Water-phase high-brightness luminous cluster-DNA compound as well as preparation method and application thereof

Abstract

The invention discloses a water-phase high-brightness luminous cluster-DNA compound and a preparation method and application thereof, and the preparation method comprises the following steps: A1, carrying out mixed incubation on single-stranded DNA with different lengths and different base sequences and hydrophobic metal nanoclusters, analyzing the product through agarose gel electrophoresis, and screening out the optimal single-stranded DNA; a2, hydrophobic metal nanoclusters and the single-stranded DNA screened in the step A1 are mixed according to a certain proportion, the hydrophobic metal nanoclusters are mediated by the single-stranded DNA to be transferred from an organic phase to a water solution phase, and the cluster-DNA compound which is good in water phase dispersity and high in brightness light emitting can be prepared. The luminous cluster-DNA compound prepared according to the invention has the advantages of good water dispersibility, small and controllable size, high luminous efficiency, strong light stability and good biocompatibility, has significant advantages in living cell imaging, and provides a new tool for biological imaging.

Description

Technical Field

[0001] The present invention relates to the field of bionanotechnology, and particularly to a cluster-DNA complex with high brightness luminescence in aqueous phase, and a preparation method and application thereof. Background Art

[0002] As a novel structure between molecules and nanoparticles, the optical properties of atomically precise metal nanoclusters can be regulated by the atomic arrangement, composition and surface ligand chemistry within the clusters, as well as the molecular motions of the inner and interfacial ligands. The precise atomic arrangement within the cluster structure and the interaction between the ligand and the metal core provide a basis for studying the relationship between the cluster structure and its photophysical properties. Currently, luminescent metal nanoclusters have a long-range spectral tunability in the visible to near-infrared range (500-1700 nm), making them widely used in fields such as bioimaging, detection and analysis, and photothermal therapy. However, compared with traditional organic dyes, fluorescent proteins, and quantum dots, the luminescence ability of metal nanoclusters in the solution state is relatively weak (usually PLQY < 10%), which severely limits their applications. Therefore, it is of great significance to develop water-dispersible and highly luminescent metal nanoclusters.

[0003] Atomically precise metal nanoclusters have a core-shell structure, mainly composed of an internal metal core and peripheral ligands. Previous studies have confirmed that restricting the intramolecular and intermolecular motions (RIM) related to the structural vibrations and rotations of metal-ligand motifs can achieve strong fluorescence emission of the clusters. The activation of the RIM process can reduce the energy loss of the photoexcited state caused by non-radiative relaxation, thereby increasing the energy released through radiative transitions. Many metal nanoclusters exhibit crystallization-induced emission enhancement or aggregation-induced emission enhancement effects, specifically manifested as problems such as greatly weakened or quenched luminescence when the clusters are completely dissolved in the solvent. The aggregation effect reduces the non-radiative transitions of the excited-state clusters by suppressing the molecular motions at the interface. However, it is difficult to control the particle size distribution during the aggregation process. Therefore, it is of great significance to develop monodisperse and strongly luminescent clusters in aqueous solution. Therefore, there has been no report on the research of regulating the luminescence behavior of metal nanoclusters through a universal and effective post-synthesis strategy for constructing water-dispersible and highly luminescent metal nanoclusters.

[0004] Therefore, there is still a lack of a method in the art to construct metal nanoclusters with good water dispersion and high brightness luminescence, which is of great significance for broadening the application scope of luminescent metal nanoclusters. Summary of the Invention

[0005] The object of the present invention is to provide a cluster-DNA complex with high brightness luminescence in aqueous phase, and a preparation method and application thereof, so as to solve the problem that the prior art still lacks a method for constructing metal nanoclusters with good water dispersion and high brightness luminescence.

