Double-DNA nanosphere probe as well as preparation method and biosensing application thereof

Abstract

The invention relates to a double DNA nanosphere probe (DDNS) as well as a preparation method and biosensing application thereof. The DDNS comprises a DNA (deoxyribonucleic acid) nanosphere 1 (DNS-1) and a DNA nanosphere 2 (DNS-2). The DNS-1 and the DNS-2 are respectively formed by self-assembling two cholesterol modified DNA single-stranded probes with hairpin structures, and are respectively used for identifying target miRNA and triggering a non-enzymatic circulating strand displacement amplification (T-SDA) reaction. The DDNS can realize high-sensitivity fluorescence detection on target miRNA and can effectively inhibit non-target miRNA interference, and the interference inhibition rate is close to 100%. Besides, DDNS has excellent nuclease stability, the system can automatically enter living cells without a transfection reagent, high signal-to-noise ratio fluorescence differentiation of cancer cells and healthy cells is realized on the premise of maintaining cell activity, the performance is equivalent to that of a transfection agent dependent system, and the biological safety is remarkably improved. The invention provides a new material and a new method for application of the DNA nanostructure in the fields of molecular diagnosis and precision medical treatment.

Description

Technical Field

[0001] This invention belongs to the field of DNA nanomaterials, specifically relating to a dual DNA nanosphere probe, its preparation method, and its application in biosensing. Background Technology

[0002] Minimal RNAs (miRNAs) are considered tumor suppressors and oncogenes in the human body, and have become potential diagnostic and prognostic biomarkers for assessing tumorigenesis, monitoring progression, and predicting human cancer prognosis. Due to their short length, high homology, low abundance, and wide dynamic range, accurate detection of miRNAs, especially those with low expression levels in living cells, remains a significant challenge. Compared to other cancer diagnostic methods (such as extracellular nucleic acid diagnostics), intracellular miRNA-based detection offers high sensitivity and the convenience of collecting cellular transformation information in the early stages of cancer. Currently, miRNA detection technologies can be broadly categorized into traditional and innovative methods. However, in most cases, traditional methods still face limitations such as complex detection procedures, low specificity, low sensitivity, poor reproducibility, and the need for expensive equipment and extensive professional training. To overcome these problems, new signal transduction strategies often rely on signal amplification processes, such as hybridization chain reaction (HCR), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), strand substitution amplification (SDA), nanomaterial-based amplification, and catalytic hairpin assembly (CHA).

[0003] DNA, as a crucial carrier of genetic information, not only encodes the genetic characteristics of organisms but also, due to its highly predictable base pairing principle, serves as a programmable molecular engineering material, widely used for assembling various structural systems for different purposes. DNA nanotechnology enables precise sequence-based molecular self-assembly, allowing the construction of one-dimensional, two-dimensional, and three-dimensional nanostructures, demonstrating broad prospects in biological and biomedical applications. Particularly in the field of cancer diagnosis, biosensing platforms constructed using DNA nanotechnology possess enormous potential for the precise detection of tumor biomarkers due to their excellent biocompatibility, high sensitivity, and high specificity. Summary of the Invention

[0004] Based on the above considerations, the present invention provides a dual DNA nanosphere probe, its preparation method and its biosensing application. The dual DNA nanosphere probe has a responsive stimulus fusion function and can be used for target miRNA signal amplification and detection.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A dual DNA nanosphere probe, comprising DNA nanosphere-1 and DNA nanosphere-2; DNA nanosphere-1 is self-assembled from an amphiphilic single-stranded DNA probe Chol-H1 via hydrophobic interactions, and DNA nanosphere-2 is self-assembled from an amphiphilic single-stranded DNA probe FB-Chol-H2 via hydrophobic interactions; both Chol-H1 and FB-Chol-H2 have hairpin structures, and their 5' ends are modified with cholesterol; the hairpin structure of FB-Chol-H2 is modified with a fluorescent group and a quenching group at both ends, respectively; Furthermore, the sequence of Chol-H1 is: 5'-Cholesterol-CATCGACTGGTCGCGTTTTCTAAACGGAACCACTAGTGACTTGAGATGTGTAGCACAAGTCACTAGTGGT-3'; the sequence of FB-Chol-H2 is: 5'-Cholesterol-CTCGGAGAAGAGATGTTTTTGTGACTTG / i6FAMdT / GCTACACATCTCAAGTCACTAGTGGTAGATGTGTAGCA-BHQ1-3'.

[0006] The above-mentioned method for preparing a dual DNA nanosphere probe includes the following steps: diluting Chol-H1 and FB-Chol-H2 with 1×TE buffer to obtain Chol-H1 solution and FB-Chol-H2 solution respectively; mixing Chol-H1 solution with 10×TE buffer... 2+ The buffer solution and ddH2O were mixed, heated at 90°C for 5 minutes, and then naturally cooled to room temperature to obtain the DNA nanosphere-1 solution; FB-Chol-H2 solution and 10× TAE-Mg were added. 2+ Mix the buffer solution and ddH2O, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-2 solution; mix DNA nanosphere-1 solution with DNA nanosphere-2 solution to obtain dual DNA nanosphere probe solution.

