CN116144737A - Porous silicon deoxyribozyme fluorescent probe and application thereof in evaluating anticancer activity of traditional Chinese medicine - Google Patents

Abstract

The invention discloses a porous silicon deoxyribozyme fluorescent probe and its application in the evaluation of the anticancer activity of traditional Chinese medicine. DNA hairpins HP1 and HP2 covalently linked to the surface of the nanoparticles; wherein, the sequence of HP1 contains the complementary sequence of the microRNA to be tested, and the sequence of HP2 contains the enzyme cleavage site of zinc ion-specific deoxyribozyme; after HP1 binds to the microRNA, The root is opened, the configuration of the HP1 loop changes and binds to the nucleotide sequence of the HP2 loop, and the zinc ion-specific deoxyribozyme is activated with the assistance of zinc ions, and then HP2 is cut off at the enzyme cleavage site, and the nanoparticle channel The medium fluorescent molecules are released, and the detection of microRNA is realized through the intensity of the fluorescent molecules. The probe can specifically and sensitively detect trace amounts of microRNA in the system.

Description

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

technical field

The invention belongs to the field of materials, and relates to a fluorescent probe and its application, in particular to a porous silicon deoxyribozyme fluorescent probe and its application in evaluating the anticancer activity of traditional Chinese medicine.

Background technique

Traditional Chinese medicine has the advantages of multiple targets and few side effects in the treatment of cancer. It can enhance the patient's own immune system, reduce adverse reactions during treatment, and improve the quality of life of patients. Among various types of breast cancer, triple negative breast cancer (TNBC) is a type of breast cancer with negative expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Metastatic, high recurrence and other characteristics, low clinical cure rate, poor prognosis.

During the occurrence and development of TNBC, the body will respond to abnormal tumor cells and cause changes in the content of certain substances in the body, including gene products, proteins, hormones, polyamines and enzymes. Among them, microRNA, as one of the reliable indicators in the clinical diagnosis of TNBC, can reflect the development process of TNBC. Many monomer components in traditional Chinese medicine, such as berberine, tanshinone Ⅱ A, quercetin, curcumin, etc., can regulate the content of microRNA in TNBC cells, inhibit cancer cell proliferation and metastasis, and reduce the high metastasis and high recurrence of TNBC. It has a significant effect in improving the clinical symptoms of patients.

At present, there are many methods for the detection of cancer marker microRNA, among which fluorescence analysis has the advantages of simple equipment, high analytical sensitivity, strong selectivity, and intuitive detection, and is the most widely used. Due to the extremely low content of microRNA in the body, it is usually necessary to combine appropriate nanomaterials to improve the sensitivity of detection. As a small molecule that can regulate gene expression, microRNA21 plays an important role in physiological activities such as normal growth and development, cell proliferation, and apoptosis. Changes in the content of microRNA21 may indicate abnormalities in signaling pathways and protein synthesis in the body, and can be used as disease markers to predict the progress of cancer, metabolic diseases, viral infections, etc., and are 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.

Contents of the invention

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

The above object of the present invention is achieved through the following technical solutions:

A porous silicon deoxyribozyme fluorescent probe for detecting microRNA, comprising amino-modified porous silica nanoparticles, fluorescent molecules loaded in the pore structure of the porous silica nanoparticles, and covalently linked in the porous silica nanoparticles Two DNA hairpins HP1 and HP2 that block the fluorescent molecules in the pore structure on the surface of the silica nanoparticles; wherein the nucleotide sequence of the HP1 contains the complementary sequence of the microRNA to be tested, and the HP2 The nucleotide sequence contains the enzyme cleavage site of zinc ion-specific deoxyribozyme; after HP1 combines with the microRNA to be tested, the root is opened, and the configuration of the HP1 loop changes and binds to the nucleotide sequence of the HP2 loop , with the assistance of zinc ions, zinc ion-specific deoxyribozymes are activated, and then HP2 is cut off at the enzyme cleavage site, the corresponding blocked pores are opened, and the fluorescent molecules loaded in the pore structure are released. Molecular strength enables detection of the microRNA to be tested.

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

(1) Chemical synthesis: take an appropriate amount of cetyltrimethylammonium bromide and dissolve it in sodium hydroxide solution, then add an appropriate amount of tetraethoxysilane and 3-aminopropyltriethoxysilane in turn, and stir the reaction. Stand still, after the solution is separated into layers, centrifuge to obtain a white precipitate, then wash with ethanol and pure water, and dry;

(2) Template removal: dissolve the precipitate in hydrochloric acid-ethanol solution, reflux extraction, centrifuge, wash the obtained white precipitate with ethanol and pure water, and dry to obtain amino-modified porous silica nanoparticle powder.

More preferably, the fluorescent molecule is ce6, and the steps of loading the fluorescent molecule in the pore structure of the porous silica nanoparticles are as follows:

Take an appropriate amount of amino-modified porous silica nanoparticle powder and evenly disperse it in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution with pH = 7.0, and add an appropriate amount of N-ε-maleimidocaproyl-oxysuccinimide ester of amino groups on silica nanoparticles modifies porous silica nanoparticles, centrifuges, and uniformly disperses the precipitate in N-2 at pH = 7.0 -Hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, add appropriate amount of ce6, mix, centrifuge, and disperse the precipitate in N-2-hydroxyethylpiperazine-N'-2- in ethanesulfonic acid buffer.

More preferably, the method for covalently linking DNA hairpins HP1 and HP2 on the surface of porous silica nanoparticles is as follows: mix porous silica nanoparticles loaded with fluorescent molecules with freshly prepared DNA hairpins HP1 and HP2, and react at room temperature for an appropriate amount of time , centrifuged, washed with pure water, and redissolved the precipitate in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer solution with pH=7.0.

More preferably, the microRNA is microRNA 21, and the DNA hairpins HP1 and HP2 are prepared by the following method: take an appropriate amount of DNA 1 and DNA2 and mix them with an appropriate amount of tris-(2-formylethyl)phosphine hydrochloride at room temperature, and reduce the dihydrochloride in the DNA. Sulfur bond; then, the reduced DNA is annealed, and allowed to stand at room temperature to obtain DNA hairpin HP1 and DNA hairpin HP2 respectively;

in:

The sequence of DNA 1 is: 5'-TTTT GATG TTGATTC TCC GAG CCG GTC GAAATAGTG GGT TTTTTT TTT TTT TTT TTT TTT TT T CAACAT CAG TCT GATAAG 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'.

A method for preparing the above-mentioned porous silicon deoxyribozyme fluorescent probe, comprising steps:

Mix amino-modified porous silica nanoparticles loaded with fluorescent molecule ce6 with freshly prepared DNA hairpin HP1 and DNA hairpin HP2, react gently at room temperature, centrifuge, collect the precipitate, wash with pure water, and redissolve the precipitate in pH=7.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer.

