Sulfonitroso near-infrared light-activated probes, methods of making and using the same

Abstract

This invention provides a near-infrared photoactivated probe for thionitrite, its preparation method, and its application. The probe is stable, selective, sensitive, and has a low detection limit (142 nM). This probe can detect physiological levels of HSNO in HCT116 cells and can be applied to fluorescence imaging of HSNO in HCT116 colon cancer xenografts in nude mice, providing a visual detection tool for revealing the physiological and pathological mechanisms of HSNO.

Description

Technical Field

[0001] This invention belongs to the field of chemistry and analytical detection, specifically relating to a near-infrared light-activated probe for thionitrous acid, its preparation method, and its application. Background Technology

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

[0003] In recent years, increasing research has shown that hydrogen sulfide can inhibit or activate NOS activity. NO affects endogenous H2S production by inhibiting or inducing the activity or expression of cystathionine-β-synthase and cystathionine-γ-lyase. It exhibits a biphasic effect on cell proliferation and apoptosis, which is related to factors such as H2S / NO levels, duration of exposure, tumor cell type, and sensitivity. In colorectal 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 antitumor activity. In colorectal cancer, H2S participates in exogenous NO-mediated antitumor migration and invasion activities, suggesting a cross-talk mechanism mediated by the cGMP / VASP signaling pathway between H2S and NO. Therefore, the expression of H2S and NO, as well as their crosstalk, influence the development of colorectal cancer. HSNO is both a source of NO and a product of H2S-NO crosstalk; its level changes are closely related to the occurrence and development of colorectal cancer, but the specific mechanism remains unclear. There is an urgent need to develop accurate HSNO detection methods, which will help to better understand its physiological role and facilitate research on the relationship between colorectal cancer and changes in HSNO levels.

[0004] Existing methods for HSNO detection include Fourier transform infrared spectroscopy and high-resolution mass spectrometry. These methods require the separation and extraction of cells or tissues and cannot provide real-time imaging. Fluorescent probe methods offer high selectivity and sensitivity, enabling real-time in-situ monitoring of HSNO. Currently, only two HSNO fluorescent probes have been reported, but they do not provide 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, and high sensitivity, suitable for in vivo HSNO detection.

[0005] Photoactivated fluorescent probes enable sensing and imaging through light modulation, providing high spatiotemporal resolution imaging. It is well known that most photoactivated protecting groups require UV / visible light excitation, leading to strong autofluorescence interference, along with phototoxicity and photodamage, limiting their imaging applications in deep tissues, organs, and in vivo. To overcome these limitations, near-infrared photoactivated probes have been developed, which can improve the tissue imaging depth of the probes while reducing phototoxicity and photodamage. Rare-earth upconversion nanoparticles are important carriers for near-infrared photoactivation / modulation; they can absorb long-wavelength near-infrared excitation light and upconvert it into visible / ultraviolet or short-wavelength near-infrared emission light. Therefore, developing near-infrared photoactivated fluorescent probes can achieve deep tissue and in vivo imaging while reducing phototoxicity and photodamage. Summary of the Invention

[0006] The purpose of this invention is to provide a near-infrared photoactivated probe for thionitrite, based on existing technologies. This probe not only has good selectivity and high sensitivity (detection limit of 142 nM), but also successfully achieves fluorescence imaging detection of HSNO at both the HCT116 cell level and in vivo level. This provides a visual detection tool for revealing the physiological and pathological mechanisms of HSNO, and is of great significance for studying diseases related to HSNO, such as cancer, cardiovascular diseases, and neurodegenerative diseases.

[0007] A second objective of this invention is to provide a method for preparing the aforementioned photoactivated probe.

[0008] A third objective of this invention is to provide the application of the aforementioned photoactivated probe in the detection of thionitrous acid.

[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 the carrier for near-infrared photoactivation and coupling photoactivation fluorescent probes. (2) The o-nitrobenzyl ester derivative is selected as the photoactivation protecting group, the rhodamine derivative is used as the near-infrared fluorescent group, and the o-mercaptobenzoate and o-phenylenediamine are used as HSNO recognition groups to construct the photoactivation fluorescent probe R-PSN. (3) The photoactivation fluorescent probe is coupled to the upconversion nanoparticles through amide bonds to construct the near-infrared photoactivation fluorescent probe UCNPs-R-PSN based on UCNPs.

[0011] A near-infrared photoactivated probe of thionitrous acid (UCNPs-R-PSN) is synthesized via the following route:

[0012]

[0013]

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

[0015] The near-infrared photoactivated probe of thionitrous acid provided by this invention includes the following steps in its preparation method:

[0016] (1) Synthesis of NaYF4:Yb / Tm core nanoparticles: YCl3 . 6H2O, YbCl3 . 6H2O, TmCl3 . 6H2O, oleic acid, and octadecene were mixed evenly, and the mixture was heated to 140-160℃ to remove the low-boiling-point solvent. After cooling to 20-30℃, an anhydrous methanol solution containing NaOH and NH4F was added and stirred. The mixture was then heated to 80-100℃ to remove the anhydrous methanol. The resulting mixture was rapidly heated to 280-320℃ at a rate of 10-20℃ / min and stirred. After cooling to 20-30℃ again, the mixture was centrifuged and separated to obtain NaYF4:Yb / Tm core nanoparticles.

[0017] (2) Synthesis of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles: YCl3 .Oleic acid and octadecene were mixed evenly and heated to 140-160℃ to remove low-boiling-point solvents. After cooling to 20-30℃, a mixed solution of NaYF4:Yb / Tm core nanoparticles obtained in step (1) dispersed in cyclohexane was added. The mixture was then heated to 80-100℃ to remove cyclohexane. After cooling to 20-30℃ again, an anhydrous methanol solution containing NaOH and NH4F was added and stirred. The mixture was then heated to 80-100℃ to remove anhydrous methanol. The resulting mixture was rapidly heated to 280-320℃ at a rate of 10-20℃ / min and stirred. After cooling to 20-30℃, the mixture was centrifuged and separated to obtain NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles.

[0018] (3) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2: CTAB was dispersed in deionized water, and a mixed solution of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles obtained in step (2) dispersed in cyclohexane was added. The mixture was stirred and reacted to form NaYF4:Yb / Tm@NaYF4-CTAB solution. Ethanol and sodium hydroxide solution were added to the solution and stirred evenly. The temperature was raised to 60-80℃, and tetraethyl silicate was added dropwise and stirred. The reaction solution was collected, centrifuged, and separated. The surfactant CTAB was removed from the product by ion exchange. The precipitate was resuspended in anhydrous ethanol and NaCl was added. The temperature was raised to 50-70℃ and stirred. The mixture was centrifuged and separated again to obtain SiO2-encapsulated upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2.

[0019] (4) Synthesis of UCNPs-SiO2-NH2: The NaYF4:Yb / Tm@NaYF4@SiO2 obtained in step (3) was dispersed in anhydrous ethanol, and (3-aminopropyl)triethoxysilane was added dropwise. The reaction was stirred at 60-80℃, centrifuged, and separated 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 mixed evenly, and 4-bromomethyl-3-nitrobenzoic acid and tert-butanol were added to it. The mixture was stirred at 25-35℃ 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℃ 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℃ 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 and stirred to prepare compound R-3;

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

[0025] (10) Synthesis of compound R-PSN: compound R-4 was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring 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. DIPEA was then added and the stirring reaction was continued to obtain 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-to-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-to-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; and 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-to-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-to-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. YCl₃ . 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℃, preferably 150℃; the temperature for removing cyclohexane is 85-95℃, preferably 90℃; the temperature for removing anhydrous methanol is 85-95℃, preferably 90℃; and the temperature is rapidly increased to 290-310℃, preferably 300℃, at a rate of 15℃ / 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-to-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-to-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-to-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; and the molar ratio of compound R-PSN to DIPEA is 1:0.8-1.5, preferably 1:1.

