CN113528423A - Method for separating plasma lipoprotein and exosome - Google Patents

Method for separating plasma lipoprotein and exosome Download PDF

Info

Publication number
CN113528423A
CN113528423A CN202110763017.2A CN202110763017A CN113528423A CN 113528423 A CN113528423 A CN 113528423A CN 202110763017 A CN202110763017 A CN 202110763017A CN 113528423 A CN113528423 A CN 113528423A
Authority
CN
China
Prior art keywords
lipoprotein
exosome
sample
channel
mixing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110763017.2A
Other languages
Chinese (zh)
Inventor
朱庆夫
娄豆豆
刘飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eye Hospital of Wenzhou Medical University
Original Assignee
Eye Hospital of Wenzhou Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eye Hospital of Wenzhou Medical University filed Critical Eye Hospital of Wenzhou Medical University
Priority to CN202110763017.2A priority Critical patent/CN113528423A/en
Publication of CN113528423A publication Critical patent/CN113528423A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention provides a method for separating plasma lipoprotein and exosome, which is characterized in that after a plasma sample is subjected to ultrafiltration pretreatment to remove most of high-abundance plasma protein, a separation carrier containing Ciba, such as Ciba modified amino magnetic beads, is adopted to separate the lipoprotein from the plasma, and the separation of the plasma lipoprotein and exosome can be realized. The process of separating the plasma lipoprotein and the exosome can be integrated on the microfluidic chip, so that corresponding operations such as separation, detection and the like are modularized, the automation degree of the process can be improved, the aim of efficiently and conveniently separating the plasma lipoprotein and the exosome is fulfilled, the exosome and the lipoprotein can be respectively detected, and the application research of the plasma lipoprotein and the exosome in disease mechanism and diagnosis can be promoted.

