Hypochlorous acid response type long afterglow nanoprobe as well as preparation method and application thereof

Abstract

The invention relates to a hypochlorous acid response type long-afterglow nanoprobe and a preparation method and application thereof, the long-afterglow nanoprobe comprises long-afterglow nanoparticles, the chemical composition general formula of the long-afterglow nanoparticles is Zn1 + xGa2-2xGexO4: Cr < 3 + > y and Eu < 3 + > z, 0.1 < = x < = 0.5, 0.005 < = y < = 0.05, and 0.005 < = z < = 0.05. The hypochlorous acid response type long-afterglow nanoprobe is good in responsiveness and selectivity, can sensitively respond to hypochlorous acid in a solution and a body, generates enhanced afterglow luminescence, is beneficial to accurate detection, has a high signal-to-noise ratio, can be used for carrying out low-background and high-response afterglow luminescence imaging detection on hypochlorous acid, and has a wide application prospect. The probe can be applied to diagnosis or imaging products of rheumatoid arthritis or tumors, research on hypochlorous acid related physiological or pathological processes, and can also be applied to the fields of information encryption and the like by utilizing response characteristics of the probe.

Description

Technical Field

[0001] The invention relates to the technical field of long afterglow materials, and in particular to a hypochlorous acid-responsive long afterglow nanoprobe and a preparation method and application 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 traps. When the light excitation of the material stops, it continues to emit light. 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 O 4 , lanthanide-doped NaYbF 4 Upconversion nanoparticles) and organic materials such as derivatives of poly [2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinyl] (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 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, wherein the matrix material is La 3 Ga 5 GeO 14 or Ca 3 Ga 2 Ge 3 O 12 or Zn 3 Ga 2 Ge 2 O 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 combined with 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 actual scenarios.

[0006] Therefore, developing new long-afterglow 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] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a hypochlorous acid-responsive long afterglow nanoprobe and its preparation method and application. The hypochlorous acid-responsive long afterglow nanoprobe can sensitively respond to hypochlorous acid in solution and in vivo, produce enhanced afterglow luminescence, and can perform 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 a first aspect, the present invention provides a hypochlorous acid responsive long afterglow nanoprobe, wherein the long afterglow nanoprobe comprises long afterglow nanoparticles, and the chemical composition general formula of the long afterglow nanoparticles is: Zn 1+x Ga 2-2x Ge x O 4 :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 value of x can be selected as 0.1, 0.2, 0.3, 0.4, 0.5, etc., the specific point value 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 value 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 O 4 As a matrix structure, it has 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.

[0012] The Zn 1+x Ga 2-2x Ge x O 4 :Cr 3+ y ,Eu 3+ z It 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 each element of the matrix structure by 0.1≤x≤0.5, 0.005≤y≤0.05, and 0.005≤z≤0.05. When Ge is doped too little, 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 afterglow nanoparticles for the excitation light energy; when Ge is doped too much, the stability of the crystal structure will be destroyed, resulting in too many defects or lattice distortions in the crystal, increasing energy loss and reducing the long afterglow performance. Moreover, too much Ge may change the chemical stability of the material, making it more prone to chemical reactions under certain environments, affecting the service life of the material; when Zn or Ga is doped too much, the distribution and depth of the trap energy level will be changed, so that the process of energy storage and release is disturbed; when Zn or Ga is doped too little, the stability of the crystal structure will be reduced, and the optical band gap of the material will be changed, affecting the uniformity and consistency of the material, thereby affecting the performance of the long afterglow.

[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 afterglow 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 good biocompatibility and biodegradability. Hyaluronic acid and long afterglow nanoparticles are electrostatically adsorbed, causing the surface potential of the probe to become negative, which helps to reduce the cell's rejection of the probe, reduce the cell-probe adhesion and reduce the cell's inflammatory response, thereby improving biocompatibility. At the same time, HA can promote cell proliferation and enhance the compatibility of nanoprobes 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) GeO 2 With 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 ammonia water 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 finds 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 afterglow luminescence enhancement.

[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 to 40° C., and the stirring time is 2 to 6 hours.

