Reagent and kit for rapidly detecting tumors and application

Abstract

The invention provides a reagent for rapidly detecting tumors, and belongs to the technical field of biochemistry. The reagent provided by the invention comprises a MnO2 (at) CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nano probe and MR21C, the MnO2 (at) CRISPR nano probe is prepared by mixing a MnO2 nano sheet with Cas12a, crRNA and an F-Q reporter probe, and the MnO2 (at) CRISPR nano probe is prepared by mixing the MnO2 nano sheet with the And the MR21C contains a sequence of an MUC1 aptamer, a sequence of a CytC aptamer, a complementary sequence of crRNA (Complementary Ribonucleic Acid) and a complementary sequence of miR-21 (Micro Ribonucleic Acid). By means of the advantages of efficient delivery, rapid release and signal output reaction acceleration of the manganese dioxide nanosheets, rapid tumor detection and imaging amplification by combining the CRISPR system with the RCA technology are completed.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a reagent, a kit and an application for rapid detection of tumors. Background Art

[0002] MicroRNA (miRNA) is an endogenous non-coding RNA containing 20-24 nucleotides, which plays an important role in various biological processes in cells. Among them, miRNA-21 (miR-21), as a member of the miRNA family, is overexpressed in many cancers, especially in the occurrence and development of triple-negative breast cancer. MiR-21 promotes the proliferation and metastasis of breast cancer by targeting LZTFL 1. Therefore, miR-21 is widely used as a biomarker for early cancer screening.

[0003] Rolling circle amplification (RCA) is an isothermal enzymatic process driven by DNA polymerase. In the presence of DNA template and short DNA primers, long single-stranded DNA molecules with mostly repetitive sequence motifs are synthesized, which can be easily detected. The RCA products can encode DNA aptamers for specific tumor cell targeting and corresponding sequences for responding to specific targets in order to enhance the target CRISPR / Cas system as a powerful tool for genetic engineering and biosensing.

[0004] Two-dimensional nanomaterials, manganese dioxide (MnO2) nanosheets, have attracted attention in biological applications due to their large specific surface area, excellent biocompatibility and low toxicity. The wide and ultrathin sheet interface has effective π-stacking interactions, and it can physically absorb various biomolecules, such as proteins and nucleic acids. In addition to the potential carrying capacity, due to the overexpression of biothiols in biological media, especially glutathione (GSH) in cancer cells, the surface-loaded substances can be quickly and efficiently released through simple reduction behavior.

[0005] However, so far, there have been no reports on the rapid detection of tumors using MnO2 nanosheets loaded with CRISPR system combined with RCA technology. Summary of the invention

[0006] The purpose of the present invention is to provide a reagent for rapid detection of tumors, including MnO2@CRISPR nanoprobe and MR21C; the MnO2@CRISPR nanoprobe is a MnO2 nanosheet loaded with a CRISPR system, and MR21C is a product of rolling circle amplification. With the advantages of efficient delivery, rapid release and accelerated signal output reaction of manganese dioxide nanosheets, the CRISPR system is combined with RCA technology to perform rapid detection and imaging amplification of tumors.

[0007] The present invention provides a reagent for rapid detection of tumors, the reagent comprising MnO2@CRISPR nanoprobe and MR21C;

[0008] The MnO2@CRISPR nanoprobe is prepared by mixing MnO2 nanosheets with Cas12a, crRNA, and FQ reporter probes;

[0009] The MR21C contains the sequence of the MUC1 aptamer, the sequence of the CytC aptamer, the complementary sequence of the crRNA, and the complementary sequence of miR-21.

[0010] Preferably, the sequence of the crRNA is as shown in SEQ ID NO.1; the FQ reporter probe is FAM-TTATT-BHQ1.

[0011] Preferably, the MR21C is obtained by rolling circle amplification using a locked DNA template with the participation of a polymerase.

[0012] Preferably, the locked DNA template is prepared using a primer and a phosphorylated DNA template.

[0013] Preferably, the primer sequence is shown as SEQ ID NO.3, and the phosphorylated DNA template is shown as SEQ ID NO.2.

[0014] The present invention also provides the use of the above reagent in preparing tumor diagnosis products.

[0015] The present invention also provides the use of the above reagent in preparing a targeted tumor diagnosis product.

[0016] The present invention also provides the use of the above reagent in preparing a product for detecting mitochondrial miR-21.

[0017] The present invention also provides a kit for amplifying mitochondrial miR-21 imaging, and the kit comprises the above reagent.

