Preparation method of a biosensor for targeted detection of microRNA in tumor-derived extracellular vesicles

Abstract

This invention discloses a method for preparing a biosensor for the targeted detection of microRNA in tumor-derived extracellular vesicles. Using mesoporous silica as a substrate, the method utilizes electrostatic adsorption to modify a probe and a phospholipid bilayer onto the mesoporous silica. Furthermore, the hydrophobic effect of cholesterol is utilized to modify an aptamer onto the phospholipid bilayer, thereby constructing a biosensor for detecting microRNA in tumor-derived extracellular vesicles. The biosensor method is simple, inexpensive, and has broad application prospects in early tumor screening.

Description

Technical Field

[0001] The present invention relates to a method for preparing a biosensor, and in particular to a method for preparing a biosensor for targeted detection of microRNA in tumor-derived extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are vesicles (30 nm to 1000 nm in diameter) secreted by cells and containing a phospholipid bilayer. As a medium for intercellular communication, EVs are widely present in various body fluids and contain many biomarkers (nucleic acids, proteins, lipids, etc.). Extracellular vesicles secreted by tumor cells contain many cancer-related biomarkers. Among these markers, many studies have shown that the expression levels of some microRNAs are significantly different from those in EVs derived from normal cells (such as microRNA-21, microRNA-16, etc.). At the same time, the phospholipid bilayer of EVs separates the internal microRNAs from complex body fluids, preventing the degradation of microRNAs. Therefore, microRNAs in EVs secreted by tumor cells are widely used as markers for early diagnosis of cancer. The prior art has not yet developed a simple and fast biosensor for the targeted detection of microRNAs in tumor-derived EVs. Summary of the Invention

[0003] Purpose of the invention: The present invention aims to provide a method for preparing a biosensor that is simple to operate, rapid in detection, and capable of targeted detection of microRNA in extracellular vesicles secreted by tumor cells.

[0004] Technical solution: The method for preparing a biosensor for targeted detection of microRNA in tumor-derived extracellular vesicles of the present invention comprises the following steps:

[0005] (1) dissolving (2,3-dioleoyl-propyl)-trimethylammonium chloride (hereinafter referred to as DOTAP), palmitoyloleoylphosphatidylcholine (hereinafter referred to as POPC) and cholesterol in a solvent, and drying the solvent by spin drying to obtain a lipid film composed of DOTAP, POPC and cholesterol;

[0006] Dulbecco's phosphate buffered saline (DPBS) was added to a round-bottom flask for hydration, and the hydrated product was extruded using an extruder to obtain liposomes composed of DOTAP, POPC, and cholesterol;

[0007] (2) heating and annealing the molecular beacon targeting microRNA in DPBS to obtain a hairpin-shaped molecular beacon;

[0008] The hairpin-shaped molecular beacon and mesoporous silica (silica spheres having a diameter of 3 μm) were mixed and vortexed, and the liposomes obtained in step (1) were added, incubated, and washed by centrifugation to obtain mesoporous silica microspheres loaded with the hairpin-shaped molecular beacon;

[0009] (3) Incubating the mesoporous silica microspheres loaded with the hairpin-shaped molecular beacon and the aptamer with cholesterol, and after centrifugation and washing, obtaining the mesoporous silica microspheres modified with the aptamer and loaded with the hairpin-shaped molecular beacon.

[0010] Furthermore, the composition ratio of the three components of the liposome in step (1) is 1:1:0.1 to 1:1:0.6. At this ratio, the supported lipid bilayer formed by the liposome on the surface of the material is more stable.

[0011] Furthermore, the hydration temperature in step (1) is 25-37° C., and the treatment time is 1-2 hours.

[0012] Furthermore, the extrusion condition in step (1) is to extrude the porous film with a pore size of 100 nm 11-21 times.

[0013] Furthermore, in step (2), the incubation temperature is 25-37°C, the treatment time is 1-2 hours, the concentration of the hairpin-shaped molecular beacon is 1-2 μM, the concentration of the mesoporous silica is 5-20 mg / ml, and the concentration of the liposome is 1.2-2.5 mg / ml. The material prepared using these concentrations exhibits better microRNA detection performance using the hairpin-shaped molecular beacon on the mesoporous silica.

[0014] Furthermore, the incubation condition in step (3) is 25-37° C. for 1-2 hours; the concentration of the cholesterol-containing aptamer is 500-1000 nanomolar, under which the sensor has the best capture effect on tumor-derived extracellular vesicles.

