Preparation method and application of a urease-responsive miRNA detection nanoprobe for bladder cancer

Abstract

The present invention discloses a preparation method and application of a urease-responsive miRNA detection nanoprobe for bladder cancer. In this method, magnetic Fe3O4 nanoparticles and urease are linked to the surface of SiO2, and H1 and H2 modified with a fluorescent group and a quenching group are respectively linked to the surfaces of gold nanorods and Fe3O4 to prepare a Janus nanoprobe that can specifically capture target miRNA and release fluorescence. This method is stable, reliable and has good repeatability. The prepared Janus nanoprobe has good stability and dispersibility, can be fully diffused in the urine environment driven by urease, and realizes the specific binding of H1 and H2 to miRNA; then, through magnetic separation and enrichment effects, signal amplification is achieved; by catalyzing urease to fully capture target miRNA, non-invasive detection is realized. In addition, the stable structure of hDNA also improves the sensitivity and specificity of detection to a certain extent, providing a new idea for the non-invasive diagnosis of bladder cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a preparation method and application of a urease-responsive miRNA detection nanoprobe for bladder cancer. Background Art

[0002] As the most common cancer in the urinary system, bladder cancer ranks ninth in the global cancer statistics report in terms of incidence and 13th in terms of mortality. Bladder cancer is the sixth most common cancer in men, with an incidence approximately four times that of women. 75% of patients present with non-muscle-invasive bladder cancer (NMIBC), and at an early stage, the prognosis is good. However, the postoperative recurrence rate of high-risk patients is as high as 80%, and the recurrence rate of low-risk patients is still as high as 50%. The 5-year survival rate after early detection and treatment is 94%, which is because the intervention measures will greatly affect the overall survival rate. It can be seen that the diagnosis of bladder cancer is of great significance for subsequent treatment.

[0003] Currently, cystoscopy is still the gold standard for diagnosing bladder cancer. It is a method of inserting a cystoscope through the urethra into the bladder to directly observe the lesions in the bladder and urethra. As an invasive examination method, cystoscopy is prone to complications such as urinary tract infection, hematuria, and pain, and it is difficult to detect smaller lesions. Urine cytology is an auxiliary means of cystoscopy. It mainly detects exfoliated cancer cells from urine or bladder wash specimens and is the first non-invasive examination method applied clinically. Urine cytology detection has high specificity, but the sensitivity varies depending on the tumor grade. Due to the high intercellular adhesion and lack of significant cellular atypia in low-grade tumors, the sensitivity of urine cytology detection is as low as 16% in low-grade tumors. Moreover, urine cytology examination can only see single or a pile of cells, cannot comprehensively observe the tissue structure of the lesion, and it is not easy to make a clear tissue typing of cancer cells, which results in the inability of urine cytology detection to determine the specific location of the tumor. In addition, the high recurrence rate of bladder cancer requires regular monitoring of high-risk patients after surgery, and frequent cystoscopy is likely to cause anxiety and pain to patients and also consume certain economic costs. It can be seen that cystoscopy has certain limitations in urine cytology examination and cannot meet the needs of bladder cancer patients in terms of diagnosis and monitoring.

[0004] To address the deficiencies in the diagnosis, treatment, and follow-up of bladder cancer, the identification of biomarkers in urine, tissues, and blood has become an important part of precision medicine. Currently, the six urine biomarker tests approved by the FDA mainly include: quantitative nuclear matrix protein, qualitative NMP22, quantitative bladder tumor-associated antigen, qualitative BTA, fluorescence in situ hybridization, and fluorescence immunohistochemistry. Although the sensitivity of these detection methods is higher than that of urine cytology, the specificity is much lower than that of urine cytology. Therefore, they can only be used as part of the monitoring and cannot be used independently without a cystoscope. Currently, miRNA has become an emerging urine biomarker. miRNA is a class of non-coding single-stranded RNA molecules encoded by endogenous genes, with a length of approximately 22 nucleotides, and is involved in the expression and regulation of multiple genes in the body. The abnormal expression of miRNA is associated with various cancers, including bladder cancer, and is widely present in human body fluids such as serum, urine, and saliva. The traditional techniques for miRNA determination mainly include Northern blotting, nucleic acid microarray technology, and quantitative reverse transcription polymerase chain reaction. However, these techniques have disadvantages such as time-consuming, low sensitivity, false positives, and difficult primer design, thus limiting their application. To overcome the limitations of traditional bladder cancer diagnosis methods, seeking new diagnostic tools is currently a research hotspot.

