Probe for detecting endogenous zinc ions in lysosome as well as preparation method and application of probe
By encapsulating zinc ion-responsive hydrophobic fluorescent dyes in DNA nanocages, probes with lysosomal targeting capabilities were prepared, solving the problem of detecting zinc ions in endogenous lysosomes of neurons and achieving highly sensitive imaging monitoring.
Patent Information
- Application Number
- CN202410762597.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-13
- Publication Date
- 2025-12-16
AI Technical Summary
Existing probes cannot effectively detect zinc ions in neuronal endogenous lysosomes due to a balance problem between hydrophilicity, brightness, and lysosomal targeting, resulting in insufficient detection sensitivity.
A zinc ion-responsive hydrophobic fluorescent dye was encapsulated in DNA nanocages. The dye was encapsulated in the hydrophobic cavity of the DNA nanocage by shaking or ultrasonic mixing, forming a probe with lysosomal targeting capability.
It achieves highly sensitive imaging monitoring of zinc ions in neuronal endogenous lysosomes, solving the problem of zinc ion detection in AD progression and significantly improving detection sensitivity and brightness.
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Figure CN121136699A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, and more particularly to a preparation method of a lysosome endogenous zinc ion detection probe and its application in the detection of lysosome zinc levels in an AD disease model. BACKGROUND
[0002] Zinc homeostasis imbalance plays a key role in the progression of Alzheimer's disease (AD). Endosome-lysosome is the main organelle that stores endogenous zinc ions, and when overloaded, it can cause an increase in lysosome membrane permeability, leading to the release of various acid hydrolases into the cytoplasm. Zinc ions can serve as a marker for monitoring the progression of AD. However, the concentration of endogenous zinc ions in neuronal cells is usually below the detection threshold of current in situ imaging techniques.
[0003] In previous studies, a large amount of work has been devoted to the development of zinc ion-sensitive probes. However, due to the balance between hydrophilicity, brightness, lysosome targeting, and zinc ion sensitivity, these probes cannot detect endogenous lysosome zinc ions in living cells. For example, organic dyes are usually small hydrophobic molecules that require covalent chemical modification to improve water solubility and specific targeting of organelles, but this can reduce their brightness and thus sensitivity. Larger-sized inorganic nanoparticles, such as quantum dots, metal nanoclusters, and upconversion nanoparticles, typically exhibit stable high brightness, and some of them show the ability to detect intracellular zinc ions when introduced into cells in vitro or exogenously, however, detecting endogenous lysosome zinc ions in cells is still not possible. Therefore, it is necessary to develop highly sensitive probes to detect and image endogenous lysosome zinc ions in neuronal cells.
[0004] Advances in DNA nanotechnology have facilitated the construction of DNA framework structures (DNFs) with programmable geometric parameters, superior hydrophilicity, and biocompatibility. These structures can serve as universal scaffolds for the precise spatial arrangement of various functional materials such as fluorescent dyes, inorganic nanoparticles, and proteins. More importantly, DNFs can be internalized by mammalian cells, enabling them to serve as transport carriers for a variety of substances. SUMMARY
[0005] The purpose of the present application is to provide a lysosome endogenous zinc ion detection probe and its preparation method, as well as its application in the detection of zinc levels in an Alzheimer's disease (AD) biological model, thereby solving the problem that the original probe cannot detect and image endogenous lysosome zinc ions in neurons.
[0006] To solve the above technical problems, the present application adopts the following technical solutions:
[0007] According to a first aspect of the present application, there is provided a method for preparing a lysosome endogenous zinc ion detection probe, comprising the following steps: 1) preparing a DNA nanocage with cell lysosome targeting ability and containing a specific hydrophobic cavity; 2) providing a hydrophobic fluorescent dye with zinc ion response ability; and 3) mixing the DNA nanocage and the hydrophobic fluorescent dye with zinc ion response ability by shaking or ultrasonic mixing, so that the hydrophobic fluorescent dye enters the hydrophobic cavity of the DNA nanocage and is encapsulated therein, thereby obtaining a lysosome endogenous zinc ion detection probe.