[0006] To solve the above problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, there is provided a method for preparing a cluster-DNA complex with high brightness luminescence in an aqueous phase, comprising the following steps: A1: Mixing and incubating single-stranded DNA with different lengths and different base sequences with hydrophobic metal nanoclusters, and analyzing the obtained product by agarose gel electrophoresis. Taking obtaining a uniform band as a standard, the optimal single-stranded DNA is screened out; A2: Mixing the hydrophobic metal nanoclusters with the single-stranded DNA screened in step A1 in a certain ratio, and transferring the hydrophobic metal nanoclusters from the organic phase to the aqueous phase through the mediation of the single-stranded DNA, thereby obtaining a cluster-DNA complex with good water dispersibility and high brightness luminescence.

[0008] According to a preferred embodiment of the present invention, step A1 includes: screening the interaction between single-stranded DNA with different lengths (T20, T30, T40, T50, T60, and T80 respectively) and different base sequences (A40, T40, and C40 respectively) and the hydrophobic clusters.

[0009] It should be understood that, depending on the different metal nanoclusters selected, the optimal single-stranded DNA sequence needs to be determined according to the results of gel electrophoresis, and the screening of DNA sequences by different clusters will vary; precisely because different clusters may bind to single-stranded DNA with different sequences, this screening step A1 is an indispensable step in the present invention.

[0010] Preferably, in step A2, the metal nanoclusters are selected from: Cu2Au2(R-C 10 H 10 S2N)4, Cu2Au2(S-C 10 H 10 S2N)4, Au8(C 21 H 28 O2)8, Ag6(S-C6H 10 S2N)6, Au4(S-C6H 10 S2N)4, Au4(S-C9H9S2N)4, Ag6(R-C9H9S2N)6, Ag6(S-C9H9S2N)6. It should be understood that these metal nanoclusters preferably selected in the present invention belong to a class of typical clusters with aggregation-induced emission effects carefully selected by the inventor, and are particularly suitable for studying the optical properties in the solution state in the later stage.

[0011] Preferably, in step A2, the molar ratio of the metal nanoclusters to the single-stranded DNA is 32:1 to 4:1. More preferably, it is 8:1.

[0012] Preferably, step A2 further includes: analyzing the obtained product by agarose gel electrophoresis to screen out the optimal ratio of metal nanoclusters to single-stranded DNA.

[0013] Preferably, in step A2, the single-stranded DNA and the hydrophobic metal nanoclusters are incubated overnight in a mixed solvent, and the solvent is replaced to obtain an aqueous-phase highly luminescent cluster-DNA complex. In the mixed solvent, the volume ratios of DMF, THF, and H2O are 64.51%, 25.81%, and 9.68% respectively.

[0014] Preferably, step A2 further includes: analyzing the obtained product by transmission electron microscopy to verify whether it exhibits a uniform size distribution.

[0015] According to the second aspect of the present invention, there is provided an aqueous-phase highly luminescent cluster-DNA complex prepared by the above preparation method.

[0016] According to the third aspect of the present invention, there is provided a method for enhancing the chirality selectivity of chiral metal cluster luminescence. The method includes: B1: Mixing and incubating single-stranded DNA with different lengths and different base sequences with hydrophobic metal nanoclusters, and analyzing the obtained product by agarose gel electrophoresis to screen out the optimal single-stranded DNA based on obtaining a single band; B2: Synthesizing a pair of chiral metal nanoclusters, mixing the chiral metal nanoclusters with the single-stranded DNA screened in step B1 in a certain ratio to obtain highly luminescent cluster-DNA complexes with different chiralities; B3: Comparing the emission intensity enhancement effect and chirality selectivity of the cluster-DNA complexes by comparing the photoluminescence spectral data.

[0017] According to a preferred embodiment of the present invention, the method is used to achieve chiral recognition of metal nanoclusters.