[0005] In recent years, probes based on nanomaterials have attracted extensive attention. In particular, Janus-structured nanomaterials have been widely used due to their advantage of integrating multiple material functional characteristics to exert synergistic functions. Compared with mechanical mixtures of single components, Janus nanoparticles can effectively break the traditional symmetry, integrate two or more compounds with different functions or properties into one system, and exhibit properties different from those of individual particles. Therefore, propeller-shaped nanomotors based on Janus structures show great application prospects in the biomedical field.

[0006] Due to their controllable size and easy characterization, gold nanorods (AuNRs) are easily combined with other materials to form carriers with multiple functions and are widely used in the fields of drug delivery, targeted therapy, and biosensing. And silica is commonly used as a coating material for AuNRs due to its high surface area and surface reactivity. This is because the composite formed by silica and AuNRs can not only retain the optical properties of AuNRs but also increase the surface area. In addition, the silanols on the surface of the composite can be coupled with various ligands, thus effectively achieving functionalization. As a commonly used magnetic material, Fe3O4 is often used to pre-enrich and separate targets, thereby enabling the test to avoid background interference.

[0007] At present, there are mainly three types of driving for nanomotors: chemical driving, physical driving, and biological driving. Among them, chemical driving is mainly driven by catalytic reactions and enzyme-catalyzed reactions, including self-electrophoresis, self-diffusion, and bubble driving. Physical driving mainly converts external driving into mechanical energy by magnetism, ultrasound, light, electricity, etc. to drive itself. Biological driving mainly uses microorganisms such as red blood cells, platelets, macrophages, sperm, and bacteria as driving sources. However, the problem of biocompatibility limits the application of many nanomotors loaded with toxic fuels. Therefore, enzyme-catalyzed reactions show great application prospects. Urea usually exists at high concentrations in multiple disease sites (such as the bladder). Designing a nanomotor using enzyme-catalyzed urea as a power source has good application prospects for the diagnosis and treatment of various urinary tract diseases such as bladder cancer. Summary of the Invention

[0008] The purpose of the present invention is to provide a preparation method and application of a urease-responsive miRNA detection nanoprobe for bladder cancer. The probe prepared by this method can fully capture the target miRNA and achieve non-invasive detection.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is: a preparation method of a urease-responsive miRNA detection nanoprobe for bladder cancer, comprising the following steps:

[0010] (1) Add the seed solution prepared from 100 - 500 mg of CTAB, 0.5 - 2 mg of chloroauric acid trihydrate, and 10 - 50 mg of sodium borohydride to the growth solution prepared from 2 - 5 g of CTAB, 0.1 - 0.5 g of sodium oleate, 15 - 40 mg of chloroauric acid trihydrate, 0.5 - 1 mL of hydrochloric acid, 10 - 15 mg of silver nitrate, and 2 - 5 mg of ascorbic acid. After the two solutions are mixed evenly, let it stand at 20 - 50 °C for 10 - 30 h to obtain the AuNR solution;

[0011] (2) Mix 400 - 600 mg of CTAB with 10 - 20 mg of the AuNR solution, 1 - 3 mL of absolute ethanol, 0.5 - 1 mL of ammonia water, and 10 - 20 μL of BTEE, and stir at 20 - 50 °C for 2 - 10 h to obtain the SiO2@AuNR solution;

[0012] (3) Mix 10 - 20 mg of SiO2@AuNR with 60 - 90 μL of APTES at 20 - 50 °C and stir for 8 - 15 h. Centrifuge to wash away the unreacted APTES. Then add 0.5 - 2 mg of carboxylated Fe3O4 to the amino-functionalized SiO2@AuNR solution and stir at 20 - 50 °C for 10 - 30 h to obtain the Fe3O4-SiO2@AuNR solution;