[0008] In step 1), the DNA nanocage with cell lysosome targeting ability and containing a specific hydrophobic cavity has hydrophobic groups facing the inside of the DNA nanocage so as to form an internal hydrophobic cavity and has a cell lysosome targeting function. It should be understood that the DNA nanostructure is negatively charged, and after being taken up by cells, it enters the early endosome through the caveolin and scavenger receptor-mediated endocytosis pathway, and gradually moves to and fuses with the lysosome, and finally locates in the lysosome. Therefore, the DNA nanocage prepared according to the present application has a cell lysosome targeting function. In addition, the size of the DNA nanostructure is preferably in the range of 5-100 nm, which is too small to form a DNA nanocage, and too large to easily cause lysosome swelling and cause cytotoxicity.
[0009] According to the present application, the inventors have prepared a plurality of DNA nanocage structures, among which the cell uptake and lysosome co-localization effects of a DNA tetrahedron with a side length of 20 bp, a DNA cube with a side length of 20 bp, and a DNA tetrahedron with a side length of 37 bp have been studied.
[0010] Preferably, the DNA tetrahedron with a side length of 20 bp has the best cell uptake and lysosome co-localization effect in nerve cells.
[0011] In step 2), the hydrophobic fluorescent dye with zinc ion response ability includes commercially available zinc ion probes, newly synthesized zinc ion probes by researchers, such as TSQ, ZnAF-1, FluoZin TM -3, NapBu-BPEA, and other hydrophobic fluorescent molecules with zinc ion response ability.
[0012] Preferably, the hydrophobic fluorescent dye with zinc ion response ability is NapBu-BPEA, which is synthesized by the research group, has an excitation wavelength of 450 nm, and an emission wavelength of 550 nm (see literature: Simultaneous Zn 2+tracking in multiple organelles using super-resolution morphology-correlated organelle identification in living cells. https: / / doi.org / 10.1038 / s41467-020-20309-7). It should be understood that this fluorescent dye is only exemplified as a preferred embodiment and is not intended to be limiting, in fact, other conventional small molecule dyes with hydrophobic properties that are responsive to zinc ions are suitable for use in the present application.
[0013] According to a second aspect of the present application, there is provided a lysosome endogenous zinc ion detection probe prepared by the above preparation method, wherein the lysosome endogenous zinc ion detection probe is prepared by encapsulating a zinc ion responsive hydrophobic fluorescent dye in a DNA nanocage with a hydrophobic cavity through hydrophobic-hydrophilic interaction, wherein the zinc ion responsive fluorescent dye has both hydrophobicity and zinc ion responsiveness.
[0014] According to a third aspect of the present application, there is provided a use of a lysosome endogenous zinc ion detection probe prepared by the above preparation method in the detection of endogenous zinc levels in an AD biological model.
[0015] Preferably, the AD biological model includes a drug-induced model (e.g., biochemical reagent-induced such as okadaic acid, D-galactose, etc.) and a transgenic model, etc. The biological model includes a cell model, an animal model (e.g., zebrafish, mouse, etc.).
[0016] The AD cell model includes a primary neuron cell, a neural stem cell, a clonal cell line from a neurogenic tumor (e.g., human neuroblastoma cell SH-SY5Y and BE(2)-M17 cell line, hybrid cell NC108-15 cell line, neuroblastoma cell Neuro-2A cell line, microglial cell BV2 cell line, hippocampal neuron cell HT22 cell line, rat pheochromocytoma cell PC12 cell line (commonly used for studying neuron cell death and neurotoxic injury), etc.).
[0017] It should be understood that primary cell culture and stem cell culture have a limited lifespan, genetic variation is easily increased between model systems and culture, different neuron populations are mixed, and it is expensive. Preferably, the clonal cell line of the neurogenic tumor is the best in vitro cell model due to its easy availability, easy growth, easy observation, rapid isolation, continuous passage, provision of a large number of cells in a short period of time, and many other advantages.
