Nucleic acid nanoprobe for detecting signal amplification of A [beta] O and preparation method of nucleic acid nanoprobe

Abstract

The invention provides a nucleic acid nanoprobe for detecting signal amplification of A [beta] O, comprising: a signal output unit QDs, which is a CdSe / ZnS quantum dot modified by surface modification streptavidin synthesized by a thermosol method; the metal organic framework MOFs is synthesized by taking a metal ion Zr as a center and porphyrin as an organic ligand through a hydrothermal method, and is used as a carrier and a quencher of the CdSe / ZnS quantum dots at the same time; an Aptamer sequence of A [beta] O; the DNA1 sequence is designed according to the AbetaO Aptamer sequence and is complementary to the AbetaO Aptamer sequence, the 5'end of the DNA1 sequence is modified with a phosphorylation group, and the 3 'end of the DNA1 sequence is modified with avidin; stability is good, cost is low and sensitivity is high. The invention also provides a preparation method of the nucleic acid nanoprobe for detecting signal amplification of AbetaO. According to the present invention, the AbetaO in the peripheral blood is quantitatively detected, and the new thought and the new means can be provided for the early diagnosis of the Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and in particular relates to a nucleic acid nanoprobe for detecting AβO signal amplification and its preparation method. Background Technology

[0002] β-amyloid oligomers (AβO) are associated with cognitive impairment and disease severity in Alzheimer's disease (AD). A possible mechanism of AβO-induced toxicity is their binding to cell membrane receptors, leading to increased membrane porosity and accelerated intracellular accumulation. Simultaneously, AβO can interact with neurons and glial cells, activating pro-inflammatory cascades such as mitochondrial dysfunction and increased oxidative stress, impairing intracellular signaling pathways, interfering with calcium metabolism, and ultimately inducing neuronal apoptosis and cell death. Therefore, AβO is currently considered a promising candidate biomarker for the early diagnosis of AD.

[0003] In recent years, researchers have developed various methods for detecting AβO levels in serum, such as enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis, resonance light scattering (RLS), electrochemical detection, and polymerase chain reaction (PCR). While these methods have successfully detected AβO, most require expensive antibodies, which are susceptible to environmental influences, resulting in excessively high background signals and affecting detection efficiency. Currently, the most commonly used method in clinical practice is enzyme-linked immunosorbent assay (ELISA) for detecting serum AβO levels. However, in ELISA, firstly, antibodies are expensive; secondly, the ELISA signal is based on a time-dependent enzyme reaction, and enzymes are highly sensitive to environmental factors (temperature, time, etc.) and easily affected by matrix components, which can easily influence the detection results and lead to inaccurate results; these factors limit the clinical application of this method.

[0004] Aptamers are oligonucleotides obtained through phylogenetic analysis (SELEX) and can specifically recognize ions, small molecules, proteins, cells, and even tissues. They are widely used in biosensing, molecular detection, and drug delivery. Compared with traditional antibodies, aptamers have significant advantages such as high affinity, good stability, low cost, and convenient automated synthesis. Tsukakoshi et al. successfully isolated an AβO-specific aptamer by combining gel transfer and competitive screening methods. This aptamer has been successfully applied to bioassays in AD-related research. However, these methods mainly output a 1:1 signal conversion, which greatly limits the detection sensitivity of AβO and ultimately restricts its application in the early diagnosis of Alzheimer's disease. Summary of the Invention

[0005] One object of the present invention is to provide a nucleic acid nanoprobe for detecting signal amplification of AβO and a method for preparing the same, and to provide at least the advantages described below.

[0006] Another objective of this invention is to provide a nucleic acid nanoprobe for detecting AβO signal amplification, which has good stability, low cost, and high sensitivity, and can quantitatively detect AβO in peripheral blood, thereby improving the early diagnosis of AD and monitoring patients' response to treatment.