[0018] The present invention also provides a miR-21 detection kit, comprising the above reagent and NE Buffer.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a reagent for rapid detection of tumors. Manganese dioxide nanosheets are used to accelerate the response of the CRISPR system to the RCA product MR21C, amplify the selective imaging of mitochondrial miR-21 in tumors, and then complete the detection of tumors. Manganese dioxide nanosheets act as "transmitters" and "accelerators" to achieve rapid delivery of the CRISPR system and rapid response of the CRISPR system to MR21C. MR21C can selectively target mitochondria in tumor cells. When miR-21 is "turned on", MR21C responds to crRNA in the CRISPR system, thereby activating Cas12a, cutting the FQ reporter probe, and achieving fluorescence recovery. The present invention uses miR-21 to turn on biosensing, from Mn 2+ Tumor cell detection and imaging are achieved through acceleration and rolling circle amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the characterization results of the MnO2 nanosheets in Example 1; wherein A is the TEM image of the MnO2 nanosheets, and B is the DLS data of the MnO2 nanosheets.

[0022] Figure 2 It is the assembly result of the MnO2@CRISPR nanoprobe in Example 1; wherein A is the ultraviolet spectrum of the MnO2@CRISPR nanoprobe, and B is the zeta potential of the MnO2@CRISPR nanoprobe.

[0023] Figure 3 This is the agarose gel electrophoresis diagram of MR21C in Example 1.

[0024] Figure 4 The feasibility test results of MnO2@CRISPR nanoprobe combined with RCA in solution in Example 1; A is the fluorescence spectrum of the CRISPR / Cas 12a-based sensing system under different conditions; B is the fluorescence intensity diagram of the CRISPR / Cas12a-based sensing system under different conditions (Ⅰ: with miR-21+1.5mM Mn 2+; Ⅱ: with miR-21; Ⅲ: without Casl2a; Ⅳ: without crRNA; Ⅴ: without FQ reporter probe: VI: without miR-21; VII: without MR21C); C is the fluorescence spectrum of the sensing system based on MnO2@CRISPR nanoprobe under different conditions; D is the fluorescence intensity diagram of the sensing system based on MnO2@CRISPR nanoprobe under different conditions (Ⅰ: withmiR-21+GSH; Ⅱ: with miR-21without GSH; Ⅲ: without Casl2a; Ⅳ: without crRNA; Ⅴ: without FQ reporter probe: VI: without miR-21; VII; without MR21C).

[0025] Figure 5 This is the fluorescence image after different sensor system response times in Example 1.

[0026] Figure 6 A is the fluorescence spectrum of the mixed solution of different RNA-responsive MnO2@CRISPR nanoprobes and MR21C in Example 1; Figure 6 B is the fluorescence intensity diagram of the mixed solution of different RNA responses MnO2@CRISPR nanoprobe and MR21C in Example 1, wherein Ⅰ: miR-21; Ⅱ: Blank; Ⅲ: miR-10b; Ⅳ: miR-141; V: miR-let-7f; Ⅵ: miR-210; Ⅶ: miR-let-7a.

[0027] Figure 7 A is the result of the fluorescence intensity at 520 nm given by the two sensing systems in Example 1, which is dependent on the miR-21 concentration; among them, red: MnO2@CRISPR nanoprobe + MR21C; purple: CRISPR + MR21C. Figure 7 B is the linear relationship between the MnO2@CRISPR nanoprobe + MR21C system and the miR-21 concentration in Example 1, y = 16.37708x + 18.22189, x represents the concentration of miR-21 (nM), and y represents the fluorescence intensity. Figure 7 C is the linear relationship between the CRISPR+MR21C system and the miR-21 concentration in Example 1, y=12.28884x+12.69777, x represents the concentration of miR-21 (nM), and y represents the fluorescence intensity.

[0028] Figure 8A is the result of the fluorescence intensity at 520 nm given by the two sensing systems in Example 1, which is dependent on the miR-21 concentration; among them, red: MnO2@CRISPR nanoprobe + MR21C; blue: MnO2@CRISPR nanoprobe + single-stranded substrate. Figure 8 B is the linear relationship between the MnO2@CRISPR nanoprobe + MR21C system and the miR-21 concentration in Example 1, y = 16.37708x + 18.22189, x represents the concentration of miR-21 (nM), and y represents the fluorescence intensity. Figure 8 C is the linear relationship between the MnO2@CRISPR nanoprobe + single-stranded substrate system and the miR-21 concentration in Example 1, y = 8.68828x + 13.93812, x represents the concentration of miR-21 (nM), and y represents the fluorescence intensity.

[0029] Fig. 9 A is the fluorescence microscope imaging based on MnO2@CRISPR nanoprobe under different conditions in Example 1 (scale bar: 20 μm); Fig. 9 B is the flow cell count based on MnO2@CRISPR nanoprobe under different conditions in Example 1, wherein, Ⅰ: blank control; Ⅱ: MnO2 / FQ reporter probe; Ⅲ: MnO2@CRISPR nanoprobe; Ⅳ: MnO2@CRISPR nanoprobe and MR21C.