Description

Method for separating plasma lipoprotein and exosome
Technical Field
The invention relates to the technical field of biology, in particular to a method for separating plasma lipoprotein and exosome.
Background
The exosome is a vesicle with a lipid bilayer structure, which is actively secreted by cells, is wrapped by a membrane, has a uniform size and a diameter of about 30nm-200nm, lipoprotein is a spherical particle consisting of an inner core of sterol ester and triglyceride and an outer shell consisting of apolipoprotein, phospholipid, cholesterol and the like, and both exist in plasma, so that valuable genetic information can be provided for the clinical diagnosis of tumors.
However, the isolation and detection of exosomes has been a problem that has not been completely solved. Firstly, due to the high overlapping of the density and size of exosome and lipoprotein and the lack of an effective separation method, a large amount of lipoprotein is often mixed in plasma exosome obtained by researchers, which greatly affects the sensitivity and accuracy of an exosome detection method; secondly, the exosome has very strong heterogeneity, the particle diameters of the exosome are different in size, the heterogeneity is very large, and specific markers are lacked, so that the current exosome separation and detection method has no unified standard, and objective judgment on the extraction effect cannot be made temporarily.
Specifically, the existing exosome extraction method comprises the following steps: ultracentrifugation, and kit extraction (Exo quick. TM. method, qEV technique). However, these techniques all have significant disadvantages in extracting exosomes from liquid biopsy biological samples for analysis. Among these, ultracentrifugation precipitation requires high initial capital costs (ultracentrifuge prices > $ 10 ten thousand), and later requires significant maintenance and operational costs, while being time consuming and labor intensive, requires the collection of higher volumes of samples, and results in lower purity products. The kit used in the kit extraction method (ExoQuickTM method, qEV technology) is expensive, the required cost is high, large-scale application in clinical and practical operation is difficult to realize, the volume of the exosome extracted by the method every time is only about 1.5mL, the concentration of the exosome is low, and if high-concentration exosome needs to be extracted, further exosome enrichment and purification work needs to be carried out.
In recent years, an exosome total isolation chip (exosome total isolation chip) based on size separation is used for extracting and separating exosomes, the method is to drive fluid by pressure so as to efficiently separate high-purity exosomes from various biological fluids, but a nanopore film required by the technology has weak pressure bearing capacity, is easy to deform and cause a pore blocking phenomenon, the damage of the nanopore film is irreversible, and the research is continued if the nanopore film is applied to the ground.
In addition, the existing method only aims at the separation and extraction of exosome, and neglects the biological significance of lipoprotein in disease research. Therefore, it is necessary to develop a more efficient and convenient separation method to separate plasma exosomes and lipoproteins and to separately detect exosomes and lipoproteins, thereby simultaneously promoting the research on the application of both exosomes and lipoproteins in disease mechanism and diagnosis.
Disclosure of Invention
Aiming at the defects of the method for separating plasma lipoprotein and exosome in the prior art, the invention provides the method for effectively separating the plasma lipoprotein and exosome by effectively separating the lipoprotein in the plasma by utilizing the excellent affinity of the Cibacron to various lipoproteins.
The technical scheme adopted by the invention is as follows:
a method of separating plasma lipoproteins and exosomes, comprising the steps of:
s1: primary separation; carrying out ultrafiltration operation on plasma to be separated to obtain an intermediate sample;
s2: secondary separation; mixing the intermediate sample with a separation vehicle comprising Cibacron and separating the lipoproteins from the intermediate sample.
In other optimized technical schemes, the separation carrier containing the Cibacron is a magnetic bead modified by the Cibacron.
In other optimized technical solutions, the magnetic beads include at least one of amino magnetic beads, silicon-based magnetic beads, carboxyl magnetic beads, epoxy magnetic beads, streptavidin magnetic beads, or biotin magnetic beads.
In other optimized technical schemes, the magnetic beads have the particle size of 1-3 μm.
In other optimized technical schemes, the modification density of the Cibacron on the magnetic beads is 2-10 mmol/L.
In other optimized technical schemes, in the step S2, a microfluidic chip is used to separate the lipoprotein and exosome; the microfluidic chip comprises a channel layer and a substrate layer, wherein the channel layer is positioned on the substrate layer;
the channel layer comprises a sample introduction area and a mixing area which are communicated in sequence; the sample introduction region comprises a sample introduction port and a sample introduction channel, and the intermediate sample and the separation carrier containing the Cibacron are injected from the sample introduction port and enter the mixing region through the sample introduction channel;