Application of the above-mentioned porous silicon deoxyribozyme fluorescent probe in the detection of microRNA.

Application of the above-mentioned porous silicon deoxyribozyme fluorescent probe in detecting microRNA21.

The application of the above-mentioned porous silicon deoxyribozyme fluorescent probe in evaluating the anti-tumor activity of traditional Chinese medicine by detecting microRNA21 in vitro, and the tumor is triple-negative breast cancer.

Technical advantages:

1. The porous silicon deoxyribozyme fluorescent probe provided by the present invention is based on amino-modified porous silicon. Two functional DNA hairpin structures are respectively loaded inside the pore and modified outside the pore to synthesize a microRNA capable of detecting cancer markers. Porous silicon deoxyribozyme fluorescent probe. The experimental results show that using microRNA 21 as the microRNA to be detected, the probe can specifically and sensitively detect trace amounts of microRNA21 in the system, and can be quantitatively analyzed by fluorescence intensity.

2. On the basis of investigating the effects of four traditional Chinese medicine monomers (tanshinone ⅡA, curcumin, berberine, and quercetin) on MDA-MB-231 cell morphology, cell proliferation, cell apoptosis, and cell migration, the synthetic The porous silicon deoxyribozyme fluorescent probe MSN@ce6-HP1/HP2 detects the changes of microRNA21 content in cells after administration. The results show that the porous silicon deoxyribozyme fluorescent probe can realize highly sensitive detection of trace microRNA21 in cells, and the decrease of microRNA21 content in cells after administration is consistent with the change trend of cancer cell behavior after administration (decreased cell viability, clone Formation ability is weakened, cell migration ability is slowed down, and the degree of cell apoptosis is increased, etc.), indicating that the constructed porous silicon-DNAzyme fluorescent probe can be used to evaluate the anticancer activity of different traditional Chinese medicine ingredients.

Description of drawings

Fig. 1 is the schematic diagram of the design of the porous silicon deoxyribozyme fluorescent probe and its detection of microRNA21;

Fig. 2 is the TEM picture of the porous silica nanoparticle of amino modification;

Fig. 3 is a nitrogen adsorption-desorption curve diagram (inset: the diameter of the porous silicon channel);

Fig. 4 is the reaction characteristic between DNA hairpin HP1, HP2 and microRNA21 determined by agarose gel electrophoresis;

Figure 5 shows the release of ce6 in the pores of porous silicon in the presence or absence of zinc ions;

Fig. 6 is the verification flow chart of zinc ion-specific enzyme cleavage reaction;

Fig. 7 is in the presence or absence of zinc ions, the change diagram of zinc ion-specific DNAzyme enzyme cleavage reaction over time;

Figure 8 is the trend of the fluorescence intensity of ce6 in the supernatant under different concentration ratios of HP1:HP2 over time;

Fig. 9 shows the trend of the fluorescence intensity of ce6 in the supernatant of the porous silicon probe reacting with different concentrations of zinc ions over time (A: 5μM-1mM, B: 100nM-5μM);

Figure 10 is a graph of the fluorescence intensity of ce6 in the supernatant after the reaction of the porous silicon probe in the presence of different metal ions;

Figure 11 is a diagram of the fluorescent signal of ce6 in the supernatant obtained by reacting the porous silicon probe with microRNA 21, single-base mismatch microRNA 21, double-base mismatch microRNA21 and three-base mismatch microRNA21 respectively;

Figure 12 is the fluorescence signal intensity of ce6 in the supernatant after the reaction of the porous silicon probe in five different buffer solutions;

Figure 13 is the concentration-fluorescence intensity standard curve obtained after the reaction of the porous silicon probe with different concentrations of microRNA21;

Figure 14 is the co-culture cell morphology of Tanshinone Ⅱ A and MDA-MB-231 cells; wherein, (a) blank control group after 24h; (b) blank control group after 48h; (c) administration group after 24h; (d) 48h post-administration group;

Figure 15 is the co-culture cell morphology of quercetin and MDA-MB-231 cells; wherein, (a) blank control group after 24h; (b) blank control group after 48h; (c) administration group after 24h; (d) Administration group after 48h;

Figure 16 is the co-culture cell morphology of curcumin and MDA-MB-231 cells; wherein, (a) blank control group after 24h; (b) blank control group after 48h; (c) administration group after 24h; (d) 48h After administration group

Figure 17 is the co-culture cell morphology of berberine and MDA-MB-231 cells; wherein, (a) blank control group after 24h; (b) blank control group after 48h; (c) administration group after 24h; (d) 48h later administration group

Figure 18 shows that four kinds of traditional Chinese medicine monomers have time and dose-dependent effects on the growth of breast cancer cell MDA-MB-231 cells; wherein, (a) berberine; (b) curcumin; (c) quercetin; ( d) Tanshinone Ⅱ A

Figure 19 is the effect of four kinds of traditional Chinese medicine monomers on the formation of MDA-MB-231 cell clones; wherein, A: crystal violet staining; B: number of clones;

Figure 20 is the effect of four kinds of traditional Chinese medicine monomers on the migration and wound healing rate of MDA-MB-231 cells over time;

Figure 21 is the DAPI fluorescent staining diagram of MDA-MB-231 cells in different groups; wherein, the control group (0M), the administration group (0.625M tanshinone Ⅱ A, 20M berberine, 25M quercetin, 15M curcumin);

Figure 22 is a histogram of DAPI staining fluorescence intensity of MDA-MB-231 cells in different groups;

Fig. 23 is a graph showing changes in microRNA21 content in cells after cultured with various concentrations of traditional Chinese medicine and MDA-MB-231 cells.

Detailed ways

The substantive content of the present invention will be described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

Experimental principle:

In this study, a porous silicon deoxyribozyme fluorescent probe MSN@ce6-HP1/HP2 was synthesized, which was based on amino-modified porous silica nanoparticles (MSN) The fluorescent small molecule ce6 is loaded inside; two kinds of DNA hairpins (HP1 and HP2) are covalently linked to block the pores by using its surface modification characteristics. Among them, the HP1 sequence contains the complementary sequence of the microRNA21 to be tested, and the HP2 sequence is designed with an enzyme cleavage site for a zinc ion-specific deoxyribozyme. If and only when microRNA 21 exists, the root of HP1 can be opened, the configuration of the HP1 ring will change and bind to the ring sequence of HP2, and the activity of zinc ion-specific DNAzyme will be activated with the assistance of zinc ions, and then in The enzyme cutting site cuts off HP2, so that the fluorescent molecule ce6 in the hole is released. Accordingly, the porous silicon deoxyribozyme fluorescent probe can realize the sensitive detection of microRNA21 through the change of the fluorescence intensity of the supernatant before and after the reaction.