[0030] Fig.10 This is a fluorescence microscope image of MR21C targeting mitochondria in Example 1, wherein red represents Cy-5-labeled MR21C; green represents Mito-Tracker Green (scale bar: 20 μm). DETAILED DESCRIPTION

[0031] The present invention provides a reagent for rapid detection of tumors, the reagent comprising MnO2@CRISPR nanoprobe and MR21C; the MnO2@CRISPR nanoprobe is prepared by mixing MnO2 nanosheets with Cas12a, crRNA, and FQ reporter probe; the MR21C contains the sequence of MUC1 aptamer, the sequence of CytC aptamer, the complementary sequence of crRNA, and the complementary sequence of miR-21. The Cas12a, crRNA, and FQ reporter probe are components of the CRISPR sensor system.

[0032] The MnO2@CRISPR nanoprobe of the present invention is prepared by directly mixing MnO2 nanosheets with components of the CRISPR sensing system, namely Cas12a, crRNA, and FQ reporter probes, and can be effectively internalized by living cells. As an implementable method, the preparation method of the MnO2 nanosheet includes mixing TMA solution and H2O2 with ultrapure water as solution A, dissolving MnCl24H2O in H2O as solution B, and then quickly mixing solution A with solution B to obtain a dark brown suspension, and stirring vigorously overnight at room temperature to obtain a precipitate of block MnO2. The prepared block MnO2 is centrifuged and washed with distilled water and methanol in sequence. Afterwards, the block MnO2 is dried and stored. The block MnO2 is dispersed in water and ultrasonically treated. The dispersion is then centrifuged, and the supernatant is retained as a MnO2 nanosheet. Preferably, the hydrodynamic diameter of the MnO2 nanosheet is 100 to 200 nm, more preferably 150 nm. As an implementation method, the sequence of the crRNA is shown in SEQ ID NO.1; the FQ reporter probe is FAM-TTATT-BHQ1; the Cas 12a is Lba Cas12a (Cpf1) purchased from Beijing NEB (Cat. No.: M0653T).

[0033] The MR21C described in the present invention is obtained by rolling circle amplification with the participation of a locked DNA template and a polymerase. The polymerase described in the present invention is preferably phi29 polymerase; the locked DNA template contains the sequence of the MUC1 aptamer, the sequence of the CytC aptamer, the complementary sequence of crRNA, and the complementary sequence of miR-21. As an implementation method, the locked DNA template is prepared using a primer and a phosphorylated DNA template. Preferably, the primer sequence is shown in SEQ ID NO.3, and the phosphorylated DNA template is shown in SEQ ID NO.2.

[0034] In the present invention, the MUC1 aptamer is encoded to selectively target MCF-7 cells, and the CytC aptamer further targets mitochondria, and the complementary sequence of crRNA is in a base pairing state with the complementary sequence of miR-21. At this time, the sensing function of the CRISPR system loaded by the MnO2 nanosheet is turned off. When miR-21 is present, it is paired with the complementary sequence of miR-21 in MR21C. At this time, MR21C is completely "opened", and the complementary sequence of crRNA is "exposed", completing the effective binding with the crRNA in the CRISPR system, thereby activating Cas 12a, cutting the FQ reporter probe, i.e., FQDNA, and realizing fluorescence recovery. The present invention utilizes the accelerated response of the MnO2 nanosheet and the signal amplification of the RCA technology to complete the "acceleration + amplification" imaging of mitochondrial miR-21, and further detects tumor cells. The tumor described in the present invention includes a tumor containing miR-21, such as any one of breast cancer, pancreatic cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, lung cancer, and kidney cancer. As an embodiment, the detected tumor cells are breast cancer tumor cells.

[0035] The present invention also provides the use of the above reagent in preparing a tumor diagnosis product. The above tumor is any one of breast cancer, pancreatic cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, lung cancer, and kidney cancer.

[0036] The present invention also provides the use of the above reagent in preparing a targeted tumor diagnosis product. The MUC1 aptamer of the present invention is encoded to selectively target tumor cells containing MUC1.

[0037] The present invention also provides the use of the above reagent in the preparation of a product for detecting mitochondrial miR-21. The MUC1 aptamer of the present invention is encoded to selectively target tumor cells containing MUC1; the CytC aptamer further targets mitochondria, using miR-21 in tumor cells to activate biosensing, and 2+ Mitochondrial miR-21 detection was achieved through both acceleration and rolling circle amplification.

[0038] The present invention also provides a kit for amplifying mitochondrial miR-21 imaging, the kit comprising the above-mentioned reagents. The kit of the present invention is based on the RCA product MR21C that accelerates the response of the CRISPR system by manganese dioxide nanosheets, and amplifies the selective imaging of mitochondrial miR-21. The MR21C contains the sequence of the MUC1 aptamer, the sequence of the CytC aptamer, the complementary sequence of crRNA, and the complementary sequence of miR-21. The MUC1 aptamer is encoded to selectively target tumor cells containing MUC1; once MR21C reaches the mitochondria, the CytC aptamer specifically binds to cytochrome c (CytC) to achieve targeted uptake of mitochondria.

