Modularized nanometer machine and preparation method and application thereof

Abstract

The invention relates to a modularized nano-machine, which comprises a single-stranded DNA, an organic dye and an inorganic nano-cluster, the organic dye is connected to the 3'end of the single-stranded DNA through chemical covalent bond modification, the inorganic nano-cluster is combined to the basic group of the single-stranded DNA through hydrophobic interaction, and pi-pi accumulation is formed between the inorganic nano-cluster and the organic dye. The invention also relates to a preparation method and application of the modularized nano machine. The modularized nanometer machine has the advantages of being high in modularization degree, high in expansibility, clear in energy conversion mechanism and design principle, adjustable in wavelength range and photo-thermal conversion efficiency and the like, has remarkable advantages in the aspect of customizing and constructing a photo-thermal conversion nanometer machine, and provides a new design principle for nanometer machine design.

Description

Technical Field

[0001] The present invention relates to bio-nanotechnology, and more particularly to a modular nano-machine and its preparation method and application. Background Art

[0002] Molecular machines at the nanoscale participate in and regulate energy conversion processes at the microscale. However, different forms of energy are suitable for different carriers, and a complete nano-machine needs to carry materials with different compositions and structures. For example, in photosynthesis, the most extensive and important energy conversion process in nature, photosynthesis-related proteins carry three components with completely different properties and functions, and each functional component cooperates with each other to precisely regulate the process of the flow of light energy into chemical energy in organic matter. In the past few decades, people have used various nanostructures to try to build energy conversion systems with different functions. Based on the advantages of different materials as carriers of different forms of energy, many different types of materials have been coupled with each other to achieve the function of energy regulation.

[0003] However, in the process of constructing artificial nano-energy conversion systems, two major challenges are faced: First, the problem of atomic-precision integration of cross-scale functional components: Traditional methods are difficult to achieve atomic-level precision when integrating functional components at different scales. For example, conventional nano-scale machines or devices usually use nanoparticles and polymer molecules as raw materials, which do not have atomic-level precision in the practical sense, significantly limiting the mechanism analysis of energy transfer and technological iteration. Second, the problem of synergistic regulation of chemical forces in existing systems: It is very difficult to precisely regulate covalent and non-covalent interactions simultaneously in the same system because there are significant differences in strength, reversibility, kinetics, thermodynamics, and regulation conditions among different interactions, resulting in competition and interference among various interactions. This complexity significantly limits the purposeful design of the interactions between components in the same system to play a functional role, resulting in low energy transfer efficiency and difficulty in refining general design principles. Summary of the Invention

[0004] In order to solve the problems such as the difficulty in achieving atomic-level precision in the above-mentioned prior art, the present invention provides a modular nano-machine and its preparation method and application.

[0005] The modular nano-machine according to the present invention includes single-stranded DNA, organic dyes, and inorganic nanoclusters. Among them, the organic dyes are covalently modified and connected to the 3'-end of the single-stranded DNA, the inorganic nanoclusters are bound to the bases of the single-stranded DNA through hydrophobic interactions, and π-π stacking is formed between the inorganic nanoclusters and the organic dyes.

[0006] In a preferred embodiment, the length of the single-stranded DNA is between 20 bp and 80 bp.

[0007] In a preferred embodiment, the organic dye has a planar rigid donor-π-electron-acceptor structure.

[0008] In a preferred embodiment, the organic dye is a cyanine fluorescent dye Cy3, Cy5, Cy7 or ICG.

[0009] In a preferred embodiment, the structure of the inorganic nanocluster is a metal core protected by an external hydrophobic ligand.

[0010] In a preferred embodiment, the inorganic nanocluster is a gold nanocluster Au2Cu2(C 10 H 10 S2N)4, Au4(C6H 10 S2N)4, Au 25 (SCH2CH2Ph) 18 , [Au8(dppp)4(TPA-C≡C)2](NO3)2 or [Au8(dppp)4(TPE-C≡C)2](NO3)2.

