Chemiluminescent nanoprobes, methods of making and using the same
The chemiluminescent nanoprobe with a water-in-oil-in-water emulsion structure solves the problem of oxalate aggregation effect in the aqueous phase, achieves efficient energy transfer and stable chemiluminescence performance, and is used for highly sensitive detection of superoxide radicals and early diagnosis of diseases.
Patent Information
- Application Number
- CN202310512284.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-09
- Publication Date
- 2025-10-17
- Estimated Expiration
- 2043-05-09
AI Technical Summary
Existing oxalate-based chemiluminescent probes have an aggregation effect in the aqueous phase, resulting in low efficiency in the generation of reaction intermediates, poor luminescence stability, difficulty in achieving efficient chemiluminescence energy transfer, and a low detection limit for superoxide radicals, which limits their application at the in vivo level.
It adopts a water-in-oil-in-water emulsion structure, with the inner layer being the first aqueous phase of red organic dye and polyvinyl alcohol, the middle layer being the oil phase of amphiphilic polymer and oxalate, and the outer layer being the second aqueous phase of superoxide dismutase and polyvinyl alcohol. Stable nanoprobes are formed through ultrasonic treatment, and efficient energy transfer is achieved by utilizing the chemiluminescent energy donor of oxalate and the energy acceptor of the red organic dye.
The chemiluminescent nanoprobe has been used to detect superoxide radicals with high sensitivity and selectivity, with a luminescence half-life of 1000 seconds and a detection limit of 0.27 micromoles, enabling early detection of superoxide radical-related diseases in vivo.
Smart Images
Figure HDA0004217772340000011 
Figure HDA0004217772340000012 
Figure HDA0004217772340000021
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biological detection, in particular to a chemiluminescence nanoprobe and a preparation method and application thereof. BACKGROUND
[0002] Chemiluminescence is a kind of light radiation phenomenon generated in the process of oxidation-reduction chemical reaction of substances. Due to its high sensitivity, wide dynamic range, small background interference and the like, it has been widely applied in the fields of biosensing, disease detection and environmental chemistry. As a typical chemiluminescence probe, the oxalate probe system mainly comprises three components: hydrogen peroxide, aromatic oxalate and fluorescent dye. The specific luminescence process is as follows: the oxalate is oxidized by hydrogen peroxide in anhydrous medium to generate unstable high-energy reaction intermediates, i.e. 1,2-dioxetanone. After its decomposition, carbon dioxide is generated and photons are released to excite the fluorescent dye to generate corresponding optical signals. The energy generated in this process can excite fluorescent dyes of different emission wavelengths and has high energy transfer efficiency.
[0003] However, the energy donor based on oxalate usually has poor water solubility. The strong dielectric shielding effect in the aqueous phase leads to aggregation of the oxalate and reduces the generation efficiency of the reaction intermediates, so it is difficult to realize efficient chemiluminescence energy transfer. Although researchers have introduced micelles and hydrophilic groups to modify the system to alleviate the aggregation effect to some extent, the poor luminescence stability and luminescence lifetime still cannot meet the real-time chemiluminescence detection at the molecular level. In addition, superoxide radicals, as typical biological active oxygen, have a close relationship with many diseases (such as acute kidney injury and acute liver injury) in the expression level in the body. The low detection limit of the chemiluminescence probe triggered by superoxide radicals further limits its application in the living body. Therefore, it is crucial to establish a probe with stable chemiluminescence characteristics and energy transfer efficiency to realize high-sensitivity disease detection triggered by superoxide radicals. SUMMARY
[0004] Therefore, the present application aims to provide a chemiluminescence nanoprobe and a preparation method thereof, so that the chemiluminescence nanoprobe has strong and stable chemiluminescence performance, and can realize high-sensitivity and high-selectivity detection of superoxide radicals.
[0005] Another object of the present application is to provide the application of the chemiluminescence nanoprobe in the preparation of a product for detecting diseases related to superoxide radicals.
