Preparation method of DNA (deoxyribonucleic acid) functionalized nanosphere, selective recognition of fluorescent probe and application of DNA functionalized nanosphere
Through the protein scaffolding, the protein scaffold DNA-Cu2+ composite nanospheres were prepared by room temperature in one-step, solving the problems of complex preparation process and harsh conditions in the existing DNA nanomaterials, and achieving multifunctional integration and selective recognition.
Patent Information
- Application Number
- CN202411894604.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-28
AI Technical Summary
During the preparation process, existing DNA nanomaterials rely on a large number of DNA strands and a variety of enzymes, requiring complex and time-consuming operation steps and precision temperature control technology, and the synthesis conditions are harsh, which limits their wide application.
The protein scaffold was used to regulate the spatial distribution of DNA hairpins, and a protein scaffold cross-linked DNA network was formed through the streptavidin-biotin system and aptamers. Combined with the coordination between Cu2+ and DNA bases, the protein scaffold DNA-Cu2+ complex nanospheres were constructed at room temperature in one-step.
It realizes nanosphere preparation without complex operational steps and does not rely on a large number of DNA strands and multiple enzymes. The synthesis conditions are mild, and multifunctional integration of target object recognition, signal amplification, transduction and output can be achieved.
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Figure CN119709953A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological detection, and specifically relates to a preparation method of a DNA functionalized nanosphere and the selective recognition and application of a fluorescent probe. Background Art
[0002] In recent years, a large number of DNA nanomaterials have been developed and applied in the fields of biosensing. Using a bottom-up self-assembly method, various DNA nanostructures such as DNA tetrahedrons, DNA tiles and DNA origami can be prepared with DNA as a building block. These nanostructures can be used as DNA scaffolds for the spatial arrangement and functional manipulation of functional elements such as aptamers, peptides and nanoparticles. Among them, composite DNA functional nanomaterials have attracted much attention due to their excellent functional programmability and integration. The nanomaterials can be prepared by rolling circle amplification (RCA), protein-DNA nanotechnology and metal-DNA coordination, and can be used as sensing elements for detecting a variety of biomarkers from ions to cells. In addition, a variety of molecular recognition elements, nucleic acid amplification technology and signal labeling technology can be organically integrated into composite DNA nanomaterials to develop powerful modular sensing elements.
[0003] However, there are still some limiting factors that restrict their further widespread application, including the reliance on large amounts of DNA chains and multiple enzymes during the preparation process, the need for multi-step precision temperature control and purification technology, complex and time-consuming operation steps, and harsh synthesis conditions.
[0004] Protein-DNA nanotechnology and metal-DNA coordination have played an important role in the preparation of composite DNA nanomaterials in recent years. The former combines these two biomacromolecules synergistically to develop composite nanostructures with unique properties. However, most composite nanostructures use DNA as a scaffold and focus on the functional role and functional regulation of proteins. Therefore, the use of proteins as scaffolds to spatially orient DNA and construct new composite nanostructures still needs further exploration. In addition, metal-DNA coordination is also a powerful technology to promote the development of composite DNA materials, but the preparation steps are complicated and time-consuming, and the harsh synthesis conditions limit further widespread application. Increasing the length of DNA can improve the synthesis efficiency and stability of composite metal DNA nanomaterials, and it is expected to achieve the synthesis of the composite material under mild conditions and promote the widespread application of materials. The increase in DNA length can be achieved through hybridization chain reaction (HCR). At present, a variety of DNA nanostructures such as DNA tetrahedrons have been developed as scaffolds to regulate the spatial distribution and spacing of DNA hairpins, and increase local concentration to promote the reaction kinetics and amplification efficiency of HCR. However, whether protein scaffolds can also be used to regulate DNA hybridization reactions and improve amplification efficiency remains to be explored. Summary of the invention
[0005] In order to solve the above problems existing in the prior art, the present invention provides a method for preparing DNA functionalized nanospheres and the selective recognition and application of fluorescent probes.
[0006] The present invention first provides a one-step method for constructing a protein scaffold DNA-Cu at room temperature. 2+ The method of the present invention uses the streptavidin-biotin system as a protein scaffold to regulate the spatial distribution of DNA hairpins on the basis of traditional HCR, and uses the aptamer as a triggering chain to form a protein scaffold cross-linked DNA network. 2+ The protein scaffold DNA-Cu is formed by the coordination with the complementary bases of DNA 2+ Functional nanospheres. The composite nanospheres organically integrate molecular recognition elements (aptamers), nucleic acid amplification technology (hybridization chain reaction) and metal ions (Cu 2+ ), achieving multifunctional integration of target recognition, signal amplification, transduction and output. Secondly, the present invention also studies the effects of various fluorescent probes such as cadmium telluride (CdTe) quantum dots (QDs) on free Cu 2+ And the selective recognition phenomenon of the composite nanospheres.
