A fluorescent nanoprobe COF-H1 / H2-Peptide and its preparation method and application

Abstract

The invention relates to the technical field of nanoprobe preparation and application, and specifically discloses a fluorescent nanoprobe COF-H1 / H2-Peptide and a preparation method and application thereof. 1,3,5-Tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthaldehyde are mixed with acetonitrile, glacial acetic acid is added, mixed and centrifuged to obtain a precipitate as COF NPs nanoparticles; the COF NPs nanoparticles are dispersed in a PBS buffer to obtain a COF NPs solution; the COF NPs solution is mixed with a hairpin DNA labeled with Texas Red to obtain a COF-HI / H2NPs solution, and then the COF-H1 / H2NPs solution is mixed with a peptide labeled with Cy5 to obtain a fluorescent nanoprobe COF-H1 / H2-Peptide. The fluorescent nanoprobe COF-H1 / H2-Peptide prepared by the present invention is used for cyclic amplification detection of miRNA-221, and realizes simultaneous detection of apoptotic protein caspase-3, has high specificity, a detection limit of 2.79 pM, good biocompatibility, and is easy for cell imaging, and realizes CHA cyclic amplification detection of low-abundance miRNA-221 in living cells.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoprobe preparation and application, and in particular to a fluorescent nanoprobe COF-H1 / H2-Peptide and a preparation method and application thereof. Background Art

[0002] In recent years, the incidence and mortality of cancer have risen sharply, and the age of onset is showing a trend of younger age, which has become the main cause of human death. At present, targeted therapy based on miRNA is a research hotspot in the field of precision tumor treatment. MiRNA is a class of endogenous, non-coding, low-abundance single-stranded small RNA molecules (18-22nt). Its abnormal expression is closely related to the proliferation, metastasis and invasion of cancer cells. It is an important biomarker for cancer diagnosis and prognosis. Different miRNAs play different roles in carcinogenesis or tumor suppression in the human body. For example, in most cancers, miRNA-221 is overexpressed and plays the role of tumor oncogenes, and let-7 is underexpressed and plays the role of tumor suppressor genes. Studies have shown that by reducing or increasing the expression of specific miRNAs, apoptosis genes can be regulated, thereby inducing cell apoptosis and achieving precision treatment of cancer. Therefore, in the development of precision therapy based on miRNA, it is of great significance to study the changes in miRNA and cell apoptosis.

[0003] Traditional miRNAs detection methods (Northern blotting, qRT-PCR, miRNA microarray) and cell apoptosis detection methods (Western blotting, immunohistochemistry analysis) all require cell fixation and lysis, and cannot intuitively monitor the expression changes of miRNA and apoptosis markers in living cells. Fluorescence technology has the advantages of good selectivity, high sensitivity, high imaging resolution, and non-invasiveness, and has become a common strategy for detecting intracellular biomarkers. Many studies have shown that due to the low abundance of miRNA, the traditional "one-to-one" mode of fluorescence imaging cannot sensitively monitor low levels of miRNA in cells. Summary of the invention

[0004] In order to provide a highly sensitive miRNAs and cell apoptosis detection product, the present invention provides a fluorescent nanoprobe COF-H1 / H2-Peptide and a preparation method and application thereof. The fluorescent nanoprobe COF-H1 / H2-Peptide prepared by the present invention is used for cyclic amplification detection of miRNA-221, and realizes simultaneous detection of apoptotic protein caspase-3, has high specificity, a detection limit of 2.79pM, good biocompatibility, and is easy for cell imaging, and realizes CHA cyclic amplification detection of low-abundance miRNA-221 in living cells.

[0005] The present invention provides a method for preparing a fluorescent nanoprobe COF-H1 / H2-Peptide, comprising the following steps:

[0006] 1,3,5-Tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthaldehyde were mixed with acetonitrile, glacial acetic acid was added, and the mixture was centrifuged to obtain the precipitate, which was COF NPs nanoparticles.

[0007] The COF NPs nanoparticles were dispersed in PBS buffer to obtain a COF NPs solution;

[0008] The COF NPs solution was mixed with the hairpin DNA labeled with Texas Red to obtain the COF-HI / H2NPs solution, and then the COF-H1 / H2 NPs solution was mixed with the Cy5-labeled peptide to obtain the fluorescent nanoprobe COF-H1 / H2-Peptide.

[0009] The hairpin DNA preparation process is as follows: labeling the ends of H1 and H2 with Texas Red respectively and then mixing them to form a mixed solution;

[0010] The H1 nucleotide sequence is shown in SEQ ID NO.1, and the H2 nucleotide sequence is shown in SEQ ID NO.2;

[0011] The sequence of Cy5-labeled peptide is: Cy5-Gly Gly Asp Glu Val Asp Gly Gly Cys.

[0012] The present invention also provides the fluorescent nanoprobe COF-H1 / H2-Peptide prepared by the above method.

[0013] The present invention also provides the use of the fluorescent nanoprobe COF-H1 / H2-Peptide in preparing a product for detecting tumor cytokines, wherein the tumor cytokines are miRNA-221 and apoptosis marker caspase-3.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. Based on COF materials and CHA detection strategy, we developed a new intelligent nanoprobe COF-H1 / H2-Peptide for cyclic amplification detection of miRNA-221 and simultaneous detection of apoptotic protein caspase-3, which can visualize the effect of miRNA expression changes on cell apoptosis.

[0016] like Figure 1As shown in the figure, due to the existence of π-π stacking and hydrogen bonding interactions, H1, H2 and peptide can be adsorbed on COF NPs step by step. At this time, the fluorescence of Texas Red labeled on H1 and H2 and Cy5 labeled on peptide are quenched by COFNPs, thus obtaining a functionalized COF-H1 / H2-Peptide nanoprobe. The principle of CHA amplification detection of target miRNA-221 is as follows: When the probe encounters the target, the target specifically hybridizes with the unpaired region b of H1 on the nanoprobe to form a T-H1 double-stranded complex. At the same time, the sticky end a' of H2 hybridizes with the newly exposed region a in H1 in the T-H1 double-stranded complex to form a T-H1-H2 semi-stable ternary complex. Then H2 replaces the target miRNA-221, resulting in a strand displacement reaction, and finally forming an H1-H2 double-stranded complex. At this time, Texas Red can stay away from COF NPs to restore fluorescence. Then, the released miRNA-221 will repeatedly trigger the next round of CHA, which allows a single miRNA-221 to generate a large number of H1-H2 double-stranded complexes, resulting in significant fluorescence enhancement. When COF-H1 / H2-Peptide NPs encounter caspase-3, caspase-3 specifically cleaves the DEVD sequence in the peptide, resulting in the recovery of Cy5 fluorescence. In summary, the COF-H1 / H2-Peptide nanoprobe we designed can become an intelligent system for intracellular nucleic acid CHA amplification detection and simultaneous and protein dual sensing, which has great potential in disease diagnosis and screening of new drugs.

