Core-shell structure nano-reactor for high-specificity nucleic acid detection and preparation method of core-shell structure nano-reactor

Abstract

The invention discloses a core-shell structure nanoreactor for high-specificity nucleic acid detection and a preparation method of the core-shell structure nanoreactor, and belongs to the technical field of nucleic acid detection. The invention solves the problem that the current miRNA detection is susceptible to interference of non-target molecules, so that the high-specificity and high-precision recognition of miRNA is limited. According to the nanoreactor, hollow meso-porous silicon balls with adjustable pore diameters are used as a shell, and Cu (I) / Cu (II)-MOF is used as a catalyst for catalyzing azide-alkyne cycloaddition reaction through copper and is packaged in cavities of the hollow meso-porous silicon balls. The hollow meso-porous silicon spheres provide a rigid and chemically stable size screening environment, and meanwhile, by adjusting an etching process, the nanoreactor has the aperture of 4-9 nm, miRNA is allowed to enter a cavity for a catalytic reaction, and macromolecules such as pre-miRNA are blocked outside HMS, so that the purpose of accurate size selection and accurate detection of miRNA are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nucleic acid detection, and particularly relates to a core-shell structure nano-reactor for high-specificity nucleic acid detection and a preparation method thereof. BACKGROUND

[0002] In the field of biosensing, the high-specificity recognition of microRNA (miRNA) is the core basis for achieving precise molecular diagnosis. Current mainstream technologies rely on the sequence complementarity binding mechanism between probes and target nucleic acids. However, when target miRNAs coexist with non-target molecules (such as analogs or precursors) that are similar in structure or sequence, traditional molecular recognition methods often face the challenge of insufficient selectivity. Taking miRNA detection as an example, its detection accuracy is often affected by pre-miRNA, because the sequence of miRNA is completely contained in pre-miRNA, making it difficult for sequence complementarity-based methods to distinguish between the two. Considering the significant size difference between miRNA and non-target molecules, utilizing this size difference for molecular recognition (i.e., size selectivity) is an effective strategy to solve this problem.

[0003] In view of the above challenges, developing a new molecular strategy that can accurately recognize and screen based on molecular size difference is crucial to improve the efficiency and specificity of biosensing and labeling. Although DNA-cage and other nanostructures based on DNA self-assembly have been explored for size-selective separation or reaction in miRNA detection in recent years, DNA-cage technology has limitations in achieving high-precision and high-stability size screening. For example, DNA-cage nanostructures are essentially dynamic molecular frameworks, and even in relatively pure in vitro solutions, their pore size can be affected by slight fluctuations in ion concentration and temperature, leading to subtle fluctuations or conformational changes. This can lead to a decrease in selectivity for applications such as miRNA detection that require nanoscale precision size screening. In addition, the large-scale and high-reproducibility preparation of DNA-cage nanostructures still faces challenges. The complex self-assembly process makes it difficult to achieve uniform and controllable pore size distribution like inorganic materials. Furthermore, integrating high-efficiency catalytic functions into DNA frameworks in a high-density and stable manner also presents technical bottlenecks. SUMMARY

[0004] The present application provides a core-shell structure nano-reactor for high-specificity nucleic acid detection and a preparation method thereof, aiming to address the problem of limited high-specificity and high-precision recognition of miRNA due to interference from non-target molecules during the miRNA detection process.

[0005] To achieve the above object, the technical scheme of the present application is as follows: One of the objects of the present application is to provide a core-shell structure nano-reactor for high specificity nucleic acid detection, which takes hollow mesoporous silica spheres (HMS) as a shell, Cu(I) / Cu(II)-MOF as a core, the Cu(I) / Cu(II)-MOF is encapsulated in the cavity of the hollow mesoporous silica spheres, the Cu(I) / Cu(II)-MOF acts as a catalyst for copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, and the mesoporous pore size of the hollow mesoporous silica spheres is adjustable.

[0006] Further limited, the mesoporous pore size is 4-9 nm.

[0007] Further limited, the copper element in the nano-reactor contains mixed valence of +1 valence copper and +2 valence copper.

[0008] Further limited, the molar percentage of +1 valence copper in the copper element of the nano-reactor is 26%.

