CN117511879B - Method for integrally realizing exosome enrichment and micromolecule extraction based on microfluidic chip - Google Patents
Method for integrally realizing exosome enrichment and micromolecule extraction based on microfluidic chip Download PDFInfo
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Abstract
The invention discloses a method for integrally realizing exosome enrichment and micromolecule extraction based on a microfluidic chip, which comprises the following steps: mixing a sample containing an exosome with a ferroferric oxide magnetic microsphere coated with a metal oxide nano coating on a digital microfluidic chip, and incubating for 5-15 min to realize enrichment of the exosome and the magnetic microsphere; attracting the magnetic ball through an external magnetic field, and introducing buffer solution to clean the enriched exosomes; then, in an alkaline liquid drop environment, eluting the exosomes from the surface of the magnetic sphere, enriching and suspending in the liquid drop; or directly in an organic solvent environment, exosome lipid is extracted from exosome on the surface of the magnetic sphere, resuspended in a droplet, and then collected by a microshutter. The invention combines the microfluidic technology with the method for enriching exosomes by the magnetic microspheres, fully plays the advantages of the two, and realizes high efficiency, rapid operation, low sample consumption, automation, high throughput, precise control and accurate analysis.
Description
Technical Field
The invention belongs to the field of biotechnology and biomedicine, and particularly relates to a method for realizing exosome enrichment and small molecule extraction based on integration of a microfluidic chip.
Background
As a vesicle secreted by cells, the exosomes encapsulate proteins, lipids and other bioactive molecules secreted by the cells. Exosomes serve as an important pathway for intercellular information transfer, with a wide range of biological functions and regulatory roles. For researching the mechanism and application potential, the method can help to understand the interaction among cells and the disease occurrence mechanism in depth and provide a new idea for disease diagnosis and treatment. Research shows that lipids in exosomes are the main components of exosome membranes, playing an important role in intercellular information transfer, compared to proteins, nucleic acids in exosomes. The lipid component is capable of modulating the formation, release, and uptake of the exosome vesicles by the target cells, thereby affecting exosome-mediated cell signaling. Intensive studies on the composition and function of exosome lipids have helped understand the fine regulatory mechanisms of intercellular communication. In addition, bioactive molecules that lipids can carry can be released to the external environment, thereby affecting the biological behavior of the recipient cells, and in addition, abnormal exosome lipid composition can be associated with a variety of diseases.
Before exosome lipids can be studied, the exosomes should first be enriched from the biological sample before extracting the lipids. The exosomes have diameters of between 30 and 150 nanometers, are tiny and diversified vesicles, have relatively low content and are easily covered by other cell secretions, so that development of exosome enrichment technology is of great significance for effectively enriching exosomes from complex biological samples. In addition, the exploration of exosomes, particularly exosome lipids, is significant for the mechanism of tumor formation, growth, and metastasis of reveal the secrets.
Currently, a number of methods have been applied to exosome enrichment, such as ultracentrifugation, filter enrichment, density gradient centrifugation, and immunocapture. However, there are common disadvantages such as complex operation, long time consumption, need for specialized equipment and reagents, and a certain influence on the integrity and function of exosomes, and difficulty in achieving high purity and high throughput exosome enrichment. Furthermore, these methods may suffer from inefficiency in sample processing when processing large volumes of samples. Thus, a new method is needed to enrich exosomes from micro-samples, followed by extraction of their lipids.