[0018] The creativity of the present application mainly lies in: 1) designing DNA structures of different sizes and screening to determine the structure with the highest lysosome co-localization efficiency; 2) the wrapping of the DNA structure effectively alleviates the aggregation and fluorescence quenching of the dye molecules in the water environment, thereby significantly improving the brightness; 3) the probe can be effectively internalized by neurons and localized in lysosomes, providing lysosome targeting ability, and showing high sensitivity of the probe to endogenous lysosome zinc ions in AD cells and AD zebrafish and other models.
[0019] In summary, the lysosome endogenous zinc ion probe provided by the present application has good brightness, cell lysosome targeting ability and zinc ion detection sensitivity, and in practical applications, the dynamic monitoring of the change of the endogenous zinc ions in the lysosomes of AD cells and AD zebrafish models is realized. Therefore, the probe can be better used for imaging and monitoring of endogenous lysosome zinc ions in neurons, and at the same time solves the problem of difficult detection of zinc ions related to AD progression. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 The figure shows the quantitative statistical results of the co-localization efficiency of tetrahedron and cube with the same size and different morphology with PC12 cell lysosomes in Example 1;
[0021] Figure 2 The figure shows the quantitative statistical results of the co-localization efficiency of tetrahedron with the same morphology and different size with PC12 cell lysosomes in Example 2;
[0022] Figure 3 The figure shows the fluorescence spectrum of NapBu-BPEA and the agarose verification of the successful wrapping of the dye in Example 3;
[0023] Figure 4 The figure shows the linear fitting of the response of NapBu-BPEA and the lysosome endogenous zinc ion probe to zinc ions in a physiological environment in Example 4;
[0024] Figure 5A The figure shows the fluorescence intensity of NapBu-BPEA in a mixture of water and DMSO with different volume ratios in Example 5;
[0025] Figure 5B The figure shows the fluorescence intensity of NapBu-BPEA and the endogenous zinc ion probe in an aqueous solution system in Example 5;
[0026] Figure 6 The figure shows the cytotoxicity data of the lysosome endogenous zinc ion probe with different concentrations in Example 6;
[0027] Figure 7AThe results of semi-quantitative analysis of zinc ion fluorescence signal in an AD cell model by the lysosomal endogenous zinc ion probe in Example 7, as well as the co-localization efficiency of zinc ion fluorescence signal with cell lysosomes, are shown.
[0028] Figure 7B The fluorescence signal of zinc ions in an AD cell model using the lysozyme endogenous zinc ion probe in Example 7 is shown.
[0029] Figure 8 The results of zinc ion fluorescence imaging of an AD cell model using NapBu-BPEA and lysozyme endogenous zinc ion probes in Example 8 are shown.
[0030] Figure 9A The semi-quantitative results of zinc ion fluorescence signal in the AD zebrafish model by the probe in Example 9 are shown, as well as the co-localization efficiency of zinc ion fluorescence signal with cell lysosomes.
[0031] Figure 9B The zinc ion fluorescence signal of the probe in Example 9 on the AD zebrafish model is shown. Detailed Implementation
[0032] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are performed according to conventional methods and conditions, or as selected according to the product instructions.
[0033] In the following specific embodiments, this invention mainly synthesizes a zinc ion-responsive fluorescent small molecule, NapBu-BPEA, as the luminescent core, and uses a tetrahedron with a hydrophobic alkyl chain as a nanocage modified for hydrophilic and hydrophobic properties of the fluorescent molecule to construct an endogenous zinc ion probe for lysozyme. This probe is then used in imaging to achieve dynamic monitoring of zinc ion levels in Alzheimer's disease (AD) model cells and zebrafish. The following examples illustrate the implementation effects of this invention.
[0034] Example: Comparison of the targeting ability of 20bp DNA tetrahedrons and cubes to lysosomes.