[0039] The present invention also provides a detection kit for miR-21, comprising the above-mentioned reagents and NE Buffer. As an implementable embodiment, the reagents include MnO2 nanosheets, Cas 12a, crRNA, FQ reporter probes, and MR21C. Preferably, the MR21C contains the sequence of the MUC1 aptamer, the sequence of the CytC aptamer, the complementary sequence of crRNA, and the complementary sequence of miR-21. As an implementable embodiment, the final concentration of the MnO2 nanosheet is 40 μg / mL; the final concentration of Cas 12a is 80 pmol; the final concentration of crRNA is 150 pmol; and the final concentration of the FQ reporter probe is 150 pmol. The MnO2 nanosheet of the present invention is used as a transmitter and accelerator of the CRISPR system for rapid response to the CRISPR system. MR21C is obtained by rolling circle amplification by designing templates and primers. MR21C contains the sequence of the MUC1 aptamer, the sequence of the CytC aptamer, the complementary sequence of crRNA, and the complementary sequence of miR-21. The MUC1 aptamer enables MR21C to specifically target MCF-7 cells, and the CytC aptamer can specifically target mitochondria. The complementary sequence of crRNA is in a base pairing state with the complementary sequence of miR-21. At this time, the sensing function of the entire CRISPR system is silent. In the presence of miR-21, MR21C is "turned on", miR-21 and the complementary sequence of miR-21 in MR21C are base paired, and the complementary sequence of crRNA is "exposed" and completes base complementation with the crRNA in the CRISPR system, thereby activating the trans-cleavage activity of Cas 12a. As a result, the CRISPR system switches from the "OFF" to the "ON" state, resulting in the breakage of the FQ reporter probe and the recovery of FAM fluorescence, completing the spatial selective imaging of miR-21 in mitochondria.

[0040] In the following embodiments of the present invention, the FQ reporter probe is the FQ DNA in the attached figure.

[0041] All DNA and RNA sequences in Table 1 used in the following examples were purchased from Shanghai Bioengineering Co., Ltd.; Lba Cas12a (Cpf 1) was purchased from Beijing NEB (M0653T); MnCl2·4H2O and tetramethylammonium hydroxide (TMA·OH) were purchased from AlfaAesar (China); glutathione (GSH) and N-ethylmaleimide (NEM) were purchased from Sigma-Aldrich Chemical Co., Ltd.; 6×DNA loading buffer, nucleic acid dye SuperRed, and DNA molecular weight standard MarkerA (50-500bp) were purchased from Solebao; Lipofectamine 3000 transfection reagent was purchased from Thermo; Trypsin / EDTA (0.25%) was purchased from Gibco (Thermo Fisher Scientific); fetal bovine serum (FBS), DMEM and double antibody (penicillin and streptomycin mixture) were provided by Solebao; DAPI was purchased from Biyuntian; MCF-7 breast cancer cells were from Wuhan Pronosai Life Science Technology Co., Ltd.

[0042] Unless otherwise specified, the test methods used in the following examples are all conventional test methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0044] Example 1

[0045] 1. Materials and Reagents

[0046] The DNA and RNA sequences involved in the examples are shown in Table 1.

[0047] Table 1 DNA and RNA sequences involved in the examples

[0048]

[0049] 2. Preparation of MnO2 nanosheets loaded with CRISPR system (MnO2@CRISPR nanoprobes)

[0050] 1. Preparation of MnO2 nanosheets

[0051] 12 mL of TMA solution (0.1 M) and 2 mL of H2O2 (30 wt.%) were mixed with 6 mL of ultrapure water as solution A, and MnCl24H2O (0.6 g) was dissolved in 10 mL of H2O as solution B. Then, solution A and solution B were quickly mixed within 15 seconds, and the color of the mixture turned dark brown, indicating that Mn 2+ Oxidized to Mn 4+. The resulting dark brown suspension was vigorously stirred at room temperature overnight to obtain bulk MnO2. The prepared bulk MnO2 was centrifuged at 2000 rpm for 10 minutes and washed three times with distilled water and methanol respectively. Thereafter, the bulk MnO2 was dried at 60 °C and stored for use. To prepare MnO2 nanosheets, 10 mg of bulk MnO2 was dispersed in 20 mL of water and ultrasonically treated for 10 h. The dispersion was then centrifuged at 2000 rpm, and the supernatant was retained as MnO2 nanosheets, which was stored for further use.

[0052] 2. Preparation of MnO2@CRISPR nanoprobes

[0053] The above 100 μL MnO2 nanosheets (40 μg / mL), 3 μL crRNA (100 nM) and 3 μL FQ reporter probe (100 nM) were stirred and mixed for 20 minutes, and then 20 μL Tris-HAc buffer (100 mM, pH 7.4) was added to prepare a mixture. Afterwards, 8 μL Cas 12a, i.e., Lba Cas12a (Cpf1) (purchased from Beijing NEB (M0653T)) (50 nM) was added to the above mixture and incubated at room temperature for another 10 minutes to obtain the MnO2@CRISPR nanoprobe. The prepared MnO2@CRISPR can be used directly in subsequent experiments.