The digital microfluidic technology is a microfluidic technology based on discrete droplet operation and is used for accurately controlling micro-liquid. On a digital microfluidic chip, a liquid is divided into discrete droplets, and the droplets are precisely positioned and operated by manipulating an electric field, mechanical force, temperature, or the like. The principle of the electric control digital micro-fluidic technology based on dielectric wetting is that the contact angle of liquid is regulated by changing the distribution of an electric field, so that the movement of liquid drops is controlled. The technology mainly utilizes an electric field to regulate and control the interaction between liquid and the solid surface, and realizes the operations of positioning, merging, separating, moving and the like of liquid drops. The dielectric wetting-based electrically controlled digital microfluidic technology has the advantages of high precision, programmability, quick response, real-time control and the like. Digital microfluidic technology has wide application in the fields of biology, chemical analysis, drug screening and the like. It can be used for various experimental operations such as sample mixing, reaction control, cell operation, enzyme reaction, gene analysis, etc. The high precision, high flexibility and automation of digital microfluidic technology provide new possibilities for experimental operation, which is helpful for improving experimental efficiency and reducing reagent waste. At present, an exosome enrichment method based on a digital microfluidic technology mainly comprises a microfluidic technology based on immunoaffinity, a microfluidic technology based on micro-filter filtration, a microfluidic technology based on external force field assistance and the like, but the problems of complex manufacturing process, relatively complex operation, insufficient recovery rate and the like generally exist.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a method for realizing exosome enrichment and small molecule extraction based on integration of a microfluidic chip, and a method for enriching exosome and extracting small molecules (such as lipid) by using metal oxide magnetic spheres on a digital microfluidic chip is developed so as to enrich trace exosome and extract small molecules (such as lipid) rapidly and efficiently. The method can reduce sample loss while maintaining high purity enrichment of exosomes, and has the advantage of simple operation.
The invention provides a microfluidic chip-based integrated exosome enrichment and micromolecule extraction method, which comprises the following steps: mixing a sample containing an exosome with a ferroferric oxide magnetic microsphere coated with a metal oxide nano coating on a digital microfluidic chip, and incubating for 5-15 min to realize enrichment of the exosome and the magnetic microsphere; attracting the magnetic ball through an external magnetic field, and introducing buffer solution to clean the enriched exosomes; then, in an alkaline liquid drop environment, eluting the exosomes from the surface of the magnetic sphere, enriching and suspending in the liquid drop; or directly in an organic solvent (such as acetonitrile) environment, exosome lipid is extracted from exosomes on the surface of the magnetic sphere, resuspended in a droplet, and then collected by a microshutter needle.
The method comprises the following specific steps:
a) Firstly, introducing ferroferric oxide magnetic microspheres coated with a metal oxide nano coating on the surface into an electrode of a digital microfluidic chip, and cleaning with PBS buffer solution;
b) Incubating the magnetic microspheres with a trace of sample containing exosomes on a digital microfluidic chip, and extracting exosome molecules in the sample by specific adsorption:
c) Washing the magnetic microspheres in step b) with PBS buffer to remove other non-specific molecules adsorbed on the magnetic microspheres; in the process, an external magnetic field is increased to gather the magnetic microspheres with the surfaces adsorbing exosomes; controlling the electrode of the digital micro-fluidic chip, controlling the movement of liquid drops, and separating washing waste liquid from the chip;
d) Introducing an alkaline solution into the digital microfluidic chip for eluting the exosomes on the surface of the magnetic microspheres aggregated in the step c) to obtain complete exosomes, and leading the complete exosomes into an EP tube;
Or, introducing an organic solvent into the digital microfluidic chip, extracting biological small molecules in the exosomes on the surfaces of the magnetic microspheres gathered in the step c) by using the organic solvent, collecting the small molecules, and leading the small molecules out of the EP tube.
Further, in said step b), said exosome-containing sample solution comprises body fluids of animal and plant origin, including serum, plasma, urine or platelet rich plasma; also included are culture supernatants of cells of different origins, including plant cells, animal somatic cells and stem cells thereof.
Taking a cell sample as an example, the cell sample solution containing exosomes needs to be used for removing cell debris, organelles, microvesicles and the like.
Further, the surface of the step b) is coated with a metal oxide nano-coating of ferroferric oxide magnetic microsphere, wherein the metal oxide can be TiO 2、ZrO2 and the like.
Further, in the step b), specifically, as shown in fig. 1 i) and ii), the magnetic microsphere suspension is dripped into the digital microfluidic chip, and a trace amount of sample containing exosomes (such as a cell culture medium or a treated tumor serum sample) is introduced into the chip and is fully mixed with the magnetic microsphere; after incubation for 5-15min at normal temperature, the exosomes with complete structures are adsorbed to the surfaces of the magnetic microspheres through nonspecific actions.
The dosage ratio of the magnetic microsphere to the trace exosome-containing sample is 50 micrograms: 2.5 microliters.