[0046] The advantages of using the technical solution of this invention are as follows:

[0047] This invention provides a near-infrared photoactivated probe for thionitrite, which not only exhibits good selectivity and high sensitivity (detection limit of 142 nM), but also successfully achieves fluorescence imaging detection of HSNO at both the HCT116 cell level and in vivo level. Intraperitoneal injection of exogenous HSNO revealed its antitumor activity against HCT116 xenografts. Using the probe to detect HSNO levels in HCT116 tumor sections, the results showed that compared with the saline group, HSNO treatment significantly increased HSNO levels in nude mouse HCT116 tumor sections. This finding preliminarily suggests a correlation between changes in HSNO levels and the occurrence and development of colon cancer, providing a visual detection tool for revealing the physiological and pathological mechanisms of HSNO. Furthermore, it is of great significance for studying HSNO-related diseases such as cancer, cardiovascular disease, and neurodegenerative diseases. Attached Figure Description

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

[0049] Figure 2 This is the identification mechanism of HSNO detection using the UCNPs-R-PSN probe;

[0050] Figure 3 It 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 It is the compound R-PSN 1 H NMR spectrum;

[0052] Figure 5 It is 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 This is a characterization of NaYF4:Yb / Tm; among which, Figure 7 A is a transmission electron microscope image of NaYF4:Yb / Tm; Figure 7B is the particle size distribution diagram of NaYF4:Yb / Tm; Figure 7 C represents the fluorescence spectrum of NaYF4:Yb / Tm; Figure 7 D is the XRD pattern of NaYF4:Yb / Tm;

[0055] Figure 8 It is a characterization of NaYF4:Yb / Tm@NaYF4; where, Figure 8 A is a transmission electron microscope image of NaYF4:Yb / Tm@NaYF4; Figure 8 B is the particle size distribution diagram of NaYF4:Yb / Tm@NaYF4;

[0056] Figure 9 This is a characterization of the probe UCNPs-R-PSN; among which, Figure 9 A is a transmission electron microscope image of UCNPs-R-PSN; Figure 9 B is the particle size distribution diagram of UCNPs-R-PSN; Figure 9 C represents the Zeta potential of UCNPs-SiO2, UCNPs-SiO2-NH2, and UCNPs-R-PSN;

[0057] Figure 10 The UV absorption spectra of 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 / cm²). 2 ,10min), probe UCNPs-R-PSN (0.1mg / mL, in DMSO) + 980nm light (3W / cm) 2 The UV absorption spectra of compound R-2 (40 μM) incubated in PBS buffer (50 mM, pH = 7.4, 20% DMSO) for 10 min were obtained. Among them, at 450 nm, the curves from high to low are those of compound R-2, probe UCNPs-R-PSN+light+HSNO, probe UCNPs-R-PSN, and probe UCNPs-R-PSN+light, with the latter two curves basically overlapping.

[0058] Figure 11 This involves determining the loading of compound R-PSN in the probe UCNPs-R-PSN; among which, Figure 11In section A, the black line (higher curve at 275 nm) represents the UV-Vis absorption spectrum of compound R-PSN (65 μM), and the red line (lower curve at 275 nm) represents the UV-Vis absorption spectrum of the remaining unreacted compound R-PSN in the reaction supernatant obtained by centrifugation (10000 rpm / min) after compound R-PSN (65 μM) was loaded onto the surface of upconversion nanoparticles. Figure 11 B is the linear fitting standard curve of the absorbance at 267 nm for 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).

[0059] Figure 12 The fluorescence spectra of the probe UCNPs-R-PSN in response to HSNO are shown; among them, the probe UCNPs-R-PSN (0.1 mg / mL), the probe UCNPs-R-PSN (0.1 mg / mL) + HSNO (100 μM), and the probe UCNPs-R-PSN (0.1 mg / mL) + 980 nm light (3 W / cm²) are also shown. 2 ,10min), probe UCNPs-R-PSN (0.1mg / mL) + 980nm light (3W / cm) 2 The fluorescence spectrum (λ) of HSNO (100 μM) in PBS buffer (50 mM, pH 7.4, 20% DMSO) incubated at 37 °C for 10 min was obtained. ex =575nm, slit: 10nm);

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

[0061] Figure 14 These are the fluorescence spectra of probes UCNPs-R-PSN incubated with HSNO for different times; among them, Figure 14 A is the probe UCNPs-R-PSN (0.1 mg / mL) passing through 980 nm (3 W / cm). 2 After irradiation for 10 min, the fluorescence spectra of HSNO (100 μM) in PBS buffer (50 mM, pH = 7.4, 20% DMSO) were obtained after incubation 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. Figure 14 B represents the change in fluorescence intensity (λ) of probe UCNPs-R-PSN incubated with HSNO (100 μM) at 37°C in PBS buffer (50 mM, pH = 7.4, 20% DMSO) over time. ex =575nm, slit: 10nm);

[0062] Figure 15 The effect of pH on the reaction of UCNPs-R-PSN with HSNO; among which, Figure 15 A probe UCNPs-R-PSN (0.1 mg / mL) was subjected to 980 nm (3 W / cm²) 2 Fluorescence spectra of HSNO (100 μM) in PBS buffers at 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) after 10 min of irradiation at 37 °C for 10 min. Figure 15 B probe UCNPs-R-PSN (0.1 mg / mL) was subjected to 980 nm (3 W / cm) 2 The fluorescence intensity after irradiation for 10 min and incubation with HSNO (100 μM) at 37 °C for 10 min varies with different pH values ​​(λ). ex =575nm, slit: 10nm);

[0063] Figure 16 It is the selectivity of the probe UCNPs-R-PSN for HSNO; among which, Figure 16 A is UCNPs-R-PSN (0.1 mg / mL) after passing through 980 nm (3 W / cm). 2After irradiation for 10 min, the fluorescence spectra of the 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 M L-His; 1 mM Hcy) were obtained after incubation in PBS buffer (50 mM, pH = 7.4, 20% DMSO) at 37 °C for 10 min. Figure 16 B represents the fluorescence intensity of UCNPs-R-PSN after irradiation at 980 nm, incubated with HSNO (100 μM) and amino acids for 10 min. Black represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after co-incubation with various amino acids, and red represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after co-incubation with a mixture of various amino acids and HSNO (100 μM). 1. Blank + HSNO (100 μM); 2. Gly (1 mM) + HSNO (100 μM); 3. D-Ala (1 mM) + HSNO (100 μM); 4. Val (1 mM) + HSNO (100 μM); 5. Ile (1 mM) + HSNO (100 μM); 6. L-Cys (1 mM) + HSNO (100 μM); 7. L-Thr (1 mM) + HSNO (100 μM) 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] Figure 17 It is the selectivity of the probe UCNPs-R-PSN for HSNO; among which, Figure 17 A is UCNPs-R-PSN (0.1 mg / mL) after passing through 980 nm (3 W / cm). 2 After irradiation for 10 min, it was reacted with HSNO (100 μM) and inorganic salt ions (blank; 1 mM Sn). 2+ 1mM Cd 2+ ;1mM Mn 2+ 1mM Co 2+ 1mM Cu 2+ 1mM Hg 2+ 1mM Zn 2+ 1mM Fe2+ 1mM Fe 3+ 1mM Ca 2+ 1mM Al 3+ 1mM Ni 2+ 1mM Mg 2+ 1mM Li + 1mM Na + 1mM K + 1mM Ag + Fluorescence spectrum of the sample after incubation at 37°C for 10 min in PBS buffer (50 mM, pH = 7.4, 20% DMSO); Figure 17 B represents the fluorescence intensity of UCNPs-R-PSN after irradiation at 980 nm, incubated with HSNO (100 μM) and inorganic salt ions for 10 min. Black represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after co-incubation with various inorganic salt ions, and red represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after co-incubation with a mixture of various inorganic salt ions and HSNO (100 μM); 1. Blank + HSNO (100 μM); 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] Figure 18 The selectivity of the probe UCNPs-R-PSN for HSNO; among which, Figure 18 A is UCNPs-R-PSN (0.1 mg / mL) after passing through 980 nm (3 W / cm). 2 After irradiation for 10 min, fluorescence spectra were obtained by incubating 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. Figure 18 BUCNPs-R-PSN was irradiated at 980 nm and then incubated with HSNO (100 μM) and active sulfur for 10 min. Black represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after co-incubation with various active sulfur compounds, and red represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after co-incubation with mixtures of various active sulfur compounds and HSNO (100 μM). The incubation reactions were: 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] Figure 19 It is the selectivity of the probe UCNPs-R-PSN for HSNO; among which, Figure 19 A is UCNPs-R-PSN (0.1 mg / mL) after passing through 980 nm (3 W / cm). 2 After irradiation for 10 min, it was reacted with HSNO (100 μM) and ROS (blank; 100 μM H2O2; 100 μM ClO) - 100μM t BuOOH; 100μM · OH; 100μM 1 O2; 100μM O2 ·- 100μM NO3 - 100μM NO2 - 100μM ONOO - Fluorescence spectra of 1 mM M-AA, 1 mM DHA, and 1 mM MGO incubated in PBS buffer (50 mM, pH 7.4, 20% DMSO) at 37°C for 10 min. Figure 19 B represents the fluorescence intensity of UCNPs-R-PSN after irradiation at 980 nm and incubation with HSNO (100 μM) and reactive oxygen species for 10 min. Black represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after co-incubation with various reactive oxygen species, and red represents the fluorescence intensity of UCNPs-R-PSN at 645 nm after co-incubation with a mixture of various reactive oxygen species and HSNO (100 μM). 1. Blank + HSNO (100 μM); 2. H₂O₂ (100 μM) + HSNO (100 μM); 3. ClO₂ - (100μM) + HSNO (100μM); 4. t BuOOH(100μM)+HSNO(100μM); 5. · OH (100 μM) + HSNO (100 μM); 6. 1 O2(100μM)+HSNO(100μM); 7.O2 ·- (100μM) + HSNO (100μM); 8NO3 - (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] Figure 20 This study investigated the effect of the probe UCNPs-R-PSN on cell viability. The effect of co-incubation of HCT116 cells with different concentrations of the probe UCNPs-R-PSN (0, 50, 100, 200, 400, 500 μg / mL) for 48 h on cell viability was studied. Data are expressed as mean ± standard deviation (n = 3).

[0068] Figure 21 This study investigated the effect of the probe UCNPs-R-PSN on cell viability; specifically, the effect of co-incubating HCT116 cells with the probe (final concentration 100 μg / mL) for different durations (0, 6, 12, 24, 48 h) on cell viability. Data are expressed as mean ± standard deviation (n = 3).