[0007] The above-mentioned dual DNA nanosphere probe is used in the preparation of biosensing platforms and cell imaging reagents.

[0008] A biosensing platform comprising the aforementioned dual DNA nanosphere probe.

[0009] The above-mentioned method for constructing a biosensing platform includes the step of co-incubating a dual DNA nanosphere probe with a target miRNA or its DNA mimicry. Furthermore, during the co-incubation process, the dual DNA nanosphere probe can promote the hybridization of Chol-H1 and FB-Chol-H2 under the stimulation of the target miRNA or its DNA mimic, and bring DNA nanosphere-1 and DNA nanosphere-2 closer together. Under the stimulation of the target miRNA or its DNA mimic, these two DNA nanospheres will recombine and gradually fuse to form a super-large DNA nanosphere and generate an amplified signal. This fusion process is a dynamic, ordered self-assembly process mediated by hydrophobic interactions and DNA hybridization. The above-mentioned biosensing platform is used for the detection of miRNA or its DNA mimics for non-disease diagnosis and treatment purposes.

[0010] A cell imaging reagent comprising the above-described dual DNA nanosphere probe; Furthermore, the cell imaging reagent is used for fluorescence imaging of intracellular target miRNAs.

[0011] The inventive principle of this invention is as follows: Figure 1 As shown: This invention develops a dual-DNA nanosphere probe composed of two DNA nanospheres (DNS-1 and DNS-2) based on hydrophobic interaction-mediated hairpin DNA sequence self-assembly. In the presence of target miRNA, the hairpin structure in DNS-1 opens due to hybridization with the target, releasing a complementary fragment that contacts and hybridizes with FB-Chol-H2 in DNS-2, opening the hairpin structure in FB-Chol-H2 and generating a fluorescent signal. Then, the more stable hybridization of Chol-H1 and FB-Chol-H2 forces the target miRNA to leave, initiating the next round of hybridization-opening-hybridization-release process. Furthermore, the hybridization of Chol-H1 and FB-Chol-H2 brings DNS-1 and DNS-2 closer together. These two DNA nanospheres recombine and gradually fuse under the stimulation of the target miRNA, forming a super-large DNA nanosphere (SLDNS) and generating an amplified signal. Throughout the reaction, the target itself is recovered without being consumed, thus enabling highly sensitive detection of the target miRNA.

[0012] The advantages of this invention are: The dual DNA nanosphere probe with responsive stimulus fusion function has the following advantages for the detection of signal activation amplification of cancer-related miRNAs: (1) High detection sensitivity and strong specificity. The detection limit of the DDNS biosensing platform of this invention is 25.7pM, and it does not respond to non-target miRNAs. (2) Convenient operation. This invention does not require special treatment of DNA sequences, nor does it require the use of enzymes or other catalysts to catalyze the reaction. The DDNS biosensing platform can directly enter living cells without the assistance of external reagents, and its ability to distinguish cancer cells from normal cells is comparable to that of similar systems mediated by commercial transfection agents, but it does not damage cell viability, thus avoiding common safety issues. (3) Good serum stability. As a DNA material with a multilayer spherical nanostructure, SLDNS has an outermost layer composed of hairpin probes arranged in an orderly upright manner, which can protect DNA ends from nuclease attack and maintain long-term stability in harsh biological media. Attached Figure Description

[0013] Figure 1 : A schematic diagram illustrating the structure of the dual DNA nanosphere probe (DDNS) and the principle of the DDNS biosensing platform.

[0014] Figure 2 : Schematic diagram of target recovery strand displacement amplification (T-SDA) and characterization of T-SDA reaction products by polyacrylamide gel electrophoresis.

[0015] Figure 3 Structural characterization of the ultra-large DNA nanospheres (SLDNS) generated during the T-SDA reaction. A, Schematic diagram of the structure; B, Dynamic light scattering (DLS) analysis; C, Confocal microscopy image.

[0016] Figure 4 Feasibility analysis of DDNS biosensor platform for detecting target miRNA mimics.

[0017] Figure 5 Sensitivity of the DDNS biosensor platform. A, Fluorescence spectral response to target miRNA mimics from 0 to 400 nM; B, Fluorescence spectra of target miRNA mimics from 0 to 1 nM; C, Dynamic response relationship between fluorescence intensity and target miRNA mimic concentration; D, Linear fitting of target miRNA mimic concentration and fluorescence intensity from 0 to 150 nM.

[0018] Figure 6 The specificity of the DDNS biosensing platform.