[0006] To solve the above technical problems or at least partially solve the above technical problems, the present application provides a method for solving the above technical problems or at least partially solving the above technical problems, as a first aspect of the present application, a chemiluminescence nanoprobes is provided, comprising a first aqueous phase as an inner layer, an oil phase as an intermediate layer and a second aqueous phase as an outer layer, forming a water-in-oil-in-water emulsion;
[0007] The first aqueous phase comprises a red organic dye and polyvinyl alcohol, the oil phase comprises an amphiphilic polymer and an oxalate-based chemiluminescence energy donor, and the second aqueous phase comprises superoxide dismutase and polyvinyl alcohol.
[0008] Optionally, the concentration of the red organic dye in the first aqueous phase is 0.01-10 mg / mL; the concentration of the amphiphilic polymer in the oil phase is 1-10 mg / mL, and the concentration of the oxalate-based chemiluminescence energy donor is 1-5 mg / mL; the concentration of the superoxide dismutase in the second aqueous phase is 0.05-5 mg / mL.
[0009] Further optionally, the red organic dye comprises one or more of chlorin, rhodamine 6G and rhodamine B.
[0010] Further optionally, the amphiphilic polymer comprises one or more of polylactic acid-glycolic acid copolymer and polyoxyethylene polyoxypropylene.
[0011] Further optionally, the oxalate-based chemiluminescence energy donor comprises bis-oxalate.
[0012] As a second aspect of the present application, the application of the chemiluminescence nanoprobes in preparing products for detecting superoxide radical related diseases is provided.
[0013] Optionally, the superoxide radical related diseases include kidney injury, brain injury and liver injury.
[0014] As a third aspect of the present application, a preparation method of the chemiluminescence nanoprobes is provided, comprising:
[0015] The amphiphilic polymer and the oxalate-based chemiluminescence energy donor are mixed and dispersed in an organic solvent to obtain an oil phase; an aqueous solution of the red organic dye and polyvinyl alcohol is prepared to obtain a first aqueous phase;
[0016] The first aqueous phase is transferred to the oil phase and ultrasonically treated to obtain a first emulsion;
[0017] The first emulsion is transferred to the polyvinyl alcohol aqueous solution and ultrasonically treated to obtain a second emulsion;
[0018] The second emulsion is co-incubated with a superoxide dismutase solution to form a second water phase as the outermost layer, thereby obtaining a water-in-oil-in-water structured chemiluminescence nanoprobe emulsion.
[0019] Optionally, the organic solvent comprises an organic solvent with a polarity less than 4; further optionally, the organic solvent comprises dichloromethane and / or toluene.
[0020] The present application utilizes oxalate-based chemiluminescence energy donors and red organic dyes as light-emitting energy acceptors; utilizes hydrophilic polymers as matrix raw materials, and prepares chemiluminescence nanoprobes through a water-in-oil-in-water complex emulsification method. The obtained chemiluminescence nanoprobes have a chemiluminescence half-life of up to 1000 seconds, a detection limit of 0.27 micromole for superoxide free radicals, and strong and stable chemiluminescence performance without interference from other ions, and can be used for monitoring diseases related to superoxide free radicals. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 The structure of the chemiluminescence nanoprobes of the present application is shown;
[0022] Figure 2 The electron microscope picture of the chemiluminescence nanoprobes of the present application is shown;
[0023] Figure 3 The spectral characterization of the chemiluminescence nanoprobes of the present application is shown; a is the chemiluminescence spectrum; b is the luminescence half-life spectrum; c is the correlation spectrum of the chemiluminescence signal intensity and the gradient concentration of superoxide free radicals; d is the chemiluminescence signal intensity spectrum under the coexistence of various interference ions;
[0024] Figure 4 The chemiluminescence in vivo imaging (a) and data statistics (b) of the chemiluminescence nanoprobes of the present application in an acute kidney injury mouse model are shown. DETAILED DESCRIPTION
[0025] The present application discloses a chemiluminescence nanoprobe, a preparation method and application thereof. Those skilled in the art can refer to the content of the present application to appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The chemiluminescence nanoprobe, the preparation method and the application thereof have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the chemiluminescence nanoprobe, the preparation method and the application thereof described in the present application without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.