[0069] Figure 22 It is a fluorescence imaging method for detecting HSNO using the UCNPs-R-PSN probe; among which, Figure 22 A is a probe (final concentration 0.1 mg / mL, 5 μL DMSO) co-incubated with HCT116 cells for 30 min; Figure 22 B is a probe (final concentration 0.1 mg / mL, 5 μL DMSO) co-incubated with HCT116 cells for 30 min, then 100 μM HSNO is added and co-incubated for 10 min; Figure 22 C is a probe (final concentration 0.1 mg / mL, 5 μL DMSO) co-incubated with HCT116 cells for 30 min, then subjected to 980 nm (1 W / cm) 2 Irradiate continuously for 10 minutes; Figure 22 D was a probe (final concentration 0.1 mg / mL, 5 μL DMSO) co-incubated with HCT116 cells for 30 min, then 100 μM HSNO was added and co-incubated for 10 min, followed by 980 nm (1 W / cm) treatment. 2 Irradiate continuously for 10 minutes; Figure 22 E was used as a probe (final concentration 0.1 mg / mL, 5 μL DMSO) and co-incubated with HCT116 cells for 30 min, followed by incubation with 150 μM Na2S for 10 min, and then subjected to 980 nm (1 W / cm) 2 Irradiate continuously for 10 minutes; Figure 22F was a probe (final concentration 0.1 mg / mL, 5 μL DMSO) co-incubated with HCT116 cells for 30 min, then 1 mM GSNO was added and co-incubated for 10 min, followed by 980 nm (1 W / cm) treatment. 2 Irradiate continuously for 10 minutes; Figure 22 G is Figure 22 The average fluorescence intensity of cells in A, 22B, 22C, 22D, 22E, and 22F;

[0070] Figure 23 yes Figure 22 Corresponding bright-field cell image;

[0071] Figure 24 It is a fluorescence imaging method for detecting HSNO using the UCNPs-R-PSN probe; among which, Figure 24 A is a probe (final concentration 0.1 mg / mL, 5 μL DMSO) co-incubated with HCT116 cells for 30 min; Figure 24 B is a probe (final concentration 0.1 mg / mL, 5 μL DMSO) co-incubated with HCT116 cells for 30 min, then subjected to 980 nm (1 W / cm) 2 Irradiate continuously for 10 minutes; Figure 24 C. Cells were co-incubated with AOAA (20 μM) for 45 min, then co-incubated with the probe (final concentration 0.1 mg / mL, 5 μL DMSO) for 30 min, and then subjected to 980 nm (1 W / cm) 2 Irradiate continuously for 10 minutes; Figure 24 D probe (final concentration 0.1 mg / mL, 5 μL DMSO) was co-incubated with CBS siRNA transfected HCT116 cells for 30 min; Figure 24 E probe (final concentration 0.1 mg / mL, 5 μL DMSO) was co-incubated with CBS siRNA transfected HCT116 cells for 30 min, followed by administration of 980 nm (1 W / cm²) 2 Irradiate continuously for 10 minutes; Figure 24 F is Figure 24 The average fluorescence intensity of cells in A, 24B, 24C, and 22D; Figure 24 G represents the expression level of CBS protein in HCT116 cells and HCT116 cells transfected with CBS siRNA; data are expressed as mean ± SD (n = 3), and compared with the control group, ****P < 0.0001;

[0072] Figure 25 yes Figure 24 Corresponding bright-field cell image;

[0073] Figure 26The probe UCNPs-R-PSN is used to detect HSNO in a nude mouse xenograft model of colorectal cancer; among which, Figure 26 A is the mouse intratumoral injection probe UCNPs-R-PSN (50 mg / mL, 10 μL, physiological saline: dimethyl sulfoxide = 9:1 v / v); Figure 26 B involves intratumoral injection of the probe UCNPs-R-PSN (50 mg / mL, 10 μL, physiological saline: dimethyl sulfoxide = 9:1 v / v) into mice, followed by administration of 980 nm (2 W / cm). 2 Irradiate continuously for 5 minutes, then at 5-minute intervals, repeating this cycle 4 times for a total of 20 minutes of irradiation. Figure 26 C represents the intratumoral injection of the probe UCNPs-R-PSN (50 mg / mL, 10 μL, physiological saline: dimethyl sulfoxide = 9:1 v / v) into mice, followed by incubation with exogenous HSNO (100 μM, 100 μL) in PBS solution (50 mM, pH = 7.4) for 30 min, and then incubation at 980 nm (2 W / cm). 2 Irradiate continuously for 5 minutes, then at 5-minute intervals, repeating this cycle 4 times for a total of 20 minutes of irradiation. Figure 26 D represents the fluorescence intensity at the tumor sites in each group of mice. Data are expressed as mean ± standard deviation (n = 3).

[0074] Figure 27 The probe UCNPs-R-PSN was used to detect the change in fluorescence intensity of HSNO in colon cancer xenografts in nude mice over time. Control group mice were injected intratumorally with the probe UCNPs-R-PSN (50 mg / mL, 10 μL, physiological saline: dimethyl sulfoxide = 9:1 v / v), followed by administration of 980 nm (2 W / cm²). 2 The mice were irradiated continuously for 5 min, then at 5 min intervals, for a total of 4 cycles, for a total of 20 min. Mice in the HSNO group were injected intratumorally with the probe UCNPs-R-PSN (50 mg / mL, 10 μL, physiological saline: dimethyl sulfoxide = 9:1 v / v), followed by incubation for 30 min in exogenous HSNO (100 μM, 100 μL) in PBS solution (50 mM, pH = 7.4), and then subjected to 980 nm (2 W / cm²) irradiation. 2 Irradiate continuously for 5 minutes, then at 5-minute intervals, repeating this cycle 4 times for a total of 20 minutes of irradiation.

[0075] Figure 28 It is a quantitative expression Figure 27 The fluorescence signal intensity of two groups of mice; data are expressed as mean ± standard deviation (n=3);

[0076] Figure 29 This relates to the effect of exogenous HSNO on tumor growth; among which, Figure 29A represents the weight change of nude mice with colon cancer xenografts over 12 days; Figure 29 B represents the change in tumor volume in nude mice with colon cancer xenografts over 12 days; Figure 29 C is a photograph of the tumor tissue in the right axilla of a nude mouse with colon cancer xenograft tumor 12 days after treatment; Figure 29 D represents nude mice with colon cancer xenografts that were sacrificed 12 days after treatment. The dissected tumor tissues were stained with H&E and were divided into the Saline group and the HSNO group (Scalebar: 50 μm). Data are expressed as mean ± standard deviation (n = 6).

[0077] Figure 30 This is confocal imaging of HSNO (High-Speed ​​Nose) sections of tumor tissue from nude mice with colon cancer xenografts; among which, Figure 30 Group A (Saline group): Nude mice with colon cancer xenografts were administered 100 μL of physiological saline via tail vein on days 0, 3, 6, 9, and 12. On day 12, the probe UCNPs-R-PSN (50 mg / mL, 10 μL, physiological saline: dimethyl sulfoxide = 9:1 v / v) was injected intratumorally, followed by administration of 980 nm (2 W / cm). 2 Irradiation was performed continuously for 5 minutes, followed by a 5-minute interval, for a total of 20 minutes. The tumor tissue was then removed and frozen sectioned. Figure 30 Group B was the HSNO group: nude mice with colon cancer xenografts were administered HSNO (1.6 mg / kg, Saline:DMSO = 99:1) via tail vein on days 0, 3, 6, 9, and 12. On day 12, the probe UCNPs-R-PSN (50 mg / mL, 10 μL, physiological saline: dimethyl sulfoxide = 9:1 v / v) was injected intratumorally, followed by administration of 980 nm (2 W / cm). 2 Irradiation was performed continuously for 5 minutes, followed by 5-minute intervals, for a total of 20 minutes. Tumor tissue was then removed and frozen sectioned. Imaging was performed using a laser confocal microscope. Figure 30 C is Figure 30 A magnified view of a portion of A; Figure 30 D is Figure 30 A magnified view of part B; Figure 30 E represents the fluorescence intensity of tumor sections in the Saline and HSNO groups; excitation wavelength was 561 nm, and emission wavelength was 600 nm–700 nm; data are expressed as mean ± standard deviation (n = 3), ***P < 0.001. Detailed Implementation

[0078] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0079] I. Implementation Methods

[0080] 1 Materials and Instruments

[0081] 1.1 Solution Preparation

[0082] (1) Preparation of probe UCNPs-R-PSN solution: Dissolve UCNPs-R-PSN (20.0 mg) in 1.0 mL of anhydrous DMSO solution to prepare a probe solution of 20 mg / mL, and store it in a -20℃ refrigerator protected from light.

[0083] (2) Preparation of GSNO stock solution: Weigh GSNO (11.37 mg, 0.034 mmol / L), 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℃.

[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. Then dilute the stock solution to a 300 μM solution for later use.