[0011] In a preferred embodiment, the molar ratio of the inorganic nanocluster to the single-stranded DNA is between 1:1 and 8:1.

[0012] According to the preparation method of the modular nanomachine of the present invention, the method includes the following steps: S1, chemically covalently modifying and connecting the organic dye to the 3'-end of the single-stranded DNA to form a single-stranded DNA labeled with the organic dye; S2, co-mixing the aqueous solution of the single-stranded DNA labeled with the organic dye and the organic phase solution of the inorganic nanocluster in an intermediate solvent, and shaking and mixing to form a homogeneous solution; S3, performing phase transfer treatment on the homogeneous solution to purify and separate the modular nanomachine.

[0013] According to the application of the modular nanomachine of the present invention, the photothermal conversion efficiency of the modular nanomachine is regulated by regulating the single-stranded DNA and / or the inorganic nanocluster.

[0014] In a preferred embodiment, a nanocluster beacon is provided by hybridization of the single-stranded DNA with a complementary strand.

[0015] According to the modular nanomachine of the present invention, its preparation method and application, it has the advantages of high modularity, strong scalability, clear energy conversion mechanism and design principle, adjustable wavelength range and photothermal conversion efficiency, etc., and has significant advantages in customizing the construction of photothermal conversion nanomachines, providing new design principles for nanomachine design. Brief Description of the Drawings

[0016] Figure 1 is a schematic structural diagram of the modular nanomachine according to the present invention.

[0017] Figure 2 Agarose gel electrophoresis characterization results of the modular nanomachine according to the present invention.

[0018] Figure 3 Transmission electron microscopy characterization results of the modular nanomachine according to the present invention.

[0019] Figure 4 UV-Vis absorption spectrum of an aqueous solution of the modular nanomachine according to the present invention.

[0020] Figure 5 UV-Vis absorption spectrum of a mixed solution of single-stranded DNA labeled with an organic dye and inorganic nanoclusters according to the present invention.

[0021] Figure 6 Shows the relative fluorescence intensity of the modular nanomachine with different inorganic nanocluster loading ratios according to the present invention.

[0022] Figure 7 Shows the fluorescence lifetime of the modular nanomachine with different inorganic nanocluster loading ratios according to the present invention.

[0023] Figure 8 Shows the fluorescence quantum yield of the modular nanomachine with different inorganic nanocluster loading ratios according to the present invention.

[0024] Figure 9 Shows the radiative and non-radiative transition rate constants of the modular nanomachine with different inorganic nanocluster loading ratios according to the present invention.

[0025] Figure 10 Is a comparison diagram of the excited-state dynamics of the modular nanomachine and single-stranded DNA labeled with an organic dye according to the present invention.

[0026] Figure 11 Is an all-atom molecular dynamics simulation diagram of the modular nanomachine according to the present invention.

[0027] Figure 12 Shows the regulation of the energy conversion efficiency of the modular nanomachine according to the present invention by the length of single-stranded DNA.

[0028] Figure 13 Shows the regulation of the energy conversion efficiency of the modular nanomachine according to the present invention by the type of inorganic nanoclusters.

[0029] Figure 14 Is a characterization diagram of a multicolor nanocluster beacon developed based on the modular nanomachine according to the present invention. Detailed implementation mode

[0030] The following will, in conjunction with the accompanying drawings, give the preferred embodiments of the present invention and describe them in detail.

[0031] The present invention is constructed based on single-stranded DNA, organic dyes, and inorganic nanoclusters. Modular nanomachine Among them, the organic dye is chemically covalently modified and connected to the 3′ end of the single-stranded DNA, the inorganic nanocluster is bound to the base of the single-stranded DNA through hydrophobic interaction (non-covalent interaction), and a π-π stacking (non-covalent interaction) is formed between the inorganic nanocluster and the organic dye.