[0017] 2. Covalent organic frameworks (COFs) have the advantages of structural diversity, large specific surface area, good stability, high quenching efficiency and good biocompatibility, and have great potential in drug delivery, bioanalysis and disease treatment and diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Schematic diagram of the analysis of the preparation principle of COF-H1 / H2-Peptide nanoprobe. In the figure, A is the preparation of COF-H1 / H2-Peptide nanoprobe, and B is the CHA cycle amplification detection of miRNA-221 in living cells by the nanoprobe.

[0020] Figure 2Characterization and EDS element mapping of the fluorescent nanoprobe COF-H1 / H2-Peptide prepared in Example 1 of the present invention; in the figure, A is a TEM image of COF NPs; B is a TEM image of COF-H1 / H2-Peptide NPs; C is a dynamic light scattering (DLS) analysis of COF NPs and COF-H1 / H2-Peptide NPs; D is a zeta potential analysis of COF, COF-H1 / H2 and COF-H1 / H2-Peptide; E is a UV-visible absorption spectrum of HI / H2, Peptide, COF, COF-H1 / H2 and COF-H1 / H2-Peptide; F is a SEM image of COF NPs from left to right, and EDS element mapping of C, N, and O; G is a SEM image of COF-H1 / H2-Peptide NPs and EDS element mapping of C, N, O, P, and S from left to right. Scale bar is 1 μm.

[0021] Figure 3 The effect of the concentration of COF NPs and COF-H1 / H2 NPs on the fluorescence spectrum and fluorescence intensity;

[0022] In the figure, A and B are the fluorescence spectra and fluorescence intensity of H1 / H2 (150 / 150nM) after adding different concentrations (0-250μg / mL) of COF NPs, respectively; C and D are the fluorescence spectra and fluorescence intensity of Peptide (2μM) after adding different concentrations (0-130μg / mL) of COF-H1 / H2 NPs, respectively; Texas Red: Ex=595nm, Em=620nm; Cy5: Ex=625nm, Em=660nm;

[0023] E is the fluorescence spectra of H1 / H2 labeled with different concentrations of Texas Red; F is the relevant standard curve of H1 / H2 labeled with different concentrations of Texas Red; Texas Red: Ex = 595nm, Em = 620nm;

[0024] G is the fluorescence spectra of peptides labeled with Cy5 at different concentrations, and H is the relevant standard curve of peptides labeled with Cy5 at different concentrations; Cy5: Ex = 625 nm, Em = 660 nm.

[0025] Figure 4 To verify the feasibility of CHA;

[0026] In the figure, A is the polyacrylamide gel electrophoresis (PAGE) image of DNA nanostructure; B is the fluorescence spectra of free H1 / H2 (curve a), COF-H1 / H2 NPs (curve b), COF-H1 / H2 NPs+miRNA-221 (curve c), COF-H1 NPs (curve d), and COF-H1 NPs+miRNA-221 (curve e). Texas Red: Ex = 595nm, Em = 620nm; C is the fluorescence spectrum of COF-H1 / H2-Peptide NPs after incubation for 2h with different concentrations of miR-221 (0, 0.001, 0.01, 0.2, 0.4, 0.6, 0.8, 1.0, 3.0, 5.0, 8.0, 10.0nM); D is the fluorescence intensity of COF-H1 / H2-Peptide NPs after incubation for 2h with different concentrations of miR-221 (0, 0.001, 0.01, 0.2, 0.4, 0.6, 0.8, 1.0, 3.0, 5.0, 8.0, 10.0nM) (the inset in B is the calibration curve of COF-H1 / H2-Peptide NPs probe); E is the fluorescence intensity of COF-H1 / H2-Peptide NPs after incubation for 2h with different concentrations of miR-221 (0, 0.001, 0.01, 0.2, 0.4, 0.6, 0.8, 1.0, 3.0, 5.0, 8.0, 10.0nM). Fluorescence spectra of NPs after incubation for 2 h with different concentrations of miR-221 (0, 0.6, 0.8, 1.0, 3.0, 5.0, 8.0, 10.0 nM); F is the fluorescence intensity of COF-H1-Peptide NPs after incubation for 2 h with different concentrations of miR-221 (0, 0.6, 0.8, 1.0, 3.0, 5.0, 8.0, 10.0 nM) (the inset in D is the calibration curve of COF-H1-Peptide NPs probe). G is the fluorescence spectrum of COF-H1 / H2-Peptide nanoprobe after incubation for 4 h with different concentrations of caspase-3 (0, 1, 2, 3, 4 Unit); H is the fluorescence intensity of COF-H1 / H2-Peptide nanoprobe after incubation for 4 h with different concentrations of caspase-3 (0, 1, 2, 3, 4 Unit). Texas Red: Ex=595nm, Em=620nm; Cy5: Ex=625nm, Em=660nm.

[0027] Figure 5 The results showed that the COF-H1 / H2-Peptide nanoprobe had a rapid response to miRNA-221, and had specificity and stability to the targets miRNA-221 and Caspase-3;

[0028] In the figure, A is the Texas Red fluorescence spectra of COF-H1 / H2-Peptide NPs at different time points (0, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160 min) in the presence of target miRNA-221 (10 nM); B is the Texas Red fluorescence spectra of COF-H1 / H2-Peptide NPs at different time points (0, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160 min) in the presence of target miRNA-221 (10 nM). Red fluorescence intensity; C is the Cy5 fluorescence spectrum of COF-H1 / H2-Peptide NPs at different time points (0, 0.5, 1.0, 2.0, 3.0, 4.0h) in the presence of target caspase-3 (2Unit); D is the Cy5 fluorescence intensity of COF-H1 / H2-Peptide NPs at different time points (0, 0.5, 1.0, 2.0, 3.0, 4.0h) in the presence of target caspase-3 (2Unit). Texas Red: Ex = 595nm, Em = 620nm; Cy5: Ex = 625nm, Em = 660nm; E is the specificity of COF-H1 / H2-Peptide nanoprobe to the targets miRNA-221, single-base mismatch miRNA-221 (MisTar-221), double-base mismatch miRNA-221 (TwoMisTar-221), triple-base mismatch miRNA-21 (ThreeMisTar-221), Target-34a, Target-21, and Target-67, and COF-H1-Peptide solution is used as the control; F is the specificity of COF-H1 / H2-Peptide probe to the targets Caspase-3, hemoglobin (HGB), bovine serum albumin (BSA), glucose (Glu), sodium chloride (NaCl), and calcium chloride (CaCl 2 ), COF-H1-Peptide solution was used as control; G is the relative fluorescence intensity change of Texas Red and Cy5 during COF-H1 / H2-Peptide storage in cell culture medium for 0-7 days; H is the relative fluorescence intensity change of Texas Red and Cy5 during COF-H1 / H2-Peptide storage in PBS buffer for 0-7 days; I is the relative fluorescence intensity change of Texas Red and Cy5 in Tris-HCl solutions with different pH values ​​(2-10). Texas Red: Ex = 595nm, Em = 620nm; Cy5: Ex = 625nm, Em = 660nm.