According to an embodiment of the present invention, in the step b), the magnetic microsphere may be a ferroferric oxide magnetic microsphere coated with a TiO 2 metal oxide nano-coating, which is available from qifeng nano-company; cargo number 103100; link https:// www.xfnano.com/product/SEARCHSEARCHKEY =xf245, diameter 1.45±0.2 μm; the original concentration of the magnetic microspheres was 25mg/mL.
Further, in the step c), specifically, by controlling the external magnetic field to agglomerate the magnetic spheres, as shown in iii) in fig. 1, a buffer solution PBS is introduced to wash the exosomes enriched on the surfaces of the magnetic microspheres.
Further, in the step d), specifically, as shown in iv) in fig. 1, the organic solvent may be an acetonitrile aqueous solution with a volume fraction of 30% -70%, and the acetonitrile aqueous solution is mixed with the magnetic microspheres with the surface enriched exosomes, so as to sufficiently extract small molecules in the exosomes on the magnetic microspheres. The small molecules of the present invention include lipids and the like.
Of course, after the above step c), if the particle size and concentration of the enriched exosomes are to be detected, the following operations may be performed: and introducing an alkaline solution into the digital microfluidic chip for eluting the exosomes enriched on the surface of the magnetic microsphere to obtain complete exosomes, and introducing the complete exosomes into the EP tube.
Further, in particular in said step d), said lye may be selected from at least one of the following: ammonia water with volume concentration of 10%, sodium hydroxide and other alkaline solutions (pH 10-12).
Further, 70% acetonitrile in IV in FIG. 1 can be replaced by 10% ammonia solution, and the 10% ammonia solution is introduced to wash the magnetic microspheres, so that exosomes are released from the surfaces of the magnetic microspheres into the solution.
Further, a surfactant (Pluronic F-127 or Tetronic 90R 4) is added to the solutions loaded on the chip in steps a) to c) to promote the smooth movement of the solutions on the chip.
Wherein the concentration of Pluronic F-127 is 0.08%; the Tetronic 90R4 concentration was 0.1%.
The invention also adopts SEM, nano-flow detector and western immunoblotting method to characterize the structure form and enrichment effect of the separated exosomes,
First, exosome solutions were collected and the morphology and structure of exosomes were assessed by SEM and nanoflow detector. Then, to optimize the effect of exosome enrichment, the size of the magnetic spheres, titanium dioxide concentration and incubation time were examined in detail. Through a magnetic sphere enrichment experiment on a digital microfluidic chip, aiming at a trace (microliter-level) exosome model sample, the control feasibility and enrichment efficiency of magnetic spheres (1.45 mu m, 4.5 mu m) with different diameters and magnetic spheres with different masses (10-100 mu g, single experiment) are analyzed and compared, and through continuous attempts, the magnetic sphere dispersion effect and the electrode driving capability are comprehensively considered, and the single magnetic sphere loading quantity is preferably 50 mu g; further, the effect of different incubation times on the exosome enrichment effect was assessed by quantitative analysis of the gray scale of the exosome marker protein CD63 by Western blotting (Western blot).
Compared with the prior art, the invention has the beneficial effects that: the invention combines the microfluidic technology with the TiO 2 magnetic sphere exosome enrichment method, can fully exert the advantages of the two methods, and realizes the aspects of high efficiency, rapid operation, low sample consumption, automation, high flux, precise control, accurate analysis and the like. The specific analysis is as follows:
(1) High-efficiency enrichment: the microfluidic technology and the magnetic sphere exosome enrichment method are combined to realize efficient exosome enrichment. The magnetic sphere is used as an exosome enrichment material, has high affinity and selectivity, and can effectively capture exosomes. Through the liquid drop operation of the microfluidic technology, the exosome sample to be enriched can be mixed with the magnetic ball, so that each exosome liquid drop is ensured to be fully contacted with the magnetic ball, and the enrichment efficiency is improved. In addition, the liquid drop manipulation and separation module on the digital microfluidic chip can realize the accurate control of the enriched exocrine liquid drops, and further improve the enrichment effect.
(2) And (3) fast operation: the microfluidic technology has the characteristics of quick response and real-time control, and can rapidly control the positions and movements of liquid drops and magnetic balls by applying an electric field or mechanical acting force. Thus, the process time of exosome enrichment can be greatly shortened, and the operation efficiency is improved. The rapid operation can save the experimental time (the total duration is about 1 h), reduce the loss and the change of samples and improve the consistency and the repeatability of the enrichment result.
