CN116903875A - Biosensor based on metal-organic framework and preparation method thereof - Google Patents

Abstract

The application provides a metal organic framework MNP/Cu-BTC MOF, preparation and application thereof as a tumor source exosome catcher. The application realizes the capturing of exosomes through the interaction of metal ions in the metal organic framework and phospholipid structures on the exosomes from tumor sources. The tumor source exosome catcher based on the metal organic framework can realize simple and convenient detection of tumor exosome with good active site and biocompatibility without structural modification. The tumor source exosome catcher based on the metal organic framework, which is developed by the application, provides a new platform for ultrasensitive and low-cost biological detection, and provides a new tool for cancer detection and continuous monitoring, and accurate medical treatment-based treatment and screening of therapeutic drug resistance markers.

Description

A biosensor based on metal-organic framework and its preparation method

Technical field

The invention belongs to the technical field of polymer composite materials and biosensing detection, specifically relates to a biosensor based on a metal organic framework and a preparation method thereof, and particularly relates to a metal organic framework MNP/Cu-BTC MOF and its preparation and its use as a tumor source. Applications of exosome traps.

Background technique

As a type of extracellular membrane vesicle with a diameter of 30-150 nm, exosomes are distributed in different biological fluids, such as blood, cerebrospinal fluid, saliva, and urine. It is considered as a potential biomarker of tumors because it is rich in proteins, mRNA and miRNA, and it is widely used in clinical applications. These vesicles (exosomes) contain phospholipid structures, which serve as intercellular messengers and actively participate in various pathophysiological processes such as inflammation, tissue regeneration, and cancer metastasis. Recently, researchers have discovered that cancer cells secrete significantly higher amounts of exosomes than normal cells, and that tumor exosomes contain unique nucleic acid or protein markers, making them potential biomarkers for non-invasive cancer diagnosis. However, the development of highly sensitive and reliable detection technologies has been hampered by the low concentrations of these species and cross-reactivity between monomers and oligomers. To this end, despite many obstacles, different methods have been developed to analyze tumor exosomes. For example, based on the structure of exosomes, ultracentrifugation technology is often used to purify exosomes from complex biological media, but the detection process is quite complex and time-consuming. In addition, immunoaffinity capture technology has high specificity, is simple to operate, and does not affect the integrity of exosomes, but its overall efficiency is still low and requires expensive antibodies. Therefore, it remains a difficult task to develop a capture device that can satisfy both simple and efficient exosome collection and subsequent highly sensitive and selective detection.

Metal-organic frameworks (MOFs) are composed of metal ions and organic linkers. Due to their advantages of easy synthesis, low cost, tunable porosity, and flexible modification, they have been widely used in gas storage, energy, drug delivery, and biosensing. The field shows broad application prospects. Especially in the field of biosensing, MOFs can be combined with different recognition elements, such as DNA, peptides and proteins, through strong interactions between metal nodes and certain groups (such as phosphates or imidazole). Therefore, the synthesized nanoconjugates can effectively identify and capture analytes from complex media.

Contents of the invention

The purpose of the present invention is to provide a metal organic framework MNP/Cu-BTC MOF and its preparation and application as a capture device for tumor-derived exosomes. Identification through the interaction of metal ions in metal-organic frameworks with phospholipid structures on tumor-derived exosomes and determination of their capture efficiency through fluorescence spectroscopy for biosensing and analytical detection. In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

First, the invention provides a metal organic framework MNP/Cu-BTC MOF, which is mainly composed of 1,3,5-benzenetricarboxylic acid (BTC), copper chloride dihydrate and superparamagnetic silica-coated magnetic beads. (MNP) composite, the overall shape is rod-shaped, with an average diameter of about 5-20 μm and a length range of 80-160 μm. Cu ²⁺ and BTC form a MOF rod-like skeleton structure, and the magnetic beads MNP are spherical and attached to The surface of the rod-like structure. The average diameter of the rod-shaped morphology of metal organic frameworks (MNP/Cu-BTC MOFs) can be 5μm, 10μm, 15μm, 20μm; the length range can be 80μm, 100μm, 120μm, 140μm, 160μm. Among them, superparamagnetic silica-coated magnetic beads (MNP) are silica magnetic beads. The particle size is 300-500 nm, preferably about 400 nm. Commercially available silica hydroxyl magnetic beads can be used, or It is obtained through common one-pot chemical synthesis using Fe ₃ O ₄ /SiO ₂ .