[0026] In a first aspect of the present application, a chemiluminescent nanoprobe is provided, comprising a first aqueous phase as an inner layer, an oil phase as a middle layer, and a second aqueous phase as an outer layer, forming a water-in-oil-in-water emulsion;
[0027] The first aqueous phase comprises a red organic dye and polyvinyl alcohol, the oil phase comprises an amphiphilic polymer and an oxalate-based chemiluminescent energy donor, and the second aqueous phase comprises superoxide dismutase and polyvinyl alcohol, as shown in the structural schematic diagram Figure 1 .
[0028] The present application realizes a stable 100-150 nanometer water-in-oil-in-water system by designing a hydrophilic polymer cavity, placing the oxalate-based energy donor and the luminescent energy acceptor (red organic dye) in the oil phase and the first aqueous phase, respectively, introducing superoxide dismutase as a catalyst in the second aqueous phase to reduce superoxide radicals to hydrogen peroxide, and further oxidizing the oxalate by the hydrogen peroxide, and the released photons excite the red organic dye to release an optical signal through energy transfer.
[0029] In some embodiments of the present application, the concentration of the red organic dye in the first aqueous phase is 0.01-10 mg / mL, and the polyvinyl alcohol acts as an emulsifier, and its amount can be adjusted according to actual needs, for example, the concentration of polyvinyl alcohol is 1-45 mg / mL; in some other embodiments of the present application, the concentration of the red organic dye in the first aqueous phase is 0.04-8.33 mg / mL, for example, 0.83 mg / mL, 1.30 mg / mL, 0.05 mg / mL, 0.32 mg / mL, 0.16 mg / mL, 0.45 mg / mL, etc., and the concentration of polyvinyl alcohol is 1.82-41.67 mg / mL, for example, 8.33 mg / mL, 8.70 mg / mL, 9.77 mg / mL, 9.68 mg / mL, 9.09 mg / mL, etc.
[0030] In some embodiments of the present application, the concentration of the amphiphilic polymer in the oil phase is 1-10 mg / mL, for example, 2.5 mg / mL, 5 mg / mL, 3.33 mg / mL, 6.67 mg / mL, etc., and the concentration of the oxalate-based chemiluminescent energy donor is 1-5 mg / mL, for example, 2.5 mg / mL, 3.33 mg / mL, 1.67 mg / mL, etc.
[0031] In some embodiments of the present application, the concentration of the second aqueous superoxide dismutase is 0.05-5 mg / mL, and polyvinyl alcohol is used as an emulsifier, the amount of which can be adjusted according to actual needs, and the concentration of polyvinyl alcohol is 1-150 mg / mL; in some other embodiments of the present application, the concentration of the second aqueous total superoxide dismutase is 0.1-2.5 mg / mL, such as 0.2 mg / mL, 0.33 mg / mL, 0.4 mg / mL, 0.83 mg / mL, 1.0 mg / mL, etc., and the concentration of polyvinyl alcohol is 50-100 mg / mL, such as 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, etc.
[0032] The present application has been tested for various emulsifiers including polyvinyl alcohol, and among the various emulsifiers tested, only polyvinyl alcohol can successfully achieve a water-in-oil-in-water emulsion and achieve stable optical luminescence properties.
[0033] In some embodiments of the present application, the red organic dye includes one or more than two of chlorin, rhodamine 6G, and rhodamine B; the amphiphilic polymer includes one or more than two of polylactic acid-glycolic acid copolymer and polyoxyethylene polyoxypropylene; and the oxalate-based chemiluminescence energy donor includes bis-oxalate and the like.
[0034] In some other embodiments of the present application, the oil phase in the chemiluminescence nanoprobes includes polylactic acid-glycolic acid copolymer + bis-oxalate, or polyoxyethylene polyoxypropylene + bis-oxalate; and the first aqueous phase includes chlorin + polyvinyl alcohol, or rhodamine 6G + polyvinyl alcohol.
