Preparation method and application of fluorescence sensing array based on MOF material

Abstract

The invention discloses a preparation method and application of a fluorescence sensing array based on an MOF material. The preparation method comprises the following specific steps: step 1, preparing Zr-MOF; step 2, obtaining single-stranded DNA1, DNA2, DNA3 and DNA4, and marking carboxyl fluorescein at 5'ends of the single-stranded DNA1, DNA2, DNA3 and DNA4; carrying out centrifugal treatment on the labeled DNA single chain, and respectively adding a TBE buffer solution to prepare a single-chain DNA1 solution, a single-chain DNA2 solution, a DNA3 solution and a DNA4 solution for later use; and 3, respectively adding the Zr-MOF prepared in the step 1 into the single-stranded DNA1 solution, the single-stranded DNA2 solution, the DNA3 solution and the DNA4 solution prepared in the step 2, and incubating at room temperature to obtain the fluorescent sensing array.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of a fluorescence sensor array based on MOF materials. Background Art

[0002] Colorectal cancer (CRC) occupies a prominent position in the global burden of disease. According to the World Health Organization, it is the third most common cancer worldwide, accounting for approximately 10% of all cancer cases and the second leading cause of cancer-related deaths. Therefore, the development of drugs targeting CRC is crucial. Detecting the concentrations of certain miRNAs in the blood can provide a reference for evaluating the efficacy of colorectal cancer (CRC) drugs, and the concentrations of some miRNAs can even indicate drug resistance. Therefore, detecting miRNA concentrations is crucial. Currently, various sensors have been designed for detecting miRNA concentrations, but these sensors suffer from numerous challenges in preparation and detection performance. Regarding nanomaterial sensors, gold nanoparticle sensors suffer from insufficient effective loading rates due to steric hindrance during preparation, and the nanoparticles are susceptible to irreversible aggregation due to changes in salt concentration, affecting detection sensitivity. To mitigate the disadvantage of protein adsorption on their surfaces, carbon-based materials require the addition of polyethylene glycol (PEG) or bovine serum albumin (BSA) to form a blocking layer, which complicates the preparation process and increases production costs. For probes: locked nucleic acid (LNA) probes must strictly match the GC content of the target miRNA and are easily affected by other miRNAs; the synthesis of stem-loop primers is difficult and requires multiple optimization of synthesis conditions, which is not only time-consuming but also costly. For signal amplification strategies: Cas13a-based sensors require the Cas protein and crRNA to be assembled into a complex in advance, and then the reporter RNA is generated by T7 RNA polymerase. The entire process involves temperature gradient control and multiple centrifugation purifications. The preparation steps are complex and time-consuming; although hybridization chain reaction (HCR) can achieve enzyme-free amplification, the triggering efficiency of its hairpin probe is significantly affected by the ionic strength of the solution. For non-enzymatic catalysis: the assembly of catalytic hairpin sensors is kinetically limited. The rate of the hairpin assembly reaction catalyzed by the AuNPs surface is limited by the diffusion efficiency of the probe. At low concentrations of targets (such as miRNA-721), the reaction equilibrium time needs to be extended to more than 2 hours, which cannot meet the needs of rapid detection. For paper-based sensors: Although the paper-based distance sensor based on DNA hydrogel simplifies the operation, the swelling behavior of the hydrogel is significantly affected by the ambient humidity. When the relative humidity is >60%, the infiltration distance error can reach 15%, and strict temperature control conditions are required for preparation. Summary of the Invention

[0003] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a preparation method and application of a fluorescence sensing array based on MOF materials, so as to solve the problems of the prior art in preparing corresponding sensors, such as numerous preparation steps, long preparation time, high production cost, low sensitivity of the prepared sensors, and susceptibility to interference from non-target miRNA.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] The first aspect of the present invention provides a method for preparing a fluorescence sensor array based on MOF materials, the specific steps are as follows:

[0006] Step 1: Preparation of Zr-MOF;