[0009] The second object of the present application is to provide a preparation method of the core-shell structure nano-reactor (Cu(I) / Cu(II)-MOF@HMS) for high specificity nucleic acid detection, which comprises the following steps: (1) Synthesis of hollow mesoporous silica spheres: ammonia and tetraethyl orthosilicate are sequentially added in a mixed solvent composed of ethanol and water, octadecyltrimethoxysilane and tetraethyl orthosilicate are added after stirring, and SiO2@HMS is obtained after centrifugation after vigorous stirring, the SiO2@HMS is divided into multiple parts, each part of the SiO2@HMS is etched in a Na2CO3 solution, and the hollow mesoporous silica spheres are obtained after washing, drying and calcining; (2) Synthesis of Cu(II)-MOF@HMS: the hollow mesoporous silica spheres are dispersed in an ethanol solution containing 1,3,5-benzenetricarboxylic acid (H3BTC), H3BTC@HMS intermediate is obtained by vacuum evaporation of ethanol, then the H3BTC@HMS intermediate is dispersed in a Cu(OAc)2 aqueous solution, N,N-dimethylformamide is added after vacuum evaporation of water, and the product is washed after reaction to obtain Cu(II)-MOF@HMS; (3) Synthesis of core-shell structure nano-reactor: hydroquinone is dissolved in water to obtain a hydroquinone solution, the Cu(II)-MOF@HMS obtained in step (2) is prepared into a suspension, the suspension is added to the hydroquinone solution, and hydrothermal reaction is carried out, and the product is sequentially centrifuged, washed and dried after the reaction to obtain the core-shell structure nano-reactor.

[0010] Further limit, the amount of Na2CO3 solution used in each SiO2@HMS etching in (1) is 26-32 mL, and the etching time is 1.5-2 h.

[0011] Further limit, the reaction temperature in (2) is 30-50 ℃, and the time is 2-4 h.

[0012] Further limit, the hydrothermal reaction temperature in (3) is 140-160 ℃, and the time is 15-17 h.

[0013] The present application has the following advantages: The present application aims to solve the problem of detection accuracy caused by the presence of pre-miRNA in miRNA detection, and further provides a nano-reactor (Cu(I) / Cu(II)-MOF@HMS) with size-selective hollow mesoporous silica sphere encapsulated Cu(I) / Cu(II)-MOF. Compared with the prior art, the present application has the following advantages: (1) The present application combines inorganic materials with high structural stability and adjustable pore size with efficient copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to construct a size-selective core-shell nano-reactor. Hollow mesoporous silica spheres (HMS) are used as carriers, i.e. shells, by adjusting the mesoporous pore size of HMS to 4-9 nm, allowing miRNA to enter the cavity and contact the catalyst, while pre-miRNA, proteins and other macromolecules are efficiently blocked outside the HMS, thereby realizing size selection of large and small size substances and precise detection of miRNA. The catalytically active sites of CuAAC reaction catalyst Cu(I) / Cu(II)-MOF are encapsulated inside the nanoscale cavity with specific pore size, thereby realizing efficient catalytic reaction inside the HMS cavity, while non-target molecules outside the cavity cannot trigger catalysis due to spatial hindrance regardless of whether they contain reaction groups. By utilizing the synergistic effect of size selection and catalytic reaction, the purpose of precise size selection and accurate miRNA detection is achieved.

[0014] (2) HMS as a shell has the following advantages: First, its unique mesoporous structure can precisely control the pore size, thereby effectively allowing miRNA to enter the active region while efficiently blocking the non-specific entry of macromolecules such as pre-miRNA and proteins; Second, unlike the dynamic nature of traditional DNA cages, HMS provides a rigid, highly controllable, and chemically stable size screening environment, which is crucial for achieving precise material size screening in vitro. Furthermore, the HMS of this invention has the ability to controllably synthesize different pore sizes. By adjusting the etching process, different pore sizes can be synthesized, facilitating the customized synthesis of nanoreactors with specific size screening capabilities according to the size requirements of the target miRNA, providing high flexibility for the detection of highly specific miRNAs.

[0015] (3) This invention synthesizes a core-shell structured nanoreactor in steps. First, HMS is synthesized by etching using a hard template method. Then, it is combined with H3BTC to synthesize an H3BTC@HMS intermediate. The intermediate is then immersed in a copper-containing precursor solution to synthesize Cu(II)-MOF in the HMS cavity, yielding Cu(II)-MOF@HMS. Based on this, under the presence of a reducing agent, Cu(II)-MOF is further grown into Cu + With Cu 2+ Mixed-state MOF@HMS materials, namely Cu(I) / Cu(II)-MOF@HMS. Cu(I) / Cu(II)-MOF@HMS can not only efficiently catalyze the CuAAC reaction, but its unique size selectivity also means that it does not have the ability to catalyze large-sized materials.

[0016] (4) In this invention, when the target miRNA enters the cavity of the nanoreactor, it serves as a template to drive two probe DNAs, modified with alkyne and azide groups respectively, to undergo a cycloaddition reaction (CuAAC) under the catalysis of a catalyst, releasing a fluorescent signal. Experimental results show that, based on the precise control of the nanoreactor pore size (e.g., 4 nm), this material can efficiently catalyze the click reaction of small nucleic acid sequences (e.g., 20 nt), while having almost no catalytic activity for large nucleic acid sequences (e.g., 50 nt), thereby achieving specific recognition and detection of small molecules such as miRNA, while effectively avoiding interference from large molecules such as pre-miRNA and proteins. This detection method is simple and inexpensive, suitable for large-scale applications. The core-shell structured nanoreactor of this invention provides a universal and effective strategy for highly specific miRNA detection. Attached Figure Description

