KR102557495B1 - Method and apparatus for isolating bioparticles derived from saliva without damage - Google Patents

Abstract

The present invention relates to a saliva-derived bioparticle separation method without damage and a device therefor. The saliva-derived bioparticle separation method according to an embodiment of the present invention includes the steps of (a) stabilizing a saliva sample (S100); (b) filtering the stabilized saliva sample using a filter (S200); and (c) separating bioparticles from the filtered saliva sample (S300).

Description

Method and apparatus for isolating bioparticles derived from saliva without damage}

The present invention relates to a method for separating bioparticles derived from saliva without damage and a device therefor, and more particularly, a method for separating/storing bioparticles from saliva samples without damage by performing optimal pretreatment, filtering, and separation/storage processes; and a device for separating/storing bioparticles capable of effectively performing the above process.

Extracellular vesicles are micro- and nano-sized particles composed of a cell-like lipid layer and containing intracellular nucleic acids and proteins, and are attracting attention as diagnostic markers of diseases because they are originally secreted with cellular characteristics. These extracellular vesicles include exosomes and microvesicles. In particular, exosomes that can be extracted from various body fluids are endoplasmic reticulum made of a lipid bilayer and are stable in the body and can be attacked by proteases, RNases, and DNases. It is free from this and can be used as a new biomarker for diagnosing specific diseases such as cancer and degenerative diseases.

Among conventional techniques for isolating extracellular vesicles, the density difference-based ultra-centrifugation isolation method has been evaluated as a highly reproducible and relatively reliable separation method due to its simple principle. However, ultracentrifugation has disadvantages such as low yield, long separation time, and the need to use expensive equipment. In addition, microfluidics chip, size exclusion, immunoaffinity isolation, and polymeric method disclosed in the patent documents of the following prior art documents have been developed, but body fluids There is a limitation in isolating a very small amount of extracellular vesicles within the cell, and problems such as damage to extracellular vesicles and reduction in yield still exist.

On the other hand, in the case of isolating bioparticles such as extracellular vesicles from saliva in the oral cavity, it can be used as a specific biomarker for oral diseases, but research and development to isolate saliva-derived bioparticles is insufficient to date. .

Accordingly, there is an urgent need for a technology for solving the problems of the conventional extracellular vesicle separation technology and for separating and recovering bioparticles from saliva samples without damage.

KR 10-2014-0050465 A

The present invention is to solve the problems of the prior art described above, and one aspect of the present invention is a saliva that separates and collects bioparticles from a saliva sample by performing a pretreatment and filtration process to stabilize the collected saliva sample at an optimal temperature and pH. It is to provide a method for separating bioparticles derived from the present invention.

Another aspect of the present invention is to provide an all-in-one saliva-derived bioparticle separation device capable of directly collecting, separating, and storing saliva samples in a clinical setting.

A saliva-derived bioparticle separation method according to an embodiment of the present invention includes the steps of (a) stabilizing a saliva sample; (b) filtering the stabilized saliva sample using a filter; and (c) separating bioparticles from the filtered saliva sample.

In addition, in the saliva-derived bioparticle separation method according to an embodiment of the present invention, in the step (a), the saliva sample may be stabilized at a temperature of 2 to 6 °C.

In addition, in the saliva-derived bioparticle separation method according to an embodiment of the present invention, in the step (a), the saliva sample may be stabilized in an acidic condition of pH 3 to 6.

In addition, in the saliva-derived bioparticle separation method according to an embodiment of the present invention, in the step (b), the filter may have a hydrophilic surface.

In addition, in the saliva-derived bioparticle separation method according to an embodiment of the present invention, the filter includes polytetrafluoroethylene (PTFE), polycarbonate (PC), polyester, and polytetrafluoroethylene (PTFE). It may be made of one or more polymers selected from the group consisting of ethersulfone (polyethersulfone, PES).

In addition, in the saliva-derived bioparticle separation method according to an embodiment of the present invention, the filter may have a pore size of 0.8 to 1.2 μm.

In addition, in the saliva-derived bioparticle separation method according to an embodiment of the present invention, in the step (c), the saliva sample may be centrifuged.

In addition, in the saliva-derived bioparticle separation method according to an embodiment of the present invention, (d) storing the separated bioparticles at a temperature of -20 to -90 ° C; may further include.

In addition, in the method for separating saliva-derived bioparticles according to an embodiment of the present invention, the bioparticles may be exosomes.

On the other hand, the saliva-derived bioparticles separation device according to an embodiment of the present invention includes a collection sample accommodating part accommodating a saliva sample therein, and a collection sample outlet inlet communicating with the collection sample accommodating part so that the saliva sample flows in and out at one end. A sample collection container having a; a filter for filtering the saliva sample discharged through the collected sample outlet; and a separated sample accommodating part having the filter disposed therein and accommodating the separated sample passing through the filter, and a separated sample outlet having one end communicating with the separated sample accommodating part so that the separated sample flows in and out. ; can be included.

In addition, in the saliva-derived bioparticle separation device according to an embodiment of the present invention, one end of the sample collection container and one end of the sample separation container may be detachably screwed together.

In addition, in the saliva-derived bioparticle separation device according to an embodiment of the present invention, the filter has a hollow tube shape with one end open so that the saliva sample flows in, and a plurality of pores on the outer circumferential surface through which the separated sample is discharged. can be provided.

In addition, in the saliva-derived bioparticle separation device according to an embodiment of the present invention, the filter may have a hydrophilic surface.

In addition, in the saliva-derived bioparticle separation device according to an embodiment of the present invention, the filter includes polytetrafluoroethylene (PTFE), polycarbonate (PC), polyester, and polytetrafluoroethylene (PTFE). It may be made of one or more polymers selected from the group consisting of ethersulfone (polyethersulfone, PES).

