A porous silicon deoxyribozyme fluorescent probe and its application in evaluating the anticancer activity of traditional Chinese medicine

Abstract

The present invention discloses a porous silicon deoxyribozyme fluorescent probe and its application in evaluating the anticancer activity of traditional Chinese medicine. The fluorescent probe comprises amino-modified porous silica nanoparticles, fluorescent molecules loaded in the nanoparticle pores, and DNA hairpins HP1 and HP2 covalently linked to the nanoparticle surface. The HP1 sequence contains a complementary sequence to the microRNA to be detected, and the HP2 sequence contains a zinc ion-specific deoxyribozyme cleavage site. After HP1 binds to the microRNA, the root is opened, the HP1 loop undergoes a conformational change, and binds to the HP2 loop nucleotide sequence. With the assistance of zinc ions, the zinc ion-specific deoxyribozyme is activated, which then cuts HP2 at the cleavage site, releasing the fluorescent molecules in the nanoparticle pores. The microRNA is detected based on the intensity of the fluorescent molecules. The probe can specifically and sensitively detect trace amounts of microRNA in a system.

Description

Technical Field

[0001] The present invention belongs to the field of materials, relates to fluorescent probes and applications, and particularly relates to a porous silicon deoxyribozyme fluorescent probe and its application in evaluating the anti-cancer activity of traditional Chinese medicine. Background Art

[0002] Traditional Chinese medicine (TCM) offers advantages in cancer treatment, such as multiple targets and minimal side effects. It can enhance the patient's immune system, reduce adverse reactions during treatment, and improve the patient's quality of life. Among various types of breast cancer, triple-negative breast cancer (TNBC) is a type of breast cancer that is negative for estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2. It is characterized by high metastatic and recurrent rates, low clinical cure rates, and poor prognosis.

[0003] During the development and progression of TNBC, the body responds to abnormal tumor cells by causing changes in the levels of certain substances in the body, including gene products, proteins, hormones, polyamines, and enzymes. Among these, microRNA, a reliable indicator in the clinical diagnosis of TNBC, can reflect the progression of TNBC. Many monomeric components in traditional Chinese medicine, such as berberine, tanshinone IIA, quercetin, and curcumin, can regulate microRNA levels in TNBC cells, inhibiting cancer cell proliferation and metastasis, and reducing the high metastatic and recurrent risk of TNBC, demonstrating significant efficacy in improving patients' clinical symptoms.

[0004] Currently, there are multiple methods for detecting the cancer marker microRNA. Among them, fluorescence analysis is the most widely used due to its advantages such as simple equipment, high analytical sensitivity, strong selectivity, and intuitive detection. Due to the extremely low content of microRNA in the body, it is usually necessary to combine it with appropriate nanomaterials to improve the sensitivity of detection. As a small molecule that regulates gene expression, microRNA21 plays an important role in normal growth and development, cell proliferation, apoptosis and other physiological activities of the body. Changes in the content of microRNA21 may indicate abnormalities in signaling pathways and protein synthesis in the body. It can be used as a disease marker to predict the progression of cancer, metabolic diseases, viral infections, etc., and is of great significance in the early prevention and treatment of diseases. Due to the low content and short sequence of microRNA21, a detection method with higher sensitivity and accuracy is needed. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a porous silicon DNAzyme fluorescent probe and its application in the evaluation of the anticancer activity of traditional Chinese medicine.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A porous silicon deoxyribozyme fluorescent probe for detecting microRNA comprises amino-modified porous silica nanoparticles, fluorescent molecules loaded in the pore structure of the porous silica nanoparticles, and two DNA hairpins HP1 and HP2 covalently attached to the surface of the porous silica nanoparticles to block the fluorescent molecules in the pore structure. The nucleotide sequence of HP1 contains a complementary sequence to the microRNA to be detected, and the nucleotide sequence of HP2 contains a zinc ion-specific deoxyribozyme cleavage site. After HP1 binds to the microRNA to be detected, the root portion is opened, the HP1 loop undergoes a conformational change and binds to the loop nucleotide sequence of HP2, activating the zinc ion-specific deoxyribozyme with the assistance of zinc ions, and then cutting HP2 at the cleavage site. The corresponding blocked pore is opened, and the fluorescent molecules loaded in the pore structure are released, and the microRNA to be detected is detected by the intensity of the fluorescent molecules.

[0008] Preferably, the amino-modified porous silica nanoparticles are prepared by the following steps:

[0009] (1) Chemical synthesis: Dissolve an appropriate amount of hexadecyltrimethylammonium bromide in sodium hydroxide solution, then add appropriate amounts of tetraethoxysilane and 3-aminopropyltriethoxysilane in sequence, stir to react, let stand, and after the solution is separated, centrifuge to obtain a white precipitate, which is then washed with ethanol and pure water and dried;

[0010] (2) Removing the template: dissolving the precipitate in a hydrochloric acid-ethanol solution, extracting by reflux, and centrifuging. The obtained white precipitate is washed with ethanol and pure water, and dried to obtain amino-modified porous silica nanoparticle powder.

[0011] More preferably, the fluorescent molecule is CE6, and the steps of loading the fluorescent molecule into the pore structure of the porous silica nanoparticles are as follows:

[0012] An appropriate amount of amino-modified porous silica nanoparticle powder is uniformly dispersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.0, and an appropriate amount of N-ε-maleimidocaproyl-oxysuccinimide ester for linking DNA to the amino groups on the porous silica nanoparticles is added to modify the porous silica nanoparticles. The mixture is centrifuged and the precipitate is uniformly dispersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.0. An appropriate amount of ce6 is added, mixed, and centrifuged, and the precipitate is dispersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.0.

[0013] More preferably, the method for covalently linking DNA hairpins HP1 and HP2 on the surface of porous silica nanoparticles is: mixing porous silica nanoparticles loaded with fluorescent molecules with freshly prepared DNA hairpins HP1 and HP2, reacting at room temperature for an appropriate amount of time, centrifuging, washing with pure water, and redissolving the precipitate in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.0.

[0014] More preferably, the microRNA is microRNA 21, and the DNA hairpins HP1 and HP2 are prepared by the following method: taking appropriate amounts of DNA 1 and DNA 2, respectively, and mixing them with appropriate amounts of tris-(2-formylethyl)phosphine hydrochloride at room temperature to reduce the disulfide bonds in the DNA; then, annealing the reduced DNA and allowing it to stand at room temperature to obtain DNA hairpins HP1 and DNA hairpins HP2, respectively;

[0015] in:

[0016] The sequence of DNA 1 is: 5′-TTTT GATG TTGATTC TCC GAG CCG GTC GAAATAGTG GGT TTTTTT TTT TTT TTT TTT TT T CAACAT CAG TCT GATAAG CTA-HS-3′;

[0017] The sequence of DNA 2 is: 5′-CGA CGA CG TTT TTT ACC CAC TAT rA G GAA T CAA C TTTTTT CG TCG TCG TTTT-HS-3′.