(3) Low sample consumption: microfluidic technology has the advantage of small sample size and reagent economy in terms of sample processing. Through the liquid drop operation module of the digital micro-fluidic chip, trace exosome samples can be precisely mixed with TiO 2 magnetic balls and other reagents, so that the consumption of the samples and the reagents is reduced. This is particularly important for the study of rare samples or expensive reagents, not only saving costs, but also making maximum use of limited sample resources.
(4) Automation and high throughput: microfluidic technology can enable automated sample processing and analysis, as well as high throughput operation. By combining with the magnetic sphere exosome enrichment method, the movement and operation flow of liquid drops can be controlled through programming, and automatic exosome enrichment and subsequent treatment can be realized. This may improve the reproducibility, stability and number of samples processed.
(5) Accurate control and accurate analysis: the microfluidic technology can precisely control the size, shape and position of liquid drops, and can ensure the specific combination of exosomes and magnetic spheres by combining a TiO 2 magnetic sphere exosome enrichment method. Thus, nonspecific binding and background interference can be reduced, and the selectivity and accuracy of exosome enrichment can be improved. At the same time, the exosome-enriched droplets can be conveniently subjected to downstream analysis and measurement such as extraction and analytical determination of lipids in a microsample.
Drawings
Fig. 1 is a schematic diagram of experimental steps for separating exosomes on a digital microfluidic chip.
Fig. 2 is a diagram of actual chip droplet motion. Wherein A is that under the action of an electrode, liquid drop movement is mixed with a magnetic ball; b is that under the action of external magnetic field and without electrode, the magnetic balls gather as shown by red arrow; after 70% acetonitrile extraction, under the action of external magnetic field fixation and electrode, magnetic balls (red arrow) are separated from the extract (white arrow).
FIG. 3 shows the aggregation of magnetic spheres on a chip using a larger diameter (4.5 microns) when no electrode is applied under the action of a magnetic field.
FIG. 4 is a surface of a ferroferric oxide magnetic microsphere characterized by SEM in example 5; a is blank control, b is sample;
FIG. 5 is a graph showing the exosome particle size distribution of each sample of example 6; a is enrichment from 1.5mL supernatant using the UC gold standard, b is enrichment from 15mL supernatant using the UC gold standard, c is enrichment from 1.5mL supernatant using on-chip magnetic beads.
FIG. 6 is a graph depicting exosomes enriched in microplates using western blotting methods for on-chip (chip) and UC methods, respectively.
FIG. 7 is a graph depicting enriched exosomes using western blotting for incubation time of different magnetic spheres with model exosome samples.
Fig. 8 is a spectrum obtained after extraction with 70% acetonitrile on a digital microfluidic chip.
Detailed Description
The digital microfluidic chip used in the following examples was prepared in the subject group laboratory, and the structure was divided into an upper plate and a lower plate. The lower polar plate comprises a patterned electrode, a dielectric layer and a hydrophobic layer which are arranged on the substrate. The upper polar plate comprises a transparent substrate, and a conductive layer and a hydrophobic layer which are arranged on the substrate. For details of the preparation process, reference is made to patent CN 116764705A).
The surface-coated TiO 2 metal oxide nanocoating ferroferric oxide magnetic microspheres used in the examples below were purchased from Jiangsu Xianfeng nanomaterial technologies Inc. under the trade designation 103100.
Example 1: experimental step of enriching exosomes and extracting lipid by digital microfluidic chip magnetic sphere
(1) Pretreatment of cell culture supernatant:
Resuscitate the cells. Thawing frozen Hela cells by heating in water bath at 40deg.C, adding culture medium (DMEM: fetal calf serum: 1% penicillin-streptomycin at a volume ratio of 100:10:1), centrifuging, and retaining precipitate. Gently blowing the sediment in a centrifuge tube by using a culture medium, transferring the sediment to a culture bottle when the sediment is not observed, and changing the culture medium after 24 hours; finally, carrying out passage on the cells once in two days; standby;
changing the culture medium (DMEM: fetal bovine serum: double antibody (penicillin-streptomycin) =100:10:1, v/v/v) prepared by DMEM, fetal bovine serum without exosomes and penicillin-streptomycin, changing the liquid of the cells, culturing for 24 hours, and centrifuging to collect the supernatant, namely cell culture supernatant for later use;
Centrifuging 15mL of the cell culture supernatant obtained in the last step, removing cell debris and the like. Sequentially carrying out 300g and 20min on the supernatant; 2000g,20min;10000g,30min, the supernatant was retained for each centrifugation.
