A two-photon fluorescence nanosensor for detecting microRNA-203 in tumor cells, its preparation method and application

Abstract

The present invention discloses a two-photon fluorescence nanosensor for detecting microRNA-203 in tumor cells, and its preparation method and application. The two-photon fluorescence nanosensor includes two-photon silicon nanoparticles, carboxyfluorescein, and hairpin DNA. The two-photon silicon nanoparticles are modified with hairpin DNA1, and the carboxyfluorescein is modified on hairpin DNA2. The two-photon silicon nanoparticles serve as an energy donor, and the carboxyfluorescein serves as a fluorescence acceptor. The present invention can achieve imaging and detection of microRNA-203 in cells by synthesizing a nucleic acid-functionalized two-photon silicon nanoprobe and combining the catalytic hairpin assembly (CHA) signal amplification strategy and fluorescence resonance energy transfer (FRET) method.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials and biosensors, and particularly relates to a two-photon fluorescence nanosensor for detecting microRNA-203 in tumor cells, a preparation method thereof, and an application thereof. Background Art

[0002] MicroRNA, as a short-chain, non-coding, endogenous RNA, not only widely participates in the regulation of various biological processes such as early development, cell proliferation, differentiation, and apoptosis, but also its expression level is closely related to various diseases. Therefore, microRNA can be used as an important biomarker for studying many diseases. In particular, the abnormal expression (up-regulation or down-regulation) of microRNA is closely related to the occurrence and development of cancer, and is considered to be a promising biomarker in the prognosis, diagnosis, and treatment of different cancers. Since the content of microRNA in cells is generally low, people are committed to developing methods for specifically and highly sensitively detecting microRNA. In the prior art, reverse transcription-polymerase chain reaction (RT-PCR) is a traditional signal amplification strategy for detecting low-concentration RNA, but it can only be operated in a buffer solution and cannot reflect the differences in expression in cells; in situ hybridization (FISH) strategy based on isothermal enzyme signal amplification, since the inward transport of the enzyme requires cell fixation, can only achieve imaging of microRNA in single cells and is not suitable for live cell imaging. In recent years, based on enzyme-free signal amplification of the target, the detection and imaging of low-concentration microRNA in live cells have become the focus of attention. Catalytic hairpin assembly (CHA) is a signal amplification strategy that can well achieve in situ trace target, and it is expected to achieve fluorescence imaging of microRNA in cells in situ.

[0003] Fluorescence imaging, as a currently commonly used technology for visualizing and dynamically monitoring the changes of live cells, has been widely used in biomedical research and molecular diagnosis. Traditional fluorescence imaging methods are easily interfered by the autofluorescence of the biological background and the external environment, while fluorescence resonance energy transfer (FRET), as a ratio fluorescence imaging method, can well eliminate the interference of background signals and is not interfered by external factors, realizing precise quantification of intracellular substances. At the same time, traditional fluorescence imaging methods have a low penetration depth for tissue imaging and cannot achieve imaging with a large tissue penetration depth, while two-photon imaging with a deeper tissue penetration depth, lower fluorescence background, and high spatiotemporal resolution can well avoid the above problems. Combining FRET and two-photon imaging can not only reduce the interference of biological autofluorescence background, but also obtain information with a large tissue penetration depth, realizing non-destructive, highly sensitive and precise quantification of biological samples.

[0004] Based on the above research background, if a nucleic acid-functionalized two-photon silicon nanocomposite probe based on triggerable CHA signal amplification and FRET strategy can be constructed, it is expected to be used to monitor the expression of endogenous microRNAs in living cells, which is of great significance for the development of tissue imaging and in vivo imaging technologies. Summary of the Invention

[0005] In view of the above problems, the object of the present invention is to construct a fluorescence probe capable of accurately quantifying trace microRNA-203 in different tumor cells, and to realize the imaging and detection of microRNA-203 in cells by combining the catalytic hairpin self-assembly (CHA) signal amplification strategy and fluorescence resonance energy transfer (FRET).

[0006] To solve the above technical problems, in the first aspect of the present invention, a two-photon fluorescence nanoprobe for detecting microRNA-203 in tumor cells is provided, which includes two-photon silicon nanoparticles, carboxyfluorescein and hairpin DNA. The two-photon silicon nanoparticles are modified with hairpin DNA1, and the carboxyfluorescein is modified on hairpin DNA2. The two-photon silicon nanoparticles serve as an energy donor, and the carboxyfluorescein serves as a fluorescence acceptor.