[0018] A method for preparing the porous silicon DNAzyme fluorescent probe comprises the following steps:

[0019] Amino-modified porous silica nanoparticles loaded with fluorescent molecules ce6 were mixed with freshly prepared DNA hairpins HP1 and HP2, reacted gently at room temperature, centrifuged, and the precipitate was collected and washed with pure water. The precipitate was redissolved in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.0.

[0020] Application of the porous silicon DNAzyme fluorescent probe in detecting microRNA.

[0021] Application of the porous silicon DNAzyme fluorescent probe in detecting microRNA21.

[0022] The porous silicon DNAzyme fluorescent probe is used to evaluate the anti-tumor activity of traditional Chinese medicine in vitro by detecting microRNA21, wherein the tumor is triple-negative breast cancer.

[0023] Technical advantages:

[0024] The porous silicon DNAzyme fluorescent probe provided by this invention is based on amino-modified porous silicon. Fluorescent molecules are loaded into the pores, while two functional DNA hairpin structures are modified on the pores. This allows for the detection of microRNA, a cancer marker. Experimental results demonstrate that the probe can specifically and sensitively detect trace amounts of microRNA 21 in a system, and can also be quantitatively analyzed using fluorescence intensity.

[0025] 2. Based on the effects of four traditional Chinese medicine monomers (tanshinone IIA, curcumin, berberine, and quercetin) on MDA-MB-231 cell morphology, cell proliferation, apoptosis, and cell migration, the synthesized porous silicon-DNAzyme fluorescent probe MSN@ce6-HP1 / HP2 was used to detect changes in intracellular microRNA21 levels after drug administration. The results demonstrated that the porous silicon-DNAzyme fluorescent probe was capable of highly sensitive detection of trace amounts of microRNA21 in cells. The decrease in microRNA21 levels in cells after drug administration was consistent with the changes in cancer cell behavior (decreased cell viability, weakened colony formation ability, slowed cell migration ability, and increased apoptosis). This suggests that the constructed porous silicon-DNAzyme fluorescent probe can be used to evaluate the anticancer activity of different traditional Chinese medicine components. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The design of the porous silicon DNAzyme fluorescent probe and its principle diagram for detecting microRNA21;

[0027] Figure 2 TEM image of amino-modified porous silica nanoparticles;

[0028] Figure 3 This is the nitrogen adsorption and desorption curve (inset: the diameter of the porous silicon pore);

[0029] Figure 4 To determine the reaction characteristics between DNA hairpins HP1, HP2 and microRNA21 by agarose gel electrophoresis;

[0030] Figure 5 The release of Ce6 in the porous silicon pores in the presence and absence of zinc ions;

[0031] Figure 6 This is a flow chart for the validation of zinc ion-specific enzyme digestion reaction;

[0032] Figure 7 The graph shows the change of zinc ion specific DNAzyme digestion reaction over time in the presence and absence of zinc ions;

[0033] Figure 8 The fluorescence intensity of ce6 in the supernatant changes with time at different HP1:HP2 concentration ratios;

[0034] Figure 9 The trend of ce6 fluorescence intensity change in the supernatant of the porous silicon probe reacting with different concentrations of zinc ions over time (A: 5μM-1mM, B: 100nM-5μM);

[0035] Figure 10 This is the fluorescence intensity diagram of ce6 in the supernatant after the porous silicon probe reacted in the presence of different metal ions;

[0036] Figure 11 The porous silicon probe reacts with microRNA 21, single-base mismatch microRNA 21, double-base mismatch microRNA 21, and triple-base mismatch microRNA 21, and the ce6 fluorescence signal in the supernatant is measured;

[0037] Figure 12 is the fluorescence signal intensity of ce6 in the supernatant after the porous silicon probe reacted in five different buffers;

[0038] Figure 13 The concentration-fluorescence intensity standard curve was obtained after the porous silicon probe reacted with different concentrations of microRNA21;

[0039] Figure 14 The morphology of cells co-cultured with Tanshinone ⅡA and MDA-MB-231 cells; (a) blank control group after 24 hours; (b) blank control group after 48 hours; (c) drug-treated group after 24 hours; (d) drug-treated group after 48 hours;

[0040] Figure 15 The cell morphology of quercetin and MDA-MB-231 cells co-cultured; (a) blank control group after 24 hours; (b) blank control group after 48 hours; (c) drug-treated group after 24 hours; (d) drug-treated group after 48 hours;

[0041] Figure 16 Cell morphology of curcumin co-cultured with MDA-MB-231 cells; (a) blank control group after 24 hours; (b) blank control group after 48 hours; (c) drug-treated group after 24 hours; (d) drug-treated group after 48 hours

[0042] Figure 17 Cell morphology of co-cultured MDA-MB-231 cells with berberine; (a) blank control group after 24 hours; (b) blank control group after 48 hours; (c) drug-treated group after 24 hours; (d) drug-treated group after 48 hours

[0043] Figure 18 The effects of four Chinese herbal medicine monomers on the growth of breast cancer MDA-MB-231 cells are time- and dose-dependent; among them, (a) berberine; (b) curcumin; (c) quercetin; (d) tanshinone ⅡA

[0044] Figure 19 The effects of four traditional Chinese medicine monomers on the clone formation of MDA-MB-231 cells; A: crystal violet staining; B: clone number;

[0045] Figure 20 The effects of four traditional Chinese medicine monomers on the migration and wound healing rate of MDA-MB-231 cells over time;

[0046] Figure 21 The DAPI fluorescence staining images of MDA-MB-231 cells in different groups; control group (0M), drug-treated group (0.625M tanshinone ⅡA, 20M berberine, 25M quercetin, 15M curcumin);

[0047] Figure 22 The DAPI staining fluorescence intensity histogram of MDA-MB-231 cells in different groups;

[0048] Figure 23 This is a graph showing the changes in intracellular microRNA21 content after culture of MDA-MB-231 cells with Chinese medicine at various concentrations. DETAILED DESCRIPTION

[0049] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0050] Experimental principle:

[0051] In this study, a porous silicon DNAzyme fluorescent probe, MSN@ce6-HP1 / HP2, was synthesized. This probe, based on amino-modified porous silica nanoparticles (MSNs), utilizes its dense pore structure to load the fluorescent small molecule ce6 within the pores. Taking advantage of the easily modifiable nature of the MSN surface, two DNA hairpins (HP1 and HP2) were covalently attached to block the pores. The HP1 sequence contains a complementary sequence to the target microRNA 21, while the HP2 sequence incorporates a zinc-specific DNAzyme cleavage site. In the presence of microRNA 21, the HP1 root is opened, and the HP1 loop undergoes a conformational change, allowing it to bind to the HP2 loop sequence. This, aided by zinc ions, activates the zinc-specific DNAzyme, which then cleaves HP2 at the cleavage site, releasing the fluorescent molecule ce6 from the pores. Consequently, the porous silicon DNAzyme fluorescent probe enables sensitive detection of microRNA 21 by measuring the fluorescence intensity changes in the supernatant before and after the reaction.

