Magnetic enzyme-MOF (Metal Organic Framework) compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological analysis, and discloses a magnetic enzyme-MOF compound, a preparation method thereof and application in preparation of a miRNAs marker detection kit.The disclosed magnetic enzyme-MOF compound is composed of magnetic materials Fe3O4 (at) MOF, AMG (at) MOF, a first aptamer chain UG and a second aptamer chain ZG, when the magnetic materials make contact with target miRNAs, the AMG (at) MOF is released from the surface of the Fe3O4 (at) MOF, and when the magnetic materials make contact with the target miRNAs, the AMG (at) MOF is released from the surface of the first aptamer chain UG and the second aptamer chain ZG; colorimetric detection and electrochemical detection can be respectively carried out on the content of the target miRNAs by reducing the content of the starch and increasing the content of the glucose.

Description

Technical Field

[0001] The invention belongs to the technical field of bioanalysis and discloses a magnetic enzyme-MOF complex and a preparation method thereof and an application thereof in preparing a miRNAs marker detection kit. Background Art

[0002] The occurrence and progression of malignant tumors are highly complex. Currently, the most effective control method is early diagnosis and treatment to reduce mortality. Diagnosing tumors primarily involves tumor marker testing, imaging, and histopathological examination. Tumor marker testing, due to its non-invasive nature, plays a crucial role in assisting diagnosis, evaluating treatment efficacy, monitoring recurrence, and guiding medication.

[0003] MicroRNAs (miRNAs) are ubiquitous in tumors and exhibit relatively specific expression profiles in different tumor tissues. Their expression levels are of great value for tumor diagnosis, prognosis, and therapeutic efficacy, making them a highly promising class of tumor markers. Furthermore, in clinical practice, miRNAs not only serve as biomarkers in tumors but also have diagnostic applications in cardiovascular diseases, such as acute myocardial infarction (AMI). Common AMI diagnostic markers are cardiac troponin T (cTnT) or high-sensitivity cardiac troponin T (hs-cTnT). However, other conditions, such as hypertensive crisis, renal failure, and heart failure, can also lead to elevated cTnT, potentially leading to misdiagnosis. Furthermore, elevations often occur 3 to 6 hours after the onset of an AMI, potentially delaying diagnosis. Because miRNA levels show significant changes in the early stages of myocardial infarction, they can serve as diagnostic markers for AMI.

[0004] Enzymes offer advantages such as high catalytic efficiency, strong specificity, and mild reaction conditions. However, natural enzymes are prone to inactivation, have low operational stability, and are difficult to recover. To overcome these limitations in practical applications, various enzyme immobilization methods have emerged. Metal-organic frameworks (MOFs) are a class of porous materials composed of metal ions chelated with organic ligands. They possess high surface area and high porosity, which endows them with excellent enzyme-loading capacity, helping to improve enzyme stability, catalytic efficiency, and reusability. Furthermore, MOFs can adsorb nucleic acid chains through interactions such as coordination, electrostatics, and π-π stacking. Some MOFs can also form covalent bonds with phosphate groups in nucleic acids, demonstrating excellent nucleic acid enrichment capabilities.

[0005] Currently, miRNA detection methods mainly include Northern hybridization, real-time quantitative reverse transcription PCR (qRT-PCR), and microarray analysis. Northern hybridization requires a large number of samples, has low sensitivity, and is time-consuming. qRT-PCR has high sensitivity and practicality, but miRNAs are short, making conventional primer design impossible and requiring reverse transcription, which affects PCR amplification efficiency. Microarrays can achieve high-throughput analysis of miRNAs, but suffer from poor reproducibility and low sensitivity, and are often used as screening tools rather than quantitative analysis platforms.

[0006] In recent years, a range of new miRNA detection technologies have demonstrated their potential. These include sensing systems based on novel nanoluminescent materials such as fluorescent metal nanoclusters and quantum dots, constant-temperature nucleic acid amplification techniques such as loop-mediated isothermal amplification and hybridization chain reaction, as well as spectroscopic and electrochemical methods such as fluorescence, Raman spectroscopy, and bioluminescence. However, most detection methods suffer from poor reproducibility, high cost, and demanding testing conditions, limiting their clinical application and limiting their industrial foundation. Therefore, efficient, selective, and sensitive detection of miRNAs for early cancer screening remains a major challenge in analytical research. Summary of the Invention

[0007] The purpose of the present invention is to provide a magnetic enzyme-MOF complex and its preparation method and application. The disclosed magnetic enzyme-MOF complex can be applied to the detection of miRNAs biomarkers.