1. Reagents and instruments

Reagents: tetraethoxysilane (TEOS); cetyltrimethylammonium bromide (CTAB); 3-aminopropyltriethoxysilane (APTES); N-2-hydroxyethylpiperazine-N '-2-Ethylsulfonic acid (HEPES); N-ε-maleimidocaproyl-oxysuccinimide 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); , quercetin; GBICO 10270-106 fetal bovine serum; DAPI kit; sterile PBS; DMEM; crystal violet staining solution; CCK-8 kit; 4% paraformaldehyde; Triton-X-100; cells Freezing solution; methanol; absolute ethanol; dimethyl sulfoxide (DMSO); isopropanol; RNAeasy ^TM animal small RNA extraction kit (spin column type), etc.

Instruments: transmission electron microscope; numerical control ultrasonic instrument; magnetic stirrer; centrifuge; high-speed low-temperature centrifuge; analytical balance; transient/steady-state fluorescence spectrometer; vortex oscillator; constant temperature oscillation incubator; rotary mixer; inverted fluorescence microscope; biological safety cabinets, etc.

2. Experimental method

1. Synthesis of porous silicon

法合成氨基修饰的多孔二氧化硅纳米粒子(MSN-NH₂)，具体步骤：(1)化学合成：称取48mL 14.5mM NaOH溶液，精密称定100.00mg CTAB加入并充分溶解，80℃搅拌30min，然后依次加入500μLTEOS和20μLAPTES，继续搅拌2h，反应结束后静置过夜。待溶液分层后，10000rpm离心20min，得到白色沉淀，再用乙醇、纯水各洗涤3次。60℃烘干，密封，室温保存。(2)去除模板：将沉淀溶解在盐酸-乙醇溶液(2.5mL:100mL)中，回流12h，共2次。离心，将得到的白色沉淀用乙醇、纯水各洗涤3次，60℃烘干，得到MSN-NH₂粉末，密封，常温保存。pass

Amino-modified porous silica nanoparticles (MSN-NH ₂ ) were synthesized by the following method: (1) Chemical synthesis: Weigh 48mL of 14.5mM NaOH solution, accurately weigh 100.00mg of CTAB, add and fully dissolve, stir at 80°C for 30min , and then sequentially added 500 μLTEOS and 20 μL APTES, continued to stir for 2 hours, and stood overnight after the reaction. After the solution was separated, it was centrifuged at 10,000 rpm for 20 min to obtain a white precipitate, which was then washed three times with ethanol and pure water. Dry at 60°C, seal and store at room temperature. (2) Removal of template: Dissolve the precipitate in hydrochloric acid-ethanol solution (2.5 mL:100 mL), and reflux for 12 h, twice in total. After centrifugation, the obtained white precipitate was washed three times with ethanol and pure water, and dried at 60°C to obtain MSN-NH ₂ powder, which was sealed and stored at room temperature.

2. Porous silicon pores are filled with fluorescent molecules ce6

Accurately weigh 10.00 mg MSN-NH ₂ and dissolve it in 950 μL 20 mM HEPES buffer (pH=7.0), sonicate for 30 min to make the MSN-NH ₂ disperse evenly. Add 50 μL of 10.00 mg/mL sulfo-EMCS (linker between DNA and MSN-NH ₂ ) to modify the porous silicon, and mix thoroughly for 30 minutes. Centrifuge at 10000rpm for 3min to remove excess sulfo-EMCS. Then redissolve the pellet in 900 μL HEPES buffer to make it evenly dispersed. Add 100 μL of 1.00 mg/mL ce6, mix overnight, centrifuge, redissolve the precipitate in 900 μL of HEPES buffer, and finally obtain porous silicon MSN@ce6 with fluorescent molecule ce6 encapsulated in the pores.

3. DNA hairpin HP1 and HP2 pretreatment

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'

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

DNA hairpin HP1: Take 20 μL of 5 μM DNA 1 and 20 μL of 500 μM TCEP and mix gently at room temperature for 2 hours to reduce the disulfide bonds in DNA and facilitate the connection of DNA to porous silicon. Then the reduced DNA was annealed at 95°C for 5min, and left at room temperature for 2h to obtain DNA hairpin HP1, which was stored at 4°C for future use.

DNA Hairpin HP2: Take 20 μL of 50 μM DNA2 and 20 μL of 5 mM TCEP and mix gently at room temperature for 2 hours to reduce the disulfide bonds in DNA and facilitate the connection of DNA to porous silicon. Then the reduced DNA was annealed at 95°C for 5min, and left at room temperature for 2h to obtain DNA hairpin HP2, which was stored at 4°C for future use.

4. Preparation of MSN@ce6/HP1HP2 probe

The MSN@ce6 synthesized above was mixed with freshly prepared DNA hairpins HP1 and HP2, and reacted gently at room temperature for 2 h. Centrifuge and wash with pure water. The precipitate was redissolved in 850 μL HEPES buffer to obtain the porous silicon deoxyribozyme fluorescent probe MSN@ce6-HP1/HP2, which was used in subsequent experiments.

5. Detection principle of microRNA21

The reaction principle of this study is shown in Figure 1. The porous silicon deoxyribozyme fluorescent probe MSN@ce6-HP1/HP2 has been synthesized. Since HP1 contains the complementary sequence of the microRNA 21 to be tested, HP1 can be opened in the presence of microRNA 21, and then HP1 undergoes a configuration change and binds to the corresponding sequence on HP2, activating the enzyme activity of the zinc ion-specific DNAzyme of HP2, cutting off the DNA enzyme on HP2. enzyme cleavage site. HP1 can cut several nearby HP2s in a walking manner, and eventually the multiple HP2s blocked on the surface of the pore are removed successively, and the fluorescent molecule ce6 in the pore is released at an accelerated rate. Therefore, only in the presence of a small amount of microRNA21, the highly sensitive detection of the analyte can be achieved by measuring the fluorescence intensity of the supernatant after the reaction.

6. Application of probes in drug efficacy evaluation

6.1 Principle

Four common traditional Chinese medicine monomers (curcumin, berberine, tanshinone ⅡA, quercetin) of different concentrations were selected to co-culture with triple-negative breast cancer cell MDA-MB-231 for a certain period of time, and microRNA 21 in the cells was extracted and analyzed. Add the probe MSN@ce6-HP1/HP2, measure the change of fluorescence intensity after the reaction, and speculate the effect of four kinds of traditional Chinese medicine monomers on the content of microRNA21 in cells, so as to evaluate the therapeutic effect of each traditional Chinese medicine monomer on triple-negative breast cancer.

6.2 Preparation of main reagents in the experiment

Tanshinone IIA stock solution: use dimethyl sulfoxide (DMSO) to prepare tanshinone IIA so that the drug concentrations are 5mM, 2.5mM, 1.25mM, 0.625mM, and 0.5mM, and store each stock solution in a refrigerator at 4°C, away from light. spare. The concentration required for the experiment was diluted with serum-free DMEM culture solution (the final concentration of DMSO was 0.1%).