[0052] 1. Reagents and Instruments

[0053] Reagents: tetraethoxysilane (TEOS); hexadecyltrimethylammonium bromide (CTAB); 3-aminopropyltriethoxysilane (APTES); N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); N-ε-maleimidocaproyloxysuccinimide ester (Sulfo-EMCS); tris-(2-formylethyl)phosphine hydrochloride (TCEP); TE solution; DEPC-treated water; sodium hydroxide; hydrochloric acid; ethanol; acetone; aldehyde-modified glass slides; human breast cancer cells (MDA-MB-231); tanshinone IIA, curcumin, berberine hydrochloride, quercetin; GBICO 10270-106 fetal bovine serum; DAPI kit; sterile PBS; DMEM; crystal violet staining solution; CCK-8 kit; 4% paraformaldehyde; Triton-X-100; cell freezing solution; methanol; anhydrous ethanol; dimethyl sulfoxide (DMSO); isopropanol; RNAeasy TM Animal small RNA extraction kit (spin column type), etc.

[0054] Instruments: transmission electron microscope; digitally controlled ultrasonic device; magnetic stirrer; centrifuge; high-speed low-temperature centrifuge; analytical balance; transient / steady-state fluorescence spectrometer; vortex oscillator; constant temperature shaking incubator; rotary mixer; inverted fluorescence microscope; biological safety cabinet, etc.

[0055] 2. Experimental Methods

[0056] 1. Synthesis of porous silicon

[0057] pass The amino-modified porous silica nanoparticles (MSN-NH2) were synthesized by the method. The specific steps are as follows: (1) Chemical synthesis: Weigh 48mL of 14.5mM NaOH solution, accurately weigh 100.00mg of CTAB, add it and dissolve it completely, stir it at 80℃ for 30min, then add 500μLTEOS and 20μLAPTES in sequence, continue stirring for 2h, and let it stand overnight after the reaction is completed. After the solution is separated, centrifuge it at 10000rpm for 20min to obtain a white precipitate, which is then washed with ethanol and pure water 3 times each. Dry it at 60℃, seal it, and store it at room temperature. (2) Template removal: Dissolve the precipitate in hydrochloric acid-ethanol solution (2.5mL:100mL) and reflux it for 12h, for a total of 2 times. Centrifuge it, wash the obtained white precipitate with ethanol and pure water 3 times each, dry it at 60℃ to obtain MSN-NH2 powder, seal it, and store it at room temperature.

[0058] 2. Fluorescent molecules ce6 are filled into the porous silicon channels

[0059] Accurately weigh 10.00 mg of MSN-NH2 and dissolve it in 950 μL of 20 mM HEPES buffer (pH 7.0). Sonicate for 30 minutes to evenly disperse the MSN-NH2. Add 50 μL of 10.00 mg / mL sulfo-EMCS (a linker between DNA and MSN-NH2) to modify the porous silica and mix thoroughly for 30 minutes. Centrifuge at 10,000 rpm for 3 minutes to remove excess sulfo-EMCS. The precipitate is then re-dissolved in 900 μL of HEPES buffer to evenly disperse it. Add 100 μL of 1.00 mg / mL ce6, mix overnight, centrifuge, and re-dissolve the precipitate in 900 μL of HEPES buffer to obtain porous silica MSN@ce6 with the fluorescent molecule ce6 encapsulated within its pores.

[0060] 3. DNA hairpin HP1 and HP2 pretreatment

[0061] DNA 1 sequence: 5'-TTTT GATG TTGATTC TCC GAG CCG GTC GAAATAGTG GGT TTT TTTTTT TTT TTT TTT TT T CAACAT CAG TCT GATAAG CTA-HS-3'

[0062] DNA2 sequence: 5'-CGACGACG TTT TTTACC CAC TATrAG GAAT CAAC TTT TTT CG TCGTCG TTTT-HS-3'

[0063] DNA Hairpin HP1: Gently mix 20 μL of 5 μM DNA 1 with 20 μL of 500 μM TCEP at room temperature for 2 hours to reduce the disulfide bonds in the DNA, facilitating its attachment to the porous silicon. Anneal the reduced DNA at 95°C for 5 minutes and let it stand at room temperature for 2 hours to obtain DNA Hairpin HP1. Store at 4°C until ready for use.

[0064] DNA Hairpin HP2: Gently mix 20 μL of 50 μM DNA2 with 20 μL of 5 mM TCEP at room temperature for 2 hours to reduce the disulfide bonds in the DNA, facilitating its attachment to the porous silicon. Anneal the reduced DNA at 95°C for 5 minutes and let it stand at room temperature for 2 hours to obtain DNA Hairpin HP2. Store at 4°C until needed.

[0065] 4. Preparation of MSN@ce6 / HP1HP2 Probe

[0066] The synthesized MSN@ce6 was mixed with freshly prepared DNA hairpins HP1 and HP2 and allowed to react gently at room temperature for 2 hours. The mixture was then centrifuged and washed with pure water. The precipitate was redissolved in 850 μL of HEPES buffer to obtain the porous silicon DNAzyme fluorescent probe MSN@ce6-HP1 / HP2, which was used in subsequent experiments.

[0067] 5. Detection principle of microRNA21

[0068] The reaction principle of this study is as follows Figure 1 As shown, a porous silicon DNAzyme fluorescent probe, MSN@ce6-HP1 / HP2, has been synthesized. Because HP1 contains a complementary sequence to the analyte, microRNA 21, microRNA 21 can open HP1 in the presence of HP1. HP1 then undergoes a conformational change and binds to the corresponding sequence on HP2, activating the enzymatic activity of the zinc-specific DNAzyme on HP2 and cleaving the enzyme site on HP2. HP1 can perform a walking cleavage of multiple nearby HP2s, ultimately removing multiple HP2s blocked on the pore surface and accelerating the release of the fluorescent molecule ce6 within the pore. Therefore, highly sensitive detection of the analyte can be achieved by measuring the fluorescence intensity of the reaction supernatant in the presence of only a small amount of microRNA 21.

[0069] 6. Application of probes in drug efficacy evaluation

[0070] 6.1 Principle

[0071] Four common Chinese herbal monomers (curcumin, berberine, tanshinone IIA, and quercetin) were co-cultured with triple-negative breast cancer cells MDA-MB-231 at different concentrations for a certain period of time. MicroRNA 21 in the cells was extracted and added to the probe MSN@ce6-HP1 / HP2. The changes in fluorescence intensity after the reaction were measured to infer the effects of the four Chinese herbal monomers on the intracellular microRNA21 content. This was used to evaluate the therapeutic effect of each Chinese herbal monomer on triple-negative breast cancer.