[0018] According to the fourth aspect of the present invention, there is provided a method for delivering water-dispersible luminescent metal nanoclusters into cells. The method includes: C1: Providing an aqueous-phase highly luminescent cluster-DNA complex as described above; C2: Mixing the cluster-DNA complex with test cells and incubating at 37 °C for 2 h; C3: Measuring the fluorescence emitted by the cluster-DNA complex under predetermined conditions and judging the cell delivery efficiency according to the fluorescence imaging results.

[0019] The present invention also provides the application of the luminescent cluster-DNA complex in cell delivery and imaging.

[0020] In a preferred embodiment, the application includes incubating the cluster-DNA complex with the cell sample to be tested at 37 °C and 5% CO2 for 2 h, and washing it twice with cell culture medium, and then the endocytosis efficiency of the sample can be detected using a laser confocal microscope. The cells are HeLa cells. Among them, the laser wavelength of the used laser confocal microscope is 405 nm.

[0021] According to the present invention, a method for preparing water-phase well-dispersed and highly bright luminescent metal nanoclusters based on amphiphilic single-stranded DNA as a template is provided, specifically involving regulating the optical behavior of water-phase metal clusters through the DNA interface confinement effect. Its working principle is that single-stranded DNA is mainly composed of hydrophilic phosphate backbones and hydrophobic bases, and it can firmly wrap the metal clusters in the hydrophobic confinement cavity through the multivalent cooperative interaction between the bases and the surface ligands of the clusters, while exposing the hydrophilic phosphate backbones outside. Therefore, the confinement cavity formed by single-stranded DNA and the interface ligands is expected to inhibit the intramolecular movement of the surface ligands of the metal clusters, thereby reducing the non-radiative energy dissipation rate, and further enhancing the luminescence efficiency of the metal clusters.

[0022] A cluster-DNA complex with high brightness luminescence in water phase, its preparation method and application provided by the present invention have the following beneficial effects compared with the prior art:

[0023] 1) A highly bright luminescent cluster-DNA complex is prepared according to the method of the present invention, which solves the problems of low luminescence efficiency of existing luminescent metal clusters, uncontrollable aggregate size, and limited application of hydrophobic clusters in aqueous solutions.

[0024] 2) The luminescent cluster-DNA complex prepared according to the present invention has the advantages of controllable size, high brightness, strong photostability, and good biocompatibility, and has significant advantages compared with existing nano-fluorescent probes, providing a new tool for biological imaging.

[0025] In summary, according to the present invention, a water-dispersible and highly bright luminescent metal nanocluster, its preparation method, and a method for realizing cell delivery and imaging through it are provided. The luminescent metal cluster prepared according to the present invention has the advantages of good water dispersibility, small and controllable size, high luminescence efficiency, strong photostability, and good biocompatibility, and has significant advantages in live cell imaging, providing a new tool for biological imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings without creative efforts based on these drawings.

[0027] Figure 1 It is a schematic diagram of the interaction between luminescent metal nanoclusters and single-stranded DNA;

[0028] Figure 2 It is the agarose gel electrophoresis result after the interaction of DNA sequences with different lengths and metal clusters;

[0029] Figure 3 It is the agarose gel electrophoresis result after the interaction of DNA sequences with different base types and metal clusters;

[0030] Figure 4 It is the gel electrophoresis result of the product prepared by mixing luminescent metal clusters and amphiphilic single-stranded DNA at different concentration ratios;

[0031] Figure 5 It is the agarose gel electrophoresis characterization result after the interaction between luminescent metal nanoclusters and single-stranded DNA;

[0032] Figure 6 It is the transmission electron microscopy characterization result after the interaction between luminescent metal nanoclusters and single-stranded DNA;

[0033] Figure 7 It is the photoluminescence spectrum result before and after the interaction between luminescent metal nanoclusters and single-stranded DNA;

[0034] Figure 8 It is the luminescence lifetime result before and after the interaction between luminescent metal nanoclusters and single-stranded DNA;

[0035] Figure 9 It is the molecular structure diagram of chiral luminescent silver clusters;

