A hypochlorous acid-responsive long-lasting glow nanoprobe and its preparation method and application

Abstract

The present invention relates to a hypochlorous acid responsive long afterglow nanoprobe and its preparation method and application. The long afterglow nanoprobe comprises long afterglow nanoparticles. The chemical composition general formula of the long afterglow nanoparticles is: Zn 1+x Ga 2‑2x Ge x O4:Cr 3+ y ,Eu 3+ z , where 0.1≤x≤0.5, 0.005≤y≤0.05, and 0.005≤z≤0.05. This hypochlorous acid-responsive long-afterglow nanoprobe has good responsiveness and selectivity, and can sensitively respond to hypochlorous acid in solutions and in the body, producing enhanced afterglow luminescence, which is conducive to precise detection. It also has a high signal-to-noise ratio and can perform low-background, high-response afterglow luminescence imaging detection of hypochlorous acid. It can be applied to the diagnosis or imaging products of rheumatoid arthritis or tumors, and to the study of hypochlorous acid-related physiological or pathological processes. Its response characteristics can also be used in fields such as information encryption.

Description

Technical Field

[0001] The present invention relates to the technical field of long afterglow materials, and in particular to a hypochlorous acid-responsive long afterglow nanoprobe, a preparation method thereof, and applications thereof. Background Art

[0002] Long afterglow materials are materials that absorb and store energy such as visible light, ultraviolet light, and X-rays. They can continue to emit light even after the energy is cut off. They are also called light-storage luminescent materials or luminous materials. Long afterglow materials can store energy in a trap. When the light excitation of the material stops, it is accompanied by the phenomenon of continuous luminescence. Due to this remarkable feature, long afterglow materials have begun to be used in lighting, emergency instructions, light energy storage, detection, transportation, and military fields. At present, the development of afterglow imaging probes mainly includes inorganic materials (such as ZnGa 1.995 Cr 0.005 O4, lanthanide-doped NaYbF4 upconversion nanoparticles) and organic materials such as derivatives of poly [2-methoxy-5- (2-ethylhexyloxy) -1,4-phenylene vinyl] (MEHPPV) .

[0003] CN110041928A discloses a Cr-doped 3+ The zinc gallate-based fluorescent material has good dispersion, excellent uniformity, emission range in the near-infrared light region, and excellent afterglow performance. It not only meets the conditions of long fluorescence lifetime and can be excited in vitro but not excited in vivo, but its fluorescence emission wavelength range is also suitable for the important condition of "near-infrared medical window".

[0004] CN105199732A discloses a near-infrared long afterglow material with dual functions of biological imaging and photothermal therapy, the matrix material of which is La3Ga5GeO 14 or Ca3Ga2Ge3O 12 or Zn3Ga2Ge2O 10 , doped with Cr 3+ As well as lanthanide ions, they can stably emit near-infrared long afterglow. When the long afterglow material is irradiated with an 808nm laser that is harmless to normal human cells, the long afterglow material absorbs the energy of the light and converts it into heat. The temperature rises rapidly, which can kill the cancer cells bound to it.

[0005] Commonly used afterglow luminescence probes require precise matching and construction of multiple response elements and luminescence units, which increases the complexity of the synthesis, reduces the repeatability of the preparation, and limits their application capabilities in practical scenarios.

[0006] Therefore, developing new long-lasting nanoprobes for HClO imaging, which have the advantages of good biosafety, sensitivity, selectivity and easy preparation, has become an urgent problem to be solved. Summary of the Invention

[0007] To address the shortcomings of the prior art, the present invention aims to provide a hypochlorous acid-responsive, long-lasting nanoprobe, its preparation method, and its application. This hypochlorous acid-responsive, long-lasting nanoprobe can sensitively respond to hypochlorous acid in solutions and in vivo, producing enhanced afterglow luminescence, enabling low-background, high-response afterglow luminescence imaging detection of hypochlorous acid.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a hypochlorous acid-responsive long-lasting glow nanoprobe, wherein the long-lasting glow nanoprobe comprises long-lasting glow nanoparticles, and the chemical composition formula of the long-lasting glow nanoparticles is: Zn 1+x Ga 2-2x Ge x O4:Cr 3+ y ,Eu 3+ z , where 0.1≤x≤0.5, 0.005≤y≤0.05, 0.005≤z≤0.05.

