DNA / metal-organic framework composite nanoscale enzyme, and preparation method and application thereof

Abstract

The present application relates to a kind of DNA / metal organic framework composite nano-enzyme and its preparation method and application, it relates to microneedle technical field.A kind of preparation method of DNA / metal organic framework composite nano-enzyme, the composite nano-enzyme is obtained by incubating metal oxide modified metal organic framework and heme / G-quadruplex DNA together.The present application also provides a kind of microneedle detection system based on the composite nano-enzyme, the detection system includes the composite nano-enzyme and the hydrogel microneedle for extracting skin interstitial fluid.The method extracts human skin interstitial fluid by hydrogel microneedle patch, and the biomarker uric acid in interstitial fluid is detected by the aid of composite nano-enzyme colorimetry.This means of detecting uric acid is compared with the method of detecting uric acid in blood, minimally invasive painless, simple and fast, intuitive and accurate.

Description

TECHNICAL FIELD

[0001] The present application relates to a DNA / metal organic framework composite nanoscale enzyme and a preparation method and application thereof, and relates to the technical field of microneedles. BACKGROUND

[0002] The increase of uric acid content in vivo is a potential risk factor for chronic kidney disease, gout, cardiovascular disease and diabetes. It is essential to monitor uric acid in human blood to prevent the occurrence of related diseases. Traditional uric acid detection is through blood detection, which not only consumes time for sampling, but also requires the use of complex laboratory equipment for analysis. Moreover, it causes mental distress to patients, brings invasive physical pain and trauma to patients, and increases the possibility of infection of patients due to the breaking of the skin barrier. The hydrogel microneedle penetrates the stratum corneum of the skin to reach the epidermis and dermis to extract the interstitial fluid of the skin, and through the detection of biomarkers in the interstitial fluid, the biological information about cells and tissues can be grasped. It is a safe, painless and minimally invasive means, and is convenient and easy to use. At the same time, the detection system of uric acid is constructed by combining the nanoscale enzyme colorimetric method. The DNA / metal organic framework composite nanoscale enzyme combines DNA enzyme and metal organic framework, has the advantages of sequence variability, information, structure designability and function controllability, and also has excellent peroxidase-like activity. The colorimetric method based on nanoscale enzyme has been widely concerned in the field of uric acid detection, which is convenient, fast, simple and intuitive, and is conducive to the clinical diagnosis of related diseases. SUMMARY

[0003] In view of the above defects or improvement needs of the prior art, the present application provides a DNA / metal organic framework composite nanoscale enzyme, and provides a method for colorimetric detection of uric acid content by using the composite nanoscale enzyme. The method extracts the interstitial fluid of human skin by a hydrogel microneedle patch, and detects the biomarker uric acid in the interstitial fluid by means of a composite nanoscale enzyme colorimetric method. The composite nanoscale enzyme has peroxidase activity and catalyzes the substrate to produce color change under the action of H2O2. The present application solves the technical problems of high cost and instability of natural enzymes, low patient compliance and susceptibility of blood detection, etc.

[0004] A preparation method of a DNA / metal organic framework composite nanoscale enzyme, the composite nanoscale enzyme is obtained by incubating a metal oxide modified metal organic framework and a hemin / G-quadruplex DNA together.

[0005] In the above technical solution, the hemin / G-quadruplex DNA solution is used as a dispersion medium, the metal oxide modified metal organic framework is ultrasonically treated with the hemin / G-quadruplex DNA solution for 5-30 min, and then incubated at a temperature of 0-40℃ for 5-300 min to obtain a DNA / metal organic framework composite nanoscale enzyme solution with a mass concentration of 0.1-10 mg / mL, wherein,

[0006] The heme / G quadruplex DNA solution is obtained by mixing a 0.4 μM heme solution and a 0.8 μM G quadruplex DNA solution in a volume ratio of 1:9 to 1:1.