[0007] Step 2: Obtain single-stranded DNA 1, DNA 2, DNA 3, and DNA 4, and label the 5' ends of the single-stranded DNA 1, DNA 2, DNA 3, and DNA 4 with carboxyfluorescein; centrifuge the labeled DNA single strands, and then add TBE buffer to prepare single-stranded DNA 1 solution, single-stranded DNA 2 solution, DNA 3 solution, and DNA 4 solution for later use;

[0008] Among them, the sequence of DNA1 is: CCCCCCCCCCCCCCCCCCCC;

[0009] The sequence of DNA2 is: AAAAAAAAAAAAAAAAAAAA;

[0010] The sequence of DNA3 is: TCTCTCTCTCTCTCTCTCTC;

[0011] The sequence of DNA4 is: AGAGAGAGAGAGAGAGAGAG:

[0012] The concentrations of the single-stranded DNA 1 solution, the single-stranded DNA 2 solution, the DNA 3 solution, and the DNA 4 solution were all 5 to 10 nmol / L;

[0013] Step 3: adding the Zr-MOF prepared in step 1 to the single-stranded DNA 1 solution, the single-stranded DNA 2 solution, the DNA 3 solution, and the DNA 4 solution prepared in step 2, respectively, and incubating at room temperature to obtain the fluorescent sensing array;

[0014] Wherein, in the incubation system, the concentration of Zr-MOF is 10-100 μg / mL, and the incubation time is 10-20 min.

[0015] Preferably, in step 1, Zr-MOF is prepared by the following steps:

[0016] Step 1: adding ZrCl4 and tetracarboxyphenylporphyrin to dimethylformamide, dissolving them by ultrasonication, and then adding dichloroacetic acid to obtain a reaction solution; wherein, in the reaction solution, the concentration of ZrCl4 is 5 to 10 mmol / L, the concentration of tetracarboxyphenylporphyrin is 0.5 to 1.0 mmol / L, and the concentration of dichloroacetic acid is 1 to 5 mg / mL;

[0017] Step 2: reacting the reaction solution obtained in step 1 at 100-150° C. for 18-24 hours;

[0018] Step 3: The reaction product obtained in step 2 was centrifuged to obtain a dark purple solid, which was washed with dimethylformamide and ethanol several times and then dried at 100-150° C. for 12 h to obtain Zr-MOF.

[0019] Preferably, in step 2, centrifugation is performed at 3000-4000 rpm for 30-60 s.

[0020] Preferably, in step 3, whether the incubation is terminated is determined by detecting changes in the fluorescence signal of the incubation system.

[0021] Preferably, in step 3, the concentration of Zr-MOF is 60 μg / mL.

[0022] Preferably, in step 3, the incubation time is 13 min.

[0023] In a second aspect, the present invention provides an application of a fluorescence sensor array based on MOF materials. The fluorescence sensor array prepared by the above preparation method is used to detect the concentrations of miR-29a, miR-92a, miRNA-21, and miRNA-17-5.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention combines Zr-MOF with a special fluorescently labeled DNA single strand. Due to the π-π stacking and electrostatic interaction between the DNA single strand and the Zr-MOF, the nucleic acid single strand can be firmly adsorbed to the Zr-MOF surface to prepare the fluorescent sensor array. The entire preparation process is simple and controllable, the reaction conditions are not harsh, and there is no need for special optimization of specific conditions, which has great application prospects.

[0026] 2. The fluorescent sensor array prepared by the present invention induces photoelectron transfer due to the adsorption of single-stranded DNA on the Zr-MOF surface, which weakens the fluorescence signal of the FAM-labeled single-stranded nucleic acid and causes fluorescence quenching. After the fluorescence intensity stabilizes, the addition of the target miRNA in the sample to be tested can restore the fluorescence. In addition, different single-stranded DNA responds significantly differently to different target miRNAs, which can effectively distinguish between single and mixed markers, greatly improving the detection accuracy compared with traditional detection methods.