[0010] Among them, the specific point values ​​of x can be selected as 0.1, 0.2, 0.3, 0.4, 0.5, etc., the specific point values ​​of y can be selected as 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, etc., and the specific point values ​​of z can be selected as 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, etc.

[0011] The present invention selects Zn 1+x Ga 2-2x Ge x O4 as a matrix structure has a certain rigidity and excellent stability, which can effectively bind the energy of the excited state and reduce the non-radiative transition of energy, thus facilitating the generation of long afterglow. 3 + and Eu3+, as luminescent center ions, can absorb and store energy after being excited. When the excitation source stops, the stored energy can be slowly released to produce long afterglow luminescence.

[0012] The Zn 1+x Ga 2-2x Ge x O4:Cr 3+ y ,Eu 3+ zIt serves as both a light-emitting element and a response element for hypochlorous acid, and can achieve a highly sensitive and specific response to hypochlorous acid without introducing external response elements.

[0013] The present invention defines the ratio of the elements in the matrix structure by 0.1≤x≤0.5, 0.005≤y≤0.05, and 0.005≤z≤0.05. When Ge doping is too low, the crystal structure and band gap cannot be fully adjusted, resulting in reduced energy transfer efficiency and affecting the absorption and storage capacity of the long-lasting nanoparticles for the excitation light energy. When Ge doping is too high, the stability of the crystal structure is destroyed, resulting in excessive defects or lattice distortion in the crystal, increasing energy loss and reducing the long-lasting performance. Moreover, excessive Ge may change the chemical stability of the material, making it more susceptible to chemical reactions under certain circumstances, affecting the service life of the material. When Zn or Ga doping is too high, the distribution and depth of the trap energy levels are changed, interfering with the energy storage and release process. When Zn or Ga doping is too low, the stability of the crystal structure is reduced and the optical band gap of the material is changed, affecting the uniformity and consistency of the material, thereby affecting the long-lasting performance.

[0014] Preferably, the ratio of y to z is 1:(1-0.1), for example, it can be 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1, etc.

[0015] The Cr 3+ and Eu 3+ When doped in the above-mentioned specific ratio, the two can work synergistically to improve the excitation efficiency of the luminescence center, help balance the energy transfer and radiation transition process between the luminescence centers, provide sufficient luminescence centers, and enhance long-lasting luminescence.

[0016] Preferably, the long-afterglow nanoprobe further comprises hyaluronic acid coated on the surface of the long-afterglow nanoparticles, and the hyaluronic acid is adsorbed on the long-afterglow nanoparticles by electrostatic adsorption.

[0017] Hyaluronic acid (HA) is a natural polysaccharide with excellent biocompatibility and biodegradability. The electrostatic adsorption of HA and long-lasting nanoparticles causes the probe's surface potential to become negative, helping to reduce cell rejection of the probe, decrease cell-probe adhesion, and reduce cellular inflammatory responses, thereby improving biocompatibility. HA can also promote cell proliferation, enhancing the compatibility of the nanoprobe in biological systems.

[0018] Preferably, the particle size of the long afterglow nanoprobe is 2 to 200 nm, for example, it can be 2 nm, 10 nm, 20 nm, 40 nm, 60 nm, 80 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, etc.

[0019] In a second aspect, the present invention provides a method for preparing the hypochlorous acid-responsive long-afterglow nanoprobe as described in the first aspect, the preparation method comprising the following steps:

[0020] (1) GeO2 and Zn 2+ Salt, Ga 3+ Salt, Eu 3+ Salt, Cr 3+ The salts are mixed in an aqueous phase in a molar ratio of (0.1-0.5):(1.1-1.5):(1-1.8):(0.005-0.05):(0.005-0.05) to obtain a solution, and then aqueous ammonia is added to adjust the pH value to 7-9;

[0021] (2) heat-treating the solution obtained in step (1) and centrifuging to obtain a precipitate;

[0022] (3) dispersing the precipitate obtained in step (2) in ultrapure water;

[0023] (4) The product obtained in step (3) is subjected to light treatment for 0.1 to 4 hours under a light source with a wavelength of 10 to 700 nm to obtain the hypochlorous acid-responsive long-afterglow nanoprobe.