[0007] In the above technical solution, the metal oxide modified metal organic framework is prepared according to the following method:

[0008] The metal oxide and the metal organic framework are dispersed in deionized water in a mass ratio of 1:1 to 10, mixed, ultrasonicated for 5 to 60 minutes, centrifuged at 3000 rpm for 5 to 30 minutes, washed, centrifuged, and dried at 40 to 80° C. to obtain a metal oxide modified metal organic framework.

[0009] Among them, the metal organic framework is a cerium metal organic framework, an iron metal organic framework, a zirconium metal organic framework, and a cobalt metal organic framework; the metal oxide is copper oxide, cuprous oxide, ferrosoferric oxide, cerium dioxide, and ruthenium oxide.

[0010] In the above technical solution, the ratio of the metal oxide and metal organic framework to deionized water is 1 mg: 1 to 10 mL.

[0011] Furthermore, the metal organic framework is prepared by the following method: dissolving a metal salt and terephthalic acid in N,N'-dimethylformamide at a mass ratio of 1:1 to 10 to obtain a mixed solution, placing the mixed solution in a reactor for hydrothermal reaction at a reaction temperature of 120°C and a reaction time of 6 to 48 hours.

[0012] The metal salt is zirconium chloride, ferric chloride, cerium nitrate, or cobalt nitrate; and the ratio of the metal salt to N,N'-dimethylformamide is 1 mg:0.1-1 mL.

[0013] In the above technical solution, the heme / G quadruplex DNA solution is prepared by the following method: the oligonucleotide sequence dry powder is centrifuged at 4000 rpm for 5 minutes, deionized water is added to prepare a 100 μM mother solution, the mother solution is diluted to 0.8 μM with 10 mM Tris-HCl buffer (pH 7.0), and the mixture is incubated at room temperature for 3 hours to obtain a G quadruplex DNA solution. 0.4 μM heme solution is added, and the heme solution and the G quadruplex DNA solution are mixed in a volume ratio of 1:9 to 1:1, and the mixture is allowed to stand for 3 hours to obtain a heme / G quadruplex DNA solution, which is stored at 4°C.

[0014] In the above technical solution, the oligonucleotide sequence is 5'-TTGGGTGGGTGGGTGGGTC-3', which can be purchased commercially or prepared using the method disclosed in the prior art.

[0015] Preferably, the heme solution and the G quadruplex DNA solution are mixed at a volume ratio of 3:7.

[0016] Furthermore, the G quadruplex is a parallel quadruplex or an antiparallel quadruplex.

[0017] Another object of the present invention is to provide a DNA / metal organic framework composite nanozyme prepared using the above method.

[0018] Furthermore, the composite nanozyme has peroxidase activity.

[0019] The DNA / metal organic framework composite nanozyme described in the present invention has peroxidase-like activity and can catalyze the chromogenic substrate TMB under the action of H2O2, causing the solution to change color.

[0020] Another object of the present invention is to provide the use of the above-mentioned DNA / metal organic framework composite nanozyme in the quantitative or qualitative detection of uric acid.

[0021] Another object of the present invention is to provide a microneedle detection system based on the composite nanozyme.

[0022] A microneedle detection system based on the composite nanozyme comprises the composite nanozyme and a hydrogel microneedle for extracting skin interstitial fluid.

[0023] In the above technical solution, the hydrogel microneedle patch can extract skin interstitial fluid. The hydrogel microneedle patch can be prepared according to the methods disclosed in the prior art, such as physical cross-linking method or chemical cross-linking method.

[0024] Furthermore, the physical cross-linking method is a freeze-thaw cycle method, and the chemical cross-linking method can use an aldehyde cross-linking agent such as glutaraldehyde.

[0025] Furthermore, the hydrogel microneedle patch can be prepared by the following method: 0.4g of polyvinyl alcohol is added to deionized water to prepare a 10wt% solution; 0.02g of sodium polyacrylate is added to deionized water to prepare a 0.5wt% solution, stirred evenly, 20μL of cross-linking agent glutaraldehyde is added, and stirred evenly to obtain a matrix solution; the matrix solution is injected into a PDMS mold, centrifuged, and dried to obtain a microneedle patch.