[0035] Cell culture. PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in RPMI 1640 medium (containing 10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine) at 37°C in a 5% CO2 incubator. 7 × 10⁶ cells were cultured. 4A certain number of cells were seeded in a confocal dish and placed in a cell incubator overnight. After the cells adhered to the dish, DNA tetrahedrons and cubes with a side length of 20 bp (final concentration 200 nM) were co-incubated with the cells for 2 hours, and then a lysosome probe was loaded. The cells were imaged using a laser confocal microscope (Leica sp8) to observe the lysosome co-localization of these two nanostructures with the same size but different morphologies in PC12 cells.
[0036] The sequences used for the DNA nanostructures are shown in Table 1 below (SEQ ID NO.1-9):
[0037] Table 1
[0038] D is a modified hydrophobic alkyl chain, and its structural formula is as follows:
[0039] Synthesis of 20-bp tetrahedron: The single-stranded DNAs, S1-D, S2-D, S3-D, and S4-D, which were accurately quantified by an ultraviolet spectrophotometer, were added to TM buffer at a stoichiometric ratio of 1:1:1:1. The final concentration of each single-stranded DNA was 1 μM. After thorough mixing, it was placed in a PCR instrument for high-temperature annealing to obtain a 20-bp tetrahedron structure modified with Cy5 fluorescence. It should be understood that the present invention is not limited to this hydrophobic alkyl chain. The hydrophobic groups include: alkyl alcohol {HO-(CH2) x -} y -, cholesterol, polycyclic aromatic hydrocarbon structure, or other molecular structures with hydrophobic properties, where x is the length of the alkyl chain, 1 < x < 100, and y is the number of alkyl chains, 1 < y < 100.
[0040] Synthesis of 20-bp cube: The single-stranded DNAs, 1AA, 2AA, 3AA, 4AA, and A’-D, which were accurately quantified by an ultraviolet spectrophotometer, were added to TAM buffer at a stoichiometric ratio of 1:1:1:1:8. The final concentration of the four AA single-stranded DNAs was 1 μM, and the final concentration of the A’-D single-stranded DNA was 8 μM. After thorough mixing, it was placed in a PCR instrument for high-temperature annealing to obtain a 20-bp cube structure modified with Cy5 fluorescence.
[0041] Results: As Figure 1 , the results of laser confocal imaging showed that both the 20-bp DNA tetrahedron and cube co-localized with the lysosomes of PC12 cells after being incubated with the cells for 2 hours, and the co-localization efficiency of the tetrahedron and cell lysosomes was higher than that of the cube.
[0042] Example 2: Comparison of lysosomal targeting ability of DNA tetrahedra with side lengths of 20 bp and 37 bp
[0043] Cell culture. PC12 cells were cultured in the same manner as in Example 1. After the cells adhered, tetrahedra with side lengths of 20 bp and 37 bp (final concentration 200 nM) were co-incubated with the cells for 2 hours, and then loaded with lysosomal probes. The cells were imaged using a laser confocal microscope (Leica sp8) to observe the co-localization of these two DNA nanostructures of different sizes with the same morphology in PC12 cells.
[0044] The 20bp DNA tetrahedron used the same sequence and preparation method as in Example 1, while the 37bp DNA tetrahedron used the sequences shown in Table 2 below (SEQ ID NO.10-17):
[0045] Table 2
[0046] Synthesis of 37bp DNA tetrahedrons: Single-stranded DNA, precisely quantified by a UV spectrophotometer, consisting of A3, A5-D, B3, B5-D, C3, C5-D, D3, and D5-D, was added to TM buffer in a stoichiometric ratio of 1:1:1:1:1:1:1:1. The final concentration of each single strand was 1 μM. After thorough mixing, the mixture was placed in a PCR instrument for high-temperature annealing to obtain a Cy5-modified tetrahedron structure with a side length of 37bp.