[0017] Figure 1 These are TEM images of HMS, Cu(II)-MOF@HMS, and Cu(I) / Cu(II)-MOF@HMS from Examples 1-3 of this invention; Figure 2 XRD patterns of HMS, Cu(II)-MOF and Cu(II)-MOF@HMS of embodiments 1-3 of the present application; Figure 3 XRD patterns of Cu(I) / Cu(II)-MOF and Cu(I) / Cu(II)-MOF@HMS of embodiments 1-3 of the present application; Figure 4 FT-IR patterns of HMS, Cu(II)-MOF and Cu(II)-MOF@HMS of embodiments 1-3 of the present application; Figure 5 FT-IR patterns of Cu(I) / Cu(II)-MOF and Cu(I) / Cu(II)-MOF@HMS of embodiment 2 of the present application; Figure 6 XPS patterns of Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS of embodiment 2 of the present application, (a) Cu(II)-MOF@HMS, (b) Cu(I) / Cu(II)-MOF@HMS; Figure 7 N2 adsorption-desorption isotherm patterns of HMS, Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS of embodiments 1-3 of the present application; Figure 8 Pore size distribution patterns of HMS, Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS of embodiments 1-3 of the present application; Figure 9 Catalytic kinetics patterns and cyclic test results of catalytic reactions of embodiments 4-6 of the present application; Figure 10 Fluorescent labeling linear patterns of Cu(I) / Cu(II)-MOF@HMS in catalytic reactions of embodiments 4-6 of the present application under different substrate concentrations and different times, (a)-(c) different times, (d)-(f) different substrate concentrations; Figure 11 Catalytic kinetics pattern of nucleic acid detection of Cu(I) / Cu(II)-MOF@HMS of embodiment 7 of the present application; Figure 12 Nucleic acid detection kinetics fitting patterns of Cu(I) / Cu(II)-MOF@HMS of embodiment 1 of the present application on different sequence miRNAs, (a) T20, (b) T30, (c) T40, (d) T50. DETAILED DESCRIPTION

[0018] In order to make the above objectives, features and advantages of the present application more obvious and comprehensible, the specific embodiments of the present application will be described in detail below with reference to the description and examples.

[0019] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other manners different from those described herein, and those skilled in the art can make similar generalizations without departing from the spirit and scope of the present application, therefore the present application is not limited to the specific embodiments disclosed below.

[0020] Secondly, the "one embodiment" or "embodiment" referred to herein means that a specific feature, structure or characteristic can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is separate or alternative to other embodiments.

[0021] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0022] In the present application, M represents mol / L, and mM represents mmol / L.

[0023] Example 1 (1) Synthesis of hollow mesoporous silica spheres (HMS): 3.14 mL of ammonia water was added in a mixed solvent composed of 71.4 mL of ethanol and 10 mL of water, then 6 mL of tetraethyl orthosilicate (TEOS) was added at 30 ℃, 3 mL of octadecyltrimethoxysilane (C 18 TMS) and 5 mL of TEOS were added after stirring vigorously for 1 h, and the reaction system was centrifuged at a speed of 11000 rpm to obtain SiO2@HMS. The SiO2@HMS was divided into 6 parts for better etching, and each part of SiO2@HMS was etched in 32 mL of 0.6 M Na2CO3 solution at 80 ℃ for 1.5 h, then centrifuged at a speed of 11000 rpm to obtain the crude product of HMS. The crude product was washed with anhydrous ethanol for 3 times and dried at 70 ℃ under vacuum for 12 h to obtain a white powder solid, which was calcined in a muffle furnace at 550 ℃ for 5 h to remove C 18 TMS, to obtain HMS with a mesoporous pore size of 4 nm, and the six parts of HMS were placed together; (2) Synthesis of Cu(II)-MOF@HMS: 0.3 g of HMS was dispersed in 2.5 mL of an ethanol solution containing 1,3,5-benzenetricarboxylic acid (10 g / L), and then ethanol was evaporated under vacuum at 35°C to obtain an H3BTC@HMS intermediate. The H3BTC@HMS intermediate was dispersed in 2.5 mL of an aqueous Cu(OAc)2 solution (25 g / L), and water was evaporated under vacuum at 55°C. Then, 50 μL of N,N-dimethylformamide (DMF) was added to the system, and the reaction was performed at 85°C for 12 h. The product was washed with methanol three times to obtain Cu(II)-MOF@HMS; (3) Synthesis of a core-shell nano-reactor (Cu(I) / Cu(II)-MOF@HMS): 0.5 g of hydroquinone was dissolved in 8 mL of H2O to obtain a hydroquinone solution. The hydroquinone solution was transferred to a 25 mL reaction kettle, and 0.1 g of Cu(II)-MOF@HMS was weighed and ultrasonically dispersed in 2 mL of H2O for 30 min to obtain a suspension. The suspension was added to the hydroquinone solution, and a hydrothermal reaction was performed at 150°C for 16 h. After the reaction, the product was centrifuged at a speed of 11000 rpm to obtain a Cu(I) / Cu(II)-MOF@HMS crude product. The crude product was washed with methanol three times and then dried under vacuum at 70°C for 8 h to obtain a black-green powder, i.e., a nano-reactor with a mesopore size of 4 nm. Before use, the nano-reactor needs to be activated by being kept at 120°C for 5 h under vacuum.