[0008] In the first aspect, the present invention provides a magnetic enzyme-MOF complex, which includes a magnetic material Fe3O4@MOF, a MOF material encapsulating an immobilized enzyme, namely AMG@MOF, a first aptamer chain UG and a second aptamer chain ZG, and utilizes the enzyme's efficient catalytic ability on the substrate to amplify the miRNAs detection signal.

[0009] The magnetic material Fe3O4@MOF is assembled from Fe3O4 nanoparticles and a first metal-organic framework material. The assembly process is as follows: using Fe3O4 as a core layer, metal ions and organic ligands are deposited on the surface of Fe3O4 to synthesize the MOF in situ; the first metal-organic framework material is a MOF capable of forming coordination bonds with nucleic acids, such as UiO-66, PCN-224, or DUT-51.

[0010] The MOF material encapsulating the immobilized enzyme in AMG@MOF, i.e., the second metal-organic framework material, is a MOF that can be synthesized under room temperature aqueous conditions, such as ZIF-90, MIL-100, or HKUST-1; the immobilized enzyme is an enzyme that uses starch as a substrate, such as saccharifying enzyme, α-amylase, or β-amylase;

[0011] Taking advantage of the nucleic acid adsorption properties of MOFs, the first aptamer chain UG and the second aptamer chain ZG were adsorbed on the surfaces of Fe3O4@MOF and AMG@MOF, respectively, to form UG-Fe3O4@MOFs and ZG-AMG@MOFs. The first aptamer chain UG and the second aptamer chain ZG have complementary sequences, and the two can specifically bind to each other. UG and ZG are used as linkers to couple to form the composite material Fe3O4@MOF+AMG@MOF. Among them, the mixing ratio of UG-Fe3O4@MOFs and ZG-AMG@MOFs ranges from (0.5:1) to (3:1).

[0012] In addition, the second aptamer chain ZG has a sequence complementarity with the target miRNAs, and the binding ability between UG and ZG is smaller than that between the target miRNAs and ZG, which is specifically manifested in the number and position of complementary bases: in terms of the number of complementary bases, the number of complementary sequence bases between ZG and UG is M, and the number of complementary bases between ZG and target miRNAs is N, and M is smaller than N; in terms of the position of complementary bases, UG and ZG are divided into three blocks respectively, the first block is used to anchor the carrier MOF, and the first block consists of a PolyA sequence with a length of 8 to 12 bases and preferably 10 bases, and the second block is a double-stranded block formed by DNA chains C8 and G8. This double-stranded structure of the second block can make the third block Maintaining an upright conformation facilitates the complementary connection of the third block, wherein the nucleotide sequence of DNA chain C8 is eight C bases, namely CCCCCCCC, and the nucleotide sequence of DNA chain G8 is eight G bases, namely GGGGGGGGG; for example, UG is divided into UG-1 (anchor block), UG-2 (double-stranded block) and UG-3 (connector block); ZG is divided into ZG-1 (anchor block), ZG-2 (double-stranded block) and ZG-3 (connector block), and there are complementary sequences between UG-3 and ZG-3 but they are not completely complementary; ZG-3 is completely complementary to the target miRNAs. Through the aptamer design method, the composite material Fe3O4@MOF+AMG@MOF is used for the recognition and detection of target miRNAs in biological samples;

[0013] In a second aspect, the present invention also provides a method for preparing the magnetic enzyme-MOF complex, such as Figure 3a As shown, the preparation method comprises the steps of:

[0014] Step 1: Take Fe3O4@MOFs with a concentration range of 0.5 mg / mL to 2.5 mg / mL and DNA chain UG with a concentration of 0.05 to 5 μM, incubate at a temperature of 4°C to 37°C for 5 to 30 minutes, then use a magnet to adsorb the complex, remove the supernatant, and obtain a complex of UG and Fe3O4@MOFs, UG-Fe3O4@MOFs; using the same incubation conditions, then obtain a complex of ZG and AMG@MOFs, ZG-AMG@MOFs, by centrifugation, the concentration ratio range of AMG@MOF and ZG is: 1 mg / mL: (50 to 250 nM); the concentration range of UG and ZG is: 0.05 to 5 μM.