[0054] 3. Detection

[0055] The morphology of MnO2 nanosheets was characterized by TEM, and the hydrodynamic diameter of MnO2 nanosheets was analyzed by DLS. The zeta potential and UV light detection of the assembled MnO2@CRISPR were also performed.

[0056] 4. Results:

[0057] By TEM( Figure 1 A) characterizes the morphology of MnO2 nanosheets and finds that the prepared MnO2 nanosheets exhibit an irregularly folded sheet structure and have good dispersibility. DLS ( Figure 1 B) Analysis shows that the hydrodynamic diameter of MnO2 nanosheets is about 150nm, which is similar to Figure 1 The A in the figure is basically consistent. Through physical adsorption, Cas12a, crRNA and FQ reporter probes can be easily assembled on the surface of MnO2 nanosheets. Specifically, the coexistence of the ultraviolet characteristic absorption of MnO2 nanosheets at 380nm and DNA (or RNA) at 260nm ( Figure 2 A in the figure), and the zeta potential of the assembled MnO2 nanosheets ( Figure 2B) decreased to (-20.3 ± 0.17 mV). Since the Cas 12a protein carries an opposite charge (23.1 ± 0.55 mV), MnO2 and the FQ reporter probe form an assembly. Through electrostatic interaction, the Cas 12a / crRNA complex can be further assembled to form a MnO2@FQ reporter probe / crRNA / Cas12a nanoprobe (abbreviated as MnO2@CRISPR nanoprobe).

[0058] 3. Preparation of MR21C

[0059] 1. Preparation of MR21C:

[0060] Mix 1 μL phosphorylated DNA template strand (10 μM) and 2 μL primer (10 μM) in 2 μL 10×T4 DNA ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP), add 15 μL DEPC water, heat at 95°C for 5 min, then gradually cool to room temperature, incubate at 25°C for 2-3 h, and add 1 μL T4 DNA ligase (1 U / μL) after annealing. React at 37°C for 3 h to generate a locked DNA template. Take 5 μL of the above locked DNA template, 1 μL 10×phi29 buffer, 0.8 μL dNTP 10 mM, 1 μL (10 U / μL) phi29 polymerase, 15 μL DEPC water, and incubate at 37°C for 1-2 h. Heat at 90°C for 10 minutes to inactivate active substances such as polymerase. Finally, the mixture was centrifuged at 12000 rpm for 20 min, washed with ultrapure water to obtain MR21C, which was stored at -20°C for future use.

[0061] To examine the efficient synthesis of MR21C, agarose gel electrophoresis was performed.

[0062] Test results:

[0063] MR21C was synthesized by rolling circle amplification using the phosphorylated DNA template and primers in Table 1 above. Figure 3 As shown, the leftmost side is a marker of 50-500 bp. The phosphorylated DNA template and primers all have corresponding bands at the corresponding molecular weights, and the locked DNA template (template and primers are combined) and high molecular weight MR21C are synthesized.

[0064] 4. Feasibility analysis of MnO2@CRISPR nanoprobe combined with RCA in solution

[0065] The 5 μg / mL MnO2 nanosheets prepared above were incubated with 0.5 mM GSH. After 5 min, 2.1 NEBuffer (1×), Cas 12a (50 nM), crRNA (100 nM), FQ reporter probe (100 nM), MR21C (100 nM), and miR-21 (100 nM) were added to obtain a mixture with a total volume of 100 μL.

[0066] The mixture was incubated at 37°C for 10 min, and the fluorescence spectrum was recorded on a three-dimensional imaging luminescence spectrometer with an excitation wavelength of 488 nm.

[0067] The samples used as controls were the samples without MnO2 nanosheets in the above mixture, the samples without miR-21 in the above mixture, the samples without Cas12a in the above mixture, the samples without crRNA in the above mixture, the samples without FQ reporter probe in the above mixture, and the samples without MR21C in the above mixture. The experiment was repeated with other conditions unchanged.

[0068] Test results:

[0069] First, the ability of the free CRISPR / Cas 12a-based sensing system to respond to miR-21 was verified by three-dimensional imaging luminescence spectrometry. miR-21 can activate the trans-cleavage activity of CRISPR / Cas 12a, such as Figure 4 As shown in A and B in Figure 1, in the absence of miR-21, the sensing system is in the "OFF" state, giving very low background fluorescence. miR-21 can activate the trans-cleavage activity of CRISPR / Cas 12a, thereby increasing FAM fluorescence, Mn 2+ The addition of further improved the luminescence intensity of FAM.

[0070] This clearly demonstrates the feasibility of the proposed sensing strategy and highlights the 2+ Sensitization effect of MnO2@CRISPR nanoprobe.