[0007] The technical solution adopted by the present invention is:
[0008] A method for preparing DNA functionalized nanospheres comprises the following steps:
[0009] (1) Biotinylated DNA hairpin binds to streptavidin to form a cross-shaped protein scaffold-DNA tetramer;
[0010] (2) Adding an aptamer, metal ions and a buffer to the cross-shaped protein scaffold-DNA tetramer, and incubating the reaction to obtain a DNA-functionalized nanosphere.
[0011] In step (1), the biotinylated DNA hairpin includes biotin-H1, biotin-H2, biotin-H3 and biotin-H4;
[0012] The cross-shaped protein scaffold-DNA tetramer consists of streptavidin-biotin-H1, streptavidin-biotin-H2, streptavidin-biotin-H3 and streptavidin-biotin-H4.
[0013] The nucleotide sequences of the four hairpins H1, H2, H3 and H4 in the biotinylated DNA hairpin are shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4 respectively.
[0014] In step (2), the aptamer is a cortisol aptamer, and the nucleotide sequence of the cortisol aptamer is shown in SEQ ID No.5.
[0015] The metal ion is Cu 2+ .
[0016] The buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.
[0017] The incubation reaction is carried out at room temperature for 2-6 hours.
[0018] The DNA functionalized nanosphere is a protein scaffold DNA-Cu 2+ Composite nanospheres.
[0019] The DNA functionalized nanosphere is used for selectively identifying fluorescent probes.
[0020] The fluorescent probes are QDs, calcein, Cu 2+ -Any of the probes.
[0021] The beneficial effects of the present invention are:
[0022] The present invention provides a protein scaffold DNA-Cu 2+ The preparation method of composite nanospheres is achieved by integrating suitable ligands, protein scaffolds, hybridization chain reactions and DNA and Cu 2+ Interaction, achieving room temperature one-step construction of protein scaffold DNA-Cu 2+ Composite nanospheres. The protein scaffold DNA-Cu 2+ The preparation method of the composite nanospheres is simple and easy, does not require complex and time-consuming operation steps, does not rely on a large number of DNA chains and multiple enzymes, does not involve precise temperature control instruments and purification technology, and has mild synthesis conditions. The composite nanospheres can achieve multifunctional integration of target recognition, signal amplification, transduction and output. A variety of fluorescent probes such as QDs, calcein and Cu 2+ -probes and other fluorescent probes can selectively identify the protein scaffold DNA-Cu 2+ Nanospheres and free Cu 2+ The protein scaffold DNA-Cu 2+ The multifunctional integration and selective recognition of nanospheres lay a favorable foundation for subsequent one-step fluorescent or electrochemical biosensors. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] 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.
[0024] Figure 1 The protein scaffold DNA-Cu 2+ Schematic diagram of the preparation strategy of composite nanospheres;
[0025] Figure 2 Display protein scaffold DNA-Cu 2+ Characterization of nanospheres and verification of the selective recognition phenomenon of fluorescent probes; (a) is the fluorescence probe for nanospheres and Cu 2+ Schematic diagram of selective recognition; (b)-(d) show that the introduction of protein scaffolds regulates the transformation of traditional hybridization chain reaction products from strips to networks under the influence of Cu 2+ Driven by the coordination effect with DNA base pairs, they further self-assemble to form nanospheres; (e) characterization of the synthesized quantum dots CdTe QDs using transmission electron microscopy; (f) Cu 2+ The cation exchange reaction with QDs leads to the quenching of QDs fluorescence, and obvious agglomeration is observed under TEM microscope; (g) The formation of nanospheres can significantly inhibit the 2+ The QDs agglomeration phenomenon caused by Cu 2+ The concentration of Cu was gradually decreased with the increase of the concentration; (i), (j), and (l) are the reaction products of aptamer, four DNA hairpins modified with biotin, or the mixture of the two for conventional HCR, which did not significantly affect the 2+ The fluorescence quenching of QDs; (k) Excess SA can significantly adsorb Cu 2+ , resulting in a significant increase in the fluorescence signal of QDs; (m) is only when the streptavidin-biotin protein scaffold regulates the HCR reaction and Cu 2+ After synergistically promoting DNA self-assembly to form nanospheres, the fluorescence of QDs gradually increased with the increase of trigger chain concentration in the HCR reaction; (n)-(o) Dynamic light scattering (DLS) and Zeta potential analysis prove that the protein scaffold and Cu 2+ Synergistically promote DNA self-assembly to form nanospheres;
[0026] Figure 3 The versatility of the selective recognition of fluorescent probes is shown in Figure 1. (a) is the characterization of synthesized quantum dots CdTe QDs using ultraviolet absorption spectroscopy; (b) is Cu 2+It can complex with organic dye calcein, causing its characteristic UV absorption peak to red-shift; (c) is the synthesized Cu 2+ -Characteristic UV absorption spectrum of the probe; (d) in Cu 2+ After synergistically promoting the formation of nanospheres with the protein scaffold, the Cu 2+ The fluorescence of calcein gradually increases after complexation with calcein; (e) The fluorescent molecule is replaced by Cu 2+ -probes to further verify the selective recognition phenomenon. DETAILED DESCRIPTION
[0027] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.