the mixing area comprises a plurality of mixing units, each mixing unit comprises at least 1 wide channel and at least 1 narrow channel, the width of each wide channel is 100-300 mu m, and the width of each narrow channel is 1/5-1/2 of the width of each wide channel.
In other optimized technical solutions, the mixing area includes not less than 100 mixing units, each mixing unit includes 1 wide channel and 1 narrow channel; the length of the wide channel is 200-500 mu m, and the length of the narrow channel is not more than that of the wide channel;
wherein the sampling flow rate is 1-10 mu L/min.
In other optimized technical solutions, the channel layer further includes a detection region, and the intermediate sample and the separation carrier containing tubbalan enter the detection region through the mixing region; the detection area comprises at least 1 chamber, and the height of the chamber is not less than 0.5 mm.
In other optimized technical schemes, the detection area comprises a lipoprotein detection chamber and an exosome detection chamber which are communicated in sequence; the mixing area with lipoprotein detects the cavity intercommunication, lipoprotein detects the cavity with exosome detects the cavity intercommunication, wherein, lipoprotein detects the height of cavity and is 0.5 ~ 1.5 mm.
In other optimized technical schemes, the microfluidic chip further comprises a magnet, the magnet is arranged below the lipoprotein detection chamber, when a mixed sample flows through the lipoprotein detection chamber, the separation carrier containing the Ciba, stays in the lipoprotein detection chamber, and other parts flow into the exosome detection chamber, wherein the height of the exosome detection chamber is 5-10 mm.
The invention has the beneficial effects that:
after most of high-abundance plasma proteins are removed through pretreatment, the lipoprotein is separated from the plasma by adopting a separation carrier containing the Cibacron, and the separation of the plasma lipoprotein and exosome can be realized.
In an optimized technical scheme, the process of separating the plasma lipoprotein and the exosome is further integrated on a microfluidic chip, so that corresponding operations such as separation, detection and the like are modularized, and the automation degree of the process can be improved.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without any creative effort.
FIG. 1 is a graph of particle size distribution of an exosome sample and a plasma sample before and after treatment with a Ciba-modified magnetic bead, (a) particle size change of a standard exosome sample before and after treatment with a Ciba-modified magnetic bead; (c) particle size change of the plasma sample before treatment with the magnetic babalan beads and after treatment in (d);
FIG. 2 is a protein-based peak diagram of a protein sample captured by Ciba balana beads after processing the plasma sample, (a) a protein-based peak diagram of residual protein in the plasma sample (b) and a comparison diagram of lipoprotein-specific protein content and exosome-specific protein content of the two (c);
FIG. 3 is a schematic view of a channel layer of the microfluidic chip in example 5;
FIG. 4 is a simulation of the mixing effect of a conventional rectangular microchannel (a) and a mixing channel (b) of example 5 with wide and narrow spaces;
FIG. 5 shows the results of staining the samples in the lipoprotein detection chamber and the exosome detection chamber with lipoprotein-targeting quantum dot Dye (Qdot 605-antimAPOE) and cell membrane green fluorescent Dye (DIO Dye) in example 2;
fig. 6 is a two-dimensional code address for storing the color original drawings of fig. 1 to 5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that, in the description of the present invention, unless otherwise specifically limited, "ultrafiltration", "mixing", etc. are to be construed broadly, and operations that can achieve the objects of the present invention are within the scope of the present invention. All directional indicators (such as up, down, left, right, front, and rear … …) in the embodiments of the present invention are only used to explain the relative positional relationship between the components, the movement, and the like in a specific state (as shown in the drawings), and if the specific state changes, the directional indicator changes accordingly. In the description of the present invention, the measurement direction of "length" is the flow direction of the sample, the measurement direction of "width" is the direction perpendicular to the flow direction of the sample and parallel to the base layer, and the measurement direction of "height" is the direction perpendicular to the base layer. In the present invention, the terms "carrier," "linked," "communicating," and the like are to be construed broadly unless otherwise explicitly specified or limited.
In addition, Cibacron Blue 3GA is an English name of Cibacron Blue 3GA, and the CAS number is 84166-13-2, and Cibacron is hereinafter collectively referred to as CB for short.
The inventor finds that the CB has good affinity to various lipoproteins. As shown in fig. 1, nanoparticle tracking was used to analyze the particle size distribution change of the exosome sample and the plasma sample before and after CB magnetic bead treatment. Wherein, fig. 1-a is a particle size distribution diagram of a standard exosome sample before CB magnetic bead treatment, and fig. 1-b is a particle size distribution diagram of a standard exosome sample after CB magnetic bead treatment; FIG. 1-c is a particle size distribution diagram of a plasma sample before CB magnetic bead treatment, and FIG. 1-b is a particle size distribution diagram of a plasma sample after CB magnetic bead treatment. Wherein, the standard exosome sample refers to HEK293t cell exosome separated by ultracentrifugation, and the plasma sample refers to plasma treated by a 300KD ultrafiltration tube for 30min under the centrifugation condition of 250 g.