[0029] Figure 6The effects of different concentrations of COF NPs or COF-H1 / H2-Peptide NPs nanoprobes on cell viability;

[0030] In the figure, A is the cell viability of HeLa cells after treatment with different concentrations of COF NPs or COF-H1 / H2-Peptide NPs nanoprobes for 6 h;

[0031] B is the cell viability of HepG-2 cells after treatment with different concentrations of COF NPs or COF-H1 / H2-Peptide NPs nanoprobes for 6 hours;

[0032] C is the cell viability of A549 cells after treatment with different concentrations of COF NPs or COF-H1 / H2-Peptide NPs nanoprobes for 6 hours.

[0033] Figure 7 CLSM images and relative fluorescence intensity of Hela cells after incubation with COF-H1-Peptide nanoprobe and COF-H1 / H2-Peptide nanoprobe for 6 hours;

[0034] In the figure, A is the CLSM image of Hela cells incubated with COF-H1-Peptide nanoprobe and COF-H1 / H2-Peptide nanoprobe for 6 hours, and the green and red channels are excited at 595 and 625 nm, respectively; B is the relative fluorescence intensity of the underlined part in A.

[0035] Figure 8 CLSM images of HepG-2 cells incubated with COF-H1-Peptide and COF-H1 / H2-Peptide nanoprobes for 6 hours, with green and red channels excited at 595 and 625 nm, respectively. The scale bar is 25 μm; B is the relative fluorescence intensity of the underlined part in A.

[0036] Fig. 9 CLSM images of A549 cells incubated with COF-H1-Peptide and COF-H1 / H2-Peptide nanoprobes for 6 hours. The green and red channels were excited at 595 and 625 nm, respectively. The scale bar is 25 μm. B is the relative fluorescence intensity of the underlined part in A.

[0037] Fig.10 For confocal imaging of miRNA-221 and caspase-3;

[0038] In the figure, A is the confocal imaging of miRNA-221 and caspase-3 in Hela cells; cells in the LPS group were pretreated with LPS for 12 hours to increase the content of miRNA-221, and cells in the STS group were pretreated with STS for 12 hours to enhance the expression of caspase-3. The pretreated cells were incubated with COF-H1 / H2-Peptide at 37°C for 4 hours, and the green and red channels were excited with 595 and 625 nm, respectively. The scale bar is 25 μm;

[0039] B is the relative fluorescence intensity of the underlined part in A after Hela cells in LPS group, STS group and control group were incubated with COF-H1 / H2-Peptide nanoprobe for 4 hours.

[0040] Fig.11 Confocal imaging of miRNA-221 and caspase-3 in HepG-2 cells;

[0041] In the figure, A is the confocal imaging of miRNA-221 and caspase-3 in HepG-2 cells; cells in the LPS group were pretreated with LPS for 12 h to increase the content of miRNA-221, and cells in the STS group were pretreated with STS for 12 h to enhance the expression of caspase-3. The pretreated cells were incubated with COF-H1 / H2-Peptide at 37°C for 4 h. The green and red channels were excited with 595 and 625 nm, respectively. The scale bar is 25 μm;

[0042] B is the relative fluorescence intensity of the underlined part in A after HepG-2 cells in LPS group, STS group and control group were incubated with COF-H1 / H2-Peptide nanoprobe for 4 hours.

[0043] Fig.12 Confocal imaging of miRNA-221 and caspase-3 in A549 cells;

[0044] In the figure, A is the confocal imaging of miRNA-221 and caspase-3 in A549 cells. The cells in the LPS group were pretreated with LPS for 12 h to increase the miRNA-221 content, and the cells in the STS group were pretreated with STS for 12 h to enhance the expression of caspase-3. The pretreated cells were incubated with COF-H1 / H2-Peptide at 37°C for 4 h, and the green and red channels were excited with 595 and 625 nm, respectively. The scale bar is 25 μm;

[0045] B is the relative fluorescence intensity of the underlined part in A after A549 cells in LPS group, STS group and control group were incubated with COF-H1 / H2-Peptide nanoprobe for 4 hours.

[0046] Fig.13 Figure 2 shows the flow cytometry results of LPS group, STS group and control group of Hela cells, HepG-2 cells and A549 cells after adding COF-H1 / H2-Peptide NPs; in the figure, A and C are the flow cytometry Texas Red average fluorescence intensity and average fluorescence intensity statistics of Hela cells treated with different drugs, respectively; B and D are the flow cytometry Cy5 average fluorescence intensity and average fluorescence intensity statistics of Hela cells treated with different drugs, respectively; E and G are the flow cytometry Texas Red average fluorescence intensity and average fluorescence intensity statistics of HepG-2 cells treated with different drugs, respectively; F and H are the flow cytometry Cy5 average fluorescence intensity and average fluorescence intensity statistics of HepG-2 cells treated with different drugs, respectively; I and K are the flow cytometry Texas Red average fluorescence intensity and statistics of A549 cells treated with different drugs, respectively; J and L are the flow cytometry Cy5 average fluorescence intensity and statistics of A549 cells treated with different drugs, respectively.

[0047] Fig.14 The effect of Caspase-3 activity and cell apoptosis in Hela cells was detected by Annexin V apoptosis detection kit. The green and red channels were excited with 500 and 587 nm, respectively. The scale bar is 25 μm.

[0048] Fig.15 The effect of Caspase-3 activity and cell apoptosis in HepG-2 cells was detected by Annexin V apoptosis detection kit. The green and red channels were excited with 500 and 587 nm, respectively. The scale bar is 25 μm.

[0049] Fig.16 The effect of Caspase-3 activity and cell apoptosis in A549 cells was detected by Annexin V apoptosis detection kit. The green and red channels were excited with 500 and 587 nm, respectively. The scale bar is 25 μm.

[0050] Fig.17 CLSM images of Hela cells treated with different concentrations of STS for 12 h and then incubated with COF-H1 / H2-Peptide (A) and COF-H1-Peptide (B). The green and red channels were excited at 595 nm and 625 nm, respectively. The scale bar is 25 μm.

[0051] Fig.18CLSM imaging of Hela cells treated with 50 nM STS for different time periods and then incubated with COF-H1 / H2-Peptide (A) and COF-H1-Peptide (B). The green and red channels were excited at 595 nm and 625 nm, respectively. The scale bar is 25 μm.

[0052] Fig.19 CLSM images of HepG-2 cells treated with different concentrations of STS for 12 h and then incubated with COF-H1 / H2-Peptide (A) and COF-H1-Peptide (B). The green and red channels were excited at 595 nm and 625 nm, respectively. The scale bar is 25 μm.