[0028] The protein scaffold DNA-Cu 2+ The reaction mechanism of the one-step synthesis of composite nanospheres is shown in the figure Figure 1 As shown. First, a specific biotinylated DNA hairpin (biotin-H1 to biotin-H4) is designed according to the aptamer sequence. Streptavidin (SA) binds to biotin with high affinity, and the DNA hairpin is anchored on SA to form a cross-shaped protein scaffold-DNA tetramer. In addition, the aptamer acts as a trigger chain to trigger a bidirectional cross-linked hybridization chain reaction (bi-cHCR) between DNA hairpins. Unlike general hybridization chain reactions, the present invention uses a protein scaffold to regulate the spatial distribution and spacing of DNA hairpins. Due to the increase in local DNA concentration and the higher probability of molecular collision in a spatially restricted environment, the reaction kinetics of the DNA chain displacement reaction is significantly enhanced, forming a spatially three-dimensional protein scaffold-DNA cross-linked network.
[0029] Finally, in Cu 2+ Self-assembly of protein scaffold DNA-Cu under coordination-driven effect mediated by DNA base pair interaction 2+ Nanospheres, the mechanism of action is as follows:
[0030]
[0031] The method described in the present invention cleverly combines functional nucleic acids, confinement theory and metal-DNA coordination effect, and finally prepares metal mineralized protein scaffold-DNA nanospheres in a one-step method at room temperature. The nanospheres can serve as powerful sensing elements to achieve multifunctional integration of target recognition, signal amplification, transduction and output.
[0032] When the target analyte is bound to the protein scaffold DNA-Cu 2+ When the nanospheres are incubated, the target induces conformational changes in the aptamers in the nanospheres. The aptamers fold into a specific secondary structure and bind to the target with high affinity. As the target concentration increases, a large number of aptamers in the nanospheres bind to the target, causing the nanospheres to gradually disaggregate and release a large amount of Cu 2+ The released Cu 2+ The electrochemical workstation can be used for direct testing, achieving one-stop testing of samples in and results out.
[0033] In addition, the released Cu 2+ It can be recognized by various fluorescent probes. Different detection modes can be achieved according to the choice of probes. For example, QDs and Cu 2+ The cation exchange reaction between calcein and Cu 2+ The complexation reaction between Cu and MgO achieves fluorescence quenching, thus realizing a turn off detection strategy. 2+ Reaction mechanism of quenching calcein fluorescence:
[0034]
[0035] In contrast, fluorescence enhancement by Cu 2+ -Probe and Cu 2+ The hydrolysis reaction between Cu and Mg realizes the turn-on detection mode. 2+ -Probe synthesis and response reaction mechanism; wherein (a) is Cu 2+ -probe synthesis reaction formula; (b) Cu 2+ Cu 2+ -Hydrolysis reaction equation of the probe.
[0036]
[0037] Oligonucleotides of different sequences were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China), and the sequences are shown in Table 1. Copper sulfate pentahydrate (CuSO4·5H2O), sodium tellurite (Na2TeO3), and 3-mercaptopropionic acid (MPA) were ordered from Aladdin Reagents (Shanghai, China). Sodium chloride (NaCl), trisodium citrate dihydrate (Na3C6H5O7·2H2O), cadmium chloride (CdCl2), potassium borohydride (KBH4), hydrochloric acid (HCl), sodium hydroxide (NaOH), and magnesium chloride (MgCl2) were purchased from Cologne Chemical Reagents (Chengdu, China). 3-(N-morpholino)propanesulfonic acid (MOPS) was purchased from Solarbio Technology Co., Ltd. (Beijing, China). DNAMarker (50-1031 bp) and 4SGelred were purchased from BBI Co., Ltd. (Shanghai, China). 6X DNA loading dye was ordered from Thermo Fisher Scientific (China) Co., Ltd. All reagents used in the present invention were analytical grade or higher and were used without further purification. All experimental water was prepared using a water purification system from Chengdu Ultrapure Technology Co., Ltd. (Chengdu, China) with a resistivity of 18.25 MΩ·cm. All working solutions were prepared using MOPS buffer solution (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4). All solutions to be used were stored at 4 °C.
