Synthesis of a nano-hollow metal-organic framework material and application thereof to nucleic acid detection

Abstract

The application relates to the technical field of nucleic acid detection, and discloses synthesis of a nano hollow metal organic framework material and application of the nano hollow metal organic framework material in nucleic acid detection, and aims to solve the problem that the size of an inorganic catalyst used in a CuAAC reaction is too large and uncontrollable, thereby restricting detection. The application has the remarkable beneficial effects that the application of the nano hollow Cu(I) metal organic material in nucleic acid detection has the following advantages. Firstly, the hollow structure formed by internal etching and external protection of the material significantly improves the catalytic efficiency, so that the efficiency of the CuAAC reaction in nucleic acid detection is greatly improved. Secondly, since Cu(I) is fixed in the material, the escape and biological toxicity of Cu(I) ions are reduced, and the biological safety is improved. In addition, the material also has the ability of fluorescent detection marking, and can realize high-sensitivity and high-specificity nucleic acid detection.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to the synthesis of a nano-hollow metal-organic framework material and its application in nucleic acid detection. Background Technology

[0002] Bioorthogonal reactions, as chemical reactions that do not interact with or interfere with biological systems, offer a novel approach to chemical biology research by achieving highly selective chemical reactions in complex samples. Click chemistry is a commonly used bioorthogonal reaction, attracting widespread attention in biosensing, DNA and RNA labeling, small molecule labeling, drug design and synthesis, and oligonucleotide functionalization materials. CuAAC reaction is one of the most frequently used click reactions. CuAAC reactions are typically highly efficient at ambient temperatures, produce no byproducts, and are simple to process and purify.

[0003] MOF materials are porous materials with metal ions as nodes and organic ligands as a framework. MOFs have attracted increasing attention in fields such as gas separation, gas storage, drug delivery, and heterogeneous catalysis. Cu(I)-MOFs are promising catalysts for the CuAAC reaction. Compared with traditional supported catalysts, the catalytic sites of Cu-MOFs are fixed at the nodes through the framework, exhibiting strong stability and resistance to leaching. However, the reported synthesis conditions of Cu(I)-MOFs are demanding, and the inability to control the material size makes it difficult to apply Cu(I)-MOF-catalyzed CuAAC reactions in biological conditions. Summary of the Invention

[0004] The purpose of this invention is to solve the problem that the inorganic catalyst used in the CuAAC reaction is too large and the size is uncontrollable, which leads to limited detection. The invention provides a method for synthesizing a nano-hollow metal-organic framework material and applying it to nucleic acid detection.

[0005] The present invention discloses a method for synthesizing a nano-hollow metal-organic framework material, which is carried out according to the following steps:

[0006] Step 1: Mix Cu(NO3)2·3H2O with polyvinylpyrrolidone, then add methanol and stir to obtain a blue solution;

[0007] Step 2: Dissolve 1,3,5-pyromellitic acid in methanol, add it dropwise to the blue solution obtained in Step 1, and then let it stand at 25-30℃; centrifuge to obtain crude Cu(II)-MOF.

[0008] Step 3: After washing the crude Cu(II)-MOF with methanol, it was dried under vacuum to obtain a blue powder;

[0009] Step 4: Synthesis of Cu(I)-MOF;

[0010] 1) Dissolve hydroquinone in water and place it in the polytetrafluoroethylene liner of the reaction vessel;

[0011] 2) Take the blue powder from step 3 and disperse it in water by ultrasonication to obtain a suspension. Add it to the polytetrafluoroethylene liner of the reaction vessel from step 1), add methanol and polyvinylpyrrolidone, and react at 140-160°C for 10-20 hours. After centrifugation, obtain crude Cu(I)-MOF, wash it with methanol, and dry it under vacuum to obtain a black powder. This completes the synthesis of the nano-hollow metal-organic framework material.

[0012] Furthermore, the mass ratio of Cu(NO3)2·3H2O to polyvinylpyrrolidone in step one is 1:1.1 to 1.5.

[0013] Furthermore, the mass-to-volume ratio of Cu(NO3)2·3H2O to methanol in step one is 1g:40-60mL.

