Preparation and application of cholesterol molecularly imprinted material

Abstract

The invention is applicable to the technical field of biomedicine, and provides preparation and application of a cholesterol molecularly imprinted material, and the preparation method comprises the following steps: adding Triton X-100, n-hexyl alcohol, cyclohexane, water and ammonia water into a beaker, and stirring by using a magnetic stirrer until the solution is transparent and clear to form a reversed-phase microemulsion; cholesterol is added into the microemulsion; mixing cyclohexane, TEOS (Tetraethyl Orthosilicate) and APTES (Aminopropyltriethoxysilane) to form an imprinting solution, gradually dropwise adding the imprinting solution into the microemulsion containing cholesterol, stirring, adding ethanol into the microemulsion, discarding supernate, and retaining precipitate; and washing the precipitate, and drying to obtain the cholesterol molecularly imprinted material (CHO-MIP). According to the method, high-flux and effective enrichment and separation of the exosomes in the body fluid are realized, the separation efficiency is greatly improved, the cost is reduced, a new path is opened up for exosome application in a body fluid biopsy technology, and particularly, the method shows remarkable clinical application potential in separation of the exosomes in human urine.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to the preparation and application of a cholesterol molecular imprinting material. Background Art

[0002] Exosomes are extracellular vesicles with a diameter of about 30-150nm wrapped by a lipid membrane. They are widely present in various body fluids of the human body, such as blood, urine, cerebrospinal fluid and saliva. Studies have shown that exosomes play an important role in intercellular signal transduction, antigen presentation, tissue repair, tumorigenesis and drug resistance. Because they contain rich biological information (nucleic acids, proteins, lipids and metabolites), exosomes have become an important component of liquid biopsy materials. Compared with traditional body fluid biopsy materials such as circulating tumor cells (CTCs) and cell-free nucleic acids (cell free-DNA), exosomes have the following advantages: 1) Relatively easy to obtain; 2) Have a stable membrane structure; 3) Reflect the biological information of parent cells at multiple levels. These characteristics make exosomes show great potential in the diagnosis of clinical diseases.

[0003] In the process of using exosomes for clinical testing, the key to whether the disease can be effectively detected lies in the extraction efficiency and quality of exosomes. At present, the methods for isolating exosomes include ultracentrifugation, precipitation, size exclusion chromatography, etc., but for body fluid biopsy, especially when facing large-scale sample testing in clinical practice, these traditional methods have their own shortcomings. Recent studies have shown that the use of nanomaterials to enrich and separate exosomes may be a promising method because it consumes less sample volume and the enrichment process is simple and fast, which provides new possibilities for isolating exosomes from large-scale clinical samples. In view of the problems of low efficiency, complex synthesis and high cost of current exosome separation technology, it is urgent to develop a high-throughput and effective exosome enrichment and separation method. To this end, the present invention proposes a preparation and application of cholesterol molecular imprinting material. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of a cholesterol molecular imprinting material, aiming to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing a cholesterol molecular imprinting material comprises the following steps:

[0007] Step 1, preparation of water / oil reverse microemulsion: 1.77 mL Trition X-100, 1.6 mL n-hexanol, 6.5 mL cyclohexane, 480 μL water and 100 μL ammonia (NH3·H2O) were added into a 100 mL beaker, and the mixture in the beaker was stirred at 700 rpm at 25°C using a magnetic stirrer until the solution became transparent and clear to form a reverse microemulsion, and the magnetic stirring was continued for 30 min;

[0008] Step 2, synthesis of molecular imprinting polymer: add 4 mg of cholesterol to the prepared microemulsion; mix 750 μL of cyclohexane, 100 μL of tetraethyl orthosilicate (TEOS) and 26 μL of 3-aminopropyltriethoxysilane (APTES) to form an imprinting solution, gradually add the imprinting solution dropwise to the microemulsion containing cholesterol, and then use a magnetic stirrer to stir at 700 rpm at 25°C for 24 hours; after stirring, add 2 mL of ethanol to the microemulsion to break the emulsion, discard the supernatant, and retain the precipitate; the precipitate is first washed once with deionized water, then washed once with a mixed solution of anhydrous methanol and acetic acid, and then washed twice with methanol, and finally dried at 70°C to obtain a cholesterol molecular imprinting material, namely CHO-MIP, for use.