[0047] Result: As Figure 2 Statistical results from laser confocal imaging showed that DNA tetrahedra with sides of 20 bp and 37 bp could co-localize with lysosomes after being incubated with PC12 cells for 2 hours, and the co-localization efficiency of the 20 bp tetrahedron with lysosomes was higher than that of the 37 bp tetrahedron.
[0048] Example 3: Comparison of the encapsulation of different zinc ion dyes by 20bp DNA tetrahedra
[0049] This invention uses NapBu-BPEA as an example, and its luminescence properties are characterized using fluorescence spectroscopy. NapBu-BPEA dissolved in dimethyl sulfoxide and 20bp tetrahedra are mixed at a molar ratio of 100:1 and incubated overnight. Excess NapBu-BPEA is removed by passing it through a Nap-5 column, and the successful encapsulation of the dye is verified by agarose gel electrophoresis.
[0050] result: Figure 3Fluorescence spectroscopy showed that the emission peak of NapBu-BPEA was at 550 nm. Under blue light gel illumination, NapBu-BPEA exhibited a distinct fluorescent band. Further gel staining with GelRed revealed that the fluorescent band co-localized with the DNA band, indicating that NapBu-BPEA was indeed encapsulated within the hydrophobic cavity of the DNA tetrahedron. Therefore, this embodiment successfully prepared a lysozyme-derived in vivo zinc ion probe.
[0051] Example 4: Evaluation of the detection sensitivity of lysozyme endogenous zinc ion probes in the physiological environment
[0052] The sensitivity of the lysosomal endogenous zinc ion probe was evaluated in an acidic environment (pH 5.0) simulating the lysosomal environment. The fluorescence emission intensity of the lysosomal endogenous zinc ion probe (1.6 μM, equivalent to 5 μM NapBu-BEPA) and NapBu-BEPA (5 μM) dye prepared in Example 3 was repeatedly measured, and the background noise of the two probes was determined. After thoroughly mixing the probes with zinc ion solutions of different concentrations (0-5 μM), the spectroscopic characterization was performed using a fluorescence spectrometer. Linear regression curves were fitted based on the emission intensity of the lysosomal endogenous zinc ion probe and NapBu-BEPA in the 0-5 μM range, as shown in the figure. Figure 4 As shown.
[0053] Results: In a simulated lysosomal environment, the detection limit of the lysosomal endogenous zinc ion probe was 31.9 nM, and the detection limit of the small molecule NapBu-BEPA was 76.5 nM. This indicates that the encapsulation of NapBu-BEPA by the DNA tetrahedral structure significantly improves its detection sensitivity for zinc ions.
[0054] Example 5: DNA tetrahedral encapsulation prevents the aggregation and quenching of NapBu-BEPA small molecules in an aqueous environment.
[0055] NapBu-BPEA at a final concentration of 5 μM was dispersed in solutions with different water volumes (water:DMSO: 0:100, 20:80, 40:60, 60:40, 80:20, and 99.9:0.1, respectively). The fluorescence intensity of NapBu-BPEA in different solution systems was detected using a fluorescence spectrometer. Furthermore, the fluorescence intensity of 1.6 μM lysozyme endogenous zinc ion probe (equivalent to 5 μM NapBu-BPEA) and 5 μM NapBu-BPEA was detected in a 99.9% aqueous solution.
[0056] result: Figure 5A The results showed that the fluorescence intensity of NapBu-BPEA molecules decreased with increasing water content in the solution system, demonstrating that NapBu-BPEA molecules exhibit an aggregation-induced quenching effect in an aqueous environment. Figure 5BIn the same aqueous system, the fluorescence intensity of the lysozyme endogenous zinc ion probe was significantly higher than that of NapBu-BPEA, indicating that the encapsulation of NapBu-BPEA by the DNA structure effectively alleviated the aggregation of dye molecules and fluorescence quenching in the aqueous environment, thereby significantly improving the brightness.