[0007] The technical solution of the present invention is as follows: Nucleic acid nanoprobes for detecting AβO signal amplification include: The signal output unit QDs are CdSe / ZnS quantum dots with surface-modified streptavidin synthesized by a hot melt method. Metal-organic frameworks (MOFs) are synthesized via a hydrothermal method with Zr ions at the center and porphyrins as organic ligands. They serve as a support and also as quenchers for CdSe / ZnS quantum dots. Aptamer sequence of AβO; The DNA1 sequence is designed based on the Aptamer sequence of AβO and is complementary to the Aptamer sequence of AβO, and the 5' end of the DNA1 sequence is modified with a phosphorylation group and the 3' end is modified with avidin. in; DNA1 sequence: 5'- GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Aptamer sequence of AβO: 5'-PO4 3--AGAGAGCCTGTGTTGGGGCGGGTGCG-3'; Signal output units (QDs) are modified onto the DNA1 sequence via a biotin-avidin reaction; The DNA1 sequence binds to the Aptamer sequence of AβO through complementary base pairing; Functionalization of the surface of metal-organic framework (MOF) nanoparticles by phosphorylation of the DNA1 sequence.

[0008] The preparation method of nucleic acid nanoprobes for detecting AβO signal amplification includes the following steps: CdSe / ZnS quantum dots with surface-modified streptavidin were synthesized by a thermosol method and used as signal output units (QDs). Metal-organic frameworks (MOFs) centered on Zr ions and with porphyrins as organic ligands were synthesized via a hydrothermal method and used as a support and quencher for CdSe / ZnS quantum dots. Based on the Aptamer sequence of AβO, a complementary DNA1 sequence was designed, and a phosphorylation group was modified at the 5' end and avidin was modified at the 3' end. Signal output units (QDs) were modified onto the DNA1 sequence via a biotin-avidin reaction, and then the DNA1 sequence was bound to the Aptamer sequence of AβO through complementary base pairing. AβO-responsive nucleic acid nanoprobes were constructed by functionalizing the surface of metal-organic framework (MOF) nanoparticles with the phosphorylation group of DNA1.

[0009] Preferably, in the method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification, DNA1 sequence: 5'- GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Aptamer sequence of AβO: 5'-PO4 3--AGAGAGCCTGTGTTGGGGCGGGTGCG-3'.

[0010] Preferably, in the method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification, surface-modified streptavidin-modified CdSe / ZnS quantum dots are synthesized by a thermosol method, including the following steps: Se powder was added to NaOH solution, stirred for 10 min under N2 protection, Al powder was added, the temperature was raised to 85℃, and sonicated for 15 min to generate Na2Se precursor solution. A CdSe quantum dot solution was prepared by adding a Na2Se precursor solution to a cadmium perchlorate solution. Ammonium methyl sulfate and zinc chloride solution were added to a CdSe quantum dot solution, and the mixture was sonicated at 85°C for 25 min. Then, activated streptavidin was added and stirred overnight. After purification, surface-modified streptavidin-modified CdSe / ZnS quantum dots were obtained.

[0011] Preferably, the method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification includes the following steps: Metal-organic frameworks (MOFs) centered on Zr ions and using porphyrin as organic ligands are synthesized via a hydrothermal method. 100 mg TCPP, 300 mg ZrOCl2 and 2.8 g phenylboronic acid reagent were added to 10 mL N,N-dimethylformamide DMF to form a mixture solution; The mixture solution was transferred to a hydrothermal reactor lined with tetrafluoroethylene and reacted at 90°C for 5 h. After the reaction was completed, the particles were centrifuged at 12,000 rpm for 5 min and washed three times with DMF to obtain MOF particles, which were then stored at 4℃ for later use.

[0012] The present invention has the following beneficial effects: By constructing nucleic acid nanoprobes responsive to β-amyloid oligomers (AβO), and applying the response analysis of nucleic acid nanoprobes to AβO and the determination of actual samples, the content of AβO in human peripheral blood was studied. This provides a theoretical basis for the early diagnosis of Alzheimer's disease (AD), fundamentally helping patients to detect and treat the disease early.

[0013] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0014] Figure 1 Characterization of the nucleic acid nanoprobe for detecting AβO signal amplification provided by this invention: 1a is a TEM image of ZrMOF, 1b is an infrared spectrum of ZrMOF, 1c is a thermogravimetric analysis diagram of ZrMOF, 1d is a comparison of the UV spectra of the nanoprobe and ZrMOF, 1e is a comparison of the particle size of the nanoprobe and ZrMOF, and 1f is a comparison of the potential of the nanoprobe and ZrMOF. Scale bar: 1 µm; Figure 2 The fluorescence recovery spectrum of the nucleic acid nanoprobe for detecting AβO signal amplification provided by the present invention; Figure 3 The time response curve of the nucleic acid nanoprobe for detecting AβO signal amplification provided by the present invention.