[0009] Furthermore, the volume ratio of anhydrous methanol to acetic acid is 4:1.

[0010] A CHO-MIP prepared according to the preparation method described above.

[0011] A use of the CHO-MIP described above in enriching and isolating exosomes.

[0012] Furthermore, the specific process is as follows:

[0013] Step 1, sample pretreatment: the collected liquid biopsy samples were centrifuged at 17000×g for 20 min; after centrifugation, the supernatant was kept to remove the bottom sediment for later use;

[0014] Step 2, exosome enrichment and metabolite extraction: 4 mg of CHO-MIP was weighed in a 2 mL EP tube, washed twice with 500 μL PBS, centrifuged at 10,000 rpm for 3 min, and a precipitate was left; 200 μL PBS was added to the precipitate to disperse it and form a turbid solution; 2 mL of fresh urine was mixed with the turbid solution, and the test tube was placed on a rotator at room temperature for 30 min, and then centrifuged at 10,000 rpm for 3 min, leaving a precipitate; washed twice with 200 μL PBS to remove residual urine, and then centrifuged at 10,000 rpm for 3 min, leaving a precipitate; the precipitate was mixed evenly with 1 mL of metabolite extract, first ultrasonically treated for 10 min, then vortexed for 10 min, and finally the mixture was repeatedly placed in liquid nitrogen and a 37°C water bath to destroy the membrane structure; the supernatant was filtered using a 0.22 μm filter on ice, and the filtered extract was blown dry with nitrogen; 50 μL The dried samples were redissolved in 50% MeOH aqueous solution and centrifuged at 14,000 × g for 15 min at 4 °C, and the supernatant was transferred to a liquid phase bottle to prepare for metabolite analysis;

[0015] Step 3. Metabolite mass spectrometry identification and analysis: LC-MS was used to analyze the exosome metabolites enriched by CHO-MIP; the sample was first separated by a chromatographic column and then entered the mass spectrometer for analysis in positive ion mode; MassLynx V4.2 and MS-DIAL software were used to process the obtained mass spectrometry data, and RStudio software was used to perform machine learning on the processed data to complete the analysis.

[0016] Furthermore, the metabolite extract is a pre-cooled 50% MeOH aqueous solution.

[0017] Furthermore, the parameter settings of the mass spectrometer include: injection time of 12 min, spray voltage of 1.50 kV, capillary temperature of 80° C., and mass-to-nuclear ratio scanning range of 50-1000 Da.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention uses reverse microemulsion combined with cholesterol imprinting template to synthesize molecular imprinting polymers, which achieves high-throughput and effective enrichment and separation of exosomes in body fluids, greatly improves separation efficiency and reduces costs, opens up a new path for the application of exosomes in body fluid biopsy technology, and especially shows significant clinical application potential in the separation of exosomes in human urine, indicating broad prospects in biomedical research and future health assessments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure is a flow chart for the preparation of CHO-MIP.

[0021] Figure 2 Flow chart for the use of CHO-MIP.

[0022] Figure 3 The enrichment results of urinary exosomes; (A) is the diameter of exosomes separated from human urine by CHO-MIP, (B) is the concentration of exosomes separated from human urine by CHO-MIP, (C) is the protein immunoblot analysis of the typical marker protein CD9, (D) is the fluorescence spectrum analysis of the exosomes remaining in the supernatant after the membrane is stained with DiO dye; (E) is the principal component analysis diagram of exosome metabolomics of healthy people (Health), patients with liver cirrhosis (Liver cirrhosis), and patients with liver cancer (HCC). DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] One embodiment of the present invention provides a method for preparing a cholesterol molecular imprinted material, comprising the following steps:

[0025] Step 1, preparation of water / oil reverse microemulsion: 1.77 mL Trition X-100, 1.6 mL n-hexanol, 6.5 mL cyclohexane, 480 μL water and 100 μL ammonia (NH3·H2O) were added into a 100 mL beaker, and the mixture in the beaker was stirred at 700 rpm at 25°C using a magnetic stirrer until the solution became transparent and clear to form a reverse microemulsion, and the magnetic stirring was continued for 30 min;