[0015] Step 2: Using the specific binding of UG and ZG, UG-Fe3O4@MOFs and ZG-AMG@MOFs were mixed and incubated in a ratio of (0.5-2.5):1 to obtain a composite material.

[0016] Preferably, in order to balance the connection steric hindrance between Fe3O4@MOFs and AMG@MOFs and the adsorption interference between target miRNAs and MOF, and comprehensively considering the significance of the signal change before and after the final detection, the mixed concentration ratio of Fe3O4@MOFs and UG is 1 mg / mL:100 nM, and the mixed concentration ratio of AMG@MOFs and ZG is also 1 mg / mL:100 nM.

[0017] In a third aspect, the present invention also provides the use of the above-mentioned magnetic enzyme-MOF complex in the preparation of a miRNAs marker detection kit; miRNAs include but are not limited to tumor-related abnormally expressed miRNAs and myocardial infarction-related abnormally expressed miRNAs, and the tumor-related abnormally expressed miRNAs include but are not limited to miR-21, miR-155, and Let-7; myocardial infarction-related abnormally expressed miRNAs include but are not limited to miR-499, miR-208, and miR-133.

[0018] As a preferred embodiment of the above application, the detection kit includes a first reagent, a second reagent and a third reagent;

[0019] The first reagent is the magnetic enzyme-MOF complex, which is used to extract target miRNAs. The complex is stored in a dry powder state and is resuspended in deionized water when used. The magnetic enzyme-MOF complex is mixed with the target miRNAs, incubated at room temperature for 20 minutes, and then an external magnetic field is applied to remove the adsorption precipitate, thereby extracting the target miRNAs.

[0020] The second reagent and the third reagent are used to amplify the first signal. The second reagent is starch, and the third reagent is acetic acid (HAc). The second reagent and the third reagent are added to the supernatant after the extraction with the first reagent, and the reaction is carried out in a 50°C water bath for 0.5 to 7 hours, preferably 1.5 hours. After the reaction, iodine solution is added for color development, thereby realizing UV detection. Alternatively, after the reaction, blood glucose meter can be used for direct detection, i.e., blood glucose test strip GI method detection.

[0021] Detection principle: Figure 3b As shown, due to the stronger binding force between the aptamer ZG and the target miRNAs, the linker breaks, the Fe3O4@MOF and AMG@MOF separate from each other, and the AMG@MOF released in the solution contacts the substrate starch. Under the efficient catalytic ability of the enzyme, the starch is hydrolyzed and a large amount of glucose is produced. The first signal amplification utilizes the efficient catalytic ability of AMG to convert the content of target miRNAs into a decrease in starch content. The colorimetric reaction between iodine and starch can be used as the output signal for colorimetric detection such as UV-visible spectrophotometry.

[0022] If more sensitive detection is required, the detection kit further includes a fourth reagent, a fifth reagent and a sixth reagent;

[0023] The fourth, fifth, and sixth reagents are used to amplify the second signal. The fourth reagent is glucose oxidase, the fifth reagent is horseradish peroxidase, and the sixth reagent is luminol. All of these reagents are dry powders and should be stored in the dark. Upon use, the fourth and fifth reagents, glucose oxidase and horseradish peroxidase, are dissolved in deionized water, while the sixth reagent, luminol, is dissolved in 0.1M NaOH. The fourth reagent, glucose oxidase, is added to the liquid obtained after the second reagent reaction, and the reaction is allowed to proceed at room temperature. The pH is adjusted to 8 with NaOH, and the fifth and sixth reagents, horseradish peroxidase and luminol, are then added. The light signal intensity is measured using electrochemiluminescence (ECL) detection. Alternatively, the pH can be adjusted to an acidic level using HCl, and detection is performed based on the color change using luminol colorimetry.

[0024] Detection principle: Figure 3b As shown, the second signal amplification is detected by electrochemiluminescence. On the basis of the first signal amplification, the "glucose oxidase-horseradish peroxidase-luminol" reaction is introduced to convert the content of target miRNAs into an increase in glucose content, further amplifying the detection signal.