[0024] Among them, the specific point values ​​in 0.1-0.5 can be selected from 0.1, 0.2, 0.3, 0.4, 0.5, etc., the specific point values ​​in 1.1-1.5 can be selected from 1.1, 1.2, 1.3, 1.4, 1.5, etc., the specific point values ​​in 1-1.8 can be selected from 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, etc., the specific point values ​​in 0.005-0.05 can be selected from 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, etc., and the pH value can be selected from 7, 7.5, 8, 8.5, 9, etc.

[0025] The wavelength of the light source can be selected from 10nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, etc., and the time of light treatment can be selected from 0.1h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, etc.

[0026] The present invention creatively discovered that light treatment for 0.1 to 4 hours under a light source with a wavelength of 10 to 700 nm can improve the specific response performance of the long afterglow nanoprobe to hypochlorous acid, showing an effect of enhanced afterglow luminescence.

[0027] Preferably, the salt in step (1) is independently selected from nitrate or acetate.

[0028] Preferably, step (1) further comprises stirring after adjusting the pH value to 7-9.

[0029] Preferably, the stirring temperature in step (1) is 20-40° C., and the stirring time is 2-6 h.

[0030] The temperature can be selected from 20°C, 25°C, 30°C, 35°C, 40°C, etc., and the time can be selected from 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, etc.

[0031] Preferably, the temperature of the heat treatment in step (2) is 150-250° C., and the heat treatment time is 20-60 h.

[0032] The temperature can be selected from 150° C., 175° C., 200° C., 225° C., 250° C., etc., and the time can be selected from 20 h, 30 h, 40 h, 50 h, 60 h, etc.

[0033] Preferably, the heat treatment in step (2) is carried out in a high pressure sterilizer.

[0034] Preferably, the step (3) further includes washing the precipitate before dispersing the precipitate in ultrapure water.

[0035] Preferably, the washing comprises washing by centrifugation three times with ethanol and ultrapure water.

[0036] Preferably, step (3) further comprises mixing the precipitate with hyaluronic acid in a mass ratio of 1:(2-5), washing, and redispersing in ultrapure water.

[0037] The 1:(2-5) can be, for example, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0038] Preferably, the mixing further includes stirring.

[0039] Preferably, the stirring temperature is 20-40° C., and the stirring time is 10-20 h.

[0040] The temperature can be selected from 20°C, 25°C, 30°C, 35°C, 40°C, etc., and the time can be selected from 10h, 12h, 14h, 16h, 18h, 20h, etc.

[0041] Preferably, the wavelength in step (4) is 200-300 nm, for example, it can be 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, etc.

[0042] In a third aspect, the present invention provides a use of the hypochlorous acid-responsive long-afterglow nanoprobe as described in the first aspect in a diagnosis or imaging product for rheumatoid arthritis or tumors.

[0043] During the pathological process of rheumatoid arthritis, a large number of immune cells, such as neutrophils, are present at the site of inflammation. Upon activation, neutrophils undergo a respiratory burst, producing a variety of reactive oxygen species, including hypochlorous acid. Using this hypochlorous acid-responsive, long-lasting nanoprobe, it is possible to monitor the physiological and pathological processes of the disease.

[0044] In tumor tissue, M1 macrophages secrete myeloperoxidase in host defense, which can catalyze hydrogen peroxide to produce hypochlorous acid. Using the above-mentioned hypochlorous acid-responsive long-afterglow nanoprobe, hypochlorous acid in tumor tissue can be detected to reflect the situation of the tumor tissue site.

[0045] In a fourth aspect, the present invention provides an application of the hypochlorous acid-responsive long-afterglow nanoprobe as described in the first aspect in information encryption processing.

[0046] By utilizing the response characteristics of the long-afterglow nanoprobe to hypochlorous acid, the afterglow luminescence enhancement at the probe position can be observed through hypochlorous acid, thereby displaying the position information, reading the hidden information, and realizing the encryption / decryption processing of the position information.