As can be seen from FIGS. 1-a and 1-b, when a standard exosome sample is treated with CB magnetic beads, the particle size and concentration of the sample are not obviously changed before and after the treatment, which indicates that the CB magnetic beads do not adsorb exosomes. With continued reference to fig. 1-c and fig. 1-d, it can be seen that after the plasma sample is processed by the CB magnetic beads, the particle size of the processed sample is significantly increased, and the total particle number is decreased, which indicates that the lipoprotein with a smaller size in the plasma is adsorbed by the CB magnetic beads.
Further, after the plasma sample is processed by the CB magnetic beads, proteins adsorbed on the magnetic beads and proteins remaining in the plasma sample are analyzed by using a liquid chromatography-mass spectrometry technology, and as a result, reference may be made to fig. 2, specifically, fig. 2-a is a protein-based peak diagram of the protein sample captured by the magnetic beads, fig. 2-b is a protein-based peak diagram of the remaining proteins in the plasma sample, and fig. 2-c is a content comparison diagram of lipoproteins captured by the magnetic beads and the remaining proteins in the sample. Wherein apolipoprotein associated with lipoproteins and cell membrane, extracellular and vesicle-associated proteins are selected.
By analyzing the protein captured on the CB magnetic beads and the residual protein in the plasma sample by a liquid chromatography-mass spectrometry combined technology, the following results are found by comparing protein-based peak maps: the difference between the protein on the CB beads and the protein species in the treated sample was very large, and the high peaks in both figures did not coincide. Further classification of the protein found: a large number of Apolipoprotein (APOs) related to the lipoprotein are captured on the magnetic beads, so that the CB magnetic beads have strong adsorbability to the lipoprotein, and the protein related to the vesicle still exists in a sample, so that the separation of plasma exosomes and the lipoprotein is realized.
Example 1:
in this example, about 0.5mL of plasma was taken and lipoproteins and exosomes were separated therefrom, and the specific method included the following steps:
s1: primary separation; carrying out ultrafiltration operation on plasma to be separated to obtain an intermediate sample;
wherein, ultrafiltration uses 300 KD's ultrafiltration pipe, and centrifugal force setting is 250 g's centrifugal force for the centrifugation condition, filters twice altogether, after first filtration, uses PBS to adjust the sample volume to 0.5mL again, after the second filtration, fixes the volume of counterfeit to 100 uL, obtains above-mentioned intermediate sample. This step removes most of the abundant proteins in plasma, such as albumin, IgG, etc., and the main components in the intermediate sample are vesicles and some foreign proteins, which facilitates the secondary separation.
S2: secondary separation; the intermediate sample is mixed with a separation carrier containing CB, and the lipoprotein is separated from the intermediate sample.
In this case, CB can be immobilized using magnetic beads as a carrier, and CB-modified magnetic beads can be obtained as the above CB-containing separation carrier. The specific modification method comprises the following steps: mixing amino magnetic beads (dispersed in a PBS solution) with the particle size of 1 mu m, CB (dispersed in deionized water) and a sodium chloride aqueous solution, reacting for 2 hours at 40 ℃ to obtain CB modified magnetic beads, wherein the modified magnetic beads are blue, and the modification density of the CB is measured by the following method: dissolving CB on the magnetic beads by using 200mmol/L HCl solution, and measuring the absorbance value QD of the dissolved solution by using a spectrophotometer due to the characteristic absorption peak of the CB at 620nm620The modification density was measured to be 5 mmol/L. In other embodiments, the density of CB modification can be adjusted to 2-10 mmol/L, and the CB can sufficiently adsorb lipoproteins in plasma.
Example 2:
in this example, one or more of carboxyl magnetic beads, silicon-based magnetic beads, epoxy magnetic beads, streptavidin magnetic beads, and biotin magnetic beads were used instead of the amino magnetic beads in example 1, and the effective separation of plasma lipoproteins and exosomes was also achieved under the same conditions as in example 1.
Example 3:
in this example, agarose beads having a particle size of about 20 μm were used as a carrier for immobilizing Ciba, and effective separation of plasma lipoproteins and exosomes was achieved under the same conditions as in example 1, with the addition of a sufficient dispersion means.
Example 4:
in this embodiment, the CB magnetic beads that can achieve the object of the present invention, which are preferably modified with 1 to 3 μm magnetic beads, can be obtained, and can ensure effective adsorption of lipoproteins in plasma while maintaining good dispersion with the plasma sample to be treated. On one hand, compared with magnetic beads with too large particle sizes, the CB magnetic beads obtained in the embodiment are not easy to settle and convenient to disperse; on the other hand, the CB magnetic beads obtained in this example do not have too weak magnetism compared to magnetic beads having too small particle sizes, and thus can efficiently and sufficiently adsorb lipoproteins.
Example 5:
in this embodiment, the step S2 in embodiment 1 is implemented using a microfluidic chip.
Specifically, the microfluidic chip comprises a channel layer and a substrate layer, wherein the channel layer is bonded on the substrate layer in a surface plasma treatment manner, the further structural characteristics of the channel layer are shown in figure 3, and the channel layer comprises a sample injection region 1, a mixing region 2 and a detection region 3 which are sequentially communicated. In this embodiment, the sample injection region 1 includes two sample injection ports, and the sample injection channel is in a "Y" shape, and both the width and the height of the "Y" shaped channel are in the micrometer scale, specifically, in this embodiment, the width of the "Y" shaped channel is designed to be 200 μm, and the height is designed to be 100 μm. The plasma sample and the CB magnetic bead sample are respectively input from two sample inlets and then enter the mixing area 2 through the sample inlet channel.
In other embodiments, the sample introduction channel may also be designed to be "T" shaped, "S" shaped, and the like, and the height and width of the channel may also be adjusted, and in a specific application scenario, as long as the shape and size of the channel that can meet the corresponding sample introduction requirement are within the protection scope of the present invention.
With continued reference to fig. 3, the mixing area 2 includes a plurality of mixing units, the specific number of the mixing units can be determined according to the mixing system in the specific application, the microfluidic chip of the embodiment includes not less than 100 mixing units, and each mixing unit includes 1 wide channel and 1 narrow channel. The width of the wide channel can be adjusted between 100-300 μm, such as 200 μm, the width of the narrow channel can be 1/5-1/2 of the width of the wide channel, such as 50 μm, the length of the wide channel is 400 μm, the height is 100 μm, the length of the narrow channel is 200 μm, and the height is 100 μm. Compared with a microchannel with a pure rectangular structure, the mixing units with wide and narrow intervals can effectively improve the mixing efficiency, and reference can be made to the simulation result shown in fig. 4.
Wherein, the attached figure 4-a is a common rectangular micro-channel with a width of 200 μm, the attached figure 4-B is a mixing channel with a wide-narrow alternate structure in the embodiment, and the mixing area of the two is a vertical channel in the figure, the sample injection area is a horizontal channel in the figure, wherein, the length of the vertical channel is 5.2mm, the length of the horizontal channel is 4.2mm (from the meeting time of the two samples), the sample injection speed of the sample A and the sample B is 5mm/s, the sample temperature is 20 ℃, the viscosity is 1 mPa.s, and the diffusion coefficient is 1e-9m2The simulation software is COMSOL Multiphysics 5.6, and the results are shown in FIG. 4: in the sample introduction area, the flow state of the fluid is laminar flow, and the sample A and the sample B are still in a layered state and are not effectively mixed. After entering the mixing region, the flow state of the fluid in fig. 4-a is still laminar flow, and the mixing is started gradually in the second half of the mixing channel, but at this time, it can still be seen that the two have a relatively obvious concentration gradient, and the mixing effect is poor; after the fluid in the attached fig. 4-b enters the wide-narrow mixing unit, a "turbulence-like flow" is formed, and after about 4 wide-narrow mixing units, better mixing is achieved, at this time, the fluid in the channel is uniformly mixed, and there is almost no concentration gradient, which shows that the mixing efficiency of the wide-narrow mixing structure in the embodiment is high.
With continued reference to fig. 3, the mixed sample of plasma and CB magnetic beads passes through the mixing region 2 and into the detection region 3. In this embodiment, the detection region 3 includes a lipoprotein detection chamber and an exosome detection chamber which are sequentially communicated, the mixing region is communicated with the lipoprotein detection chamber, the lipoprotein detection chamber is communicated with the exosome detection chamber, wherein the height of the lipoprotein detection chamber can be adjusted between 0.5mm and 1.5mm so as to accommodate a plasma sample transiting from the mixing region, specifically, in this embodiment, the height of the lipoprotein detection chamber is 1mm, and the volume is designed to be 5 μ L to 10 μ L; the height of the exosome detection chamber can be adjusted between 5-10 mm, and in the embodiment, the volume of the exosome detection chamber is designed to be large and can reach 140-300 mu L, so that the exosome detection chamber can fully contain a processed mixed sample. However, the above design of the size and capacity of the chamber is determined according to the application requirements, and does not limit the scope of the technical solution to be protected by the present invention.