[0026] Furthermore, the matrix material includes polyvinyl alcohol, polyvinyl pyrrolidone, sodium polyacrylate, chitosan, sodium alginate, and hyaluronic acid.

[0027] Furthermore, the needle tip is pyramidal or conical, the needle tip length is 100 to 1000 μm, and the distance between two adjacent needle tips is 100 to 1500 μm.

[0028] A uric acid detection method based on the microneedle detection system of the composite nanozyme comprises the following steps: transferring the microneedle to a centrifuge tube containing 1 mL of PBS solution, placing the microneedle in a water bath at 30-80°C for 5-30 minutes or centrifuging at a speed of 1000-3000 rpm for 5-30 minutes; detecting and analyzing the recovered uric acid using a DNA / metal organic framework composite nanozyme colorimetric solution, wherein the volume ratio of the recovered liquid to the colorimetric solution is 1:1-10, reacting at room temperature for 5-30 minutes, and then testing.

[0029] In the above technical solution, the DNA / metal organic framework composite nanozyme provided by the present invention has peroxidase-like activity, and under the action of H2O2, it can change the color of the substrate to obtain a stable color-developing solution.

[0030] The color developing solution consists of the following components: 75 μL of 1 mg / mL DNA / metal organic framework composite nanozyme solution, 25 μL of 10 mM TMB solution, 125 μL of 10 mM H2O2 solution, and 50 μL of pH 5.5 sodium acetate buffer solution.

[0031] The present invention also provides applications of the composite nanozyme combined with microneedle detection system in the fields of disease detection, food detection or environmental monitoring.

[0032] The beneficial effects of the present invention are as follows: the DNA / metal organic framework composite nanozyme provided by the present invention has peroxidase activity, catalyzes the substrate to produce a color change under the action of H2O2, and can be used to detect uric acid. The present invention further provides a composite nanozyme combined with a microneedle detection system, which can be used for the visual detection of uric acid in interstitial fluid. This method extracts interstitial fluid from human skin through a hydrogel microneedle patch and detects the biomarker uric acid in the interstitial fluid using a composite nanozyme colorimetric method. Compared with the method of detecting uric acid in blood, this method of detecting uric acid is minimally invasive and painless, simple and rapid, intuitive and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the detection principle of the microneedle patch for colorimetric detection of uric acid concentration provided in Example 1 of the present invention. As shown in the figure, when the microneedle patch provided by the present invention is used for colorimetric detection of uric acid concentration, the microneedles extract interstitial fluid in the skin, and the uric acid in the interstitial fluid is recovered; the DNA enzyme composite metal-organic framework with peroxidase-like activity oxidizes the chromogenic substance under the action of H2O2 to produce a color change, and uric acid inhibits this process, thereby realizing the detection of uric acid in the interstitial fluid.

[0034] Figure 2 is a microscope image of the microneedle provided in Example 1 of the present invention;

[0035] Figure 3Schematic diagram of the swelling performance of the microneedle patch provided in Example 1 of the present invention in agarose gel;

[0036] Figure 4 Schematic diagram of the mechanical strength of the microneedle patch provided in Example 1 of the present invention;

[0037] Figure 5 The enzyme activity of the composite nanozyme prepared in Example 1 of the present invention was investigated;

[0038] Figure 6 The composite nanozyme prepared in Example 1 of the present invention is used to detect uric acid.

[0039] Figure 7 The composite nanozyme prepared in Example 1 of the present invention and the uric acid meter are used to perform in vivo detection of uric acid. DETAILED DESCRIPTION

[0040] The following non-limiting examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are all commercially available unless otherwise specified.

[0042] Example 1

[0043] 1. Preparation of hydrogel microneedle patches

[0044] A method for preparing a hydrogel microneedle patch for colorimetric detection of uric acid concentration, the method comprising the following steps:

[0045] 1. Add 0.4 g of polyvinyl alcohol to deionized water to make a 10 wt% solution; add 0.02 g of sodium polyacrylate to deionized water to make a 0.5 wt% solution, stir evenly, add 20 μL of crosslinking agent glutaraldehyde, and stir evenly to obtain a matrix solution.