[0007] Further, the sequence of the hairpin DNA1 is: 5'-TTT AGG ACC ACT AGG GTG TGT GTG GGCTAG TGG TCC TAAACATTTCAC-NH2-3', and the sequence of the hairpin DNA2 is: 5'-GGT GTG TGT GGG (T-FAM) TTA GGACCACTAGCC CAC ACA CAC CCT AGT GGT-3'.

[0008] In the second aspect of the present invention, a preparation method of the above two-photon fluorescence nanoprobe for detecting microRNA-203 in tumor cells is provided, which includes the following steps:

[0009] S1. Synthesize two-photon silicon nanoparticles TP-SiNPs;

[0010] S2. Modify the two-photon silicon nanoparticles with hairpin DNA1 to prepare TP-SiNPs@H1;

[0011] S3. Modify carboxyfluorescein FAM on hairpin DNA2 to obtain H2-FAM.

[0012] Further, the step S1 specifically includes:

[0013] S11. Add 2-naphthoic acid to N,N-dimethylformamide (DMF) containing N,N-diisopropylethylamine (DIPEA) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), react in an ice bath for 0.5 - 1.5 hours, add γ-aminopropyltriethoxysilane (APTES) and stir at room temperature for 1 - 2 hours;

[0014] S12. Pour the mixture obtained in step S11 into ice water to form a precipitate. The precipitate is separated by a vacuum filter and purified by silica gel column chromatography to obtain a solid crude product;

[0015] S13. Dissolve tetraethoxysilane (TEOS) in ethanol, add it dropwise to the solid crude product obtained in step S12, then add triethanolamine and stir to react for 1 - 2 hours. Centrifuge to collect the reactant and wash it several times with ethanol;

[0016] S14. Dissolve the reactant obtained in step S13 in ethanol, add glutaric anhydride, stir for 3 - 5 hours, centrifuge and wash away the unreacted glutaric anhydride, and freeze-dry to obtain two-photon silicon nanoparticles TP-SiNPs.

[0017] Further, step S2 specifically includes:

[0018] S21. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) to 2-(N-morpholino)ethanesulfonic acid (MES) buffer containing two-photon silicon nanoparticles TP-SiNPs, react for 8 - 12 hours, and ultrafiltrate and centrifuge to remove the excess EDC and sulfo-NHS to obtain carboxyl-activated TP-SiNPs;

[0019] S22. Disperse the carboxyl-activated TP-SiNPs in HEPES buffer and mix with H1, and react at 3 - 7 °C for 40 - 56 hours;

[0020] S23. Ultrafiltrate and centrifuge and wash several times with tris(hydroxymethyl)aminomethane (Tris) buffer to obtain hairpin DNA1-modified two-photon silicon nanoparticles TP-SiNPs@H1.

[0021] Further, step S3 specifically includes:

[0022] After obtaining hairpin DNA2, add a hexyl-deoxythymidine-amino linker (C6-dT amino-linker) to the thymine residue, with the amino group 10 atoms away from the main chain after modification. Label the carboxyfluorescein FAM on the sequence of hairpin DNA2 by organic synthesis method to obtain H2-FAM.

[0023] Furthermore, the hairpin DNA1 and hairpin DNA2 are synthesized by the phosphoramidite method, and the synthesis direction is: from the 3'-end to the 5'-end of the primer to be synthesized, and adjacent nucleotides are linked by 3'-5' phosphodiester bonds.

[0024] The third aspect of the present invention provides an application of the above-mentioned two-photon fluorescence nanoprobe for detecting microRNA-203 in tumor cells in tissue imaging / in vivo imaging.

[0025] In summary, the beneficial effects of the present invention are as follows:

[0026] 1. The present invention realizes the imaging and detection of microRNA-203 in cells by synthesizing a nucleic acid-functionalized two-photon silicon nanoprobe and combining the catalytic hairpin assembly (CHA) signal amplification strategy and fluorescence resonance energy transfer (FRET) method.