On the other hand, the present invention also provides a method for preparing the above-mentioned metal organic framework MNP/Cu-BTC MOF, which is to add BTC to MNP, then add copper chloride dihydrate, and react at room temperature; magnetic separation Wash and dry. The molar ratio of BTC to copper chloride dihydrate is 1:1-3.

As an implementation of the above preparation method, preferably, 1 mL, 0.1-0.2 M BTC ethanol solution can be added to 10-50 μL, 30-50 μg/mL MNPs ethanol solution, stir continuously, and then 1 mL, Slowly add 0.1-0.3M CuCl ₂ solution to the above solution to prepare a reaction solution with a total volume of 2 mL, and stir at room temperature for 10-60 min.

As a specific implementation of the above preparation method, 1 mL, 0.125 M BTC ethanol solution can be added to 50 μL, 40 μg/mL MNPs ethanol solution, stir continuously, and then slowly add 1 mL, 0.2 M CuCl ₂ solution Add to the above solution to prepare a reaction solution with a total volume of 2 mL, and stir at room temperature for 0.5 h.

Preferably, the magnetic separation, washing and drying can be performed by magnetically separating the prepared MNP/Cu-BTC MOF solution for 3-8 minutes, separating the supernatant and precipitate, and washing the bottom precipitate with pure water 2-5 times. The final product is obtained; the final product solution is placed in a vacuum drying box and dried to obtain the finished product.

Second, the inventor of the present application discovered through research that the metal organic framework MNP/Cu-BTCMOF of the present invention can be used as a trap for tumor-derived exosomes.

In the above-mentioned applications, preferably, tumor-derived exosomes need to be obtained through an exosome extraction kit and labeled with a fluorescent dye to prepare fluorescently labeled tumor-derived exosomes. Specifically, the preparation of fluorescently labeled tumor-derived exosomes can adopt the following method steps:

Collect the supernatant in the tumor cell culture bottle into a test tube, and collect exosomes from tumor cells according to the extraction kit; incubate the fluorescent dye with the extracted exosomes to obtain fluorescently labeled exosomes; through dialysis Fluorescent dye-labeled exosomes were purified and stored in a -20--40°C refrigerator.

Among the above-mentioned applications, as a preferred application scheme, the application can adopt the following steps: using the metal organic framework MNP/Cu-BTC MOF as the trap, and combining it with fluorescently labeled tumor-derived exosomes Co-incubation is performed at room temperature, and the capture efficiency is determined by fluorescence spectrum, which can be used for biosensing analysis and detection.

In the above-mentioned applications, preferably, after co-incubation at room temperature, it is necessary to perform magnetic sorting, discard the supernatant, and resuspend the sediment to obtain a tumor-derived exosome trap based on a metal-organic framework, and then use it for tumor-derived exosomes. Fluorescence spectrum analysis and detection of clinical samples; a more preferred application method is to incubate 5 μL of fluorescently labeled tumor-derived exosomes with 45 μL of MNP/Cu-BTC MOFs solution (50 μg/mL) for 15 min at room temperature in the dark, and perform magnetic separation. Select 5 minutes and resuspend in HEPES buffer solution to obtain a metal-organic framework-based tumor-derived exosome capture device, which can be further used for analysis and detection of tumor-derived clinical samples.

In the above-mentioned applications, preferably, the tumor-derived exosomes can be exosomes derived from various tumors, including breast cancer cell MCF-7 exosomes and breast cancer cell SK-BR-3 exosomes. , human malignant melanoma cell A-375 exosomes, pancreatic cancer cell line BxPC-1 exosomes, etc.; more preferably, breast cancer cell SK-BR-3 exosomes, the particle size range is 30-150nm, Displays red fluorescence.