[0032] The present invention replaces the nanoparticles and polymer molecules in the traditional method by introducing single-stranded DNA and inorganic nanoclusters with precise molecular structures, so as to achieve atomic precision, that is, to construct an atomically precise nanomachine. The advantage of this precision enables the present invention to accurately model and analyze the nanomachine, clarify the coupling interaction mechanism between each module at the atomic-molecular level, deeply study the energy transfer mechanism, regulate the wavelength and efficiency of the microscale photothermal conversion process, and provide general design principles. Specifically, there is an inherent energy flow process in the organic dye, that is: the organic dye molecule absorbs light energy, and a part of the energy is converted into the energy in the emitted fluorescence through the radiative transition pathway; another part of the energy is converted into heat in the system or environment through the non-radiative transition pathway. The present invention can regulate the ratio of the two parts of energy involved in the energy conversion process of the organic dye molecule by adjusting the length of the single-stranded DNA and the type of inorganic nanocluster. Specifically, the non-radiative transition of the organic dye is related to its vibrational energy level. Due to the π-π stacking interaction between the inorganic nanocluster and the organic dye, and this interaction only acts on one end of the organic dye due to spatial limitation, this asymmetric effect leads to the splitting of the vibrational energy level, thereby intensifying the non-radiative transition of the organic dye molecule. Moreover, by regulating the strength of the π-π interaction between the organic dye and the inorganic nanocluster through the single-stranded DNA with an adjustable length and precise structure, the ratio of the radiative / non-radiative transition energy of the organic dye can be regulated, and targeted design can be carried out, so as to refine general design principles.

[0033] There is an inherent photothermal conversion path in the organic dye. The ligand of the inorganic nanocluster perturbs the microenvironment of the organic dye through non-covalent interaction (π-π stacking), thereby realizing the controllability of the microscale photothermal conversion process. By adjusting the length of the single-stranded DNA, the types of organic dyes and inorganic nanoclusters, the absorption wavelength and photothermal conversion efficiency of the modular nanomachine can be regulated.

[0034] The flexible single-stranded DNA winds around the surface of the hydrophobic inorganic nanocluster, improving the stability of the inorganic nanocluster in aqueous solution while enriching the inorganic nanocluster in the nanomachine system. The organic dye acting as the energy input end is affected by the ligand of the inorganic nanocluster and changes its energy dissipation pathway.

[0035] Since single-stranded DNA has a relatively unified structure (a flexible molecular chain that is hydrophilic on one side and hydrophobic on the other side) and its length and sequence are controllable, organic dye molecules have a relatively unified structure (a planar rigid donor-π-electron-acceptor structure, i.e., D-π-A structure) and their wavelengths are adjustable, and inorganic nanoclusters have a relatively unified structure (a metal core protected by an external hydrophobic ligand) and their types are adjustable. The nanomachines of the present invention have the advantages of high modularity, strong scalability, clear energy conversion mechanisms and design principles, adjustable wavelength ranges and photothermal conversion efficiencies, etc. They have significant advantages in customizing the construction of photothermal conversion nanomachines and provide new design principles for the design of nanomachines.

[0036] The preparation method of the modular nanomachine according to the present invention first includes separately providing single-stranded DNA with an organic dye label and inorganic nanoclusters Among them, through conventional biochemical methods, organic dyes can be covalently modified and linked to the 3′ end of single-stranded DNA to form single-stranded DNA with organic dye labels.

[0037] In a preferred embodiment, the length of the single-stranded DNA is between 20 bp and 80 bp, such as 20 bp, 40 bp, 60 bp, 80 bp, etc. In a preferred embodiment, the DNA sequence of the single-stranded DNA is shown in Table 1 below. It should be understood that this length and specific sequence are only examples and not limitations, and the base sequence can be a randomized sequence.

[0038] Table 1

[0039] In a preferred embodiment, the organic dye is a cyanine fluorescent dye, such as Cy3, Cy5, Cy7, ICG, and its structural formula is shown in Table 2 below. It should be understood that this dye molecule is only an example and not a limitation.