[0027] 2. Hairpin DNA1 (H1) is modified on the two-photon silicon nanoparticles (TP-SiNPs), and carboxyfluorescein (FAM) is modified on the hairpin DNA2 (H2). TP-SiNPs serve as the energy donor, and FAM serves as the fluorescence acceptor. When the target microRNA-203 is added, H1 modified on the TP-SiNPs is opened, triggering H2 to displace the target, and fluorescence resonance energy transfer occurs. At the same time, the displaced target can be used for the next round of cyclic amplification. Since the distance between the fluorescence of the two-photon silicon nanoparticles and FAM becomes smaller after the combination of TP-SiNPs@H1 and H2-FAM, the FRET phenomenon occurs. This enzyme-free signal amplification CHA strategy can highly sensitively detect ultra-low levels of microRNA-203, and through FRET and two-photon fluorescence imaging, the background interference in biological imaging can be effectively overcome, realizing imaging and information collection with a relatively large tissue penetration depth.

[0028] 3. The cyclic amplification nanoprobe constructed by the present invention can be used to monitor the expression of endogenous microRNAs in living cells, and has good application prospects in tissue imaging and in vivo imaging technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the structural and working principle diagram of the two-photon fluorescence nanoprobe;

[0030] Figure 2 is the transmission electron microscope image of TP-SiNPs and TP-SiNPs@H1;

[0031] Figure 3 is the particle size distribution diagram of TP-SiNPs and TP-SiNPs@H1;

[0032] Figure 4 is the Zeta potential diagram of TP-SiNPs and TP-SiNPS@H1;

[0033] Figure 5 is the infrared spectrum diagram of TP-SiNPs and TP-SiNPs@H1;

[0034] Figure 6 is the ultraviolet absorption spectrum diagram of H1, TP-SiNPs and TP-SiNPs@H1;

[0035] Figure 7 is the gel electrophoresis image of H1, H2, and microRNA-203;

[0036] Figure 8 is the change in the fluorescence spectra of TP-SiNPs and FAM after adding different concentrations of the target microRNA-203;

[0037] Figure 9 is the fluorescence intensity ratio (F A / F D ) of the acceptor to the donor in the two-photon fluorescence nanoprobe under different concentrations of microRNA-203;

[0038] Figure 10 is the fluorescence intensity ratio (F A / F D ) of the acceptor to the donor in the two-photon fluorescence nanoprobe after adding various analytes;

[0039] Figure 11 is the kinetic process of the two-photon fluorescence nanoprobe in the absence and presence of microRNA-203;

[0040] Figure 12 is the fluorescence intensity ratio (F A / F D ) of the acceptor to the donor in the two-photon fluorescence nanoprobe in the absence and presence of microRNA-203 under different pH environments;

[0041] Figure 13 is the cell viability of MCF-7 cells after incubation with different concentrations of TP-SiNPs and TP-SiNPs@H1 for 24 h and 48 h;

[0042] Figure 14It is the fluorescence confocal imaging of microRNA-203 in MCF-7 cells by a two-photon nanocomposite probe; (A-D) MCF-7 cells without adding the probe; (E-H) Imaging effect after incubating MCF-7 cells with the probe for 3 h; (I-L) Imaging effect after incubating MCF-7 cells with miR-203 minic for 24 h and then incubating with the probe for 3 h; (M-P) Imaging effect after incubating MCF-7 cells with miR-203 Antagomir for 24 h and then incubating with the probe for 3 h (λ ex = 405 nm, λ em1 = 420 - 480 nm, λ em2 = 500 - 550 nm); scale bar = 25 μm;

[0043] Figure 15 It is the imaging depth of TP-SiNPs in mouse liver tissue sections at different depths measured by a fluorescence confocal microscope (λ ex = 405 nm, λ em = 420 - 480 nm); Scale bar: 50 μm;

[0044] Figure 16 It is the imaging depth of TP-SiNPs in mouse liver tissue sections at different depths measured by a two-photon confocal microscope (λ ex = 740 nm, λ em = 420 - 480 nm); Scale bar: 100 μm. Detailed implementation manners

[0045] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings.

[0046] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0049] The mimics microRNA-203, microRNA-141, microRNA-151, and microRNA-21 used in the following examples were synthesized and purified by Shanghai GenePharma Co., Ltd. using high performance liquid chromatography, and their sequences are as follows:

[0050] microRNA-203 5'-GUG AAA UGU UUA GGA CCA CUA G-3';

[0051] microRNA-141 5'-UAA CAC UGU CUG GUA AAG AUG G-3';

[0052] microRNA-151 5'-UUA AUG CUA AUC GUG AUA GGG GU-3';

[0053] microRNA-21 5'-UAG CUUAUC AGA CUG AUG UUG A-3'.