[0014] Furthermore, the mass-to-volume ratio of 1,3,5-pyromellitic acid to methanol in step one is 1 g: 80-100 mL.

[0015] Furthermore, the vacuum drying conditions described in steps one and two are both 70°C drying for 5 to 10 hours.

[0016] Further, the mass-to-volume ratio of hydroquinone to water in step 1) is 1 g: 8-10 mL.

[0017] Furthermore, in step 2), the mass-to-volume ratio of the blue powder to water is 1g:15-20mL.

[0018] Furthermore, the ultrasonic dispersion time in step 2) is 30 min.

[0019] Furthermore, in step 2), the volume-to-mass ratio of methanol to polyvinylpyrrolidone is 1 mL: 3–5 g.

[0020] The present invention relates to an application of a nano-hollow metal-organic framework material, wherein the nano-hollow metal-organic framework material is used for nucleic acid detection.

[0021] This invention aims to synthesize nanoscale Cu(I)-MOFs with controllable size via a stepwise method to catalyze the CuAAC reaction for bioorthogonal catalysis. First, Cu(II)-MOF materials of different sizes and dispersions are synthesized under mild conditions. The Cu(II)-MOFs are then grown into mixed Cu(I) and Cu(II) MOF materials in the presence of a reducing agent. Precursors of different sizes are synthesized by varying the molecular weight and amount of polyvinylpyrrolidone (PVP). The reduction of these precursors yields materials of different sizes. The Cu(I)-MOFs possess nanoscale dimensions and a hollow structure that provides a larger specific surface area, resulting in excellent catalytic activity. Furthermore, the Cu(II) in the framework maintains material stability and improves catalyst cycling performance. The superior catalytic performance of Cu(I)-MOFs enables the catalysis of the CuAAC reaction for applications in nucleic acid detection sensing and labeling imaging.

[0022] The present invention has the following beneficial effects:

[0023] The nano-hollow Cu(I) organometallic material of this invention exhibits significant beneficial effects in nucleic acid detection. Firstly, the hollow structure formed through internal etching and external protection significantly improves catalytic efficiency, greatly enhancing the efficiency of the CuAAC reaction in nucleic acid detection. Secondly, since Cu(I) is immobilized within the material, Cu(I) ion escape and biotoxicity are reduced, improving biosafety. Furthermore, this material possesses fluorescent labeling capabilities, enabling highly sensitive and specific nucleic acid detection. In summary, this invention provides an efficient, low-cost, and large-scale nucleic acid detection method with broad application prospects. Attached Figure Description

[0024] Figure 1 This is an XRD diagram of the nano-hollow Cu(I) metal-organic material of the present invention;

[0025] Figure 2 These are TEM images of the nano-hollow Cu(I) metal-organic materials and precursors of this invention.

[0026] Figure 3 This is a SEM image of the nano-hollow Cu(I) metal-organic material of this invention;

[0027] Figure 4 The attached diagram shows the FT-IR and N2 adsorption / desorption of the nano-hollow Cu(I) metal-organic material of this invention.

[0028] Figure 5 TEM image of Cu(I) organometallic material controlled in Example 2;

[0029] Figure 6 This is a catalytic kinetic diagram for nucleic acid detection in Example 3;

[0030] Figure 7 This is a linear range graph for nucleic acid detection in Example 3;

[0031] Figure 8 This is a linear graph of the fluorescent labeling in Example 4. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0033] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0034] Example 1

[0035] 0.9 g of Cu(NO3)2·3H2O and 1.2 g of polyvinylpyrrolidone (PVP) (MW = 1,300,000) were weighed and placed in a round-bottom flask. 50 mL of methanol was added and the mixture was stirred vigorously for 5 min to obtain a blue solution. 0.43 g of 1,3,5-pyromellitic acid was weighed and dissolved in 50 mL of methanol. This solution was then slowly added dropwise to the blue solution and allowed to stand at 27 °C for 24 h. The mixture was centrifuged at 11,000 rpm to obtain crude Cu(II)-MOF1 products of different sizes. After washing three times with methanol, the products were dried under vacuum at 70 °C for 8 h to obtain a blue powder. Before use, the powder was activated under vacuum at 120 °C for 5 h.