[0072] 6.2 Preparation of main reagents in the experiment

[0073] Tanshinone IIA stock solution: Prepare Tanshinone IIA in dimethyl sulfoxide (DMSO) at concentrations of 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, and 0.5 mM. Store each stock solution at 4°C in the dark until ready for use. Dilute the desired concentration in serum-free DMEM (final DMSO concentration of 0.1%).

[0074] Quercetin stock solution: Prepare quercetin in dimethyl sulfoxide (DMSO) at concentrations of 100 mM, 50 mM, 25 mM, 5 mM, and 1 mM. Store each stock solution in a 4°C refrigerator protected from light until use. Dilute the solution to the desired concentration in serum-free DMEM (final DMSO concentration of 0.1%).

[0075] Curcumin stock solutions: Curcumin was prepared in dimethyl sulfoxide (DMSO) at concentrations of 25 mM, 20 mM, 15 mM, 10 mM, and 5 mM. Each stock solution was stored in a 4°C refrigerator, protected from light until use. The desired concentration was diluted in serum-free DMEM (final DMSO concentration of 0.1%).

[0076] Berberine hydrochloride stock solution: Prepare berberine hydrochloride in dimethyl sulfoxide (DMSO) at concentrations of 100 mM, 80 mM, 40 mM, 20 mM, and 10 mM. Store each stock solution at 4°C in the dark until ready for use. Dilute the solution to the desired concentration in serum-free DMEM (final DMSO concentration of 0.1%).

[0077] 6.3 Effects of Traditional Chinese Medicine on Cell Viability

[0078] The cell concentration of MDA-MB-231 selected in the experiment was logarithmic growth phase tumor cells, diluted to 3×10 4 / mL, 100 μL per well was inoculated into 4 96-well culture plates, and cultured in a 37°C, 5% CO2 incubator for 24 h and 48 h. Each plate was then added with different concentrations of tanshinone ⅡA (final concentrations of 0, 0.5, 0.625, 1.25, 2.5, and 5 μM), quercetin (final concentrations of 0, 1, 5, 25, 50, and 100 μM), and curcumin (final concentrations of 0, 5, 10, 15, and 2 0, 25 μM) and berberine hydrochloride (final concentrations of 0, 10, 20, 40, 80, 100 μM, respectively) were added to 100 μL of serum-free fresh culture medium, and 0 μM of tanshinone Ⅱ A, 0 μM of quercetin, 0 μM of curcumin, and 0 μM of berberine hydrochloride were set as blank controls (0.1% DMSO was added to the culture medium as the solvent control group). Four replicates were set for each concentration. After 24 hours and 48 hours of action, 10 μL of CCK-8 was added, placed in a 37 ° C, 5% CO2 incubator, protected from light for 1 hour, and placed in a microplate reader for detection. The detection wavelength was selected at 450 nm, and the absorbance value A of each group was measured. The cell viability and IC were calculated by averaging the absorbance values ​​of the four replicates. 50 .

[0079] Formula: Cell viability (%) = (A(drug added) - A(blank)) / (A(0 drug added) - A(blank)) × 100%

[0080] A(drug added): absorbance of wells containing cells, CCK8 solution and drug solution; A(blank): absorbance of wells containing culture medium and CCK8 solution but no cells; A(0 drug added): absorbance of wells containing cells, CCK8 solution but no drug solution.

[0081] 6.4 Effects of Traditional Chinese Medicine on Cell Clone Formation

[0082] The MDA-MB-231 cells selected in the experiment were all tumor cells in the logarithmic growth phase, and the cell concentration was diluted to 5×10 2 / mL, seeded at 1000 cells / well in 6-well plates and cultured overnight to allow attachment. The next day, experimental cells were treated with tanshinone IIA (0.625μM), quercetin (25μM), curcumin (15μM), and berberine hydrochloride (20μM), respectively, while control cells were treated with 0μM of the drug (i.e., the culture medium contained a final concentration of 0.1% DMSO). The cells were cultured in a 37°C, 5% CO2 incubator for 14 days. When colonies were visible, the plates were removed and the culture medium discarded. The plates were washed three times with PBS, fixed with 1mL of 4% paraformaldehyde for 20 minutes, discarded, and washed three times with pure water. The plates were then stained with 1mL of 10% Giemsa stain for 10 minutes, washed thoroughly with pure water, and allowed to dry. Cell colony formation was then observed under a low-power microscope (≥50 cells were considered the standard), and the number of breast cancer cell colonies in each well was counted.

[0083] 6.5 Effects of Traditional Chinese Medicine on Cell Morphology

[0084] MDA-MB-231 cells were co-cultured with four traditional Chinese medicine monomers at varying concentrations for 24 and 48 hours. The culture medium was discarded, and the morphology of the cells in each experimental group was observed under a microscope. The types and final concentrations of the traditional Chinese medicines in each group were: Tanshinone IIA (final concentrations of 0, 0.5, 0.625, 1.25, 2.5, and 5 μM); Quercetin (final concentrations of 0, 1, 5, 25, 50, and 100 μM); Curcumin (final concentrations of 0, 5, 10, 15, 20, and 25 μM); and Berberine Hydrochloride (final concentrations of 0, 10, 20, 40, 80, and 100 μM).

[0085] 6.6 Effects of Traditional Chinese Medicine on Cell Migration

[0086] MDA-MB-231 breast cancer cells were seeded at a specific density in a 6-well plate. After 24 hours of growth, cells adhered to the plate and reached approximately 90% confluency. A 10 μL pipette tip was used to gently scratch the cell monolayer, perpendicular to the bottom of the wells, with the scratch distance equal to the diameter of the tip. After scratching, the plate was gently washed three times with PBS to remove detached cells. Freshly prepared drug-containing medium (tanshinone IIA (final concentrations of 0, 0.625, 1.25, and 2.5 μM), quercetin (final concentrations of 0, 5, 25, and 50 μM), curcumin (final concentrations of 0, 10, 15, and 20 μM), and berberine hydrochloride (final concentrations of 0, 10, 20, and 40 μM)) containing 1% serum was added to each well. Cells were grown for 24 and 48 hours, then washed twice with sterile PBS. Finally, 2 mL of PBS was added. Images were taken under a microscope to observe and record changes in cell fusion over time. The scratch area was obtained using ImageJ to calculate the cell scratch healing rate. The calculation formula is: Scratch healing rate (%) = (initial scratch area – scratch area at a certain time) / initial scratch area × 100%.