[0054] Example 1

[0055] Based on the triggerable CHA signal amplification and FRET strategy, this example constructs a nucleic acid-functionalized two-photon fluorescence nanoprobe for ratio-based highly sensitive detection of the microRNA content in tumor cells. The preparation method of the two-photon fluorescence nanoprobe is as follows:

[0056] S1. Synthesize two-photon silicon nanoparticles TP-SiNPs

[0057] 2-Naphthoic acid (215.2 mg, 1.0 mmol) was added to 5.0 mL of DMF containing 0.5 mL of DIPEA and HATU (456.2 mg, 1.2 mmol). After reacting for 30 minutes in an ice bath, APTES (221 mg, 1.0 mmol) was added and the mixture was stirred at room temperature for 1 hour.

[0058] The mixture was poured into ice water to produce a precipitate, which was separated by a vacuum filter and purified by silica gel column chromatography (DCM / EtOH = 50:1, v / v) to obtain a crude solid product.

[0059] 1.0 mL of TEOS was dissolved in 1.0 mL of ethanol, and it was added dropwise to 20 mg of the above-mentioned crude solid product. A few drops of triethanolamine were then added, and the reaction mixture was stirred for 1 hour. The reactants were collected by centrifugation and washed several times with ethanol.

[0060] The surface of silica nanoparticles was carboxyl-functionalized by co-condensation polymerization. The obtained reactants were fully dissolved in ethanol, and then glutaric anhydride was added. The mixture was stirred for 3 hours, centrifuged, and the unreacted glutaric anhydride was washed away. The carboxyl-functionalized two-photon silicon nanoparticles TP-SiNPs were obtained by freeze-drying.

[0061] Preparation of TP-SiNPs@H1 by modifying two-photon silicon nanoparticles with S2 and hairpin DNA1

[0062] EDC (10 mg) and sulfo-NHS (3.0 mg) were added to MES buffer (0.1 M, pH 5.5) containing 1.5 mg of two-photon silicon nanoparticles (TP-SiNPs). The mixture was stirred at room temperature for 8 hours, and the excess EDC and sulfo-NHS were removed by ultrafiltration centrifugation to obtain carboxyl-activated TP-SiNPs.

[0063] The carboxyl-activated TP-SiNPs were dispersed in HEPES buffer (0.1 M, 1.5 mL, pH 7.4) and mixed with hairpin DNA1 solution (0.33 μM). The reaction was carried out at 4.0 °C for 48 hours.

[0064] It was washed several times by ultrafiltration centrifugation with Tris buffer (0.1 M, pH 7.4) to obtain hairpin DNA1-modified two-photon silicon nanoparticles TP-SiNPs@H1.

[0065] Modifying carboxyfluorescein FAM on hairpin DNA2 to obtain H2-FAM

[0066] After obtaining the hairpin DNA2, C6-dT amino-linker was added to the thymine residue. After modification, the amino group was 10 atoms away from the main chain. Carboxyfluorescein FAM was labeled on the sequence of hairpin DNA2 by organic synthesis to obtain H2-FAM.

[0067] The sequence of hairpin DNA1 (H1) is: 5'-TTT AGG ACC ACT AGG GTG TGT GTG GGC TAG TGGTCC TAAACATTTCAC-NH2-3';

[0068] The sequence of hairpin DNA2 (H2) is: 5'-GGT GTG TGT GGG (T-FAM)TTA GGACCACTAGCC CACACA CAC CCT AGT GGT-3'.

[0069] Hairpin DNA1 and hairpin DNA2 were synthesized by the phosphoramidite method. The phosphoramidite method is to fix DNA on a solid-phase carrier to complete the synthesis of the DNA strand. The synthesis direction is: from the 3’ end to the 5’ end of the primer to be synthesized, and adjacent nucleotides are connected by 3’-5’ phosphodiester bonds.

[0070] The synthesis steps of the DNA sequence are as follows:

[0071] S01. React the nucleotide with a protected active group pre-connected to the solid-phase carrier CPG with trichloroacetic acid to remove the protecting group DMT of its 5’-hydroxyl group to obtain a free 5’-hydroxyl group;

[0072] S02. Mix the raw material for DNA synthesis, phosphoramidite-protected nucleotide monomer, and activator tetrazole to obtain a nucleoside phosphite-activated intermediate, whose 3’ end is activated and the 5’-hydroxyl group is still protected by DMT, and it undergoes a condensation reaction with the free 5’-hydroxyl group in the solution;

[0073] S03. Capping reaction. In the condensation reaction, there are very few 5’-hydroxyl groups that do not participate in the reaction, and the reaction is terminated with acetic anhydride and 1-methylimidazole;

[0074] S04. Under the action of the oxidant iodine, the phosphite form is converted into a more stable phosphate triester.