[0013] (4) Slowly add 1 - 3 mL of 25% glutaraldehyde solution dropwise to 10 - 20 mg of Fe3O4 - SiO2@AuNR solution, stir at 20 - 50 °C for 2 - 4 h, then add 40 - 80 mg of urease to the solution and stir at 20 - 50 °C for 10 - 12 h. Through the immobilization of urease, Fe3O4 / urease - SiO2@AuNR with urease only on one side is obtained;

[0014] (5) Add 20 - 40 μL of H1 with a concentration of 5 - 20 uM and 40 - 70 μL of H2 with a concentration of 5 - 20 μM to 10 - 20 mL of an aqueous solution of Fe3O4 / urease - SiO2@AuNR with a concentration of 1 - 2 mg / mL, stir in the dark at 20 - 50 °C for 8 - 12 h to prepare a urease - driven Janus H2 - Fe3O4 / urease - SiO2@AuNR - H1 nanoprobe.

[0015] Preferably, in step (1), the aspect ratio of the prepared AuNR is 50 - 100 nm for the long diameter and 10 - 50 nm for the short diameter.

[0016] Preferably, in step (2), the particle size of the prepared SiO2@AuNR is 70 - 150 nm, the surface charge is 15 - 25 mv, and the molar ratio of SiO2 to AuNR is 1:1 - 1:5.

[0017] Preferably, in step (3), the particle size of Fe3O4 is 5 - 15 nm, and the mass ratio of Fe3O4 to SiO2@AuNR is 1:2 - 1:10.

[0018] Preferably, in step (3), the particle size of the prepared Fe3O4 - SiO2@AuNR is 90 - 150 nm, and the surface charge is -5 - 10 mv.

[0019] Preferably, in step (4), the mass ratio of the Fe3O4 - SiO2@AuNR solution to urease is 1:2 - 1:6.

[0020] Preferably, in step (4), the particle size of the prepared Fe3O4 / urease - SiO2@AuNR is 100 - 180 nm, and the surface charge is -16 - -25 mv.

[0021] Preferably, in step (5), the cross - linking rates of H1 and H2 are 20 - 40% and 25 - 50% respectively.

[0022] Preferably, in step (5), the particle size of the prepared Janus H2 - Fe3O4 / urease - SiO2@AuNR - H1 nanoprobe is 110 - 200 nm, and the surface charge is 10 - -25 mv.

[0023] To achieve the above object, the present invention also provides an application of the urease-responsive miRNA detection nanoprobe for bladder cancer prepared by the above method in detecting urinary biomarker miRNA. The nanoprobe uses gas driving to diffuse and capture target miRNA in a liquid environment, and uses magnetic separation to enrich the probe, thereby achieving signal amplification and improving the accuracy of detection, which is of great significance for non-invasive diagnosis of bladder cancer.

[0024] The nanoprobe prepared by the present invention uses Janus nanoparticles with an asymmetric structure as a carrier, and innovatively connects urease and carboxylated Fe3O4 to the surface of amino-functionalized SiO2. Through Au-S and amidation reactions, thiol-modified H1 and amino-modified H2 are respectively connected to the surface of gold nanorods and Fe3O4 to prepare a self-driven Janus nanoprobe that can specifically capture target miRNA and release fluorescence. Driven by the gas generated by the catalytic action of urease, the Janus nanoprobe moves sufficiently in urine, specifically recognizes and captures target miRNA, and through the magnetic separation and enrichment of Fe3O4, background interference is avoided, signal amplification is achieved, and the sensitivity and specificity of detection are improved, providing a new idea for non-invasive diagnosis of bladder cancer.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) Urease in the present invention has high specificity and can directly catalyze the hydrolysis of urea (present at high concentrations in diseased sites such as the bladder) into ammonia and carbon dioxide in a urine environment, generating gas driving, thereby promoting the nanoprobe to capture target miRNA more fully and improving the detection accuracy;