[0053] Fig. 20 CLSM imaging of HepG-2 cells treated with 50 nM STS for different time periods and then incubated with COF-H1 / H2-Peptide (A) and COF-H1-Peptide (B). The green and red channels were excited at 595 nm and 625 nm, respectively. The scale bar is 25 μm.

[0054] Fig.21 CLSM images of A549 cells treated with different concentrations of STS for 12 h and then incubated with COF-H1 / H2-Peptide (A) and COF-H1-Peptide (B). The green and red channels were excited at 595 nm and 625 nm, respectively. The scale bar is 25 μm.

[0055] Fig. 22 CLSM imaging of A549 cells treated with 50 nM STS for different time periods and then incubated with COF-H1 / H2-Peptide (A) and COF-H1-Peptide (B). The green and red channels were excited at 595 nm and 625 nm, respectively. The scale bar is 25 μm. DETAILED DESCRIPTION

[0056] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0057] The present invention provides a fluorescent nanoprobe COF-H1 / H2-Peptide and a preparation method and application thereof. The main experimental materials and reagents are as follows:

[0058] 1. Experimental Materials

[0059] 1,3,5-Tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthaldehyde were purchased from Shanghai Anaiji Chemical Co., Ltd. All oligonucleotides were synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd. All peptides were synthesized and purified by GenScript Biotech Co., Ltd. The sequence information of oligonucleotides and peptides is shown in Table 1.

[0060] Table 1 Sequence information of DNA and peptide

[0061]

[0062] 2. Fluorescence Quenching Experiment

[0063] In order to prepare nanoprobes with good performance, COF materials, H1 / H2 (mixed concentration of H1 and H2) and peptide concentration were optimized by quenching experiments. First, COF NPs solutions with different gradient concentrations (final concentration: 0-250 μg / mL, 20 mM MgCl 2 , pH 7.4) and a mixture of H1 and H2 labeled with Texas Red were shaken at room temperature for 1 hour to obtain COF-H1 / H2 NPs solutions of different concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 μg / mL). The fluorescence intensity of Texas Red (Ex / Em=595 / 620nm) in the solution was then recorded using a fluorescence spectrophotometer. The optimal concentration of COF NPs nanoparticles was determined based on the fluorescence quenching curve.

[0064] Next, in order to study the fluorescence quenching effect of COF-H1 / H2 NPs on peptide, Cy5-labeled peptide (2 μM) was added into COF-H1 / H2 NPs solutions of different concentrations (0-130 μg / mL, 20 mM MgCl 2 , pH 7.4) and shaken at room temperature for 1 hour to obtain COF-H1 / H2-Peptide NPs solutions of different concentrations. Then, the fluorescence intensity of Cy5 (Ex / Em=625 / 660nm) in the COF-H1 / H2-Peptide NPs solution was recorded by a fluorescence spectrophotometer. According to the fluorescence quenching curve, the optimal concentration of the COF-H1 / H2 NPs solution was determined.

[0065] Example 1: A fluorescent nanoprobe COF-H1 / H2-Peptide and a preparation method thereof.

[0066] 1. Experimental Methods

[0067] 1. Synthesis of COF NPs

[0068] Weigh 14.06 mg 1,3,5-Tris(4-aminophenyl)benzene, weigh 11.65 mg 2,5-dimethoxyterephthaldehyde, and then mix with 20 mL acetonitrile, and sonicate for 1 min to completely dissolve the mixture. Then add 500 μL of glacial acetic acid, stir rapidly at room temperature for 1 min, and centrifuge at 9500 rpm for 10 min to obtain precipitated COFNPs nanoparticles, and wash the COF NPs nanoparticles with acetonitrile and ethanol for 3 times. Finally, disperse the nanoparticles COF NPs into PBS buffer to obtain a 1 mg / mL COF NPs solution for subsequent use.

[0069] 2. Preparation of fluorescent nanoprobe COF-H1 / H2-Peptide:

[0070] Add MgCl to the 1 mg / mL COF NPs solution. 2 To a final concentration of 20mM, pH 7.4, then 30uL COF NPs solution was mixed with hairpin DNA and oscillated at room temperature for 1h to obtain COF-HI / H2 NPs solution. Then 107uL 150ug / mL COF-H1 / H2 NPs solution was further mixed with 4uL 100uM Cy5-labeled peptide (final concentration 2μM) and oscillated at room temperature for 1h. Then washed 3 times with PBS buffer to obtain the final fluorescent nanoprobe COF-H1 / H2-Peptide.

[0071] The hairpin DNA is: after labeling the ends of H1 and H2 with the dye Texas Red, 1.5uL of 20uM each is taken and mixed to form a mixed solution (also written as H1 / H2), and the final concentrations of H1 and H2 are both 150nM, recorded as H1 / H2 final concentration 150 / 150nM.

[0072] Comparative Example 1: COF-H1-PeptideNPs nanoprobe was synthesized as a comparative example.

[0073] Add MgCl to the 1 mg / mL COF NPs solution. 2To a final concentration of 20mM, pH 7.4, then 30uL COFNPs solution was mixed with 3uL 20uM Texas Red end-labeled H1 (final concentration of 300nM), and oscillated at room temperature for 1h to obtain COF-H1 NPs. The COF-H1 solution was then further mixed and oscillated with 4uL 100uM Cy5-labeled peptide (Cy5-Peptide) for 1h to prepare COF-H1-Peptide nanoprobes.

[0074] The feasibility and characteristics of the above examples were tested, and the fluorescence standard curves of Texas Red-H1 / H2 (abbreviated as H1 / H2) and Cy5-Peptide were drawn. Taking Example 1 as an example, the prepared fluorescent nanoprobe COF-H1 / H2-Peptide was characterized, and the ability of the fluorescent nanoprobe COF-H1 / H2-Peptide to detect miRNA-221 and caspase-3 outside the cell was tested. The specific method and results are as follows:

[0075] 1. Experimental Methods

[0076] 1. Fluorescence standard curve of Texas Red-H1 / H2 and Cy5-Peptide

[0077] In order to draw the fluorescence standard curve of Texas Red-H1 / H2, different concentrations of hairpin DNA were prepared (mixed solution of H1 and H2), with final concentrations of H1 / H2 being 0, 50 / 50nM, 100 / 100nM, 150 / 150nM, 200 / 200nM, 250 / 250nM, 300 / 300nM, 350 / 350nM), respectively. The fluorescence intensity of Texas Red (Ex / Em=595 / 620nm) was then recorded using a fluorescence spectrophotometer to draw the H1 / H2 fluorescence standard curve.

[0078] In order to draw the fluorescence standard curve of Cy5-Peptide, peptide solutions with different final concentrations (0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00 μM) were prepared, and then the fluorescence intensity of Cy5 (Ex / Em=625 / 660 nm) was recorded using a fluorescence spectrophotometer to draw the peptide fluorescence standard curve.