[0040] Table 2

[0041] In a preferred embodiment, the inorganic nanocluster is a gold nanocluster, such as Au2Cu2(C 10 H 10 S2N)4, Au4(C6H 10 S2N)4, Au 25 (SCH2CH2Ph) 18 、[Au8(dppp)4(TPA-C≡C)2](NO3)2、[Au8(dppp)4(TPE-C≡C)2](NO3)2, and its structural formula is shown in Table 3 below. It should be understood that this gold nanocluster is only an example and not a limitation. Different ligands L of it have different interactions with organic dyes, so different regulation effects can be produced.

[0042] Table 3

[0043] The preparation method of the modular nanomachine according to the present invention next includes co-mixing an aqueous solution of single-stranded DNA with an organic dye label and an organic phase solution of inorganic nanoclusters in an intermediate solvent, shaking and mixing evenly to form a homogeneous solution solution Among them, the surface of the inorganic nanocluster is a hydrophobic group and can itself dissolve in the organic phase.

[0044] In a preferred embodiment, the organic phase is acetonitrile and / or tetrahydrofuran (THF).

[0045] In a preferred embodiment, the intermediate solvent is N,N-dimethylformamide solvent. It should be understood that this solvent is only an example and not a limitation, as long as the intermediate solvent can mix the aqueous solution and the organic phase solution system into one phase.

[0046] In a preferred embodiment, in a metal bath, a homogeneous solution is formed by constant temperature oscillation at 20 °C - 30 °C for more than 8 hours.

[0047] The preparation method of the modular nanomachine according to the present invention finally includes performing phase transfer treatment on the homogeneous solution to purify and separate the modular nanomachine Through phase transfer treatment, the inorganic nanocluster binds to the base of single-stranded DNA through hydrophobic interaction.

[0048] In a preferred embodiment, the phase transfer treatment includes solvent replacement. Specifically, a small amount of water is added to the homogeneous solution to replace acetonitrile or N,N-dimethylformamide in the homogeneous solution with water. In a preferred embodiment, 1 mL of ultrapure water is added to every 500 μL of the homogeneous solution.

[0049] In a preferred embodiment, the phase transfer treatment further includes ultrafiltration (ultracentrifugal filtration). Specifically, an ultrafiltration device is used to filter the homogeneous solution to retain the modular nanomachine, and the smaller unreacted substances, by-products, and impurities passing through the filter membrane are removed.

[0050] In a preferred embodiment, the steps of adding a small amount of water and ultrafiltration are repeated until the solvent in the homogeneous solution is completely replaced with water.

[0051] Example 1 Preparation of Modular Nanomachine

[0052] The T40 single-stranded DNA labeled with Cy5 organic dye (i.e., ssDNA-Cy5) was dissolved in ultrapure water and quantified to 100 μM using a UV-visible spectrophotometer. The [Au8(dppp)4(TPA-C≡C)2](NO3)2 inorganic nanocluster (i.e., Au NC) was dissolved in an acetonitrile solvent and quantified to 200 μM using a UV-visible spectrophotometer.

[0053] An aqueous solution of single-stranded DNA T40 labeled with Cy5 organic dye and an acetonitrile solution of inorganic nanocluster [Au8(dppp)4(TPA-C≡C)2](NO3)2 were respectively mixed in N,N-dimethylformamide solvent. Among them, the molar ratios of inorganic nanoclusters to single-stranded DNA were 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1. They were placed in a metal bath and shaken at a constant temperature of 25 °C for 10 hours to form a homogeneous solution.

[0054] Five volumes of ultrapure water were added to the homogeneous solution, followed by ultrafiltration and discarding the filtrate. After adding ultrapure water again, ultrafiltration was repeated multiple times to obtain a modular nanomachine solution with water as the solvent.

[0055] The local structure of the modular nanomachine obtained in this example is as Figure 1 shown, including single-stranded DNA T40, Cy5 organic dye covalently modified at the 3′ end of the single-stranded DNA, and inorganic nanocluster [Au8(dppp)4(TPA-C≡C)2](NO3)2 hydrophobically bound to the base side of the single-stranded DNA.