[0027] (2) Fe3O4 nanoparticles in the present invention have good biocompatibility and are easy to surface-modify; after modification, the present invention not only has higher stability, enhanced dispersibility and biocompatibility in a liquid environment, but also the magnetism is not affected by surface modification, and can move directionally under the action of an external magnet, which is easy for magnetic separation and enrichment to achieve signal amplification;

[0028] (3) hDNA designed by fluorescence method in the present invention forms a stable structure through intrastrand base complementary pairing, and only opens the double strand when binding to target miRNA, separating the fluorescent group and quenching group at both ends and releasing fluorescence, which helps to improve the specificity and sensitivity of detection;

[0029] (4) The preparation method of the present invention is stable, reliable and has good repeatability. The prepared Janus nanoprobe has good stability and dispersibility, and can fully capture the target miRNA through urease catalysis. At the same time, with the help of magnetic Fe3O4 nanoparticles, magnetic separation and enrichment are easy to achieve, realizing signal amplification. In addition, the stable structure of hDNA also improves the detection sensitivity and specificity to a certain extent, providing new ideas for non-invasive diagnosis of bladder cancer. Description of the Drawings

[0030] Figure 1 Transmission electron microscopy images of AuNR, SiO2@AuNR, and Fe3O4-SiO2@AuNR prepared in the examples of the present invention, (a) AuNR, (b) SiO2@AuNR, (c) Fe3O4-SiO2@AuNR;

[0031] Figure 2 Element mapping diagram of Fe3O4-SiO2@AuNR prepared in the examples of the present invention;

[0032] Figure 3 Hydrated particle size diagram of Fe3O4 / urease-SiO2@AuNR prepared in the examples of the present invention;

[0033] Figure 4 Surface charge diagram of different nanoparticles involved in the examples of the present invention;

[0034] Figure 5 Ultraviolet spectrum diagram of different nanoparticles involved in the examples of the present invention;

[0035] Figure 6 Standard curve for BCA protein concentration determination when Fe3O4-SiO2@AuNR in the present invention is connected with urease;

[0036] Figure 7 Standard curves of ultraviolet of different concentrations of H1 and H2 in the present invention, (A) H1, (B) H2;

[0037] Figure 8 Catalytic efficiency of different concentrations of Fe3O4 / urease-SiO2@AuNR on different concentrations of urea in the present invention, (A) different concentrations of Fe3O4 / urease-SiO2@AuNR (0.02 - 0.3 mg / mL), (B) different concentrations of urea (25 - 600 mM);

[0038] Figure 9 Gel electrophoresis diagram of the binding of hDNA and target miRNA in the present invention;

[0039] Figure 10Specific binding diagram of the nanoprobe prepared in the embodiment of the present invention with the target miRNA and fluorescence gain diagrams before and after enzymatic cleavage. (A) Fluorescence analysis of the probe specifically capturing miRNA-21. (B) Fluorescence analysis of the probe specifically capturing miRNA-182. (C) Fluorescence analysis of the probe before and after enzymatic cleavage after capturing miRNA.

[0040] Figure 11 Fluorescence comparison diagram before and after enrichment of the probe prepared in the embodiment of the present invention after capturing the target miRNA. (A) Fluorescence comparison diagram before and after enrichment of the probe after capturing miRNA-182. (B) Fluorescence comparison diagram before and after enrichment of the probe after capturing miRNA-21.

[0041] Figure 12 Detection limit of the probe prepared in the embodiment of the present invention for capturing miRNA-182 with / without driving. (A) Detection limit of the probe for capturing miRNA-182 without driving. (B) Detection limit of the probe for capturing miRNA-182 with driving.

[0042] Figure 13 Detection limit of the probe prepared in the embodiment of the present invention for capturing miRNA-21 with / without driving. (A) Detection limit of the probe for capturing miRNA-21 without driving. (B) Detection limit of the probe for capturing miRNA-21 with driving. Detailed implementation manners

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] The technical solutions in the embodiments of the present invention will be further described below with reference to the drawings in the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0045] All raw materials and reagents in the embodiments of the present application are purchased on the market or prepared by conventional methods well-known to those skilled in the art.