[0079] 2. Polyacrylamide gel electrophoresis (PAGE)

[0080] In order to study the feasibility of CHA reaction, the amplification products were first characterized by 20% polyacrylamide gel electrophoresis. Seven samples were prepared for electrophoresis: sample 1: miRNA-221 (100nM); sample 2: H1 (300nM); sample 3: H2 (300nM); sample 4: H1 / H2 (150 / 150nM); sample 5: H1 (300nM), miRNA-221 (100nM); sample 6: H2 (300nM), miRNA-221 (100nM); sample 7: H1 / H2 (150 / 150nM), miRNA-221 (100nM). Samples 5-7 were incubated at 37°C for 2h. Then 10μL of sample was mixed with 2μL of 6× loading buffer and transferred to 20% polyacrylamide gel. It was run at 50V for 2h in 1×TBE buffer. Subsequently, the gel was stained with GelRed for 10 min and then transferred to the FluorChemHD2 Gel Imaging System for analysis.

[0081] 3. Fluorescence experiment

[0082] Fluorescence analysis was further used to verify the feasibility of CHA strategy for amplified detection of miRNA. The following five samples were prepared for fluorescence testing: sample a: H1 / H2 (150 / 150nM); sample b: COF-H1 / H2 NPs (150μg / mL); sample c: COF-H1 / H2NPs (150μg / mL), miRNA-221 (100nM); sample d: COF-H1 NPs (150μg / mL); sample e: COF-H1 NPs (150μg / mL), miRNA-221 (100nM). Samples c and e were incubated at 37°C for 2h, and then sample be was centrifuged at low speed, and the fluorescence changes of Texas Red (Ex / Em=595 / 620nm) in the supernatant were recorded by fluorescence spectrophotometer.

[0083] 4. Extracellular detection of miRNA-221 and caspase-3

[0084] In order to investigate the ability of CHA method to amplify the detection of miRNA-221, the nanoprobe COF-H1 / H2-Peptide NPs (80 μg / mL, 10 mM CaCl 2, pH 7.4) were incubated with different concentrations of target miRNA-221 (0, 0.001, 0.01, 0.2, 0.4, 0.6, 0.8, 1.0, 3.0, 5.0, 8.0, 10.0 nM) at 37 ° C for 2 h. As a control, the probe COF-H1-Peptide NPs were also incubated with different concentrations of target miRNA-221 (0, 0.6, 0.8, 1.0, 3.0, 5.0, 8.0, 10.0 nM) at 37 ° C for 2 h. All samples were centrifuged and the fluorescence signal of TexasRed (Ex / Em=595 / 620 nm) in the supernatant was recorded by a fluorescence spectrophotometer.

[0085] Detection of Caspase-3. When studying the feasibility of the probe COF-H1 / H2-Peptide NPs in detecting caspase-3, the probe COF-H1 / H2-Peptide NPs (80 μg / mL, 50 mM Hepes, 50 mM NaCl, 10 mM EDTA, 10 mM DTT, 0.1% Chaps, 5% glycerol) were incubated with different concentrations of target caspase-3 (0, 1, 2, 3, 4 Unit) at 37°C for 4 h. After centrifugation, the fluorescence signal of Cy5 (Ex / Em=625 / 660 nm) in the supernatant was recorded using a fluorescence spectrophotometer.

[0086] 5. Kinetic analysis

[0087] In order to optimize the response time of the probe to miRNA-221, 10 nM miRNA-221 was added to the COF-H1 / H2-Peptide NPs solution, incubated at 37 °C, and the fluorescence signals of Texas Red (Ex / Em=595 / 620 nm) at different time points (0, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160 min) were recorded by a fluorescence spectrophotometer.

[0088] Similarly, in order to optimize the response time of the probe to caspase-3, 2Unit of caspase-3 was added to the COF-H1 / H2-Peptide NPs solution, incubated at 37°C, and the fluorescence signals of Cy5 (Ex / Em=625 / 660nm) at different time points (0, 0.5, 1.0, 2.0, 3.0, 4.0h) were recorded using a fluorescence spectrophotometer.

[0089] 6. Specificity Experiment

[0090] In order to evaluate the specificity of probe COF-H1 / H2-Peptide NPs in detecting target miRNA-221, the fully matched Target-221 and other non-matching different targets MisTar-221, TwoMisTar-221, ThreeMisTar-221, Target-34a, Target-67 and Target-21 were added to the COF-H1 / H2-Peptide NPs solution. After incubation at 37°C for 2 h, the fluorescence signal of Texas Red (Ex / Em=595 / 620 nm) was recorded by a fluorescence spectrophotometer.

[0091] In order to evaluate the specificity of the probe COF-H1 / H2-Peptide NPs in detecting caspase-3, HGB, BSA, Glu, NaCl, CaCl 2 Different targets including β-actin and caspase-3 were added to the COF-H1 / H2-Peptide NPs solution. After incubation at 37°C for 4 h, the fluorescence signal of Cy5 (Ex / Em=625 / 670 nm) was recorded using a fluorescence spectrophotometer.

[0092] 6. Storage stability

[0093] When evaluating the storage stability of COF-H1 / H2-Peptide NPs, COF-H1 / H2-Peptide NPs were dispersed in cell culture medium and PBS buffer, respectively, and the fluorescence changes of Texas Red (Ex / Em=595 / 620nm) and Cy5 (Ex / Em=625 / 660nm) within one week were recorded using a fluorescence spectrophotometer.

[0094] Next, the effect of different solution pH on the stability of COF-H1 / H2-Peptide NPs was studied. COF-H1 / H2-Peptide NPs were dispersed in Tris-HCl solutions with different pH values ​​(2-10), and the fluorescence changes of Texas Red (Ex / Em=595 / 620nm) and Cy5 (Ex / Em=625 / 660nm) were recorded.

[0095] 7. Cell culture

[0096] The cells were cultured in DMEM containing 10% fetal bovine serum, 1% penicillin, and 100 U / mL streptomycin at 37°C and 5% CO. 2 HeLa, HepG-2, and A549 cells were cultured in a culture dish in a 95% air incubator.

[0097] 8. MTT assay

[0098] In evaluating the biotoxicity of the nanoprobes, the biotoxicity of COF NPs and COF-H1 / H2-Peptide NPs in Hela, HepG-2, and A549 cells was determined by MTT assay. 5 The cells were placed in a 96-well plate with a total volume of 200 μL per well and incubated at 37°C with 5% CO 2 / 95% air incubator for 24h. Remove the culture medium, add different concentrations (0, 20, 40, 60, 80, 100μg / mL) of COF NPs or COF-H1 / H2-Peptide NPs, and culture for 6 hours respectively. Then directly add 10μL 5mg / mL MTT solution and incubate for another 4h, remove MTT, and add 150μL DMSO to dissolve the formed formazan crystals. Finally, record the absorbance at 490nm with a microplate reader.