[0036] The synthesis of Cu(I)-MOF was achieved via a hydrothermal reduction method. 1 g of hydroquinone was dissolved in 8 mL of H₂O and transferred to a 25 mL polytetrafluoroethylene (PTFE) liner in a reaction vessel. 0.1 g of Cu(II)-MOF was weighed and ultrasonically dispersed in 2 mL of H₂O for 30 min. The suspension was added to the PTFE liner, along with 2 mL of methanol and 8 mg of PVP (MW = 1300000). The reaction was carried out at 150 °C for 16 h. Centrifugation at 11000 rpm yielded crude Cu(I)-MOF. After washing three times with methanol, the product was dried under vacuum at 70 °C for 8 h to obtain a blackish-green powder, yielding a nano-hollow metal-organic framework material used as an inorganic catalyst. Before use, the catalyst was activated under vacuum at 120 °C for 5 h.

[0037] Cu(I)-MOF was characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), nitrogen adsorption-desorption isotherms, and Fourier transform infrared spectroscopy (FT-IR), confirming its nano-hollow structure and high specific surface area. Figures 1 to 4It is understood that the nano-hollow metal-organic framework material of this embodiment is used to catalyze the CuAAC reaction, linking DNA and fluorescent labeling, while maintaining high catalytic efficiency and reducing the amount of Cu(I) in the system, thus reducing biotoxicity.

[0038] Example 2

[0039] 0.9 g of Cu(NO3)2·3H2O and 0.4 g of polyvinylpyrrolidone (PVP) (MW = 8000) were weighed and placed in a round-bottom flask. 50 mL of methanol was added and the mixture was stirred vigorously for 5 min to obtain a blue solution. 0.43 g of 1,3,5-pyromellitic acid was weighed and dissolved in 50 mL of methanol. This solution was then slowly added dropwise to the blue solution and allowed to stand at 27 °C for 24 h. The mixture was centrifuged at 11000 rpm to obtain crude Cu(II)-MOF1 products of different sizes. After washing three times with methanol, the products were dried under vacuum at 70 °C for 8 h to obtain a blue powder. Before use, the powder was activated under vacuum at 120 °C for 5 h.

[0040] The synthesis of Cu(I)-MOF was achieved via a hydrothermal reduction method. 1 g of hydroquinone was dissolved in 8 mL of H₂O and transferred to a 25 mL polytetrafluoroethylene (PTFE) liner in a reaction vessel. 0.1 g of Cu(II)-MOF was weighed and ultrasonically dispersed in 2 mL of H₂O for 30 min. The suspension was added to the PTFE liner, along with 2 mL of methanol and 8 mg of PVP (MW = 1300000). The reaction was carried out at 150 °C for 16 h. Centrifugation at 11000 rpm yielded crude Cu(I)-MOF. After washing three times with methanol, the product was dried under vacuum at 70 °C for 8 h to obtain a blackish-green powder, thus yielding a nano-hollow metal-organic framework material. Before use, the material was activated under vacuum at 120 °C for 5 h. Figure 5 As can be seen, this embodiment achieves size control of nano-hollow metal-organic framework materials.

[0041] Example 3

[0042] In Example 3, the nucleic acid detection capability of the nano-hollow Cu(I) metal-organic material of Example 1 was tested.

[0043] Denaturing polyacrylamide gel electrophoresis (PAGE) assay using Cu(I)-MOF (Cu(I)-MOF)-catalyzed CuAAC reaction of DNA template. First, 200 nM P2FAM probe, 200 nM P1 probe, and 200 nM template DNA were mixed (nucleic acid fragments are shown in Table 3-5), and then 400 μg·mL⁻¹ Cu(I)-MOF was added to Phys2 buffer. All control groups were performed without template DNA or Cu(I)-MOF. A 20 μL sample of the reaction solution was immediately mixed with 10 μL of 8M urea (50%, v / v) and 10 μL of DNA loading buffer (6×), and loaded onto a 20% denaturing polyacrylamide gel for PAGE (8M urea in 1× TBE buffer). The denaturing gel was imaged using a fluorescence imager.