[0046] In the present invention, CTAB represents cetyltrimethylammonium bromide, AuNR represents gold nanorods, BTEE represents 1,2-bis(triethoxysilyl)ethane, SiO2@AuNR represents silica semi-coated gold nanorods, APTES represents 3-aminopropyltriethoxysilane, H1 represents thiol-modified hDNA1, H2 represents amino-modified hDNA2, GA represents glutaraldehyde, HAuCl4·3H2O represents chloroauric acid trihydrate, and NaBH4 represents sodium borohydride.

[0047] Example

[0048] A preparation method of a urease-responsive miRNA detection nanoprobe for bladder cancer, comprising the following steps:

[0049] (1) Preparation of AuNR: Mix 10 mL of CTAB with a concentration of 100 mM and 250 μL of HAuCl4·3H2O with a concentration of 10 mM evenly at 30 °C. Add 0.6 mL of 10 mM NaBH4 prepared with ice water to the solution and stir vigorously for 2 min. After the solution changes from yellow to brown, age the mixture at 30 °C for 30 min to obtain seed solution A. Dissolve 2.7 g of CTAB and 0.3702 g of sodium oleate in 142.5 mL of deionized water and mix evenly at room temperature. Then, slowly add 7.5 mL of HAuCl4·3H2O solution with a concentration of 10 mM to the solution, and the mixture changes from yellow to colorless. Finally, add 0.45 mL of HCl, 2.88 mL of AgNO3 solution with a concentration of 10 mM, and 0.375 mL of L-AA with a concentration of 64 mM to the solution, and obtain growth solution B after mixing. Take 30 μL of solution A and slowly drop it into solution B. After stirring vigorously for 2 min, let the mixture stand overnight at 30 °C. Finally, centrifuge to collect the precipitate and dissolve the precipitate in water to form a 1 mL AuNR solution. As shown in Figure 1 (a), the major axis of AuNR is about 70 - 80 nm, and the minor axis is about 20 - 30 nm, with uniform size.

[0050] (2) Preparation of SiO2@AuNR: Disperse 1 mL of the pre-prepared AuNR solution in 19 mL of deionized water, and add 27.33 mg of CTAB to the solution and ultrasonically mix evenly. Then, add 1.5 mL of CTAB solution with a concentration of 45 mg / mL, 1.5 mL of absolute ethanol, and 0.55 mL of 28 wt% ammonia water to the mixed solution in sequence. After stirring at 1600 rpm for 40 min, continue to add 15 μL of BTEE to the solution and stir at 25 °C for 4 h. Finally, centrifuge to collect the semi-coated SiO2@AuNR. As shown in Figure 1 (b), the particle size of SiO2@AuNR is about 100 - 110 nm, with a uniform and regular morphology and good dispersibility.

[0051] (3) Preparation of Fe3O4-SiO2@AuNR: Dissolve 20 mg of AuNR@SiO2 in 8 mL of water, and sequentially add 0.4 mL of 28 wt% ammonia water and 80 μL of APTES. After the solution reacts at room temperature for 10 h, centrifuge to collect AuNR@SiO2-NH2. After dissolving the obtained AuNR@SiO2-NH2 in water to 1.5 mL, add 500 μL of carboxylated Fe3O4 nanoparticles to the solution. Stir overnight at room temperature, and centrifuge to collect the precipitate Fe3O4-SiO2@AuNR. As Figure 1 (c) shows, Fe3O4 was successfully modified onto the surface of SiO2@AuNR, and the obtained Fe3O4-SiO2@AuNR had a particle size of about 100 - 110 nm. Its elemental mapping is shown in Figure 2 . The uniform distribution of elements such as gold, silicon, and oxygen indicates the successful synthesis of Janus-structured nanoparticles, and the appearance of iron (Fe) element further proves that Fe3O4 was successfully adsorbed onto the SiO2 surface.