[0099] 9. Confocal fluorescence imaging

[0100] When studying the feasibility of CHA strategy to amplify the detection of intracellular miRNA-221, COF-H1-PeptideNPs was first used as a control probe. HeLa, HepG-2, and A549 cells were inoculated in confocal dishes and cultured for 24 hours. Each cell was incubated with COF-H1 / H2-Peptide NPs probe (30μg / mL) and COF-H1-Peptide NPs probe (30μg / mL) for 4 hours and then CLSM imaging was performed. Texas Red fluorescence images were excited at 595nm and fluorescence was monitored between 600-620nm.

[0101] Next, the effect of changes in the expression level of miRNA-221 detected by nanoprobe visualization on cell apoptosis was studied. HeLa, HepG-2, and A549 cells were inoculated in confocal dishes and cultured for 24 hours. Each cell was divided into three groups, one group was not treated as the control group, and the other two groups were treated with lipopolysaccharide (LPS, 10μg / mL) and staurosporine (STS, 100nM) for 12 hours. The cell culture medium was removed, and then the probe COF-H1 / H2-Peptide NPs (30μg / mL) was added and incubated for another 4 hours before CLSM imaging. Texas Red fluorescence images were excited at 595nm and fluorescence was monitored between 600-620nm; Cy5 fluorescence images were excited at 625nm and fluorescence was monitored between 655-700nm.

[0102] Then, the nanoprobes were studied for imaging of miRNA-221 and caspase-3 in cells treated with different concentrations of STS and different times of STS treatment. HeLa, HepG-2, and A549 cells were inoculated in confocal dishes and cultured for 24 hours. Each cell was treated with different concentrations of STS (0, 50, 100, and 150 nM) for 12 hours; 50 nM STS was treated for different times (0, 12, 24, 36, and 48 hours). Then, each cell was co-incubated with COF-H1 / H2-Peptide NPs probes (30 μg / mL) and COF-H1-Peptide NPs probes (30 μg / mL) for 4 hours and then CLSM imaging was performed. Texas Red fluorescence images were excited at 595 nm and fluorescence was monitored between 600 and 620 nm; Cy5 fluorescence images were excited at 625 nm and fluorescence was monitored between 655 and 700 nm.

[0103] 10. Flow cytometry analysis

[0104] To further study the changes in the expression levels of miRNA-221 and caspase-3 in drug-treated cells, HeLa, HepG-2, and A549 cells were inoculated in 6-well plates and cultured for 24 hours. Each cell type was divided into three groups, and the drug-treated conditions of the three groups of cells were the same as those of the confocal experiment. The drug-containing cell culture medium was removed, and then COF-H1 / H2-Peptide NPs (30μg / mL) were added and incubated for 4 hours. All cells were digested and washed three times with PBS buffer, and the cells were analyzed for fluorescence using a flow cytometer.

[0105] 11. Apoptosis Detection Analysis

[0106] The cell apoptosis was investigated using a cell apoptosis detection kit. HeLa, HepG-2, and A549 cells were inoculated in confocal dishes and cultured for 24 hours. Each cell type was divided into three groups, and the drug treatment conditions of the three groups of cells were the same as the above experiment. The cell culture medium was removed and PBS buffer was added to wash once. Then 194μL Annexin V-mCherryBinding Buffer, 5μL Annexin V-mCherry, and 1μL GreenNuc TM Caspase-3 Substrate (1mM), gently mix. Incubate at room temperature in the dark for 20-30min, then perform CLSM imaging. TM The fluorescence images were excited at 500 nm and monitored between 515 and 570 nm. The Annexin V-mCherry fluorescence images were excited at 587 nm and monitored between 600 and 700 nm.

[0107] 2. Experimental Results

[0108] 1. Characterization of the fluorescent nanoprobe COF-H1 / H2-Peptide prepared in the present invention

[0109] TEM and SEM observations of COF NPs and fluorescent nanoprobe COF-H1 / H2-Peptide were performed. Figure 2 A and B) and scanning electron microscopy (SEM) ( Figure 2 It can be observed from the images (F and G) that the fluorescent nanoprobe COF-H1 / H2-Peptide prepared in the present application has a uniform spherical structure with an average diameter of 160 nm. DLS size distribution ( Figure 2 The results of C) also showed that the average diameter of the probe COF-H1 / H2-PeptideNPs prepared after COF NPs were modified with DNA and Peptide increased slightly. In addition, the EDS elemental analysis results showed that COF-H1 / H2-Peptide NPs showed obvious P and S elements compared with COFNPs, indicating the successful loading of DNA and Peptide. Zeta potential characterization ( Figure 2 D) showed that after step-by-step loading of functional DNA molecules and peptides, the potential of COF NPs dropped from -22.1mV to -31.1mV, and then to -44.1mV. In addition, the material was subjected to ultraviolet-visible (UV-Vis) spectroscopy ( Figure 2 E) analysis showed that compared with COF, COF-H1 / H2-Peptide had three new absorption peaks at 260nm, 595nm and 650nm, corresponding to the characteristic absorption peaks of DNA, Texas Red modified on H1 / H2 and Cy5 modified on Peptide, respectively, indicating that DNA and Peptide were successfully loaded on the surface of COFNPs. The above results all illustrate the successful preparation of COF-H1 / H2-Peptide nanoprobes.

[0110] 2. Quenching curve study

[0111] The concentrations of COF NPs and COF-H1 / H2 NPs were optimized by fluorescence quenching experiments. Figure 3As shown in A and B, COF-H1 / H2 NPs were prepared by adding different concentrations of COF NPs to the mixture of H1 and H2, and the fluorescence quenching curve of Texas Red was plotted. The results showed that with the increase of COF NPs concentration, the fluorescence signal weakened rapidly. When the concentration of COF NPs solution was 150 μg / mL, the fluorescence of Texas Red was almost completely quenched. Then Peptide (2 μM) was added to COF-H1 / H2NPs solutions of different concentrations. Figure 3 As shown in C and D, 80 μg / mL COF-H1 / H2 NPs solution almost completely quenched the fluorescence of Cy5 on Peptide. Therefore, in the following experiments, 150 μg / mL COF NPs were first used to prepare COF-H1 / H2 NPs, and then 80 μg / mL COF-H1 / H2 NPs were used to prepare COF-H1 / H2-Peptide probes. In addition, the unloaded H1 / H2 and Peptide in the supernatant were collected by centrifugation, and according to HI / H2 ( Figure 3 E and F) and Peptide( Figure 3 The fluorescence standard curves of G and H) were used for quantification, and the loading density of HI / H2 and Peptide on COF NPs was calculated. The results showed that the loading capacity of COF NPs for HI / H2 was 0.793 nmol / mg, and the loading capacity for Peptide was 16.87 nmol / mg.