[0075] After the above four steps, a deoxynucleotide is linked to the nucleotide on the solid-phase carrier. Then, the protecting group DMT on its 5’-hydroxyl group is removed with trichloroacetic acid, and the above steps are repeated until the synthesized base is linked. Through high-temperature treatment with ammonia water, the primer linked to the CPG is cut off, and the primer is purified by means such as OPC and HPLC. The finished primer is used with C 18Reverse-phase chromatography column for concentration, desalting, and precipitation. The precipitated primers are suspended in water and the OD is measured. 260 Quantification. The required H1 and H2 sequences are synthesized by this method.

[0076] In this example, the structure and working principle of the two-photon fluorescence nanoprobe are as Figure 1 shown. Hairpin DNA1 (H1) is modified on the two-photon silicon nanoparticles (TP-SiNPs), and carboxyfluorescein (FAM) is modified on the hairpin DNA2 (H2). TP-SiNPs serve as the energy donor and FAM serves as the fluorescence acceptor. When the target microRNA-203 is added, H1 modified on the TP-SiNPs is opened, triggering H2 to displace the target, resulting in fluorescence resonance energy transfer. At the same time, the displaced target can be used for the next round of cyclic amplification. Since the distance between the fluorescence of the two-photon silicon nanoparticles and FAM becomes smaller after the binding of TP-SiNPs@H1 and H2-FAM, the FRET phenomenon occurs. This enzyme-free signal amplification CHA strategy can highly sensitively detect ultra-low levels of microRNA-203, and the background interference in bioimaging can be effectively overcome through FRET and two-photon fluorescence imaging.

[0077] Example 2

[0078] In this example, the TP-SiNPs and TP-SiNPs@H1 prepared in Example 1 are tested and analyzed. The analysis contents are as follows:

[0079] (1) Morphology analysis

[0080] The morphologies of TP-SiNPs and TP-SiNPs@H1 are observed using a transmission electron microscope (TEM), as Figure 2 shown, Figure 2 (A, B) are the TEM images of TP-SiNPs, Figure 2 (C, D) are the TEM images of TP-SiNPs@H1. The results show that the TP-SiNPs and TP-SiNPs@H1 nanoparticles exhibit a uniform and monodisperse spherical morphology.

[0081] (2) Particle size analysis

[0082] The particle sizes of TP-SiNPs and TP-SiNPs@H1 are measured by dynamic light scattering (DLS). The results are as Figure 3 shown. The average particle size of TP-SiNPs is about 130 nm, and the average particle size of TP-SiNPs@H1 is about 150 nm. The increase in the particle size of TP-SiNPs@H1 indicates that H1 has successfully bound to the TP-SiNPs.

[0083] (3) Zata potential analysis

[0084] The Zeta potentials of TP-SiNPs and TP-SiNPs@H1 were measured, and the results are as Figure 4 shown. The Zeta potential of TP-SiNPs was -26.4 ± 1.1 mV, and that of TP-SiNPs@H1 was -54.4 ± 1.4 mV. Since H1 is negatively charged, the Zeta potential of TP-SiNPs@H1 is lower than that of TP-SiNPs, indicating that H1 was successfully modified on TP-SiNPs.

[0085] (4)Infrared spectroscopy and ultraviolet absorption spectroscopy analysis

[0086] Figure 5 are the infrared spectra of TP-SiNPs and TP-SiNPs@H1, indicating that TP-SiNPs can be coupled with amino-modified H1 through an amide reaction.

[0087] Figure 6 are the ultraviolet absorption spectra of H1, TP-SiNPs and TP-SiNPs@H1. The maximum absorption wavelengths of H1 and TP-SiNPs are 260 nm and 330 nm respectively, further verifying the covalent binding of H1 and TP-SiNPs.

[0088] Example 3

[0089] This example verifies the feasibility of the CHA reaction of the hairpin structure DNA in Example 1. The verification method and results are as follows:

[0090] The feasibility of the CHA reaction of hairpin DNA1 (H1) and hairpin DNA2 (H2) was verified by gel electrophoresis. The concentrations of H1, H2-FAM and microRNA-203 used were 0.3 μM, 0.3 μM and 1 μM respectively. The reaction was carried out at 37 °C for 40 min and then electrophoresed. The results are as Figure 7 shown. The complementary hairpins H1 and H2 were slowly annealed from 95 °C to 25 °C. Channels 1, 2, and 3 are the bands of H1, H2, and the target microRNA-203 respectively. When H1 and H2 were incubated, the bands of H1 and H2 overlapped (channel 4), and no new bands appeared, indicating that H1 and H2 did not react. In channel 5, when the target was added, new bands appeared, indicating that due to the presence of the target, H1 and H2 underwent a CHA reaction, proving the feasibility of CHA, and signal amplification of target recycling can occur in this system.