[0056] The results of analyzing the modular nanomachine by agarose gel electrophoresis are as Figure 2 shown. The modular nanomachines at each molar ratio formed uniform bands. The band representing the inorganic nanoclusters in the bright field and the band representing the dye-labeled single-stranded DNA in the ultraviolet light channel after Gel-Red staining showed good colocalization, indicating that the inorganic nanoclusters and the single-stranded DNA with organic dye labels are closely connected in the modular nanomachine. The migration rate of the modular nanomachine with a high proportion of inorganic nanoclusters was slightly slower than that of the modular nanomachine with a low proportion of inorganic nanoclusters, which was due to the increase in the molecular weight of the modular nanomachine caused by loading more inorganic nanoclusters.

[0057] The results of analyzing the modular nanomachine by scanning transmission electron microscopy are as Figure 3 shown. The modular nanomachine has good monodispersity, and the size of a single modular nanomachine is between 2 nm and 3 nm, meeting the expectations, indicating that the modular nanomachine was successfully prepared.

[0058] Example 2 Verification of the photothermal conversion mechanism of the modular nanomachine

[0059] The relative intensities of modular nanomachines dissolved in water with different inorganic nanocluster loadings were characterized using an ultraviolet-visible spectrophotometer and a fluorescence spectrometer, and compared with a mixed solution of single-stranded DNA labeled with an organic dye and inorganic nanoclusters dissolved in acetonitrile or tetrahydrofuran at the same concentration ratio (the single-stranded DNA and inorganic nanoclusters in the mixed solution exist independently in the organic phase).

[0060] As Figure 4 and Figure 5 shown, in the ultraviolet-visible spectrum, λ abs ≈510 nm is the characteristic ultraviolet absorption peak of the inorganic nanoclusters. The absorption peak of the inorganic nanoclusters shows an expected linear increase, and the position of the maximum absorption peak remains unchanged, indicating that the inorganic nanoclusters are stably present in the modular nanomachines and their structures remain stable. As Figure 4 shown, in the aqueous solution of the modular nanomachines, the absorption peak intensity of the organic dye decreases with the increase of the inorganic nanocluster loading ratio, and the position of the absorption peak shows an obvious red-shift trend. As Figure 5 shown, in the mixed solution of single-stranded DNA labeled with an organic dye and inorganic nanoclusters at the corresponding ratio, no red-shift phenomenon is observed in the absorption peak intensity of the organic dye. This trend indicates a significant change in the local environment of the organic dye molecules inside the modular nanomachines. The interaction between the organic dye and the inorganic nanoclusters significantly changes the electron cloud distribution of the organic dye molecules, resulting in a decrease in their photon absorption ability. At the same time, this interaction causes the ground state of the organic dye molecules to deviate from the undisturbed equilibrium state, and the increase in its energy makes the energy level difference between the ground state and the excited state decrease, ultimately leading to the red-shift of the absorption peak.

[0061] As Figure 6 shown, for modular nanomachines with different inorganic nanocluster loadings, as the inorganic nanocluster loading ratio increases, the relative fluorescence intensity of the organic dye decreases significantly, indicating the feasibility of regulating the energy pathway by the inorganic nanocluster loading ratio inside the modular nanomachines.

[0062] The modular nanomachines were placed in a quartz cuvette with a 1 cm optical path length, and the modular nanomachines in the cuvette were excited with a 645 nm laser emitted by a fluorescence spectrometer. The organic dye molecules were excited to a high-energy excited state and emitted fluorescence during the process of returning from the excited state to the ground state. The data of the fluorescence intensity changing with time were recorded to calculate the fluorescence lifetime τ f , and the results are as Figure 7 shown. The fluorescence intensity at a specific wavelength was measured to calculate the fluorescence quantum yield φ f , and the results are as Figure 8 shown.