[0015] Furthermore, in step (2), the molecular beacon is annealed in DPBS at 95° C. for 10 minutes. The hairpin-shaped molecular beacon obtained under this condition has the best signal-to-background ratio and specificity.

[0016] Principle of the invention: The biosensor in the present invention recognizes tumor-derived extracellular vesicles through aptamers, achieves targeted capture and fusion of tumor-derived extracellular vesicles, and then uses molecular beacons loaded in microspheres to detect microRNA in tumor-derived extracellular vesicles.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0018] (1) The biosensor can directly target and identify microRNA in tumor-derived extracellular vesicles in human serum without the need for prior separation or lysis, is easy to operate, and has a fast detection speed. The present invention self-assembles hairpin-shaped molecular beacons onto mesoporous silica to modify aptamers, which is simple to operate and does not require complex purification steps.

[0019] (2) Using liposomes to load hairpin-shaped molecular beacons and aptamers onto mesoporous silica, the sensor has excellent stability due to the good stability of the supported lipid bilayer on silica;

[0020] (3) Using cationic liposomes and utilizing the electrostatic attraction between liposomes and hairpin-shaped molecular beacons, the encapsulation efficiency of hairpin-shaped molecular beacons is greatly improved;

[0021] (4) By utilizing the hydrophobic interaction between cholesterol and phospholipid bilayers, the aptamer was modified on the sensor surface to achieve targeted recognition of extracellular vesicles derived from tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a fluorescence microscope image of the biosensor;

[0023] Figure 2 is the detection of microRNA-21 in extracellular vesicles of A375 and U251 cells by the biosensor;

[0024] Figure 3 The biosensor detects microRNA-21 in extracellular vesicles in DPBS and plasma. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below with reference to specific examples.

[0026] Example 1

[0027] 1. Preparation of liposome suspension:

[0028] Liposomes were prepared by thin film hydration and extrusion. (2,3-Dioleoylpropyl)-trimethylammonium chloride (DOTAP), (2-oleoyl-1-palmitanin-glycero-3-phosphocholine) POPC, and cholesterol (molar ratio = 1:1:0.6) were dissolved in chloroform. The chloroform was removed by rotary evaporation, forming a lipid film at the bottom of a round-bottom flask. The film was hydrated in DPBS (37°C, 1 hour) and then extruded 21 times through a 100 nm film to form liposomes (2.5 mg / mL).

[0029] 2. Preparation of biosensors:

[0030] 1) Annealing of molecular beacons:

[0031] The molecular beacon targeting microRNA-21 was annealed in DPBS at 95°C for 10 minutes to obtain a hairpin-shaped molecular beacon.

[0032] 2) Preparation of biosensor:

[0033] The biosensor consists of four parts: molecular beacon, mesoporous microspheres with a diameter of 3 microns, phospholipid bilayer and aptamer.

[0034] Molecular beacons with 5'-terminal FAM and 3'-terminal quencher modifications were loaded into microspheres through a self-assembly process. 10 μL of annealed molecular beacons (1 μM) was mixed with 100 μL of microspheres (20 mg / mL) and shaken for 10 seconds.

[0035] 100 μL of liposome solution (2.5 mg / mL) was added and incubated for 1 hour at 37° C. Excess molecular beacons and liposomes were removed by centrifugation (6,000 rpm, 5 minutes) and washing with DPBS at least 10 times.

[0036] 500 nM cholesterol-terminated EpCAM aptamers were added to the suspension and gently shaken at 37°C for 1 hour. Excess aptamers were removed by centrifugation and the resulting fused microspheres were washed with binding buffer (containing 5 mM Mg 2+ The cells were washed with DPBS and stored at 4 °C for further use.

[0037] Depend on Figure 1 It can be seen that the phospholipid bilayer and the aptamer are mainly distributed on the surface of the microspheres, while the molecular beacon fills the entire microspheres, which indicates that the biosensor was successfully constructed.

[0038] Example 2

[0039] Referring to the preparation method of Example 1, except that the concentration of liposomes was 1.5 mg / mL and the concentration of EpCAM aptamer was 1 μM, the prepared biosensor was mixed with extracellular vesicles derived from U251 and A375 cell lines and incubated at 37° C. for 2 hours.