The cell culture supernatant was concentrated. Cell culture supernatants were centrifuged (5000 g,10 min) using ultrafiltration tubes and concentrated supernatants (model exosomes) were collected, approximately 500 microliters.
(2) The magnetic microspheres are utilized to enrich exosomes on the digital microfluidic chip, 2 experiments can be carried out on the same chip in parallel, and the single experiment steps are as follows:
Step 1: 2 microliter of the magnetic microsphere suspension (25 mg/mL, dispersion solvent is ultrapure water, diameter is 1.45 microns) and 1 microliter of surfactant Pluronic F-127 (0.08% w/v, dispersion solvent is ultrapure water) are mixed in an EP tube in a vortex oscillator, all of which are loaded into a digital microfluidic chip, then the mixture is driven by an electrode (a driving electric field is formed in the solution to drive the liquid in a microchannel), and the mixture is moved for 2 minutes according to the path (the path, voltage, time and the like are designed in advance on computer software corresponding to the digital microfluidic chip, and the liquid drops move according to the path after the key is started). The magnetic microspheres are then separated from the droplets and removed from the droplets under the influence of a magnetic field.
Step 2: 12 microliters of 1 XPBS buffer (pH 7.2-7.4) was mixed with 4 microliters of surfactant Tetronic 90R4 (0.1% v/v, dispersion solvent is ultra pure water) in an EP tube in a vortex shaker, 3 microliters were loaded onto a digital microfluidic chip, and the magnetic beads of step 1 were washed according to the designed path (closed path can be designed in the chip) for 5min under electrode drive. Finally, under the action of a magnetic field, the magnetic microspheres are separated from the liquid drops and the liquid drops are removed.
Step 3: 2.5 microliters of the supernatant (model exosome sample) from the above example was mixed with 1 microliter of surfactant Tetronic 90R4 in an EP tube in a vortex shaker. All samples are loaded on the digital microfluidic chip, and under the drive of an electrode, the magnetic microspheres in the step 2 are mixed for 10min according to the designed path (inconsistent with the path in the step 2) so as to avoid pollution, and the closed path is also adopted, so that the exosomes in the supernatant are enriched. Finally, under the action of a magnetic field, the magnetic microspheres are separated from the liquid drops and the liquid drops are removed.
Step 4: the remaining 12. Mu.l of the mixture (mixture of buffer and surfactant) from step 2 was applied in three separate injections of 3. Mu.l each. The magnetic microspheres are washed according to step 2 to wash impurities on the magnetic microspheres due to nonspecific adsorption.
Step 5: and (3) completely loading 4 microliters of 70% acetonitrile water solution on a digital microfluidic chip, and mixing the acetonitrile water solution with the volume fraction of 70% with the magnetic microspheres in the step (3) for 10 minutes according to a designed path (which is not repeated with the previous path, avoids pollution and closes the path) under the driving of an electrode so as to fully extract the lipid in the exosome on the magnetic microspheres. And finally, under the action of a magnetic field, separating the magnetic microspheres from the liquid drops, and removing the liquid drops, wherein the liquid drops are the obtained lipid sample.
If the whole exosomes obtained are used for analysis of particle size or concentration, step 5 above is as follows: 2.5. Mu.l of 10% strength by volume ammonia was mixed with 0.5. Mu.l of the surfactant Tetronic 90R4 in an EP tube in a vortex shaker, all loaded on a digital microfluidic chip, and mixed with the magnetic microspheres of step 3 for 10min according to the designed path (no repetition of the previous path, avoidance of contamination, closed path) under electrode drive to elute the exosomes on the magnetic beads. And finally, under the action of a magnetic field, separating the magnetic microspheres from the liquid drops, and removing the liquid drops, wherein the liquid drops are the obtained exosome sample.