[0085] (4) Preparation of HSNO stock solution: Under light-protected, room temperature, and nitrogen protection conditions, place freshly prepared 1 mM GSNO solution and 0.3 mM Na2S solution in PBS buffer (50 mM, pH = 7.4) to obtain a 300 μM HSNO stock solution. Then, dilute the stock solution to 100 μM and use 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 to 1.0 mM and 10.0 μM solutions for later use.

[0087] (6) Preparation of L-cysteine ​​(L-Cys) stock solution: Add Cys (6.05 mg, 0.05 mmol) to deionized water (10.0 mL) to prepare a 5.0 mM stock solution. Then dilute the stock solution 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 10 mL of 0.01 M sodium hydroxide solution 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), and 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 the 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 t BuOOH (5mM).

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

[0093] (12) 1 Preparation of O2 stock solution: Methylene blue (31.98 mg, 0.10 mmol) was irradiated with an LED lamp, and 5 mL of NaClO4 (1 mM) solution and 5 mL of H2O2 (30%) solution were added to prepare a 10 mM stock solution, which was prepared and used immediately.

[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, which should be prepared and used immediately.

[0095] (14) NO3 - Preparation of stock solution: Add NaNO3 (8.50 mg, 0.1 mmol) to deionized water (10 mL) to prepare a 10 mM NO solution. 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 10 mM NO solution. 2- Stock solution.

[0097] (16)ONOO - Preparation of the stock solution: Add NaNO₂ (0.6 M, 10 mL) and H₂O₂ (0.7 M, 10 mL) to deionized water and stir vigorously. Add HCl (0.6 M, 10 mL) at 0 °C, then quickly add NaOH solution (1.5 M, 20 mL). Determine the ONOO₂ concentration by UV analysis. - The concentration of , with a molar extinction coefficient of 1670 M -1 cm -1 The calculation formula is c = A / (bε).

[0098] In addition to the substances mentioned above, other stock solutions may be prepared using 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 prepared by directly dissolving them in deionized water.

[0099] 1.2 Cells

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

[0101] 1.3 Experimental Animals

[0102] The strain is a healthy male Balb / C nude mouse, weighing between 20-25g, sourced from Changzhou Cavens Laboratory Animal Co., Ltd.

[0103] 2. Method

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

[0105] NaYF4:Yb / Tm@NaYF4 core-shell nanomaterials were synthesized by high-temperature organic thermal decomposition. Subsequently, silica and (3-aminopropyl)triethoxysilane were coated on the surface of NaYF4:Yb / Tm@NaYF4 to obtain amino-functionalized upconversion nanoparticles UCNPs@SiO2-NH2, which served as carriers for near-infrared photoactivation and coupling fluorescent probes. (2) o-nitrobenzyl derivatives were selected as photoactivation protecting groups, and rhodamine derivatives were selected as near-infrared fluorescent groups. o-mercaptobenzoic acid and o-phenylenediamine were selected as recognition groups for HSNO. After esterification, nucleophilic addition elimination, and nucleophilic substitution reactions, photoactivation fluorescent probes R-PSN were synthesized. (3) The photoactivation fluorescent probes R-PSN were coupled to upconversion nanoparticles UCNPs by amide condensation reaction to construct a near-infrared photoactivation fluorescent probe UCNPs-R-PSN based on UCNPs.

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

[0107] (1) Synthesis of NaYF4:Yb / Tm nuclear nanoparticles: ① Weigh YCl3 . 6H2O (241.00mg, 0.79mmol), YbCl3 . 6H2O (77.00mg, 0.20mmol), TmCl3 . ① 1.910 mg (0.005 mmol) of 6H₂O was added to a 100 mL three-necked flask and stirred slowly until homogeneous. Then, 7 mL of oleic acid (OA) and 15 mL of octadecene (1-ODE) were added. Under a nitrogen purging stream, the resulting mixture was heated to 150 °C and held for 1 h to remove low-boiling-point solvents, yielding a pale yellow clear solution. ② After the pale yellow clear solution cooled naturally to 20-30 °C, 10 mL of anhydrous methanol solution containing 100.00 mg (2.50 mmol) of NaOH and 148.00 mg (4.00 mmol) of NH₄F was quickly added, and the mixture was stirred at 30 °C for 1 h. ③ Under a nitrogen purging stream, the temperature was further increased to 90 °C and held for 1 h to remove anhydrous methanol. ④ Under a circulating cooling water system, the obtained mixed solution was rapidly heated to 300℃ at a rate of 15℃ / min and maintained at 300℃ for 1 hour to obtain a yellow solution. ⑤ After the clear yellow solution cooled naturally to 20-30℃, anhydrous ethanol was added, and the mixture was centrifuged at 10000 rpm / min for 5 minutes to obtain the centrifuged product. ⑥ A 1:1 volume ratio of cyclohexane and anhydrous ethanol was added to the obtained product, and the mixture was ultrasonically washed for 5 minutes and centrifuged at 10000 rpm / min for 5 minutes to obtain a precipitate. The washing and centrifugation steps were repeated three times to obtain a white solid, which was then dispersed in 10 mL of cyclohexane.

[0108] (2) Synthesis of rare earth nanoparticles with core-shell structure of NaYF4:Yb / Tm@NaYF4: ① Weigh YCl3 . 6H2O (303.00 mg, 1.00 mmol) was added to a 100 mL three-necked flask and stirred slowly until homogeneous. Then, 7 mL of oleic acid (OA) and 15 mL of octadecene (1-ODE) were added. The resulting mixed solution was heated to 150 °C and kept at this temperature for 1 h under a nitrogen stream to remove low-boiling-point solvents, resulting in a pale yellow clear solution. ② After the pale yellow clear solution cooled naturally to 20-30 °C, 10 mL of the cyclohexane solution of NaYF4:Yb / Tm nanoparticles obtained in step (1) was added to the above pale yellow clear solution. The solution was stirred at 30 °C for 30 min, then heated to 90 °C and kept at this temperature for 1 h to remove cyclohexane. ③ After the cyclohexane is completely removed, allow the mixed solution to cool naturally to 20-30℃, then quickly add 10 mL of anhydrous methanol solution containing NaOH (100.00 mg, 2.50 mmol) and NH4F (148.00 mg, 4.00 mmol), and stir at 30℃ for 1 h. ④ Under a nitrogen purging stream, continue heating to 90℃ and hold for 1 h to remove anhydrous methanol. ⑤ Under a circulating cooling water system, rapidly heat the resulting mixed solution to 300℃ at a rate of 15℃ / min and maintain the reaction at 300℃ for 1 h to obtain a yellow solution. ⑥ After the clear yellow solution cools naturally to 20-30℃, add anhydrous ethanol, and centrifuge at 10000 rpm / min for 5 min to obtain the centrifuged product. ⑦ Add a 1:1 volume ratio of cyclohexane and anhydrous ethanol to the obtained product, sonicate for 5 min, centrifuge at 10000 rpm / min for 5 min to obtain the product precipitate, repeat the washing and 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. Heat the solution to 60 °C until it becomes clear and transparent. ② Add 1 mL of cyclohexane solution containing 20.00 mg NaYF4:Yb / Tm@NaYF4 to the CTAB solution. Stir the resulting mixture at 1000 rpm / min for 12 h at 30 °C until the cyclohexane is completely evaporated, forming a clear and transparent NaYF4:Yb / Tm@NaYF4-CTAB solution. ③ Add 3 mL of ethanol and 150 μL of 2 M sodium hydroxide solution to the resulting clear and transparent solution. Stir the mixture at 1000 rpm / min until homogeneous. Heat the solution to 70 °C. After the temperature stabilizes, add 60 μL of tetraethyl orthosilicate (TEOS) dropwise in increments of 5 μL. Continue stirring for 2 h. ④ After the reaction, the reaction solution was quickly collected and washed with anhydrous ethanol by centrifugation at 10,000 rpm / min for 5 min. This operation was repeated 5 times. ⑤ The surfactant CTAB was removed using a rapid and efficient ion exchange method. The precipitate was resuspended in 30 mL of anhydrous ethanol and NaCl (6.00 g, 0.10 mol) was added. The mixture was stirred at 1000 rpm / min for 6 h at 60 °C. After the reaction, the reaction solution was allowed to stand for 10 min, the supernatant was collected, and washed with anhydrous ethanol by centrifugation at 10,000 rpm / min for 5 min. This operation was repeated 5 times. Finally, SiO2-encapsulated upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2 (UCNPs-SiO2) were obtained.

[0110] (4) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2-NH2: 10.00 mg of UCNPs-SiO2 (NaYF4:Yb / Tm@NaYF4@SiO2) was weighed and dissolved in 10 mL of anhydrous ethanol. (3-aminopropyl)triethoxysilane (20 μL) was added dropwise, and the resulting mixture was rapidly stirred at 70 °C for 3 h. After the reaction, the mixture was washed with anhydrous ethanol by centrifugation at 10000 rpm / min for 5 min. This process was repeated 5 times. Finally, amino-functionalized upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2-NH2 (UCNPs-SiO2-NH2) were obtained.