[0040] The specific operation is as follows: 10 μL of extracellular vesicles (20 μg / mL) from the cell line is mixed with 10 μL of biosensor (microsphere concentration is 2 mg / mL) and incubated for 2 hours at 37°C. The mixture is transferred to a 384-well plate and the fluorescence of FAM is read using a microplate reader.

[0041] like Figure 2As shown, no signal appeared when the biosensor was incubated with extracellular vesicles derived from U251 cells, while a signal appeared when the biosensor was incubated with extracellular vesicles derived from A375 cells, indicating that the biosensor has the selective detection ability for extracellular vesicles derived from tumor cells.

[0042] Example 3

[0043] The preparation method of Example 1 was referred to, except that the microsphere concentration was 5 mg / mL. Extracellular vesicles derived from A375 cells were dispersed in DPBS and plasma, and incubated with the prepared biosensor.

[0044] Specifically, 200 μg / mg, 40 μg / mg, 20 μg / mg, 4 μg / mg, 2 μg / mg, 0.4 μg / mg, 0.2 μg / mg, and 0.04 μg / mg of extracellular vesicles were dispersed in 100 μL of DPBS and plasma, respectively, mixed with 10 μL of biosensor (microsphere concentration was 2 mg / mL), and incubated at 37°C for 2 hours to detect the FAM signal.

[0045] like Figure 3 As shown in the figure, the biosensor can detect microRNA-21 in extracellular vesicles in both DPBS and plasma environments, and the detection limit of the biosensor for extracellular vesicles is 1.4 μg / mL in both environments.

[0046] It can be seen that the biosensor electrode prepared by the present invention has high stability and can detect microRNA-21 in extracellular vesicles in the complex environment of plasma. It is expected to be applied to the detection of microRNA in extracellular vesicles of human plasma and is expected to be applied to the clinical detection of cancer.

Claims

1. A method for preparing a biosensor for targeted detection of microRNA in tumor-derived extracellular vesicles, characterized in that: The following steps are involved: (1) Dissolve (2,3-dioleoyl-propyl)-trimethylammonium chloride, palmitoyloleoylphosphatidylcholine and cholesterol in a solvent, spin-dry the solvent to obtain a lipid film; then add Dulbecco's phosphate buffer solution to hydrate, and extrude the hydrated product using an extruder to obtain liposomes; The molar ratio of (2,3-dioleoyl-propyl)-trimethylammonium-chloride, palmitoyloleoylphosphatidylcholine, and cholesterol is 1:1:0.1 to 1:1:0.6; (2) heating and annealing the molecular beacon targeting microRNA in Dulbecco's phosphate buffered saline solution to obtain a hairpin-shaped molecular beacon; mixing the hairpin-shaped molecular beacon and silica, vortexing the mixture, adding the above-mentioned liposomes, incubating, centrifuging and washing, and obtaining mesoporous silica microspheres loaded with the hairpin-shaped molecular beacon; The diameter of the mesoporous silica microspheres is 3 microns; The concentration of the hairpin molecular beacon is 1-2 μM, the concentration of the mesoporous silica is 5-20 mg / ml, and the concentration of the liposome is 1.2-2.5 mg / ml; (3) Incubate the mesoporous silica microspheres loaded with hairpin-shaped molecular beacons and the cholesterol-containing aptamer, and then centrifuge and wash to obtain the biosensor; The molecular beacon has a 5'-terminal FAM and a 3'-terminal quencher modification.

2. The method for preparing a biosensor for detecting microRNA in tumor-derived extracellular vesicles according to claim 1, characterized in that: The hydration temperature in step (1) is 25-37 °C and the treatment time is 1-2 hours.

3. The method for preparing a biosensor for detecting microRNA in tumor-derived extracellular vesicles according to claim 1, characterized in that: The porous film is extruded 11-21 times under the extrusion conditions described in step (1).

4. The method for preparing a biosensor for detecting microRNA in tumor-derived extracellular vesicles according to claim 1, characterized in that: The incubation temperature in step (2) is 25-37 °C and the treatment time is 1-2 hours.

5. The method for preparing a biosensor for detecting microRNA in tumor-derived extracellular vesicles according to claim 1, characterized in that: The incubation conditions in step (3) are 25-37 °C for 1-2 hours.

6. The method for preparing a biosensor for detecting microRNA in tumor-derived extracellular vesicles according to claim 1, characterized in that: The concentration of the cholesterol-containing aptamer in step (3) is 500-1000 nanomolar.

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