[0015] Figure 4 The synthesis efficiency diagram of the nucleic acid nanoprobe for detecting AβO signal amplification provided by the present invention; Figure 5 The standard curve of the response of the nucleic acid nanoprobe for detecting AβO signal amplification provided by the present invention to miR-16 is shown in Figure 5a; the fluorescence spectra of different concentrations of miR-16 and the nanoprobe are shown in Figure 5b; the standard curve of the response of the nanoprobe to miR-16 is shown in Figure 5b. Figure 6 The specificity diagram of the nucleic acid nanoprobe for detecting AβO signal amplification provided by the present invention is shown in Figure 6a; agarose gel electrophoresis is shown in Figure 6b; fluorescence spectrum is shown in Figure 6b. Figure 7 The diagram shows the cytotoxicity detection analysis of the nucleic acid nanoprobe for detecting AβO signal amplification provided by this invention. Figure 8Laser confocal imaging of the nucleic acid nanoprobe for detecting AβO signal amplification and miR-16 provided by the present invention; Figure 9 A plot showing the p53 protein levels after miR-16 treatment as revealed by Western blot. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0017] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not imply the presence or addition of one or more other elements or combinations thereof.

[0018] This invention provides a nucleic acid nanoprobe for detecting signal amplification of AβO, comprising: The signal output unit QDs are CdSe / ZnS quantum dots with surface-modified streptavidin synthesized by a hot melt method. Metal-organic frameworks (MOFs) are synthesized via a hydrothermal method with Zr ions at the center and porphyrins as organic ligands. They serve as a support and also as quenchers for CdSe / ZnS quantum dots. Aptamer sequence of AβO; The DNA1 sequence is designed based on the Aptamer sequence of AβO and is complementary to the Aptamer sequence of AβO, and the 5' end of the DNA1 sequence is modified with a phosphorylation group and the 3' end is modified with avidin. in; DNA1 sequence: 5'- GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Aptamer sequence of AβO: 5'-PO4 3--AGAGAGCCTGTGTTGGGGCGGGTGCG-3'; Signal output units (QDs) are modified onto the DNA1 sequence via a biotin-avidin reaction; The DNA1 sequence binds to the Aptamer sequence of AβO through complementary base pairing; Functionalization of the surface of metal-organic framework (MOF) nanoparticles by phosphorylation of the DNA1 sequence.

[0019] Quantum dots (QDs) typically refer to semiconductor fluorescent nanocrystals confined in a three-dimensional scale (2-20 nm) by elements of groups II-VI, III-V, and IV-VI. Recently, CdSe / ZnS quantum dots have attracted widespread attention due to their structural stability, high quantum yield, and low toxicity. Surface-modified streptavidin-modified CdSe / ZnS quantum dots were synthesized via a thermosol method, and CdSe / ZnS served as signal output units in the constructed probes. Metal-organic frameworks (MOFs) are organic-inorganic hybrid materials with intramolecular pores, assembled from organic ligands and metal ions through coordination bonds. MOFs centered on the metal ion Zr and using porphyrin (TCPP) as the organic ligand were synthesized via a hydrothermal method. MOFs served not only as carriers in the constructed nucleic acid nanoprobes but also as quenchers for quantum dots. Based on the Aptamer sequence of AβO, a complementary DNA1 sequence was designed, and a phosphorylation group was modified at its 5' end. To enable the signal output units (QDs) to be modified onto the Aptamer sequence, avidin needs to be modified at the 3' end of the Aptamer. The proposed method is to modify the QDs onto DNA1 via a biotin-avidin reaction, and then DNA1 binds to the Aptamer of AβO through base complementarity pairing. Finally, the phosphorylation group of DNA1 is functionalized onto the surface of MOF nanoparticles to construct AβO-responsive nucleic acid nanoprobes.