[0046] 2. The matrix solution was evenly poured into the PDMS microneedle mold. After centrifugation, the matrix solution was completely filled into the mold. After drying, the mold was removed to obtain a microneedle patch. The microneedle patch measures 12.4 x 12.4 mm, with a needle length of 800 μm and an array of 11 x 11.

[0047] The obtained microneedle patch was observed under a microscope. Figure 2 It can be seen that the microneedle patch prepared in this embodiment is pyramid-shaped and the microneedle tips are complete.

[0048] The microneedle patch prepared by the present invention was inserted into agarose gel (1.4 wt%) after being pierced through the sealing film to test its swelling performance. The results are as follows: Figure 3As shown in the figure, the microneedle patch reached swelling equilibrium in 15 minutes and had a good ability to extract interstitial fluid.

[0049] The mechanical strength of the microneedle patch prepared by the present invention was investigated, and the results were as follows: Figure 4 As shown, the microneedle patch has good mechanical strength.

[0050] 2. Preparation of DNA / metal-organic framework composite nanozymes

[0051] A method for preparing a composite nanozyme for colorimetric detection of uric acid concentration, the method comprising the following steps:

[0052] Add 0.13g of copper chloride and 3.0g of polyvinyl pyrrolidone to 100mL of deionized water and incubate in a 25°C water bath until fully dissolved. Once dissolved, add 10mL of sodium hydroxide (2M concentration) to form a turbid blue solution. Continue stirring for 30 minutes. Add 10mL of ascorbic acid and stir at 60°C for 3 hours. Centrifuge, wash, and dry to obtain cuprous oxide.

[0053] 0.14g of zirconium chloride and 0.10g of terephthalic acid were dissolved in 25mL of N,N'-dimethylformamide and the reaction mixture was incubated at 120°C for 24h, centrifuged, washed, and dried to obtain the zirconium metal-organic framework (MOF). 20mg of cuprous oxide and 20mg of the MOF were dispersed in 40mL of deionized water, sonicated for 40min, centrifuged at 3000rpm for 15min, washed, and dried to obtain the modified MOF. 1mg of the modified MOF was dispersed in 1mL of a heme / G-quadruplex DNA solution (0.8μM G-quadruplex DNA solution and 0.4μM heme solution in a volume ratio of 7:3), sonicated at room temperature for 20min, and incubated at 4°C for 30min to obtain the DNA / MOF composite nanozyme solution.

[0054] The enzymatic activity of the resulting DNA / MOF composite nanozyme was compared with that of a single nanozyme. 1 mg of modified MOF, 1 mg of metal oxide, and 1 mg of MOF were each dispersed in 1 mL of deionized water to obtain a single nanozyme solution.

[0055] 75 μL of 1 mg / mL DNA / metal organic framework composite nanozyme solution, 25 μL of 10 mM TMB solution, 125 μL of 10 mM H2O2 solution, and 50 μL of pH 5.5 sodium acetate buffer solution were reacted at room temperature for 30 min to obtain solution 1.

[0056] 75 μL of 1 mg / mL modified metal organic framework solution, 25 μL of 10 mM TMB solution, 125 μL of 10 mM H2O2 solution, and 50 μL of pH 5.5 sodium acetate buffer solution were reacted at room temperature for 30 min to obtain solution 2.

[0057] 75 μL of 1 mg / mL metal oxide solution, 25 μL of 10 mM TMB solution, 125 μL of 10 mM H2O2 solution, and 50 μL of pH 5.5 sodium acetate buffer solution were reacted at room temperature for 30 min to obtain solution 3.

[0058] 75 μL of 1 mg / mL metal organic framework solution, 25 μL of 10 mM TMB solution, 125 μL of 10 mM H2O2 solution, and 50 μL of pH 5.5 sodium acetate buffer solution were reacted at room temperature for 30 min to obtain solution 4.

