A method for preparing MOFs-DNA chip

Abstract

The application belongs to the field of biochemistry and medical diagnosis, and discloses a preparation method of MOFs-DNA chips. The application utilizes the fact that 1,5-diaminonaphthalene (DAN) can be combined with active metal porphyrin-based organic framework (PCN) in the form of "π-π" bond, and the amino group (-NH2) of DAN reacts with the aldehyde group (-CHO) of a glass slide, so that PCN and DAN are connected to the aldehyde glass slide; the amino group (-NH2) at the end of DAN reacts with the active ester (-NHS) of active ester polyethylene glycol azide (NHS-PEG-N3), to generate a stable amide bond, that is, a NH2-PEG-N3 layer is introduced on the surface of the glass slide, which can undergo Click reaction with Dibenzocyclooctyne (DBCO) modified DNA, so as to fix the DNA on the surface of MOFs, thereby constructing a MOFs-DNA chip with a detection function, and the purpose is to realize high-sensitivity and multi-sample detection of target objects.

Description

Technical Field

[0001] This invention belongs to the fields of biochemistry and medical diagnostic technology, and relates to a detection method, especially a method for preparing MOFs-DNA chips used in the detection. Background Technology

[0002] Metal-organic frameworks (MOFs) are crystalline porous materials composed of metal ions and organic ligands linked by coordination bonds. MOFs possess advantages such as nanoscale framework structure, tunable pore size, large specific surface area, and good chemical stability. They can be used as signal probes for various detection methods, as well as as signal probe carriers for optical, electrochemical, and photoelectrochemical detection. They have been widely applied in biosensors, biomedicine, electrocatalysis, energy storage, and conversion.

[0003] Organic ligands with abundant functional groups make MOFs (Metal-Organic Facility) readily functionalizable with various molecules and materials, including nucleic acids, enzymes, and nanoparticles. Researchers have combined nucleic acids with MOFs for biosensing. The uniform crystal structure of MOFs facilitates a high specific surface area, allowing various DNA, RNA, and aptamers to attach to them. Furthermore, due to the tunable pore size of MOFs, different types of nucleic acids with varying specificities can be immobilized on or inside the MOFs. Nucleic acid aptamer-functionalized MOFs specifically bind to target metal ions, achieving high sensitivity and selectivity. Therefore, MOFs can serve as signal probes for various methods.

[0004] Currently, most detection methods, such as enzyme-linked adsorption assay (ELISA), high-performance liquid chromatography (HPLC), and immunofluorescence assay (IFA), suffer from problems such as long processing time, complex procedures, low sensitivity, and radioactive contamination. They cannot effectively detect trace substances, and some detection processes require secondary labeling, posing potential radiation hazards. This not only increases the cost and difficulty of detection but also hinders further improvements in detection efficiency.

[0005] Compared to other detection methods, biochips utilize probe biomolecules attached to a solid matrix to identify target biomolecules. Various techniques (radioactive, fluorescent, or electrochemical) are then used to screen for biorecognition elements. Biochips offer high throughput, large scale, and parallelism, enabling simultaneous detection of multiple samples. They can be used for disease diagnosis, drug screening, gene diagnosis, and screening, and their application prospects in medicine are undoubtedly very broad. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention utilizes the ability of 1,5-diaminonaphthalene (DAN) to bind with active metalloporphyrin-based organic frameworks (PCN) via a π-π bond. The amino group (-NH2) of DAN reacts with the aldehyde group (-CHO) of the glass slide, linking PCN and DAN to the aldehyde-based glass slide. The amino group (-NH2) at the end of DAN reacts with the active ester (-NHS) of the active ester polyethylene glycol azide (NHS-PEG-N3) to generate a stable amide bond, thus introducing an NH2-PEG-N3 layer on the glass slide surface. This layer can undergo a Click reaction with dibenzocyclooctyne (DBCO)-modified DNA, immobilizing the DNA on the MOF surface, thereby constructing a MOF-DNA chip with detection capabilities. The aim is to achieve highly sensitive, multi-sample detection of target analytes.