[0087] 6.7 Effects of Traditional Chinese Medicine on Cell Apoptosis

[0088] (1) Dilute MDA-MB-231 cells to a cell density of 4×10 4 / mL, 8000 cells / well were seeded in a 6-well plate and cultured in an incubator for 24 hours to allow the cells to adhere to the wall. The culture medium was discarded and different concentrations of drug-containing culture medium were added (tanshinone ⅡA at 0, 0.625, 1.25, 2.5μM; quercetin at 0, 5, 25, 50μM; curcumin at 0, 10, 15, 20μM; berberine hydrochloride at 0, 10, 20, 40μM). (2) After 48 hours of culture, the culture medium was discarded and 1mL PBS was added for washing twice. (3) 1mL 4% paraformaldehyde was added and reacted at room temperature for 20 minutes to increase the permeability of the cell membrane. (4) 4% paraformaldehyde was discarded and 1mL PBS was added for washing three times to remove excess 4% paraformaldehyde residue. (5) 1mL 0.1% Triton-X-100 was added and reacted at room temperature for 20 minutes to denature some membrane proteins and further enhance the permeability. (6) Discard Triton-X-100 and wash three times with 1 mL of PBS. (7) Add 200 μL of DAPI staining solution to each well and incubate at 37°C for 15 min. Discard the staining solution and gently wash three times with 1 mL of PBS. (8) Finally, resuspend the cells in 1 mL of PBS and observe the staining under an inverted fluorescence microscope.

[0089] 6.8 Effects of Traditional Chinese Medicine on Intracellular MicroRNA21 Content

[0090] MDA-MB-231 cells were all in logarithmic growth phase and cultured overnight to allow attachment and 80%-90% confluency. The next day, cells were treated with equal volumes of different concentrations of tanshinone IIA (final concentrations of 0, 0.5, 0.625, and 1.25 μM), quercetin (final concentrations of 0, 1, 5, and 25 μM), curcumin (final concentrations of 0, 5, 10, and 15 μM), and berberine hydrochloride (final concentrations of 0, 10, 20, and 40 μM). The control group received 0 μM drug concentration (i.e., culture medium containing 0.1% DMSO). The cells were incubated at 37°C, 5% CO₂, for 48 hours. The cell culture supernatant was then collected, and microRNA was extracted from each cell group using an RNA extraction kit. Finally, the extracted microRNA was reacted with a multi-porous silicon DNAzyme fluorescent probe to measure microRNA levels in the cells.

[0091] The specific steps are as follows: (1) Collect 1×10 6 10 cells, add 300 μL lysis buffer to dissolve them, transfer to a clean centrifuge tube, then add 300 μL binding solution I and gently invert to mix; (2) transfer the mixture in (1) to the first purification column, centrifuge at 12000g for 1 minute, recover the supernatant to a new centrifuge tube, add 700 μL binding solution II and mix; (3) transfer the mixture in (2) to the second purification column twice, centrifuge at 12000g for 1 minute, and discard the liquid in the tube; (4) add 600 μL washing solution to the purification column, centrifuge at 12000g for 1 minute, twice in total, discard the liquid in the tube; centrifuge at 16000g for 2 minutes to remove the residual liquid; (5) place the purification column in the RNA elution tube, add 30 μL elution buffer, let it stand at room temperature for 2 to 3 minutes, and centrifuge at 16000g for 1 minute. The resulting solution is the purified microRNA. (6) The purified microRNA of each group was mixed with the probe, the CE6 fluorescence was measured, and the content of microRNA21 in each group of cells was calculated.

[0092] 7. Statistical analysis

[0093] Data are presented as mean ± SEM (x ± s). An independent sample t-test was used to compare the two groups, and P < 0.05 was considered statistically significant.

[0094] 3. Experimental Structure

[0095] 1. Characterization of MSN-NH2

[0096] The appearance and size of the prepared MSN-NH2 were observed using a transmission electron microscope (TEM). Figure 2The results showed that the porous silica nanoparticles were spherical or quasi-spherical, with diameters between 100 and 120 nm. The pores of MSN-NH2 were dense and uniform, with pore diameters of approximately 2 to 3 nm. The surface area and pore size distribution of the porous silicon were determined by nitrogen adsorption-desorption (BET) spectroscopy. Figure 3 It can be seen that the IV type adsorption isotherm can be obtained after the sample is obtained in this study. The synthesized porous silicon material has uniform mesopores. The specific parameters are: specific surface area 797.0492m 2 / g, pore diameter 3.7296nm.

[0097] 2. Verification of the reactivity of HP1 / HP2 with microRNA21

[0098] Agarose gel electrophoresis was used to characterize the reaction characteristics between HP1, HP2 and microRNA21. Figure 4 As shown in Figure 3 , lanes 1, 2, and 3 correspond to the distances traveled by HP1, HP2, and microRNA21 of different molecular weights in the same time period, respectively. In lane 4, HP1 and HP2 are located at their respective molecular weight positions, indicating that the HP1 and HP2 hairpins are structurally stable and that cross-binding does not occur upon mixing. Similarly, lane 6 shows that microRNA21 and HP2 are located at their respective positions, with no new light spots generated. This indicates that microRNA21 and HP2 hairpins do not bind when present alone. Therefore, the presence of HP2 does not interfere with microRNA21, and the reaction system is free of byproducts. Lane 5 shows that a new, brighter, larger spot is generated upon mixing HP1 and microRNA21, indicating that a reaction occurs only when HP1 and microRNA21 are present together, with microRNA21 opening the HP1 hairpin and binding. This spot represents the new complex formed by the binding of microRNA21 and HP1. Lane 7 also shows two spots: one representing HP2 alone and the other representing a complex of HP1 and microRNA21. This indicates that HP2 does not interfere with the reaction between HP1 and microRNA21. These analyses demonstrate that the DNA hairpins designed in this study, HP1 / HP2, exhibit a highly specific reaction with microRNA21.

[0099] 3. Verification of zinc ion-specific DNAzyme reactivity

[0100] Firstly, the release of Ce6 in porous silicon in the presence and absence of zinc ions was investigated. Figure 5As shown, in the absence of zinc ions, only a small amount of Ce6 is released. This phenomenon occurs because a small amount of microRNA21 opens HP1, allowing a small number of Ce6 molecules in the pore to slowly release, but it is insufficient to open the pore and quickly release Ce6. When microRNA21 and zinc ions are present simultaneously, Ce6 release increases continuously over time, but slows down around 2 hours. These results indicate that only with the assistance of zinc ions can the zinc-specific DNAzyme be activated, cleaving the HP2 cleavage site and leading to the massive release of Ce6 from the pore.

[0101] This experiment further verified the changes in the enzyme digestion reaction of zinc ion-specific DNAzyme over time. One end of HP2 is fixed to the glass slide substrate through a covalent bond, and the other end is modified with the fluorescent molecule cy3. The change in the fluorescence intensity of cy3 on the substrate will be used to indicate the enzyme digestion efficiency of HP2. The principle is as follows Figure 6 As shown. In the presence of microRNA21 and zinc ions, HP1 opens and undergoes a conformational change, binding to a complementary sequence on HP2. This activates the enzymatic activity of the zinc-specific DNAzyme, which cleaves HP2 and breaks it. Because HP2 is modified with Cy3 at its tail end, the amount of Cy3 on the slide decreases after cleavage and washing, leading to a decrease in fluorescence intensity. As the reaction time increases, HP1 can continue to walk on the slide, reacting with surrounding HP2 and cleaving more HP2. This results in a decreasing amount of Cy3 on the slide, and the fluorescence intensity also exhibits a dynamic decrease.