[0063] Combined with the fluorescence lifetime τf With the fluorescence quantum yield φ f The data can be used to quantitatively evaluate the radiative transition rate constant k r and the non-radiative transition rate constant k nr of the modular nanomachine, that is, according to the formula to obtain the photophysical properties of the modular nanomachine. As Figure 9 shown, as the loading ratio of the inorganic nanoclusters increases, the radiative transition rate constant k r remains basically unchanged, while the non-radiative transition rate constant k nr increases to about 6 times that of the Cy5-labeled single-stranded DNA. This quantitative result indicates that inside the constructed modular nanomachine, the introduction of inorganic nanoclusters mainly affects the non-radiative transition process of organic dye molecules. By regulating the loading ratio of inorganic nanoclusters, this non-radiative transition process can be regulated within a certain range (for example, 4×10 -10 s -1 ~1.9×10 -9 s -1 ), so as to realize the control of the excited state energy flow inside the modular nanomachine.

[0064] Example 3 Characterization of the excited state kinetic properties of the modular nanomachine

[0065] Place the modular nanomachine in a quartz cuvette with a 2-mm optical path length, and use a 647-nm femtosecond pulsed laser emitted by a femtosecond transient absorption spectrometer to excite the modular nanomachine in the cuvette. The organic dye molecules are excited to a high-energy excited state, and the kinetic behavior of the modular nanomachine in the excited state is traced and recorded. The excited state absorption values at corresponding wavelengths are extracted at different time points to analyze the excited state kinetics of the modular nanomachine. The exponential decay model is used to fit the collected kinetic data to obtain the excited state kinetic parameters. Replace the modular nanomachine with the single-stranded DNA labeled with the organic dye for comparison.

[0066] As Figure 10 shown, the excited state of the organic dye in the modular nanomachine exhibits a more rapid decay kinetic characteristic compared to the excited state of the organic dye on the single strand, and its half-life is reduced from ~500 ps to ~200 ps, which indicates that the excited state lifetime of the organic dye in the modular nanomachine is significantly shortened due to the presence of the inorganic nanoclusters. In addition, no other excited state absorption (ESA) signals appear in the observable wavelength range, indicating that its energy dissipation pathway does not originate from electron transfer or energy transfer, but from the spontaneous energy dissipation process originally existing in the organic dye.

[0067] Example 4 Atomic-molecular level mechanism of photothermal conversion of the modular nanomachine

[0068] The modular nanomachine was modeled at the all-atom level using molecular modeling software and molecular dynamics simulations were performed to observe non-covalent interactions at the molecular level and to interpret the mechanism in combination with photophysical properties and excited-state kinetic properties.

[0069] As Figure 11 shown, the organic dyes in the modular nanomachine approach each other and form distinct non-covalent interactions (π-π interactions) within 80 ns. Specifically, a shows the relative positions and distance changes among the inorganic nanocluster, the organic dye, and the single-stranded DNA at different time points (0 ns, 21.3 ns, 52.0 ns). It can be seen that the inorganic nanocluster moves on the single-stranded DNA and gradually approaches the organic dye; the upper part of b shows the change in the distance between the center of the inorganic nanocluster and the center of the organic dye over time, and the lower part shows the change in the number of non-covalent interaction contacts (such as π-π stacking) between the inorganic nanocluster and the organic dye over time. From the increase and stability of the number of non-covalent interaction contacts, it can be seen that the inorganic nanocluster and the organic dye tend to be stable after approaching each other; c shows the detailed process of the interaction between the inorganic nanocluster and the organic dye, and it can be seen that the ligand of the inorganic nanocluster changes its orientation to form a stable π-π interaction with the organic dye.

[0070] The coupling process between the inorganic nanocluster and the organic dye of the modular nanomachine can be roughly divided into three stages. In the initial stage, the inorganic nanocluster binds to the bases of the single-stranded DNA driven by hydrophobic interactions. In the wandering stage, the inorganic nanocluster wanders on the single-stranded DNA until it moves near the organic dye. In the coupling stage, the ligand on the inorganic nanocluster changes its orientation to establish a stable π-π interaction with the organic dye and maintains this thermodynamically stable state in the subsequent time. Due to conformational constraints, the π-π interaction between the organic dye and the ligand of the inorganic nanocluster usually occurs on the aromatic ring far from the single-stranded DNA coupling site. The non-radiative energy dissipation of the organic dye is mainly achieved through molecular vibrations. The asymmetric intermolecular interaction between the organic dye and the inorganic nanocluster disrupts the originally symmetric vibration mode of the organic dye, thus changing the non-radiative energy dissipation pathway of the organic dye.