[0111] 2.1.2 Synthetic route of compound R-PSN

[0112] (1) 4-Bromomethyl-3-nitrobenzoic acid was selected as the starting material. Compound a was esterified with tert-butanol to obtain compound T-1. Compound T-1 and o-mercaptobenzoic acid were then subjected to a nucleophilic substitution reaction to obtain compound T-2. (2) 4-Diethylaminoketo acid and 1,6-dihydroxynaphthalene were selected as the starting materials. Compound R-2 was obtained through a condensation reaction. Compound R-2 was then reacted with compound T-1 through an amide condensation reaction to obtain compound R-3. Compound R-3 was condensed with oxalyl chloride and o-phenylenediamine to obtain compound R-4. Compound R-4 was deprotected with trifluoroacetic acid to generate 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, followed by anhydrous magnesium sulfate (1.81 g, 15.00 mmol). The mixture was stirred at 30 °C for 15 min, then 4-bromomethyl-3-nitrobenzoic acid (1.00 g, 3.75 mmol) and tert-butanol (2.00 mL, 18.75 mmol) were added. The mixture was 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, followed by washing with dichloromethane (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a brown oily substance. Purification was performed by silica gel column chromatography (silica, PE:EtOAc, 3:1 v / v) to give 850.00 mg of white solid compound T-1, yield 71.95%. 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, followed by the addition of anhydrous potassium carbonate (439.20 mg, 3.17 mmol), and then a catalytic amount of DMF (2 mL). The mixture was 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 give a pale yellow solid. Purification was performed by silica gel column chromatography (silica, DCM:MeOH, 20:1 v / v) to give 905.00 mg of white solid compound T-2, with a yield of 86.49%.f =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. The resulting mixture was stirred at 90 °C for 8 h. The reaction solution was naturally cooled to 20-30 °C and then transferred to ice water with thorough stirring. Finally, the mixture was filtered and the residue was collected to obtain a dark red solid. Purification was performed by silica gel column chromatography (silica, DCM:MeOH, 30:1 v / v) to give 2.40 g of red solid compound R-2, yield 85.95%. 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 reacted with the solution in an ice-water bath for 5 min. Then, compound R-2 (1.20 g, 2.75 mmol) was added and the mixture was 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 give 1.47 g of dark red solid compound R-3, in 66.16% yield.f =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. Oxaloyl chloride (695.00 mg, 5.48 mmol) was then added dropwise, and the mixture was 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 a 15 mL anhydrous dichloromethane solution containing o-phenylenediamine (590.00 mg, 5.46 mmol) and triethylamine (550.00 mg, 5.46 mmol). The reaction was carried out 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 oily substance. Purification was performed by silica gel column chromatography (silica, DCM:MeOH, 80:1 v / v) to give 860.00 mg of a pale yellow solid, compound R-4, in 52.62% yield. 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, and trifluoroacetic acid (2 mL) was added dropwise. The mixture was stirred for 3 h in the dark under ice-water bath conditions. 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 give 340.00 mg of pale 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. See related spectra for details. Figure 4-6 .

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

[0120] Synthesis of the 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), and HOBt (8.00 mg, 0.059 mmol) were dissolved in 8 mL of anhydrous DMF and stirred in the dark at 20-30 °C under nitrogen protection for 5 min. Then, DIPEA (11.00 μL, 0.06 mmol) was added to the reaction solution and stirred in the dark at 20-30 °C for 12 h. After the reaction was complete, the reaction was quenched with deionized water and centrifuged at 10000 rpm / min for 10 min. The resulting precipitate was washed with anhydrous ethanol by centrifugation at 10000 rpm / min for 10 min, and the above operation was repeated 5 times. Finally, a pale yellow solid UCNPs-R-PSN was obtained.

[0121] 2.2 Mechanism verification of UCNPs-R-PSN for identifying HSNO

[0122] Under light-protected conditions, the probe UCNPs-R-PSN (final concentration 0.1 mg / mL) was first incubated at 980 nm (3 W / cm²). 2 The sample was irradiated for 10 min, then incubated with HSNO (final concentration 300 μM) in PBS (pH = 7.4, 50 mM, 1 mL) buffer at 37 °C for 10 min. Extraction was performed with ethyl acetate (3 × 1 mL), the organic phase was concentrated under reduced pressure, and the product was separated. The reaction product was then confirmed by high-resolution mass spectrometry to verify the mechanism by which the probe UCNPs-R-PSN recognizes HSNO.

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

[0124] The loading of compound R-PSN on 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-Vis spectrophotometer. Then, a standard curve of R-PSN at a wavelength of 267 nm was fitted.

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

[0126]

[0127] 2.4 Fluorescence Spectroscopy Measurement

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

[0129] Fluorescence spectrophotometer detection conditions: For the reaction of probe UCNPs-R-PSN with 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, and the emission spectral range was set to 600-750 nm. The photomultiplier tube voltage 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 is passed through 980nm (3W / cm). 2The sample was irradiated and then incubated for 10 min 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. The fluorescence intensity was then measured. The linear equation between the fluorescence intensity and the HSNO concentration was calculated. The detection probe UCNPs-R-PSN was detected only at 980 nm (3 W / cm²). 2 The fluorescence intensity was measured 10 times in PBS buffer (containing no HSNO) after irradiation, and the standard deviation of the 10 results was calculated. The limit of detection (LOD) was calculated as: LOD = 3σ / k; where σ is the standard deviation of the blank sample, and k is the slope of the linear equation between 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. Cells were placed in a cell culture incubator and cultured at 37°C and 5% CO2. Cells were passaged every two days, and cells were transplanted into new culture dishes at a ratio of 1:3.

[0134] 2.7 CCK8 assay for the cytotoxicity of probe UCNPs-R-PSN

[0135] Preparation of cell suspension: Collect cells from T25 cell culture flasks, and dilute an appropriate amount of cells to a suitable concentration (1×10⁻⁶). 5 (cells / mL).

[0136] The prepared cell suspension was prepared at approximately 1 × 10⁻⁶ cells per well. 4 Cells were seeded into 96-well plates with a volume of 100 μL per well, and each group had three parallel replicate wells. The 96-well plates were then placed in a cell culture incubator and cultured for 24 h to promote full cell adhesion and proliferation.

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

[0138] After co-incubation for 48 hours, discard the original culture medium in each well. Under light-protected conditions, gently add serum-free medium (McCOY's 5A medium) containing 10% CCK-8 enhanced solution to each well, handling carefully to prevent air bubble formation. Then, wrap the plate in aluminum foil and incubate for another 30 minutes. Finally, measure the absorbance at 450 nm using a microplate reader.

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

[0140] As: Absorbance of the experimental wells (containing probes, CCK8, and cell culture medium);

[0141] Ac: Absorbance of the control well (containing CCK8 and cell culture medium, but without probe);

[0142] Ab: Blank wells (containing no cells, only the absorbance of CCK8 and cell culture medium).

[0143] 2.8 Cell-level fluorescence imaging

[0144] HCT116 cells were collected from T25 cell culture flasks, and an appropriate amount of cells was diluted to a suitable concentration to prepare a cell suspension. The prepared cell suspension was then diluted with approximately 1×10⁻⁶ cells / mL. 5 The cells were seeded into a confocal microplate and cultured.

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

[0146] The cells were divided into six groups: ① probe+cell group: HCT116 cells were co-incubated with the probe UCNPs-R-PSN (final concentration 0.1 mg / mL, 5 μL DMSO) for 30 min. ② probe+cell+HSNO group: HCT116 cells were co-incubated with the probe UCNPs-R-PSN (final concentration 0.1 mg / mL, 5 μL DMSO) for 30 min, followed by incubation with 100 μM HSNO for 10 min. ③ probe+cell+980nm light: HCT116 cells were co-incubated with the probe UCNPs-R-PSN (final concentration 0.1 mg / mL, 5 μL DMSO) for 30 min, followed by incubation with 980nm light (1 W / cm²). 2④ AOAA+probe+cell+980nmlight: First, incubate cells with AOAA (20μM) for 45min, then incubate with probe UCNPs-R-PSN (final concentration 0.1mg / mL, 5μL DMSO) for 30min, and then administer 980nm (1W / cm²) light. 2 ⑤ Cell + Probe + Na2S + 980nm Light: First, incubate HCT116 cells with the probe UCNPs-R-PSN (final concentration 0.1mg / mL, 5μL DMSO) for 30min, then add 150μM Na2S and incubate for another 10min. Finally, irradiate with 980nm (1W / cm²) light. 2 ⑥ Irradiate continuously for 10 min. ⑥ Cell + Probe + GSNO + 980nm Light: First, co-incubate HCT116 cells with the probe UCNPs-R-PSN (final concentration 0.1 mg / mL, 5 μL DMSO) for 30 min, then add 1 mM GSNO and co-incubate for 10 min. Finally, irradiate with 980nm light (1 W / cm²). 2 ⑦ Irradiate continuously for 10 min. ⑦ Cell + probe + HSNO + 980nm light: First, co-incubate HCT116 cells with the probe UCNPs-R-PSN (final concentration 0.1 mg / mL, 5 μL DMSO) for 30 min, then add 100 μM HSNO and co-incubate for 10 min. Finally, irradiate with 980nm light (1 W / cm²). 2 Irradiate continuously for 10 minutes. Rinse the confocal dish three times with PBS buffer before imaging. A Leica STELLARIS 5 laser confocal microscope (63× oil immersion) was used for imaging.