Example 2: testing selection of surfactants on chip to facilitate droplet movement
For the loading and selection problems of the surfactant (Pluronic F-127 or Tetronic 90R 4) in the above examples, we found that,
(1) In step 1 of example 1, when the suspension of magnetic spheres and the surfactant were introduced on the chip, when the surfactant was Tetronic 90R4, the movement of the magnetic spheres on the chip was slightly tailing and the clusters of magnetic spheres could not be dispersed in the droplets. When Pluronic is used for turning to F-127, the magnetic ball moves smoothly.
(2) Example 1 steps 2 to 4 when PBS buffer, supernatant, etc. were introduced on the chip and mixed with the surfactant, there was a slight tailing phenomenon when the surfactant Pluronic F-127 was selected. When the Tetronic 90R4 is used, the magnetic ball moves smoothly.
(3) The loading amounts of the surfactants described in example 1 are the lowest loading amounts under the premise of ensuring smooth movement of the magnetic ball.
Example 3: testing influence of different particle sizes of magnetic balls on chip on exosome enrichment effect
Different diameter magnetic spheres (1.45 microns and 4.5 microns) were tested to select the preferred diameter spheres for exosome enrichment. It was found that when larger diameter magnetic spheres (e.g., 4.5 microns) were chosen, the effect of the magnetic spheres on the chip was not better than that of smaller magnetic spheres. As shown in fig. 3, under the action of a magnetic field and without an electrode, the magnetic balls with larger diameters cannot be fully gathered, so that the separation of the magnetic balls from the liquid drops is affected. Therefore, we selected magnetic spheres with a diameter of about 1.45 microns for a series of exosome enrichment and lipid extraction.
Example 4: selection of different organic solvents during lipid extraction
When exosome lipid is extracted on a digital microfluidic chip, a group of mixed liquid (methanol/chloroform/water, 2:1:0.8, v: v) is tested for extracting the lipid on the chip, and the mixed liquid is introduced into the chip to be fully mixed with the magnetic ball adsorbed with exosome, but the liquid drop volatilizes 80% in 3 minutes due to the volatility of methanol, and the rest liquid drop can not be driven by an electrode successfully. After new organic solvent is continuously tested, and finally 70% acetonitrile is loaded, no obvious evaporation exists and the liquid drop can be driven within 10 minutes of driving the liquid drop to move by the electrode.
Example 5: characterization of magnetic microsphere surface by Scanning Electron Microscopy (SEM)
For the magnetic beads obtained in step 4 of example 1 above, the pellet was first left to stand in 1ml of 2.5% glutaraldehyde in PBS buffer (ph=7.2-7.4) for 2 hours, and then washed three times with PBS, and centrifuged to retain the pellet. Then dehydrated with gradient ethanol (30% aqueous ethanol solution, 1×5 min; 50% aqueous ethanol solution, 1×5 min; 75% aqueous ethanol solution, 1×5 min; 95% aqueous ethanol solution, 1×5 min; 100% anhydrous, 3××10 min), and then the samples were dried overnight. The dried sample was placed in 30 microliters of absolute ethanol, which was dropped onto a silicon wafer with a pipette, and coated with gold using a sputtering apparatus for 90s. Finally, SEM is used for observation and characterization of the magnetic ball surface. As shown in fig. 4, a is a blank control, and b is a magnetic sphere sample (red arrow pointing) with surface enriched exosomes. As can be seen from fig. 4, the incubated and repeatedly washed magnetic spheres were enriched with vesicles approximately similar to the exosomes in size (exosomes diameter of 30-150 nm) compared to the blank control. It should be noted that the purchased magnetic ball is not very smooth due to the magnetic surface.
Comparative example 1: two common exosome separation methods enrich exosomes
(1) UC gold standard enrichment from 1.5mL supernatant
1.5ML of the cell culture supernatant was first centrifuged (10000 g,4 ℃ C., 30 minutes), and the supernatant was retained after centrifugation and centrifuged again (14000 g,4 ℃ C., 30 minutes). After centrifugation, the supernatant was retained, transferred to an ultracentrifuge tube, leveled on a balance with a pipette using PBS buffer, and placed in a high-speed centrifuge for centrifugation (110000 g,4 ℃ C., 120 minutes); the pellet was retained after centrifugation, resuspended using PBS buffer, trimmed, and centrifuged (110000 g,4 ℃ C., 60 min). And after the centrifugation, discarding the supernatant, wherein the obtained precipitate at the bottom of the centrifuge tube is the exosome, blowing the centrifuge tube precipitate by using PBS, and transferring the centrifuge tube precipitate into an EP tube.