[0052] (4) Preparation of Fe3O4 / urease-SiO2@AuNR: Disperse 10 mg of AuNR@SiO2-Fe3O4 in 9 mL of PBS with a pH of 6.5 and sonicate for 15 min. Then, add 1 mL of GA to the solution and stir at room temperature for 2 h. After washing three times with PBS and centrifuging to collect the precipitate, resuspend the precipitate in 10 mL of PBS and add 30 mg of urease to it. After mechanical stirring for 16 h, centrifuge to separate the precipitate and wash it three times with deionized water. Finally, redisperse the prepared Fe3O4 / urease-SiO2@AuNR in 10 mL of deionized water and store it at 4 °C for later use. After measuring the particle size of Fe3O4 / urease-SiO2@AuNR by TEM, it can be known that ( Figure 3 ), the size distribution of these nanoparticles is uniform, and the particle size is 100 - 120 nm.

[0053] (5) Preparation of H2-Fe3O4 / urease-SiO2@AuNR-H1: Sequentially add 33 μL of H1 with a concentration of 10 μM and 50 μL of H2 with a concentration of 10 μM to the prepared 10 mL of Fe3O4 / urease-SiO2@AuNR, and stir overnight at room temperature. After the reaction is completed, centrifuge to collect the precipitate, and redisperse the precipitate in 10 mL of buffer solution containing some (sodium ions, magnesium ions, etc.) ions, and store it in the dark at 4 °C for later use.

[0054] To further verify the successful coating of SiO2 and the successful connection of urease and hDNA, the present invention used a dynamic light scattering instrument to detect the surface charges of SiO2@AuNR, NH2-SiO2@AuNR, Fe3O4-SiO2@AuNR, Fe3O4 / urease-SiO2@AuNR, urease, H1, H2, and H2-Fe3O4 / urease-SiO2@AuNR-H1 respectively. The results are as Figure 4 shown. The surface potential of SiO2@AuNR is positive, and it remains positive after amination. However, after connecting Fe3O4 nanoparticles with a negative surface potential, the overall potential of the nanoprobe becomes negative. After modifying with negatively charged urease, H1, and H2, the surface potential of the nanoprobe remains negative. The change in surface charge indicates the successful preparation of the probe.

[0055] To further verify the successful preparation of the H2-Fe3O4 / urease-SiO2@AuNR-H1 probe, the present invention tested the ultraviolet absorption spectra of SiO2@AuNR, Fe3O4-SiO2@AuNR, Fe3O4 / urease-SiO2@AuNR, H2-Fe3O4 / urease-SiO2@AuNR-H1, urease, H1, and H2 respectively. The results are as Figure 5 shown. AuNR shows absorption peaks at 500 nm and 808 nm, while Fe3O4 has no obvious peak in the ultraviolet region. Therefore, the ultraviolet spectrum of Fe3O4-SiO2@AuNR after connecting Fe3O4 has no obvious change. Urease and DNA show absorption peaks at 270 nm and 260 nm respectively, which results in Fe3O4 / urease-SiO2@AuNR showing characteristic absorption peaks at 270 nm after connecting urease. However, the small interval between 270 nm and 260 nm wavelengths cannot be completely converted into two characteristic peaks. Therefore, H2-Fe3O4 / urease-SiO2@AuNR-H1 after modifying with H1 and H2 only shows a broad peak, which indicates the successful connection of urease, H1, and H2, that is, the successful preparation of the H2-Fe3O4 / urease-SiO2@AuNR-H1 probe.

[0056] To calculate the optimal connection ratio of Fe3O4-SiO2@AuNR and urease, the present invention first tested different concentrations of urease and made a standard curve ( Figure 6 ). According to the standard curve of urease, the loading amounts of Fe3O4-SiO2@AuNR and urease at different ratios were calculated. The results are shown in Table 1. When the mass ratio of Fe3O4-SiO2@AuNR to urease is 1:4, the maximum loading of urease can be achieved, avoiding material waste.