Further, the microfluidic chip can be prepared by the following method:
firstly, respectively preparing a channel layer and a substrate layer, and assembling the channel layer and the substrate layer to obtain a target microfluidic chip; the preparation method of the channel layer comprises the following steps:
s1: printing the microstructure on a piece of resin by using a 3D printing technology, and determining the size of the cavity by controlling the height and the width of the microstructure to obtain the microstructure of the channel layer;
s2: the microstructure is replicated using a reverse-mode method. Specifically, the channel layer can be obtained by copying the microstructure onto Polydimethylsiloxane (PDMS) in the form of a PDMS reverse mold.
And finally, bonding the PDMS chip on a glass plate through surface plasma treatment so as to finish the manufacture of the target microfluidic chip. The preparation method has the following remarkable advantages: the method can be used for preparing a cavity with a higher height (such as a cavity with a height of 1-10 mm), and channels or cavities with different heights are more efficient and convenient to use through 3D printing, so that the method is very suitable for manufacturing a micro-channel structure mold to be protected by the invention.
With continued reference to fig. 3, the microfluidic chip further includes a magnet disposed below the lipoprotein detection chamber, and when the mixed sample flows through the lipoprotein detection chamber, the CB magnetic beads stay in the lipoprotein detection chamber, and the other parts flow into the exosome detection chamber. Furthermore, the substrate layer of the microfluidic chip can be made of a glass plate or a polyethylene plate, in this embodiment, the glass plate is used as a material for making the substrate layer, and distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG) is modified on the surface of the glass plate.
Next, plasma lipoproteins and exosomes were separated and detected separately using the microfluidic chip in this example.
With reference to fig. 3, the plasma sample and the CB magnetic bead sample pretreated in step S1 are respectively injected from two injection ports at the same flow rate (1-10 μ L/min), and enter the mixing region 2 through the injection region 1, and are sufficiently mixed in the mixing region 2, so that the CB magnetic bead can better capture the lipoprotein, and then the plasma sample and the lipoprotein captured by the magnetic bead enter the lipoprotein detection chamber 31 along with the magnetic bead, in the lipoprotein detection chamber, the magnetic bead is fixed by a magnetic tape disposed under the microfluidic chip, and the plasma continues to flow and enter the following exosome detection chamber 32, the sample is accumulated in the exosome detection chamber 32, wherein the exosome in the plasma sample is captured by distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) modified on the surface of the substrate layer, and the lipoprotein and the exosome in the plasma are separated and detected by using the microfluidic chip, an amount of about 100. mu.L of plasma can be treated at one time.
Finally, a sample in the lipoprotein detection chamber 31 is taken and is marked as sample 1, a sample in the exosome detection chamber 32 is taken and is marked as sample 2, and lipoprotein and exosome in the sample 1 and the sample 2 are stained by using a cell membrane green fluorescent Dye (DIO Dye) and a quantum dot Dye (Qdot605-anti apoe) targeting the lipoprotein, and are subjected to quantitative analysis, and the result is shown in fig. 5.
Wherein, fig. 5-a is a representation image of a sample 1 after Qdot605-anti APOE staining, fig. 5-b is a representation image of a sample 2 after Qdot605-anti APOE staining, and fig. 5-c is a Qdot signal intensity histogram of the sample 1 and the sample 2 after Qdot605-anti APOE staining; fig. 5-d is a characterization image of the sample 1 after DIO Dye staining, fig. 5-e is a characterization image of the sample 2 after DIO Dye staining, and fig. 5-f is a DIO signal intensity histogram of the sample 1 and the sample 2 after DIO Dye staining.
As can be seen from FIG. 5, the Qdot605 fluorescence signal on sample 1 is strong and the Qdot605 fluorescence signal on sample 2 is weak, demonstrating that a large amount of lipoproteins are captured on the CB beads, while almost no signals of lipoproteins are found on the DSPE. The DIO Dye staining results prove that vesicles with membrane structures are found on the magnetic beads in the first cavity and the DSPE in the second cavity, the lipoprotein mainly exists on the magnetic beads, and the exosome is mainly captured by the DSPE, so that the purpose of efficiently and conveniently separating plasma lipoprotein and exosome is achieved, the exosome and the lipoprotein can be respectively detected, and the application research of the exosome and the exosome in disease mechanism and diagnosis can be promoted.
To more clearly illustrate the technical solutions and the technical effects achieved by the embodiments of the present invention, the two-dimensional code viewing addresses of the color original drawings of fig. 1 to 5 are attached, as shown in fig. 6.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the specification and drawings of the present invention or other related technical fields directly/indirectly using the technical idea of the present invention shall fall within the scope defined by the claims of the present invention without departing from the spirit of the present invention.