[0091] Example 4

[0092] This example examines the performance of the two-photon fluorescence nanoprobe in Example 1. The analysis method is as follows:

[0093] (1)Sensitivity test of two-photon fluorescence nanoprobe

[0094] TP-SiNPs@H1 (50 nmol / g), H2-FAM (50 nM) and microRNA-203 (0 - 250 nM) were reacted in a PBS solution with a total volume of 200 μL for 40 min, and the fluorescence spectrum and the peak signals of the acceptor (FAM) and donor (TP-SiNPs) (F A / F D ) were obtained from an RF-6000 fluorescence spectrometer. The results are shown in Figure 8 and Figure 9 . Figure 8 The results show that as the concentration of the target microRNA-203 increases from 0 to 250 nM, the fluorescence intensity of the emission peak of the acceptor FAM (F A ) at 520 nm gradually increases, while the fluorescence intensity of the emission peak of the donor TP-SiNPs (F D ) at 440 nm gradually decreases. Figure 9 The results show that the ratio of fluorescence intensity F A / F D increases by 4 times as the concentration of microRNA-203 increases. When the concentration of miR-203 is within 0 - 2.0 nM, F A / F D shows a linear relationship with the concentration of miR-203 ( Figure 9 inset). The limit of detection (LOD) is approximately 33 pM (3σ / slope). The results prove that the two-photon fluorescence nanoprobe has excellent sensitivity for the target microRNA-203.

[0095] (2)Specificity test of two-photon fluorescence nanoprobe

[0096] To further verify the specificity of the two-photon fluorescence nanoprobe, the F A / F D values of microRNA-203 and other interfering microRNAs, including microRNA-141, microRNA-155 and microRNA-21, were compared. TP-SiNPs@H1 (50 nmol / g), H2-FAM (50 nM) and microRNA (250 nM) were reacted in a PBS solution with a total volume of 200 μL for 40 min, and the fluorescence spectrum and the peak signals of the acceptor (FAM) and donor (TP-SiNPs) (F A / F D ) were obtained from an RF-6000 fluorescence spectrometer. The results are shown in Figure 10 . The F A / F Dsignificantly lower than that of microRNA-203 at the same concentration, F A / F D . The results show that the two-photon fluorescence nanoprobe has high selectivity for the detection of miR-203.

[0097] (3) Reaction kinetics of the two-photon fluorescence nanoprobe

[0098] To verify the effect of the two-photon fluorescence nanoprobe, after mixing the probe with the target microRNA-203 (250 nM), the F A / F D value changing with time was measured every 5 minutes, and a control group without the target microRNA-203 was also tested. The results are as Figure 11 shown. The reaction between the two-photon fluorescence nanoprobe and microRNA-203 reached equilibrium at 35 min, indicating that the probe responds rapidly to miR-203.

[0099] (4) Influence of pH on the two-photon fluorescence nanoprobe

[0100] To evaluate the influence of pH on the two-photon fluorescence nanoprobe, after mixing the probe with the target microRNA-203 (250 nM), the F A / F D value was tested under different pH environments. The results are as Figure 12 shown. When the pH ranges from 3 to 8, the influence of pH on the fluorescence intensity of the two-photon fluorescence nanoprobe is not significant; when microRNA-203 is present, when the pH is 7 - 8, the response of the probe to microRNA-203 is relatively good, indicating that the two-photon silicon nanocomposite probe can detect microRNA-203 in the physiological environment.