[0036] Figure 10 It is the molecular structure diagram of chiral luminescent copper-gold alloy clusters;

[0037] Figure 11 It is the statistical result diagram of the enhanced luminescence after the phase transition of chiral silver clusters mediated by single-stranded DNA;

[0038] Figure 12 It is the statistical result diagram of the enhanced luminescence after the phase transition of chiral copper-gold alloy clusters mediated by single-stranded DNA;

[0039] Figure 13 It is the fluorescence imaging result of aqueous-phase luminescent metal clusters and template-free aggregates for cell delivery. Specific implementation manners

[0040] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The raw materials and instruments used in the following embodiments are all commercially available; the equipment and preparation processes used are all conventional equipment and conventional processes unless otherwise specified.

[0041] Example 1

[0042] According to this embodiment, a preparation method of an aqueous high-brightness luminescent cluster-DNA complex constructed based on DNA nanotechnology is provided, including the following steps:

[0043] 1.1 Screening of single-stranded DNA. Single-stranded DNAs with different lengths (T20, T30, T40, T50, T60 and T80) and different base sequences (A40, T40 and C40) were used for experiments respectively. The specific DNA sequences are shown in Table 1 below.

[0044] Dissolve various DNA single strands in ultrapure water and quantify to 100 μM with a UV spectrophotometer. Incubate the luminescent metal cluster Cu2Au2(R-C 10 H 10 S2N)4 (dissolved in an organic solvent) and the amphiphilic single-stranded DNA solution overnight in a mixed solvent (where the volume ratios of DMF, THF and H2O are 64.51%, 25.81%, 9.68% respectively) and perform solvent replacement to obtain an aqueous high-brightness luminescent cluster-DNA complex (the interaction principle between the luminescent metal nanocluster and the single-stranded DNA is as Figure 1 shown). Analyze the obtained aqueous high-brightness luminescent cluster-DNA complex by agarose gel electrophoresis (running conditions: running at 100 V for 20 min) and transmission electron microscopy respectively.

[0045] Table 1 DNA sequences (SEQ ID No. 1-8)

[0046] Results: The agarose gel electrophoresis results after the interaction of DNA sequences with different lengths and base types with the metal cluster (as Figures 2 - 3 shown) indicate that T40 is the optimal base sequence corresponding to the alloy cluster Cu2Au2(R-C10H 10 S2N)4.

[0047] 1.2 Screening of concentration ratios. Incubate the luminescent metal cluster (dissolved in an organic solvent) and the amphiphilic single-stranded DNA solution overnight in a mixed solvent at different molar concentration ratios (32∶1; 16∶1; 8∶1; 4∶1) and perform solvent replacement to obtain an aqueous high-brightness luminescent cluster-DNA complex.

[0048] Results: The gel electrophoresis results of the products prepared with different concentration ratios (as Figure 4 ) showed that 8:1 was the optimal concentration ratio, and a relatively uniform band was obtained at this time.

[0049] 1.3 Preparation of an aqueous-phase highly luminescent cluster-DNA complex constructed based on DNA nanotechnology. The hydrophobic metal nanoclusters were mixed with the single-stranded DNA (T40) screened above at a molar concentration ratio of 8:1. Through the transfer of hydrophobic metal nanoclusters from the organic phase to the aqueous phase mediated by single-stranded DNA, a cluster-DNA complex with good water dispersibility and high luminescence can be prepared.

[0050] Compared with the formation of aggregates of naked clusters in the aqueous phase, the cluster-DNA complex showed relatively uniform bands, and the migration speed of this band was slower than that of free T40 ( Figure 5 ). The results of transmission electron microscopy further showed that the cluster-DNA had a uniform size distribution, and the particle diameter was 2.04 ± 0.42 nm ( Figure 6 ). The above results indicate that hydrophobic clusters can be efficiently transferred to the aqueous solution and have good dispersibility in the aqueous solution.