[0059] The results were as follows: Figure 5 As shown, DNA / metal organic framework composite nanozymes have higher enzymatic activity.

[0060] 3. Uric acid detection method

[0061] The DNA / metal organic framework composite nanozyme solution prepared by the present invention is used to detect uric acid and establish a standard curve , the method comprises the following steps:

[0062] 75 μL of 1 mg / mL DNA / metal organic framework composite nanozyme solution, 25 μL of 10 mM TMB solution, 125 μL of 10 mM H2O2 solution, and 50 μL of pH 5.5 sodium acetate buffer solution were reacted at room temperature for 30 min to obtain a color development solution. 100 μL of the reaction solution was added to a 96-well plate and the absorbance was measured at 652 nm. The value was A 空 Prepare 10, 25, 100, 200, and 500 μM uric acid solutions, add 50 μL of uric acid solution and 100 μL of color development solution to a centrifuge tube, vortex mix, react at room temperature for 20 minutes, take 100 μL of the reaction solution and add it to a 96-well plate, measure the absorbance at 652 nm, and the value is A. 测 A standard curve was established with the concentration C of uric acid solution as the horizontal axis and the absorbance difference ΔA (ΔA=A 空 -A 测 ) is the vertical axis, and the result is as follows Figure 6 As shown, the standard curve y=0.0012x+0.0108(R 2 =0.9997).

[0063] The DNA / metal organic framework composite nanozyme solution prepared by the present invention is used to detect uric acid in vitro, and the method comprises the following steps:

[0064] Using pig skin as an in vitro model, the skin was thawed in saline and soaked in uric acid solutions of varying concentrations (0.1, 0.5, 1, 3, and 5 mM) for 30 minutes. The skin was then removed and dried with filter paper. After microneedles were applied to the skin for 10 minutes, the microneedles were transferred to a centrifuge tube, 1 mL of PBS was added, and the tube was placed in a 40°C waterbath for 10 minutes to recover the uric acid extracted by the microneedles. The recovered liquid was transferred to a new centrifuge tube and detected using the composite nanozyme. A 1 mg / mL DNA / metal-organic framework composite nanozyme solution (75 μL), 25 μL of 10 mM TMB, 125 μL of 10 mM H₂O₂, and 50 μL of pH 5.5 sodium acetate buffer were reacted at room temperature for 30 minutes to produce a chromogenic solution (blue). 50 μL of the recovered liquid and 100 μL of the chromogenic solution were added to the centrifuge tube, vortexed, and allowed to react at room temperature for 20 minutes. The chromogenic solution faded as the uric acid concentration in the recovered solution increased.

[0065] The DNA / metal organic framework composite nanozyme solution prepared by the present invention is used to detect uric acid in vivo, and the method comprises the following steps:

[0066] Using SD rats as a model, the SD rats were divided into three groups. The first group was injected with 250 mg / kg uric acid and 300 mg / kg potassium oxonate (UA+Potassiumoxonate); the second group was injected with 250 mg / kg uric acid (UA); and the third group was injected with normal saline (Saline).

[0067] After pressing the hydrogel microneedle on the rat's back skin for 10 minutes, the microneedle was transferred to a centrifuge tube, 1 mL of PBS solution was added, and the tube was placed in a 40°C water bath for 10 minutes to recover the uric acid extracted by the microneedle. The recovered liquid was transferred to a new centrifuge tube, and the recovered uric acid was detected with the help of the composite nanozyme. 75 μL of 1 mg / mL DNA / metal organic framework composite nanozyme solution, 25 μL of 10 mM TMB solution, 125 μL of 10 mM H2O2 solution, and 50 μL of pH 5.5 sodium acetate buffer solution were reacted at room temperature for 30 minutes to obtain a colorimetric solution. 50 μL of the recovered liquid and 100 μL of the colorimetric solution were added to the centrifuge tube, vortexed to mix, and reacted at room temperature for 20 minutes. 100 μL of the reaction solution was added to a 96-well plate, the absorbance was measured at 652 nm, and the in vivo concentration was calculated by substituting it into the standard curve. The results were compared with the uric acid meter, as shown in the figure. Figure 7 As shown, the calculated in vivo results of the first group (266 μM) were basically consistent with the uric acid meter (273 μM). The uric acid meter showed that L0 represented a uric acid value lower than 180 μM. The second group (188 μM) and the third group (125 μM) also confirmed this result through calculation.