[0020] This invention also provides a method for preparing a nucleic acid nanoprobe for detecting AβO signal amplification, comprising the following steps: CdSe / ZnS quantum dots with surface-modified streptavidin were synthesized by a thermosol method and used as signal output units (QDs). Metal-organic frameworks (MOFs) centered on Zr ions and with porphyrins as organic ligands were synthesized via a hydrothermal method and used as a support and quencher for CdSe / ZnS quantum dots. Based on the Aptamer sequence of AβO, a complementary DNA1 sequence was designed, and a phosphorylation group was modified at the 5' end and avidin was modified at the 3' end. Signal output units (QDs) were modified onto the DNA1 sequence via a biotin-avidin reaction, and then the DNA1 sequence was bound to the Aptamer sequence of AβO through complementary base pairing. AβO-responsive nucleic acid nanoprobes were constructed by functionalizing the surface of metal-organic framework (MOF) nanoparticles with the phosphorylation group of DNA1.

[0021] In one embodiment of the method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification provided by the present invention, DNA1 sequence: 5'- GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Aptamer sequence of AβO: 5'-PO4 3--AGAGAGCCTGTGTTGGGGCGGGTGCG-3'.

[0022] In one embodiment of the method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification provided by the present invention, surface-modified streptavidin-modified CdSe / ZnS quantum dots were synthesized by a thermosol method, including the following steps: Se powder was added to NaOH solution, stirred for 10 min under N2 protection, Al powder was added, the temperature was raised to 85℃, and sonicated for 15 min to generate Na2Se precursor solution. A CdSe quantum dot solution was prepared by adding a Na2Se precursor solution to a cadmium perchlorate solution. Ammonium methyl sulfate and zinc chloride solution were added to a CdSe quantum dot solution, and the mixture was sonicated at 85°C for 25 min. Then, activated streptavidin was added and stirred overnight. After purification, surface-modified streptavidin-modified CdSe / ZnS quantum dots were obtained.

[0023] In one embodiment of the method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification provided by the present invention, metal-organic frameworks (MOFs) centered on Zr ions and using porphyrins as organic ligands are synthesized by a hydrothermal method, comprising the following steps: 100 mg TCPP, 300 mg ZrOCl2 and 2.8 g phenylboronic acid reagent were added to 10 mL N,N-dimethylformamide DMF to form a mixture solution; The mixture solution was transferred to a hydrothermal reactor lined with tetrafluoroethylene and reacted at 90°C for 5 h. After the reaction was completed, the particles were centrifuged at 12,000 rpm for 5 min and washed three times with DMF to obtain MOF particles, which were then stored at 4℃ for later use.

[0024] This invention designs a DNA1 sequence complementary to the Aptamer sequence of AβO and modifies it with a phosphorylation group at the 5' end. To enable the signal output units (QDs) to be modified onto the Aptamer sequence, avidin needs to be modified at the 3' end of the Aptamer. The specific DNA1 sequence and the Aptamer sequence of AβO are as follows: DNA1 sequence: 5'- GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Aptamer sequence of AβO: 5'-PO43--AGAGAGCCTGTGTTGGGGCGGGTGCG-3'.

[0025] Specific embodiments of the present invention are as follows: Table 1 Reagents and Materials reagents company benzoic acid Shanghai Aladdin Biochemical Technology Co., Ltd. Zirconium oxychloride, Octahydrate Shanghai Aladdin Biochemical Technology Co., Ltd. Meso-tetra(4-carboxyphenyl)porphyrin Shanghai Aladdin Biochemical Technology Co., Ltd. N,N-dimethylamide Tianjin Fuyu Fine Chemical Co., Ltd. PBS buffer Gibco Bovine serum albumin Solarborg HeLa cells, SY5Y cells Central laboratory provides DMEM medium Beijing Thermo Fisher SDS-PAGE gel preparation kit Azure Sky P53 antibody Shanxi Sai'ao Biotechnology Co., Ltd. BCA reagent kit Azure Sky Western and IP cell lysates Azure Sky CCK-8 reagent kit Solarborg Hoechst 3328 Azure Sky Table 2 Instruments and Models instrument model Biosafety cabinet 11229 BBC 86 CO2 incubator Thermo centrifuge 5424R Ultraviolet spectrophotometer UV-2600i Fluorescence spectrophotometer RF-6000 Fourier transform infrared spectrometer Irinmstat Nanoparticle size potentiometer Zatasizer Nano ZSE Simultaneous thermal analyzer; multi-functional imaging analysis system; laser scanning confocal microscope TG-DTA8122; Bio-Rad ChemiDoc(TM) MP; LEICA SP8 1. Synthesis of nanoprobes Accurately weigh 30 mg of zirconium oxychloride, ZrOCl2 octahydrate, 10 mg of racemic tetra(4-carboxyphenyl)porphyrin (TCPP), and 280 mg of benzoic acid, and dissolve them in 10 mL of N,N-dimethylamide (DMF) in a 20 mL glass bottle. Incubate the solution in an oil bath at 90 °C for 5 hours, then centrifuge three times at 25 °C at 13,000 rpm for 10 min. Collect the product and dry it to obtain 85 mg of a purple solid.