[0047] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The present invention creatively develops a hypochlorous acid-responsive long-afterglow nanoprobe that can specifically respond to hypochlorous acid and produce enhanced afterglow luminescence with good selectivity, which is conducive to the accurate detection of hypochlorous acid; the electrostatic adsorption of hyaluronic acid and long-afterglow nanoparticles can also improve the biocompatibility of the probe.

[0050] (2) The synthesis steps of the signal-enhanced long-afterglow nanoprobe in the present invention are simple and easy to operate, without the need to introduce external response elements. The prepared nanoprobe has uniform particle size, good stability, and stable chemical properties.

[0051] (3) The hypochlorous acid-responsive long-afterglow nanoprobe developed in the present invention can be used for diagnosis or imaging products of rheumatoid arthritis or tumors, and can also be used for encryption processing of position information, and has a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a flow chart of the preparation of a hypochlorous acid-responsive long-afterglow nanoprobe according to Example 1;

[0053] Figure 2 This is a transmission electron microscopy characterization result of the hypochlorous acid-responsive long-afterglow nanoprobe prepared in Example 1;

[0054] Figure 3 : This is a structural characterization diagram of the hypochlorous acid-responsive long-afterglow nanoprobe prepared in Example 1 (where A is an X-ray diffraction diagram and B is an X-ray photoelectron spectrum diagram);

[0055] Figure 4 Graphs showing the optical performance of the hypochlorous acid-responsive long-lasting nanoprobe prepared in Example 1 (A is an absorption spectrum graph, and B is a fluorescence graph);

[0056] Figure 5 These are morphological characterization diagrams of the hypochlorous acid-responsive long-afterglow nanoprobe prepared in Example 1 and the hypochlorous acid-responsive long-afterglow nanoprobe adsorbed with hyaluronic acid prepared in Example 2 (where A is the particle size characterization result diagram, and B is the zeta potential characterization result diagram);

[0057] Figure 6 Graphs showing the afterglow luminescence performance test results of the hypochlorous acid-responsive long-afterglow nanoprobe prepared in Example 1 (A is a graph showing the afterglow attenuation of the long-afterglow nanoprobe, B is a graph showing repeated activation of the long-afterglow nanoprobe, and C is a quantitative comparison graph showing the afterglow intensity of the long-afterglow nanoprobe after repeated activation);

[0058] Figure 7 Graphs showing the hypochlorous acid response performance verification results of the hypochlorous acid-responsive long-afterglow nanoprobe prepared in Example 1 (A is a comparison of PLNPs fluorescence change and afterglow luminescence enhancement, B is a comparison of the response selectivity of different experimental groups to the long-afterglow nanoprobe, and C is a quantitative comparison of the afterglow signal enhancement of different experimental groups to the long-afterglow nanoprobe);

[0059] Figure 8 This is a flow chart of the rheumatoid arthritis position imaging experiment in Application Example 1;

[0060] Figure 91. The results of the rheumatoid arthritis position imaging experiment in Application Example 1 (A is the afterglow luminescence imaging image of the rheumatoid arthritis position imaging experiment, and B is the afterglow intensity quantification comparison image of the rheumatoid arthritis position imaging experiment);

[0061] Figure 10 This is a flow chart of the tumor resection guidance experiment in Application Example 2;

[0062] Figure 11 This is the afterglow luminescence imaging image of the resection process of normal mice and hepatocellular carcinoma mice in the tumor resection guidance experiment of Application Example 2;

[0063] Figure 12 This is a comparison of afterglow luminescence imaging of normal liver tissue and tumor tissue in the tumor resection guidance experiment of Application Example 2 (where A is the afterglow luminescence imaging image and B is the afterglow intensity quantification comparison image);

[0064] Figure 13 This is a schematic diagram of position processing for different experimental groups in the hidden information reading experiment of Application Example 3;

[0065] Figure 14 These are the results of the hidden information reading experiment in Application Example 3 (A is the afterglow luminescence imaging image of Group I, B is the afterglow luminescence imaging image of Group II, C is the afterglow intensity quantification comparison image of Group I, D is the afterglow intensity quantification comparison image of Group I after hypochlorous acid treatment, E is the afterglow intensity quantification comparison image of Group II, and F is the afterglow intensity quantification comparison image of Group II after hypochlorous acid treatment). DETAILED DESCRIPTION