[0051] Example 2

[0052] Characterization of the optical properties of the luminescent cluster-DNA complex before and after phase transition, including the following steps:

[0053] Use a fluorescence spectrometer and a quantum yield spectrometer to measure the fluorescence intensity and quantum yield of the luminescent metal clusters before and after the reaction with single-stranded DNA (T40), and compare them with the cluster monomers.

[0054] Results: The results of photoluminescence spectra and quantum yields showed that for the alloy cluster Cu2Au2(R-C 10 H 10 S2N)4, the cluster-DNA complex was 1865 times higher than the cluster monomer ( Figure 7 ), and the quantum yield increased by 89 times. In addition, the luminescence lifetime realized the transition from fluorescence (the average fluorescence lifetime in the organic phase was 4.50 ns) to phosphorescence (the average fluorescence lifetime in the aqueous phase was 2.99 μs) compared with the cluster monomer ( Figure 8 ).

[0055] Among them, the luminescence quantum yields of the cluster-DNA complex and the cluster monomer are shown in Table 2 below.

[0056] Table 2 Luminescence quantum yields of cluster monomers and cluster-DNA complexes

[0057] Example 3

[0058] According to this embodiment, a method for enhancing the selectivity of chiral metal cluster luminescence is provided, including the following steps:

[0059] First, synthesize two pairs of different types of chiral metal nanoclusters: silver clusters Ag6(R-C9H9S2N)6 / Ag6(S-C9H9S2N)6( Figure 9 ) and copper-gold alloy clusters Cu2Au2(R-C 10 H 10 S2N)4 / Cu2Au2(S-C 10 H 10 S2N)4( Figure 10 ), and mix the chiral metal nanoclusters with the optimized single-stranded DNA (i.e., C40 and T40) at a molar ratio of 8:1. Comparative photoluminescence spectral data are used to compare the effects of enhanced emission intensity and chiral selectivity of the aqueous-phase clusters after confinement mediated by single-stranded DNA. It should be understood that the purpose of synthesizing two pairs of different types of chiral metal nanoclusters here is to preliminarily verify the universality of this enhanced chiral selectivity, and specific optical characterization shows that both pairs of chiral clusters have a better right-handed enhancement effect than the left-handed one.

[0060] Results: Comparative analysis of the luminescence properties with monodisperse clusters (dissolved in the organic phase) shows that the emission intensity of the aqueous-phase clusters after confinement mediated by single-stranded DNA is significantly enhanced. Relevant statistics show that the luminescence intensity increases by 18 times and 9 times Figure 11 ) and 1865 times and 305 times Figure 12 ) respectively compared to the organic phase. Thus, it can be obtained that single-stranded DNA can be used to identify the chirality of the clusters, showing that the luminescence enhancement effect of the metal clusters induced by the right-handed helical ligand is significantly better than that of the left-handed one. This phenomenon may be related to the chirality matching between DNA itself and the chirality of the clusters, resulting in different degrees of restricted movement of the surface ligands of different chiral clusters.

[0061] Example 4

[0062] According to this embodiment, an application of a luminescent cluster-DNA complex in cell delivery and imaging is provided, including the following steps:

[0063] In this embodiment, HeLa cells are used as the research object. Then, the samples (luminescent cluster-DNA complex and template-free aggregates, with a cluster concentration of 10 μM) are incubated with HeLa cells at 37 °C for 2 h, and washed twice with cell culture medium to remove free samples. Subsequently, fluorescence signals in the 405 nm channel of the cells are collected using a fluorescence confocal microscope.