Claims (10)

1. A method of separating plasma lipoproteins from exosomes, comprising:
the method comprises the following steps:
s1: primary separation; carrying out ultrafiltration operation on plasma to be separated to obtain an intermediate sample;
s2: secondary separation; mixing the intermediate sample with a separation vehicle comprising Cibacron and separating the lipoproteins from the intermediate sample.
2. A method of separating plasma lipoproteins and exosomes according to claim 1, characterized in that:
the separation carrier containing the Cibacron is a magnetic bead modified by Cibacron.
3. A method of separating plasma lipoproteins and exosomes according to claim 2, characterized in that:
the magnetic beads comprise at least one of amino magnetic beads, carboxyl magnetic beads, silicon-based magnetic beads, epoxy magnetic beads, streptavidin or biotin magnetic beads.
4. A method of separating plasma lipoproteins and exosomes according to claim 3, characterized in that:
the magnetic beads have a particle size of 1-3 μm.
5. A method of separating plasma lipoproteins and exosomes according to claim 4, characterized in that:
the modification density of the Cibacron on magnetic beads is 2-10 mmol/L.
6. A method according to any one of claims 1 to 5, wherein said step of separating comprises the steps of:
in the S2 step, separating the lipoprotein and exosome using a microfluidic chip; the microfluidic chip comprises a channel layer and a substrate layer, wherein the channel layer is positioned on the substrate layer;
the channel layer comprises a sample introduction area and a mixing area which are communicated in sequence; the sample introduction region comprises a sample introduction port and a sample introduction channel, and the intermediate sample and the separation carrier containing the Cibacron are injected from the sample introduction port and enter the mixing region through the sample introduction channel;
the mixing area comprises a plurality of mixing units, each mixing unit comprises at least 1 wide channel and at least 1 narrow channel, the width of each wide channel is 100-300 mu m, and the width of each narrow channel is 1/5-1/2 of the width of each wide channel.
7. A method of separating plasma lipoproteins and exosomes according to claim 6, characterized in that:
the mixing region comprises not less than 100 mixing units, each mixing unit comprises 1 wide channel and 1 narrow channel; the length of the wide channel is 200-500 mu m, and the length of the narrow channel is not more than that of the wide channel;
wherein the sampling flow rate is 1-10 mu L/min.
8. A method of separating plasma lipoproteins and exosomes according to claim 6, characterized in that:
the channel layer further comprises a detection area, and the intermediate sample and the separation carrier containing the Cibacron enter the detection area through the mixing area; the detection area comprises at least 1 chamber, and the height of the chamber is not less than 0.5 mm.
9. A method of separating plasma lipoproteins and exosomes according to claim 8, characterized in that:
the detection area comprises a lipoprotein detection chamber and an exosome detection chamber which are communicated in sequence; the mixing area with lipoprotein detects the cavity intercommunication, lipoprotein detects the cavity with exosome detects the cavity intercommunication, wherein, lipoprotein detects the height of cavity and is 0.5 ~ 1.5 mm.
10. A method of separating plasma lipoproteins and exosomes according to claim 9, characterized in that:
the micro-fluidic chip further comprises a magnet, wherein the magnet is arranged below the lipoprotein detection chamber, a mixed sample flows through the lipoprotein detection chamber, the separation carrier containing the Baylan stays in the lipoprotein detection chamber, other parts flow into the exosome detection chamber, and the height of the exosome detection chamber is 5-10 mm.
CN202110763017.2A 2021-07-06 2021-07-06 Method for separating plasma lipoprotein and exosome Pending CN113528423A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110763017.2A CN113528423A (en) 2021-07-06 2021-07-06 Method for separating plasma lipoprotein and exosome