[0019] Figure 7Serum stability of the DDNS biosensor platform. A, Gel electrophoresis patterns after incubation in a solution containing 10% fetal bovine serum for different times; B, Quantitative analysis of the band intensities corresponding to the gel electrophoresis patterns.

[0020] Figure 8 Intracellular target miRNA fluorescence imaging using the DDNS biosensing platform. A, Fluorescence images and relative fluorescence intensities of MCF-7 cells after incubation with different probes; B, Fluorescence images and relative fluorescence intensities of L02 cells after incubation with different probes; C, Quantitative fluorescence intensity recorded by confocal fluorescence microscopy of MCF-7 and L02 cells.

[0021] Figure 9 Comparison of biocompatibility between DDNS biosensing platform and Lip-DDNS biosensing platform. Detailed Implementation

[0022] To make the content of this invention easier to understand, the technical solution of this invention will be further described below in conjunction with specific embodiments. However, the following examples are merely examples of this invention and do not represent the scope of protection of this invention. The scope of protection of this invention is determined by the claims.

[0023] The sequences of the single-stranded DNA probes involved in this embodiment of the invention are as follows: Chol-H1: 5'-Cholesterol-CATCGACTGGTCGCGTTTTCTAAACGGAACCACTAGTGACTTGAGATGTGTAGCACAAGTCACTAGTGGT-3' FB-Chol-H2: 5'-Cholesterol-CTCGGAGAAGAGATGTTTTTGTGACTTG / i6FAMdT / GCTACACATCTCAAGTCACTAGTGGTAGATGTGTAGCA-BHQ1-3' H1: 5'-CATCGACTGGTCGCGTTTTCTAAACGGAACCACTAGTGACTTGAGATGTGTAGCACAAGTCACTAGTGGT-3' H2: 5'-CTCGGAGAAGAGATGTTTTTGTGACTTGGCTACACATCTCAAGTCACTAGTGGTAGATGTGTAGCA-3' FB-H2: 5'-CTCGGAGAAGAGATGTTTTTGTGACTTG / i6FAMdT / GCTACACATCTCAAGTCACTAGTGGTAGATGTGTAGCA-BHQ1-3' The sequences of the miRNA mimics (DNA mimics) involved in the embodiments of the present invention are as follows: miR-224D analogue: 5'-CAAGTCACTAGTGGTTCCGTTTA-3'.

[0024] miR-155D emulator: 5'-TTAATGCTAATCGTGATAGGGGT-3'.

[0025] miR-21D analogue: 5'-TAGCTTATCAGACTGATGTTGA-3'.

[0026] miR-221D analogue: 5'-AGCTACATTGTCTGCTGGGTTTC-3'.

[0027] miR-222D analogue: 5'-CTCAGTAGCCAGTGTAGATCCT-3'.

[0028] Let-7aD analogue: 5'-TGAGGTAGTAGGTTGTATAGTT-3'.

[0029] 1-Mut analogue: 5'-CAAGTCACTAGTGGTTCTGTTTA-3'.

[0030] 2-Mut analogue: 5'-CAAGTCACTAGTGATTCTGTTTA-3'.

[0031] 3-Mut analogue: 5'-CAAGTCACTGGTGATTCTGTTTA-3'.

[0032] The formulation of the 1×TE buffer involved in the embodiments of the present invention is: 10mM Tris, 1mM EDTA; pH 8.0.

[0033] The 10× TAE-Mg involved in the embodiments of the present invention 2+ The buffer solution was formulated as follows: 20 mM Tris, 2 mM EDTA, 12.5 mM MgCl2; pH 8.0.

[0034] Example 1: The preparation of dual DNA nanosphere probes includes the following steps: S1: Dilute DNA single-stranded probe Chol-H1 and DNA single-stranded probe FB-Chol-H2 with 1× TE buffer to obtain Chol-H1 solution and FB-Chol-H2 solution with a concentration of 10 µM.

[0035] S2: Add 2 µL of 10 µM Chol-H1 solution and 2 µL of 10× TAE-Mg 2+ Add buffer to 16 µL ddH2O, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-1 (DNS-1) solution. Add 2 µL 10 µM FB-Chol-H2 solution and 2 µL 10× TAE-Mg... 2+ Add the buffer solution to 16 µL ddH2O, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-2 (DNS-2) solution.

[0036] S3: Mix 7.5 µL of DNS-1 solution from S2 with 10 µL of DNS-2 solution from S2 thoroughly to obtain a dual DNA nanosphere probe (DDNS) solution.

[0037] Example 2: The reaction products were characterized by polyacrylamide gel electrophoresis, including the following steps: S1: Dilute DNA single-stranded probe H1, DNA single-stranded probe H2, and miR-224D mimic with 1× TE buffer to obtain H1 solution, H2 solution, and miR-224D mimic solution with a concentration of 10 µM.

