CN114323863B - Preparation method and application of exosome array capture probe - Google Patents

Abstract

The invention is suitable for the field of exosome capturing and detecting, and discloses an array exosome capturing and detecting method based on amphiphilic supermolecule modification. Importantly, the probe molecules are coated on the nitrocellulose membrane, so that the requirement of clinical mass sample detection can be met, and the exosomes in trace body fluid can be detected in an array mode with high flux. The exosome enrichment method based on the amphiphilic supermolecular probe has the characteristics of strong specificity, short detection time, low required sample size, high detection flux and the like, and is expected to provide an emerging platform for the discovery and screening of disease biomarkers.

Description

Preparation method and application of exosome array capture probe

Technical field

The invention belongs to the field of exosome capture and detection, and in particular relates to a preparation method and application of an exosome array capture probe.

Background technique

Exosomes are extracellular vesicles with a lipid bilayer membrane structure ranging from 40 to 160 nm in diameter. At present, the important role played by exosomes in intercellular communication and cellular immune response has been gradually discovered. They not only carry active ingredients (lipids, proteins, genetic materials, etc.) that perform biological functions, but also carry components involved in cell communication. , angiogenesis, cell migration, cardiovascular disease, neurodegenerative diseases, tumor cell growth and other physiological and pathological processes, the capture and detection of exosomes is of high research significance.

In clinical cancer diagnosis, tissue biopsy is a necessary tool for pathological examination, which is invasive and highly painful for patients. The form of tissue biopsy is not only unsuitable for dynamic monitoring of patients, but also increases the patient's physical and psychological pressure. Therefore, liquid biopsy, a non-invasive biopsy method, is necessary to obtain the required diagnostic information from biological fluids. In the context of liquid biopsy, analyzing the contents of exosomes can obtain information about the presence and distribution of cancer. Therefore, exosomes are used as biomarkers in liquid biopsies with real-time monitoring of tumors and therapeutic effects.

A number of technologies have been developed for exosome isolation to date, including those based on differences in exosome density and size compared with cells, cell debris, and other biological interferences; Surface-specific proteins or specific structures can be used to separate exosomes through the force of antibody immunoaffinity; as well as some technologies that simulate the interaction between antigens and antibodies and capture and separate through molecular recognition. However, each method cannot be widely used due to its own limitations, and there is no method that can satisfy the parallel capture and detection of exosomes in large batches of clinical samples.

Contents of the invention

The purpose of the embodiments of the present invention is to provide a preparation method and application of an exosome array capture probe, aiming to solve the problem of isolating non-exosome components from sufficient quantities and purity and achieving in-situ detection.

The embodiment of the present invention is achieved as follows. The synthesis and membrane modification of amphiphilic dendritic supramolecular probes include the following steps:

1) Use dimethyl sulfoxide to dissolve amphotericin B, and use ultrasound to promote dissolution;

2) Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in MES buffer;

3) Mix the two solutions obtained in steps 1) and 2), and mix and stir in a 37°C environment to activate the carboxyl groups in the AMB molecules;

4) Add the dendrimer solution to the solution obtained in step 3) and react overnight at room temperature;

5) Use a dialysis tube to dialyze the solution in step 4), remove excess reactants by replacing water, and collect the dialyzed probe solution;

6) Activate the nitrocellulose membrane in water, add probe molecule solution and incubate overnight. After the incubation is completed, wash it with MES buffer and phosphate buffer respectively, and dry it at room temperature;

Array detection of body fluids using amphipathic dendritic supramolecular probe-modified membranes includes the following steps:

1) Pre-process the body fluid samples. The collected body fluid samples are stored at -80°C, thawed at 4°C, and then centrifuged at low speed to collect the supernatant for spotting;

2) Spot 1 μL of each body fluid sample from different patients on the NC membrane modified with amphipathic dendritic supramolecular probes in array form;

3) The spotted membrane was dried at room temperature and washed twice with phosphate buffer for 5 minutes each time;

4) Block the membrane in phosphate buffer containing 5% skim milk powder for 20-30 minutes, and then wash it 3 times with phosphate buffer containing Tween, 5 minutes each time;

4) Select antibodies corresponding to proteins highly expressed in cancer for incubation. The incubation conditions are room temperature for 2 hours, or incubation at 4°C overnight. After incubation, wash 3 times with phosphate buffer containing Tween, 10 minutes each time;

5) Incubate with the secondary antibody corresponding to the primary antibody , wash 3 times with phosphate buffer containing Tween for 10 minutes each time, and use an imaging system to quantitatively scan the protein signal.