[0035] In the second aspect of the present application, under the action of hydrogen peroxide or potassium superoxide (which can be used to generate superoxide radicals), the chemiluminescence nanoprobes of the present application obtain a chemiluminescence signal at 680 nm, and the luminescence half-life is about 1000 seconds; the chemiluminescence signal intensity is positively correlated with the concentration of superoxide radicals in the system (32.5-500 micromoles), and sensitive detection of superoxide radicals can be achieved (detection limit: 0.27 micromole). At the same time, the probe can still maintain good selectivity in the presence of different interfering ions. In addition, the present application establishes a cisplatin-induced acute kidney injury model (with superoxide radicals as a marker), and the probe is injected into the living body through the tail vein, and through continuous imaging, early detection (12 hours) of acute kidney injury disease can be achieved. Based on the above various excellent technical effects, the present application proposes the use of the chemiluminescence nanoprobes in the preparation of products for detecting superoxide radical-related diseases.
[0036] In some embodiments of the present application, the superoxide radical-related diseases include acute kidney injury and acute liver injury.
[0037] In a third aspect of the present application, a preparation method of the chemiluminescent nanoprobe is also provided, comprising:
[0038] mixing the amphiphilic polymer and the oxalate-based chemiluminescent energy donor to disperse in an organic solvent to obtain an oil phase; preparing an aqueous solution of a red organic dye and polyvinyl alcohol to obtain a first aqueous phase;
[0039] transferring the first aqueous phase to the oil phase and ultrasonically treating to obtain a first emulsion;
[0040] transferring the first emulsion to an aqueous solution of polyvinyl alcohol and ultrasonically treating to obtain a second emulsion;
[0041] co-incubating the second emulsion with a solution of superoxide dismutase to form a second aqueous phase as the outermost layer, to obtain a water-in-oil-in-water structured chemiluminescent nanoprobe emulsion.
[0042] In some embodiments of the present application, the organic solvent comprises dichloromethane and toluene; the ultrasonic treatment time is 10-20 minutes; and the co-incubation is room temperature co-incubation for 2-12 hours.
[0043] In some other embodiments of the present application, the preparation method comprises:
[0044] S1, mixing 5-20 mg of amphiphilic polymer (such as polylactic acid-glycolic acid copolymer, polyoxyethylene polyoxypropylene, etc.) and 5-10 mg of bisoxalate in 2-5 ml of organic solvent (such as dichloromethane, toluene) to obtain an oil phase solution by shaking and mixing for 10-30 minutes;
[0045] adding 20-50 μl of 1-20 mg / ml aqueous solution of red organic dye (such as chlorin, rhodamine 6G, rhodamine B, etc.) to 100-500 μl of 1% polyvinyl alcohol aqueous solution to obtain a first aqueous phase solution;
[0046] S2, slowly transferring the first aqueous phase solution prepared in S1 to the oil phase solution and ultrasonically treating under ice water bath conditions for 10-20 minutes to obtain a first emulsion solution;
[0047] S3, adding the first emulsion solution obtained in S2 to 2-5 ml of 5-10% polyvinyl alcohol solution, and ultrasonically treating under ice water bath conditions for 10-20 minutes to obtain a second emulsion;
[0048] S4, co-incubating the reaction completed solution in S3 with 1-5 mg / ml superoxide dismutase for 1-2 hours to form a second aqueous phase as the outer layer, to obtain a water-in-oil-in-water structured chemiluminescent nanoprobe emulsion.
[0049] Unless otherwise specified, the experimental environment and parameter conditions of each group in the specific embodiment test are consistent except for the explicit distinction.
[0050] The chemical luminescence nanoprobes provided by the present application, the preparation method and application thereof are further described below.