In the above-mentioned application, when the tumor-derived exosomes are breast cancer cell SK-BR-3 exosomes, a method for preparing fluorescently labeled tumor-derived exosomes or an extraction method for fluorescently labeled tumor-derived exosomes The following steps can be included:

Step (2.1) Collect the supernatant from the breast cancer cell (SK-BR-3) cell culture bottle into a test tube, and collect exosomes secreted from SK-BR-3 breast cancer cells according to the extraction kit;

Step (2.2) obtains fluorescently labeled exosomes by incubating the extracted exosomes with a fluorescent dye (Dil);

Step (2.3) Purify Dil-labeled exosomes by dialysis and store them in a -20°C refrigerator.

Preferably among the above extraction methods, in step (2.1), use a pipette to absorb 5 mL of SK-BR-3 breast cancer cell culture supernatant, add 1.25 mL of Hieff ^TM Quick exosome extraction reagent, and let stand at 4°C. 5h; then centrifuge the above mixture at 4℃, 10000rpm for 1h, collect the bottom precipitate, resuspend it into a 2mL EP tube with PBS buffer solution; then centrifuge at 4℃, 14000rpm for 1min, collect the supernatant to -20 Store in refrigerator at ℃;

Preferably, in the above extraction method, in step (2.2), 99 μL of the exosome solution collected above is incubated with 1 μL of Dil at low temperature for 15 min under light-proof conditions. Then, the mixture was ultracentrifuged at 100,000 rpm and 4°C for 1 hour, and the tube wall and bottom sediment were pipetted with PBS buffer solution to obtain a resuspended solution;

Preferably, in the above extraction method, in step (2.3), the above fluorescently labeled exosome sample is placed in a dialysis bag and allowed to react overnight to remove the free Dil dye and finally obtain a pure fluorescently labeled exosome solution.

Among the above-mentioned applications, it is preferred to use a metal organic framework (MNP/Cu-BTC MOF) and fluorescently labeled tumor-derived exosomes as raw materials to prepare a metal organic framework-based tumor-derived exosome trap, Its capture efficiency is 80-96%.

beneficial effects

On the one hand, the present invention can achieve ultra-sensitive capture of exosomes by interacting with the phospholipid structure on tumor-derived exosomes through the interaction of metal ions Cu ²⁺ in the metal-organic framework, and at the same time based on the good binding properties of MNP to organisms. Adsorption. In addition, the present invention can magnetically sort the complex through the MNP magnetism of the metal organic framework itself to achieve quick and easy separation. It can further achieve the detection of tumor-derived exosomes through the fluorescence spectrum displayed by fluorescent molecules. intuitive analysis.

The tumor-derived exosome trap based on metal organic frameworks of the present invention can achieve simple and convenient adsorption of metal organic frameworks on the surface of exosomes without structural modification, and has good active sites and biocompatibility. Therefore, the developed tumor-derived exosome trap provides a new idea for an ultrasensitive and low-cost detection platform. This opens new avenues for cancer detection and continuous monitoring, precision medicine-based treatment, and screening of therapeutic resistance markers.

Description of the drawings

Figure 1 is an electron microscope image of the MNP/Cu-BTC MOF provided in Embodiment 1 of the present invention;

Figure 2 is an electron microscope image of exosomes provided in Embodiment 2 of the present invention;

Figure 3 is a correlation analysis diagram between exosome particle size and concentration provided in Embodiment 2 of the present invention;

Figure 4 is the fluorescence spectrum of fluorescently labeled exosomes captured by MNP/Cu-BTC MOF provided in Example 3 of the present invention;

Figure 5 is a laser confocal microscope of MNP/Cu-BTC MOF capturing fluorescently labeled exosomes provided in Example 3 of the present invention;

Figure 6 shows the fluorescence spectrum of fluorescently labeled exosomes captured by the MNP/Cu-BTC MOF provided in Example 3 of the present invention under different biological backgrounds.

Figure 7 is a schematic diagram of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below. However, it should be understood that the specific embodiments described here are only used to explain the present invention and are not used to limit the scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the technical field of the present invention. The terms used herein in the description of the present invention are only for describing specific implementations. The examples are not intended to limit the invention.