[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 to 250° C., and the time of the heat treatment is 20 to 60 hours.

[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 20h, 30h, 40h, 50h, 60h, etc.

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

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

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

[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] In the pathological process of rheumatoid arthritis, there are a large number of immune cells, such as neutrophils, in the inflammatory site. After being activated, neutrophils will undergo a respiratory burst and produce a variety of reactive oxygen species, including hypochlorous acid. The above-mentioned hypochlorous acid-responsive long-afterglow nanoprobe can be used to detect the physiological and pathological process of the disease.

[0044] In tumor tissues, 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 tissues 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 not only includes the point values ​​listed above, but also includes 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, which 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, and no external response elements need to be introduced. 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 microscope characterization result diagram of the hypochlorous acid responsive long afterglow nanoprobe prepared in Example 1;

[0054] Figure 3 It 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 This is a test diagram of the optical performance of the hypochlorous acid-responsive long afterglow nanoprobe prepared in Example 1 (where A is an absorption spectrum diagram and B is a fluorescence diagram);

[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 adsorbing hyaluronic acid prepared in Example 2 (where A is a particle size characterization result diagram, and B is a Zeta potential characterization result diagram);

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

[0058] Figure 7It is a hypochlorous acid response performance verification result diagram of the hypochlorous acid responsive long afterglow nanoprobe prepared in Example 1 (wherein A is a comparison diagram of PLNPs fluorescence change and afterglow luminescence enhancement, 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);

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

[0060] Fig. 9 It is a result diagram of the rheumatoid arthritis position imaging experiment in Application Example 1 (where A is an afterglow luminescence imaging diagram of the rheumatoid arthritis position imaging experiment, and B is a quantitative comparison diagram of the afterglow intensity of the rheumatoid arthritis position imaging experiment);

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

[0062] Fig.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 in Application Example 2;

[0063] Fig.12 It is a comparison diagram 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 diagram, and B is the afterglow intensity quantification comparison diagram);

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

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

[0066] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only 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 nanoprobe

[0069] Zn(OAc) 2Aqueous solution (2.2 mL, 0.5 M), Ga(NO 3 ) 3 Aqueous solution (3.6 mL, 0.5 M), GeO 2 Ammonia solution (1 mL, 0.1 M), Eu(NO 3 ) 3 Aqueous solution (0.45 mL, 0.02 M), Cr(OAc) 3 A mixed aqueous solution of aqueous solution (0.45mL, 0.02M) and ultrapure water (7.2mL) was vigorously stirred to obtain a uniform solution, and then 800μL of 14.84mol / L ammonia water was added, the pH value was adjusted to 8, and stirred at 25℃ for 4h. The solution was then transferred to a 30mL stainless steel autoclave lined with teflon and heated at 170℃ for 48h. The precipitate was obtained by centrifugation, washed three times by centrifugation with ethanol and ultrapure water, and then dispersed in ultrapure water.

[0070] The product dispersed in ultrapure water was treated with light for 1 h under a 254 nm ultraviolet light source 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-Afterglow Nanoprobe Adsorbed with Hyaluronic Acid

[0073] Zn(OAc) 2 Aqueous solution (2.2 mL, 0.5 M), Ga(NO 3 ) 3 Aqueous solution (3.6 mL, 0.5 M), GeO 2 Ammonia solution (1 mL, 0.1 M), Eu(NO 3 ) 3 Aqueous solution (0.45 mL, 0.02 M), Cr(OAc) 3 A mixed aqueous solution of aqueous solution (0.45mL, 0.02M) and ultrapure water (7.2mL) was vigorously stirred to obtain a uniform solution, and then 800μL of 14.84mol / L ammonia water was added, the pH value was adjusted to 8, and stirred at 25℃ for 4h. The solution was then transferred to a 30mL stainless steel autoclave lined with teflon and heated at 170℃ for 48h. The precipitate was obtained by centrifugation, washed three times by centrifugation with ethanol and ultrapure water, and 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 by centrifugation with ultrapure water, and redispersed in ultrapure water.