[0007] A method for preparing a MOFs-DNA microarray includes the following steps:

[0008] (1) Immerse the glass slide in a mixture of concentrated sulfuric acid and hydrogen peroxide for 2-3 hours, then rinse it thoroughly with ultrapure water 2-3 times, place it on a slide rack, and let it air dry naturally.

[0009] (2) Place the dried slide from step (1) into an ethanol solution containing 2-3 v% APTES, shake for 25-35 minutes, rinse thoroughly with ultrapure water 2-3 times, place on a slide rack, and air dry naturally to obtain an aminated slide.

[0010] (3) Place the aminated glass slide obtained in step (2) in a 5% glutaraldehyde solution and soak for 2-3 hours. Then rinse thoroughly with ultrapure water 2-3 times, place on a glass slide rack, and air dry naturally to obtain the aldehyde-modified glass slide.

[0011] (4) Add PCN & DAN mixture to the aldehyde-modified glass slide, incubate overnight at 4°C, and wash slowly with ultrapure water to remove the unfixed PCN-DAN mixture.

[0012] (5) Add NHS-PEG-N3 to a glass slide containing the PCN&DAN complex, incubate at 4°C for 10-12 hours, and slowly wash the surface of the glass slide with ultrapure water to remove unreacted NHS-PEG-N3.

[0013] (6) On the glass slide processed in step (5), the DNA modified by DBCO is linked by the Click reaction to obtain the MOFs-DNA chip.

[0014] In step (1), the volume ratio of concentrated sulfuric acid to hydrogen peroxide is 7:3, and the concentration of hydrogen peroxide is 30% (v).

[0015] In step (2), the purity of ethanol is 95% v%.

[0016] In step (4), the concentration of PCN in the PCN & DAN mixture is 200 nM and the concentration of DAN is 70 ug / mL. PCN is a MOF material.

[0017] In step (5), the concentration of NHS-PEG-N3 is 500 ug / mL.

[0018] This invention selects DNA sequences that can specifically identify the target analyte based on the type of the target analyte, prepares a specific MOFs-DNA chip, and then uses it for the detection of the target analyte.

[0019] The MOFs-DNA chip prepared according to this invention was used for the detection of heavy metal ions in solution. Specifically, a solution containing heavy metal ions was dropped onto the surface of the MOFs-DNA chip sensor, reacted at room temperature for 20 min, washed with ultrapure water, and the fluorescence intensity was measured under a microarray scanner at 650 nm.

[0020] In one step, the MOFs-DNA chip prepared by this invention is used in Hg solution. 2+ Detection; when the target analyte is Hg 2+ At that time, the DNA sequence was: 5'-GGGTGGGTGGGGTGGGT-3'.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) The glass slides used to prepare MOFs in this invention are easy to obtain and have the advantages of low cost, smooth surface and low background fluorescence signal. They can be widely used as carriers in gene chips.

[0023] (2) The method for preparing MOFs-DNA chips in this invention is simple and the reaction conditions are mild. The amino group (-NH2) at the end of the DNA reacts with the active ester (-NHS) of NHS-PEG-N3 to form a stable amide bond. Further introducing an NH2-PEG-N3 layer on the glass slide surface can increase its stability. A Click reaction occurs with DBCO-modified DNA under mild reaction conditions. Stable thioether bonds are formed, and the DNA is fixed on the MOF surface using the thioether bond interaction.

[0024] (3) In this invention, DBCO is modified and DNA with fluorescent labeling at the complementary sequence ends can achieve high-sensitivity detection at low concentrations. After the target analyte is added, the target analyte binds to the DNA, thereby changing the conformation of the DNA, changing the distance between the DNA and the surface of MOFs, and changing the fluorescence intensity, thereby achieving detection.

[0025] (4) MOFs have advantages such as nanoscale framework structure, tunable pore size, large specific surface area and good chemical stability, and have been widely used in biosensors, biomedicine, electrocatalysis, energy storage and conversion. Attached Figure Description

[0026] Figure 1 For the detection of different concentrations of Hg using MOFs-DNA chip sensor 2+ The fluorescence intensity change graph;

[0027] Figure 2 For Hg 2+ Sensitivity detection graph;

[0028] Figure 3 The fluorescence intensity changes of various metal ions in the MOFs-DNA chip sensor are shown. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Example 1

[0031] (1) Immerse the glass slide in a mixture of concentrated sulfuric acid and hydrogen peroxide for 2 hours, then rinse it thoroughly with ultrapure water 3 times, place it on a slide rack and let it air dry naturally.