[0038] S2: Preparation of reaction products in each lane: ① Add 1.5 µL of 10 µM H1 solution and 3 µL of 10× TAE-Mg 2+ The buffer solution and 25.5 µL ddH2O were thoroughly mixed, heated at 90°C for 5 minutes, and then naturally cooled to room temperature to obtain the H1 reaction product, which is also the reaction product of lane 1.

[0039] ② Add 1.5 µL of 10 µM H2 solution and 3 µL of 10× TAE-Mg 2+ The buffer solution and 25.5 µL ddH2O were thoroughly mixed, heated at 90°C for 5 minutes, and then naturally cooled to room temperature to obtain the H2 reaction product, which is also the reaction product of lane 2.

[0040] ③ Add 1 µL of 10 µM miR-224D simulant solution and 3 µL of 10× TAE-Mg 2+The buffer solution and 26 µL ddH2O were thoroughly mixed, heated at 90°C for 5 minutes, and then naturally cooled to room temperature to obtain the reaction product of lane 3.

[0041] ④ Add 1.5 µL of 10 µM H1 solution and 1 µL of 10× TAE-Mg 2+ The buffer solution and 7.5 µL ddH2O were thoroughly mixed, heated at 90°C for 5 minutes, and then naturally cooled to room temperature to obtain the H1 reaction product; 1.5 µL of the H2 solution from step S1 and 1 µL of 10× TAE-Mg were added. 2+ The buffer solution and 7.5 µL ddH2O were thoroughly mixed, heated at 90°C for 5 minutes, and then naturally cooled to room temperature to obtain the H2 reaction product. The resulting H1 reaction product and H2 reaction product were then mixed, 10 µL ddH2O was added, and the mixture was thoroughly mixed and incubated at 37°C for 4 hours to obtain the lane 4 reaction product.

[0042] ⑤ Add 1.5 µL of 10 µM H1 solution, 1 µL of 10 µM miR-224D simulant solution, and 3 µL of 10×TAE-Mg 2+ The buffer solution and 24.5 µL ddH2O were thoroughly mixed, heated at 90°C for 5 minutes, and then naturally cooled to room temperature to obtain the reaction product in lane 5.

[0043] ⑥ Add 1.5 µL of 10 µM H1 solution and 1 µL of 10× TAE-Mg 2+ The buffer solution and 7.5 µL ddH2O were thoroughly mixed, heated at 90 °C for 5 minutes, and then naturally cooled to room temperature to obtain the H1 reaction product; 1.5 µL 10 µM H2 solution and 1 µL 10× TAE-Mg were then added. 2+ The buffer solution and 7.5 µL ddH2O were thoroughly mixed, heated at 90 °C for 5 minutes, and then naturally cooled to room temperature to obtain the H2 reaction product. The resulting H1 reaction product and H2 reaction product were then mixed, and 1 µL of 10 µM miR-224D simulant solution and 9 µL ddH2O were added. After thorough mixing, the mixture was incubated at 37 °C for 4 hours to obtain the lane 6 reaction product.

[0044] S3: Take 8 µL of reaction products from lanes 1 to 6 and mix them with 2 µL of DNA loading buffer and 2 µL of SYBR Green I, respectively. Use freshly prepared 12% non-denaturing polyacrylamide gel and electrophoresis in 0.5× TBE electrophoresis buffer at a constant voltage of 100 V for 1 hour. After electrophoresis, use the ChemiDox XRS imaging system to acquire gel images.

[0045] The results are as follows Figure 2As shown, the migration position of the band in lane 6 indicates a significant increase in its molecular weight. Based on this, it is speculated that after the addition of the target miRNA mimic, the hairpin structure of the DNA single-stranded probe H1 opens and binds to H2 via a strand displacement reaction, forming the H1-H2 complex.

[0046] Example 3: Structural characterization of the ultra-large DNA nanospheres generated during the target strand replacement amplification reaction includes the following steps: S1: Dilute DNA single-stranded probe H1, DNA single-stranded probe FB-H2, DNA single-stranded probe Chol-H1, DNA single-stranded probe FB-Chol-H2, and miR-224D mimic solution with 1× TE buffer to obtain H1 solution, FB-H2 solution, Chol-H1 solution, FB-Chol-H2 solution, and miR-224D mimic solution, each with a concentration of 10 µM.

[0047] S2: Add 5 µL of 10× TAE-Mg to 2.5 µL of 10 µM H1 solution. 2+ The buffer solution was adjusted to 50 μL with ddH2O, thoroughly mixed, heated at 90°C for 5 minutes, and then allowed to cool naturally to room temperature to obtain the H1 reaction product. 5 µL of 10× TAE-Mg was added to 2.5 µL of 10 µM FB-H2 solution. 2+ The buffer solution was adjusted to 50 μL with ddH2O, thoroughly mixed, heated at 90°C for 5 minutes, and then allowed to cool naturally to room temperature to obtain the FB-H2 reaction product. 5 µL of 10× TAE-Mg was added to 2.5 µL of 10 µM Chloro-H1 solution. 2+ Add ddH2O to the buffer solution to a final volume of 50 μL. Mix thoroughly and heat at 90°C for 5 minutes, then allow to cool naturally to room temperature to obtain the DNA nanosphere-1 (DNS-1) solution. Add 5 µL of 10×TAE-Mg to 2.5 µL of 10 µM FB-Chol-H2 solution. 2+ Add ddH2O to the buffer solution to bring the volume to 50 μL. Mix thoroughly and heat at 90 °C for 5 minutes. Then cool naturally to room temperature to obtain DNA nanosphere-2 (DNS-2) solution.