[0102] The fluorescence intensity of cy3 after different reaction times was photographed and recorded using an inverted fluorescence microscope. Figure 7 As shown. At the beginning of the reaction, the fluorescence intensity is the strongest, because the DNAzyme has not yet taken effect and HP2-cy3 has not been cut; as the reaction time increases, the fluorescence intensity shows a decreasing trend, because HP2 is gradually cut off and cy3 is gradually removed from the slide. At about 50 minutes, the fluorescence intensity is weak, almost zero, indicating that the enzyme cutting is sufficient. This phenomenon verifies Figure 6 The principle that "one molecule of miRNA21 can induce the shearing of multiple molecules of HP2 with the help of zinc ions under the same conditions" was used as a gated porous silicon release fluorescent probe in this experiment ( Figure 1 ) signal amplification basis.

[0103] 4. Optimization of reaction conditions

[0104] 4.1 Optimization of the ratio of HP1 to HP2

[0105] To investigate the optimal ratio of HP1 to HP2 on the porous silicon surface, equal volumes of HP1 and HP2 at concentration ratios of 1:5, 1:10, 1:15, 1:20, and 1:25 were selected for encapsulating MSN@ce6. All other reaction conditions remained the same. After adding 5 μM microRNA 21 and 5 μM zinc ion solution, the fluorescence intensity of ce6 in the supernatant was measured. The results are shown in Figure 2. Figure 8 As shown, the reaction efficiency is best when HP1:HP2=1:10.

[0106] 4.2 Optimization of zinc ion concentration

[0107] The fluorescence intensity of ce6 released by the probe after reaction with different zinc ion concentrations was measured to investigate the effect of zinc ion concentration on enzyme cleavage efficiency. Figure 9 As shown, at high zinc ion concentrations (5 μM to 100 μM), ce6 release gradually increased over time, indicating that increasing zinc ion concentration accelerates enzymatic cleavage efficiency. However, as zinc ion concentrations continued to increase (>100 μM), ce6 release decreased, likely because higher zinc ion concentrations disrupt the conditions required to maintain DNA stability, hindering the reaction. At lower zinc ion concentrations (100 nM to 5 μM), ce6 release increased with increasing zinc ion concentration. These results demonstrate that the enzymatic cleavage efficiency of zinc ion-specific DNAzymes is concentration-dependent within a certain range. Therefore, a 5 μM zinc ion concentration was selected for subsequent experiments to achieve optimal enzymatic cleavage efficiency.

[0108] 5. Specificity investigation

[0109] 5.1 Interference from other metal ions

[0110] This experiment investigated the effects of other interfering ions such as calcium, potassium, magnesium, manganese and sodium on the reaction system under the same experimental conditions. The interference of other metal ions on the reaction system was evaluated by comparing the CE6 fluorescence intensity after the reaction. Figure 10 As shown, the reaction system can release a large amount of Ce6 only in the presence of zinc ions, while other metal ions have little effect on the reaction and will not interfere with the reaction.

[0111] 5.2 Investigation of microRNA21 Specificity

[0112] This experiment compared the fluorescence intensity of microRNA21 and other mismatched microRNAs after reaction with the porous silicon DNAzyme fluorescent probe to verify whether the probe has good selectivity for microRNA21. Figure 11As shown, the fluorescence signal after reaction with the mismatched microRNA is weak, indicating that less Ce6 is released from the pore. However, the Ce6 fluorescence intensity is strongest after reaction with microRNA21, indicating that only microRNA21 can open HP1 and further trigger the subsequent enzymatic cleavage reaction, leading to the release of more Ce6. Furthermore, although a single-base mismatch sequence triggers a significant amount of Ce6 release, this concentration, as high as 10 μM, is far higher than the concentration of non-mismatched microRNA21, indicating that single-base mismatches have minimal interference in a normal reaction system. These experimental results demonstrate that the porous silicon DNAzyme fluorescent probe synthesized in this experiment has good specificity for detecting microRNA21.

[0113] 5.3 Effects of different buffers on the reaction system

[0114] This experiment investigated the reaction of porous silicon DNAzyme fluorescent probes in different buffer solutions. The probes were dispersed in a variety of different systems (pure water, PBS, HEPES, DMEM and RPMI 1640 culture medium), and then HP1 and HP2 blocking and release experiments were performed in these different systems, and the changes in fluorescence intensity during the reaction were recorded. The results are shown in Figure 2. Figure 12 As shown in the figure, in pure water medium, the fluorescent molecules are released slowly due to the lack of salt ions necessary to maintain the hybridization reaction between DNA and microRNA. In other media, the fluorescence release curve is basically consistent with the trend in HEPES buffer, which proves the controllable release of the probe in different buffer media and indicates that the probe has good stability in different solutions such as PBS, HEPES, DMEM and RPMI 1640 culture medium.

[0115] 6. Determination of miRNA21 standard curve

[0116] In this experiment, different concentrations of microRNA21 (0, 50, 100, 500, 1000, 2500, 5000 pM) were selected to react with the porous silicon probe. After a certain period of time, the fluorescence intensity of ce6 in the supernatant was detected and a concentration-fluorescence intensity relationship curve was drawn. Figure 13 As shown in Figure 2, the fluorescence intensity changes corresponding to microRNA21 in the range of 100pM to 5000pM have a good linear relationship, R 2 The detection limit was 0.9915 and 23 pM.

[0117] 7. Application of probes in drug efficacy evaluation

[0118] 7.1 Effects of Traditional Chinese Medicine on Cell Morphology

[0119] This study investigated the effects of four Chinese herbal monomers (tanshinone ⅡA, curcumin, quercetin, and berberine hydrochloride) on the growth and morphology of breast cancer cells MDA-MB-231. After co-culture of MDA-MB-231 cells with each concentration of Chinese herbal monomer for 24 hours and 48 hours, the changes in the number and morphology of MDA-MB-231 cells were observed under a microscope, and the cell inhibition rate (%) at each concentration was calculated. Figures 14 to 17 As can be seen, the breast cancer MDA-MB-231 cells in the blank control group grew well, and no morphological changes were observed. Compared with the blank control group, different concentrations of tanshinone IIA, curcumin, quercetin, and berberine hydrochloride in each experimental group inhibited the growth of breast cancer MDA-MB-231 cells to varying degrees, and were concentration- and time-dependent (see Tables 1 to 4). The results show that after co-culture of tanshinone IIA, curcumin, quercetin, and berberine hydrochloride with MDA-MB-231 cells, breast cancer cells gradually showed irregular shapes, smaller cell shrinkage, increased intercellular spaces, a tendency to grow alone, and an increase in suspended cells. In addition, with the increase in the concentration of each traditional Chinese medicine, the above cell morphological changes became more obvious, and under the same microscopic field of view, it was found that the number of tumor cells also showed a decreasing trend with the increase in traditional Chinese medicine concentration.