[0057] Example 6: Biosafety Assessment of Lysozyme In vivo Zinc Ion Probes
[0058] The cytotoxicity of the probe was measured using the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. PC12 cells were cultured at 3 × 10⁶ cells per well. 3 PC12 cells were seeded at a density of 1000 μL in 24-well plates and incubated for 12 hours to allow cell adhesion. Different concentrations of lysozyme endogenous zinc ion probes were then incubated with PC12 cells for 12 hours. The cells were then repeatedly washed with PBS to remove unbound probes, and fresh cell culture medium was added to each well. 500 μL of medium containing 0.5% MTT was added to each well, and after 4 hours of incubation, 500 μL of triple lysis buffer was added to each well. The cells were then incubated at 37°C for 4 hours, and the absorbance was measured at 570 nm using a microplate reader.
[0059] result: Figure 6 The MTT assay results showed that even at the highest exposure dose of 600 nM, the lysozyme endogenous zinc ion probe had almost no toxicity to cells.
[0060] Example 7: Detection of zinc ions in an AD cell model using an in vivo lysozyme zinc ion probe.
[0061] The PC12 cell culture method is the same as in Example 1, with PC12 cells cultured at 8 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 nM in confocal culture dishes and incubated overnight to allow cell adhesion. Okaya acid (OKA) is a protein phosphatase 2A inhibitor that can induce neurodegenerative diseases. To construct AD cell models of different disease progression processes, PC12 cells were exposed to OKA (100 nM) for 2 hours and 6 hours, respectively. After washing three times with PBS, the cells were incubated with an endogenous zinc ion probe (300 nM) on a lysosome for 2 hours. Excess probe was removed, and the cells were loaded with lysosomal probes. The images were then captured using a confocal fluorescence microscope, and the colocalization of the probe and lysosomes was calculated using ImageJ software.
[0062] result: Figure 7A (The white bright spots represent the fluorescent signal of zinc ions labeled with the probe.) Figure 7B (White bars) show that the fluorescence intensity of the lysosomal endogenous zinc ion probe increases with the extension of OKA incubation time, indicating that the zinc ion signal intensity is positively correlated with AD progression, and approximately 90% of the zinc ion fluorescence signal is consistently colocalized with lysosomes. Figure 7B(Slash bar). This demonstrates that the probe has the ability to target lysosomes in AD cell models and can perform imaging monitoring of endogenous lysosomal zinc ions in AD cell models.
[0063] Example 8: Comparison of zinc ion detection in an AD cell model using lysozyme endogenous zinc ion probes and NapBu-BPEA dye.
[0064] The PC12 cell culture and drug treatment methods were the same as in Example 7. After cell adhesion, the PC12 cells were exposed to OKA (100nM) for 2 hours and 6 hours, respectively. After washing three times with PBS, the cells were incubated for 2 hours in lysozyme endogenous zinc ion probe (300nM) and NapBu-BEPA (900nM), respectively, and imaged by confocal fluorescence microscopy.
[0065] result: Figure 8 The confocal fluorescence imaging results shown indicate that the fluorescence signal intensity in AD cells labeled with lysozyme endogenous zinc ion probes is significantly higher than that labeled with NapBu-BPEA small molecule probes (white bright spots represent the zinc ion fluorescence signal labeled with probes).
[0066] Example 9: Detection of zinc ions in an AD zebrafish model using an endogenous zinc ion probe from lysozyme.
[0067] To construct zebrafish models of Alzheimer's disease (AD) at different disease progression stages, zebrafish were exposed to OKA (200 nM), with the OKA water being replaced every other day. Zebrafish models at different AD development stages were obtained after day 3 and day 15. Zebrafish were anesthetized in culture dishes with a final concentration of 0.016% (v / v) ethyl 3-aminobenzoate methanesulfonate, and a micro-incision of approximately 200 μm was made in the skull using a fine needle. 500 nL of lysozyme-based endogenous zinc ion probe solution dispersed in physiological saline was injected using a thin glass capillary. One hour after the zebrafish awoke, they were euthanized and incubated overnight in 4% paraformaldehyde at 4°C. Brain tissue was dissected and frozen at -80°C, and 6 μm sections were prepared. Lysosomes were stained with LAMP1 primary antibody (1:1000), and fluorescence imaging was performed using confocal fluorescence microscopy. ImageJ software was used to calculate the colocalization of the probe with lysosomes in the brain tissue sections.