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

[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 by 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 spectrometer. 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, A is the absorption spectrum of PLNPs, showing 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, 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 afterglow luminescence performance and hypochlorous acid response performance of the long afterglow nanoprobe

[0083] The afterglow luminescence performance of the PLNPs prepared in Example 1 was verified, and the results were as follows: Figure 6As shown. A is the afterglow attenuation of PLNPs. PLNPs were activated under 254nm ultraviolet light for 1 minute and then the excitation was stopped. When the ultraviolet light excitation stopped, the near-infrared afterglow of PLNPs decayed rapidly in the initial stage and gradually remained stable after 6 minutes. B and C are the repeated activation diagrams of the long afterglow nanoprobe and the quantitative comparison diagram of the repeated activation afterglow intensity. After the afterglow decayed for 12 minutes, the PLNPs were activated again with 254nm ultraviolet light for 1 minute. The results showed that the afterglow intensity of the reactivated PLNPs can still reach the same luminescence level as the first activation, indicating that the prepared nanoprobe has good repeated activation characteristics.

[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. A is a comparison diagram of PLNPs fluorescence change and afterglow luminescence enhancement. A control group (i.e., before hypochlorous acid treatment) and an experimental group (i.e., after hypochlorous acid treatment) were set up respectively, and the fluorescence intensity and afterglow intensity of the two were measured respectively. The signal enhancement multiple was obtained by using the signal intensity after hypochlorous acid treatment / the signal intensity before hypochlorous acid treatment. The results showed that the fluorescence intensity of the nanoprobe 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 nanoprobe in the control group and the experimental group (i.e., before and after hypochlorous acid treatment) was significantly enhanced, and the afterglow signal enhancement could reach about 4 times, indicating that PLNPs have excellent response ability to hypochlorous acid.

[0085] The PLNPs prepared in Example 1 were subjected to specific validation of hypochlorous acid response, and 15 parallel experiments were set up to simulate the concentration under physiological environment, and the total reaction volume was 200 μL. The PLNPs with a final concentration of 500 μg / mL were respectively treated with a final concentration of 2.5 mM K 2 SO 4 , 2.5 mM K 2 CO 4 , 38 mg / mL BSA, 250 mg / mL GLU, PBS (phosphate buffer, pH = 7.4), pure cell culture medium PAN, pure cell culture medium DMEM, 0.9% NaCl, 100 μM ClO - , 100 μM H 2 O 2 、100μM OH - , 100μM 1 O 2 , 100 μM ONOO - , 100 μM TBHP, ddH 2 O was processed and the afterglow intensity of each experimental group was tested. The results are as follows Figure 7As shown, 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, showing that the afterglow intensity of PLNPs treated with HClO is significantly higher than that of other experimental groups, and PLNPs has a specific response ability to HClO.

[0086] Application Example 1

[0087] Using the PLNPs obtained in Example 1 to perform rheumatoid arthritis position imaging

[0088] In the pathological process of rheumatoid arthritis, there are a large number of immune cells, such as neutrophils, in the inflammatory site. After being activated, neutrophils will undergo a respiratory burst and produce a variety of reactive oxygen species, including hypochlorous acid. Using this probe, the physiological and pathological process of the disease can be detected.

[0089] First, a rheumatoid arthritis model was constructed: 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 ultraviolet light for 1 min. After injection, they were imaged under the IVIS small animal imaging system. The processing flow of mice is as follows: Figure 8 shown.

[0091] The imaging results are as follows Fig. 9 As shown in the figure, A is the afterglow luminescence imaging image of the rheumatoid arthritis position imaging experiment, which shows that after circulating in the body, the afterglow luminescence enhancement at the joint of the right hind leg of the mouse can be clearly observed, 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 process of the disease can be detected.

[0092] Application Example 2

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

[0094] In tumor tissue, there are many kinds of immune cells infiltrating, and macrophages are one of them. M1 macrophages secrete myeloperoxidase in host defense, which is an oxidase that catalyzes hydrogen peroxide to produce hypochlorous acid and other reactive oxygen species. Using the PLNPs prepared in Example 1, it is possible to detect hypochlorous acid in tumor tissue, thereby more clearly reflecting the situation of the tumor tissue site, and using its response characteristics to help surgical removal of tumor tissue.