[0048] S3: Thoroughly mix the DNS-1 solution and DNS-2 solution from S2 to obtain the dual DNA nanosphere probe (DDNS) solution, i.e., DNS-1+DNS-2 solution.

[0049] S4: Add 2 μL of 10 µM miR-224D mimic solution to the DDNS solution in S3, bring the volume to 1 mL with ddH2O, mix thoroughly, and incubate at 25 °C for 4 hours to obtain a super-large DNA nanosphere (DDNS) solution.

[0050] S5: The H1 reaction product, FB-H2 reaction product, DNS-1 solution, DNS-2 solution, DDNS solution, and SLDNS solution were subjected to dynamic light scattering (DLS) detection using a Malvern Zetasizer Nano ZS90 instrument equipped with a helium-neon laser, and bright-field imaging was performed using a Leica SP8 laser scanning confocal microscope (Leica GmbH, Germany).

[0051] The results are as follows Figure 3 As shown, no nanoscale spherical structures were observed in the H1 reaction products and the FB-H2 reaction products; the particle sizes of DNS-1, DNS-2, and SLDNS were 1480±51.5 nm, 1610±64.3 nm, and 4800±98.7 nm, respectively. Confocal microscopy images showed that all three were regular spherical structures, and their sizes were basically consistent with the particle size measurements. Under the activation of the target miRNA mimic, DNS-1 and DNS-2 can fuse and assemble into SLDNS; however, in the absence of the target miRNA mimic, due to steric hindrance and electrostatic repulsion, DNS-1 and DNS-2 do not undergo significant expansion or fusion and can coexist stably in DDNS solution.

[0052] Example 4: The feasibility analysis of the DDNS biosensor platform for detecting target miRNAs includes the following steps: S1: Dilute DNA single-stranded probe Chol-H1, DNA single-stranded probe FB-Chol-H2, and miR-224D mimic with 1× TE buffer to obtain Chol-H1 solution, FB-Chol-H2 solution, and miR-224D mimic solution, respectively.

[0053] S2: Add 2 µL of 10 µM Chol-H1 solution and 2 µL of 10× TAE-Mg 2+ Add buffer to 16 µL ddH2O, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-1 (DNS-1) solution. Add 2 µL 10 µM FB-Chol-H2 solution and 2 µL 10× TAE-Mg... 2+ Add the buffer solution to 16 µL ddH2O, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-2 (DNS-2) solution.

[0054] S3: Mix 15 µL of 5 µM DNS-1 solution with 20 µL of 5 µM DNS-2 solution thoroughly to obtain the dual DNA nanosphere probe (DDNS) solution.

[0055] S4: Add 4 µL of 10 µM miR-224D simulant solution and 20 µL of 10×TAE-Mg to the DDNS solution in S3. 2+ Add ddH2O to the buffer solution to bring the volume to 200 µL, mix thoroughly, and incubate at 37°C for 4 hours to obtain an ultra-large DNA nanosphere (SLDNS) solution.

[0056] S5: The fluorescence spectra of the above FB-Chol-H2 solution, DDNS solution and SLDNS solution between 500 and 600 nm were collected using a Hitachi F-7000 spectrophotometer. The excitation wavelength was set to 492 nm, and the excitation and emission slits were both set to 5.0 nm. The fluorescence intensity at λem = 520 nm was recorded.

[0057] The results are as follows Figure 4 As shown, a strong fluorescent signal is only generated when DNS-1, DNS-2, and the target miRNA mimic are all present simultaneously.

[0058] Example 5: The sensitivity analysis of the DDNS biosensor platform for detecting target miRNAs includes the following steps: S1: Same as Example 4 S1.

[0059] S2: Same as Example 4 S2.

[0060] S3: Same as Example 4 S3.

[0061] S4: Add 4 µL of miR-224D simulant solutions of different concentrations and 20 µL of 10×TAE-Mg to the DDNS solution in S3. 2+ Add ddH2O to the buffer solution to bring the volume to 200 µL, mix thoroughly, and incubate at 25°C for 4 hours to obtain an ultra-large DNA nanosphere (SLDNS) solution.