Quercetin stock solution: Quercetin was prepared with dimethyl sulfoxide (DMSO) to make drug concentrations of 100mM, 50mM, 25mM, 5mM, and 1mM respectively, and each stock solution was placed in a refrigerator at 4°C, and stored in the dark for future use. The concentration required for the experiment was diluted with serum-free DMEM culture solution (the final concentration of DMSO was 0.1%).

Curcumin stock solution: dimethyl sulfoxide (DMSO) was used to prepare curcumin into drug concentrations of 25mM, 20mM, 15mM, 10mM, and 5mM, and each stock solution was placed in a refrigerator at 4°C, and stored in the dark for future use. The concentration required for the experiment was diluted with serum-free DMEM culture solution (the final concentration of DMSO was 0.1%).

Berberine hydrochloride stock solution: use dimethyl sulfoxide (DMSO) to prepare berberine hydrochloride into drug concentrations of 100mM, 80mM, 40mM, 20mM, and 10mM, and store each stock solution in a refrigerator at 4°C, away from light. spare. The concentration required for the experiment was diluted with serum-free DMEM culture solution (the final concentration of DMSO was 0.1%).

6.3 The effect of traditional Chinese medicine on cell viability

The MDA-MB-231 selected in the experiment is the tumor cells in the logarithmic growth phase at the cell concentration, diluted to 3×10 ⁴ /mL, inoculated in 4 96-well culture plates at 100 μL per well, and kept at 37°C, 5% CO ₂ After culturing in the incubator for 24 h and 48 h, each plate was added with different concentrations of tanshinone Ⅱ A (final concentrations were 0, 0.5, 0.625, 1.25, 2.5, 5 μM), quercetin (final concentrations were 0, 1, 5 μM, respectively). , 25, 50, 100 μM), curcumin (the final concentrations were 0, 5, 10, 15, 20, 25 μM) and berberine hydrochloride (the final concentrations were 0, 10, 20, 40, 80, 100 μM) 100 μL of serum-free fresh medium, and set 0 μM of tanshinone Ⅱ A, 0 μM of quercetin, 0 μM of curcumin and 0 μM of berberine hydrochloride as blank control (adding 0.1% DMSO to the medium as solvent control group), and set 4 samples for each concentration. After 24 hours and 48 hours of treatment, 10 μL of CCK-8 was added to the duplicate wells, placed in a 37°C, 5% CO ₂ incubator, and reacted in the dark for 1 hour, and placed in a microplate reader for detection. Select the detection wavelength of 450nm, and measure the absorbance value A of each group. Cell viability and IC ₅₀ were calculated by the average of the absorbance values of 4 duplicate wells.

Formula: cell viability (%) = (A (dosing) - A (blank)) / (A (0 dosing) - A (blank)) × 100%

A (dosing): absorbance of wells with cells, CCK8 solution and drug solution; A (blank): absorbance of wells with medium, CCK8 solution, but no cells; A (0 dosing): cells, CCK8 solution, but the absorbance of wells without drug solution.

6.4 The effect of traditional Chinese medicine on the ability of cell clone formation

The MDA-MB-231 cells selected for the experiment are all tumor cells in the logarithmic growth phase. The cell concentration was diluted to 5×10 ² /mL, and 1000 cells/well were seeded in a 6-well plate, and cultured overnight to allow the cells to adhere to the wall. The next day The cells in the experimental group were treated with tanshinone Ⅱ A (0.625 μM), quercetin (25 μM), curcumin (15 μM) and berberine hydrochloride (20 μM), while the drug concentration in the cells of the control group was 0 μM (that is, the medium was the final concentration 0.1% DMSO solution), placed in a 37°C, 5% CO ₂ incubator for 14 days, when colonies were visible to the naked eye, the plate was taken out, and the culture solution was discarded. Wash 3 times with PBS, add 1 mL of 4% paraformaldehyde to fix for 20 min, discard, and wash with pure water three times. Then add 1 mL of 10% Giemsa staining solution to stain for 10 min, wash thoroughly with pure water, and let stand to dry. Then observe the formation of cell clones under a low-power microscope (with more than 50 cells as the standard), and count the number of clones of breast cancer cells in each well.

6.5 Effects of traditional Chinese medicine on cell morphology changes

MDA-MB-231 cells were co-cultured with four kinds of traditional Chinese medicine monomers at different concentrations for 24 hours and 48 hours, discarded the culture medium, and placed them on a microscope to observe the morphology of cells in each experimental group. The types and final concentrations of traditional Chinese medicine in each group were: tanshinone Ⅱ A (final concentrations were 0, 0.5, 0.625, 1.25, 2.5, 5 μM); quercetin (final concentrations were 0, 1, 5, 25, 50, 100 μM ); curcumin (final concentrations were 0, 5, 10, 15, 20, 25 μM); berberine hydrochloride (final concentrations were 0, 10, 20, 40, 80, 100 μM).

6.6 The effect of traditional Chinese medicine on cell migration ability

Breast cancer cells MDA-MB-231 were inoculated into a 6-well plate at a certain density, and after 24 hours of growth, the cells adhered to the wall, and the cell fusion degree reached about 90%. Use a 10 μL pipette tip to gently scratch between the cultured cells in the monolayer. The pipette tip is perpendicular to the bottom of the plate well, and the scratch distance is equal to the diameter of the end of the pipette tip. After scratching, gently wash the well plate 3 times with PBS to remove detached cells. Add freshly prepared drug-containing medium containing 1% serum (tanshinone Ⅱ A (final concentrations are 0, 0.625, 1.25, 2.5 μM), quercetin (final concentrations are 0, 5, 25, 50 μM) , curcumin (the final concentrations were 0, 10, 15, 20 μM) and berberine hydrochloride (the final concentrations were 0, 10, 20, 40 μM)). The cells were grown for 24h and 48h, and the cells were washed twice with sterile PBS. Finally 2 mL of PBS was added. Shoot under a microscope, observe and record the changes in the degree of cell fusion over time, and use ImageJ to obtain the scratch area to calculate the cell scratch healing rate. Calculation formula: scratch healing rate (%)=(initial scratch area-scratch area at a certain moment)/initial scratch area×100%.