[0112] 3. Feasibility study of amplification detection of extracellular miRNA-221

[0113] Native-PAGE analysis was used to determine the structure formation and feasibility of the CHA reaction. Figure 4 A shows that H1 (lane 2) and H2 (lane 3) have a good metastable state, and the mixture of H1 and H2 (lane 4) does not react in the absence of target miRNA-221. When the target is added to H1, a new band is observed (lane 5), indicating the formation of the T-H1 double-stranded complex. However, no new band is observed when the target is added to H2 (lane 6), indicating that the target miRNA-221 cannot specifically recognize H2. When the target is added to the mixed solution of H1 and H2 (lane 7), a new band is generated due to the formation of the H1-H2 double-stranded complex, indicating that miRNA-221 can successfully trigger CHA.

[0114] Then, the feasibility of CHA strategy to amplify the response to miRNA-221 was further verified by fluorescence analysis ( Figure 4B). After incubation with COF NPs, the fluorescence of free H1 / H2 decreased significantly. In the presence of miRNA-221, a CHA cyclic reaction occurred, generating and releasing a large amount of H1-H2 double-stranded complexes, restoring 13.5 times the fluorescence. As a control, the same total amount of H1 was incubated with COF NPs to synthesize the COF-H1 probe. After adding miRNA-221, the restored fluorescence signal of the probe was only 5.2 times that of COF-H1 NPs, proving that CHA can greatly enhance the fluorescence signal of miRNA-221 detection.

[0115] 4. Fluorescence response of extracellular nanoprobes to miRNA-221 and caspase-3

[0116] The COF-H1-Peptide NPs probe was used as a control. Figure 4 As shown in Figures C and E, the Texas Red fluorescence intensity of the two probes gradually increased with the increase of miRNA-221 concentration. The fluorescence intensity of the COF-H1-Peptide NPs probe was linearly related to the miRNA-221 concentration in the range of 1-10 nM, with a detection limit (LOD) of 242 pM ( Figure 4 The linear range of the amplified probe COF-H1 / H2-Peptide NPs we designed was 0.01-1.0nM, and the LOD was 2.79pM ( Figure 4 D). It should be noted that when the concentration of target chain miRNA-221 is the same, the fluorescence intensity of COF-H1 / H2-Peptide NPs is significantly higher than that of COF-H1-Peptide NPs. Next, we studied the fluorescence response of COF-H1 / H2-Peptide NPs probe to caspase-3. Figure 4 As shown in G and H, with the increase of caspase-3 concentration (0-4Unit), the fluorescence signal of Cy5 gradually increased. The above results show that the probe COF-H1 / H2-Peptide NPs designed by us has a good fluorescence response to the two targets of miRNA-221 and caspase-3. In addition, the kinetic results show that ( Figure 5 ), COF-H1 / H2-Peptide nanoprobe responded quickly to miRNA-221 and reached equilibrium at 2 hours. The fluorescence response of the probe to caspase-3 also gradually increased over time, with a significant response at 2 hours. The above results show that the probe can detect miRNA-221 and caspase-3 simultaneously.

[0117] 5. Study on the specificity and stability of COF-H1 / H2-Peptide nanoprobe

[0118] In order to study the ability of COF-H1 / H2-Peptide nanoprobe to specifically detect miRNA-221 and caspase-3, the fully matched target miRNA-221, caspase-3 and other non-matching targets were added to the COF-H1 / H2-Peptide nanoprobe solution for incubation. Figure 5 As shown in the figure, only the perfect match of miRNA-221 and caspase-3 can make COF-H1 / H2-Peptide nanoprobe produce significantly enhanced Texas Red and Cy5 fluorescence signals. These results indicate that COF-H1 / H2-Peptide nanoprobe is highly specific for the detection of miRNA-221 and caspase-3.

[0119] The stability of the COF-H1 / H2-Peptide nanoprobe was further evaluated. First, the fluorescence intensity changes of the probe in 4°C cell culture medium and PBS buffer over time were detected. Figure 5 ). The results showed that the fluorescence of Texas Red and Cy5 of the nanoprobes hardly changed, showing stability in vitro. In addition, in Tris-HCl solutions with different pH values ​​(2-10), it was found that the fluorescence intensity of the two dyes did not change significantly. These results all indicate that the prepared probes have excellent stability in complex physiological environments and are suitable for biological imaging.

[0120] 6. MTT assay for cytotoxicity of COF nanoparticles and COF-H1 / H2-Peptide

[0121] Different concentrations of COF and COF-H1 / H2-Peptide (0-100 μg / mL) were incubated with HeLa, HepG-2, and A549 cells. The results showed that even after incubation with 100 μg / mL COF NPs or COF-H1 / H2-Peptide NPs, the cell survival rate exceeded 90% ( Figure 6 ), indicating that COF-H1 / H2-Peptide NPs have good biocompatibility and are suitable for cell imaging.

[0122] 7. Imaging detection of intracellular miRNA-221 and caspase-3

[0123] Hela cells were selected as model cells and incubated with the control probe COF-H1-Peptide and the CHA amplification probe COF-H1 / H2-Peptide for 4 h before fluorescence confocal imaging (CLSM). Figure 7As shown in A, compared with the cells incubated with COF-H1-Peptide probe, the green fluorescence of miRNA-221 in Hela cells incubated with COF-H1 / H2-Peptide probe was significantly enhanced. The fluorescence intensity of the cells in the marked part of CLSM fluorescence imaging was statistically analyzed ( Figure 7 B), the data show that in Hela cells, the green fluorescence of the COF-H1 / H2-Peptide amplified probe incubated is 4.7 times that of the COF-H1-Peptide control probe. The exact same experiment was performed in HepG-2 and A549 cells, and it was found that the COF-H1 / H2-Peptide probe we designed also produced a significantly enhanced fluorescence signal relative to the COF-H1-Peptide probe ( Figure 8 , Fig. 9 ). The above results all indicate that the probe we designed can effectively realize CHA cycle amplification and detection of low-abundance miRNA-221 in living cells.

[0124] 8. COF-H1 / H2-Peptide probe visualizes the effect of changes in intracellular miRNA-221 expression on cell apoptosis

[0125] Hela cells were divided into three groups, two of which were pretreated with LPS and STS for 12 hours, respectively, and the control group was not treated with any treatment. Then, the probe COF-H1 / H2-Peptide was added and incubated for another 4 hours before CLSM imaging.