[0026] Prepare a ZrMOF solution with a concentration of 5.1 mg·mL⁻¹. Take 100 µL of the 5.1 mg·mL⁻¹ ZrMOF solution and add 4 µL of 10 µM fluorescently labeled aptamer (MB-miR-16). Shake at 37 °C for 5 h. Centrifuge at 12000 rpm for 3 min. Discard the supernatant. Add 400 µL of PBS buffer with pH 7.4 to the precipitate. The concentration of the prepared nanoprobe is 100 nM.

[0027] 2. Characterization of nanoprobes Prepare a ZrMOF solution of appropriate concentration, use DMF solution as a blank control, measure its absorbance, and plot the ultraviolet absorption spectrum; compress ZrMOF powder into tablets, use KBr as background, and plot the infrared spectrum; plot the thermogravimetric curve using a simultaneous thermal analyzer.

[0028] A fluorescence spectrum of a 100 nM nanoprobe was plotted at an excitation wavelength of 488 nm and an emission wavelength of 509–600 nm. The ultraviolet absorption of the nanoprobe was measured in DMF solution. The particle size and potential of the nanoprobe were measured by dissolving ZrMOF solution in PBS buffer and using the nanoprobe instrument.

[0029] Figure 2 a) shows ZrMOF nanoparticles synthesized in DMF. TEM images indicate that the size of the ZrMOF nanoparticles is approximately 100 nm. Figure 2 As shown in figure b, the stretching vibrations of alkyl groups are observed around 2800–3000 cm⁻¹; the stretching vibration peak of carbonyl groups (C=O) is around 1650 cm⁻¹; the stretching vibration peak of cyano groups (C≡N) is around 1440 cm⁻¹; and the stretching vibration peak of Zr−O is around 720 cm⁻¹. Figure 2 As shown in c, ZrMOF is relatively stable at 160 ℃, indicating that ZrMOF particles have good thermal stability; Figure 2 The nanoprobe in d showed a characteristic peak of Aptamer at 250 nm; Figure 2 In e, the particle size of the nanoprobe is larger than that of ZrMOF; Figure 2 ZrMOF itself carries a positive potential, while Aptamer carries a negative potential, so the resulting nanoprobe carries a negative potential. These characteristics demonstrate the synthesis of the nanoprobe.

[0030] 3. Solution-phase detection using nanoprobes 3.1 Binding rate of MOF to aptamers The ZrMOF concentration was fixed at 5.1 mg·mL⁻¹, and the aptamer concentrations were set from low to high as 20, 30, 40, 50, and 100 nM. After centrifugation and discarding the supernatant, 400 µL of PBS buffer was added, followed by 4 µL of 10 µM miR-16. Fluorescence recovery was measured, a standard curve was plotted, and the binding rate was calculated.

[0031] like Figure 2 As shown, the fluorescence of ZrMOF is quenched after being attached to Aptamer, and the fluorescence is restored when the nanoprobe binds to miR-16, proving that the two can bind and that the nanoprobe can be used for the detection of miR-16.

[0032] To determine the optimal time for the nanoprobe to react with miR-16, a time gradient experiment was conducted. The specific procedure was as follows: 100 nM nanoprobe solution and 10 μM miR-16 (final volume added: 4 μL) were mixed in a quartz cuvette and immediately placed in a constant temperature (25 ± 0.5 ℃) fluorescence spectrophotometer, and the fluorescence intensity changes were continuously scanned from 0 to 180 min.