[0024] Example 2 The difference between this example and Example 1 is that: (1) each part was etched in 28 mL of a 0.6 M Na2CO3 solution at 80°C for 1.5 h, and the mesopore size of the HMS was 5 nm. The mesopore size of the final nano-reactor Cu(I) / Cu(II)-MOF@HMS was also 5 nm. The remaining steps and process parameters were the same as in Example 1.

[0025] Example 3 The difference between this example and Example 1 is that: (1) each part was etched in 26 mL of a 0.6 M Na2CO3 solution at 80°C for 2 h, and the mesopore size of the HMS was 9 nm. The mesopore size of the final nano-reactor Cu(I) / Cu(II)-MOF@HMS was also 9 nm. The remaining steps and process parameters were the same as in Example 1.

[0026] In addition, Cu(II)-MOF, Cu(I)-MOF and Cu(I) / Cu(II)-MOF were synthesized for comparison with HMS, Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS of the present application in characterization experiments.

[0027] Preparation of Cu(II)-MOF: 0.299 g Cu(OAc)2·H2O was dissolved in 25 mL deionized water, followed by the addition of 0.21 g polyvinylpyrrolidone (PVP, molecular weight 1300000). After the mixture was refluxed for 1 hour, 25 mL of 40 mM H3BTC ethanol solution was slowly added at 60 °C. Stirring was continued for 1 hour, and then stirring was carried out at room temperature for 12 hours. The product was recovered by centrifugation at 11000 rpm for 15 minutes, washed with 1:1 deionized water / ethanol solution for 6 times, dried at 70 °C under vacuum for 12 hours, and activated at 120 °C for 12 hours before use.

[0028] Preparation of Cu(I) / Cu(II)-MOF: Cu(I) / Cu(II)-MOF was synthesized using the above Cu(II)-MOF as precursor. 100 mg of the precursor Cu(II)-MOF was dispersed in 2 mL deionized water, and ultrasonic treatment was carried out. The solution was added to a mixture of 113 mM hydroquinone solution and 20 mg PVP, and then transferred to a 25 mL polytetrafluoroethylene-lined reaction kettle. Heating was carried out at 150 °C for 16 hours. The product was recovered by centrifugation at 11000 rpm for 15 minutes, washed with methanol and deionized water alternately for 4 times, dried at 70 °C for 12 hours, and activated at 120 °C before use.

[0029] Cu(II)-MOF, Cu(I)-MOF, Cu(I) / Cu(II)-MOF and the nanoreactor of the present application were characterized and tested by X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), nitrogen adsorption-desorption isotherm and Fourier transform infrared spectroscopy (FT-IR), and the results are as follows.

[0030] Figure 1 TEM images of HMS, Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS of Examples 1-3 of the present application. From the TEM images, it can be seen that the HMS, Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS of the present application have a uniform particle size, and the particle size is in the range of 50-100 nm. Figure 1It can be seen that, regardless of the mesopore diameter of HMS is 4 nm, 5 nm or 9 nm, but the diameter of HMS is about 200 nm, the shell thickness is about 10 nm, and there is no product in the ball center, which is a hollow spherical structure. The Cu (II) -MOF is generated in the HMS by immersing the H3BTC@HMS intermediate in the copper-containing precursor solution, and the blocky product appears in the hollow ball of Cu (II) -MOF@HMS, and there is no product outside the ball, which shows that Cu (II) -MOF is successfully formed in the cavity of HMS by immersion, and Cu (II) -MOF is encapsulated in HMS, and there is no Cu (II) -MOF outside the ball, which can provide effective size-selective catalysis for small-size nucleic acid detection. No blocky material is found in the Cu (I) / Cu (II) -MOF@HMS obtained after the hydrothermal reduction reaction, which shows that the reduction process does not cause Cu (I) / Cu (II) -MOF to escape from the HMS.

[0031] Figure 2 For the XRD patterns of Cu (II) -MOF and HMS of embodiments 1-3 of the present application, Cu (II) -MOF@HMS, it can be found that there are both the wide peaks of HMS and the characteristic diffraction peaks of Cu (II) -MOF at 11.7°, 6.8°, 9.5° and 13.5° in Cu (II) -MOF@HMS with different mesopore diameters, which proves that Cu (II) -MOF and HMS exist simultaneously in Cu (II) -MOF@HMS.