[0032] (2) Place the dried slide from step (1) into an ethanol solution containing 3v% APTES, shake for 30 minutes, rinse thoroughly with ultrapure water 3 times, place on a slide rack, and air dry naturally to obtain an aminated slide.

[0033] (3) Place the aminated glass slide obtained in step (2) in a 5% glutaraldehyde solution and soak for 2 hours. Then rinse thoroughly with ultrapure water 3 times, place it on a glass slide rack, and air dry naturally to obtain the aldehyde-modified glass slide.

[0034] (4) Add the optimal concentration of PCN (60ug / mL) & DAN (200nM) mixture to the aldehyde-modified glass slide, incubate overnight at 4°C, and wash slowly with ultrapure water to remove the unfixed PCN & DAN mixture.

[0035] (5) On a glass slide containing the PCN&DAN complex, NHS-PEG-N3 with a final concentration of 500ug / mL was added, and the slide was incubated at 4℃ for 12 hours. The slide surface was then slowly washed with ultrapure water to remove unreacted NHS-PEG-N3.

[0036] (6) Based on (5), the final concentration of 50 nM DNA modified with DBCO was ligated by Click reaction, with the sequence 5'-DBCO-GGGTGGGTGGGTGGGT-3'. The DNA was incubated overnight at 4°C and then slowly washed three times with ultrapure water to remove unligated DNA; thus obtaining the MOFs-DNA chip.

[0037] The MOFs-DNA chip prepared above was used to detect Hg. 2+ :

[0038] (7) Add Hg at different final concentrations 2+ (0 nM, 5 nM, 10 nM, 20 nM, 30 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM) were applied to the surface of the MOFs-DNA chip sensor, and Hg was aspirated after reacting at room temperature for 20 min. 2+ The reaction solution was washed three times with ultrapure water, and the fluorescence intensity was measured under a microarray scanner at 650 nm. Figure 1 As shown. Figure 1 To add different concentrations of Hg 2+ The changes in fluorescence intensity caused by Hg can be seen as follows: 2+ With increasing concentration, the fluorescence intensity measured by the MOFs-DNA chip showed a decreasing trend.

[0039] Figure 2 For Hg 2+ Sensitivity analysis results, when Hg 2+ When the concentration is between 0-50 nM, the fluorescence intensity is similar to that of Hg. 2+ The concentration showed a clear linear relationship, with the linear equation being y = -9.4293 × C. Hg 2+ +827.29, R² = 0.9871.

[0040] Example 2

[0041] (1) Immerse the glass slide in a mixture of concentrated sulfuric acid and hydrogen peroxide for 2 hours, then rinse it thoroughly with ultrapure water 3 times, place it on a slide rack and let it air dry naturally.

[0042] (2) Place the dried slide from step (1) into an ethanol solution containing 3v% APTES, shake for 30 minutes, rinse thoroughly with ultrapure water 3 times, place on a slide rack, and air dry naturally to obtain an aminated slide.

[0043] (3) Place the aminated glass slide obtained in step (2) in a 5% glutaraldehyde solution and soak for 2 hours. Then rinse thoroughly with ultrapure water 3 times, place it on a glass slide rack, and air dry naturally to obtain the aldehyde-modified glass slide.

[0044] (4) Add PCN (60ug / mL) & DAN (200nM) mixture to the aldehyde-modified glass slide, incubate overnight at 4°C, and wash slowly with ultrapure water to remove the unfixed PCN-DAN mixture.

[0045] (5) On a glass slide containing the PCN&DAN complex, NHS-PEG-N3 with a final concentration of 600ug / mL was added, and the slide was incubated at 4°C for 12 hours. The slide surface was then slowly washed with ultrapure water to remove unreacted NHS-PEG-N3.

[0046] (6) Based on (5), the final concentration of 50 nM DNA modified with DBCO was ligated by Click reaction, with the sequence 5'-DBCO-GGGTGGGTGGGTGGGT-3'. The DNA was incubated overnight at 4°C and then slowly washed three times with ultrapure water to remove unligated DNA; thus obtaining the MOFs-DNA chip.