The preparation method and application of an exosome array capture probe provided by the embodiments of the present invention have the following beneficial effects:

Amphipathic supramolecules can be inserted into the interior of a single-layer membrane and combined with lipids and sterols, utilizing the multivalent synergistic effect of the supramolecular to achieve efficient capture of exosomes. This new strategy can be combined with cellulose membranes to capture exosomes in body fluid samples in the form of reversed-phase microarrays, and can also perform high-throughput analysis of large batches of samples for different disease marker proteins with differential expression in exosomes. Quantity testing. The supramolecular probe analysis method we developed is easy to operate, has high sensitivity and fast analysis speed. It is expected to become a new platform for high-throughput enrichment and detection of exosomes, providing strong technical support for early warning and tracking of diseases.

Description of the drawings

Figure 1 is a diagram of high-throughput detection of exosomes in clinical samples using an amphiphilic dendritic supramolecular probe modified nitrocellulose membrane provided by an embodiment of the present invention;

Figure 2 is a high-throughput array detection diagram of amphiphilic dendritic supramolecular probe-modified nitrocellulose membrane for exosomes in clinical samples and a modified NC membrane for clinical samples according to the embodiment of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

Example 1:

Preparation and application of ADSP molecular probe-coated nitrocellulose membrane:

1) Use 100 μL dimethyl sulfoxide (DMSO) to dissolve 2 mg of AMB, and perform ultrasonic treatment to promote dissolution;

2) Dissolve 80 mg of EDC and 48 mg of NHS in 2-(N-morpholino)ethanesulfonic acid (100 mM MES, pH 6.0) buffer;

3) Mix the two solutions in 1) 2) and mix and stir at 37°C for 20 minutes to activate the carboxyl groups in the AMB molecules;

4) Add 10 μL of dendrimer solution to the above solution and react at room temperature overnight;

5) Then use a dialysis tube to dialyze the above reaction solution, remove excess reactants by replacing water (dialysis times are 1h, 2h, 4h, 4h respectively), and collect the dialyzed probe solution;

6) Activate the nitrocellulose membrane in water, and incubate the membrane with the probe molecule solution at room temperature overnight;

7) After incubation, wash twice with MES buffer for 10 minutes each time, then wash once with phosphate buffer for 10 minutes, and dry the membrane at room temperature;

Example 2:

ADSP molecular probe coated nitrocellulose membrane is used to capture exosomes from body fluid samples (taking plasma samples as an example):

1) Pre-process the plasma samples, store the collected body fluid samples at -80°C, thaw them at 4°C, and centrifuge them at 5000 × g at 4°C for 10 minutes, then take the supernatant for spotting;

2) Preparation of ultra-centrifuged plasma samples: centrifuge body fluid samples at 2000g for 30 minutes, take the supernatant and centrifuge at 20000g for 45 minutes, take the supernatant and centrifuge at 110000g for 70 minutes. After discarding the supernatant, the precipitate is redissolved by pipetting with PBS and filtered using a 0.22 filter. Then centrifuge at 110000g for 70 minutes to precipitate exosomes, and collect the supernatant as a negative control;

3) Use PBS buffer equivalent to the amount of plasma before ultrasonic separation to redissolve the pellet to maintain the same concentration as the original plasma;

4) Plasma samples, exosomes obtained by ultracentrifugation and supernatant were directly spotted on the ADSP functionalized NC membrane in a gradient of 0.2-2µL;

5) After spotting the sample onto the ADSP-modified NC membrane, wait for the sample to dry at room temperature, and then wash it twice with 1% BSA PBS buffer for 5 minutes each time;