[0126] The results are as follows Fig.10 As shown in A, compared with untreated Hela cells, the green fluorescence signal of miRNA-221 in the LPS group increased significantly, and the red fluorescence signal of caspase-3 did not change significantly. However, the red fluorescence signal in the STS group was significantly enhanced, and the green fluorescence intensity was significantly reduced. The fluorescence intensity of the cells in the marked part of the CLSM fluorescence imaging was statistically analyzed ( Fig.10 B), the results also confirmed the above changes in fluorescence signals. Flow cytometry was used to investigate the changes in the expression of miRNA-221 and caspase-3 in three groups of Hela cells after treatment with different drugs. The results showed that after the cells were treated with STS drugs, the fluorescence intensity of miRNA-221 was significantly reduced, while the fluorescence signal of caspase-3 was significantly enhanced ( Fig.13). In cells treated with LPS, the fluorescence signal of miRNA-221 was significantly enhanced. The results of flow cytometry analysis further verified the expression changes of miRNA-221 and caspase-3 in the CLSM imaging experiment. Finally, the apoptosis of each group of cells was further verified using a cell apoptosis detection kit. The results showed that only the cells treated with STS showed apoptosis, with bright green fluorescence in the cell nucleus and red fluorescence in the cell membrane, accompanied by obvious cell fragmentation. No fluorescence signal was observed in the other two groups of cells. HepG-2 and A549 cells were selected for further research. After being treated under exactly the same conditions as Hela cells, the cells were subjected to CLSM imaging experiments ( Fig.11 , Fig.12 ), flow cytometry analysis experiment ( Fig.13 ) and apoptosis kit staining assay ( Fig.15 , Fig.16 ). The results of CLSM imaging and flow cytometry analysis were basically consistent with those of Hela cells. The fluorescence signal of miRNA-221 in LPS group cells increased significantly, the fluorescence signal of caspase-3 in STS group cells increased significantly, and the fluorescence signal of miRNA-221 decreased significantly. The apoptosis imaging results also observed that HepG-2 and A549 cells treated with STS had obvious fragmentation, bright green fluorescence in the nucleus, and red fluorescence in the cell membrane.

[0127] 9. Ability of COF-H1 / H2-Peptide probe imaging to detect low-level miRNA-221 in cells treated with STS drug

[0128] Hela cells were treated with different concentrations of STS for 12 h, and then incubated with CHA amplification probe COF-H1 / H2-Peptide and control probe COF-H1-Peptide for 4 h before fluorescence confocal imaging (CLSM).

[0129] The results are as follows Fig.17 As shown in the figure, with the increase of STS concentration, the red fluorescence signal of caspase-3 of the two probes gradually increased, and the green fluorescence signal of miRNA-221 gradually weakened. However, we can see that cells co-incubated with COF-H1 / H2-Peptide can observe green fluorescence even when treated with 150nM STS, while cells co-incubated with the control probe only did not observe fluorescence when treated with a low concentration of 50nM STS. Then, Hela cells were treated with STS for different time periods and then incubated with the amplification probe and the control probe, respectively. Fig.18As shown in the figure, as the STS treatment time increases, the green fluorescence signal of miRNA-221 gradually weakens, and the red fluorescence signal of caspase-3 gradually increases. No miRNA-221 signal was observed in cells co-incubated with the control probe after 12 hours of STS treatment, while the green fluorescence of miRNA-221 was still observed in cells co-incubated with the amplification probe after 36 hours of STS treatment. It is worth noting that at the same concentration of STS or the same STS treatment time, the green fluorescence intensity of cells incubated with COF-H1 / H2-Peptide was significantly higher than that of cells incubated with the control probe. The above results show that as the concentration of anticancer drugs increases and the treatment time increases, the amplification probe we prepared can still successfully detect the gradual decrease in the expression of oncogenes and the gradual increase in cell apoptosis. Finally, we selected HepG-2( Figure 19-20 ) and A549( Figure 21-22 ) cells, and observed two signals after being treated with the same conditions as Hela cells. We also found that compared with the control probe COF-H1-Peptide, the amplification probe COF-H1 / H2-Peptide can well observe the green fluorescence signal of miRNA-221 and the red fluorescence signal of caspase-3 even in cells treated with high concentration and long-term STS. These results show that the amplification probe COF-H1 / H2-Peptide designed by us is suitable for detecting low levels of miRNA-221 in cells, and optimizing the treatment conditions of anticancer drugs on cells according to the relative changes of miRNA-221 and apoptosis signals.

[0130] It should be noted that, in the present invention, "COF-H1 / H2-Peptide NPs", "COF-H1 / H2-Peptide nanoprobe" and "fluorescent nanoprobe COF-H1 / H2-Peptide" all have the same meaning, and all refer to the fluorescent nanoprobe COF-H1 / H2-Peptide prepared in Example 1. The concentration in brackets in this application is the final concentration.

[0131] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0132] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a fluorescent nanoprobe COF-H1 / H2-Peptide, characterized in that: The steps include: 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxyterephthalaldehyde are mixed with acetonitrile, and the mixture is completely dissolved by ultrasonic treatment for 1 min, and glacial acetic acid is added to mix well, and the mixture is rapidly stirred at room temperature for 1 min, and then centrifuged at 9500 rpm for 10 min to obtain a precipitate of COF NPs nanoparticles; the amount ratio of 1,3,5-tris(4-aminophenyl)benzene, 2,5-dimethoxyterephthalaldehyde and acetonitrile is 12-15 g: 10-13 g: 10-30 mL; Dispersing COF NPs nanoparticles into PBS buffer to obtain a COF NPs solution; the mixing ratio of the COF NPs nanoparticles and the PBS buffer is 1-2 g: 2-3 mL; The COF NPs solution is mixed with the hairpin DNA labeled with Texas Red to obtain a COF-H1 / H2 NPs solution, and then the COF-H1 / H2 NPs solution is mixed with the peptide labeled with Cy5 to obtain the fluorescent nanoprobe COF-H1 / H2-Peptide; the mixing ratio of the COF NPs solution and the hairpin DNA labeled with Texas Red is 30-40uL:3-4uL; the mixing ratio of the COF-H1 / H2 NPs solution and the peptide labeled with Cy5 is 100-120 uL:3-4 uL; The hairpin DNA preparation process is as follows: using Texas Red to label the ends of H1 and H2 respectively and then mixing them to form a mixed solution; the concentration of H1 is 20uM; the concentration of H2 is 20uM; The H1 nucleotide sequence is shown in SEQ ID NO.1, and the H2 nucleotide sequence is shown in SEQ ID NO.2; The sequence of Cy5-labeled peptide is: Cy5-Gly Gly Asp Glu Val Asp Gly Gly Cys.

2. A fluorescent nanoprobe COF-H1 / H2-Peptide prepared by the preparation method according to claim 1.

3. Use of the fluorescent nanoprobe COF-H1 / H2-Peptide according to claim 2 in the preparation of a product for detecting tumor cytokines, characterized in that: The tumor cell factors are miRNA-221 and apoptosis marker caspase-3; the product has the function of detecting low-level miRNA-221 in cells treated with STS drugs, with a detection linear range of 0.01-1.0 nM and a detection limit of 2.79 pM.

Images