[0033] like Figure 3 As shown, the fluorescence signal of the system showed a significant increasing trend within 0–150 min, indicating that the probe and miR-16 underwent a continuous hybridization reaction; when the reaction time exceeded 150 min, the fluorescence intensity entered a plateau phase. Therefore, 150 min was selected as the standard reaction time for subsequent experiments to ensure maximum detection signal and good reproducibility.

[0034] like Figure 4As shown in figure a, to investigate the efficiency of nanoprobe synthesis, different concentrations of Aptamer were designed to bind with ZrMOF, and finally, a certain concentration of miR-16 was added for fluorescence intensity measurement. Figure 4 In b, we can calculate that the binding rate of ZrMOF to Aptamer is 59.1%.

[0035] To determine the linear relationship between miR-16 concentration and fluorescence intensity, a series of miR-16 solutions with different concentrations were prepared and reacted with the nanoprobe. Figure 5 The explanation states that when the miR-16 concentration is in the range of 0.1~80 nM, the fluorescence intensity of the probe increases with increasing miR-16 concentration. However, once the miR-16 concentration exceeds 80 nM, the fluorescence intensity no longer increases. Figure 5 As shown in b, within the range of 0.1~80 nM, the fluorescence intensity of the probe gradually increases with the increase of miR-166 concentration and shows a good linear relationship, with a detection limit of 0.001 nM.

[0036] 3.2 Specificity of Nanoprobes To better study the targeting ability, anti-interference ability, and application reliability of the nanoprobes, agarose gel electrophoresis and fluorescence spectroscopy were used for verification.

[0037] Weigh 1.2 g agarose and add it to 60 mL of 1×TBE buffer solution. Heat in a microwave oven for 2 min while shaking. After it is completely dissolved, remove it from the microwave oven and cool it to 45~60 ℃. Add 30 µL of 2000× dye solution, pour it into a mold, insert a comb, and let it stand. Prepare 9 EP tubes and label them. Add 10 µL of 10 µM MB-miR-16 to tube 1, 10 µL of 10 µM miR-16 to tube 2, 10 µL of a mixed solution of MB-miR-16 and miR-16 of equal concentration and volume to tube 3, 10 µL of 10 µM miR-34a to tube 4, 10 µL of a mixed solution of MB-miR-16 and miR-34a of equal concentration and volume to tube 5, 10 µL of 10 µM miR-138 to tube 6, 10 µL of a mixed solution of MB-miR-16 and miR-138 of equal concentration and volume to tube 7, 10 µL of 10 µM miR-1980 to tube 8, and 10 µL of a mixed solution of MB-miR-16 and miR-1980 of equal concentration and volume to tube 9. Carefully remove the agarose gel, add electrophoresis buffer just enough to cover the gel to the electrophoresis tank, load 10 µL of sample into each well, electrophore for 40 min, and then perform imaging experiments.

[0038] Add 4 µL of 10 µM miR-16, miR-34a, miR-261, miR-1980, and miR-138 to 400 µL of 100 nM nanoprobe, respectively, and measure the fluorescence intensity to plot the fluorescence spectrum.

[0039] exist Figure 6 In diagram a, solution 1 is MB-miR-16, solution 2 is miR-16, solution 3 is a mixture of MB-miR-16 and miR-16, solution 4 is miR-34a, solution 5 is a mixture of MB-miR-166 and miR-34a, solution 6 is miR-138, solution 7 is a mixture of MB-miR-166 and miR-138, solution 8 is miR-1980, and solution 9 is a mixture of MB-miR-16 and miR-1980. It can be seen from the diagram that solution 3 has the largest molecular weight. Figure 6 The nanoprobe with added miR-16 showed the highest fluorescence intensity, which proves that the nanoprobe has specificity.