[0025] As an embodiment of the application described in the present application, the target miRNAs is miR-21, and correspondingly, UG is composed of a mixture of U1 and G8, and ZG is composed of a mixture of Z1 and G8. The nucleotide sequence of U1 is shown in SEQ ID NO.1, specifically:

[0026] 5'-AAAAAAAAAACCCCCCCCTAGCTTATCAGACTG-3'; The nucleotide sequence of Z1 is shown in SEQ ID NO.2, specifically:

[0027] 5'-AAAAAAAAAACCCCCCCCTCCAACATCAGTCTGATAAGCTA-3'.

[0028] The complex constructed in the first aspect of the present invention is formed by connecting Fe3O4@MOF and AMG@MOF through a nucleic acid chain linker containing miRNAs aptamers. After the enzyme is immobilized, the operational stability is enhanced and it can be recycled by magnetic separation, which is beneficial for practical applications.

[0029] The application described in the third aspect of the present invention, the detection kit provided by the present invention can be used for graded diagnosis according to different needs, which helps to achieve personalized diagnosis. If it is detected whether tumor-related miRNAs are abnormally expressed, only one signal amplification can be performed to immediately generate a diagnostic result; as a preferred embodiment of the application described in the third aspect of the present invention, if sensitive detection of miRNA levels is required, a multi-enzyme cascade system can be introduced to achieve dual signal amplification, that is, a detection kit constructed based on the miRNA detection idea of ​​"an enzyme-MOF complex, two-fold signal amplification, three enzymes involved, and four signal output means" has the advantages of simple operation and low cost. It does not require the use of large instruments and can be separated only by an external magnetic field, which reduces the requirements for the environment and operators and helps to achieve miniaturized and home-based detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a SEM image of the magnetic enzyme-MOF complex obtained in Example 1;

[0031] Figure 2 The particle size potential distribution diagram of the materials involved in Example 1, wherein Figure a is a comparison diagram of particle size results, and Figure b is a comparison diagram of potential results;

[0032] Figure 3a The present invention is a preparation process of the composite UZ;

[0033] Figure 3b Schematic diagram of the principle of detecting miRNAs;

[0034] Figure 4 This is the electrophoresis result of competitive binding of nucleic acid chains in Example 3, where lanes 1-5 are UG, ZG, UZ, UZ+miR-21, and Marker, respectively.

[0035] Figure 5This is the ratio screening result diagram of UG-Fe3O4@UiO-66 and ZG-AMG@ZIF-90 in Example 4.

[0036] Figure 6 This is a graph showing the concentration screening results of luminol in Example 5.

[0037] Figure 7 This is a graph showing the sensitivity test results of ultraviolet detection of miR-21 in Example 6.

[0038] Figure 8 This is a graph showing the sensitivity test results of detecting miR-21 by electrochemiluminescence in Example 7.

[0039] Figure 9 Graphs showing the specificity of the detection method in Example 8, wherein Graph a corresponds to the UV method, and Graph b corresponds to the electrochemiluminescence (ECL) method.

[0040] Figure 10 These are the results of detecting miR-21 in serum samples of normal and colon cancer mice in Example 9. The method corresponding to Figure a is the UV method, and the method corresponding to Figure b is the electrochemiluminescence (ECL) method. Figure c is a comparison of the ROC curves of the four detection methods. DETAILED DESCRIPTION

[0041] The following examples are used to further illustrate the present invention, but should not be considered as limiting the scope of protection of the present invention.

[0042] The main raw materials used in the following examples and test examples are:

[0043] Ferric chloride hexahydrate, anhydrous sodium acetate, and ethylene glycol required for the preparation of Fe3O4 were obtained from Sinopharm Chemical Reagent Co., Ltd., and trisodium citrate dihydrate was obtained from Nanjing Chemical Reagent Co., Ltd.