[0120] Table 1 Inhibitory rate of Tanshinone ⅡA on MDA-MB-231 in different dose groups and at different time points (%)

[0121]

[0122] a :P<0.05, vs 24h control group, b :P<0.05, vs 48h control group

[0123] Table 2 Inhibitory rate of quercetin on MDA-MB-231 in different dose groups and at different time points (%)

[0124]

[0125]

[0126] a :P<0.05, vs 24h control group, b :P<0.05, vs 48h control group

[0127] Table 3 Inhibitory effect of curcumin on MDA-MB-231 in different dose groups and at different time points (%)

[0128]

[0129] a:P<0.05, vs 24h control group, b :P<0.05, vs 48h control group

[0130] Table 4 Inhibitory effect of berberine on MDA-MB-231 in different dose groups and at different time points (%)

[0131]

[0132] a :P<0.05, vs 24h control group, b :P<0.05, vs 48h control group

[0133] 7.2 Effects of Traditional Chinese Medicine on Cell Viability

[0134] In this experiment, four Chinese herbal medicine monomers at different concentrations were co-cultured with breast cancer cells MDA-MB-231 for 24 hours and 48 hours, and the CCK-8 method was used to determine the inhibitory effects of the four Chinese herbal medicine monomers at different concentrations on breast cancer cell proliferation. Figure 18 As shown in the figure, the four Chinese herbal medicine monomers have a dose- and time-dependent effect on the cell growth of MDA-MB-231 cells. As the drug concentration increases, the growth of MDA-MB-231 cancer cells is significantly inhibited, and this phenomenon becomes more significant as time goes by. The concentration that inhibits cell growth is calculated by the median lethal concentration (IC) of each Chinese herbal medicine monomer. 50 It was found that tanshinone IIA was 1.213 μM, curcumin was 16.43 μM, berberine hydrochloride was 30.87 μM, and quercetin was 49.90 μM.

[0135] 7.3 Effects of Traditional Chinese Medicine on Cell Clone Formation

[0136] Four Chinese herbal medicine monomers were co-cultured with breast cancer cells MDA-MB-231. After administration, the cells grew for 14 days. When obvious cell clusters appeared, crystal violet staining was performed to compare the differences in the number of MDA-MB-231 cell clones in each group. Figure 19As shown, the colony-forming ability of MDA-MB-231 cells was significantly reduced, and the number of colonies decreased, after treatment with the four traditional Chinese medicines. The average number of colony-forming cells in the blank control group was 946. After treatment with 0.625 μM tanshinone IIA, 25 μM quercetin, 15 μM curcumin, and 20 μM berberine, the average number of colony-forming cells was 152, 114, 76, and 48, respectively. These differences were statistically significant compared with the blank control group (P < 0.05). In summary, the effects of four traditional Chinese medicines (tanshinone IIA, curcumin, quercetin, and berberine) on the colony-forming ability of breast cancer MDA-MB-231 cells were analyzed in a colony-forming assay. The results showed that the colony-forming ability of cells treated with these traditional Chinese medicines was significantly reduced compared with the control group, indicating that tanshinone IIA, curcumin, quercetin, and berberine hydrochloride can significantly inhibit the colony formation of MDA-MB-231 cells.

[0137] 7.4 Effects of Traditional Chinese Medicine on Cell Migration

[0138] This study selected breast cancer cells MDA-MB-231 for imaging at 0h, 24h, and 48h after administration. To ensure that the cells maintained a certain level of viability, the following concentrations were selected: 0.625μM Tanshinone IIA, 25μM Quercetin, 15μM Curcumin, and 20μM Berberine Hydrochloride to investigate the migration properties of MDA-MB-231 cells. The results are shown in Figure 2. Figure 20 As shown, cells not treated with traditional Chinese medicine gradually healed their scratches over time, and their migration speed was faster. However, after treatment with traditional Chinese medicine, cell migration slowed and healing was reduced. Comparing the migration results of the four traditional Chinese medicines, although the inhibition rates varied, with berberine and curcumin showing stronger effects and quercetin and tanshinone IIA showing more moderate effects, all showed that the four traditional Chinese medicines, tanshinone IIA, curcumin, berberine, and quercetin, could inhibit cancer cell migration and reduce their ability to spread to non-tumor areas, thereby slowing the progression of cancer.

[0139] 7.5 Effects of Traditional Chinese Medicine on Cell Apoptosis

[0140] In this study, MDA-MB-231 cells in different groups were stained with DAPI, photographed under an inverted fluorescence microscope, and the fluorescence intensity was calculated using ImageJ. Figure 21 and 22As shown, in the control group (i.e., MDA-MB-231 cells that did not interact with the traditional Chinese medicine), uniform blue fluorescence was observed throughout the nucleus. In the treated groups, DAPI staining revealed a distinct blue-white fluorescence in the nucleus, and the nuclei became rounded. The fluorescence brightness and nuclear shape can be used to assess the state of apoptosis: early-stage apoptotic cells exhibit nuclear condensation, darker staining, or crescent-shaped nuclear chromatin accumulation near the nuclear membrane. Late-stage apoptotic cells exhibit nuclear fragmentation into round bodies of varying sizes, surrounded by the cell membrane. Comparing the staining results after the four drug treatments, curcumin treatment significantly increased the degree of apoptosis in MDA-MB-231 cells, with chromatin condensation, a large number of round nuclei, blebbing of the cell membrane, and brighter fluorescence. Tanshinone IIA had the most moderate effect, with only a small number of nuclei exhibiting bright blue fluorescence. In summary, while the four traditional Chinese medicines exhibit distinct effects on the apoptosis of breast cancer cells, all of them affect the apoptotic process.

[0141] 7.6 Effects of Traditional Chinese Medicine on Changes in Intracellular MicroRNA21 Content

[0142] Four kinds of Chinese medicine with different concentrations were co-cultured with MDA-MB-231 cells, and 10 6 The microRNA21 in each group of cells was extracted using an RNA extraction kit, and then added to the porous silicon-DNAzyme fluorescent probe for reaction. The changes in microRNA21 content after Chinese medicine treatment were measured by ce6 fluorescence intensity. Figure 23 As shown in the results, compared with the control group, the levels of microRNA21 in MDA-MB-231 cells treated with tanshinone IIA, berberine, quercetin, and curcumin were all reduced in a concentration-dependent manner, indicating that all four Chinese herbs can inhibit the growth of microRNA21 in cells. Comparing the effects of the four Chinese herbs, berberine had a relatively strong effect, reducing the level of microRNA21 in cells to almost zero. Furthermore, these results indicate that the reduction in microRNA21 levels is consistent with the trends observed in cell experiments on triple-negative breast cancer cells after treatment with Chinese herbs, including decreased cell viability, weakened colony formation, slowed cell migration, and increased apoptosis. This suggests that changes in microRNA21 levels can reflect the progression of triple-negative breast cancer.