Example 1

The preparation method of metal organic framework MNP/Cu-BTC MOF includes the following steps:

Add 50 μL of ethanol solution containing MNPs (40 μg/mL) to 1 mL of BTC (0.125 M) dissolved in ethanol, and continue stirring. Then 1 mL CuCl ₂ (0.2 M) was slowly added to the above solution to prepare a reaction solution with a total volume of 2 mL, and stirred at room temperature for 0.5 h. The prepared MNP/Cu-BTC MOF solution was separated by magnetic separation for 5 minutes, and the supernatant and precipitate were separated. The bottom precipitate was washed three times with pure water to obtain the final product, which was dried in a vacuum drying oven and finally dissolved in HEPES. A MNP/Cu-BTC MOF solution with a concentration of 50 μg/mL was obtained. The electron microscope image of the finally prepared MNP/Cu-MOFs is shown in Figure 1.

Example 2

Preparing fluorescently labeled exosomes includes the following steps:

Use a pipette to absorb 5 mL of SK-BR-3 breast cancer cell (ATCC) culture supernatant, add 1.25 mL of Hieff ^TM Quick exosome extraction reagent and let stand at 4°C for 5 hours; then, the above mixture is incubated at 4°C. Centrifuge at 10,000 rpm for 1 hour, collect the bottom precipitate, resuspend in a 2 mL EP tube with PBS buffer solution, and then centrifuge at 4°C, 14,000 rpm for 1 min. Take 99 μL of exosome solution collected above and incubate it with 1 μL Dil at low temperature for 15 min in the dark. The mixture was then ultracentrifuged at 100,000 rpm and 4°C for 1 hour, and the walls and bottom of the tube were pipetted with PBS buffer solution to obtain a resuspended solution; the above fluorescently labeled exosome samples were placed in a dialysis bag, reacted overnight, and removed The free Dil dye is finally used to obtain a pure fluorescently labeled exosome solution with a concentration of (1-3)×10 ¹⁰ particles/mL, and the particles are the number of fluorescent spots. The electron microscopy image of the finally prepared exosomes is shown in Figure 2, and the nanoparticle tracking analysis results are shown in Figure 3 (the concentration of the fluorescently labeled exosome solution is diluted to E7 level, that is, 10 ⁷ ), proving that particles can be obtained according to the extraction and separation method. exosomes with uniform diameter and distribution.

Example 3

The steps to obtain a tumor-derived exosome trap based on a metal-organic framework are as follows:

45 μL of the MNP/Cu-BTC MOF solution prepared in Example 1 at 50 μg/mL was mixed with 5 μL of (1-3)×10 ¹⁰ particles/mL fluorescently labeled exosome solution (i.e., fluorescently labeled tumor-derived exosomes in Example 2 Incubate in the dark at room temperature for 15 minutes, magnetically sort for 5 minutes, and resuspend the supernatant in HEPES buffer solution. As shown in Figure 4, the maximum wavelength of the fluorescent dye in the supernatant was measured with a fluorescence spectrophotometer to obtain the respective fluorescence values. Compared with the initial fluorescence value, the fluorescence signal in the supernatant after magnetic separation was significant. The decrease corresponds to the capture rate of fluorescently labeled exosomes by the MNP/Cu-BTC MOF material being approximately 95.2%; as shown in Figure 5, laser confocal microscopy results show that the MNP/Cu-BTC MOF bright field and Dil red fluorescence background are almost completely coincide. The above results verify the capture and adsorption effect of the above materials on exosomes. Finally, as shown in Examples 4-8 below, different biological samples (such as DMEM, serum, plasma and saliva) and HEPES buffer were added to exosomes, and the same magnetic separation method was used, as shown in Figure 6. Detection performance of MNP//Cu-BTC MOF in these complex biological media.

Example 4

A method for preparing a tumor-derived exosome trap based on a metal-organic framework is similar to Example 3. The difference is that in step (3), MNP/Cu-BTC MOF and biological sample-DMEM (Thermo Fisher Scientific) are added. ) exosome incubation can still achieve effective adsorption of exosomes.

Example 5

A method for preparing a tumor-derived exosome trap based on a metal-organic framework is similar to Example 3, except that in step (3), MNP/Cu-BTC MOF and biological sample-serum (human serum) are added Incubation of exosomes can still achieve effective adsorption of exosomes.