[0066] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0067] Example 1

[0068] Preparation of hypochlorous acid-responsive long-lasting glow nanoprobes

[0069] A mixture of Zn(OAc)2 aqueous solution (2.2 mL, 0.5 M), Ga(NO3)3 aqueous solution (3.6 mL, 0.5 M), GeO2 ammonia solution (1 mL, 0.1 M), Eu(NO3)3 aqueous solution (0.45 mL, 0.02 M), Cr(OAc)3 aqueous solution (0.45 mL, 0.02 M), and ultrapure water (7.2 mL) was vigorously stirred to obtain a homogeneous solution. 800 μL of 14.84 mol / L ammonia was then added to adjust the pH to 8. The mixture was stirred at 25°C for 4 h. The solution was then transferred to a 30 mL Teflon-lined stainless steel autoclave and heated at 170°C for 48 h. The precipitate was obtained by centrifugation and washed three times with ethanol and ultrapure water, then dispersed in ultrapure water.

[0070] The product dispersed in ultrapure water was treated with light under a 254 nm ultraviolet light source for 1 h to obtain hypochlorous acid-responsive long-lasting nanoprobes (PLNPs). The preparation process of PLNPs is as follows: Figure 1 shown.

[0071] Example 2

[0072] Preparation of Hypochlorous Acid-Responsive Long-Persistence Nanoprobes Adsorbed with Hyaluronic Acid

[0073] A mixture of Zn(OAc)2 aqueous solution (2.2 mL, 0.5 M), Ga(NO3)3 aqueous solution (3.6 mL, 0.5 M), GeO2 ammonia solution (1 mL, 0.1 M), Eu(NO3)3 aqueous solution (0.45 mL, 0.02 M), Cr(OAc)3 aqueous solution (0.45 mL, 0.02 M), and ultrapure water (7.2 mL) was vigorously stirred to obtain a homogeneous solution. 800 μL of 14.84 mol / L ammonia was then added to adjust the pH to 8. The mixture was stirred at 25°C for 4 h. The solution was then transferred to a 30 mL Teflon-lined stainless steel autoclave and heated at 170°C for 48 h. The precipitate was obtained by centrifugation and washed three times with ethanol and ultrapure water, then dispersed in ultrapure water.

[0074] The precipitate was mixed with hyaluronic acid at a mass ratio of 1:3, stirred at 25°C for 12 h, washed three times with ultrapure water by centrifugation, and redispersed in ultrapure water.

[0075] The product dispersed in ultrapure water was phototreated under a 254 nm ultraviolet light source for 1 h to obtain hypochlorous acid-responsive long-lasting hyaluronic acid-adsorbed nanoprobes (PLNPs@HA).

[0076] Verification Example 1

[0077] Verification of the structural characteristics of the long-lasting nanoprobe responsive to hypochlorous acid

[0078] The morphology of the PLNPs prepared in Example 1 was characterized using transmission electron microscopy. Figure 2 The size of PLNPs is about 10 nm. The crystal structure and elemental composition were characterized by X-ray diffractometer and X-ray photoelectron spectroscopy. Figure 3 , where A is the X-ray diffraction pattern and B is the X-ray photoelectron spectrum, proving that the target PLNPs nanoprobe was successfully constructed.

[0079] The optical properties of PLNPs were tested, and the results were as follows: Figure 4 As shown in the figure, A is the absorption spectrum of PLNPs, which shows that PLNPs can absorb a wide range of light from ultraviolet light to near-infrared light, and the absorbed light energy is stored in the matrix for further continuous emission; B is the fluorescence imaging of PLNPs using a fluorescence instrument, and it can be observed that the target probe produces fluorescence emission near 695nm.

[0080] The particle size and Zeta potential of the PLNPs prepared in Example 1 and the PLNPs@HA prepared in Example 2 were characterized by a Zeta potential analyzer. Figure 5 As shown in the figure, A is the particle size characterization result diagram, which shows that the size distribution of the prepared nanoprobes is concentrated and the synthesis is relatively uniform; B is the Zeta potential characterization result diagram, which shows that the potential of the nanoprobe loaded with HA changes from +40mV to about -10mV, indicating that PLNPs@HA has better hydrophilicity.