[0047] Selectivity experiments for other metal ions:

[0048] (7) Add metal ions (Na) + K + Mg 2+ Cu 2+ Hg 2+ The metal ion reaction solution was applied to the surface of the MOFs-DNA chip sensor and reacted at room temperature for 20 minutes. The solution was then removed and washed with ultrapure water. After washing three times, the fluorescence intensity was measured under a microarray scanner at 650 nm.

[0049] Figure 3 To add different metal ions (Na) at a concentration of 100 nM + K + Mg 2+ Cu 2+ Hg 2+ The fluorescence changes after reaction with 30 nM DNA, such as Figure 3 As shown, compared to other metal ions, Hg in this MOFs-DNA chip sensor... 2+ The corresponding fluorescence was strong, thus proving that the MOFs-DNA chip was effective against Hg. 2+ It has good selectivity.

Claims

1. A method for preparing a MOFs-DNA chip, characterized in that: Includes the following steps: (1) Immerse the glass slide in a mixture of concentrated sulfuric acid and hydrogen peroxide. After immersion, rinse thoroughly with ultrapure water, place on a slide rack, and let it air dry naturally. (2) Place the dried slide from step (1) into an ethanol solution containing APTES, shake, rinse thoroughly with ultrapure water again, place on a slide rack, and air dry naturally to obtain an aminated slide. (3) Place the aminated glass slide obtained in step (2) in glutaraldehyde solution, soak it, wash it thoroughly with ultrapure water, place it on a glass slide rack, and air dry it naturally to obtain the aldehyde-modified glass slide. (4) Add PCN & DAN mixture to the aldehyde-modified glass slide, incubate overnight, and then slowly wash with ultrapure water to remove the unfixed PCN-DAN mixture. (5) On a glass slide containing the PCN&DAN complex, NHS-PEG-N3 was added dropwise. After incubation, the surface of the glass slide was slowly washed with ultrapure water to remove unreacted NHS-PEG-N3. (6) On the glass slide processed in step (5), the DNA modified by DBCO is linked by the Click reaction to obtain the MOFs-DNA chip.

2. The preparation method according to claim 1, characterized in that: In step (1), the volume ratio of concentrated sulfuric acid to hydrogen peroxide is 7:3, and the concentration of hydrogen peroxide is 30v%. Soak for 2-3 hours and wash 2-3 times.

3. The preparation method according to claim 1, characterized in that: In step (2), the ethanol solution containing APTES has an APTES content of 2-3 v% and an ethanol purity of 95 v%. The shaking time is 25-35 minutes, and the ultrapure water is used to rinse the product 2-3 times.

4. The preparation method according to claim 1, characterized in that: In step (3), the concentration of glutaraldehyde solution is 5v%; the soaking time is 2-3 hours; and the ultrapure water is used to rinse the solution 2-3 times.

5. The preparation method according to claim 1, characterized in that: In step (4), the concentration of PCN in the PCN & DAN mixture is 200 nM and the concentration of DAN is 70 ug / mL; the incubation temperature is 4℃.

6. The preparation method according to claim 1, characterized in that: In step (5), the concentration of NHS-PEG-N3 is 500ug / mL; the incubation temperature is 4℃ and the incubation time is 10-12 hours.

7. The preparation method according to claim 1, characterized in that: In step (6), the DNA is selected based on the type of the target analyte, choosing a DNA sequence that can specifically identify the target analyte.

8. The use of the MOFs-DNA chip prepared by the preparation method according to any one of claims 1 to 7 for the detection of heavy metal ions in solution.

9. The use as described in claim 8, characterized in that, The specific steps are as follows: A solution containing heavy metal ions was dropped onto the surface of the MOFs-DNA chip sensor. After reacting at room temperature for 20 minutes, the sensor was washed with ultrapure water, and the fluorescence intensity was measured under a microarray scanner at 650 nm.

10. The use as described in claim 8, characterized in that, When the heavy metal ion is Hg 2+ hour; In the MOFs-DNA chip, the DNA sequence is: 5'-GGGTGGGTGGGTGGGT-3'.

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