[0071] Example 5 Regulation of the Photothermal Conversion Efficiency of the Modular Nanomachine

[0072] Modular nanomachines were constructed using single-stranded DNAs of different lengths (20 bp, 40 bp, 60 bp, 80 bp) but loaded with the same inorganic nanoclusters. The ultraviolet-visible absorption intensity and fluorescence emission intensity of each modular nanomachine were measured separately to obtain their relative fluorescence intensities The relative fluorescence intensity was compared with single-stranded DNA labeled with organic dyes without loaded inorganic nanoclusters. The percentage decrease in relative fluorescence intensity compared to the single strand (i.e., quenching efficiency) was used as a measure of the photothermal conversion regulation ability. As Figure 12 shown, when the ratio of inorganic nanoclusters to organic dyes remained unchanged, the shorter the length of the single-stranded DNA, the higher its quenching efficiency, that is, the higher the proportion of the photothermal process. This indicates that shorter single-stranded DNA (such as 20 bp) can more effectively promote non-radiative energy dissipation and significantly improve the photothermal conversion efficiency.

[0073] The same single-stranded DNA (such as 40 bp) was used, but different types of inorganic nanoclusters (Au2Cu2L4, Au4L4, Au 25 L 18 and Au8-TPE) were used to construct modular nanomachines. The ultraviolet-visible absorption intensity and fluorescence emission intensity of each modular nanomachine were measured separately to obtain its relative fluorescence intensity (relative fluorescence intensity = ultraviolet-visible absorption intensity / fluorescence emission intensity). The relative fluorescence intensity was compared with single-stranded DNA labeled with organic dyes without loaded inorganic nanoclusters. The percentage decrease in relative fluorescence intensity compared to the single strand (i.e., quenching efficiency) was used as a measure of the photothermal conversion regulation ability. As Figure 13 shown, different types of inorganic nanoclusters exhibited different regulation abilities. For example, some inorganic nanoclusters could significantly improve the quenching efficiency, while others had weaker effects. This indicates that by selecting different inorganic nanoclusters, the precise proportion of the photothermal conversion process in the system can be freely customized.

[0074] Therefore, the photothermal conversion efficiency of the modular nanomachines of the present invention can be effectively regulated by adjusting the length of the single-stranded DNA and the type of inorganic nanoclusters, which can further expand their applications in the field of photothermal conversion.

[0075] Example 6 Multicolor Nanocluster Beacons Developed Based on Modular Nanomachines

[0076] The modular nanomachines labeled with four organic dyes with different wavelengths were mixed with an aqueous solution of their complementary strands and annealed in a PCR instrument. The annealing program was to maintain at 95 °C for 5 minutes first, and then decrease by 0.1 °C every 6 seconds. This process promoted the hybridization between the complementary strands to form a stable double-stranded structure. After hybridization, the detached inorganic nanoclusters were separated by centrifugation, and the supernatant was collected for subsequent spectral tests. The ultraviolet-visible absorption spectrum and the emission spectrum at the maximum absorption wavelength of the supernatant were measured to obtain its relative fluorescence intensity, and the sensitivity of the inorganic nanocluster beacon was judged based on the relative fluorescence intensity results. Single-stranded DNA labeled with a single organic dye was used as a control.

[0077] As Figure 14As shown, the four organic dyes exhibit a consistent quenching effect after hybridization, indicating the effectiveness of regulating energy flow through the interaction between inorganic nanoclusters and organic dyes. Specifically, when an organic dye molecule absorbs an excitation light, its energy can be released through two pathways: radiative transition (emitting fluorescence) or non-radiative transition (releasing in the form of heat). The inorganic nanoclusters promote non-radiative transition through the interaction with the organic dye, thereby improving the photothermal conversion efficiency.