[0071] When any of Cas 12a, crRNA, FQ reporter probe, MR21C, and miR-21 was lacking, there was no fluorescence signal, indicating that these components are essential. At the same time, it was found that in the presence of GSH, the MnO2@Cas12a nanoprobe gave the same fluorescence signal as the above Mn 2+ Approximate fluorescence intensity of the accelerated CRISPR / Cas 12a system ( Figure 4 C and D in Figure 3). That is, the decomposition of MnO2 nanosheets by GSH released the CRISPR / Cas 12a system, and the generated Mn 2+Promoted the accelerated response of CRISPR / Cas 12a to miR-21.

[0072] 5. Temporal fluorescence changes of different sensing systems in response to miR-21

[0073] 5 μg / mL MnO2 nanosheets were incubated with 0.5 mM GSH. After 5 min, 2.1 NEBuffer (1×), Cas 12a (50 nM), crRNA (100 nM), FQ reporter probe (100 nM), MR21C (100 nM), and miR-21 (100 nM) were added to obtain a total volume of 100 μL of the mixture.

[0074] The mixture was incubated at 37°C for 10, 20, 30, 40, 50, and 60 min, respectively, and the fluorescence spectra were recorded on a three-dimensional imaging luminescence spectrometer (FL-4700, Hitachi) with an excitation wavelength of 488 nm.

[0075] The samples used as controls were samples without MnO2 nanosheets, samples without GSH, and samples without miR-21, and the experiment was repeated with other conditions unchanged. The fluorescence changes of miR-21 under different conditions over time were monitored by a three-dimensional imaging luminescence spectrometer.

[0076] Results: From Figure 5 It can be found that free CRISPR / Cas 12a showed a slow fluorescence response to miR-21, and the slow fluorescence growth even lasted for 60 minutes. In contrast, the GSH-assisted MnO2@CRISPR nanoprobe greatly accelerated the fluorescence increase, and the fluorescence increased to a very high level after 10 minutes of reaction. In the absence of miR-21 or GSH, no fluorescence signal was generated. These results indicate that Mn 2+ The fluorescence reaction of CRISPR / Cas 12a can be greatly accelerated, thereby achieving rapid analysis of miR-21.

[0077] 6. Specific response of miR-21

[0078] 5 μg / mL MnO2 nanosheets were incubated with 0.5 mM GSH. After 5 min, 2.1 NEBuffer (1×), Cas 12a (50 nM), crRNA (100 nM), FQ reporter probe (100 nM), MR21C (100 nM), and miR-21 (100 nM) were added to obtain a total volume of 100 μL of the mixture.

[0079] The mixture was incubated at 37°C for 10, 20, 30, 40, 50, and 60 min, respectively, and the fluorescence spectra were recorded on a three-dimensional imaging luminescence spectrometer (FL-4700, Hitachi) with an excitation wavelength of 488 nm.

[0080] As a control sample, miR-21 was replaced with other RNAs, and the experiment was repeated with other conditions unchanged. Among them, Ⅰ: miR-21; Ⅱ: Blank; Ⅲ: miR-10b; Ⅳ: miR-141; V: miR-let-7f; Ⅵ: miR-210; Ⅶ: miR-let-7a;

[0081] The fluorescence signals of different RNAs responding to CRISPR / Cas12a were measured separately using a three-dimensional imaging luminescence spectrometer.

[0082] Results: The fluorescence signals of different RNA responses to CRISPR / Cas12a were measured by a three-dimensional imaging luminescence spectrometer. Figure 6 In A and B, the FAM fluorescence signal only appears in the presence of miR-21. This is because the presence of miR-21 prompts MR21C to "open", and then MR21C complements with CRISPR / crRNA, activates CRISPR / Cas12a, cuts the FQ reporter probe, and restores the signal. Other RNAs do not produce fluorescent signals, indicating that this scheme has good specificity for responding to miR-21.

[0083] VII. Sensing performance of miR-21 in solution

[0084] In order to study the performance of different concentrations of miR-21 in different sensing systems, the sensing performance of free CRISPR / Cas12a on miR-21 was compared.

[0085] 5 μg / mL MnO2 nanosheets were incubated with 0.5 mM GSH. After 5 min, 2.1 NEBuffer (1×), Cas 12a (50 nM), crRNA (100 nM), FQ reporter probe (100 nM), MR21C (100 nM), and different concentrations of miR-21 (0.1-200 nM concentration range) were added to make the total volume 100 μL. The mixture was incubated at 37 ° C for 10 min, and the fluorescence spectrum was recorded in a three-dimensional imaging luminescence spectrometer with an excitation wavelength of 488 nm.

[0086] Control: Preparation steps of free CRISPR (without MnO2 nanosheets):

[0087] 2.1NEBuffer (1×), Cas 12a (50 nM), crRNA (100 nM), and FQ reporter probe (100 nM) were added to a total volume of 100 μL and mixed to obtain the CRISPR system.