[0062] S5: The fluorescence spectrum of the above SLDNS solution between 500 and 600 nm was collected using a Hitachi F-7000 spectrophotometer. The excitation wavelength was set to 492 nm, and both the excitation and emission slits were set to 5.0 nm. The fluorescence intensity at λem = 520 nm was recorded.

[0063] The results are as follows Figure 5 As shown, the fluorescence intensity gradually increased with increasing target miRNA mimicry concentration, and in the 0–150 nM range, the fluorescence intensity showed a good linear relationship with the target miRNA mimicry concentration. 2=0.9920, and the regression equation is F=232.46+16.977C, where F represents fluorescence intensity and C represents the concentration of the target miRNA mimic; the limit of detection is 25.7pM. This fully demonstrates that the DDNS biosensor platform can accurately and stably identify the target miRNA mimic within a low concentration range.

[0064] Example 6: The DDNS biosensing platform performs specificity analysis on target miRNAs, including the following steps: S1: Dilute miR-155D mimic, miR-21D mimic, miR-221D mimic, miR-222D mimic, miR-224D mimic, 1-Mut mimic, 2-Mut mimic, 3-Mut mimic, Let-7aD mimic, DNA single-stranded probe Chol-H1, and DNA single-stranded probe FB-Chol-H2 with 1× TE buffer to obtain 10 μM solutions of miR-155D mimic, miR-21D mimic, miR-221D mimic, miR-222D mimic, miR-224D mimic, 1-Mut mimic, 2-Mut mimic, 3-Mut mimic, Let-7aD mimic, Chol-H1, and FB-Chol-H2.

[0065] S2: Same as Example 4 S2.

[0066] S3: Same as Example 4 S3.

[0067] S4: Add 4 µL of 10 μM miRNA mimic solution to the DDNS solution in S3, and bring the total volume to 200 µL with ddH2O. After thorough mixing, incubate at 25°C for 4 hours to obtain a super-large DNA nanosphere (SLDNS) solution.

[0068] S5: The fluorescence spectrum of the above SLDNS solution between 500 and 600 nm was collected using a Hitachi F-7000 spectrophotometer. The excitation wavelength was set to 492 nm, and both the excitation and emission slits were set to 5.0 nm. The fluorescence intensity at λem = 520 nm was recorded.

[0069] The results are as follows Figure 6 As shown, the DDNS biosensing platform only produced a high-intensity fluorescence response to the target miR-224D mimic. The signals produced by a series of non-target miRNA mimics used as controls remained at background levels, indistinguishable from the blank sample. This fully demonstrates the superior specific recognition capability of the DDNS biosensing platform, with negligible non-specific adsorption.

[0070] Example 7: Serum stability analysis of the DDNS biosensor platform includes the following steps: S1: Dilute DNA single-stranded probe H1, DNA single-stranded probe H2, DNA single-stranded probe Chol-H1, DNA single-stranded probe FB-Chol-H2, and miR-224D mimic solution with 1× TE buffer to obtain H1 solution, H2 solution, Chol-H1 solution, FB-Chol-H2 solution, and miR-224D mimic solution, each with a concentration of 10 μM.

[0071] S2: Mix 0.75 µL of 10 µM H1 solution with 2 µL of 10× TAE-Mg 2+ Mix the buffer solution, add ddH2O to a final volume of 20 µL, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain the H1 reaction product. Add 0.75 µL of 10 µM H2 solution and 2 µL of 10× TAE-Mg... 2+ Mix the buffer solutions, add ddH2O to a final volume of 20 µL, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain the H2 reaction product. Add 2 µL of 10 µM Chol-H1 solution and 2 µL of 10× TAE-Mg... 2+ Mix the buffer solutions, add ddH2O to a final volume of 20 µL, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain the DNA nanosphere-1 (DNS-1) solution. Add 2 µL of 10 µM FB-Chol-H2 solution and 2 µL of 10× TAE-Mg... 2+ Mix the buffer solution, add ddH2O to make up to 20 µL, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-2 (DNS-2) solution.

[0072] S3: Mix 7.5 µL of DNS-1 solution from S2 with 10 µL of DNS-2 solution from S2 to obtain a dual DNA nanosphere probe (DDNS) solution.

[0073] S4: Add 0.4 µL of 10 µM miR-224D mimic solution to the DDNS solution in S3, and make up to 20 µL with ddH2O to obtain the ultra-large DNA nanosphere (SLDNS) precursor solution.

[0074] S5: Add 2 µL of fetal bovine serum (FBS) to the H1 reaction product, H2 reaction product, DNS-1 solution, DNS-2 solution, and SLDNS precursor solution respectively, and mix thoroughly. Incubate at 37°C for 0–24 hours. Transfer 8 µL to a centrifuge tube, add 2 µL of 6× DNA loading buffer and 2 µL of SYBR Green I, mix thoroughly, and then add to the corresponding lane of a 10% nPAGE gel. Then run in 0.5× TBE buffer at a constant voltage of 100 V for 1 hour. Acquire gel images on a ChemiDox XRS imaging system and perform band quantification analysis on the obtained gel images.