[0027] 3. The fluorescent sensor array prepared by the present invention can detect multiple target miRNAs simultaneously, overcoming the defect of insufficient detection information of a single target object, and thus can detect the target miRNA more comprehensively and accurately; at the same time, the fluorescent sensor array also has good quantitative analysis capabilities, and can achieve good quantitative analysis of miRNA-17-5 within the concentration range of 0.25-1μM; it also has good time stability. The fluorescence intensity of the fluorescent sensor array can be maintained at more than 92% within 30 days and can be maintained at more than 74% within three months. It shows that the fluorescent sensor array has good stability and can still have a detection function even after being stored for a period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the SEM image of the Zr-MOF prepared in the present invention.

[0029] Figure 2 This is the TEM image of the Zr-MOF prepared in the present invention.

[0030] Figure 3 This is the FT-IR graph of Zr-MOF prepared in the present invention.

[0031] Figure 4 The fluorescence spectrum changes of four different DNA single chains reacting with Zr-MOF and miRNA-92a respectively.

[0032] Figure 5 This is a fluorescence titration curve of the fluorescence sensing array obtained by incubating the Zr-MOF prepared by the present invention with four types of FAM-labeled ss-DNA.

[0033] Figure 6 This is a graph showing the fluorescence change over time of the fluorescent sensor arrays NP1, NP2, NP3, and NP4 prepared in the present invention after incubation with miRNA-29a.

[0034] Figure 7The fluorescence response patterns of the fluorescence sensor arrays NP1, NP2, NP3, and NP4 prepared in the present invention to four miRNAs; wherein A is a multi-factor bar graph; B is a fluorescence response pattern heat map; and C is a fluorescence response pattern PCA graph.

[0035] Figure 8 Detection of miRNA-17-5 (0.001-1 μM) by the fluorescence sensor array constructed with NP1, NP2, NP3, and NP4 prepared in the present invention; wherein A is the PCA score diagram of the first and second principal components of miRNA-17-5 at different concentrations; B is the relationship diagram between miRNA-17-5 concentration and PC1.

[0036] Figure 9 This is a graph showing the fluorescence intensity changes of the fluorescence sensor arrays NP1, NP2, NP3, and NP4 prepared in the present invention within 90 days. DETAILED DESCRIPTION

[0037] The present invention will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the present invention are within the scope of protection of the present invention.

[0038] Unless otherwise indicated in specific cases, the numerical ranges listed herein include the upper and lower limits, and all integers and fractions within the range, and are not limited to the specific values ​​listed when defining the range.

[0039] 1. A method for preparing a fluorescence sensing array based on MOF materials

[0040] Step 1: Preparation of Zr-MOF;

[0041] Step 2: Obtain single-stranded DNA 1, DNA 2, DNA 3, and DNA 4, and label the 5' ends of the single-stranded DNA 1, DNA 2, DNA 3, and DNA 4 with carboxyfluorescein; centrifuge the labeled DNA single strands, and then add TBE buffer to prepare single-stranded DNA 1 solution, single-stranded DNA 2 solution, DNA 3 solution, and DNA 4 solution for later use;

[0042] Among them, the sequence of DNA1 is: CCCCCCCCCCCCCCCCCCCC;

[0043] The sequence of DNA2 is: AAAAAAAAAAAAAAAAAAAA;

[0044] The sequence of DNA3 is: TCTCTCTCTCTCTCTCTCTC;

[0045] The sequence of DNA4 is: AGAGAGAGAGAGAGAGAGAG:

[0046] The concentrations of the single-stranded DNA 1 solution, the single-stranded DNA 2 solution, the DNA 3 solution, and the DNA 4 solution were all 5 to 10 nmol / L;

[0047] Step 3: adding the Zr-MOF prepared in step 1 to the single-stranded DNA 1 solution, the single-stranded DNA 2 solution, the DNA 3 solution, and the DNA 4 solution prepared in step 2, respectively, and incubating at room temperature to obtain the fluorescent sensor arrays NP1, NP2, NP3, and NP4;

[0048] Wherein, in the incubation system, the concentration of Zr-MOF is 10-100 μg / mL, and the incubation time is 10-20 min.

