160°C to remove the low boiling point solvent, cooled to 20-30°C, added with the mixed solution prepared by dispersing the NaYF4:Yb / Tm core nanoparticles obtained in step (1) in cyclohexane, heated to 80-100°C to remove the cyclohexane, cooled to 20-30°C again, added with anhydrous methanol solution containing NaOH and NH4F to react with stirring, heated to 80-100°C to remove the anhydrous methanol, and the obtained mixture is rapidly heated to 280-320°C at a rate of 10-20°C / min to react with stirring, cooled to 20-30°C, centrifuged, and separated to obtain NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles; (3) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2: CTAB is dispersed in deionized water, and a mixed solution prepared by dispersing the NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles obtained in step (2) in cyclohexane is added thereto for stirring reaction to form a NaYF4:Yb / Tm@NaYF4-CTAB solution, to which ethanol and sodium hydroxide solution are added, stirred evenly, heated to 60-80°C, tetraethyl silicate is added dropwise for stirring reaction, the reaction solution is collected, centrifuged, and separated, the surfactant CTAB is removed from the obtained product by ion exchange, the obtained precipitate is resuspended in anhydrous ethanol and NaCl is added, the temperature is raised to 50-70°C for stirring reaction, centrifuged again, and separated to obtain SiO2-coated upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2; (4) Synthesis of UCNPs-SiO2-NH2: Disperse the NaYF4:Yb / Tm@NaYF4@SiO2 obtained in step (3) in anhydrous ethanol, add (3-aminopropyl)triethoxysilane dropwise, stir and react at 60-80°C, centrifuge and separate to obtain amino-functionalized upconversion nanoparticles UCNPs-SiO2-NH2; (5) Synthesis of compound T-1: Concentrated sulfuric acid, dichloromethane and anhydrous magnesium sulfate were uniformly mixed, 4-bromomethyl-3-nitrobenzoic acid and tert-butyl alcohol were added thereto, and the mixture was stirred and reacted at 25-35° C. to prepare compound T-1; (6) Synthesis of compound T-2: Compound T-1 and o-mercaptobenzoic acid were mixed, anhydrous potassium carbonate and DMF were added, and the mixture was stirred at 25-35° C. to react to prepare compound T-1; (7) Synthesis of compound R-2: Compound R-1 and 1,6-dihydroxynaphthalene were dissolved in methanesulfonic acid, and stirred at 80-90° C. to prepare compound R-2; (8) Synthesis of compound R-3: Compound T-2, 4-dimethylaminopyridine and EDCI were dissolved in dichloromethane, and compound R-2 was added to react by stirring to prepare compound R-3; (9) Synthesis of compound R-4: Compound R-3 was dissolved in dichloromethane, and DMF and oxalyl chloride were added to react with stirring to prepare an intermediate product; the intermediate product was dissolved in dichloromethane, and added to an anhydrous dichloromethane solution containing o-phenylenediamine and triethylamine, and reacted with stirring to prepare compound R-4; (10) Synthesis of compound R-PSN: Compound R-4 was dissolved in dichloromethane, trifluoroacetic acid was added dropwise, and the mixture was stirred to react to prepare compound R-PSN; (11) Synthesis of probe UCNPs-R-PSN: Compound R-PSN, UCNPs@SiO2-NH2, HBTU and HOBt were mixed and stirred for reaction. DIPEA was then added and the stirring reaction was continued to obtain the probe UCNPs-R-PSN.

10. Use of the thionitrite near-infrared light-activated probe according to claim 1 in detecting thionitrite.

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