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

[0096] Next, the PLNPs nanoprobe prepared in Example 1 was activated under ultraviolet light for 1 min, and then 100 μL of 5 mg / mL PLNPs was injected into G1 liver orthotopic 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 injection was performed under the IVIS small animal imaging system. The injection process is as follows Fig.10 shown.

[0097] like Fig.11 As shown in the figure, compared with the liver tissue of G2 normal mice, the orthotopic transplanted tumor of G1 mouse hepatocellular carcinoma showed obvious signal enhancement. The mouse was imaged after tumor tissue removal, and the afterglow luminescence signal of the liver tissue was observed to be restored to the same level as that of normal mice, which also indicated 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. Fig.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 Fig.13As shown, a light treatment group and a non-light treatment group are set respectively, and according to the setting position of the light treatment group, they are divided into group I and group II. The light treatment group is the PLNPs prepared in Example 1, and the non-light treatment group is the product in Example 1 that is not light treated under a 254nm ultraviolet light source for 1h.

[0102] The nanoparticles with a volume of 200uL and a final concentration of 1mg / 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 for encryption. At this time, regardless of the light-treated group or the non-light-treated group, the different wells of "E" showed consistent afterglow luminescence (such as Fig.14 As shown, the left side of Figure A is the afterglow luminescence imaging of Group I after 1 minute of activation under ultraviolet light, and the left side of Figure B is the afterglow luminescence imaging of Group II after 1 minute of activation under ultraviolet light). The fluorescence intensity is consistent (such as Fig.14 As 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, using the strong response characteristics of PLNPs to hypochlorous acid, an equal amount of 20 μL 1 mM hypochlorous acid was added to all wells to make the hypochlorous acid concentration 100 μM / well for decryption treatment. Fig.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 (such as Fig.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 illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope 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 suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A hypochlorous acid-responsive long afterglow nanoprobe, characterized in that: The long afterglow nanoprobe comprises long afterglow nanoparticles, and the general 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 hypochlorous acid-responsive long afterglow nanoprobe according to claim 1, characterized in that: The ratio of y to z is 1:(1-0.1).

3. The hypochlorous acid-responsive long afterglow nanoprobe according to claim 1 or 2, 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 hypochlorous acid-responsive long afterglow nanoprobe according to any one of claims 1 to 3, characterized in that: The particle size of the long afterglow nanoprobe is 2-200nm.

5. A method for preparing a hypochlorous acid-responsive long afterglow nanoprobe according to any one of claims 1 to 4, characterized in that: The preparation method comprises 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 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 ammonia water 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 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.

6. The preparation method according to claim 5, characterized in that: The salt in step (1) is independently selected from nitrate or acetate; Preferably, step (1) further comprises stirring after adjusting the pH value to 7 to 9; The stirring temperature is 20 to 40°C and the stirring time is 2 to 6 hours; Preferably, the temperature of the heat treatment in step (2) is 150 to 250° C., and the heat treatment time is 20 to 60 hours; Preferably, the heat treatment in step (2) is carried out in a high pressure sterilization pot; Preferably, the step (3) further comprises washing the precipitate before dispersing the precipitate in ultrapure water; Preferably, the washing comprises centrifugal washing with ethanol and ultrapure water three times.

7. The preparation method according to claim 5 or 6, 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; Preferably, the mixing further comprises stirring; The stirring temperature is 20-40° C., and the stirring time is 10-20 hours.

8. The preparation method according to any one of claims 5 to 7, characterized in that: The wavelength in step (4) is 200 to 300 nm.

9. Use of the hypochlorous acid-responsive long afterglow nanoprobe according to any one of claims 1 to 4 in a diagnosis or imaging product for rheumatoid arthritis or tumors.

10. Use of the hypochlorous acid responsive long afterglow nanoprobe as claimed in any one of claims 1 to 4 in information encryption processing.

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