[0049] After an in-depth study of the prior art, the present invention found that the current detection technology for miRNA generally has the problem of poor sensitivity and specificity, resulting in the inability to accurately identify the target miRNA. For this reason, the present invention conceives and designs a new fluorescent sensor array, which is constructed based on a fluorescent sensor and a metal organic framework. The present invention found that when Zr-MOF is selected as a substrate, when the substrate Zr-MOF is combined with a special DNA single strand, its fluorescence intensity will be quenched, and after further adding the target miRNA, the fluorescence intensity will recover. Therefore, the present invention considers using this idea to prepare a fluorescent sensor array and use it for the detection of target miRNA. Subsequently, the present invention further found that the sensor units constructed based on different DNA single strands and Zr-MOF will have significant differences in response to different target miRNAs, and can effectively distinguish between single and mixed target miRNAs. Compared with traditional detection methods, the fluorescent sensor array prepared by the present invention greatly improves the detection accuracy.

[0050] In some embodiments of the present invention, in step 1, Zr-MOF is prepared by the following steps:

[0051] Step 1: adding ZrCl4 and tetracarboxyphenylporphyrin to dimethylformamide, dissolving them by ultrasonication, and then adding dichloroacetic acid to obtain a reaction solution; wherein, in the reaction solution, the concentration of ZrCl4 is 5 to 10 mmol / L, the concentration of tetracarboxyphenylporphyrin is 0.5 to 1.0 mmol / L, and the concentration of dichloroacetic acid is 1 to 5 mg / mL;

[0052] Step 2: reacting the reaction solution obtained in step 1 at 100-150° C. for 18-24 hours;

[0053] Step 3: The reaction product obtained in step 2 was centrifuged to obtain a dark purple solid, which was washed with dimethylformamide and ethanol several times and then dried at 100-150° C. for 12 h to obtain Zr-MOF.

[0054] In some embodiments of the present invention, in step 2, centrifugation is performed at 3000-4000 rpm for 30-60 s.

[0055] In some embodiments of the present invention, in step 3, whether the incubation is terminated is determined by detecting changes in the fluorescence signal of the incubation system.

[0056] In some embodiments of the present invention, in step 3, the concentration of Zr-MOF is 60 μg / mL.

[0057] In some embodiments of the present invention, in step 3, the incubation time is 13 minutes.

[0058] 2. Application of a fluorescence sensing array based on MOF materials

[0059] The fluorescent sensor array prepared by the preparation method of the present invention is used to detect the concentrations of miR-29a, miR-92a, miRNA-21, and miRNA-17-5.

[0060] III. Specific implementation

[0061] 1. Preparation of Zr-MOF

[0062] 37.5 mg of ZrCl₄ and 6.5 mg of tetracarboxyphenylporphyrin were accurately weighed and dissolved in 16.25 mL of dimethylformamide under ultrasonication for approximately 3 minutes. 0.25 mL of dichloroacetic acid was then added. The mixture was placed on a polytetrafluoroethylene liner and heated at 130°C for 18 hours. After the reaction, the dark purple solid was collected by centrifugation at 10,500 rpm for 10 minutes. The solid was washed several times with dimethylformamide and ethanol and dried at 130°C for 12 hours, successfully preparing the Zr-MOF.

[0063] 2. Characterization of Zr-MOF

[0064] The Zr-MOF microstructure was observed using a scanning electron microscope (SEM). The Zr-MOF aqueous solution was ultrasonically dispersed, 5 μL was dropped on a polished silicon wafer, and then dried and sprayed with gold for observation. Figure 1 The SEM images show that Zr-MOF is mostly rod-shaped or nearly rod-shaped, with densely aggregated particles, a length of 0.3-0.8 μm, and a width of 0.05-0.15 μm.

[0065] The internal structure and size were analyzed by transmission electron microscopy (TEM). The powder sample was made into a thin film or dispersed in water, dropped onto a copper grid, and then dried for examination. Figure 2 The TEM image shows that Zr-MOF is a complete, slender rod-shaped crystal with no internal pores.