[0044] Zirconium oxychloride octahydrate and terephthalic acid required for the preparation of Fe3O4@UiO-66 were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd., N,N-dimethylformamide was obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd.; amylase was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd.; miR-21, miR-21-SM, miR-155, and miR-191 were all obtained from Sangon Biotech (Shanghai) Co., Ltd.; the sequence of miR-21 is 5'-UAGCUUAUCAGACUGAUGUUGA-3';

[0045] Glucose oxidase was obtained from Sangon Biotechnology (Shanghai) Co., Ltd.;

[0046] Horseradish peroxidase was from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0047] Luminol was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0048] DNA chain U1 is: 5'-AAAAAAAAAACCCCCCCCTAGCTTATCAGACTG-3'; Z1 is: 5'-AAAAAAAAAACCCCCCCCTCAACATCAGTCTGATAAGCTA-3'; DNA chain G8 is: 5'-GGGGGGGG', and was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. according to the above sequence; DNA chain UG is a mixture of U1 and G8, and DNA chain ZG is a mixture of Z1 and G8;

[0049] Serum samples from normal and colon cancer mice were obtained from 5-week-old healthy BALB / c nude mice purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd. Blood samples were collected via orbital bleeding. Colon cancer mice were modeled by subcutaneous transplantation of HCT116 cells. Blood samples were placed in tubes without anticoagulants and allowed to naturally clot at room temperature for 60 minutes. After coagulation, the blood was centrifuged at 3000 rpm for 10 minutes, and the supernatant was obtained as serum.

[0050] Example 1 Preparation of a composite material with miRNAs responsive properties

[0051] (1) Preparation of Fe3O4: Weigh 0.301 g of ferric chloride hexahydrate and 0.18 g of trisodium citrate dihydrate, sonicate until completely dissolved in 13.5 mL of ethylene glycol; add 0.87 g of anhydrous sodium acetate to the solution, stir vigorously for 30 min, transfer to a reactor and react at 200°C for 8 h. After collecting with a magnet, wash with anhydrous ethanol and deionized water three times, respectively, and evaporate to obtain Fe3O4;

[0052] (2) Preparation of Fe3O4@UiO-66: 0.156 g zirconium oxychloride octahydrate, 0.132 g terephthalic acid, and 0.2 g Fe3O4 were weighed and dispersed in 50 mL N,N-dimethylformamide. 4 ml acetic acid was added to the suspension, and after ultrasonic dissolution, the suspension was transferred to a reactor and reacted at 130 °C for 12 h. After being collected by a magnet, the suspension was washed three times with anhydrous ethanol and deionized water, respectively, and evaporated to obtain Fe3O4@UiO-66.

[0053] (3) Preparation of AMG@ZIF-90: 0.166 g of imidazole-2-carboxaldehyde and 0.317 g of zinc acetate were weighed and dissolved in 100 mL of water. Imidazole-2-carboxaldehyde solution, 20 mL of amylase (5 U / mL), and zinc acetate solution were added in that order. The mixture was stirred at room temperature for 7 h, centrifuged at 8000 rpm for 10 min, and washed three times with deionized water to obtain the product.

[0054] (4) Fe3O4@UiO-66 adsorbs DNA chain UG: 10 μL Fe3O4@UiO-66 was mixed with 10 μL 1 μM DNA chain UG. The concentration ratio of Fe3O4@UiO-66 to UG was 1 mg / mL:100 nM. After incubation at room temperature for 20 min, the complex was adsorbed by a magnet and the supernatant was removed to obtain UG-Fe3O4@UiO-66.

[0055] (5) AMG@ZIF-90 adsorbs DNA chain ZG: 10 μL AMG@ZIF-90 and 10 μL 1 μM DNAZG were mixed, and the ratio of AMG@ZIF-90 to ZG was 1 mg / mL:100 nM. After incubation at room temperature for 20 min, the supernatant was removed by centrifugation to obtain ZG-AMG@ZIF-90.

[0056] (6) Two MOFs are connected by DNA chains: 15 μL UG-U and 10 μL ZG-Z, that is, the ratio of UG-U to ZG-Z is 1.5:1, and after incubation at room temperature for 20 min, the complex is adsorbed by a magnet and the supernatant is removed. After washing twice, the composite material UZ with miRNAs responsive properties is obtained, as shown in FIG. Figure 1 Shown is the SEM image of the magnetic enzyme-MOF complex UZ obtained in Example 1.

[0057] Figure 2 These are the particle size potential distribution diagrams of the materials involved in the preparation process of Example 1, where Figure a is a comparison diagram of particle size results and Figure b is a comparison diagram of potential results.