[0032] Figure 3 For the XRD patterns of Cu (I) / Cu (II) -MOF and Cu (I) / Cu (II) -MOF@HMS of embodiments 1-3 of the present application, it can be seen that the characteristic diffraction peaks in Cu (I) / Cu (II) -MOF@HMS coincide with those of Cu (I) / Cu (II) -MOF, and the same characteristic diffraction peaks appear at 11.1°, 5.8°, 12.5° and 13.1°, which shows that Cu (II) -MOF@HMS is successfully reduced to Cu (I) / Cu (II) -MOF@HMS.

[0033] Figure 4 For the FT-IR patterns of Cu (II) -MOF and HMS of embodiments 1-3 of the present application, Cu (II) -MOF@HMS, it can be found that, regardless of the mesopore diameter of HMS is 4 nm, 5 mn or 9 nm, Cu (II) -MOF@HMS exists Si-O bond of HMS at 1100 cm -1 , and the sharp characteristic peak belonging to the asymmetric C=O stretching vibration of carboxylate group is observed at 1645 cm -1 , while 1445 cm -1The absorption band corresponds to the symmetric stretching vibration mode of the coordinated carboxylate. At the same time, the COO - symmetric stretching vibration peak appears at 1374 cm -1 The characteristic peaks appearing above are consistent with the paddle-wheel type Cu (II) coordination structure, and also prove that the Cu (II)-MOF@HMS contains HMS and Cu (II)-MOF, which is consistent with the conclusion of the XRD of Figure 2

[0034] Figure 5 The FT-IR graph of Cu (I) / Cu (II) -MOF and Cu (I) / Cu (II) -MOF@HMS of Example 2 of the present application can be seen that, compared with Cu (II) -MOF@HMS, the Si-O bond of HMS in the nanoreactor still exists, and the 1374 cm -1 peak of the original Cu (II) -MOF splits into 1370 and 1382 cm -1 doublet peaks, confirming the coexistence of Cu (I) -O and Cu (II) -O coordination environments in Cu (I) / Cu (II) -MOF, which proves the existence of mixed valence copper. In addition, the appearance of 1495 and 1503 cm -1 doublet peaks on the Cu (I) / Cu (II) -MOF curve indicates that the Cu + mediated electronic redistribution triggers the ligand distortion effect. While the 1445 cm -1 absorption band characteristic of the symmetric stretching vibration of the carboxylate remains essentially unchanged in Cu (I) / Cu (II) -MOF. These infrared spectral changes reflect the change in the coordination symmetry of the carboxylate caused by the transformation of the Cu 2+ bidentate coordination to Cu + monodentate coordination, which indicates that the Cu (II) -MOF is reduced, resulting in a change in the carboxylate coordination state.

[0035] Figure 6 The XPS graph of Cu (II) -MOF@HMS and Cu (I) / Cu (II) -MOF@HMS of Example 2 of the present application is shown, and the element type and valence state in the nanoreactor are characterized by XPS. In Figure 6 (a) In the XPS Cu spectrum of Cu (II) -MOF@HMS, characteristic signals of Cu 2+ are observed at 953.8 eV and 934.2 eV, corresponding to Cu 2p1 / 2 and Cu 2p3 / 2 peaks, respectively, confirming that only Cu 2+ exists in Cu (II) -MOF@HMS, and the satellite peaks formed by the red dots also confirm the existence of Cu 2+ In Figure 6 ​(b) XPS Cu spectral analysis of Cu(I) / Cu(II)-MOF@HMS shows that part of Cu signal shifts to 934.2 eV and 932.2 eV, indicating Cu 2+ is partially reduced to Cu + , and there is also a satellite peak, the existence of the satellite peak further confirms the existence of Cu 2+ in Cu(I) / Cu(II)-MOF@HMS, indicating that the reduction is incomplete. Peak fitting analysis shows that Cu(I) / Cu(II)-MOF@HMS contains about 26% Cu + and 74% Cu 2+ .

[0036] Figure 7 N2 adsorption-desorption isotherm graphs of HMS, Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS of Examples 1-3 of the present application. Nitrogen adsorption at 77 K was used to detect the pore properties of each substance. The Brunauer-Emmet-Teller (BET) method was used to calculate the surface area and pore volume of each substance, and the results are shown in Table 1. The results show that the specific surface area of HMS-4 nm is 159.82 m² / g, and the specific surface area of Cu(II)-MOF@HMS-4 nm decreases to 82.27 m² / g. This decrease in surface area is attributed to the impregnation and encapsulation of Cu(II)-MOF in HMS, which indicates that Cu(II)-MOF is successfully synthesized in the cavity of HMS. The specific surface area of Cu(I) / Cu(II)-MOF@HMS-4 nm after reduction of Cu(II)-MOF@HMS decreases to 32.65 m² / g. This is because the reduction reaction can cause local micropore collapse, resulting in a decrease in surface area. The same phenomenon also exists in Cu(I) / Cu(II)-MOF@HMS-5 nm and Cu(I) / Cu(II)-MOF@HMS-9 nm. During the synthesis of Cu(I) / Cu(II)-MOF@HMS-5 nm, the surface area of HMS-5 nm is 92.39 m 2 / g, Cu(II)-MOF@HMS-5 nm decreases to 80.54 m² / g, and Cu(I) / Cu(II)-MOF@HMS-5 nm decreases to 20.13 m² / g. During the synthesis of Cu(I) / Cu(II)-MOF@HMS-9 nm, the surface area of HMS-9 nm is 105.38 m² / g, Cu(II)-MOF@HMS-9 nm decreases to 74.38 m² / g, and Cu(I) / Cu(II)-MOF@HMS-9 nm further decreases to 41.45 m² / g.