[0078] In addition, the stability of inorganic nanoclusters in aqueous solution is also a key issue. Since the surface of inorganic nanoclusters is hydrophobic, they tend to aggregate and precipitate in water, thus losing their optical properties. However, through the interaction between the hydrophobic side chains of single-stranded DNA and inorganic nanoclusters, the inorganic nanoclusters can maintain their stability in aqueous solution.

[0079] By hybridizing single-stranded DNA with its complementary strand to form a double strand, the originally exposed bases can be closed, thereby stripping the inorganic nanoclusters from the single-stranded DNA. When the inorganic nanoclusters are stripped, the fluorescence of the organic dye is restored because the organic dye is no longer promoted by the non-radiative transition of the inorganic nanoclusters. Using the biorecognition ability of single-stranded DNA, this kind of nanocluster beacon can construct a series of probes from visible to near-infrared for identifying and detecting various target substances, such as specific DNA sequences, proteins, or other biomolecules. Therefore, by precisely regulating the interaction between inorganic nanoclusters and organic dyes, we can not only improve the photothermal conversion efficiency but also develop nanocluster beacons with high sensitivity and specificity, which have broad application prospects in fields such as biological detection and imaging.

[0080] 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. That is, all simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of the claims of the present invention patent. Experimental methods without specific conditions are usually carried out under conventional conditions and / or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

Claims

1. A modular nanomachine, characterized in that, The modular nanomachine includes single-stranded DNA, organic dyes, and inorganic nanoclusters. Among them, the organic dyes are covalently modified and linked to the 3′ end of the single-stranded DNA, the inorganic nanoclusters are bound to the bases of the single-stranded DNA through hydrophobic interactions, and π-π stacking is formed between the inorganic nanoclusters and the organic dyes.

2. The modular nanomachine according to claim 1, characterized in that, The length of the single-stranded DNA ranges between 20 bp and 80 bp.

3. The modular nanomachine according to claim 1, characterized in that, The organic dye has a planar rigid donor-π-electron-acceptor structure.

4. The modular nanomachine according to claim 3, wherein The organic dye is a cyanine fluorescent dye Cy3, Cy5, Cy7, or ICG.

5. The modular nanomachine according to claim 1, characterized in that, The structure of the inorganic nanocluster is a metal core protected by an external hydrophobic ligand.

6. The modular nanomachine according to claim 5, characterized in that, Inorganic nanoclusters are gold nanoclusters Au2Cu2(C 10 H 10 S2N)4, Au4(C6H 10 S2N)4, Au 25 (SCH2CH2Ph) 18 , [Au8(dppp)4(TPA-C≡C)2](NO3)2 or [Au8(dppp)4(TPE-C≡C)2](NO3)2.

7. The modular nanomachine according to claim 1, characterized in that, The molar ratio of the inorganic nanocluster to the single-stranded DNA ranges between 1:1 and 8:

1.

8. The method for preparing the modular nanomachine according to any one of claims 1-7, characterized in that, The preparation method includes the following steps: S1, covalently modifying and linking the organic dye to the 3′ end of the single-stranded DNA to form single-stranded DNA labeled with the organic dye; S2, co-mixing the aqueous solution of the single-stranded DNA labeled with the organic dye and the organic phase solution of the inorganic nanocluster in an intermediate solvent, and shaking and mixing to form a homogeneous solution; S3, performing phase transfer treatment on the homogeneous solution to purify and separate the modular nanomachine.

9. Use of the modular nanomachine according to any one of claims 1-7, characterized in that, The photothermal conversion efficiency of the modular nanomachine is regulated by regulating the single-stranded DNA and / or the inorganic nanocluster.

10. The application according to claim 9, wherein A nanocluster beacon is provided by hybridizing the single-stranded DNA with a complementary strand.

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