[0058] Example 2 Application of composite material UZ in miRNAs detection

[0059] (1) Competitive binding of miRNAs: The complex UZ prepared in Example 1 was mixed with miRNAs, incubated at room temperature for 20 min, and then subjected to an external magnetic field for adsorption and precipitation;

[0060] (2) Signal Amplification I: Add 100 μL of 1% HAc and 20 μL of 0.1% starch to the supernatant, react in a 50°C water bath for 1.5 h, and add 10 μL of iodine solution for color development (UV detection). Alternatively, after the reaction in a water bath, use a blood glucose meter for direct detection (GI test strip method).

[0061] (3) Signal Amplification II: If more sensitive detection is required, add 80 μL of 5 mg / mL glucose oxidase to the liquid obtained in (2) and react at room temperature for 15 min. Adjust the pH to 8 with 0.25 M NaOH, add 150 μL of 50 μg / mL horseradish peroxidase and 50 μL of 10 mM luminol, and measure the light signal intensity, i.e., electrochemiluminescence (ECL) detection. Alternatively, adjust the pH to acidic (pH 5) with 0.1 M HCl, and detect by color change, i.e., luminol colorimetry.

[0062] Example 3 Electrophoresis Verification of Competitive Binding of miRNAs

[0063] Prepare polyacrylamide gel according to the ratio of 4mL 30% acrylamide solution, 2.5mL 5×TBE buffer, 3.3mL deionized water, 100μL 10% ammonium persulfate solution, and 10μL N',N'-tetramethylethylenediamine. Shake to mix and pour into a mold with comb teeth inserted in advance. After cooling for about 30 minutes, pull out the comb teeth, place the gel in the electrophoresis tank, and add 1000mL 1×TBE buffer. When loading, take 10μL of sample and add 2μL 6× DNA loading buffer, mix well and then load. The voltage is 120V, the current is 300mA, and the electrophoresis time is 40min. After the electrophoresis is completed, use a gel imager to image the nucleic acid bands on the gel, and the observation results are as follows. Figure 4 The electrophoresis results of competitive binding of nucleic acid chains are shown in Figure 1. Lanes 1 to 5 represent UG, ZG, UZ, UZ + miR-21, and Marker, respectively. Lane 1 represents UG; Lane 2 represents ZG; Lane 3 represents UZ; Lane 4 represents UZ + miRNA-21; and Lane 5 represents Marker. The figure shows that UG and ZG combine to form UZ. Upon addition of target miRNAs, the miRNAs competitively bind to ZG.

[0064] Example 4 Optimization of the ratio of UG-Fe3O4@UiO-66 and ZG-AMG@ZIF-90

[0065] According to the preparation process of Example 1, different mixing ratios of UG-Fe3O4@UiO-66 (UG-U) and ZG-AMG@ZIF-90 (ZG-Z) were set to prepare different composites UZ for comparison. The mixing ratios of UG-U and ZG-Z were set to (0.5:1), (1:1), (1.5:1), (2:1), and (2.5:1), respectively.

[0066] The five complexes were used to extract miR-21. Specifically, miR-21 (final concentration 1 nM) was added and incubated for 20 minutes. The five UZ-miR groups were separated by applying a magnetic field. After adjusting the pH to 4, 20 μL of 0.1% starch was added and incubated at 50°C for 1.5 hours. After the reaction was complete, iodine solution was added for color development and the absorbance value was measured. Figure 5 The figure shows the results of the ratio screening of UG-Fe3O4@UiO-66 and ZG-AMG@ZIF-90. The results show that when the ratio of UG-U to ZG-Z is 1.5:1, the signal difference before and after the addition of miR-21 is the largest, and a ratio of UG-U to ZG-Z of 1.5:1 is the optimal setting. The UV-visible spectrophotometer used in this invention is a UV-1800PC.

[0067] Example 5 Optimization of Luminol Concentration in Electrochemiluminescence Detection of miR-21

[0068] The complex UZ was prepared according to the method in Example 1. MiR-21 (final concentration 1 nM) was added and incubated for 20 minutes. UZ-miR was separated by applying an external magnetic field. After adjusting the pH to 4, 20 μL of 0.1% starch was added and incubated at 50°C for 1.5 hours. 80 μL of 5 mg / mL glucose oxidase was added to the resulting liquid and reacted at room temperature for 15 minutes. The pH was adjusted to 8 with 0.25 M NaOH. 150 μL of 50 μg / mL horseradish peroxidase and different concentrations of luminol (0.25 mM to 1 mM) were added. The optical signal intensity was measured, as shown in Figure 2. Figure 6 The figure shows the results of the luminol concentration screening. As can be seen from the results, the signal difference before and after the addition of miR-21 was the largest at a final luminol concentration of 0.5 mM. Therefore, the final luminol concentration was determined to be 0.5 mM. The electrochemiluminescence analysis system used in the present invention is an MPI-E multifunctional chemiluminescence detector.