[0088] The fluorescence responses of MnO2@CRISPR nanoprobe+MR21C and CRISPR / Cas12a+MR21C to miR-21 were measured by a three-dimensional imaging luminescence spectrometer, and the fluorescence spectral data at 520 nm were recorded. The fluorescence data were analyzed by Origin software, and linear equations were obtained respectively.

[0089] like Figure 7 As shown in A, without the assistance of MnO2 nanosheets, the fluorescence response of CRISPR / Cas12a to miR-21 is much slower. The MnO2@CRISPR nanoprobe has a good linear relationship with miR-21 in the concentration range of 0.1-20nM, and the linear equation is: 16.37708x+18.22189( Figure 7 B); while the fluorescence signal output of CRISPR / Cas 12a to miR-21 was greatly reduced, and CRISPR / Cas 12a had a good linear relationship with miR-21 in the concentration range of 1-20nM, and the linear equation was y=12.28884x+12.69777( Figure 7 C), indicating that the MnO2@CRISPR nanoprobe has a more sensitive response to miR-21.

[0090] In order to highlight the role of RCA, the single-stranded substrate without RCA amplification was used as a control to study the single-stranded substrate without RCA amplification in response to miR-21 for comparison. The fluorescence response of MnO2@CRISPR nanoprobe + MR21C and MnO2@CRISPR nanoprobe + single-stranded substrate to miR-21 was measured by a three-dimensional imaging luminescence spectrometer, and the fluorescence spectrum data at 520nm was recorded. The fluorescence data were analyzed by Origin software to obtain linear equations.

[0091] Preparation of MnO2@CRISPR nanoprobe + single-stranded substrate

[0092] 100 μL MnO2 nanosheets (40 μg / mL), 3 μL crRNA (100 nM), 3 μL F-Q reporter probe (100 nM) and 2 μL single-stranded substrate (10 μM) were stirred and mixed for 20 minutes, and then 20 μL Tris-HAc buffer (100 mM, pH 7.4) was added to prepare. After that, 8 μL Cas 12a (50 nM) was added to the above mixture and incubated at room temperature for another 10 minutes to obtain MnO2@CRISPR nanoprobe + single-stranded substrate.

[0093] like Figure 8 As shown in A, without the help of MR21C, the fluorescence response of miR-21 by relying solely on the single-stranded substrate is much slower. The MnO2 / CRISPR nanoprobe has a good linear relationship with miR-21 in the concentration range of 0.1-20nM, and the linear equation is: y=16.37708x+18.22189( Figure 8 B); MnO2@CRISPR+ single-stranded substrate has a good linear relationship with miR-21 in the concentration range of 1-20nM, and the equation is: y=8.68828x+13.93812( Figure 8 C).

[0094] In general, the above experimental results show that the MnO2@CRISPR nanoprobe as a sensing platform has a very sensitive ability to recognize miR-21.

[0095] 8. Cell Culture

[0096] MCF-7 cells were cultured in a constant temperature and humidity chamber at 37°C, with a humidity of 95% and a CO2 concentration of 5%. The culture medium used for the cells was DMEM containing 10% fetal bovine serum and 1% diabody (penicillin-streptomycin).

[0097] IX. Feasibility analysis of MnO2@CRISPR nanoprobe combined with RCA in MCF-7 cells

[0098] In order to verify whether MnO2 / CRISPR / MR21C can achieve fluorescence recovery in MCF-7 cells, different material solutions were co-incubated with MCF-7 cells, and the fluorescence recovery ability of MnO2 / CRISPR in response to MR21C was detected by fluorescence microscopy and flow cytometry.

[0099] Different material solutions:

[0100] Group I: blank control;

[0101] Group II: MnO2 / FQ reporter probe: 100 μMnO2 nanosheets (40 μg / mL), 3 μL F-Q reporter probe (100 nM) were stirred and mixed for 20 minutes, and then 20 μL Tris-HAc buffer (100 mM, pH 7.4) was added to prepare.

[0102] Group III: MnO2@CRISPR nanoprobe: 30 μMnO2 nanosheets, Cas 12a, crRNA and FQ reporter probe (i.e., MnO2@CRISPR nanoprobe); the final concentration of each component of MnO2 nanosheets, Cas 12a, crRNA and FQ reporter probe in cell growth medium (i.e., DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin)) is 40 μg / mL of MnO2 nanosheets; 80 pmol of Cas 12a; 150 pmol of crRNA and 150 pmol of FQ reporter probe

[0103] Group IV: MnO2@CRISPR nanoprobe and MR21C: Based on the above group III, 30 μL of MR21C was added.