[0147] The excitation wavelength for channel 1 was 561 nm, and the emission wavelength range was set to 600-750 nm. Analysis was performed using Leica software. 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 20-25g, provided by Changzhou Cavens Laboratory Animal Co., Ltd. The animal experimental protocol was approved by the Animal Protection and Use Committee of Xuzhou Medical University and complies with relevant Chinese laws. The nude mice were allowed one week to acclimatize to the environment before the experiment. Under natural diurnal light conditions, they were housed in separate cages, with the ambient temperature maintained at 22±2℃ and humidity at 50±10%, and free access to food and water.

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

[0151] HCT116 cells were cultured in T75 flasks until they reached the logarithmic growth phase, then digested with trypsin and transferred to serum-free McCoy's 5A medium, resulting in a cell suspension with a concentration of approximately 4 × 10⁻⁶. 7 Cells / mL. Next, select 4-6 week old, 20-25g Balb / C nude mice, and administer approximately 4 × 10⁶ cells / mL. 6 One cell (100 μL cell suspension) was inoculated subcutaneously in the middle and posterior part of the left upper limb axilla of nude mice.

[0152] 2.10 Fluorescence imaging of a nude mouse xenograft model

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

[0154] Imaging was performed using the LB983 NightOWL II LB983 small animal live imaging system. Imaging conditions: excitation wavelength 580nm, emission wavelength 610nm. Image and data analysis were performed using Living Image software.

[0155] 2.11 Data Processing

[0156] Each experimental data set was measured at least three times. Final results were expressed as mean ± standard deviation (Mean ± SD) and statistically analyzed using SPSS 16.0 software. One-way ANOVA with a completely randomized design was used to compare differences among multiple groups. P < 0.05 was considered statistically significant.

[0157] Preparation and characterization of 3 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+ Rare-earth upconversion nanoparticles, NaYF4:Yb / Tm, with luminescence centers were synthesized. The synthesized NaYF4:Yb / Tm nanoparticles were then characterized by transmission electron microscopy (TEM), fluorescence spectroscopy, and X-ray diffraction (XRD). Figure 7 A and Figure 7 As shown in Figure B, the transmission electron microscopy (TEM) image reveals that NaYF4:Yb / Tm consists of monodisperse, uniformly sized, and regularly shaped spherical nanoparticles with an average particle size of 30.24 ± 0.22 nm. The fluorescence spectrum of NaYF4:Yb / Tm was measured under 980 nm near-infrared light excitation. Figure 7 As shown in Figure C, the synthesized nanoparticles exhibit strong fluorescence emission peaks at 365 nm, 450 nm, and 475 nm. X-ray diffraction (XRD) characterization of NaYF4:Yb / Tm indicates that ( Figure 7 D) The spectrum of the nanoparticles has a flat baseline and narrow half-peak width, which perfectly matches the standard PDF card (PDF#28-1192) of NaYF4:Yb / Tm and has no impurity peaks, indicating that the synthesized nanoparticles have high crystallinity. Based on the above results, this invention successfully constructed NaYF4:Yb / Tm core nanoparticles.

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

[0161] First, NaYF4 was encapsulated on the outside using epitaxial growth to prepare NaYF4:Yb / Tm@NaYF4 nanoparticles with a core-shell bilayer 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 indicate that the synthesized NaYF4:Yb / Tm@NaYF4 consists of monodisperse, uniformly sized, and regularly shaped short rod-shaped nanoparticles with an average particle size of 39.89 ± 0.31 nm. The diameter of the NaYF4:Yb / Tm@NaYF4 nanoparticles is significantly increased compared to NaYF4:Yb / Tm, indicating the successful construction of NaYF4:Yb / Tm@NaYF4 nanoparticles with a core-shell bilayer structure.

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

[0163] First, silica-encapsulated NaYF4:Yb / Tm@NaYF4 nanoparticles were prepared using an organic template method to obtain UCNPs@SiO2 (NaYF4:Yb / Tm@NaYF4@SiO2) nanoparticles. Then, the surface was aminated using an aminosilane coupling agent to obtain NaYF4:Yb / Tm@NaYF4@SiO2-NH2 (UCNPs-SiO2-NH2). Finally, a photoactivated fluorescent probe R-PSN was loaded onto the UCNPs surface via an amide condensation reaction to obtain the final product, UCNPs-R-PSN nanoparticles. Figure 9 A shows the TEM characterization results of UCNPs-R-PSN nanoparticles, revealing a distinct core-shell layered structure with an average particle size of 53.90 ± 0.54 nm. Figure 9 B). Zeta potential results showed that the SiO2-modified UCNPs surface exhibited a negative charge (-28.32 mV) due to the presence of surface hydroxyl groups. Subsequent APTES modification resulted in a significantly greater number of amino groups than hydroxyl groups on the UCNPs-SiO2-NH2 surface, leading to a positive charge (+14.93 mV). Further coupling with the compound R-PSN yielded UCNPs-R-PSN, which showed a significant increase in its Zeta potential. Figure 9 C). The above experimental results demonstrate that compound R-PSN was successfully coupled to the surface of NaYF4:Yb / Tm@NaYF4@SiO2-NH2.

[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 ultraviolet absorption spectrum of the probe UCNPs-R-PSN before and after the reaction with HSNO were investigated. For example... Figure 10As shown, the probe UCNPs-R-PSN itself has no absorption peak between 450-550 nm; while the probe UCNPs-R-PSN first passes through 980 nm (3W / cm). 2 After irradiation for 10 min, and after the reaction with HSNO was complete, the maximum ultraviolet absorption peak appeared at 545 nm, which is basically consistent with the ultraviolet absorption peak of compound R-2. This further indicates that compound R-PSN is loaded onto the surface of upconversion nanomaterials, and the mechanism of fluorescence generation by the reaction of probe UCNPs-R-PSN with HSNO 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 compound R-PSN on the surface of upconversion nanoparticles, the loading performance of the upconversion nanoparticles was investigated. Figure 11 In diagram A, the black line represents the UV-Vis absorption spectrum of compound R-PSN (65 μM), and the red line represents the reaction supernatant obtained after loading R-PSN (65 μM) onto the surface of upconversion nanoparticles via amide condensation and centrifugation (10000 rpm / min, 5 min). The supernatant contains unreacted R-PSN, and its absorbance value was measured by UV-Vis absorption spectroscopy. Compared to before the amide condensation reaction, the absorbance of R-PSN in the reaction supernatant at 267 nm decreased significantly. Figure 11 As shown in Figure B, the UV absorbance of compound R-PSN in the reaction supernatant after centrifugation is 0.967. Substituting this into the standard curve of R-PSN at 267 nm, the concentration of R-PSN in the loaded solution is calculated to be 29.02 μM; the initial concentration of R-PSN in the reaction solution is 65 μM. Therefore, based on the change in R-PSN concentration in the supernatant before and after the reaction, the loading amount of R-PSN on the upconversion nanoparticles can be determined to be 55.35%.

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

[0170] To investigate the response performance of the probe UCNPs-R-PSN to HSNO, such as Figure 12As shown, the UCNPs-R-PSN probe itself exhibits almost no fluorescence. When irradiated only at 980 nm without incubation with HSNO, the UCNPs-R-PSN probe shows almost no fluorescence. Only after irradiation at 980 nm and subsequent incubation with HSNO does the UCNPs-R-PSN probe exhibit a significantly enhanced fluorescence signal at 645 nm. These results indicate that after irradiation at 980 nm, incubation with HSNO by the UCNPs-R-PSN probe produces a significantly enhanced fluorescence signal.

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

[0172] The response of the probe UCNPs-R-PSN to HSNO was further investigated. For example... Figure 13 As shown, the probe UCNPs-R-PSN itself does not exhibit fluorescence. After irradiation at 980 nm, and incubation with different concentrations of HSNO (0-80 μM), the fluorescence intensity at 645 nm gradually increases. Within the HSNO concentration range of 0-2 μM, the fluorescence intensity shows a good linear relationship with the HSNO concentration. Furthermore, in PBS buffer, the detection limit of the probe is 142 nM, indicating that the probe UCNPs-R-PSN possesses high detection sensitivity.

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

[0174] This invention further explores the reaction time for incubating the probe UCNPs-R-PSN with HSNO. For example... Figure 14 As shown, after irradiation at 980 nm, the fluorescence intensity reached its peak after incubation with HSNO (100 μM) for approximately 10 min. Furthermore, the fluorescence intensity did not significantly decrease after incubation for 90 min, indicating the stability of the generated fluorescence response. These results demonstrate that the fluorescence response generated by the probes UCNPs-R-PSN and HSNO exhibits good stability and is suitable for long-term imaging.

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

[0176] To investigate the effect of different pH environments on the reaction between UCNPs-R-PSN and HSNO, the probe was first passed through a 980nm (3W / cm) probe. 2Irradiate for 10 min, then incubate with HSNO (100 μM) in PBS buffers at 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). Figure 15 As shown, in the absence of HSNO, the probe is relatively stable at pH 4.0–9.0, exhibiting no fluorescence emission. When the probe reacts with HSNO, the fluorescence signal decreases slightly with increasing pH. This is presumably because the SN single bond in HSNO is more stable under acidic conditions, while it is more easily decomposed into nitrous acid and hydrogen sulfide under neutral and alkaline conditions. These experimental results suggest that the UCNPs-R-PSN probe is suitable for the detection of HSNO under physiological conditions (pH = 7.4).