[0075] The results are as follows Figure 7 As shown, the H1 and H2 reaction products maintained stability for only about 2 hours and 4 hours, respectively, in the FBS environment, while the residual amounts of DNS-1 solution, DNS-2 solution, and SLDNS precursor solution remained close to 100% after incubation under the same conditions for 24 hours. This indicates that cholesterol modification significantly enhances the stability of the DNA probe and its assembly product SLDNS in the serum environment, providing effective protection.

[0076] Example 8: The steps for studying intracellular target miRNA fluorescence imaging based on the DDNS biosensing platform are as follows: S1: Dilute DNA single-stranded probe H1, DNA single-stranded probe FB-H2, DNA single-stranded probe Chol-H1, and DNA single-stranded probe FB-Chol-H2 with 1× TE buffer to obtain H1 solution, FB-H2 solution, Chol-H1 solution, and FB-Chol-H2 solution with a concentration of 10 μM.

[0077] S2: Mix 2 µL of 10 µM H1 solution with 2 µL of 10× TAE-Mg 2+ The buffer solution was added to 16 µL of ddH₂O, mixed thoroughly, heated at 90 °C for 5 minutes, and then allowed to cool naturally to room temperature to obtain the H₁ reaction product. 2 µL of 10 µM FB-H₂ solution and 2 µL of 10× TAE-Mg were then added... 2+ The buffer solution was added to 16 µL of ddH2O, mixed thoroughly, heated at 90 °C for 5 minutes, and then allowed to cool naturally to room temperature to obtain the FB-H2 reaction product. 2 µL of 10 µM Chol-H1 solution and 2 µL of 10× TAE-Mg were then added... 2+Add the buffer solution to 16 µL ddH2O, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain the DNA nanosphere-1 (DNS-1) solution. Add 2 µL of 10 µM FB-Chol-H2 solution and 2 µL of 10× TAE-Mg... 2+ Add the buffer solution to 16 µL ddH2O, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-2 (DNS-2) solution.

[0078] S3: Thoroughly mix 11.25 µL of 10 µM DNS-1 solution with 15 µL of 10 µM DNS-2 solution to obtain a dual DNA nanosphere probe (DDNS) solution. Thoroughly mix 11.25 µL of 10 µM H1 reaction product with 15 µL of 10 µM FB-H2 reaction product to obtain the H1+FB-H2 reaction product.

[0079] S4: After culturing MCF-7 or L02 cells in 24-well plates for 24 hours, the original culture medium was aspirated and the cells were washed three times with PBS buffer. Then, 26.25 µL of the above-mentioned DDNS solution, DNS-2 solution, H1+FB-H2 reaction product or FB-H2 reaction product was added, and the volume was made up to 300 μL with pure DMEM culture medium. After thorough mixing, the cells were incubated at 37°C for 4 hours, and finally, the cells were washed three times again with PBS buffer.

[0080] S5: After culturing MCF-7 or L02 cells in 24-well plates for 24 hours, the original culture medium was aspirated and the cells were washed three times with PBS buffer. Then, 26.25 µL of the above-mentioned DDNS solution, DNS-2 solution, H1+FB-H2 reaction product or FB-H2 reaction product was added, along with 2 μL of Lipo-8000. The volume was then brought up to 300 μL with pure DMEM culture medium. After thorough mixing, the cells were incubated at 37°C for 2 hours. Finally, the cells were washed three more times with PBS buffer.

[0081] S6: Acquire confocal laser scanning microscope images and collect fluorescence spectra between 500 and 600 nm using a Hitachi F-7000 spectrophotometer. Set the excitation wavelength to 492 nm, and set both the excitation and emission slits to 5.0 nm. Record the fluorescence intensity at λem = 520 nm.

[0082] Among them, MCF-7 cells expressed miR-224 at a relatively high level, while L02 cells expressed miR-224 at a basal low level; miR-224: 5'-CAAGUCACUAGUGGUUCCGUU-3'.

[0083] The results are as follows Figure 8As shown, the DDNS biosensing platform can sensitively and specifically identify cancer cells without transfection agents, achieving fluorescence imaging of intracellular target miRNAs. This demonstrates its significant advantages as a cancer diagnostic tool: high cost-effectiveness and ease of operation.

[0084] Example 9: The biocompatibility of the DDNS biosensing platform and the Lip-DDNS biosensing platform was compared as follows: S1: Dilute DNA single-stranded probe Chol-H1 and DNA single-stranded probe FB-Chol-H2 with 1× TE buffer to obtain Chol-H1 solution and FB-Chol-H2 solution with a concentration of 10 μM.