[0051] Example 1: Preparation of the chemical luminescence nanoprobes of the present application
[0052] (1) 5 milligrams of polylactic acid-glycolic acid copolymer and 5 milligrams of bisoxalate were mixed and dispersed in 2 milliliters of dichloromethane to obtain an oil phase solution;
[0053] (2) 20 microliters of 5 milligrams per milliliter of a water solution of chlorin was added to 100 microliters of a 1% polyvinyl alcohol aqueous solution to obtain an aqueous phase solution;
[0054] (3) The aqueous phase solution prepared in step (2) was slowly transferred to the oil phase solution prepared in step (1), and ultrasonic treatment was performed for 15 minutes under ice water bath conditions to obtain a first emulsion solution;
[0055] (4) The first emulsion solution obtained in step (3) was added to 3 milliliters of a 5% polyvinyl alcohol solution, and ultrasonic treatment was performed for 15 minutes under ice water bath conditions to obtain a second emulsion;
[0056] (5) The solution after the reaction in step (4) was stirred overnight, and 1 milliliter of 1 milligram per milliliter of superoxide dismutase was incubated to obtain the target product.
[0057] Example 2: Preparation of the chemical luminescence nanoprobes of the present application
[0058] (1) 10 milligrams of polyoxyethylene polyoxypropylene and 5 milligrams of bisoxalate were mixed and dispersed in 2 milliliters of dichloromethane to obtain an oil phase solution;
[0059] (2) 30 microliters of 10 milligrams per milliliter of a water solution of rhodamine B was added to 200 microliters of a 1% polyvinyl alcohol aqueous solution to obtain an aqueous phase solution;
[0060] (3) The aqueous phase solution prepared in step (2) was slowly transferred to the oil phase solution prepared in step (1), and ultrasonic treatment was performed for 15 minutes under ice water bath conditions to obtain a first emulsion solution;
[0061] (4) The first emulsion solution obtained in step (3) was added to 5 milliliters of a 5% polyvinyl alcohol solution, and ultrasonic treatment was performed for 15 minutes under ice water bath conditions to obtain a second emulsion;
[0062] (5) The solution of step (4) was stirred overnight, and then incubated with 0.5 mL of 2 mg / mL superoxide dismutase to obtain the target product.
[0063] Example 3: Preparation of the chemiluminescent nano-probe of the present application
[0064] (1) 10 mg of polylactic acid-glycolic acid copolymer and 10 mg of bisoxalate were mixed and dispersed in 3 mL of dichloromethane to obtain an oil phase solution;
[0065] (2) 5 μL of 2 mg / mL rhodamine 6G aqueous solution was added to 200 μL of polyvinyl alcohol aqueous solution with a mass concentration to obtain an aqueous phase solution;
[0066] (3) The aqueous phase solution prepared in step (2) was slowly transferred to the oil phase solution prepared in step (1), and ultrasonic treatment was performed for 15 minutes under ice water bath condition to obtain a first emulsion solution;
[0067] (4) The first emulsion solution obtained in step (3) was added to 3 mL of polyvinyl alcohol solution with a mass concentration of 5%, and ultrasonic treatment was performed for 15 minutes under ice water bath condition to obtain a second emulsion;
[0068] (5) The solution of step (4) was stirred overnight, and then incubated with 0.5 mL of 2 mg / mL superoxide dismutase to obtain the target product.
[0069] Example 4: Preparation of the chemiluminescent nano-probe of the present application
[0070] (1) 10 mg of polyoxyethylene polyoxypropylene and 10 mg of bisoxalate were mixed and dispersed in 3 mL of toluene to obtain an oil phase solution;
[0071] (2) 10 μL of 10 mg / mL rhodamine 6G aqueous solution was added to 300 μL of polyvinyl alcohol aqueous solution with a mass concentration to obtain an aqueous phase solution;
[0072] (3) The aqueous phase solution prepared in step (2) was slowly transferred to the oil phase solution prepared in step (1), and ultrasonic treatment was performed for 15 minutes under ice water bath condition to obtain a first emulsion solution;
[0073] (4) The first emulsion solution obtained in step (3) was added to 5 mL of polyvinyl alcohol solution with a mass concentration of 5%, and ultrasonic treatment was performed for 15 minutes under ice water bath condition to obtain a second emulsion;
[0074] (5) The solution of step (4) was stirred overnight, and then incubated with 1 mL of 5 mg / mL superoxide dismutase to obtain the target product.