[0177] 4.7 Selectivity of HSNO detection by probe UCNPs-R-PSN

[0178] Considering the complex environment within organisms, high selectivity of probes for analytes is crucial for accurate detection. To verify the selectivity of the probe UCNPs-R-PSN for HSNO, the probe UCNPs-R-PSN was first subjected to 980nm (3W / cm²) light. 2 Irradiate for 10 minutes, then react 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), and 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 + ), reactive sulfur (Na2S, Na2S2, NaHS, Na2SO3, NaHSO3, CH3SSSCH3, CysSSCys, GSSG, S8, GSH), reactive oxygen (H2O2, ClO) - , t BuOOH、 · OH、 1 O2, O2 ·- ), reactive nitrogen (NO3)- NO2 - ONOO - After incubation with DEA·NONOate and other interfering substances (L-AA, DHA, MGO) for 10 min, the fluorescence intensity was measured. Only after irradiation at 980 nm and incubation with HSNO did the probe UCNPs-R-PSN produce a significantly enhanced fluorescence signal, while it showed almost no response to other substances. Figures 16-19 The above experimental results demonstrate that the probe UCNPs-R-PSN exhibits good selectivity for HSNO and is not affected 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 assay. Human colon cancer cells HCT116 were selected and co-incubated with different concentrations of the probe UCNPs-R-PSN (0, 50, 100, 200, 400, 500 μg / mL) for 48 h. The results are as follows: Figure 20 As shown, the cell viability remained above 85%, indicating that the probe had low cytotoxicity.

[0182] To observe the changes in cell viability over time after incubation with the probe, cell viability was further examined after incubation with the probe (final concentration 100 μg / mL) for different times (0, 6, 12, 24, 48 h). Figure 21 As shown, after 48 hours of incubation with the probe, the cell survival rate was still above 90%, indicating that the probe had low toxicity to cells and had little impact on cell survival.

[0183] 5.2 Fluorescence imaging of exogenous HSNO in cells

[0184] The high sensitivity and good selectivity of the probe UCNPs-R-PSN for HSNO were successfully verified through in vitro experiments. This article further explores the fluorescence imaging performance of the probe for intracellular HSNO. First, no fluorescence signal was observed when the probe UCNPs-R-PSN was co-incubated with cells for only 10 min. Figure 22 A). Subsequently, the probe was incubated with cells for 10 min before HSNO (100 μM) was added, and no fluorescence signal was observed again. Figure 22 B). When the probe was co-incubated with cells and then exposed to 980nm light, only a weak red fluorescence appeared in the cells. Figure 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 as the source of HSNO) was introduced into cells that had been co-incubated with the probe, followed by 980 nm illumination, a significant increase (100-fold) in intracellular red fluorescence signal was observed. Figure 22 D). In contrast, when the probe was incubated with cells before adding Na2S (150 μM) or GSNO (1 mM) and then irradiated with 980 nm light, only weak fluorescence signals were observed in both groups. Figure 22 E and Figure 22 F). This indicates that the probe can effectively detect exogenous HSNO without interference from Na2S and GSNO. Figure 23 yes Figure 22 The corresponding bright-field cell images show that the cell morphology remained normal during confocal imaging. This demonstrates that the probe can detect exogenous HSNO in living cells.

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

[0186] Thionidium thionitrite, as a significant crosstalk product of H2S and NO, is typically regulated by both 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 crucial role in the generation of endogenous HSNO in cells. When the CBS gene is knocked out, intracellular H2S levels significantly decrease, leading to a decline in endogenous HSNO levels. This invention uses CBS-siRNA transfection of HCT116 cells to reduce CBS protein expression, thereby reducing the content of endogenous HSNO. Figure 24 As shown in G, compared with the control group, CBS protein expression was significantly reduced in HCT116 cells transfected with CBS-siRNA. Figure 24 A and Figure 24 As shown in Figure D, no fluorescence signal was observed when the probe UCNPs-R-PSN was co-incubated with HCT116 cells or HCT116 cells transfected with CBS-siRNA. Figure 24 B and Figure 24 As shown in E, compared with HCT116 cells irradiated at 980 nm, the fluorescence intensity of CBS-siRNA transfected cells after incubation with the probe was significantly reduced after irradiation at 980 nm, indicating a decrease in intracellular HSNO levels.

[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, cells were pretreated with AOAA (20 μM) for 45 min to inhibit the production of endogenous H2S, thereby inhibiting the reaction between endogenous H2S and NO, reducing the level of HSNO. After incubation with the probe and subsequent 980 nm illumination, the red fluorescence signal was also significantly reduced. Figure 24 C). Explanation Figure 24 The red fluorescence signal in B was induced by HSNO at physiological levels within the cell. These results indicate that the probe can detect both endogenous and physiological levels of HSNO with high sensitivity.

[0188] 6-Probe UCNPs-R-PSN Animal-Level Fluorescence Imaging

[0189] This invention further investigates the imaging performance of the probe UCNPs-R-PSN for HSNO detection at the in vivo level. Experimental results show that mice injected with only the probe UCNPs-R-PSN intratumorally show almost no fluorescence signal. Figure 26 A), mice that underwent intratumoral probe injection followed by 980nm irradiation also showed almost no fluorescence signal. Figure 26 B). Compared with the first two groups, mice that received intratumoral injection of probe UCNPs-R-PSN followed by administration of HSNO (100 μM) and then irradiated at 980 nm showed a significant increase in fluorescence intensity (19-fold). Figure 26 C). For example Figure 27 As shown, after administration of exogenous HSNO, the fluorescence signal in mice gradually increased, reaching a peak at around 25 min, and still exhibiting a strong signal at 120 min. Figure 28 Therefore, the probe UCNPs-R-PSN has good stability and is suitable for detecting HSNO in mice.

[0190] 7. The effect of exogenous HSNO administration on tumor growth

[0191] To evaluate the effect of HSNO on tumor growth, HCT116 tumor-bearing mice were randomly divided into two groups: (1) saline group: 100 μL of saline was administered via tail vein on days 0, 3, 6, 9, and 12; (2) HSNO group: 1.6 mg / kg, Saline:DMSO = 99:1, was administered via tail vein on days 0, 3, 6, 9, and 12. Tumor volume was recorded every 3 days for a 12-day treatment period. Changes in body weight and tumor volume in each group were recorded over the 12 days to assess the treatment effect. Results are as follows: Figure 29As shown in Figure A, during the treatment, the tumor volume in the saline group mice increased rapidly, while the HSNO group showed a significant inhibitory effect on tumor growth. Figure 29 As shown in Figure B, after 12 days of treatment, there was no significant difference in body weight between the HSNO treatment group and the saline group. Subsequently, mice in both the saline and HSNO groups were euthanized, and tumor tissue was removed from the tumor-bearing mice. The excised tumor tissue was weighed and photographed for comparison. Figure 29 C) Simultaneously, the tumor was subjected to H&E pathological staining and frozen section processing. Figure 29 Results showed that the tumor cells in the saline group were densely packed, with deeply stained nuclei exhibiting significant atypia, rich blood vessels, and no necrotic foci. In the HSNO group, numerous cells showed nuclear atypia, an increased nucleocytic-to-cytoplasmic ratio, significant inflammatory cell infiltration, and proliferation of fibrous connective tissue, along with extensive cell necrosis. These results indicate that exogenous HSNO possesses antitumor activity. This study further investigated the performance of the probe in detecting HSNO in tumor sections. Figure 30 As shown, the fluorescence intensity of tumor sections in the saline group was lower, indicating lower HSNO levels. However, after treatment with exogenous HSNO, the fluorescence intensity of the tumor sections increased significantly, suggesting a significant increase in HSNO levels. This preliminarily suggests a correlation between changes in HSNO levels and the occurrence and development of colorectal cancer.