[0037] Table 1. Specific surface area test results of HMS with different pore sizes, Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS

[0038] Figure 8 The pore size distribution diagrams of HMS, Cu(II)-MOF@HMS, and Cu(I) / Cu(II)-MOF@HMS in Examples 1-3 of this invention are shown below. The mesopore size distribution was determined using the Barrett-Joyner-Halenda (BJH) method, and the results are shown in [Figure 1]. Figure 8 The pore size distribution represents the proportion of different pore sizes. The most probable pore size was also tested, representing the pore size with the highest proportion. The results are shown in Table 2. The most probable diameter of HMS-4 nm was 4.2 nm. While the most probable diameter of Cu(II)-MOF@HMS-4 nm showed a slight change, the pore size distribution changed significantly. This is because during the Cu(II)-MOF impregnation process, the pore size of HMS was partially occupied by Cu(II)-MOF, leading to a change in the pore size distribution. The most probable diameter of Cu(I) / Cu(II)-MOF@HMS-4 nm after Cu(II)-MOF@HMS reduction did not change significantly. This indicates that the impregnation and reduction processes do not significantly alter the most probable pore size of HMS, which is beneficial for subsequent size-selective catalytic screening in small molecule nucleic acid detection. The same phenomenon was observed in the synthesis of Cu(I) / Cu(II)-MOF@HMS-5 nm and Cu(I) / Cu(II)-MOF@HMS-9 nm. During the synthesis of Cu(I) / Cu(II)-MOF@HMS-5 nm, the most probable diameter of HMS-5 nm was 5.1 nm, which decreased to 5.4 nm for Cu(II)-MOF@HMS-5 nm, and further decreased to 4.8 nm for Cu(I) / Cu(II)-MOF@HMS-5 nm. During the synthesis of Cu(I) / Cu(II)-MOF@HMS-9 nm, the most probable diameter of HMS-9 nm was 8.8 nm, which decreased to 9.2 nm for Cu(II)-MOF@HMS-9 nm, and further decreased to 8.9 nm for Cu(I) / Cu(II)-MOF@HMS-9 nm.

[0039] Table 2. Test results of the most probable diameters of HMS, Cu(II)-MOF@HMS and Cu(I) / Cu(II)-MOF@HMS with different pore sizes.

[0040] Example 4 The Cu(I) / Cu(II)-MOF@HMS of Example 1 was tested for catalytic performance in the CuAAC reaction of phenylacetylene and azidobenzyl.

[0041] A 3-azido-7-hydroxycoumarin DMSO solution of 8 mM and a phenylacetylene DMSO solution of 8 mM were configured, 6.25 μL of the 3-azido-7-hydroxycoumarin DMSO solution and 12.5 μL of the phenylacetylene DMSO solution were added in 5 mL of H2O, and then 500 μg of Cu(I) / Cu(II)-MOF@HMS with a mesoporous pore size of 4 nm was added, and the reaction was carried out for 5-75 min, followed by centrifugation of Cu(I) / Cu(II)-MOF@HMS in the reaction system at 11000 rpm, and the fluorescence labeling linear graph of Cu(I) / Cu(II)-MOF@HMS with different pore sizes at different times in the catalytic reaction was tested. On the other hand, according to the above method, 6.25 μL of the 3-azido-7-hydroxycoumarin DMSO solution was added in 5 mL of H2O, and then different volumes of the phenylacetylene solution were added to change the final concentration thereof, and the reaction solution was detected by a fluorescence spectrophotometer to test the relationship between the fluorescence intensity and the concentration of phenylacetylene (i.e. the concentration of the substrate). The reaction solution was detected by a fluorescence spectrophotometer.

[0042] Example 5 The Cu(I) / Cu(II)-MOF@HMS of Example 2 was tested for catalytic performance in the CuAAC reaction of phenylacetylene and azidobenzyl. The difference between this example and Example 4 is that the mesoporous structure of the HMS in the Cu(I) / Cu(II)-MOF@HMS has a pore size of 5 nm, and the remaining steps and process parameters are the same as those of Example 1.