Example 6

A method for preparing a tumor-derived exosome trap based on a metal-organic framework is similar to Example 3, except that in step (3), MNP/Cu-BTC MOF and biological sample-plasma (human plasma) are added Incubation of exosomes can still achieve effective adsorption of exosomes.

Example 7

A method for preparing a tumor-derived exosome trap based on a metal organic framework is similar to Example 3, except that in step (3), MNP/Cu-BTC MOF and biological sample-saliva (normal human saliva) are added ) exosome incubation can still achieve effective adsorption of exosomes.

Example 8

A method for preparing a tumor-derived exosome trap based on a metal organic framework is similar to Example 3, except that in step (3), MNP/Cu-BTC MOF and biological sample-HEPES buffer (A Latin) exosome incubation can still achieve effective adsorption of exosomes.

Example 9

A method for preparing a tumor-derived exosome trap based on a metal-organic framework, similar to Example 3, except that the material is replaced by MNP/Fe-BTC MOF, MNP/Fe-BTC MOF, MNP/Fe-BTC MOF The preparation method is the same as MNP/Cu-BTC MOF. Specific: Add 1mL BTC (0.125M) dissolved in ethanol to the solution containing MNPs (40μg/mL), and continue stirring. Then 1 mL FeCl ₃ (0.2 M) was slowly added to the above solution, and stirred at room temperature for 0.5 h. The obtained precipitate was magnetically separated for 5 min, and the final product was washed three times with purified water. The maximum wavelength of the fluorescent dye in the supernatant was measured with a fluorescence spectrophotometer to obtain the respective fluorescence values. Compared with the initial fluorescence value, the decrease in fluorescence signal in the supernatant after magnetic separation corresponds to MNP/Fe-BTC. The capture rate of fluorescently labeled exosomes by MOF materials is about 85.7%; laser confocal microscopy MNP/Fe-BTC MOF bright field and Dil red fluorescence background overlap effect is not as good as MNP/Cu-BTC MOF. Only replacing anhydrous copper chloride CuCl ₂ with FeCl ₃ can achieve the adsorption of exosomes, but the adsorption effect is not as good as MNP/Cu-BTC MOF.

Example 10

Similar to Example 3, the difference is that the material is MNP/Zn-BTC MOF instead of MNP/Cu-BTC MOF, and the preparation method of MNP/Zn-BTC MOF is the same as MNP/Cu-BTC MOF. The maximum wavelength of the fluorescent dye in the supernatant was measured with a fluorescence spectrophotometer to obtain the respective fluorescence values. Compared with the initial fluorescence value, the decrease in fluorescence signal in the supernatant after magnetic separation corresponds to MNP/Zn-BTC. The capture rate of fluorescently labeled exosomes by MOF materials is about 74.6%; laser confocal microscopy MNP/Zn-BTC MOF bright field only partially overlaps with the Dil red fluorescent background, which is obviously inferior to MNP/Cu-BTC MOF. Only replacing anhydrous copper chloride CuCl ₂ with ZnCl ₂ can achieve the adsorption of exosomes, but the adsorption effect is not as good as MNP/Cu-BTC MOF.

Example 11

A method for preparing a tumor-derived exosome trap based on a metal-organic framework is similar to Example 3. The difference is that MNP/DA is used as the material instead of MNP/Cu-BTC MOF. Preparation of MNPs-DA:

Add MNP (40 μg/mL) solution to the Tris-HCl solution with a pH of 8.5. After stirring, add dopamine solution to a final concentration of 1 mM. React in the dark at 4°C for 10 hours. The resulting precipitate is magnetically Separate for 5 minutes, and wash the final product three times with purified water. The test found that the material MNP/DA obtained in this example has poor adsorption effect on exosomes.

The invention discloses a tumor-derived exosome trap based on a metal-organic framework, in which a metal-organic framework (MNP/Cu-BTC MOF) is used as a trap, and breast cancer cell (SK-BR-3)-derived exosomes are used as a research The adsorption and capture of exosomes can be achieved through the interaction between metal ions in the metal-organic framework and the phospholipid structure on tumor-derived exosomes.