6.7 The effect of traditional Chinese medicine on cell apoptosis

(1) Dilute MDA-MB-231 cells to a cell density of 4×10 ⁴ /mL, inoculate 8000 cells/well in a 6-well plate, culture in an incubator for 24 hours to make the cells adhere to the wall, discard the medium, and add Drug-containing medium with different concentrations (0, 0.625, 1.25, 2.5 μM for tanshinone Ⅱ A; 0, 5, 25, 50 μM for quercetin; 0, 10, 15, 20 μM for curcumin; 10, 20, 40 μM). (2) After culturing for 48 hours, the culture medium was discarded, and 1 mL of LPBS was added to wash twice. (3) Add 1 mL of 4% paraformaldehyde and react at room temperature for 20 min to increase the permeability of the cell membrane. (4) Discard 4% paraformaldehyde, add 1 mL of PBS to wash 3 times, and remove excess 4% paraformaldehyde residual solution. (5) Add 1 mL of 0.1% Triton-X-100 and react at room temperature for 20 minutes to denature a part of the membrane protein and further enhance the permeability. (6) Discard Triton-X-100, add 1mL LPBS to wash 3 times. (7) Add 200 μL of DAPI staining solution to each well, react at 37°C for 15 minutes, discard the staining solution, add 1 mL PBS and wash gently 3 times. (8) Finally, 1 mL of PBS was added to each well to resuspend the cells, and the staining was observed and recorded under an inverted fluorescent microscope.

6.8 The effect of traditional Chinese medicine on the content of microRNA21 in cells

The MDA-MB-231 cells selected for the experiment were all tumor cells in the logarithmic growth phase, and were cultured overnight to allow the cells to adhere to the wall, and the confluence reached 80% to 90%. The next day, the same volume of different concentrations of tanshinone Ⅱ A (final concentrations were 0, 0.5, 0.625, 1.25 μM), quercetin (final concentrations were 0, 1, 5, 25 μM), curcumin (final concentrations were 0, 5, 10, 15 μM) and berberine hydrochloride (final concentrations were 0, 10, 20, 40 μM) to treat the cells, wherein, the drug concentration in the cells of the control group was 0 μM (that is, the medium was the final concentration of 0.1% DMSO solution), and cultivated in a 37°C, 5% CO ₂ incubator for 48 hours. Then the cell culture supernatant was collected, and the microRNA in each group of cells was extracted according to the requirements of the RNA extraction kit. Finally, the extracted microRNA was mixed with the porous silicon deoxyribozyme fluorescent probe to measure the microRNA content in the cells.

The specific steps are: (1) collect 1×10 ⁶ cells in each group, add 300 μL of lysate to dissolve them, transfer them to a clean centrifuge tube, then add 300 μL of binding solution I and mix by inversion; (2) Transfer the mixture in (1) to the first purification column, centrifuge at 12000g for 1min, recover the supernatant into a new centrifuge tube, add 700μL binding solution II and mix well; (3) divide the mixture in (2) Transfer to the second purification column twice, centrifuge at 12000g for 1min, discard the liquid in the tube; (4) add 600μL of washing solution to the purification column, centrifuge at 12000g for 1min, a total of 2 times, discard the liquid in the tube; centrifuge at 16000g for 2min, remove Residual liquid; (5) Put the purification column in the RNA elution tube, add 30 μL of eluent, leave it at room temperature for 2-3 minutes, centrifuge at 16000g for 1 minute, and the obtained solution is the purified microRNA. (6) Mix the purified microRNA of each group with the probe, measure the fluorescence of ce6, and calculate the content of microRNA21 in the cells of each group.

7. Statistical analysis

Data are expressed as mean±SEM (x±s). An independent sample t-test was used between the two groups, and P<0.05 was considered statistically significant.

3. Experimental structure

1. Characterization of MSN-NH ₂

The appearance and size of the prepared MSN-NH ₂ were observed with a transmission electron microscope (TEM). Figure 2 shows that the porous silica nanoparticles are spherical or quasi-spherical, with a diameter between 100 and 120 nm. The pores of MSN-NH ₂ are dense and uniform, and the diameter of the pores is about 2-3nm. The surface area and pore size distribution of porous silicon were reflected by nitrogen adsorption-desorption measurement (BET). It can be seen from Fig. 3 that type IV adsorption isotherms can be obtained after obtaining samples in this study. The synthesized porous silicon material has uniform mesopores, and the specific parameters are: specific surface area 797.0492m ² /g, pore diameter 3.7296nm.

2. Verification of the reaction ability of HP1/HP2 and microRNA21

The reaction characteristics between HP1, HP2 and microRNA21 were characterized by agarose gel electrophoresis, and the results are shown in FIG. 4 . Among them, lanes 1, 2, and 3 respectively correspond to the distances that HP1, HP2, and microRNA21 with different molecular weights move in the same time. In lane 4, HP1 and HP2 are at their corresponding molecular weight positions, indicating that hairpin HP1 and hairpin HP2 are structurally stable, and cross-combination will not occur after the two are mixed; similarly, the phenomenon in lane 6 shows that microRNA21 and HP2 are in their respective positions At the corresponding position, no new light spots were generated, indicating that when only microRNA21 and hairpin HP2 exist, the two will not combine, so the existence of HP2 will not interfere with microRNA21, and the reaction system has no by-products. The phenomenon in lane 5 shows that the mixture of HP1 and microRNA21 produces a new spot with larger molecular weight and brighter, indicating that the reaction will only occur when HP1 and microRNA21 co-exist, and microRNA21 opens the cardinal HP1 and binds, the spot It is a new complex generated after the combination of microRNA21 and HP1. In addition, the results of lane 7 showed a total of 2 spots, one was HP2 alone, and the other was the complex after HP1 combined with microRNA21, which indicated that HP2 did not interfere with the reaction between HP1 and microRNA21. Based on the above analysis, the DNA hairpin HP1/HP2 designed in this study has a good specific reaction with microRNA21.

3. Reactivity verification of zinc ion-specific DNAzyme

Firstly, the release of ce6 in porous silicon was investigated in the presence or absence of zinc ions. As shown in Figure 5, only a small amount of ce6 was released in the absence of zinc ions. This phenomenon is because a small amount of microRNA21 turns on HP1. A small amount of ce6 molecules in the pores were released slowly, but it was not enough to open a large number of pores and release ce6 quickly; when microRNA21 and zinc ions were present at the same time, the release of ce6 continued to increase with time, and the release slowed down at about 2h. The above results indicated that only with the assistance of zinc ions, the zinc ion-specific deoxyribozyme could be activated to cut the enzyme cleavage site of HP2, resulting in a large release of ce6 in the pore.

This experiment further verified the change of enzyme cleavage reaction of zinc ion-specific deoxyribozyme over time. One end of HP2 is covalently fixed on the glass slide substrate, and the other end is modified with fluorescent molecule cy3, then the change in the fluorescence intensity of cy3 on the substrate will be used to indicate the enzymatic cleavage efficiency of HP2, the principle is shown in Figure 6. In the presence of microRNA21 and zinc ions, HP1 opens and changes its configuration, binds to the complementary sequence on HP2, activates the enzymatic activity of zinc ion-specific deoxyribozyme, cuts HP2, and breaks HP2. Due to the modification of cy3 at the end of HP2, the cy3 on the glass slide will be reduced after enzyme digestion and washing, and the fluorescence intensity will be reduced. As the reaction time increases, HP1 can also walk on the slide, continue to react with surrounding HP2, and cut off more HP2, so that there will be less and less Cy3 on the slide, and the fluorescence intensity will also show lower and lower Dynamic changes.