[0104] MCF-7 cells were seeded in a 12-well plate and incubated at 37°C, 5% CO2 for 24 h. After washing three times with PBS buffer, 500 μL of fresh cell growth medium (DMEM with 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin)) was added, and the cell growth medium was supplemented, that is, different material solutions were added to the cell growth medium. After incubation for 3 h, the cells were washed three times with 1×PBS buffer, stained with DAPI for 15 min, and fluorescence microscopy imaging and flow cytometry counting were performed.

[0105] Results: After the four groups of solutions with different materials were co-incubated with MCF-7 cells, the fluorescence recovery ability of the MnO2@CRISPR nanoprobe in response to MR21C was detected by fluorescence microscopy and flow cytometry. Fig. 9A includes blank control (Ⅰ); MnO2 / FQ reporter probe (Ⅱ); MnO2@CRISPR nanoprobe (Ⅲ); MnO2@CRISPR nanoprobe and MR21C (Ⅳ); It can be seen that groups Ⅱ and Ⅲ are the same as the blank control, and no green fluorescence is produced. This is due to the lack of a certain condition in Cas12a-crRNA and MR21C, which leads to CRISPR / Cas12a not being effectively activated and unable to cut the FQ reporter probe, so the fluorescence is not restored. In group IV, there is obvious green fluorescence. This is because miR-21 in MCF-7 cells causes MR21C to "turn on" and specifically pair with crRNA, effectively activating the trans-cutting ability of Cas12a, forming a complete CRISPR / Cas12a system, cutting the FQ reporter probe, and restoring fluorescence. The above data are consistent with Fig. 9 The flow cytometry experimental results in B are consistent. Only when both MnO2@CRISPR nanoprobe and MR21C are present will the corresponding fluorescence signal be generated in the FAM channel.

[0106] 10. Analysis of MR21C targeting to mitochondria

[0107] Preparation of Cy5-labeled MR21C (Cy5-MR21C):

[0108] The primers used in the preparation of MR21C were replaced with Cy5-primers. Cy5-primers are primers with Cy5 fluorescent labeling groups added to them. Other reaction conditions remain unchanged. The experiment was repeated. Cy5-MR21C, i.e., Cy5-labeled MR21C, was obtained after RCA for the analysis of MR21C targeting mitochondria.

[0109] MCF-7 cells were seeded in 12-well plates and incubated at 37°C, 5% CO2 for 24 h. After washing three times with PBS buffer, Cy5-labeled MR21C (30 μL) was co-incubated with MCF-7 cells. After 3 h, the cells were washed three times with 1× PBS buffer, co-stained with DAPI and Mito-Tracker for 15 min, and imaged by fluorescence microscopy.

[0110] The ability of the CytC aptamer encoded in MR21C to target mitochondria was examined by using fluorescence microscopy imaging. Once MR21C reaches mitochondria, they can undergo further internalization, fusion with the outer mitochondrial membrane or uptake through mitochondria-specific import systems. Mitochondria were stained with green Mito-Tracker and nuclei were stained blue with DAPI. After co-incubation of Cy5-labeled MR21C (containing the CytC aptamer) with MCF-7 cells, co-localization of MR21C with mitochondria was observed.

[0111] like Fig.10As shown, MR21C was taken up by MCF-7 cells and almost all of MR21C was colocalized with mitochondria, suggesting an important role for MR21C in encoding the CytC aptamer for targeting to cellular mitochondria.

[0112] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A reagent for rapid detection of tumors, characterized in that: The reagents include MnO2@CRISPR nanoprobe and MR21C; The MnO2@CRISPR nanoprobe is prepared by mixing MnO2 nanosheets with Cas12a, crRNA, and FQ reporter probes; The MR21C contains the sequence of the MUC1 aptamer, the sequence of the CytC aptamer, the complementary sequence of the crRNA, and the complementary sequence of miR-21.

2. The reagent according to claim 1, characterized in that The sequence of the crRNA is shown in SEQ ID NO.1; the FQ reporter probe is FAM-TTATT-BHQ1.

3. The reagent according to claim 1, characterized in that The MR21C is obtained by rolling circle amplification with the participation of a polymerase using a locked DNA template.

4. The reagent according to claim 1, characterized in that The locked DNA template is prepared using a primer and a phosphorylated DNA template.

5. The reagent according to claim 4, characterized in that The primer sequence is shown in SEQ ID NO.3, and the phosphorylated DNA template is shown in SEQ ID NO.

2.

6. Use of the reagent according to any one of claims 1 to 5 in the preparation of a tumor diagnosis product.

7. Use of the reagent according to any one of claims 1 to 5 in the preparation of a targeted tumor diagnosis product.

8. Use of the reagent according to any one of claims 1 to 5 in the preparation of a product for detecting mitochondrial miR-21.

9. A kit for amplifying mitochondrial miR-21 imaging, characterized in that: The kit comprises the reagent according to any one of claims 1 to 5.

10. A detection kit for miR-21, characterized in that: The kit comprises the reagent according to any one of claims 1 to 5 and NE Buffer.

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