[0075] Example 5: Preparation of the chemiluminescent nanoprobes of the present application
[0076] (1) 20 mg of polylactic acid-glycolic acid copolymer and 10 mg of bis-oxalate were mixed and dispersed in 3 mL of toluene to obtain an oil phase solution;
[0077] (2) 10 μL of 5 mg / mL aqueous solution of chlorin was added to 300 μL of polyvinyl alcohol aqueous solution with a mass concentration of 5% to obtain an aqueous phase solution;
[0078] (3) The aqueous phase solution prepared in step (2) was slowly transferred to the oil phase solution prepared in step (1), and ultrasonic treatment was performed for 15 minutes under ice water bath conditions to obtain a first emulsion solution;
[0079] (4) The first emulsion solution obtained in step (3) was added to 5 mL of polyvinyl alcohol solution with a mass concentration of 5%, and ultrasonic treatment was performed for 15 minutes under ice water bath conditions to obtain a second emulsion;
[0080] (5) The solution after completion of the reaction in step (4) was stirred overnight, and 0.5 mL of 4 mg / mL superoxide dismutase was incubated to obtain the target product.
[0081] Example 6: Preparation of the chemiluminescent nanoprobes of the present application
[0082] (1) 10 mg of polyoxyethylene polyoxypropylene and 5 mg of bis-oxalate were mixed and dispersed in 3 mL of dichloromethane to obtain an oil phase solution;
[0083] (2) 30 μL of 5 mg / mL aqueous solution of chlorin was added to 300 μL of polyvinyl alcohol aqueous solution with a mass concentration of 5% to obtain an aqueous phase solution;
[0084] (3) The aqueous phase solution prepared in step (2) was slowly transferred to the oil phase solution prepared in step (1), and ultrasonic treatment was performed for 15 minutes under ice water bath conditions to obtain a first emulsion solution;
[0085] (4) The first emulsion solution obtained in step (3) was added to 10 mL of polyvinyl alcohol solution with a mass concentration of 5%, and ultrasonic treatment was performed for 15 minutes under ice water bath conditions to obtain a second emulsion;
[0086] (5) The solution after completion of the reaction in step (4) was stirred overnight, and 0.8 mL of 5 mg / mL superoxide dismutase was incubated to obtain the target product.
[0087] Experimental Example: Characterization experiment of the chemiluminescent nanoprobes
[0088] The chemiluminescent nanoprobes prepared in Example 1 were analyzed, and other examples were similar to Example 1.
[0089] 1. Morphology characterization: The chemiluminescent nanoprobes were prepared on carbon film copper grid, and were characterized by 200kv field emission transmission electron microscopy.
[0090] It can be seen from the transmission electron microscopy image that the prepared chemiluminescent nanoprobes have good dispersity, and the diameter is about 100-150 nanometers. Figure 2
[0091] 2. Optical characterization:
[0092] A certain concentration of hydrogen peroxide or potassium superoxide was mixed with the chemiluminescent nanoprobes, and the fluorescence spectrum and fluorescence intensity-time curve of the mixed system were tested. By controlling the concentration of hydrogen peroxide or potassium superoxide, the influence of the same on the luminescence intensity of the chemiluminescent nanoprobes was investigated.
[0093] (1) blank, (2) Cu 2+ , (3) Cr 3+ , (4) MnO4 - , (5) Fe 2+ , (6) Fe 3+ , (7) Cr2O4 2- , (8) ClO - were selected as interference ions to evaluate the detection selectivity of the chemiluminescent nanoprobes.