[0026] Step 2, synthesis of molecular imprinting polymer: 4 mg of cholesterol was added to the prepared microemulsion; 750 μL of cyclohexane, 100 μL of tetraethyl orthosilicate (TEOS) and 26 μL of 3-aminopropyltriethoxysilane (APTES) were mixed to form an imprinting solution, the imprinting solution was gradually added dropwise to the microemulsion containing cholesterol, and then stirred at 700 rpm at 25° C. for 24 h using a magnetic stirrer; after the stirring was completed, 2 mL of ethanol was added to the microemulsion to break the emulsion, the supernatant was discarded, and the precipitate was retained; the precipitate was first washed once with deionized water, then washed once with a mixed solution of anhydrous methanol and acetic acid (v:v=4:1), and then washed twice with methanol, and finally dried at 70° C. to obtain a cholesterol molecular imprinting material, namely CHO-MIP, for later use.

[0027] One embodiment of the present invention provides an application of CHO-MIP prepared by the above-mentioned preparation method in enriching and isolating exosomes, and the specific process is as follows:

[0028] Step 1, sample pretreatment: the collected liquid biopsy samples were centrifuged at 17000×g for 20 min; after centrifugation, the supernatant was kept to remove the bottom sediment for later use;

[0029] Step 2, exosome enrichment and metabolite extraction: 4 mg of CHO-MIP was weighed in a 2 mL EP tube, washed twice with 500 μL PBS, centrifuged at 10000 rpm for 3 min, and a precipitate was left; 200 μL PBS was added to the precipitate to disperse it and form a turbid solution; 2 mL of fresh urine was mixed with the turbid solution, and the test tube was placed on a rotator at room temperature for 30 min, and then centrifuged at 10000 rpm for 3 min, leaving a precipitate; washed twice with 200 μL PBS to remove residual urine, and then centrifuged at 10000 rpm for 3 min, leaving a precipitate; the precipitate was mixed evenly with 1 mL of metabolite extract (pre-cooled 50% MeOH aqueous solution), first ultrasonically treated for 10 min, then vortexed for 10 min, and finally the mixture was repeatedly placed in liquid nitrogen and a 37°C water bath to destroy the membrane structure; the supernatant was filtered using a 0.22 μm filter on ice, and the filtered extract was blown dry with nitrogen; 50 μL The dried samples were redissolved in 50% MeOH aqueous solution and centrifuged at 14,000 × g for 15 min at 4 °C, and the supernatant was transferred to a liquid phase bottle to prepare for metabolite analysis;

[0030] Step 3, metabolite mass spectrometry identification and analysis: Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the exosome metabolites enriched by CHO-MIP; the sample was first separated by a chromatographic column and then entered the mass spectrometer for analysis in positive ion mode. The parameters of the mass spectrometer were as follows: injection time of 12 min, spray voltage of 1.50 kV, capillary temperature of 80 ° C, and mass-to-nuclear ratio (m / z) scanning range of 50-1000 Da. MassLynx V4.2 and MS-DIAL software were used to process the obtained mass spectrometry data, and RStudio software was used to perform machine learning on the processed data to complete the analysis.

[0031] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0032] Example 1: Enrichment of urine exosomes;

[0033] CHO-MIP was used to separate exosomes from urine and to identify the exosomes. The results are as follows:

[0034] Nano-flow cytometric analysis results showed that CHO-MIP could separate particles with a diameter of about 30-150 nm from human urine with a concentration of about 1.47×10 7 / mL( Figure 3 Western blotting results showed that the isolated vesicles contained the typical exosome marker protein CD9 ( Figure 3 C). Subsequently, after enriching exosomes in human urine using CHO-MIP and ultracentrifugation (UC), the supernatant and original urine (Urine) were stained with DiO fluorescent dye for fluorescence spectroscopy analysis. The results are shown in Figure 3 As shown in D, CHO-MIP has a higher enrichment efficiency. Finally, the LC-MS method was used to perform metabolic analysis of exosomes in urine of healthy people (Health), patients with liver cirrhosis (Liver cirrhosis), and patients with liver cancer (HCC). The analysis results are shown in Figure 3 As shown in E, the three groups of people were successfully separated by principal component analysis (PCA). In summary, CHO-MIP can separate exosomes from human urine and has clinical application value.