[0066] The molecular structure and chemical bonds were determined using Fourier transform infrared absorption spectrometry (FT-IR). Figure 3 In the FT-IR image, 3394.83 cm -1 Corresponding to the OH stretching vibration in the carboxyl group, 1605.83 cm -1 The stretching vibration peaks of the C=C double bonds in the benzene ring or porphyrin ring confirm the successful synthesis of Zr-MOF and its structural characteristics.

[0067] 3. Preparation of the fluorescence sensor array

[0068] Step 1: Obtain 4 specific DNA single strands from Shanghai Sangon Biotechnology Co., Ltd.:

[0069] The single-strand sequence of DNA1 is: CCCCCCCCCCCCCCCCCCCC;

[0070] The single-stranded sequence of DNA2 is: AAAAAAAAAAAAAAAAAAAA;

[0071] The single-stranded sequence of DNA3 is: TCTCTCTCTCTCTCTCTCTC;

[0072] The single-stranded sequence of DNA4 is: AGAGAGAGAGAGAGAGAGAG.

[0073] The 5' end of the DNA single strand is labeled with carboxyfluorescein (5-FAM), the labeled DNA single strand is centrifuged at 4000 rpm for 50 seconds, and an appropriate amount of TBE buffer is added to mix and set aside.

[0074] Step 2: Prepare four 10 nM concentrations of the aforementioned FAM-labeled single-stranded DNA (ss-DNA) and record the initial fluorescence signal. The 10 nM labeled ss-DNA was incubated with 100 μg / ml of Zr-MOF, and the fluorescence intensity changes were monitored and recorded in real time. Once the fluorescence intensity stabilized, the four fluorescent sensor arrays were obtained.

[0075] 4. Verification of the feasibility of target miRNA detection

[0076] Four 10 nM FAM-labeled single-stranded DNA (ss-DNA) solutions were prepared, and the initial fluorescence signal was recorded. The 10 nM labeled ss-DNA was incubated with 100 μg / ml Zr-MOF. Due to π-π stacking and electrostatic interactions between the nucleic acid single strands and the Zr-MOF, the nucleic acid single strands adsorbed to the Zr-MOF surface, triggering photoelectron transfer. This weakened the fluorescence signal of the FAM-labeled nucleic acid single strand, resulting in fluorescence quenching. Fluorescence intensity changes were monitored and recorded in real time.

[0077] After the fluorescence intensity stabilizes, the target detection substance miRNA-92a is added. After the addition of miRNA-92a, the FAM-labeled nucleic acid single strand is released from the surface of the MOF structure and interacts with miRNA-92a, and the fluorescence intensity of the system gradually recovers. Figure 4 It can be seen that after the four DNA single strands reacted with Zr-MOF and miRNA-92a respectively, the changes in fluorescence intensity showed obvious differences, indicating that the constructed sensing system is feasible for detecting miRNA.

[0078] 5. Optimize the preparation method

[0079] (1) Zr-MOF at different concentrations

[0080] Taking DNA2 single chain as an example, 10nM FAM-labeled DNA2 chain was incubated with 6 concentration gradients (10, 20, 30, 40, 50, 60μg / ml) of Zr-MOF solution for 20 minutes to form a single sensing unit (NP2), and the fluorescence intensity was measured at 518nm using a fluorescence spectrophotometer. Figure 5 It shows that as the Zr-MOF concentration increases from 10 to 60 μg / mL, the fluorescence intensity of FAM-SS in the system decreases significantly. At 60 μg / mL, the fluorescence can be quenched to the maximum extent, and too high a concentration will affect the fluorescence recovery. Therefore, the optimal concentration of Zr-MOF is 60 μg / mL.

[0081] (2) Different incubation times

[0082] Four different FAM-labeled nucleic acid single strands with a concentration of 10 nM were incubated with 60 μg / ml Zr-MOF nanosolutions, and the degree of fluorescence quenching at 518 nm was measured at 1, 3, 6, 9, 11, 13, and 15 minutes. Figure 4-6 The results showed that the quenching of NP1, NP2 and NP3 was basically completed at 11 minutes, and the fluorescence intensity of the entire sensor array dropped to the lowest at 13 minutes. 13 minutes was determined as the fluorescence quenching time point, that is, the optimal incubation time was 13 minutes.