(2) UC gold standard enrichment from 15mL supernatant
15ML of the cell culture supernatant was first centrifuged (10000 g,4 ℃ C., 30 minutes), and the supernatant was retained after centrifugation and centrifuged again (14000 g,4 ℃ C., 30 minutes). After centrifugation, the supernatant was retained, transferred to an ultracentrifuge tube, leveled on a balance with a pipette using PBS buffer, and placed in a high-speed centrifuge for centrifugation (110000 g,4 ℃ C., 120 minutes); the pellet was retained after centrifugation, resuspended using PBS buffer, trimmed, and centrifuged (110000 g,4 ℃ C., 60 min). And after the centrifugation, discarding the supernatant, wherein the obtained precipitate at the bottom of the centrifuge tube is the exosome, blowing the centrifuge tube precipitate by using PBS, and transferring the centrifuge tube precipitate into an EP tube.
Example 6: nanoFCM nanometer flow detector for analyzing exosome particle size distribution
The supernatant of the chip of example 1 was concentrated to a volume of 1.5mL before the concentration, the supernatant of comparative example 1 (1) to a volume of 1.5mL before the concentration, and the supernatant of comparative example 1 (2) to a volume of 15mL before the concentration. The exosome samples obtained in the above three examples are hereinafter referred to as sample 1 (complete exosome eluted by ammonia in step 5), control 1, and control 2, respectively. And taking out a proper amount of the sample 1, the control 1 and the control 2, diluting to a proper multiple, and detecting the information of the particle size and the concentration of the exosome after the instrument tests the sample to be qualified.
The exosome concentration information of each sample is shown in table 1, and the particle size distribution is shown in fig. 5.
(1) As can be seen from Table 1, the exosomes enriched by the method are more than UC gold standard, and the separation time is greatly reduced.
(2) From the particle size distribution chart 5, it can be observed that the exosome particle size obtained by the present invention conforms to the normal distribution of exosome particle sizes.
Example 7: protein immunoblotting method for characterizing exosomes enriched by different methods
Exosome suspensions eluted with ammonia water, i.e., sample 1 and control 1 in example 3 above, were lysed using exosome-specific lysis solution (Yeasen next holy, cat No. 41211ES 20), centrifuged, and loading buffer (lanbordete, cat No. G2526) was added. It was then placed in a metal water bath to boil (95 degrees celsius, 10 min), the exosome proteins were separated by using a 12-well pre-gel (lanboride, cat No. P01212) and transferred onto PVDF membranes. Blocking with nonfat dry milk at 5% for 2 hours at room temperature, incubating overnight at 4deg.C using CD63 (1:2000, soy pal, cat# K007602P), after rinsing, incubating PVDF membrane with secondary antibody (goat anti-rabbit IgG-HRP,1:10000, soy pal, cat# SE 134) for 2 hours at room temperature, and immunoblotting with an imaging system (iBright ™ FL1500 IMAGING SYSTEM). As shown in fig. 6, it can be seen that the on-chip enriched exosomes are able to efficiently enrich exosomes in the trace samples compared to the UC gold standard.
Example 8: protein immunoblotting method for characterizing enriched exosomes under different incubation times
For the incubation times of the supernatant from step 3 with the magnetic beads in example 1, the optimal incubation time was assessed by western blotting and a set of extracted exosome samples (corresponding to 4 different incubation times, respectively) were tested, with the other conditions unchanged.
The exosome suspensions from 4 groups eluted with ammonia water were separately lysed using exosome-specific lysates (Yeasen next holy, cat No. 41211ES 20), centrifuged, and loading buffer (ebolite, cat No. G2526) was added. It was then placed in a metal water bath to boil (95 degrees celsius, 10 min), the exosome proteins were separated by using a 12-well pre-gel (lanboride, cat No. P01212) and transferred onto PVDF membranes. Blocking with nonfat dry milk at 5% for 2 hours at room temperature, incubating overnight at 4deg.C using CD63 (1:2000, soy pal, cat# K007602P), after rinsing, incubating PVDF membrane with secondary antibody (goat anti-rabbit IgG-HRP,1:10000, soy pal, cat# SE 134) for 2 hours at room temperature, and immunoblotting with an imaging system (iBright ™ FL1500 IMAGING SYSTEM). As shown in fig. 7, the exosome protein greyscale was maximized when the incubation time was 10 minutes. When the incubation time is too long (e.g. 15 minutes), we speculate that the heat generated by the chip itself is due to the long-time movement of the electrodes on the microfluidic chip, which has a bad effect on the droplets containing the exosomes on the chip, and seriously affects the enrichment of the exosomes.