[0081] Verification Example 2

[0082] Verification of the persistent luminescence performance and hypochlorous acid response performance of the long-lasting nanoprobe

[0083] The PLNPs prepared in Example 1 were tested for their afterglow luminescence performance. Figure 6 Figure A shows the afterglow decay of PLNPs. PLNPs were activated under 254nm UV light for 1 minute and then the excitation was stopped. After the UV excitation was stopped, the near-infrared afterglow of the PLNPs rapidly decayed at the beginning and gradually stabilized after 6 minutes. Figures B and C show the repeated activation image and the quantitative comparison of the afterglow intensity of the long-afterglow nanoprobe, respectively. After the afterglow decayed for 12 minutes, the PLNPs were reactivated with 254nm UV light for 1 minute. The results showed that the afterglow intensity of the reactivated PLNPs still reached the same luminescence level as the first activation, indicating that the prepared nanoprobe has good repeated activation properties.

[0084] The hypochlorous acid response performance of the PLNPs prepared in Example 1 was verified, and the results were as follows: Figure 7 As shown in Figure 1. A is a comparison of the fluorescence change and afterglow luminescence enhancement of PLNPs. A control group (i.e., before hypochlorous acid treatment) and an experimental group (i.e., after hypochlorous acid treatment) were set up, and the fluorescence intensity and afterglow intensity of the two were measured respectively. The signal enhancement factor was obtained by dividing the signal intensity after hypochlorous acid treatment by the signal intensity before hypochlorous acid treatment. The results showed that the fluorescence intensity of the nanoprobes in the control group and the experimental group (i.e., before and after hypochlorous acid treatment) was almost unchanged, while the afterglow intensity of the nanoprobes in the control group and the experimental group (i.e., before and after hypochlorous acid treatment) was significantly enhanced. The afterglow signal enhancement can reach about 4 times, indicating that PLNPs have excellent response to hypochlorous acid.

[0085] The PLNPs prepared in Example 1 were subjected to hypochlorous acid response specificity verification. Fifteen parallel experiments were set up to simulate the concentration under physiological conditions. The total reaction volume was 200 μL. PLNPs with a final concentration of 500 μg / mL were treated with 2.5 mM K2SO4, 2.5 mM K2CO4, 38 mg / mL BSA, 250 mg / mL GLU, PBS (phosphate buffer with pH = 7.4), pure cell culture medium PAN, pure cell culture medium DMEM, 0.9% NaCl, 100 μM ClO - 、100μM H2O2、100μM OH - , 100μM 1 O2, 100 μM ONOO - , 100μM TBHP, ddH2O were used to treat the afterglow intensity of each experimental group. The results are as follows Figure 7 As shown, B is a comparison diagram of the response selectivity of different experimental groups to the long afterglow nanoprobe, and C is a quantitative comparison diagram of the afterglow signal enhancement of different experimental groups to the long afterglow nanoprobe, showing that the afterglow intensity of PLNPs after HClO treatment is significantly higher than that of other experimental groups, and PLNPs have a specific response ability to HClO.

[0086] Application Example 1

[0087] Rheumatoid arthritis position imaging using the PLNPs obtained in Example 1

[0088] During the pathological process of rheumatoid arthritis, a large number of immune cells, such as neutrophils, are present at the site of inflammation. Upon activation, neutrophils undergo a respiratory burst, producing a variety of reactive oxygen species, including hypochlorous acid. This probe can be used to monitor the physiological and pathological processes of the disease.

[0089] First, a rheumatoid arthritis model was established: 100 μL of 10 mg / mL carrageenan was injected into the right leg joint of mice (BALB / c mice, 6-8 weeks old, purchased from Shanghai Slake Laboratory Animal Co., Ltd.) to induce inflammation.

[0090] Next, 100 μL of 5 mg / mL PLNPs prepared in Example 1 were injected through the tail vein. Before injection, they were activated under UV light for 1 min. After injection, imaging was performed under the IVIS small animal imaging system. The processing flow of mice is as follows: Figure 8 shown.