[0068] result: Figure 9A (The white area represents the fluorescent signal of zinc ions labeled with the probe.) Figure 9B (White bars) show that the fluorescence intensity of the lysosomal endogenous zinc ion probe increases with the extension of the incubation days of zebrafish OKA, indicating that the zinc ion signal intensity is positively correlated with AD progression, and approximately 90% of the zinc ion fluorescence signal is consistently colocalized with lysosomes. Figure 9BThe slash bar (slash bar) demonstrates that the probe can image and monitor endogenous lysosomal zinc ions in brain neurons of the AD zebrafish model.
[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.
Claims
1. A method for preparing a lysozyme endogenous zinc ion detection probe, characterized in that, Includes the following steps: 1) Modify DNA nanostructures with hydrophobic groups to prepare a DNA nanocage with specific hydrophobic cavities that has cell lysosome targeting capability; 2) Provide a hydrophobic fluorescent dye with zinc ion responsiveness; as well as 3) The DNA nanocage and the hydrophobic fluorescent dye with zinc ion responsiveness are mixed by shaking or sonication, so that the hydrophobic fluorescent dye enters the hydrophobic cavity of the DNA nanocage and is encapsulated therein, thereby obtaining a lysozyme endogenous zinc ion detection probe.
2. The preparation method according to claim 1, characterized in that, In step 1), the DNA nanocage containing a specific hydrophobic cavity is formed by thermal annealing of 4-N DNA chains with hydrophobic alkyl chains at the ends, where N is an integer greater than 4.
3. The preparation method according to claim 1, characterized in that, In step 1), the DNA nanocage includes DNA nanostructures of various morphologies and sizes, including: DNA tetrahedron, DNA cube, DNA triangular pyramid, and DNA triangular bipyramid.
4. The preparation method according to claim 1, characterized in that, In step 2), the zinc ion-responsive hydrophobic fluorescent dyes include: NapBu-BPEA, TSQ, ZnAF-1, and FluoZin. TM -3.
5. The preparation method according to claim 1, characterized in that, In step 3), the endogenous zinc ion detection probe has the ability to target lysosomes.
6. A lysozyme endogenous zinc ion detection probe prepared by the preparation method according to any one of claims 1-5, characterized in that, The lysozyme endogenous zinc ion detection probe is prepared by encapsulating a zinc ion-responsive hydrophobic fluorescent dye in a DNA nanocage with a hydrophobic cavity through hydrophilic-hydrophobic interactions. The zinc ion-responsive fluorescent dye is both hydrophobic and zinc ion responsive.
7. The application of the lysozyme endogenous zinc ion detection probe according to claim 6 in the detection of zinc levels in a biological model of Alzheimer's disease.
8. The application according to claim 7, characterized in that, The Alzheimer's disease biological models are divided into: Alzheimer's disease cell models and Alzheimer's disease animal models. The Alzheimer's disease biological models include: drug-induced models and transgenic models. The drugs in the drug-induced models include: okadaic acid and D-galactose.
9. The application according to claim 8, characterized in that, The Alzheimer's cell model includes: primary neurons, neural stem cells, and clonal cell lines derived from neurogenic tumors. The clonal cell lines include: human neuroblastoma cells SH-SY5Y and BE(2)-M17, mouse hybrid cells NC108-15, neuroblastoma cells Neuro-2A, microglia BV2, hippocampal neurons HT22, and rat adrenal pheochromocytoma cells PC12.
10. The application according to claim 8, characterized in that, The animal models of Alzheimer's disease include zebrafish, mice, and rats.