[0069] Example 6 UV sensitivity test

[0070] The optimized reaction system was used for the reaction. The reaction conditions were: a MOFs to nucleic acid ratio of 1 mg / mL:100 nM; a Fe3O4@UiO-66 to AMG@ZIF-90 ratio of 1.5:1; an incubation time of 20 minutes for each UZ preparation step; and enzymatic starch hydrolysis at 50°C for 1.5 hours. miR-21 was detected over a concentration range of 0.1-1 nM, with three replicates at each concentration. Good linearity was observed within this range, as shown in Figure 2. Figure 7 Shown is the sensitivity test result of ultraviolet detection of miR-21.

[0071] Example 7 Electrochemiluminescence Sensitivity Test

[0072] Under the optimized reaction conditions of Example 6, a "glucose oxidase-horseradish peroxidase-luminol" signal amplification system was introduced. The reaction conditions were: glucose oxidase (GOD) concentration of 0.4 mg / mL; horseradish peroxidase (HRP) concentration of 7.5 μg / mL; incubation time of GOD and substrate for 20 minutes; luminol concentration of 0.5 mM; and reaction pH of 8. The detection concentration range of miR-21 was 0-240 pM, and the test was repeated three times at each concentration. Good linearity was observed within this detection range. Figure 8 The sensitivity test results of electrochemiluminescence detection of miR-21 are shown in the figure.

[0073] Example 8 Specificity Test

[0074] Under the reaction conditions of Examples 6 and 7, miR-21 was replaced with a variety of miRNAs, such as miR-21-SM, miR-155, miR-191, and NC. Other conditions remained unchanged for detection. Each miRNA was tested in parallel three times. Figure 9 The figure shows the specificity test results, where the method corresponding to Figure a is the UV method, and the method corresponding to Figure b is the electrochemiluminescence ECL method. It is verified that the method used in the present invention has good specificity and has a good recognition effect on target miRNAs.

[0075] Example 9 Serum sample testing

[0076] Under the reaction conditions of Examples 6 and 7, the miR-21 standard was replaced with serum samples of normal mice and colon cancer mice, and other conditions remained unchanged for detection. Each group of samples was tested in parallel 3 times. Figure 10 Figure 3 shows the results of detecting miR-21 in serum samples of normal and colon cancer mice. Figure a corresponds to the UV method, Figure b corresponds to the electrochemiluminescence (ECL) method, and Figure c is a comparison of the ROC curves of the four detection methods, verifying that the method used in the present invention can effectively distinguish normal and cancer samples with good accuracy.

Claims

1. A magnetic enzyme-MOF complex, characterized in that: The composite is composed of a magnetic material Fe3O4@MOF, a MOF material encapsulating an immobilized enzyme, namely AMG@MOF, a first aptamer chain UG and a second aptamer chain ZG. The magnetic material Fe3O4@MOF is assembled from Fe3O4 nanoparticles and a first metal organic framework material. The assembly process is: taking Fe3O4 as a core layer, depositing metal ions and organic ligands on the surface of Fe3O4 to synthesize MOF in situ; the first metal organic framework material is a MOF that can form a coordination bond with nucleic acid; The MOF material encapsulating the immobilized enzyme in the AMG@MOF, i.e., the second metal organic framework material, is a MOF that can be synthesized under room temperature aqueous conditions, and the immobilized enzyme is an enzyme that uses starch as a substrate; The first aptamer chain UG and the second aptamer chain ZG were adsorbed on the surface of Fe3O4@MOF and AMG@MOF to form UG-Fe3O4@MOFs and ZG-AMG@MOFs, respectively. There was a complementary sequence between the first aptamer chain UG and the second aptamer chain ZG. UG and ZG were used as connecting chains to couple to form the composite material Fe3O4@MOF+AMG@MOF. The mixing ratio of UG-Fe3O4@MOFs and ZG-AMG@MOFs ranged from (0.5:1) to (3:1). The second aptamer chain ZG has a sequence complementarity with the target miRNAs, and the binding ability between UG and ZG is smaller than that between the target miRNAs and ZG. The number of complementary sequence bases between ZG and UG is M, and the number of complementary bases between ZG and the target miRNAs is N, where M is smaller than N. UG and ZG are divided into three blocks respectively, the first block is used to anchor the carrier MOF, the second block is a double-stranded block formed by DNA chain C8 and DNA chain G8, wherein the nucleotide sequence of DNA chain C8 is CCCCCCCC, and the nucleotide sequence of DNA chain G8 is GGGGGGGG; the third block is used for complementary connection.