The fluorescence intensity of Cy3 after different reaction times was photographed and recorded with an inverted fluorescence microscope, and the results are shown in FIG. 7 . At the beginning of the reaction, the fluorescence intensity was the strongest, because the deoxyribozyme had not acted yet, and HP2-cy3 was not cleaved; as the reaction time increased, the fluorescence intensity showed a downward trend, because HP2 was gradually cut off, and cy3 was released from the load. gradually removed from the slide. At about 50 minutes, the fluorescence intensity was weak, almost zero, indicating that the digestion was sufficient. This phenomenon verifies the principle in Figure 6 that "under the same conditions, a molecule of miRNA21 can cause multi-molecule HP2 shearing with the help of zinc ions", and it serves as the signal amplification of the gated porous silicon-released fluorescent probe (Figure 1) in this experiment Base.

4. Optimization of reaction conditions

4.1 The ratio optimization of HP1 and HP2

In order to investigate the optimal ratio between HP1 and HP2 on the porous silicon surface, HP1 and HP2 with equal volumes and concentration ratios of 1:5, 1:10, 1:15, 1:20 and 1:25 were selected for wrapping MSN @ce6, other reaction conditions are 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 8, when HP1:HP2=1:10, the reaction efficiency is the best.

4.2 Zinc ion concentration optimization

The fluorescence intensity of ce6 released by the probe after reacting at different zinc ion concentrations was measured to investigate the effect of zinc ion concentration on enzyme cleavage efficiency. The results are shown in Figure 9, when the concentration of zinc ions was higher (5 μM to 100 μM), the release of ce6 gradually increased with time, indicating that as the concentration of zinc ions increased, the enzyme cleavage efficiency accelerated; when the concentration of zinc ions continued to increase (> 100 μM), the release of ce6 decreased instead, probably because the higher zinc ion concentration destroyed the conditions required to maintain DNA stability in the system, which was not conducive to the reaction. When the concentration of zinc ions is small (100nM～5μM), the release of ce6 increases with the increase of the concentration. The above results indicated that the enzymatic cleavage efficiency of zinc ion-specific DNAzyme was concentration-dependent within a certain range. Therefore, for a suitable concentration and better digestion efficiency, a zinc ion concentration of 5 μM was selected for subsequent experiments.

5. Specificity investigation

5.1 Other metal ion interference

This experiment investigated the influence of other interfering ions such as calcium ions, potassium ions, magnesium ions, manganese ions and sodium ions on the reaction system under the same experimental conditions, and evaluated the interference of other metal ions on the reaction system by comparing the ce6 fluorescence intensity after the reaction . The results are shown in Figure 10, the reaction system can only release a large amount of ce6 in the presence of zinc ions, while other metal ions have little effect on the reaction and will not interfere with the reaction.

5.2 microRNA21 specificity investigation

In this experiment, the fluorescence intensity of microRNA21 and other mismatched microRNAs reacted with the porous silicon deoxyribozyme fluorescent probe was compared to verify whether the probe has good selectivity for the recognition of microRNA21. The results are shown in Figure 11. The fluorescent signal after reacting with mismatched microRNA is weak, indicating that less ce6 is released in the hole; after reacting with microRNA21, the fluorescence intensity of ce6 is the strongest, indicating that only microRNA 21 can open HP1, and further Trigger the subsequent enzyme cleavage reaction to realize the release of more ce6. In addition, although the single-base mismatch sequence triggered more ce6 release, the concentration was as high as 10 μM, much higher than the concentration of non-mismatched microRNA21, indicating that single-base mismatch has little interference in the normal reaction system. The above experimental results show that the porous silicon deoxyribozyme fluorescent probe synthesized in this experiment has good specificity for detecting microRNA21.

5.3 Effect of different buffers on the reaction system

In this experiment, the reaction of porous silicon deoxyribozyme fluorescent probe in different buffer solutions was investigated. Disperse the probe in a variety of different systems (pure water, PBS, HEPES, DMEM and RPMI 1640 medium), and then perform the blocking and releasing experiments of HP1 and HP2 in these different systems, and record the fluorescence in the reaction Changes in intensity. The results are shown in Figure 12. In pure water medium, due to the lack of salt ions necessary to maintain DNA and microRNA hybridization reactions, the release of fluorescent molecules is slow; while in other media, the fluorescence release curves are basically the same as those in HEPES buffer. Consistent, proving the controllable release of the probe in different buffer media, indicating that the stability of the probe in different solutions such as PBS, HEPES, DMEM and RPMI 1640 medium is good.

6. Determination of miRNA21 standard curve

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

7. Investigation on the application of probes in drug efficacy evaluation

7.1 The effect of traditional Chinese medicine on cell morphology

This experiment investigated the effects of four traditional Chinese medicine monomers (tanshinone ⅡA, curcumin, quercetin and berberine hydrochloride) on the growth and morphology of breast cancer cell MDA-MB-231. After co-cultivating with MDA-MB-231 cells for 24 hours and 48 hours at different concentrations of traditional Chinese medicine monomers, observe and record the changes in the number and shape of MDA-MB-231 cells with a microscope, and calculate the cell inhibition rate (%) at each concentration. It can be seen from Figures 14 to 17 that the breast cancer cells MDA-MB-231 in the blank control group grew well, and no morphological changes were observed. Compared with the blank control group, different concentrations of tanshinone Ⅱ A, curcumin, quercetin and berberine hydrochloride in each experimental group inhibited the growth of breast cancer MDA-MB-231 cells to varying degrees, and the concentration- and time-dependent Sex (see Table 1 to Table 4). It can be seen from the results that after the co-culture of tanshinone ⅡA, curcumin, quercetin and berberine hydrochloride with MDA-MB-231 cells, the shape of breast cancer cells gradually became irregular, the cells shrank and became smaller, and the intercellular spaces increased. Tend to single growth, increased suspension cells and other characteristics. In addition, with the increase of the concentration of each traditional Chinese medicine, the above-mentioned changes in cell morphology became more obvious, and under the same microscope field of view, it was found that the number of tumor cells also showed a decreasing trend with the increase of the concentration of traditional Chinese medicine.