[0043] Example 6 The Cu(I) / Cu(II)-MOF@HMS of Example 3 was tested for catalytic performance in the CuAAC reaction of phenylacetylene and azidobenzyl. The difference between this example and Example 4 is that the mesoporous structure of the HMS in the Cu(I) / Cu(II)-MOF@HMS has a pore size of 9 nm, and the remaining steps and process parameters are the same as those of Example 1.

[0044] The results of the catalytic performance of Cu(I) / Cu(II)-MOF@HMS in the CuAAC reaction of phenylacetylene and azidobenzyl in Examples 4-6 are shown in Table 3. As can be seen from the data in the table, Cu(I) / Cu(II)-MOF@HMS with different mesopore diameters all exhibit good catalytic efficiency. Among them, 4 nm pore diameter has the strongest adsorption capacity, which helps the rapid diffusion of the substrate, and the conversion rate can reach 98.4% and the TOF value can reach 264.4 h -1 -1 in 30 min. The TOF values of 5 nm and 9 nm Cu(I) / Cu(II)-MOF@HMS are reduced to 253.1 and 241.4, respectively, due to the slow diffusion of large pore diameters. The catalytic kinetic test and recycling test results are shown in Figure 9 (a) and Figure 9 (b), respectively. As can be seen from Figure 9 (a), 4 nm Cu(I) / Cu(II)-MOF@HMS has a faster kinetics, and k reaches 0.195 min -1 -1. The catalytic reaction rates of 5 nm and 9 nm Cu(I) / Cu(II)-MOF@HMS are reduced, and k is 0.154 min -1 -1 and k=0.133 min -1 -1, respectively. This result proves that compared with 4 nm pore diameter, the adsorption capacity of larger pore diameter is weak, and the diffusion rate of the substrate is slow. Figure 9 In (b), it can be seen that in the catalytic CuAAC reaction of 10 cycles, the catalytic effect does not change significantly.

[0045] Table 3. Catalytic performance results of Cu(I) / Cu(II)-MOF@HMS in the CuAAC reaction of phenylacetylene and azidobenzyl in Examples 4-6

[0046] Figure 10 The fluorescence-labeled linear graph of Cu(I) / Cu(II)-MOF@HMS in the catalytic reaction of Examples 4-6 under different substrate concentrations and times, and the catalytic activity in the CuAAC reaction between phenylacetylene and 3-azidobenzyl-7-hydroxycoumarin is evaluated by fluorescence measurement. 3-azidobenzyl-7-hydroxycoumarin is a water-soluble fluorescent dye, which can only emit strong fluorescence after the formation of triazole in the CuAAC reaction. From Figure 10As can be seen in (a)-(c), at different times, Cu(I) / Cu(II)-MOF@HMS with 4 nm, 5 nm, and 9 nm all showed rapid fluorescence recovery, and Cu(I) / Cu(II)-MOF@HMS-4 nm, 5 nm, and 9 nm all reached saturation within 45 minutes, Figure 10 (d)-(f) also quantitatively revealed a highly linear relationship between fluorescence intensity and substrate concentration.

[0047] Example 7 In Example 7, the nucleic acid detection capability of the nanoreactor of Example 1-3 of the present application for miRNAs of different lengths was tested. Denaturing polyacrylamide gel electrophoresis (PAGE) test of Cu(I) / Cu(II)-MOF@HMS catalyzed DNA template CuAAC reaction. First, 200 nM of P2FAM probe with alkyne modification, 200 nM of P1 probe with azido group, and 200 nM of template DNA were mixed (nucleic acid fragments are shown in Table 4), and then 400 μg·mL -1 Cu(I) / Cu(II)-MOF@HMS, this embodiment detects miRNAs with lengths of 20 nt, 30 nt, 40 nt, and 50 nt (i.e., T20, T30, T40, and T50 in Table 3). All control groups are performed without template DNA or Cu(I) / Cu(II)-MOF@HMS. The reaction solution is sampled 20 μL, which is then immediately mixed with 10 μL of 8 M urea (50%, v / v) and 10 μL of DNA loading buffer (6x), and loaded onto a 20% denaturing polyacrylamide gel electrophoresis (PAGE, 8 M urea in 1xTBE buffer). The denaturing gel is imaged by a fluorescence imager, and the results are shown in Figure 11 As shown, the Cu(I) / Cu(II)-MOF@HMS nanoreactor shows a rapid catalytic rate and can be efficiently applied to the DNA template ligation reaction. As the length of the template DNA sequence increases, the amount of template reaction of the 4 nm pore size nanoreactor is greatly reduced, while the 5 nm and 9 nm are not affected. As the length of the template DNA sequence continues to increase, the amount of template reaction of the 5 nm pore size nanoreactor also begins to decrease greatly, while the 9 nm nanoreactor is not affected.