[0101] Example 5

[0102] This example verifies whether the two-photon fluorescence nanoprobe in Example 1 can detect microRNA-203 in living cells, and a cell viability experiment was carried out to explore it. The experimental method is as follows:

[0103] MCF-7 cells were seeded in a 96-well plate at a seeding density of 1×10 5 / well (100.0 μL). After 24 hours of seeding, the overnight culture medium was aspirated, and a mixture containing different concentrations of TP-SiNPs, TP-SiNPs@H1 (0 - 500 μg•mL -1The culture medium was placed in an incubator at 37 °C and 5.0% CO2 and incubated for 24 hours and 48 hours respectively. Then, the overnight culture medium was aspirated, and 1× DPBS was added to each well to remove the excess mixture, and the washing was repeated three times to prevent interference with subsequent detections. Subsequently, 90.0 μL of culture medium and 10.0 μL of CCK-8 reagent solution were added to each well using a multi-channel pipette. After incubating in the incubator for 4 hours, the absorbance of each well was measured using a microplate spectrophotometer at a wavelength of 450.0 nm.

[0104] The experimental results are as Figure 13 shown. When the concentrations of TP-SiNPs and TP-SiNPs@H1 were within 500 μg / mL and the incubation times were 24 h and 48 h, the cell viability remained above 80%. This indicates that the biocompatibility of this two-photon fluorescence nanoprobe is good and cell-related experiments can be carried out.

[0105] Example 6

[0106] This example is a cell imaging experiment of the two-photon fluorescence nanoprobe in Example 1. The experimental method is as follows:

[0107] MCF-7 cells were seeded in a 24-well plate at an inoculation density of 400 μL 5×10 5 / well. One day after inoculation, the overnight culture medium was aspirated, and the culture medium containing TP-SiNPs@H1 was added. The plate was placed in an incubator at 37 °C and 5.0% CO2 and incubated for six hours. Then, the culture medium was aspirated, and 1× DPBS solution was added to remove the excess mixture, and the washing was repeated three times to prevent interference with subsequent detections.

[0108] Meanwhile, H2-FAM encapsulated in liposomes in advance was prepared for standby. The preparation steps are as follows: Take 100.0 μL of the prepared liposomes and 100.0 μL of serum-free DMEM containing H2-FAM to make the concentration of H2-FAM 5.0 μM.

[0109] Take 10.0 μL of TP-SiNPs@H1 (where the concentration of H1 is 5.0 μM) and 30.0 μL of H2-FAM (5.0 μM) and add them to 360.0 μL of the above culture medium to keep the total volume at 400 μL all the time. Add the mixture to the washed cells and incubate in the incubator. After incubating for 3 hours, 1× DPBS solution was added to remove the excess mixture, and the washing was repeated three times to prevent interference with subsequent detections. After the sample treatment was completed, single-photon and two-photon imaging were carried out. To make the detection phenomenon more obvious, four groups were set for comparison during the incubation process, namely: blank group, control group, promotion group (extra addition of mimic microRNA-203), and inhibition group (addition of microRNA-203 inhibitor).

[0110] The results are as Figure 14As shown, the confocal imaging of cells incubated under different conditions shows certain changes in fluorescence signals. Among them, the cell imaging of the blank group has no obvious background fluorescence signal; from the superimposed image, it can be observed that the signal of the green fluorescence (FAM) in the promotion group is the strongest, indicating that the addition of the mimic of microRNA-203 can trigger the signal amplification of the probe and the occurrence of FRET between the two-photon silicon nanoparticles and FAM in the cells; followed by the control group; then it can be seen from the inhibition group that the fluorescence of FAM becomes significantly weaker, indicating that the inhibitor down-regulates the content of microRNA-203 in the cells, reducing the amount of the original trace target, thereby inhibiting the triggering of the signal amplification strategy. The above embodiments prove that the two-photon fluorescence nanoprobe can well detect different concentrations of microRNA-203 in cells.

[0111] Example 7

[0112] In this example, the penetration depth of two-photon silicon nanoparticles TP-SiNPs into mouse liver tissue was tested. The experimental method is as follows:

[0113] Mouse liver tissue sections were incubated in a medium containing TP-SiNPs. Single-photon and two-photon tissue imaging depths were obtained respectively with a fluorescence confocal microscope (λ ex = 405 nm) and a two-photon confocal microscope (λ ex = 740 nm). The results are as Figure 15 and Figure 16 shown. The depth of single-photon tissue imaging is 90 μm, and the depth of two-photon tissue imaging is 190 μm. The results show that the substances in cells can be monitored in real time through two-photon confocal imaging. These advantages make the two-photon fluorescence nanoprobe have great prospects in medical diagnosis and clinical applications.