[0094] It can be seen from the chemiluminescence spectrum that the prepared chemiluminescent nanoprobes can obtain a chemiluminescence signal at 680 nanometers under the action of hydrogen peroxide or potassium superoxide (which can be used to generate superoxide free radicals). Figure 3 The luminescence half-life of the chemiluminescent nanoprobes is about 1000 seconds. Figure 3 The chemiluminescence signal intensity is positively correlated with the concentration of superoxide free radicals in the system (32.5-500 micromoles), and the sensitive detection of superoxide free radicals can be realized (detection limit: ~ 0.27 micromole). Figure 3 In addition, the probe still has good selectivity in the presence of different interference ions (same concentration) (only hydrogen peroxide and superoxide free radicals can trigger significant chemiluminescence). Figure 3
[0095] In addition, the present application also attempts to replace the selected materials of the present application with other materials to prepare the probe according to the method of Example 1, such as using methoxy-polyethylene glycol-polycaprolactone, polyacrylamide, polylactic acid-polyethylene glycol, etc. as the amphiphilic polymer, using fluorescein sodium, coumarin, 1,8-naphthalimide, etc. as the fluorescent dye, but the chemiluminescence spectrum of these probes cannot achieve stable optical emission characteristics.
[0096] A drug-induced acute kidney injury model based on the mouse in vivo level was established. After drug (cisplatin) treatment for different times (8-72 hours), the obtained probe was injected into the mouse body through the tail vein, and the small animal imaging equipment was used for continuous (0-90 minutes) chemiluminescence in vivo imaging. The results show that the kidney site exhibits significant optical properties (Fig. 6) 12 hours after drug treatment of the mouse, indicating that the probe has the ability to realize early detection of diseases related to superoxide free radicals. Figure 4 ). It is shown that the probe has the ability to realize early detection of diseases related to superoxide free radicals.
[0097] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A chemiluminescent nanoprobe, characterized in that: The invention comprises a first aqueous phase as an inner layer, an oil phase as a middle layer and a second aqueous phase as an outer layer, forming a water-in-oil-in-water emulsion; The first aqueous phase includes an organic dye and polyvinyl alcohol, the oil phase includes an amphiphilic polymer and an oxalate-based chemiluminescent energy donor, and the second aqueous phase includes superoxide dismutase and polyvinyl alcohol; the organic dye includes one or more of dihydrochlorin, rhodamine 6G, and rhodamine B, the amphiphilic polymer includes one or more of polylactic acid-glycolic acid copolymer and polyoxyethylene polyoxypropylene, and the oxalate-based chemiluminescent energy donor includes bisoxalate.
2. The chemiluminescent nanoprobe according to claim 1, characterized in that The concentration of the organic dye in the first aqueous phase is 0.01-10 mg / mL; the concentration of the amphiphilic polymer in the oil phase is 1-10 mg / mL, and the concentration of the oxalate-based chemiluminescent energy donor is 1-5 mg / mL; the concentration of superoxide dismutase in the second aqueous phase is 0.05-5 mg / mL.
3. Use of the chemiluminescent nanoprobe according to any one of claims 1 to 2 in the preparation of a product for detecting superoxide radical-related diseases.
4. The use according to claim 3, characterized in that The superoxide radical-related diseases include kidney damage, brain damage and liver damage.
5. The method for preparing the chemiluminescent nanoprobe according to claim 1, characterized in that: include: The amphiphilic polymer and the oxalate-based chemiluminescent energy donor are mixed and dispersed in an organic solvent to obtain an oil phase; preparing an aqueous solution of an organic dye and polyvinyl alcohol to obtain a first aqueous phase; Transferring the first aqueous phase to the oil phase for ultrasonic treatment to obtain a first emulsion; transferring the first emulsion into a polyvinyl alcohol aqueous solution for ultrasonic treatment to obtain a second emulsion; The second emulsion is co-incubated with a superoxide dismutase solution to form an outermost second aqueous phase, thereby obtaining a chemiluminescent nanoprobe emulsion with a water-in-oil-in-water structure.
6. The preparation method according to claim 5, characterized in that The organic solvent includes an organic solvent with a polarity less than 4.
7. The preparation method according to claim 6, characterized in that The organic solvent includes dichloromethane and / or toluene.