[0057] Table 1 shows the connection ratio of Fe3O4-SiO2@AuNR to urease determined in the present invention

[0058]

[0059] To calculate the crosslinking rates of Fe3O4 / urease-SiO2@AuNR with H1 and H2, the present invention first systematically tested different concentrations of H1 and H2 solutions and made corresponding standard curves, and the results are as Figure 7 shown in A and B. Then, after centrifugation, the concentrations of H1 and H2 in the supernatant were measured, and the crosslinking rates of Fe3O4 / urease-SiO2@AuNR with H1 and H2 were calculated to be 31% and 38% respectively. To enable the nanoprobes to maintain continuous and efficient driving even at low urea concentrations, the present invention co-incubated different concentrations of Fe3O4 / urease-SiO2@AuNR with 100 mM urea and 0.08 wt% p-nitrophenol for different times. Since urease can catalyze the hydrolysis of urea into ammonia and carbon dioxide, the production of ammonia can turn the color of the solution yellow. After co-incubation for different times, the solution was rapidly titrated with 10 mM hydrochloric acid until the solution became colorless. Therefore, the amount of ammonium ions can be evaluated by the volume of hydrochloric acid in the neutralized solution, and then the content of generated ammonia can be calculated to evaluate the catalytic activity of urease. As Figure 8 shown in A, the optimal concentration of Fe3O4 / urease-SiO2@AuNR is 0.1 mg / mL. To verify and explore the urea concentration at which Fe3O4 / urease-SiO2@AuNR can maintain continuous driving, the present invention co-incubated Fe3O4 / urease-SiO2@AuNR with different concentrations of urea for different times. As Figure 8 shown in B, when the urea concentration is greater than 100 mM, Fe3O4 / urease-SiO2@AuNR can all achieve continuous driving.

[0060] hDNA is a "hairpin" structure formed by intrastrand base complementary pairing of single-stranded DNA and spontaneously forming head-to-tail connection. It usually has a fluorescent group designed at one end and a quenching group designed at the other end. When specifically hybridized with the target sequence, the fluorescent group and the quenching group in it are separated, and then fluorescence is emitted. To verify the feasibility of the H2-Fe3O4 / urease-SiO2@AuNR-H1 probe for miRNA detection, the present invention co-incubated H1 and H2 with the target miRNA respectively and performed gel electrophoresis. As Figure 9 shown, the bands of the hybridized hDNA and the target miRNA lag behind the bands of the single hDNA or miRNA alone, indicating the successful binding of the two. One end of H1 is modified with the fluorescent group Cy5 and the other end is modified with the quenching group BHQ3. And one end of H2 is modified with the fluorescent group FAM and the other end is modified with the quenching group BHQ1. When both H1 and H2 specifically bind to the target miRNA, the hDNA double strand unfolds into a single strand, and the fluorescent group and the quenching group are separated, resulting in fluorescence release.

[0061] To verify the specific binding of the H2-Fe3O4 / urease-SiO2@AuNR-H1 probe to the target miRNA, in the present invention, H1 and H2 were respectively incubated with the corresponding miRNA with single-base mismatch, three / four-base mismatch, and other miRNAs with partially similar fragments, and the fluorescence intensity was measured. As Figure 10 shown in A and B, the fluorescence intensity of the hDNA incubated with the target miRNA was significantly higher than that of other non-target miRNAs, indicating that the probe could specifically capture the target miRNA, providing a basis for subsequent experiments.

[0062] As Figure 10 shown in C, Fe3O4 itself had a certain impact on fluorescence. Therefore, DSN enzyme was used to cleave hDNA from the nanocarrier before measuring fluorescence. The strong magnetism of Fe3O4 was hardly affected by surface modification. Therefore, magnetic separation technology could be used to enrich fluorescence. After comparison, it was found that the fluorescence intensity after enrichment was about 4 - 10 times that without enrichment ( Figure 11 ), which fully demonstrated that the magnetic enrichment effect achieved by Fe3O4 could effectively amplify the fluorescence signal. As Figure 12 and 13 shown, when urea was not added as a driving force, the detection limits of the nanoprobes for miRNA-182 and miRNA-21 were 10 -12 M and 10 -13 M respectively, while in the presence of urea, the detection limits were 10 -13 M and 10 -14 M respectively, which further demonstrated the promoting effect of urease catalysis on the probe to capture the target miRNA.