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110763017.2A CN113528423A (en) 2021-07-06 2021-07-06 Method for separating plasma lipoprotein and exosome

Publications (1)

Publication Number Publication Date
CN113528423A true CN113528423A (en) 2021-10-22

Family

ID=78126919

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110763017.2A Pending CN113528423A (en) 2021-07-06 2021-07-06 Method for separating plasma lipoprotein and exosome

Country Status (1)

Country Link
CN (1) CN113528423A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102728424A (en) * 2012-07-18 2012-10-17 哈尔滨工业大学 Microfluidic chip with symmetrical semi-cylindrical grooves on side wall
CN110079457A (en) * 2019-06-04 2019-08-02 苏州大学 Micro-fluidic chip and excretion body extracting method
CN110343664A (en) * 2018-04-03 2019-10-18 中国人民解放军军事科学院军事医学研究院 The method for extracting excretion body and excretion body protein
RU2741776C1 (en) * 2020-02-25 2021-01-28 Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр онкологии имени Н.Н. Петрова" Министерства здравоохранения Российской Федерации Method for plasma exosome release

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102728424A (en) * 2012-07-18 2012-10-17 哈尔滨工业大学 Microfluidic chip with symmetrical semi-cylindrical grooves on side wall
CN110343664A (en) * 2018-04-03 2019-10-18 中国人民解放军军事科学院军事医学研究院 The method for extracting excretion body and excretion body protein
CN110079457A (en) * 2019-06-04 2019-08-02 苏州大学 Micro-fluidic chip and excretion body extracting method
RU2741776C1 (en) * 2020-02-25 2021-01-28 Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр онкологии имени Н.Н. Петрова" Министерства здравоохранения Российской Федерации Method for plasma exosome release

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMTHAUER R, ET AL.,: "Interaction of cibacron blue and anilinonaphthalenesulphonate with lipoproteins provides a new means for simple isolation of these plasma proteins", 《BIOCHEM BIOPHYS RES COMMUN,》 *
PIETROWSKA, M., ET AL.: "《 Emerging Sample Treatments in Proteomics》", 25 June 2019 *
洪少力等: "磁控微流控芯片的研究现状及应用", 《分析科学学报》 *

Similar Documents

Publication Publication Date Title
Kang et al. High-purity capture and release of circulating exosomes using an exosome-specific dual-patterned immunofiltration (ExoDIF) device
Wei et al. Combination of size-exclusion chromatography and ultracentrifugation improves the proteomic profiling of plasma-derived small extracellular vesicles
EP2717989B1 (en) Vesicle capturing devices and methods for using same
US11684893B2 (en) Method for exosome separation and extraction by stacked centrifugal filtration
US8313906B2 (en) Methods and systems for microfluidic DNA sample preparation
US20210311025A1 (en) Exosome-Total-Isolation-Chip (ExoTIC) Device for Isolation of Exosome-Based Biomarkers
CN113101737B (en) Affinity tangential flow filtration system, construction method thereof, exosome extraction method and application
EP4299178A1 (en) Microfluidic chip, and automatic separation and detection system and method for circulating tumor cell
US20220347686A1 (en) Size-based asymmetric nanopore membrane (anm) filtration for high-efficiency exosome isolation, concentration, and fractionation
CN108841777A (en) The extracting method and device of extracellular vesica based on Electrostatic Absorption and its content
CN211771270U (en) Micro-fluidic chip for separating and enriching exosomes in peripheral blood
CN113976195B (en) Microfluidic chip for exosome separation and enrichment and exosome surface protein analysis method
Omrani et al. Global trend in exosome isolation and application: an update concept in management of diseases
CN109082400A (en) A method of excretion body being separated from biological sample using DEAE magnetic nano particle
CN112831457A (en) Method for separating and concentrating exosome
US20220364076A1 (en) Microfluidic platform for selective exosome isolation
CN113528423A (en) Method for separating plasma lipoprotein and exosome
Shami-shah et al. Advances in extracellular vesicle isolation methods: a path towards cell-type specific EV isolation
US20220305405A1 (en) System and method for isolation of intact extracellular vesicles with near-single-vesicle resolution coupled with on-line characterization
CN108088831B (en) Vesicle separation efficiency evaluation and quality control method based on liposome
CN110551680A (en) Method and system for extracting pleural effusion exosomes
CN116773500B (en) Method for separating and identifying extracellular vesicles
Morani Novel electrokinetic and analytical approaches for isolation and detection of extracellular vesicles
CN103805504B (en) Membrane type separating chips
CN113522384A (en) Microfluidic chip and preparation and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20211022