[0192] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions 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 near-infrared light-activated probe for thionitrous acid, characterized in that, The method for preparing the photoactivated probe includes the following steps: (1) Synthesis of NaYF4:Yb / Tm core nanoparticles: YCl3·6H2O, YbCl3·6H2O, TmCl3·6H2O, oleic acid and octadecene were mixed evenly and heated to 140-160℃ to remove low-boiling-point solvents. After cooling to 20-30℃, an anhydrous methanol solution containing NaOH and NH4F was added and stirred for reaction. The temperature was then raised to 80-100℃ to remove anhydrous methanol. The resulting mixture was rapidly heated to 280-320℃ at a rate of 10-20℃ / min and stirred for reaction. After cooling to 20-30℃ again, centrifuged and separated to obtain NaYF4:Yb / Tm core nanoparticles. (2) Synthesis of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles: Oleic acid and octadecene were mixed evenly with YCl3·6H2O, and the temperature was raised to 140-160℃ to remove the low-boiling solvent. After cooling to 20-30℃, a mixed solution of NaYF4:Yb / Tm core nanoparticles obtained in step (1) dispersed in cyclohexane was added. The temperature was raised to 80-100℃ to remove cyclohexane. After cooling to 20-30℃ again, an anhydrous methanol solution containing NaOH and NH4F was added and stirred. The temperature was raised to 80-100℃ to remove anhydrous methanol. The resulting mixture was rapidly heated to 280-320℃ at a rate of 10-20℃ / min and stirred. After cooling to 20-30℃, the mixture was centrifuged and separated to obtain NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles. (3) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2: CTAB was dispersed in deionized water, and a mixed solution of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles obtained in step (2) dispersed in cyclohexane was added. The mixture was stirred to form NaYF4:Yb / Tm@NaYF4-CTAB solution. Ethanol and sodium hydroxide solution were added to the solution, and the mixture was stirred evenly. The temperature was raised to 60-80℃, and tetraethyl silicate was added dropwise. The reaction solution was collected, centrifuged, and separated. The surfactant CTAB was removed from the product by ion exchange. The precipitate was resuspended in anhydrous ethanol and NaCl was added. The temperature was raised to 50-70℃ and the mixture was stirred. The mixture was centrifuged again and separated to obtain SiO2-encapsulated upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2. (4) Synthesis of UCNPs-SiO2-NH2: The NaYF4:Yb / Tm@NaYF4@SiO2 obtained in step (3) was dispersed in anhydrous ethanol, and (3-aminopropyl)triethoxysilane was added dropwise. The reaction was carried out by stirring at 60-80℃, centrifuged, and separated to obtain amino-functionalized upconversion nanoparticles UCNPs-SiO2-NH2. (5) Synthesis of compound T-1: Concentrated sulfuric acid, dichloromethane and anhydrous magnesium sulfate were mixed evenly, and 4-bromomethyl-3-nitrobenzoic acid and tert-butanol were added to it. The mixture was stirred at 25-35℃ 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℃ to prepare compound T-2; (7) Synthesis of compound R-2: Compound R-1 and 1,6-dihydroxynaphthalene were dissolved in methanesulfonic acid and stirred at 80-90℃ 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 and stirred to prepare compound R-3; (9) Synthesis of compound R-4: Compound R-3 was dissolved in dichloromethane, DMF and oxalyl chloride were added and stirred to prepare an intermediate product; the obtained intermediate product was dissolved in dichloromethane, added to an anhydrous dichloromethane solution containing o-phenylenediamine and triethylamine, and stirred to prepare compound R-4. (10) Synthesis of compound R-PSN: compound R-4 was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring 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, then DIPEA was added and stirred again to obtain probe UCNPs-R-PSN. The structural formulas of compounds T-1, T-2, R-1, R-2, R-3, R-4, and R-PSN are shown below:

2. The near-infrared light-activated probe for thionitrous acid according to claim 1, characterized in that, In step (1), the molar ratio of YCl3·6H2O, YbCl3·6H2O and TmCl3·6H2O is 0.7-0.9:0.1-0.3:0.003-0.007; the mass-volume ratio of YCl3·6H2O to oleic acid is 235-245:7 mg / mL; the mass-volume ratio of YCl3·6H2O to octadecene is 235-245:15 mg / mL; the molar ratio of YCl3·6H2O to NaOH is 235-245:100; the molar ratio of YCl3·6H2O to NH4F is 235-245:148; the temperature for removing low-boiling-point solvents is 145-155℃; the temperature for removing anhydrous methanol is 85-95℃; and the temperature is rapidly increased to 290-310℃ at a rate of 15℃ / min.

3. The near-infrared light-activated probe for thionitrous acid according to claim 2, characterized in that, In step (1), the molar ratio of YCl3·6H2O, YbCl3·6H2O and TmCl3·6H2O is 0.79:0.2:0.005; the mass-volume ratio of YCl3·6H2O to oleic acid is 241:7 mg / mL; the molar ratio of YCl3·6H2O to NaOH is 241:100; the molar ratio of YCl3·6H2O to NH4F is 241:148; the temperature for removing low-boiling-point solvents is 150℃; the temperature for removing anhydrous methanol is 90℃; and the temperature is rapidly increased to 300℃ at a rate of 15℃ / min.

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

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

6. The near-infrared light-activated probe for thionitrous acid 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; the mass-to-volume ratio of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles to tetraethyl silicate is 4-6:3 mg / μL; in step (4), the mass-to-volume ratio of NaYF4:Yb / Tm@NaYF4@SiO2 to (3-aminopropyl)triethoxysilane is 1-1.5:2.5 mg / μL.

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

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

5.

9. The near-infrared light-activated probe for thionitrous acid according to claim 8, characterized in that, In step (5), the molar ratio of 4-bromomethyl-3-nitrobenzoic acid to tert-butanol is 1:5; in step (6), the molar ratio of compound T-1 to o-mercaptobenzoic acid is 1:1; in step (7), the molar ratio of compound R-1 to 1,6-dihydroxynaphthalene is 1:

1.

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

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

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

5.

13. The near-infrared light-activated probe for thionitrous acid according to claim 12, characterized in that, In step (11), the mass ratio of compound R-PSN to UCNPs@SiO2-NH2 is 5:1; the molar ratio of compound R-PSN to HBTU is 1:1; the molar ratio of compound R-PSN to HOBt is 1:1; and the molar ratio of compound R-PSN to DIPEA is 1:

1.

14. The method for preparing the near-infrared photoactivated probe of thionitrous acid according to claim 1, characterized in that, Includes the following steps: (1) Synthesis of NaYF4:Yb / Tm core nanoparticles: YCl3·6H2O, YbCl3·6H2O, TmCl3·6H2O, oleic acid and octadecene were mixed evenly and heated to 140-160℃ to remove low-boiling-point solvents. After cooling to 20-30℃, an anhydrous methanol solution containing NaOH and NH4F was added and stirred for reaction. The temperature was then raised to 80-100℃ to remove anhydrous methanol. The resulting mixture was rapidly heated to 280-320℃ at a rate of 10-20℃ / min and stirred for reaction. After cooling to 20-30℃ again, centrifuged and separated to obtain NaYF4:Yb / Tm core nanoparticles. (2) Synthesis of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles: Oleic acid and octadecene were mixed evenly with YCl3·6H2O, and the temperature was raised to 140-160℃ to remove the low-boiling solvent. After cooling to 20-30℃, a mixed solution of NaYF4:Yb / Tm core nanoparticles obtained in step (1) dispersed in cyclohexane was added. The temperature was raised to 80-100℃ to remove cyclohexane. After cooling to 20-30℃ again, an anhydrous methanol solution containing NaOH and NH4F was added and stirred. The temperature was raised to 80-100℃ to remove anhydrous methanol. The resulting mixture was rapidly heated to 280-320℃ at a rate of 10-20℃ / min and stirred. After cooling to 20-30℃, the mixture was centrifuged and separated to obtain NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles. (3) Synthesis of NaYF4:Yb / Tm@NaYF4@SiO2: CTAB was dispersed in deionized water, and a mixed solution of NaYF4:Yb / Tm@NaYF4 core-shell nanoparticles obtained in step (2) dispersed in cyclohexane was added. The mixture was stirred to form NaYF4:Yb / Tm@NaYF4-CTAB solution. Ethanol and sodium hydroxide solution were added to the solution, and the mixture was stirred evenly. The temperature was raised to 60-80℃, and tetraethyl silicate was added dropwise. The reaction solution was collected, centrifuged, and separated. The surfactant CTAB was removed from the product by ion exchange. The precipitate was resuspended in anhydrous ethanol and NaCl was added. The temperature was raised to 50-70℃ and the mixture was stirred. The mixture was centrifuged again and separated to obtain SiO2-encapsulated upconversion nanoparticles NaYF4:Yb / Tm@NaYF4@SiO2. (4) Synthesis of UCNPs-SiO2-NH2: The NaYF4:Yb / Tm@NaYF4@SiO2 obtained in step (3) was dispersed in anhydrous ethanol, and (3-aminopropyl)triethoxysilane was added dropwise. The reaction was carried out by stirring at 60-80℃, centrifuged, and separated to obtain amino-functionalized upconversion nanoparticles UCNPs-SiO2-NH2. (5) Synthesis of compound T-1: Concentrated sulfuric acid, dichloromethane and anhydrous magnesium sulfate were mixed evenly, and 4-bromomethyl-3-nitrobenzoic acid and tert-butanol were added to it. The mixture was stirred at 25-35℃ 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℃ to prepare compound T-2; (7) Synthesis of compound R-2: Compound R-1 and 1,6-dihydroxynaphthalene were dissolved in methanesulfonic acid and stirred at 80-90℃ 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 and stirred to prepare compound R-3; (9) Synthesis of compound R-4: Compound R-3 was dissolved in dichloromethane, DMF and oxalyl chloride were added and stirred to prepare an intermediate product; the obtained intermediate product was dissolved in dichloromethane, added to an anhydrous dichloromethane solution containing o-phenylenediamine and triethylamine, and stirred to prepare compound R-4. (10) Synthesis of compound R-PSN: compound R-4 was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring 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, then DIPEA was added and stirred again to obtain probe UCNPs-R-PSN. The structural formulas of compounds T-1, T-2, R-1, R-2, R-3, R-4, and R-PSN are shown below:

15. The application of the near-infrared light-activated probe for thionitrite according to claim 1 in the detection of thionitrite, wherein the application is for the purpose of non-disease diagnosis or treatment.

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