[0035] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for preparing a cholesterol molecular imprinted material, characterized in that: The following steps are involved: Step 1, preparation of water / oil reverse microemulsion: 1.77 mL TritionX-100, 1.6 mL n-hexanol, 6.5 mL cyclohexane, 480 μL water and 100 μL NH3·H2O were added into a 100 mL beaker, and the mixture in the beaker was stirred at 700 rpm at 25°C using a magnetic stirrer until the solution became transparent and clear to form a reverse microemulsion, and the magnetic stirring was continued for 30 min; Step 2, synthesis of molecular imprinting polymer: add 4 mg of cholesterol to the prepared microemulsion; mix 750 μL of cyclohexane, 100 μL of TEOS and 26 μL of APTES to form an imprinting solution, gradually add the imprinting solution dropwise to the microemulsion containing cholesterol, and then use a magnetic stirrer to stir at 700 rpm for 24 hours at 25°C; after stirring, add 2 mL of ethanol to the microemulsion to break the emulsion, discard the supernatant, and retain the precipitate; the precipitate is first washed once with deionized water, then washed once with a mixed solution of anhydrous methanol and acetic acid, and then washed twice with methanol, and finally dried at 70°C to obtain a cholesterol molecular imprinting material, namely CHO-MIP, for use.

2. The preparation method according to claim 1, characterized in that: The volume ratio of anhydrous methanol to acetic acid is 4:

1.

3. A CHO-MIP prepared according to the preparation method of claim 1 or 2.

4. A use of the CHO-MIP according to claim 3 in enriching and isolating exosomes.

5. The use according to claim 4, characterized in that: The specific process is as follows: Step 1, sample pretreatment: the collected liquid biopsy samples were centrifuged at 17000×g for 20 min; after centrifugation, the supernatant was kept to remove the bottom sediment for later use; Step 2, exosome enrichment and metabolite extraction: 4 mg CHO-MIP was weighed in a 2 mL EP tube, washed twice with 500 μL PBS, centrifuged at 10,000 rpm for 3 min, and a precipitate was left; 200 μL PBS was added to the precipitate to disperse it and form a turbid solution; 2 mL of fresh urine was mixed with the turbid solution, and the test tube was placed on a rotator at room temperature for 30 min, and then centrifuged at 10,000 rpm for 3 min, leaving a precipitate; washed twice with 200 μL PBS to remove residual urine, and then centrifuged at 10,000 rpm for 3 min, leaving a precipitate; the precipitate was mixed evenly with 1 mL of metabolite extract, first ultrasonically treated for 10 min, then vortexed for 10 min, and finally the mixture was repeatedly placed in liquid nitrogen and a 37°C water bath to destroy the membrane structure; the supernatant was filtered using a 0.22 μm filter on ice, and the filtered extract was blown dry with nitrogen; The dried samples were re-dissolved with 50 μL of 50% MeOH aqueous solution and centrifuged at 14,000 × g for 15 min at 4 °C, and the supernatant was transferred to a liquid phase bottle to prepare for metabolite analysis; Step 3. Metabolite mass spectrometry identification and analysis: LC-MS was used to analyze the exosome metabolites enriched by CHO-MIP; the sample was first separated by a chromatographic column and then entered the mass spectrometer for analysis in positive ion mode; MassLynx V4.2 and MS-DIAL software were used to process the obtained mass spectrometry data, and RStudio software was used to perform machine learning on the processed data to complete the analysis.

6. The use according to claim 5, characterized in that: The metabolite extraction solution is a pre-cooled 50% MeOH aqueous solution.

7. The use according to claim 5, characterized in that: The parameter settings of the mass spectrometer include: injection time of 12 min, spray voltage of 1.50 kV, capillary temperature of 80° C., and mass-to-nuclear ratio scanning range of 50-1000 Da.

Images