2. The magnetic enzyme-MOF complex according to claim 1, characterized in that: The first metal organic framework material is any one of UiO-66, PCN-224, and DUT-51.

3. The magnetic enzyme-MOF complex according to claim 1, characterized in that: The second metal organic framework material is any one of ZIF-90, MIL-100 and HKUST-1.

4. The magnetic enzyme-MOF complex according to claim 1, characterized in that: The immobilized enzyme is any one of saccharifying enzyme, α-amylase and β-amylase.

5. The method for preparing the magnetic enzyme-MOF complex according to claim 1, comprising the steps of: Step 1, taking Fe3O4@MOFs with a concentration range of 0.5mg / mL to 2.5mg / mL and DNA chain UG with a concentration of 0.05 to 5μM, incubating at a temperature of 4°C to 37°C for 5 to 30min, then using a magnet to adsorb the complex, removing the supernatant, and obtaining a complex of UG and Fe3O4@MOFs, UG-Fe3O4@MOFs; using the same incubation conditions, and then obtaining a complex of ZG and AMG@MOFs, ZG-AMG@MOFs, by centrifugation, the concentration ratio of AMG@MOF and ZG is in the range of: 1mg / mL: (50 to 250nM); the concentration range of UG and ZG is: 0.05 to 5μM; Step 2, utilizing the specific binding of UG and ZG, UG-Fe3O4@MOFs and ZG-AMG@MOFs were mixed and incubated in a ratio of (0.5-2.5):1 to obtain a composite material.

6. According to the preparation method of claim 5, the mixed concentration ratio of Fe3O4@MOFs and UG is 1 mg / mL:100 nM, and the mixed concentration ratio of AMG@MOFs and ZG is also 1 mg / mL:100 nM.

7. Use of the magnetic enzyme-MOF complex according to claim 1 in preparing a miRNAs marker detection kit.

8. According to the use of claim 7, the detection kit comprises a first reagent, a second reagent and a third reagent; the first reagent is the magnetic enzyme-MOF complex, which is used to extract target miRNAs; the magnetic enzyme-MOF complex is mixed with the target miRNAs, incubated at room temperature for 20 minutes, and then adsorbed and precipitated by an external magnetic field to extract the target miRNAs; the second reagent and the third reagent are used for amplifying the first signal, the second reagent is starch, and the third reagent is acetic acid (HAc), the second reagent and the third reagent are added to the supernatant after the extraction with the first reagent, and the reaction is carried out in a water bath at 50°C for 0.5 to 7 hours; after the reaction, iodine solution is added for color development to realize UV ​​detection; or after the reaction, it is directly detected with a blood glucose meter, that is, blood glucose test paper GI method detection.

9. The use according to claim 7, characterized in that: The detection kit also includes a fourth reagent, a fifth reagent and a sixth reagent; the fourth reagent, the fifth reagent and the sixth reagent are used for amplifying the second signal, the fourth reagent is glucose oxidase, the fifth reagent is horseradish peroxidase, and the sixth reagent is luminol; the fourth reagent glucose oxidase is added to the liquid obtained after the second reagent reacts to react at room temperature, the pH is adjusted to 8 with NaOH, the fifth reagent horseradish peroxidase and the sixth reagent luminol are added, and the light signal intensity is detected, that is, electrochemical luminescence (ECL) detection; HCl can also be used to adjust the pH to acidity, and the luminol colorimetric method is used for detection according to color change.

10. The use according to claim 7, characterized in that: The target miRNAs is miR-21, and correspondingly, UG is a mixture of U1 and G8, and ZG is a mixture of Z1 and G8. The nucleotide sequence of U1 is shown in SEQ ID NO.1, and the nucleotide sequence of Z1 is shown in SEQ ID NO.2.

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