[0044] Figure 6 Figure 7 This indicates that the nano-hollow metal-organic framework material of this embodiment can rapidly connect DNA, and the connection efficiency is related to the concentration of the DNA target, thus it can be used as a catalyst for nucleic acid detection.

[0045] Table 1. Nucleic acid fragments in denaturing polyacrylamide gel electrophoresis.

[0046]

[0047] Example 4

[0048] In Example 4, the specific effect of pH on the performance of the CHIT-Fc electrochemical sensor was explored.

[0049] Fluorescence was observed in the CuAAC reaction catalyzed by the Cu(I) organometallic material (Cu(I)-MOF) of Example 1. An 8 mM solution of 3-azido-7-hydroxycoumarin DMSO and an 8 mM solution of phenylacetylene DMSO were prepared. 6.25 μL of 3-azido-7-hydroxycoumarin DMSO and 12.5 μL of phenylacetylene DMSO were added to 5 mL of H₂O, followed by the addition of 500 μg of Cu(I)-MOF. The reaction was allowed to proceed for 5–75 min. After centrifugation at 11000 rpm to separate the Cu(I)-MOF, the reaction solution was analyzed using a fluorescence spectrophotometer. Figure 8 This indicates that Cu(I) organometallic materials (Cu(I)-MOF) can efficiently catalyze fluorescent reactions, and since the materials themselves do not exhibit fluorescence, accurate quantitative fluorescent labeling can be performed.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the implementation of the present invention. Those skilled in the art can easily make corresponding modifications or alterations based on the main concept and spirit of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of protection claimed in the claims.

Claims

1. A method for synthesizing a nano-hollow metal-organic framework material, characterized in that... It is done according to the following: Step 1: Mix Cu(NO3)2·3H2O with polyvinylpyrrolidone, then add methanol and stir to obtain a blue solution; the mass ratio of Cu(NO3)2·3H2O to polyvinylpyrrolidone is 1:1.1~1.5; the mass-volume ratio of Cu(NO3)2·3H2O to methanol is 1g:40~60mL. Step 2: Dissolve 1,3,5-pyromellitic acid in methanol, add it dropwise to the blue solution obtained in Step 1, and then let it stand at 25~30 ℃; centrifuge to obtain crude Cu(II)-MOF; Step 3: After washing the crude Cu(II)-MOF with methanol, it was dried under vacuum to obtain a blue powder; Step 4: Synthesis of Cu(I)-MOF; 1) Dissolve hydroquinone in water and place it in the polytetrafluoroethylene liner of the reaction vessel; 2) Take the blue powder from step 3 and disperse it in water by ultrasonication to obtain a suspension. Add the suspension to the polytetrafluoroethylene liner of the reaction vessel from step 1), add methanol and polyvinylpyrrolidone, and react at 140~160℃ for 10~20h. After centrifugation, obtain crude Cu(I)-MOF, wash with methanol, and vacuum dry to obtain black powder, thus completing the synthesis of the nano-hollow metal-organic framework material. The mass-volume ratio of blue powder to water is 1g:15~20mL.

2. The method for synthesizing a nano-hollow metal-organic framework material according to claim 1, characterized in that... The mass-to-volume ratio of 1,3,5-pyromellitic acid to methanol in step two is 1 g: 80~100 mL.

3. The method for synthesizing a nano-hollow metal-organic framework material according to claim 1, characterized in that... The vacuum drying conditions described in steps 3 and 2) are both 70 ℃ for 5~10 h.

4. The method for synthesizing a nano-hollow metal-organic framework material according to claim 1, characterized in that... The mass-to-volume ratio of hydroquinone to water in step 1) is 1g:8~10mL.

5. The method for synthesizing a nano-hollow metal-organic framework material according to claim 1, characterized in that... The ultrasonic dispersion time in step 2) is 30 min.

6. The method for synthesizing a nano-hollow metal-organic framework material according to claim 1, characterized in that... In step 2), the volume-to-mass ratio of methanol to polyvinylpyrrolidone is 1 mL: 3~5 g.

7. The application of the nano-hollow metal-organic framework material synthesized as described in claim 1, characterized in that... The aforementioned nano-hollow metal-organic framework material is used for nucleic acid detection.

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