Table 1 Tanshinone Ⅱ A inhibits MDA-MB-231 at different dose groups and different time points (%)

^a : P<0.05, vs 24h control group, ^b : P<0.05, vs 48h control group

Table 2 Quercetin inhibits MDA-MB-231 rate (%) in different dose groups and different time points

^a : P<0.05, vs 24h control group, ^b : P<0.05, vs 48h control group

Table 3 Curcumin inhibits MDA-MB-231 rate (%) in different dosage groups and different time points

^a : P<0.05, vs 24h control group, ^b : P<0.05, vs 48h control group

Table 4 berberine inhibits MDA-MB-231 rate (%) in different dose groups and different time points

^a : P<0.05, vs 24h control group, ^b : P<0.05, vs 48h control group

7.2 The effect of traditional Chinese medicine on cell viability

In this experiment, four kinds of traditional Chinese medicine monomers with different concentrations were selected to co-culture with breast cancer cell MDA-MB-231 for 24h and 48h respectively, and the proliferation inhibition of breast cancer cells by four kinds of traditional Chinese medicine monomers with different concentrations was determined by CCK-8 method. effect. The results are shown in Figure 18, the four traditional Chinese medicine monomers have a dose- and time-dependent effect on the cell growth of MDA-MB-231 cells. With the increase of drug concentration, the growth of MDA-MB-231 cancer cells was significantly inhibited, and this phenomenon became more significant with time. The concentration for inhibiting cell growth was obtained by calculating the _IC50 of each traditional Chinese medicine monomer. Tanshinone Ⅱ A was 1.213 μM, curcumin was 16.43 μM, berberine hydrochloride was 30.87 μM, and quercetin was 49.90 μM.

7.3 The effect of traditional Chinese medicine on the formation of cell clones

The four kinds of traditional Chinese medicine monomers were co-cultured with breast cancer cells MDA-MB-231, and the cells were grown for 14 days after administration. When obvious cell clusters appeared, they were stained with crystal violet, and the number of MDA-MB-231 cell clones in each group was compared. difference. The results are shown in Figure 19, after the four kinds of traditional Chinese medicines were used, the ability of MDA-MB-231 cells to form colonies was significantly reduced, and the number of colonies decreased. The average number of colony formation of MDA-MB-231 cells in the blank control group was 946, and after treatment with 0.625 μM tanshinone Ⅱ A, 25 μM quercetin, 15 μM curcumin, and 20 μM berberine, the average number of colony formation was 152, 114, 76, 48. Compared with the blank control group, the differences were statistically significant (P<0.05). To sum up, the effect of four traditional Chinese medicines (tanshinone Ⅱ A, curcumin, quercetin and berberine) on the colony formation ability of breast cancer cell MDA-MB-231 cells was analyzed by selective clone formation experiment. In contrast, the colony formation ability of cells treated with traditional Chinese medicine was significantly reduced, indicating that the traditional Chinese medicine tanshinone ⅡA, curcumin, quercetin and berberine hydrochloride can all significantly inhibit the colony formation of MDA-MB-231 cells.

7.4 The effect of traditional Chinese medicine on cell migration behavior

In this study, images were taken at 0h, 24h and 48h after the administration of breast cancer cells MDA-MB-231. In order to ensure that the cells maintain a certain viability, the selected concentrations were: 0.625 μM tanshinone Ⅱ A, 25 μM quercetin, 15 μM curcumin and 20 μM berberine hydrochloride to investigate the migration properties of MDA-MB-231 cells. The results are shown in Figure 20, the cells that were not treated with traditional Chinese medicine, as time went on, the scratches gradually healed, and the migration speed was faster. When treated with traditional Chinese medicine, cell migration slowed down and healing speed decreased. Comparing the migration results of the four traditional Chinese medicines, although the inhibition rates are different, the effects of berberine and curcumin are stronger, and the effects of quercetin and tanshinone Ⅱ A are milder. Alkali and quercetin can inhibit the migration of cancer cells and reduce the ability of cancer cells to spread to non-tumor areas to slow down the progression of cancer.

7.5 The effect of traditional Chinese medicine on apoptosis

In this study, different groups of MDA-MB-231 cells were stained with DAPI, photographed under an inverted fluorescence microscope, and the fluorescence intensity was calculated with ImageJ. The results are shown in Figures 21 and 22. For the control group, that is, the MDA-MB-231 cells that have not interacted with traditional Chinese medicine, uniform blue fluorescence can be observed in the entire nucleus; Obvious blue-white fluorescence, and the nuclei become round. The state of apoptosis can be judged by the fluorescence brightness and the shape of the nucleus: early apoptotic cells show nuclear condensation, staining deepens, or nuclear chromatin gathers in a crescent shape on one side of the nuclear membrane; late apoptotic cells show nuclear fragmentation into different sizes Equal round bodies surrounded by cell membranes. Comparing the cell staining results after four administrations, the degree of apoptosis of MDA-MB-231 cells treated with the traditional Chinese medicine curcumin was significantly increased, the chromatin condensed a large number of nuclei, the shape was round, the cell membrane was bubbling, and the fluorescence was brighter; Tanshinone IIA has the mildest effect, and only a small number of nuclei show brighter blue fluorescence. In summary, although the four traditional Chinese medicines have significantly different effects on the apoptosis of breast cancer cells, they all affect the apoptosis process of breast cancer cells.

7.6 The effect of traditional Chinese medicine on the change of microRNA21 content in cells

Four kinds of traditional Chinese medicines with different concentrations were co-cultured with MDA-MB-231 cells, 10 ⁶ cells in each group were extracted, and microRNA21 in each group of cells was extracted with RNA extraction kit, and then porous silicon-DNAzyme fluorescent detection was added The reaction was carried out in the needle, and the change of microRNA21 content after the cells were treated with traditional Chinese medicine was measured by the fluorescence intensity of ce6. The results are shown in Figure 23. Compared with the control group, the content of microRNA21 in MDA-MB-231 cells treated with tanshinone Ⅱ A, berberine, quercetin and curcumin all decreased in a concentration-dependent manner. It shows that all four traditional Chinese medicines can inhibit the growth of microRNA21 in cells. Comparing the effects of the four traditional Chinese medicines, it can be seen that the effect of berberine is relatively strong, and the content of microRNA21 in cells is almost reduced to zero. At the same time, the above results show that the reduction of microRNA21 content is related to the change trend of cell experiment results such as decreased cell viability, weakened clone formation ability, slowed cell migration ability, and increased cell apoptosis after the traditional Chinese medicine acts on triple-negative breast cancer cells. It is consistent, indicating that the change of microRNA21 content can reflect the development process of triple-negative breast cancer.

Explanation of the experimental results:

In this study, based on amino-modified porous silicon, two functional DNA hairpin structures were loaded on the inside of the pore and modified on the outside of the pore, and a porous silicon deoxyribozyme fluorescent probe capable of detecting cancer marker microRNA 21 was synthesized. The experimental results show that the probe can specifically and sensitively detect trace amounts of microRNA 21 in the system, and can be quantitatively analyzed by fluorescence intensity.