[0038] Table 1 - Oligonucleotide sequences of aptamers and four DNA hairpins
[0039]
[0040] Example 1
[0041] This embodiment provides a protein scaffold DNA-Cu 2+ The room temperature one-step synthesis method of nanospheres is as follows:
[0042] 10 μM Cortisol aptamer (5 μL), 10 μM streptavidin-biotin-H1 to H4 (5 μL) and 50 μM Cu 2+ The precipitate (10 μL) was mixed with 70 μL 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4). The mixture was then incubated at room temperature for 2 hours.
[0043] Embodiment 2-4
[0044] The protein scaffold DNA-Cu provided in this embodiment 2+ A one-step room-temperature preparation strategy for nanospheres allows for tunable DNA, Cu 2+The dosage and reaction time can be used to obtain nanospheres of different sizes and properties. The specific operation is as follows:
[0045] 15 μM Cortisol aptamer (5 μL), 10 μM streptavidin-biotin-H1 to H4 (5 μL) and 40 μM Cu 2+ The precipitate (10 μL) was mixed with 70 μL of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4). The mixture was then incubated at room temperature for 4 hours.
[0046] Example 3
[0047] This example provides another room temperature one-step method for preparing protein scaffold DNA-Cu 2+ The nanosphere method was as follows: 20 μM cortisol aptamer (5 μL), 15 μM streptavidin-biotin-H1 to H4 (5 μL) and 65 μM Cu 2+ The precipitate (10 μL) was mixed with 70 μL of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4) and the mixture was then incubated at room temperature for 6 hours.
[0048] Experimental example
[0049] Protein Scaffold DNA-Cu 2+ Characterization of nanospheres and verification of selective recognition Figure 2 shown.
[0050] Protein Scaffold DNA-Cu 2+ Self-assembly process of nanospheres and fluorescence probes for nanospheres and Cu 2+ Selective recognition, such as Figure 2 a. The traditional HCR product is in the shape of a strip, but with the introduction of the streptavidin-biotin scaffold, the shape changes to a mesh. 2+ The addition of self-assembly formed nanospheres by coordinating with DNA base pairs ( Figure 2 bd). Further study of protein scaffolds and Cu 2+ Role in the self-assembly of DNA nanospheres. Zeta potential analysis of the changes in particle size distribution and surface charge during the assembly of nanospheres using dynamic light scattering (DLS). 2+ The gradual addition of reduced the surface charge and increased the size, which means that the protein scaffold and Cu 2+ Can synergistically promote DNA self-assembly ( Figure 2 no)
[0051] First, CdTe QDs were selected as the representative of luminescent nanomaterials to study the fluorescence probe on Cu 2+ and selective recognition of nanospheres. CdTe QDs were prepared using a conventional method previously reported. First, a 50 mL solution containing CdCl2 (0.5 mM) and trisodium citrate (0.2 g) was prepared. Then, mercaptopropionic acid (MPA, 52 μL) was immediately added to the above solution, and the pH of the solution was adjusted to 10.5 with NaOH. Subsequently, Na2TeO3 (0.1 mM) and KBH4 (50 mg) were added to the above solution, and CdTeQDs were refluxed in batches. Subsequently, high-purity CdTe QDs were obtained by precipitation with n-propanol and centrifugation (11000 rpm). The purified red CdTe QDs were redispersed in ultrapure water before use.