[0064] Results: Fluorescence imaging results showed that DNA-mediated luminophore clusters-DNA complexes could be rapidly taken up by cells. The DNA-protected clusters Cu2Au2(R-C 10 H 10 S2N)4@T40 and Au4(S-C6H 10 S2N)4@C40( Figure 13 c and e in) showed relatively high internalization efficiency and maintained cell ductility similar to that of the blank control group( Figure 13 a in), while the template-free aggregates showed minimal cell uptake and significantly affected cell status, causing cell shrinkage( Figure 13 b and d in). This result indicates that the present invention can deliver luminescent metal clusters into cells, promoting the effectiveness and universality of the strategy for transporting hydrophobic clusters into cells.

[0065] The above are only the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. Any simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application shall fall within the scope of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for preparing a water-phase high-brightness luminescent cluster-DNA complex, characterized in that: The following steps are involved: A1: Mix and incubate single-stranded DNAs of different lengths and base sequences with hydrophobic metal nanoclusters, analyze the resulting products by agarose gel electrophoresis, and use a uniform band as the standard to screen out the best single-stranded DNA; A2: The hydrophobic metal nanoclusters are mixed with the single-stranded DNA screened in step A1 in a certain proportion, and the hydrophobic metal nanoclusters are transferred from the organic phase to the aqueous solution phase mediated by the single-stranded DNA, so as to prepare a high-brightness luminescent cluster-DNA complex with good water phase dispersibility.

2. The preparation method according to claim 1, characterized in that: The metal nanoclusters are selected from: Cu2Au2(RC 10 H 10 S2N)4, Cu2Au2(SC 10 H 10 S2N)4、Au8(C 21 H 28 O2)8、Ag6(S-C6H 10 S2N)6、Au4(S-C6H 10 Any one of Au(S-C9H9S2N)4, Au4(S-C9H9S2N)4, Ag6(R-C9H9S2N)6, and Ag6(S-C9H9S2N)6.

3. The preparation method according to claim 1, characterized in that: In step A2, the molar ratio of the metal nanoclusters to the single-stranded DNA is 32:1 to 4:

1.

4. The preparation method according to claim 3, characterized in that: Step A2 also includes: analyzing the obtained product by agarose gel electrophoresis to screen out the optimal ratio of metal nanoclusters to single-stranded DNA.

5. The preparation method according to claim 1, characterized in that: In step A2, single-stranded DNA and hydrophobic metal nanoclusters are incubated overnight in a mixed solvent and replaced with the solvent to obtain an aqueous high-brightness luminescent cluster-DNA complex, in which the volume ratios of DMF, THF, and H2O are 64.51%, 25.81%, and 9.68%, respectively.

6. The preparation method according to claim 1, characterized in that: Step A2 also includes: analyzing the obtained product by transmission electron microscopy to verify whether it presents a uniform size distribution.

7. An aqueous high-brightness luminescent cluster-DNA complex prepared according to the preparation method according to any one of claims 1 to 6.

8. A method for enhancing the selectivity of chiral metal cluster luminescence, characterized in that: The method comprises: B1: Mix and incubate single-stranded DNAs of different lengths and base sequences with hydrophobic metal nanoclusters, analyze the resulting products by agarose gel electrophoresis, and screen out the best single-stranded DNA by obtaining a uniform band as the standard; B2: synthesize a pair of chiral metal nanoclusters, mix the chiral metal nanoclusters with the single-stranded DNA screened in step B1 in a certain ratio, and obtain high-brightness luminescent cluster-DNA complexes of different chirality; B3: The emission intensity enhancement effect and chiral selectivity of the cluster-DNA complex can be compared by comparing the photoluminescence spectral data.

9. The method according to claim 8, characterized in that The method is used to realize chiral recognition of metal nanoclusters.

10. A method for delivering water-dispersible luminescent metal nanoclusters into cells, characterized in that: The method comprises: C1: providing an aqueous high-brightness luminescent cluster-DNA complex as described in claim 7; C2: the cluster-DNA complex was mixed with the test cells and incubated at 37°C for 2 h; C3: The fluorescence emitted by the cluster-DNA complex is measured under predetermined conditions, and the cell delivery efficiency is determined based on the fluorescence imaging results.

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