[0040] 4. Cellular Intervention Experiments Using Nanoprobes 4.1 Cell Culture Table 3 Preparation of culture medium Cell Name Preparation of culture medium SY5Y 45 mL DMEM basal medium + 5 mL fetal bovine serum albumin + 500 μL penicillin antibiotics 4.2 Cell passage Remove the SY5Y cell cryopreservation tubes from the -80 ℃ freezer, thaw them in a 37 ℃ water bath for 2 min, transfer them to a centrifuge tube, centrifuge, remove the supernatant, add 1 mL of culture medium to resuspend them, add them to a culture flask, add another 4 mL of culture medium, place the flask in an incubator, and observe them regularly.

[0041] Remove the cells from the incubator and observe their condition to determine if the passage density has been reached. For adherent cells like SY5Y, the culture medium in the culture flask can be discarded directly. Add 2 mL of PBS buffer to rinse and discard the PBS. Then add 2 mL of trypsin and digest in the incubator for 3 min. Afterward, observe the cell suspension under a microscope. In a biosafety cabinet, use a pipette to agitate the bottom of the culture flask to detach the cells. Centrifuge at 1100 rpm for 4 min, discard the supernatant, add 1 mL of culture medium, and passage at a ratio of 1 to 3.

[0042] 5. Cytotoxicity detection of nanoprobes To investigate the cytotoxicity of this nanoprobe, this study used CCK-8 reagent to detect the viability of SY5Y cells at different concentrations of the probe. One day in advance, cells were seeded into 96-well plates, with 100 µL of PBS buffer added around the perimeter. After cell adhesion, the culture medium was aspirated, and 100 µL of a series of nanoprobe solutions at different concentrations were added. After culturing for 24 h, the supernatant was discarded, and 10% CCK-8 reagent was added. Cells were cultured for another 4 h, and the absorbance was measured at 450 nm. The relative cell viability was calculated using a formula. Figure 7 As shown, the probe concentrations were 20 nM for probe 1, 40 nM for probe 2, 60 nM for probe 3, 80 nM for probe 4, 100 nM for probe 5, 120 nM for probe 6, 140 nM for probe 7, 160 nM for probe 8, 180 nM for probe 9, and 200 nM for probe 10. As the concentration of the nanoprobes increased, the activity of SY5Y cells remained relatively stable. When the probe concentration was 200 nM, the cell survival rate reached as high as 93%.

[0043] 6. Laser confocal imaging SY5Y cells were passaged and placed in confocal dishes and incubated in an incubator. When the cell density reached 80%–90%, 100 nM nanoprobe solution was added to the confocal dish, and the dish was incubated for another 5 h. After that, the liquid in the confocal dish was discarded, 1 mL of PBS buffer was added for rinsing, and an appropriate amount of Hoechst 3328 staining solution was added. After 30 min, the liquid was discarded, and PBS buffer was added. 2 mL of tissue cell fixation solution was added and the dish was fixed in an incubator for 5 min. The cell fixation solution was discarded, and 1 mL of PBS was added. The dish was then placed on a confocal microscope to observe the fluorescence imaging of the probes in the cells.

[0044] like Figure 8 As shown, SY5Y cells exhibit strong red fluorescence, indicating that the nanoprobe can enter the cell and exert its function. In SY5Y cells, blue fluorescence is emitted when the cell nucleus is stained with the dye solution, indicating intact nucleus morphology and good cell growth. The presence of abundant miR-16 and the probe within the cell also contributes to the red fluorescence. These experimental results are consistent with theoretical predictions, further validating that the synthesized nanoprobe can enter cells and bind to miR-16, demonstrating strong feasibility.

[0045] To investigate the intervention mechanism of miR-16 in Alzheimer's disease at the cellular level, Western blot was used to detect the protein level of the apoptosis protein p53 in SY5Y cells treated with miR-16, exploring the correlation between miR-16 and p53 protein expression levels, thereby revealing the key role of miR-16 in regulating the cell apoptosis process and its potential molecular mechanism. Figure 9 The protein levels revealed by Western blot analysis of p53 in miR-16-treated SY5Y cells indicate that miR-16 reduces the expression of the apoptosis protein p53.