[0063] For those skilled in the art, the present invention is not limited to the details of the above exemplary embodiments. The embodiments are exemplary and non-restrictive. The protection scope of the present invention is defined by the appended claims rather than the above description. Therefore, any reference signs in the claims should not be regarded as limiting the claimed rights. The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Therefore, any minor modifications, equivalent replacements, and improvements made to the above embodiments based on the technical essence of the present invention should be included in the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe, characterized in that: The following steps are involved: (1) Add a seed solution prepared by 100-500 mg CTAB, 0.5-2 mg chloroauric acid trihydrate, and 10-50 mg sodium borohydride to a growth solution prepared by 2-5 g CTAB, 0.1-0.5 g sodium oleate, 15-40 mg chloroauric acid trihydrate, 0.5-1 mL hydrochloric acid, 10-15 mg silver nitrate, and 2-5 mg ascorbic acid. After the two solutions are evenly mixed, stand at 20-50°C for 10-30 h to obtain an AuNR solution. (2) Mix 400-600 mg CTAB with 10-20 mg AuNR solution, 1-3 mL anhydrous ethanol, 0.5-1 mL ammonia water, and 10-20 μL BTEE, and stir at 20-50 °C for 2-10 h to obtain SiO2@AuNR solution; (3) Mix 10-20 mg SiO2@AuNR and 60-90 uL APTES at 20-50 °C and stir for 8-15 h, centrifuge to remove unreacted APTES, then add 0.5-2 mg carboxylated Fe3O4 to the amination SiO2@AuNR solution and stir at 20-50 °C for 10-30 h to obtain Fe3O4-SiO2@AuNR solution; (4) Slowly add 1-3 mL of 25% glutaraldehyde solution to 10-20 mg Fe3O4-SiO2@AuNR solution, stir at 20-50°C for 2-4 h, then add 40-80 mg urease to the solution and stir at 20-50°C for 10-12 h. Fe3O4 / urease-SiO2@AuNR containing urease on only one side is obtained by fixing urease. (5) 20-40 μL of 5-20 uM H1 and 40-70 μL of 5-20 μM H2 were added to 10-20 mL of 1-2 mg / mL Fe3O4 / urease-SiO2@AuNR aqueous solution, and stirred at 20-50 °C in the dark for 8-12 h to prepare urease-driven Janus H2-Fe3O4 / urease-SiO2@AuNR-H1 nanoprobe; H1 represents thiol-modified hDNA1, and H2 represents amino-modified hDNA2.

2. The method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe according to claim 1, characterized in that: The AuNR prepared in step (1) has a long diameter of 50 to 100 nm and a short diameter of 10 to 50 nm.

3. The method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe according to claim 1, characterized in that: The particle size of SiO2@AuNR prepared in step (2) is 70-150 nm, the surface charge is 15-25 mv, and the molar ratio of SiO2 to AuNR is 1:1-1:

5.

4. The method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe according to claim 1, characterized in that: In step (3), the particle size of Fe3O4 is 5 to 15 nm, and the mass ratio of Fe3O4 to SiO2@AuNR is 1:2 to 1:

10.

5. The method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe according to claim 1, characterized in that: The Fe3O4-SiO2@AuNR prepared in step (3) has a particle size of 90 to 150 nm and a surface charge of -5 to 10 mv.

6. The method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe according to claim 1, characterized in that: In step (4), the mass ratio of Fe3O4-SiO2@AuNR solution to urease is 1:2 to 1:

6.

7. The method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe according to claim 1, characterized in that: The Fe3O4 / urease-SiO2@AuNR prepared in step (4) has a particle size of 100 to 180 nm and a surface charge of -16 to -25 mv.

8. The method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe according to claim 1, characterized in that: In step (5), the cross-linking rates of H1 and H2 are 20-40% and 25-50%, respectively.

9. The method for preparing a urease-responsive bladder cancer miRNA detection nanoprobe according to claim 1, characterized in that: The particle size of the Janus H2-Fe3O4 / urease-SiO2@AuNR-H1 nanoprobe prepared in step (5) is 110-200 nm, and the surface charge is 10-25 mv.

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