[0052] The synthesized quantum dots were characterized by UV absorption spectroscopy and transmission electron microscopy (TEM). The results showed that the UV characteristic absorption peak was located at 570nm ( Figure 3 a), quantum dots are uniformly distributed and have an average particle size of 5 nm ( Figure 2 e) Cu 2+ It can undergo cation exchange reaction with QDs, thus leading to the quenching of QDs fluorescence. Obvious agglomeration phenomenon was observed under TEM microscope ( Figure 2 f), and the fluorescence of QDs increases with the Cu 2+ The concentration gradually decreases ( Figure 2 h). Next, the protein scaffold DNA-Cu 2+ The various components of the nanospheres have an impact on the Cu 2+ The results showed that the reaction products of aptamers, four biotin-modified DNA hairpins, or a mixture of the two for conventional HCR did not significantly affect the Cu 2+ Fluorescence quenching of quantum dots ( Figure 2 i, j and l). It is worth noting that excess SA can significantly adsorb Cu 2+ , resulting in a significant increase in the fluorescence signal of QDs ( Figure 2 k). When preparing nanospheres, the SA concentration was 2.5 μM to avoid interference from excessive SA. It can be found that only when the streptavidin-biotin protein scaffold regulates the HCR reaction and Cu 2+ After synergistically promoting DNA self-assembly to form nanospheres, the fluorescence of QDs gradually increased with the increase of the trigger chain concentration of the HCR reaction ( Figure 2 m). This result is further supported by TEM microscopy, where it can be seen that the formation of nanospheres can significantly inhibit the 2+ The agglomeration phenomenon caused by Figure 2g). The above results show that protein scaffold and Cu 2+ In the process of promoting DNA self-assembly to form nanospheres, QDs can selectively recognize protein scaffolds DNA-Cu 2+ Nanospheres and Cu 2+ This can lay the foundation for the future use of the nanospheres for rapid fluorescence sensing detection of disease biomarkers.
[0053] To further demonstrate the selective recognition phenomenon, Cu 2+ Other fluorescent probes that responded confirmed the universality of this phenomenon. First, we selected the commonly used organic dye calcein. The ultraviolet absorption spectrum showed that the characteristic absorption peak of calcein was around 440nm. 2+ It can complex calcein to cause fluorescence quenching, and the characteristic peak of ultraviolet absorption is also found to be red-shifted by about 50nm ( Figure 3 b) In Cu 2+ After synergistically promoting the formation of nanospheres with the protein scaffold, as the concentration of the trigger chain increases, more nanospheres are formed and the adsorption of Cu 2+ The more, the more Cu 2+ The fluorescence of calcein gradually increases after quenching of calcein ( Figure 3 d) Replace the fluorescent molecule with Cu 2+ -probe further verified the selective recognition phenomenon and found that Cu 2+ -The probe has a characteristic UV absorption peak at 340nm ( Figure 3 c) After the nanospheres are formed, the free Cu 2+ Reduction, hydrolysis of Cu 2+ -The amount of fluorophores generated by the probe also decreases accordingly, and the fluorescence gradually decreases as the concentration of the trigger chain increases ( Figure 3 e). The present invention selects Cu 2+ Multiple fluorescent probes with different reaction mechanisms were verified multiple times. The above data illustrate the protein scaffold DNA-Cu 2+ The reliability and robustness of the selective recognition of nanospheres by fluorescent probes strongly support the development of related fluorescence sensing strategies in the future.
[0054] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.
Claims
1. A method for preparing DNA functionalized nanospheres, characterized in that: The steps include: (1) Biotinylated DNA hairpin binds to streptavidin to form a cross-shaped protein scaffold-DNA tetramer; (2) Adding an aptamer, metal ions and a buffer to the cross-shaped protein scaffold-DNA tetramer, and incubating the reaction to obtain a DNA-functionalized nanosphere.
2. The preparation method according to claim 1, characterized in that: In step (1), the biotinylated DNA hairpin includes biotin-H1, biotin-H2, biotin-H3 and biotin-H4; The cross-shaped protein scaffold-DNA tetramer consists of streptavidin-biotin-H1, streptavidin-biotin-H2, streptavidin-biotin-H3 and streptavidin-biotin-H4.
3. The preparation method according to claim 2, characterized in that: The nucleotide sequences of the four hairpins H1, H2, H3 and H4 in the biotinylated DNA hairpin are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively.
4. The preparation method according to claim 1, characterized in that: In step (2), the aptamer is a cortisol aptamer, and the nucleotide sequence of the cortisol aptamer is shown in SEQ ID No.
5.
5. The preparation method according to claim 1, characterized in that: The metal ion is Cu 2+ .
6. The preparation method according to claim 1, characterized in that: The buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.
7. The preparation method according to claim 1, characterized in that: The incubation reaction is carried out at 20-28° C. for 2-6 hours.
8. The DNA functionalized nanosphere prepared by the method according to any one of claims 1 to 7, characterized in that: The DNA functionalized nanosphere is a protein scaffold DNA-Cu 2+ Composite nanospheres.
9. Use of the DNA functionalized nanospheres prepared according to the method of any one of claims 1 to 7 and the DNA functionalized nanospheres according to claim 8 in selective recognition of fluorescent probes.
10. The use according to claim 9, characterized in that: The fluorescent probes are QDs, calcein, Cu 2+ -Any of the probes.