[0085] S2: Add 2 µL of 10 µM Chol-H1 solution and 2 µL of 10× TAE-Mg 2+ Add buffer to 16 µL ddH2O, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-1 (DNS-1) solution. Add 2 µL 10 µM FB-Chol-H2 solution and 2 µL 10× TAE-Mg... 2+ Add the buffer solution to 16 µL ddH2O, mix thoroughly, heat at 90°C for 5 minutes, and then cool naturally to room temperature to obtain DNA nanosphere-2 (DNS-2) solution.

[0086] S3: Mix 3.75 μL of 10 μM DNS-1 solution with 5 μL of 10 μM DNS-2 solution, add ddH2O to a final volume of 10 μL and mix thoroughly to obtain the dual DNA nanosphere probe (DDNS) solution. Alternatively, mix 3.75 μL of 10 μM DNS-1 solution with 5 μL of 10 μM DNS-2 solution, add 0.35 μL of Lipo-8000, add ddH2O to a final volume of 10 μL and mix thoroughly to obtain the Lip-DDNS solution.

[0087] S4: After culturing MCF-7 and L02 cells in 96-well plates for 24 hours, the cells were washed three times with PBS buffer. Then, 10 μL of the above-mentioned DDNS solution or Lip-DDNS solution was mixed with 90 μL of DMEM medium and added to the wells. The cells were incubated at 37°C for 4 hours and washed again with PBS. Finally, a fresh solution prepared by mixing 10 μL of CCK-8 reagent with 90 μL of DMEM was added and the cells were incubated at 37°C in the dark for 2 hours. After incubation, the absorbance at 450 nm of each well was measured using a nanoscale microplate reader (TecanInfinite M200 PRO, Austria).

[0088] The results are as follows Figure 9 As shown. Compared to Lip-DDNS, DDNS has superior biocompatibility.

[0089] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A dual DNA nanoball probe, characterized in that: The double DNA nanosphere probe comprises a DNA nanosphere-1 and a DNA nanosphere-2; the DNA nanosphere-1 is self-assembled by amphiphilic DNA single-stranded probe Chol-H1 through hydrophobic interaction, and the DNA nanosphere-2 is self-assembled by amphiphilic DNA single-stranded probe FB-Chol-H2 through hydrophobic interaction; the Chol-H1 and the FB-Chol-H2 both have hairpin structures, and the 5' end of each is modified with cholesterol; the two ends of the hairpin structure of the FB-Chol-H2 are modified with a fluorescent group and a quenching group, respectively.

2. The dual DNA nanoball probe of claim 1, wherein: The sequence of the Chol-H1 is 5'-Cholesterol-CATCGACTGGTCGCGTTTTCTAAACGGAACCACTAGTGACTTGAGATGTGTAGCACAAGTCACTAGTGGT-3'; and the sequence of the FB-Chol-H2 is 5'-Cholesterol-CTCGGAGAAGAGATGTTTTTGTGACTTG / i6FAMdT / GCTACACATCTCAAGTCACTAGTGGTAGATGTGTAGCA-BHQ1-3'.

3. The method for preparing the dual DNA nanosphere probe as described in claim 1, characterized in that: The preparation method comprises the following steps: diluting Chol-H1 and FB-Chol-H2 respectively by using 1× TE buffer to obtain Chol-H1 solution and FB-Chol-H2 solution; mixing the Chol-H1 solution, 10× TAE-Mg 2+ buffer and ddH2O, heating at 90°C for 5 minutes, and then naturally cooling to room temperature to obtain DNA nanosphere-1 solution; mixing the FB-Chol-H2 solution, 10× TAE-Mg 2+ buffer and ddH2O, heating at 90°C for 5 minutes, and then naturally cooling to room temperature to obtain DNA nanosphere-2 solution; and mixing the DNA nanosphere-1 solution and the DNA nanosphere-2 solution to obtain a double-DNA nanosphere probe solution.

4. Application of the double DNA nanosphere probe of claim 1 in preparation of a biosensing platform and preparation of a cell imaging reagent.

5. A biosensing platform characterized by: The biosensing platform comprises the double DNA nanosphere probe of claim 1.

6. The method of claim 5, wherein: The construction method comprises the step of co-incubating the double DNA nanosphere probe with the target miRNA or the DNA mimic thereof.

7. The method of construction of claim 6, wherein: In the co-incubation process, the target miRNA or the DNA mimic thereof stimulates the double DNA nanosphere probe to self-assemble under the joint driving of hydrophobic interaction and DNA hybridization, to form a super-large DNA nanosphere, accompanied by signal amplification, thereby realizing detection of the target miRNA or the DNA mimic thereof.

8. Application of the biosensing platform of claim 5 in detection of the miRNA or the DNA mimic thereof for non-disease diagnosis and treatment purposes.

9. A cellular imaging reagent, characterized by: The cell imaging reagent comprises the double DNA nanosphere probe of claim 1.

10. The cellular imaging reagent of claim 9, wherein: The cell imaging reagent is used for fluorescence imaging of intracellular target miRNA.

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