Claims

1. A method for preparing a DNA / metal organic framework composite nanozyme, characterized by: The composite nanozyme is obtained by incubating a metal oxide-modified metal organic framework with heme / G quadruplex DNA, as follows: Using a heme / G-quadruplex DNA solution as a dispersion medium, the metal oxide-modified metal organic framework and the heme / G-quadruplex DNA solution were ultrasonically incubated for 5-30 min at a temperature of 0-40°C for 5-300 min to obtain a DNA / metal organic framework composite nanozyme solution with a mass concentration of 0.1-10 mg / mL. The heme / G-quadruplex DNA solution was prepared by mixing 0.4 µM heme solution and 0.8 µM G-quadruplex DNA solution in a volume ratio of 1:9-1:

1. The heme / G-quadruplex DNA solution was prepared as follows: oligonucleotide sequence dry powder was centrifuged at 4000 rpm for 5 minutes, deionized water was added to prepare a 100 µM stock solution, the stock solution was diluted to 0.8 µM with 10 mM Tris-HCl buffer at pH 7.0, and the mixture was incubated at room temperature for 3 hours to obtain a G-quadruplex DNA solution, 0.4 µM heme solution was added, and the heme solution and G-quadruplex DNA solution were mixed at a volume ratio of 1:9 to 1:1, and the mixture was incubated for 3 hours to obtain a heme / G-quadruplex DNA solution, which was stored at 4°C. The metal oxide modified metal organic framework is prepared according to the following method: the metal oxide and the metal organic framework are dispersed in deionized water and mixed in a mass ratio of 1:1 to 10, ultrasonicated for 5 to 60 minutes, centrifuged at 3000 rpm for 5 to 30 minutes, washed, centrifuged, and dried at 40 to 80°C, wherein the metal organic framework is a cerium metal organic framework, an iron metal organic framework, a zirconium metal organic framework, or a cobalt metal organic framework; and the metal oxide is cuprous oxide, ferrous oxide, cerium dioxide, or ruthenium oxide.

2. The method according to claim 1, wherein: The oligonucleotide sequence is 5'-TTGGGTGGGTGGGTGGGTC-3'.

3. The DNA / metal organic framework composite nanozyme prepared by the method of claim 1 or 2, characterized in that: The composite nanozyme has peroxidase activity and can catalyze the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) under the action of hydrogen peroxide (H2O2), causing the solution to change color.

4. Use of the composite nanozyme according to claim 3 in constructing a microneedle detection system for quantitative or qualitative detection of uric acid.

5. A microneedle detection system based on the composite nanozyme according to claim 3, the detection system comprising the composite nanozyme according to claim 3 and a hydrogel microneedle for extracting skin interstitial fluid.

6. A method for detecting uric acid based on the microneedle detection system of the composite nanozyme according to claim 5, characterized in that: The method is a colorimetric method, specifically: Transfer the microneedle to a centrifuge tube containing 1 mL of PBS solution and incubate in a 30-80°C water bath for 5-30 minutes or centrifuge at 1000-3000 rpm for 5-30 minutes. Detect and analyze the recovered uric acid using a DNA / metal organic framework composite nanozyme colorimetric solution. The volume ratio of the recovered liquid to the colorimetric solution is 1:1-10. React at room temperature for 5-30 minutes and wait for detection. The color development solution was prepared by reacting 75 µL of 1 mg / mL DNA / metal organic framework composite nanozyme solution, 25 µL of 10 mM TMB solution, 125 µL of 10 mM H2O2 solution, and 50 µL of pH 5.5 sodium acetate buffer solution at room temperature for 30 min.

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