[0143] Experimental results:

[0144] This study synthesized a porous silicon DNAzyme fluorescent probe capable of detecting the cancer marker microRNA 21 by loading the fluorescent molecule ce6 inside the pores and modifying the pores with two functional DNA hairpin structures. Experimental results demonstrated that the probe can specifically and sensitively detect trace amounts of microRNA 21 in the system and quantitatively analyze it using fluorescence intensity.

[0145] Secondly, based on the effects of four Chinese herbal medicine monomers (tanshinone ⅡA, curcumin, berberine, and quercetin) on MDA-MB-231 cell morphology, cell proliferation, cell apoptosis, and cell migration, the synthesized porous silicon-DNAzyme fluorescent probe MSN@ce6-HP1 / HP2 was used to detect the changes in the content of microRNA21 in cells after drug administration.

[0146] The deoxyribozyme fluorescent probe can achieve highly sensitive detection of trace amounts of microRNA 21 in cells. The decrease in microRNA21 content in cells after administration is consistent with the changing trend of cancer cell behavior after administration (decreased cell viability, weakened clone formation ability, slowed cell migration ability, and increased cell apoptosis, etc.), indicating that the constructed porous silicon-deoxyribozyme fluorescent probe can be used to evaluate the anti-cancer activity of different traditional Chinese medicine ingredients.

[0147] Furthermore, the porous silicon-DNAzyme fluorescent probe synthesized in this study can be further modified with targeted aptamers or biofilms that facilitate endocytosis, allowing the porous silicon complex to enter cells and be used for real-time fluorescence imaging to monitor intracellular microRNA21 levels. To enhance the efficacy of traditional Chinese medicine (TCM), the porous silicon-DNAzyme fluorescent probe can be combined with membrane-based carriers with specific response properties to load TCMs of different structural types, achieving the orderly release of different drugs within cells, achieving the combined administration of different TCM components and enhancing the efficacy of different drugs. This approach has broad application prospects.

[0148] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A porous silicon DNAzyme fluorescent probe for detecting microRNA, comprising amino-modified porous silica nanoparticles, fluorescent molecules loaded into the pore structure of the porous silica nanoparticles, and two DNA hairpins HP1 and HP2 covalently attached to the surface of the porous silica nanoparticles to block the fluorescent molecules within the pore structure; wherein: The nucleotide sequence of HP1 contains a complementary sequence to the microRNA to be tested, and the nucleotide sequence of HP2 contains a cleavage site for a zinc ion-specific deoxyribozyme. After HP1 binds to the microRNA to be tested, the root portion is opened, the HP1 loop undergoes a conformational change and binds to the loop nucleotide sequence of HP2, activating the zinc ion-specific deoxyribozyme with the assistance of zinc ions. HP2 is then cut at the cleavage site, the corresponding blocked pore is opened, and the fluorescent molecule loaded in the pore structure is released, thereby detecting the microRNA to be tested through the intensity of the fluorescent molecule. The microRNA is microRNA 21, and the DNA hairpins HP1 and HP2 are prepared by the following method: taking appropriate amounts of DNA 1 and DNA 2, respectively, and mixing them with appropriate amounts of tris-(2-formylethyl)phosphine hydrochloride at room temperature to reduce the disulfide bonds in the DNA; then, annealing the reduced DNA and allowing it to stand at room temperature to obtain DNA hairpins HP1 and HP2, respectively; in: The sequence of DNA 1 is: 5′ - TTTT G ATG TTG A TTC TCC GAG CCG GTC GAA ATA GTG GGTTTT TTT TTT TTT TTT TTT TT T CAA CAT CAG TCT GAT AAG CTA - HS - 3′; The sequence of DNA 2 is: 5' - CGA CGA CG TTT TTT ACC CAC TAT rA G GAA T CAA C TTTTTT CG TCG TCG TTTT - HS - 3' 2. The porous silicon DNAzyme fluorescent probe according to claim 1, characterized in that: The amino-modified porous silica nanoparticles are prepared by the following steps: (1) Chemical synthesis: Take an appropriate amount of hexadecyltrimethylammonium bromide and dissolve it in sodium hydroxide solution. Then add appropriate amounts of tetraethoxysilane and 3-aminopropyltriethoxysilane in sequence. Stir the reaction and let it stand. After the solution is separated, centrifuge it to obtain a white precipitate. Wash it with ethanol and pure water and dry it. (2) Removal of the template: The precipitate was dissolved in a hydrochloric acid-ethanol solution, refluxed for extraction, and centrifuged. The obtained white precipitate was washed with ethanol and pure water, and dried to obtain amino-modified porous silica nanoparticle powder.

3. The porous silicon DNAzyme fluorescent probe according to claim 2, characterized in that: The fluorescent molecule is ce6, and the steps of loading the fluorescent molecule into the pore structure of the porous silica nanoparticles are as follows: An appropriate amount of amino-modified porous silica nanoparticle powder was uniformly dispersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.0, and an appropriate amount of N-ε-maleimidocaproyloxysuccinimide ester for linking DNA to the amino groups on the porous silica nanoparticles was added to modify the porous silica nanoparticles. The mixture was centrifuged and the precipitate was uniformly dispersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.

0. An appropriate amount of ce6 was added, mixed, and centrifuged, and the precipitate was dispersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.

0.

4. The porous silicon DNAzyme fluorescent probe according to claim 3, characterized in that The method for covalently linking DNA hairpins HP1 and HP2 on the surface of the porous silica nanoparticles is as follows: porous silica nanoparticles loaded with fluorescent molecules are mixed with freshly prepared DNA hairpins HP1 and HP2, reacted at room temperature for an appropriate amount of time, centrifuged, washed with pure water, and the precipitate was redissolved in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.

0.

5. A method for preparing the porous silicon DNAzyme fluorescent probe according to claim 4, characterized in that: The method comprises the following steps: mixing amino-modified porous silica nanoparticles loaded with fluorescent molecules ce6 with freshly prepared DNA hairpins HP1 and HP2, reacting them gently at room temperature, centrifuging, collecting the precipitate, washing with pure water, and redissolving the precipitate in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at pH = 7.

0.

6. Use of the porous silicon DNAzyme fluorescent probe according to claim 4 in in vitro evaluation of the anti-tumor activity of traditional Chinese medicine by detecting microRNA 21, wherein the tumor is triple-negative breast cancer.

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