6) Place the membrane in phosphate buffer containing 5% skimmed milk powder for blocking for 30 minutes, and wash 3 times with phosphate buffer containing Tween for 10 minutes each time;

7) Incubate with primary antibodies using exosome marker proteins (CD9, CD81, CD63, TSG101, etc.), incubate at room temperature for 2 hours or overnight at 4°C, and wash 3 times with phosphate buffer containing Tween, 5 minutes each time ;

8) Then, use the secondary antibody corresponding to the primary antibody to incubate for 1 hour, wash with PBST three times, 5 minutes each time, and use an imaging system to quantitatively scan the protein signal. (As shown in Figure 1)

Example 3:

High-throughput detection of cancer marker proteins in large batches of clinical samples using ADSP molecular probes coated on nitrocellulose membranes:

1) Preprocess liver cancer plasma samples. The collected plasma samples are stored at -80°C, thawed at 4°C, and centrifuged at 5000×g for 10 minutes at 4°C. The supernatant is taken for spotting;

2) Place 1 μL of plasma from liver cancer patients and non-cancer controls directly on the ADSP-modified NC membrane, dry at room temperature, and wash twice with 1% PBST for 5 minutes each time;

3) Then place the membrane in PBS buffer containing 5% skimmed milk powder for 30 min, and wash with PBST 3 times, 10 min each time.

4) Incubate with primary antibodies using proteins highly expressed in cancer. The incubation time is 2 hours at room temperature or overnight at 4°C. Wash 3 times with PBST for 5 minutes each time;

5) Then, use the secondary antibody corresponding to the primary antibody to incubate for 1 hour, and use an imaging system to quantitatively scan the protein signal. (as shown in picture 2)

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (2)

1. A method for preparing an exosome array capture probe, characterized in that the preparation method of the exosome array capture probe includes: Amphiphilic supramolecular probes are used to couple amphiphilic molecules to dendrimers, and the amphiphilic supramolecular probes are used to bind to lipid membrane structures; The amphiphilic supramolecular probe is coated on a nitrocellulose membrane for capturing exosomes in body fluids; The amphiphilic supramolecular probe is coated on a nitrocellulose membrane for high-throughput detection of clinical body fluid samples; The synthesis of the amphiphilic dendritic supramolecular probe includes the following steps: 1) Use dimethyl sulfoxide to dissolve amphotericin B, and use ultrasound to promote dissolution; 2) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in 0.1M MES buffer; 3) Mix the two solutions obtained in steps 1) and 2), and mix and stir in a 37°C environment to activate the carboxyl groups in the amphotericin B molecule; 4) Add the dendrimer solution to the solution obtained in step 3) and react overnight at room temperature; 5) Use a dialysis tube to dialyze the solution in step 4), remove excess reactants by replacing water, and collect the dialyzed probe solution; 6) The nitrocellulose membrane is activated in water, and the probe molecule solution is added and incubated overnight. After the incubation is completed, it is washed twice with MES buffer, then once with phosphate buffer, and dried at room temperature; 7) Pre-process the body fluid samples, store the collected body fluid samples and thaw them at 4°C, centrifuge them at low speed and take out the supernatant for spotting; 8) The body fluid is directly spotted on the ADSP-modified nitrocellulose membrane, dried at room temperature, and washed twice with phosphate buffer for 5 minutes each time; 9) Block the membrane in phosphate buffer containing 5% skimmed milk powder for 30 minutes, and then wash it with phosphate buffer 3 times, 10 minutes each time; 10) Incubate the highly expressed proteins in exosomes with primary antibodies and wash 3 times with phosphate buffer containing Tween for 10 minutes each time; 11) Incubate with the corresponding secondary antibody, wash 3 times with phosphate buffer containing Tween for 10 minutes each time, and use the imaging system to detect the protein signal; In the dendrimers, the dendrimers have multiple free amino groups for coupling with amphiphilic molecules to form amphiphilic supramolecular probes; Body fluid samples are directly spotted in an array format on a nitrocellulose membrane coated with amphipathic supramolecular probes and incubated for capturing particles with cell membrane structures in body fluids. 2. Application of the exosome array capture probe according to claim 1 in capturing exosomes.