[0114] Although the present invention is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will all fall within the protection scope of the present invention. Sequence Listing <120> Two-Photon Fluorescent Nanoprobe for Detecting microRNA-203 in Tumor Cells, Its Preparation Method and Application <140> 2021116244932 <141> 2021-12-28 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 48 <212> DNA <213> Hairpin DNA1 (synthetic construct) <400> 1 tttaggacca ctagggtgtg tgtgggctag tggtcctaaa catttcac 48 <210> 2 <211> 46 <212> DNA <213> Hairpin DNA2 (synthetic construct) <400> 2 ggtgtgtgtg ggtttaggac cactagccca cacacaccct agtggt 46

Claims

1. A two-photon fluorescence nanosensor for detecting microRNA-203 in tumor cells, characterized in that, It includes two-photon silicon nanoparticles, carboxyfluorescein, and hairpin DNA. Hairpin DNA1 is modified on the two-photon silicon nanoparticles, and carboxyfluorescein is modified on hairpin DNA2. The two-photon silicon nanoparticles serve as an energy donor, and the carboxyfluorescein serves as a fluorescence acceptor. The sequence of hairpin DNA1 is: 5'-TTT AGG ACC ACT AGG GTG TGT GTG GGC TAG TGG TCC TAAACATTTCAC-NH2-3', and the sequence of hairpin DNA2 is: 5'-GGT GTG TGT GGG(T-FAM)TTA GGACCACTAGCC CAC ACA CAC CCT AGT GGT-3'.

2. A method for preparing a two-photon fluorescence nanoprobe for detecting microRNA-203 in tumor cells as described in claim 1, characterized in that, It includes the following steps: S1. Synthesize two-photon silicon nanoparticles TP-SiNPs; S2. Prepare TP-SiNPs@H1 by modifying two-photon silicon nanoparticles with hairpin DNA1; S3. Modify carboxyfluorescein FAM on hairpin DNA2 to obtain H2-FAM.

3. The preparation method of the two-photon fluorescence nanosensor for detecting microRNA-203 in tumor cells according to claim 2, wherein The specific steps of S1 include: S11. Add 2-naphthoic acid to DMF containing DIPEA and HATU, react in an ice bath for 0.5 - 1.5 hours, add APTES and stir at room temperature for 1 - 2 hours; S12. Pour the mixture obtained in step S11 into ice water to generate a precipitate. The precipitate is separated by a vacuum filter and purified by silica gel column chromatography to obtain a solid crude product; S13. Dissolve TEOS in ethanol, dropwise add the solid crude product obtained in step S12, then add triethanolamine, stir and react for 1 - 2 hours, centrifuge to collect the reactant and wash it with ethanol several times; S14. Dissolve the reactant obtained in step S13 in ethanol, add glutaric anhydride, stir for 3 - 5 hours, centrifuge and wash away the unreacted glutaric anhydride, and freeze-dry to obtain two-photon silicon nanoparticles TP-SiNPs.

4. The preparation method of the two-photon fluorescence nanoprobe for detecting microRNA-203 in tumor cells according to claim 2, wherein, The specific steps of S2 include: S21. Add EDC and sulfo-NHS to the MES buffer solution containing two-photon silicon nanoparticles TP-SiNPs, react for 8 - 12 hours, and ultrafiltration centrifuge to remove the excessive EDC and sulfo-NHS to obtain carboxyl-activated TP-SiNPs; S22. Disperse the carboxyl-activated TP-SiNPs in HEPES buffer solution and mix it with the hairpin DNA1 solution, and react at 3 - 7 °C for 40 - 56 hours; S23. Ultrafiltration centrifuge and wash several times with Tris buffer solution to obtain hairpin DNA1-modified two-photon silicon nanoparticles TP-SiNPs@H1.

5. The preparation method of the two-photon fluorescence nanosensor for detecting microRNA-203 in tumor cells according to claim 2, wherein, The specific steps of S3 include: After obtaining hairpin DNA2, add C6-dT amino-linker to the thymine residue, with the modified amino group being 10 atoms away from the main chain, and label carboxyfluorescein FAM on the sequence of hairpin DNA2 by organic synthesis method to obtain H2-FAM.

6. The preparation method of the two-photon fluorescence nanoprobe for detecting microRNA-203 in tumor cells according to claim 2, wherein, The hairpin DNA1 and hairpin DNA2 are synthesized by the phosphoramidite triester method, and the synthesis direction is: from the 3'-end to the 5'-end of the primer to be synthesized, and adjacent nucleotides are connected by 3'-5' phosphodiester bonds.

7. Use of the two-photon fluorescence nanoprobe for detecting microRNA-203 in tumor cells in tissue imaging / in vivo imaging as claimed in claim 1.

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