Example 9: lipid sample loading analysis
After extracting the small molecule suspension on the chip using 70% acetonitrile for step 5 of example 1, the suspension was aspirated and placed into a 1.5mL centrifuge tube, which was placed into a freeze dryer and dried (1500 r, -5 degrees celsius). After the suspension was dried to a solid, it was removed and loaded to TOF-IMS-MS analysis.
Preprocessing the acquired signal data: removing background, smoothing and centering to obtain a data list. The data is patterned as shown in fig. 8. The raw data are searched in HMDB, the primary mass error is 0.05Da, the ion mode adopts positive ion mode, and the obtained lipid comprises phospholipid such as glycerophospholipid, ceramide (CERAMIDES), sphingomyelin such as Sphingomyelin (SM), phosphoglycerate (PG), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI) and the like, and participates in maintaining the integrity and functions (such as intercellular communication, immunoregulation and the like) of exosomes.
Claims (2)
1. A method for realizing exosome enrichment and biological micromolecule extraction based on integration of a microfluidic chip comprises the following steps:
a) Firstly mixing a ferroferric oxide magnetic microsphere suspension coated with a metal oxide nano coating on the surface with a surfactant Pluronic F-127, loading the mixture into a digital microfluidic chip, and then under the drive of an electrode, moving and mixing according to a path, separating magnetic microspheres from liquid drops and removing the liquid drops; after mixing PBS buffer solution and surfactant Tetronic90R4, partially loading the mixture onto a digital microfluidic chip, and cleaning the magnetic microspheres;
b) Mixing 2.5 microliters of a sample containing exosomes with 1 microliter of surfactant Tetronic90R4, loading the mixture onto a digital microfluidic chip, incubating the magnetic microspheres with the sample containing exosomes, and extracting exosome molecules in the sample through specific adsorption;
c) Washing the magnetic microspheres in step b) with a mixture of the PBS buffer remaining in step a) and the surfactant Tetronic90R4 to remove other non-specific molecules adsorbed on the magnetic microspheres; in the process, an external magnetic field is increased to gather the magnetic microspheres with the surfaces adsorbing exosomes; controlling the electrode of the digital micro-fluidic chip, controlling the movement of liquid drops, and separating washing waste liquid from the chip;
d) Mixing an alkaline solution with a surfactant Tetronic90R4, loading the mixture to a digital microfluidic chip, eluting the exosomes on the surface of the magnetic microspheres aggregated in the step c), and collecting the exosomes to obtain complete exosomes;
Or, introducing an organic solvent into the digital microfluidic chip, extracting biological small molecules in exosomes on the surfaces of the magnetic microspheres gathered in the step c) by using the organic solvent, and collecting the biological small molecules;
The surface of the step a) is coated with ferroferric oxide magnetic microspheres coated with a metal oxide nano coating, wherein the metal oxide is TiO 2; the diameter of the magnetic microsphere is 1.45+/-0.2 mu m;
in the step b), dripping the magnetic microsphere suspension into a digital microfluidic chip, and fully mixing 2.5 microliters of a sample containing exosomes with the magnetic microspheres; after incubation for 5-10min at normal temperature, the exosomes with complete structures are adsorbed to the surfaces of the magnetic microspheres through nonspecific actions;
the dosage ratio of the magnetic microsphere to the sample containing exosomes is 50 micrograms: 2.5 microliters;
In the step d), the organic solvent is acetonitrile water solution with the volume fraction of 70%;
in the step d), the alkaline solution is ammonia water with the volume concentration of 10%;
The biological small molecule is lipid.
2. The method according to claim 1, characterized in that: in said step b), said exosome-containing sample comprises cells of animal and plant origin.
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