The inventor's research found that if the metal framework model is not used, similar literature reports use heavy-duty cytometry to electroporate heavy metal ion reagents into exosomes to track exosomes at the single-cell level, but the steps are cumbersome and The cost is expensive; the inventor also found that although other metal ions such as Fe ³⁺ and Zn ²⁺ can also form MOF framework materials with MNP and BTC, their adsorption effect on tumor-derived exosomes cannot be as good as that of Cu ²⁺ Compare. At the same time, metal ions can be adsorbed on the surface of exosomes through metal-organic frameworks, and effective adsorption of exosomes can still be achieved by changing the structure of the material (such as MNP/Cu-BTCMOF). If the MNP/Cu-BTC MOF is replaced by other nanomaterials such as MNP/DA, effective adsorption of tumor-derived exosomes cannot be achieved.

It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be regarded as illustrative and non-restrictive from any point of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is therefore intended that all claims falling within the claims All changes within the meaning and scope of equivalent elements are encompassed by the present invention, and any sign in a claim should not be construed as limiting the claim involved.

Claims (10)

1. The metal organic frame MNP/Cu-BTC MOF has a rod-shaped overall structure, an average diameter of 5-20 μm and a length of 80-160 μm, wherein Cu is ²⁺ The magnetic beads MNP are spherical and are attached to the surface of the rod-shaped framework structure.

2. A preparation method of metal organic frame MNP/Cu-BTC MOF comprises adding BTC into MNP, adding copper chloride dihydrate, and reacting at room temperature; magnetic sorting, washing and drying; BTC to copper chloride dihydrate molar ratio 1:1-3.

3. The method according to claim 2, wherein 10-50. Mu.L of an ethanol solution of MNPs, 30-50. Mu.g/mL, is added to 1mL of an ethanol solution of BTC, 0.1-0.2M, followed by stirring, and then 1mL of CuCl, 0.1-0.3M is added ₂ Slowly adding the solution into the mixed solution, and stirring at room temperature for 10-60min.

4. The method according to claim 3, wherein the MNP ethanol solution containing 40. Mu.g/mL is added to 1mL of a 0.125M BTC ethanol solution, and stirring is continued, and then 1mL of 0.2M CuCl is added ₂ Slowly adding the solution into the above solution, and stirring at room temperature for 30min.

5. Use of a metallo-organic framework MNP/Cu-BTC MOF according to claim 1 or obtainable by the method of any one of claims 2-4 for the preparation of a tumor-derived exosome trap.

6. Use according to claim 5, wherein the tumor-derived exosomes are obtained by means of an exosome extraction kit and labeled with a fluorescent dye to prepare a fluorescently labeled tumor-derived exosome, and then the metallo-organic framework MNP/Cu-BTC MOF is co-incubated with the fluorescently labeled tumor-derived exosome.

7. The use according to claim 6, wherein the preparation of fluorescently labelled tumor derived exosomes employs the following method steps:

collecting the supernatant in the tumor cell culture flask into a test tube, and collecting exosomes from tumor cells according to an extraction kit; incubating the extracted exosomes with a fluorescent dye to obtain fluorescently labeled exosomes; the fluorescently labeled exosomes were purified by dialysis and stored in a-20-40 ℃ refrigerator.

8. The application according to claim 6, wherein the application employs the following steps: the metal organic framework MNP/Cu-BTC MOF and the tumor source exosome marked by fluorescence are incubated at room temperature, and the capturing efficiency is determined by fluorescence spectrum for biological sensing analysis and detection.

9. The use according to claim 8, further comprising the steps of magnetic sorting, discarding supernatant, and sediment resuspension after said room temperature co-incubation.

10. The use according to claim 8, wherein,

mu.L of the fluorescently labeled tumor-derived exosomes were incubated with 45. Mu.L of MNP/Cu-BTC MOFs solution at 50. Mu.g/mL at room temperature in the absence of light for 15min, magnetically sorted for 5min, the supernatant discarded, and resuspended with HEPES buffer solution to give a metal organic framework MNP/Cu-BTC MOF-based tumor-derived exosome trap.