[0048] Table 4 Nucleic acid fragments in denaturing polyacrylamide gel electrophoresis

[0049] Figure 12The nucleic acid detection kinetics fitting diagrams of Cu(I) / Cu(II)-MOF@HMS with different mesopore diameters of embodiments 1-3 of the present application for different sequences of miRNA in Table 4 are shown in Figures (a)-(d), which are T20, T30, T40 and T50, respectively. From the kinetic analysis in the figures, it can be seen that the Cu(I) / Cu(II)-MOF@HMS nanoreactors with different mesopore diameters all have fast catalytic kinetics in the short-chain DNA template ligation reaction. The apparent rate constant k (i.e., k in the figure) of the nanoreactor with a mesopore diameter of 4 nm is 0.031 s⁻¹, the k of the nanoreactor with a mesopore diameter of 5 nm is 0.047 s⁻¹, and the k of the nanoreactor with a mesopore diameter of 9 nm is 0.056 s⁻¹. With the increase of the sequence of the template DNA chain, the reaction kinetics of Cu(I) / Cu(II)-MOF@HMS-4 nm decreases rapidly to 0.002 s⁻¹. This indicates that the nanoreactor with a mesopore diameter of 4 nm has high selectivity and specificity for short-sequence miRNA, the reaction of the nanoreactor with a mesopore diameter of 5 nm decreases in rate with the increase of the sequence of the template, but the reaction still occurs, and the nanoreactor with a mesopore diameter of 9 nm is not significantly affected. obs obs obs

[0050] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.​​​

Claims

1. A core-shell structured nanoreactor for highly specific nucleic acid detection, characterized in that, This core-shell structured nanoreactor uses hollow mesoporous silicon spheres as the shell and Cu(I) / Cu(II)-MOF as the core. The Cu(I) / Cu(II)-MOF is encapsulated within the cavity of a hollow mesoporous silicon sphere; The Cu(I) / Cu(II)-MOF serves as a catalyst for the copper-catalyzed azido-yne cycloaddition reaction.

2. The core-shell structured nanoreactor according to claim 1, characterized in that, The mesopore size of hollow mesoporous silicon spheres is 4~9 nm.

3. The core-shell structured nanoreactor according to claim 1, characterized in that, The copper element in the nanoreactor contains a mixed valence state of +1 and +2 copper.

4. The core-shell structured nanoreactor according to claim 3, characterized in that, The molar percentage of +1 valent copper in the copper element of the nanoreactor is 26%.

5. A method for preparing a core-shell structured nanoreactor according to any one of claims 1 to 4, characterized in that, The preparation method includes the following steps: (1) Synthesis of hollow mesoporous silica spheres: Ammonia and tetraethyl silicate were added sequentially to a mixed solvent consisting of ethanol and water. After stirring, octadecyltrimethoxysilane and tetraethyl silicate were added. After vigorous stirring, SiO2@HMS was obtained by centrifugation. The SiO2@HMS was divided into multiple portions. Each portion of SiO2@HMS was etched in Na2CO3 solution, washed, dried and calcined to obtain hollow mesoporous silica spheres. (2) Synthesis of Cu(II)-MOF@HMS: Hollow mesoporous silica spheres were dispersed in an ethanol solution containing 1,3,5-pyromellitic acid. The ethanol was evaporated under vacuum to obtain H3BTC@HMS intermediate. The H3BTC@HMS intermediate was then dispersed in Cu(OAc)2 aqueous solution. After the water was evaporated under vacuum, N,N-dimethylformamide was added to carry out the reaction. The product was washed after the reaction to obtain Cu(II)-MOF@HMS. (3) Synthesis of core-shell structured nanoreactors: Hydroquinone was dissolved in water to obtain a hydroquinone solution. Cu(II)-MOF@HMS obtained in step (2) was prepared into a suspension. The suspension was added to the hydroquinone solution to carry out a hydrothermal reaction. After the reaction was completed, the product was centrifuged, washed and dried in sequence to obtain a core-shell structured nanoreactor.

6. The preparation method according to claim 5, characterized in that, (1) The amount of Na2CO3 solution used for each SiO2@HMS etching is 26~32 mL.

7. The preparation method according to claim 5, characterized in that, (1) The etching time is 1.5~2 h.

8. The preparation method according to claim 5, characterized in that, (2) The reaction temperature is 30~50 ℃ and the time is 2~4 h.

9. The preparation method according to claim 5, characterized in that, (3) The hydrothermal reaction temperature is 140-160℃ and the time is 15-17 h.

10. A method for nucleic acid detection using the nanoreactor of claim 1, characterized in that, The detection method includes the following steps: (1) The nanoreactor was dispersed in a solution containing P2FAM probe and P1 probe; (2) The target nucleic acid is added to the solution. The target nucleic acid serves as a template to guide the P1 probe with an azide group and the P2FAM probe with an alkyne group to connect in the nanoreactor via a copper-catalyzed azide-alkyne cycloaddition reaction, thereby generating a signal.

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