[0091] Imaging results such as Figure 9 As shown in the figure, A is the afterglow luminescence imaging image of the rheumatoid arthritis position imaging experiment, which shows that after circulation in the body, the afterglow luminescence enhancement can be clearly observed at the joint of the right hind leg of the mouse, indicating that there is a large amount of hypochlorous acid that can respond to PLNPs in this area, thereby detecting inflammation in this area; the quantitative comparison results of the afterglow intensity of the rheumatoid arthritis position imaging experiment are shown in B. It can be seen from the figure that after the injection of PLNPs, the right leg shows a significantly enhanced afterglow intensity than the left leg, indicating that the inflammation site is located in the joint of the right leg of the mouse, rather than the joint of the left leg. Using this probe, the physiological and pathological processes of the disease can be detected.

[0092] Application Example 2

[0093] Tumor resection guidance using the PLNPs obtained in Example 1

[0094] Tumor tissue is infiltrated by a variety of immune cells, including macrophages. M1 macrophages, as part of host defense, secrete myeloperoxidase, an oxidase that catalyzes hydrogen peroxide to produce hypochlorous acid and other reactive oxygen species. Using the PLNPs prepared in Example 1, hypochlorous acid in tumor tissue can be detected, providing a clearer picture of the tumor's location. Their responsiveness can also aid in surgical removal of tumor tissue.

[0095] First, a liver orthotopic tumor mouse model was constructed: pre-cultured Hepa 1-6 cells were injected into the liver of mice (BALB / c mice, 6-8 weeks old, purchased from Shanghai Slake Laboratory Animal Co., Ltd.) at a rate of 350,000 cells per mouse. After culturing for 3 weeks, the liver tumor mouse model was successfully constructed.

[0096] Next, the PLNPs nanoprobes prepared in Example 1 were activated under ultraviolet light for 1 min, and then 100 μL of 5 mg / mL PLNPs were injected into G1 orthotopic liver mice and G2 normal mice (BALB / c mice, 6-8 weeks old, purchased from Shanghai Slake Laboratory Animal Co., Ltd.) through the tail vein. After injection, the images were taken under the IVIS small animal imaging system. The injection process is as follows: Figure 10 shown.

[0097] like Figure 11 As shown, compared to the liver tissue of G2 normal mice, the orthotopic transplanted tumors of G1 mice with hepatocellular carcinoma showed a significant signal enhancement. After tumor tissue resection and imaging of the mice, the afterglow luminescence signal in the liver tissue returned to the same level as in normal mice, indicating that the tumor tissue was completely removed.

[0098] G1 liver tumor mice were dissected, and tumor tissue and normal liver tissue were taken out for imaging. Figure 12 As shown, A is the afterglow luminescence imaging image of normal liver tissue and tumor tissue, the left side is normal liver tissue, and the right side is the resected tumor tissue. B is a quantitative comparison of the afterglow intensity of normal liver tissue and resected tumor tissue, showing that the normal liver tissue exhibits negligible afterglow luminescence signal, while the tumor tissue exhibits obvious strong afterglow luminescence.

[0099] Application Example 3

[0100] Reading of hidden information using the PLNPs obtained in Example 1

[0101] The schematic diagram of the information reading process is as follows Figure 13 As shown, a light-treated group and a non-light-treated group were set up respectively, and divided into Group I and Group II according to the location of the light-treated group. The light-treated group was the PLNPs prepared in Example 1, and the non-light-treated group was the product in Example 1 that was not light-treated under a 254 nm ultraviolet light source for 1 hour.