[0083] 6. Practical Application

[0084] (1) Detection and analysis

[0085] Zr-MOF nanomaterials at a concentration of 60 μg / mL were incubated with four FAM-labeled nucleic acid single strands for 13 minutes to form four sensing units, NP1, NP2, NP3, and NP4. The fluorescence intensity at 518 nm was measured using a fluorescence spectrophotometer at an excitation wavelength of 480 nm and recorded as I0.

[0086] Fetal bovine serum (FBS) was diluted 10-fold as the base serum solution. Chemically synthesized miRNA lyophilized powder was stored in a brown bottle and centrifuged at 4000 rpm for 40 seconds before opening. TBE buffer was added to the bottle according to the desired concentration and vortexed to mix thoroughly to obtain the miRNA solution. Different concentrations of miRNA solutions (miR-29a, miR-92a, miRNA-21, and miRNA-17-5) were added to the diluted FBS to complete the simulated serum sample preparation.

[0087] Target analytes (miR-29a, miR-92a, miRNA-21, and miRNA-17-5) prepared in blood-simulating fluid were added to each of the four fluorescence sensor arrays (NP1-NP4) at 10 nM. The cells were incubated for 17 minutes. The fluorescence intensity was recorded as I, and the relative fluorescence intensity change (ΔI = I - I0) was calculated. Each experiment was repeated six times.

[0088] Multi-factor histogram ( Figure 7 A) shows that the fluorescence intensity changes of each sensor unit after interacting with different miRNAs are significantly different. For example, the fluorescence intensity change when NP1 interacts with miRNA-29a is significantly higher than that of NP2 and NP3. Fluorescence response pattern heat map ( Figure 7 In B), the correlation values ​​of different samples and miRNA combinations are significantly different. Figure 7 C) shows that the comprehensive characteristics of different miRNAs after interacting with samples are different, indicating that the fluorescent sensor array of the present invention can effectively distinguish the four miRNAs.

[0089] (2) Quantitative detection

[0090] Taking miRNA-17-5 as an example, the target marker was detected in a blood simulation fluid at a concentration of 1 nM-1 μM, the fluorescence response changes were recorded, and PCA analysis and linear fitting were performed. Figure 8 In A, as the concentration of miRNA-17-5 increases, the PCA scatter plot gradually shifts to the right in the PC1 direction, and confusion classification occurs within the concentration range of 1nM-25nM. Within the concentration range of 25nM-1μM, the fitting equation is y=-2.17232+1.23348x, R 2=0.98537, indicating that the fluorescent sensor array has a good linear relationship with miRNA-17-5 (25nM-1μM) and can achieve quantitative analysis.

[0091] (3) Temporal stability

[0092] miRNA-21 was selected as the target for time stability detection, and the response of the fluorescence sensor array to miRNA-21 was tested at 0, 1, 3, 5, 7, 10, 15, 20, 30, 60, and 90 days respectively. Figure 9 The results show that the fluorescence intensity of the fluorescent sensor array (NP1-NP4) prepared by the present invention decreases over time within 90 days, but remains above 92% within 30 days and above 74% within three months, indicating that the sensor array has good temporal stability.

[0093] (4) Comparison of existing detection technologies

[0094] The present invention is compared with the existing detection technology, as shown in Table 1:

[0095] Table 1

[0096]