[0046] Metal-organic frameworks (MOFs), due to their high specific surface area and tunable pore size, serve as ideal carriers for nucleic acid probes. By functionalizing aptamers to construct probes, detection sensitivity and specificity can be further enhanced. This invention designs a nucleic acid nanoprobe for intervention in Alzheimer's disease, consisting of a molecular beacon linked to a fluorescent group (FAM) and a ZrMOF quenching group. First, we synthesized a ZrMOF framework using TCPP as the photosensitizing unit and ZrOCl2 and benzoic acid as raw materials. Then, we coupled a molecular beacon with FAM to its surface, constructing an "off-on" type nanoprobe that specifically recognizes miR-16, laying the foundation for subsequent detection and intervention.

[0047] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A nucleic acid nanoprobe for detecting signal amplification of AβO, characterized in that, include: The signal output unit QDs are CdSe / ZnS quantum dots with surface-modified streptavidin synthesized by a hot melt method. Metal-organic frameworks (MOFs) are synthesized via a hydrothermal method with Zr ions at the center and porphyrins as organic ligands. They serve as a support and also as quenchers for CdSe / ZnS quantum dots. Aptamer sequence of AβO; The DNA1 sequence is designed based on the Aptamer sequence of AβO and is complementary to the Aptamer sequence of AβO, and the 5' end of the DNA1 sequence is modified with a phosphorylation group and the 3' end is modified with avidin. in; DNA1 sequence: 5'- GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Aptamer sequence of AβO: 5'-PO4 3--AGAGAGCCTGTGTTGGGGCGGGTGCG-3'; Signal output units (QDs) are modified onto the DNA1 sequence via a biotin-avidin reaction; The DNA1 sequence binds to the Aptamer sequence of AβO through complementary base pairing; The surface of metal-organic framework (MOF) nanoparticles was functionalized by phosphorylation of the DNA1 sequence.

2. A method for preparing nucleic acid nanoprobes for detecting AβO signal amplification, characterized in that, include: CdSe / ZnS quantum dots with surface-modified streptavidin were synthesized by a thermosol method and used as signal output units (QDs). Metal-organic frameworks (MOFs) centered on Zr ions and with porphyrins as organic ligands were synthesized via a hydrothermal method and used as a support and quencher for CdSe / ZnS quantum dots. Based on the Aptamer sequence of AβO, a complementary DNA1 sequence was designed, and a phosphorylation group was modified at the 5' end and avidin was modified at the 3' end. Signal output units (QDs) were modified onto the DNA1 sequence via a biotin-avidin reaction, and then the DNA1 sequence was bound to the Aptamer sequence of AβO through complementary base pairing. AβO-responsive nucleic acid nanoprobes were constructed by functionalizing the surface of metal-organic framework (MOF) nanoparticles with the phosphorylation group of DNA1.

3. The method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification as described in claim 2, characterized in that, DNA1 sequence: 5'- GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Aptamer sequence of AβO: 5'-PO4 3--AGAGAGCCTGTGTTGGGGCGGGTGCG-3'.

4. The method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification as described in claim 3, characterized in that, CdSe / ZnS quantum dots modified with streptavidin were synthesized via a thermosol method, including the following steps: Se powder was added to NaOH solution, stirred for 10 min under N2 protection, Al powder was added, the temperature was raised to 85 ℃, and sonicated for 15 min to generate Na2Se precursor solution. A CdSe quantum dot solution was prepared by adding a Na2Se precursor solution to a cadmium perchlorate solution. Ammonium methyl sulfate and zinc chloride solution were added to a CdSe quantum dot solution, and the mixture was sonicated at 85°C for 25 min. Then, activated streptavidin was added and stirred overnight. After purification, surface-modified streptavidin-modified CdSe / ZnS quantum dots were obtained.

5. The method for preparing the nucleic acid nanoprobe for detecting AβO signal amplification as described in claim 4, characterized in that, The synthesis of metal-organic frameworks (MOFs) centered on Zr ions and using porphyrins as organic ligands via a hydrothermal method includes the following steps: 100 mg TCPP, 300 mg ZrOCl2 and 2.8 g phenylboronic acid reagent were added to 10 mL N,N-dimethylformamide DMF to form a mixture solution; The mixture solution was transferred to a hydrothermal reactor lined with tetrafluoroethylene and reacted at 90°C for 5 h. After the reaction was completed, the particles were centrifuged at 12,000 rpm for 5 min and washed three times with DMF to obtain MOF particles, which were then stored at 4℃ for later use.

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