Secondly, on the basis of investigating the effects of four traditional Chinese medicine monomers (tanshinone ⅡA, curcumin, berberine, and quercetin) on MDA-MB-231 cell morphology, cell proliferation, cell apoptosis, and cell migration, the synthetic The porous silicon-DNAzyme fluorescent probe MSN@ce6-HP1/HP2 detects the changes of microRNA21 content in cells after administration. The results showed that the porous silicon

The deoxyribozyme fluorescent probe can achieve highly sensitive detection of trace microRNA 21 in cells, and the reduction of microRNA 21 content in cells after administration is consistent with the change trend of cancer cell behavior after administration (decreased cell viability, weakened colony formation ability, Cell migration slowed down, and the degree of cell apoptosis increased, etc.), indicating that the constructed porous silicon-DNAzyme fluorescent probe can be used to evaluate the anticancer activity of different traditional Chinese medicine ingredients.

In addition, the porous silicon-DNAzyme fluorescent probe synthesized in this study can be further modified to target aptamers or biomembranes that are easy to endocytosis, so that the porous silicon complex can enter the interior of the cell, and it can be applied to real-time fluorescence imaging to monitor intracellular microRNA21 content. In terms of enhancing the curative effect of traditional Chinese medicine, it can be combined with membrane carriers with specific response properties to load different structural types of traditional Chinese medicine, so as to realize the orderly release of different drugs in cells, achieve the combination of different ingredients of traditional Chinese medicine and enhance the efficacy of drugs. role and has broad application prospects.

The purpose of the above embodiments is to specifically introduce the substantive 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 the specific embodiments.

Claims (10)

1. A porous silicon deoxyribose nucleic enzyme fluorescent probe for detecting microRNA comprises amino modified porous silica nanoparticles, fluorescent molecules loaded in pore channel structures of the porous silica nanoparticles, and two DNA hairpin HP1 and HP2 which are covalently connected to the surfaces of the porous silica nanoparticles and block the fluorescent molecules in the pore channel structures; wherein the nucleotide sequence of the HP1 comprises a complementary sequence of microRNA to be detected, and the nucleotide sequence of the HP2 comprises an enzyme cleavage site of zinc ion specific deoxyribozyme; after the HP1 is combined with the microRNA to be detected, the root is opened, the ring part of the HP1 is subjected to configuration change and combined with the ring part nucleotide sequence of the HP2, the zinc ion specific deoxyribozyme is activated under the assistance of zinc ions, then the HP2 is cut off at an enzyme cutting site, a corresponding blocked pore canal is opened, fluorescent molecules loaded in the pore canal structure are released, and the detection of the microRNA to be detected is realized through the fluorescent molecular intensity.

2. The porous silica deoxyribose enzyme fluorescent probe according to claim 1, wherein the amino group-modified porous silica nanoparticle is prepared by the steps of:

(1) Chemical synthesis: dissolving a proper amount of cetyl trimethyl ammonium bromide in a sodium hydroxide solution, sequentially adding a proper amount of tetraethoxysilane and 3-aminopropyl triethoxysilane, stirring for reaction, standing, centrifuging after layering the solution to obtain white precipitate, washing with ethanol and pure water, and drying;

(2) Removing the template: dissolving the precipitate in hydrochloric acid-ethanol solution, reflux-extracting, centrifuging, washing the obtained white precipitate with ethanol and pure water, and oven drying to obtain amino-modified porous silica nanoparticle powder.

3. The porous silica deoxyribose enzyme fluorescent probe according to claim 2, wherein the fluorescent molecule is ce6, and the step of loading the fluorescent molecule in the pore structure of the porous silica nanoparticle is as follows:

uniformly dispersing a proper amount of amino modified porous silica nanoparticle powder in N-2-hydroxyethyl piperazine-N ' -2-ethanesulfonic acid buffer solution with pH value of 7.0, adding a proper amount of N-epsilon-maleimidocaproyl-oxasuccinimidyl ester for connecting DNA and amino on the porous silica nanoparticle to modify the porous silica nanoparticle, centrifuging, uniformly dispersing the precipitate in the N-2-hydroxyethyl piperazine-N ' -2-ethanesulfonic acid buffer solution with pH value of 7.0, adding a proper amount of ce6, mixing, centrifuging, and dispersing the precipitate in the N-2-hydroxyethyl piperazine-N ' -2-ethanesulfonic acid buffer solution with pH value of 7.0.

4. The porous silica deoxyribose nucleic acid fluorescent probe according to claim 3, wherein the method for covalently connecting the DNA hairpin HP1, HP2 on the surface of the porous silica nanoparticle is as follows: mixing the porous silica nanoparticle loaded with fluorescent molecules with freshly prepared DNA hairpin HP1 and HP2, reacting for a proper amount of time at normal temperature, centrifuging, washing with pure water, and redissolving the precipitate in N-2-hydroxyethyl piperazine-N' -2-ethanesulfonic acid buffer with pH=7.0.

5. The porous silicon deoxyribose nucleic acid fluorescent probe according to claim 4, wherein the microRNA is microRNA21, and the DNA hairpin HP1 and HP2 are prepared by the following method: mixing a proper amount of DNA 1 and DNA2 with a proper amount of tris- (2-formylethyl) phosphine hydrochloride at normal temperature, and reducing disulfide bonds in the DNA; then, annealing the reduced DNA, and standing at normal temperature to obtain DNA hairpin HP1 and DNA hairpin HP2 respectively;

wherein:

the sequence of DNA 1 is: 5'-TTTT GATG TTGATTC TCC GAG CCG GTC GAAATAGTG GGT TTT TTT TTT TTT TTT TTT TT T CAACAT CAG TCT GATAAG CTA-HS-3';

the sequence of DNA2 is: 5'-CGA CGA CG TTT TTT ACC CAC TAT rA G GAA T CAA C TTT TTT CG TCG TCG TTTT-HS-3'.

6. A method of preparing the porous silicon deoxyribose nucleic acid fluorescent probe of claim 5, comprising the steps of: mixing the amino modified porous silica nano particles loaded with fluorescent molecules ce6 with freshly prepared DNA hairpin HP1 and DNA hairpin HP2, performing mild reaction at normal temperature, centrifuging, collecting precipitate, washing with pure water, and redissolving the precipitate in N-2-hydroxyethyl piperazine-N' -2-ethanesulfonic acid buffer solution with pH=7.0.

7. The use of the porous silicon deoxyribose nucleic acid fluorescent probe according to any one of claims 1 to 4 for detecting microRNA.

8. The use of the porous silicon deoxyribose nucleic acid fluorescent probe according to claim 5 in detecting microRNA 21.

9. The application of the porous silicon deoxyribose nucleic acid fluorescent probe in-vitro evaluation of the anti-tumor activity of the traditional Chinese medicine by detecting microRNA 21.

10. The use of claim 9, wherein the tumor is a triple negative breast cancer.

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