[0102] Nanoparticles with a volume of 200 μL and a final concentration of 1 mg / mL in the light-treated group and the non-light-treated group were added to different wells of the 96-well plate containing the hidden information "E". After treatment, they were placed under ultraviolet light for 1 minute to activate the encryption process. At this time, regardless of the light-treated group or the non-light-treated group, the different wells of "E" showed consistent afterglow (such as Figure 14 As shown, the left side of Figure A is the afterglow luminescence imaging of Group I after 1 minute of activation under UV light, and the left side of Figure B is the afterglow luminescence imaging of Group II after 1 minute of activation under UV light). The fluorescence intensity is consistent (as shown in Figure 4). Figure 14As shown, C is the quantitative comparison of the afterglow intensity of group I, and E is the quantitative comparison of the afterglow intensity of group II). Afterwards, taking advantage of the strong response of PLNPs to hypochlorous acid, an equal amount of 20 μL of 1 mM hypochlorous acid was added to all wells to make the hypochlorous acid concentration 100 μM / well for decryption treatment. Figure 14 As shown, the right side of Figure A is the afterglow luminescence imaging of group I after hypochlorous acid treatment, and the right side of Figure B is the afterglow luminescence imaging of group II after hypochlorous acid treatment. It can be observed that the afterglow luminescence of the light-treated group is significantly stronger than that of the non-light-treated group, and the signal is enhanced by more than 1.5 times (as shown in Figure 4). Figure 14 As shown, D is a quantitative comparison diagram of the afterglow intensity of group I after hypochlorous acid treatment, and F is a quantitative comparison diagram of the afterglow intensity of group II after hypochlorous acid treatment), thereby distinguishing the position information of the different treatment methods of the light treatment group and the non-light treatment group, and realizing the reading of hidden information.

[0103] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0104] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0105] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. Application of a long afterglow nanoprobe in detecting hypochlorous acid, characterized in that: The long afterglow nanoprobe includes long afterglow nanoparticles, and the chemical composition formula of the long afterglow nanoparticles is: Zn 1+x Ga 2−2x Ge x O4:Cr 3+ y , Eu 3+ z , where 0.1≤x≤0.5, 0.005≤y≤0.05, 0.005≤z≤0.

05.

2. The use according to claim 1, characterized in that The ratio of y to z is 1:(1~0.1).

3. The use according to claim 1, characterized in that The long afterglow nanoprobe further comprises hyaluronic acid coated on the surface of the long afterglow nanoparticles, and the hyaluronic acid is adsorbed on the long afterglow nanoparticles by electrostatic adsorption.

4. The use according to claim 1, characterized in that The particle size of the long afterglow nanoprobe is 2-200 nm.

5. The use according to claim 1, characterized in that The long afterglow nanoprobe is prepared by a preparation method comprising the following steps: (1) GeO2 and Zn 2+ Salt, Ga 3+ Salt, Eu 3+ Salt, Cr 3+ The salts are mixed in an aqueous phase at a molar ratio of (0.1-0.5):(1.1-1.5):(1-1.8):(0.005-0.05):(0.005-0.05) to obtain a solution, and then aqueous ammonia is added to adjust the pH value to 7-9; (2) heat-treating the solution obtained in step (1) and centrifuging to obtain a precipitate; (3) dispersing the precipitate obtained in step (2) in ultrapure water; (4) The product obtained in step (3) is subjected to light treatment under a light source with a wavelength of 10 to 700 nm for 0.1 to 4 h to obtain the hypochlorous acid-responsive long-afterglow nanoprobe.

6. The use according to claim 5, characterized in that The salt in step (1) is independently selected from nitrate or acetate.

7. The use according to claim 5, characterized in that After the pH value is adjusted to 7-9 in step (1), stirring is also included; The stirring temperature is 20-40° C., and the stirring time is 2-6 h.

8. The use according to claim 5, characterized in that The heat treatment temperature in step (2) is 150-250°C, and the heat treatment time is 20-60 h.

9. The use according to claim 5, characterized in that The heat treatment in step (2) is carried out in a high-pressure sterilizer.

10. The use according to claim 5, characterized in that Step (3) also includes washing the precipitate before dispersing the precipitate in ultrapure water.

11. The use according to claim 10, characterized in that The washing comprises centrifugal washing with ethanol and ultrapure water three times.

12. The use according to claim 5, characterized in that Step (3) further comprises mixing the precipitate with hyaluronic acid in a mass ratio of 1:(2-5), washing, and redispersing in ultrapure water.

13. The use according to claim 12, characterized in that The mixing further comprises stirring; The stirring temperature is 20-40° C., and the stirring time is 10-20 h.

14. The use according to claim 5, characterized in that The wavelength in step (4) is 200-300 nm.

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