[0097] As can be seen from Table 1, the present invention achieves rapid, purification-free, and amplification-free detection of multiple miRNAs (such as miR-29a and miR-92a) through Zr-MOF functionalized fluorescence array technology. Compared with existing technologies, the fluorescence sensor array constructed in the present invention has excellent anti-interference capabilities. The pore structure and selective adsorption properties of Zr-MOF can directly shield the interference of proteins and impurities in complex biological samples (such as serum and plasma), avoiding the reliance of traditional methods on RNA purification. Moreover, sample processing only requires 10-15 minutes, and the reaction time is simultaneously shortened to the same level, far lower than RT-qPCR (2-3 hours) and microarray (6-24 hours), significantly improving detection efficiency. At the same time, the fluorescence sensor array of the present invention has multi-target adaptability and can support the parallel detection of multiple miRNAs, overcoming the limitation of terahertz sensors that are limited to a single target. In addition, the fluorescence sensor array has high clinical practicality. Based on the fluorescence detection principle, it does not require expensive equipment and is suitable for point-of-care testing (POCT). Although its sensitivity (25nM-1μM) is slightly lower than the picomolar level of RT-qPCR, its "one-step" detection characteristics have greater application potential in rapid diagnosis and complex sample analysis.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the technical solutions. Those skilled in the art should understand that modifications or equivalent replacements of the technical solutions of the present invention that do not depart from the purpose and scope of the technical solutions of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a fluorescence sensing array based on MOF materials, characterized in that: The specific steps are as follows: Step 1: Preparation of Zr-MOF; Step 2: Obtain single-stranded DNA 1, DNA 2, DNA 3, and DNA 4, and label the 5' ends of the single-stranded DNA 1, DNA 2, DNA 3, and DNA 4 with carboxyfluorescein; centrifuge the labeled DNA single strands, and then add TBE buffer to prepare single-stranded DNA 1 solution, single-stranded DNA 2 solution, DNA 3 solution, and DNA 4 solution for later use; Among them, the sequence of DNA1 is: CCCCCCCCCCCCCCCCCCCC; The sequence of DNA2 is: AAAAAAAAAAAAAAAAAAAA; The sequence of DNA3 is: TCTCTCTCTCTCTCTCTCTC; The sequence of DNA4 is: AGAGAGAGAGAGAGAGAGAG: The concentrations of the single-stranded DNA 1 solution, the single-stranded DNA 2 solution, the DNA 3 solution, and the DNA 4 solution were all 5 to 10 nmol / L; Step 3: adding the Zr-MOF prepared in step 1 to the single-stranded DNA 1 solution, the single-stranded DNA 2 solution, the DNA 3 solution, and the DNA 4 solution prepared in step 2, respectively, and incubating at room temperature to obtain the fluorescent sensing array; Wherein, in the incubation system, the concentration of Zr-MOF is 10-100 μg / mL, and the incubation time is 10-20 min.

2. The preparation method according to claim 1, characterized in that In step 1, Zr-MOF was prepared by the following steps: Step 1: adding ZrCl4 and tetracarboxyphenylporphyrin to dimethylformamide, dissolving them by ultrasonication, and then adding dichloroacetic acid to obtain a reaction solution; wherein, in the reaction solution, the concentration of ZrCl4 is 5 to 10 mmol / L, the concentration of tetracarboxyphenylporphyrin is 0.5 to 1.0 mmol / L, and the concentration of dichloroacetic acid is 1 to 5 mg / mL; Step 2: reacting the reaction solution obtained in step 1 at 100-150° C. for 18-24 hours; Step 3: The reaction product obtained in step 2 was centrifuged to obtain a dark purple solid, which was washed with dimethylformamide and ethanol several times and then dried at 100-150° C. for 12 h to obtain Zr-MOF.

3. The preparation method according to claim 1, characterized in that: In step 2, centrifuge at 3000-4000 rpm for 30-60 seconds.

4. The preparation method according to claim 1, characterized in that In step 3, whether the incubation is terminated is determined by detecting changes in the fluorescence signal of the incubation system.

5. The preparation method according to claim 1, characterized in that: In step 3, the concentration of Zr-MOF was 60 μg / mL.

6. The preparation method according to claim 1, characterized in that: In step 3, the incubation time was 13 min.

7. An application of a fluorescence sensing array based on MOF materials, characterized in that: The fluorescent sensor array prepared by the preparation method of claims 1 to 6 is used to detect the concentrations of miR-29a, miR-92a, miRNA-21, and miRNA-17-5.

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