In addition, in the device for separating bioparticles derived from saliva according to an embodiment of the present invention, the filter may have a pore size of 0.8 to 1.2 μm.

In addition, in the saliva-derived bioparticle separation device according to an embodiment of the present invention, the collected sample receiving unit Accepted within, a buffer solution in which the saliva sample is mixed; may further include.

In addition, in the apparatus for separating bioparticles derived from saliva according to an embodiment of the present invention, exosomes may be centrifuged from the separated sample.

Features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.

Prior to this, the terms or words used in this specification and claims should not be interpreted in a conventional and dictionary sense, and the inventor may appropriately define the concept of the term in order to explain his or her invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

According to the present invention, saliva-derived bioparticles can be separated in a high yield without damage by performing stabilization and filtration processes of saliva samples, and the separated bioparticles can be provided as specific biomarkers that can be used for diagnosing diseases in the oral cavity. can

In addition, according to the saliva-derived bioparticle separation device according to the present invention, saliva collection, pretreatment, and separation and storage of bioparticles are integrally performed at the clinical site, so saliva samples are transferred to the laboratory and separated, stored for a certain period of time and It is possible to prevent a decrease in the recovery rate and loss of characteristics of saliva-derived bioparticles that may occur during the maintenance process.

1 is a flowchart of a method for separating bioparticles derived from saliva according to an embodiment of the present invention.
2 is a flowchart of a saliva-derived bioparticle separation method according to another embodiment of the present invention.
3 is an exploded perspective view of an apparatus for separating saliva-derived bioparticles according to an embodiment of the present invention.
4 is a combined cross-sectional view of an apparatus for separating saliva-derived bioparticles according to an embodiment of the present invention.
5 is a nanoparticle tracking analysis (NTA) analysis result according to an experimental example.
6 is a Western blot analysis result according to an experimental example.

Objects, specific advantages and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments taken in conjunction with the accompanying drawings. In adding reference numerals to components of each drawing in this specification, it should be noted that the same components have the same numbers as much as possible, even if they are displayed on different drawings. In addition, terms such as “first” and “second” are used to distinguish one component from another component, and the components are not limited by the terms. Hereinafter, in describing the present invention, detailed descriptions of related known technologies that may unnecessarily obscure the subject matter of the present invention will be omitted.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a flowchart of a method for separating bioparticles derived from saliva according to an embodiment of the present invention.

As shown in FIG. 1 , the saliva-derived bioparticle separation method according to an embodiment of the present invention includes the steps of (a) stabilizing a saliva sample (S100); (b) filtering the stabilized saliva sample using a filter (S200); and (c) separating bioparticles from the filtered saliva sample (S300).

The present invention relates to a method for separating bioparticles from saliva without damage. Conventional techniques for separating extracellular vesicles from body fluids have limitations in isolating very small amounts of extracellular vesicles, and the yield of extracellular vesicles is reduced. And it has a problem caused by damage, and it has been devised as a solution for this.

Extracellular vesicles are classified into exosomes (30 to 200 nm), microvesicles (200 to 1000 nm), and large oncosomes (1 to 10 μm) according to their sizes. Here, the bioparticles include not only extracellular vesicles but also saliva-derived bioparticles, and may typically be exosomes. These bioparticles can be used as biomarkers for diagnosing various diseases, and can be used as specific biomarkers for oral diseases because they are isolated in saliva. Here, saliva may include not only bodily fluids obtained from humans, but also those obtained from other animals.

Specifically, the saliva-derived bioparticle separation method according to an embodiment of the present invention includes a saliva sample stabilization step (S100), a filtration step (S200), and a bioparticle separation step (S300).

The saliva sample stabilization step (S100) is a process of collecting a saliva sample collected from an animal, particularly a human, and performing pretreatment thereon. The pretreatment of the saliva sample is to separate the bioparticles from the saliva without damage, and implements an environment capable of stabilizing the saliva sample during separation of the bioparticles. Here, the saliva sample may be stabilized under predetermined temperature and pH conditions.

A suitable temperature condition for stabilizing the saliva sample is 2 to 6 °C. However, the stabilization temperature is not necessarily limited thereto, and may be specifically determined according to the type of bioparticles. In one embodiment, in the case of exosome separation, stabilization is possible within the above temperature range, and it is appropriate to maintain the temperature preferably at 3 to 5° C., more preferably at about 4° C.

The pH conditions that stabilize the saliva sample may be acidic conditions. Here, the pH may be adjusted by adding and mixing the saliva sample to a buffer. At this time, acidic conditions are suitable within the range of pH 3 ~ 6. However, since the pH range is determined in consideration of separated bioparticles, the scope of the present invention should not necessarily be limited thereto. On the other hand, in the embodiment of exosome separation, the above pH range is appropriate, and preferably pH 4 to 5 can be set.

The filtration step (S200) is a process of filtering out some of the components in the stabilized saliva sample according to the size, and the saliva sample is filtered using a porous filter. At this time, only substances of a certain size or less pass through the filter. Since bioparticles are micro- or nano-sized bioparticles, the pores of the filter must be of a size that allows bioparticles to pass through. In one embodiment, the pore size of the filter for separating exosomes may be 0.8 to 1.2 μm. If the pore size is less than 0.8 μm, exosomes may not pass through the pores and may be lost, and if the pore size exceeds 1.2 μm, impurities other than exosomes are not filtered out, and the yield of exosomes is reduced.

On the other hand, non-specific binding to bioparticles occurs during the separation process by the filter, and as a result, a yield reduction is expected. As a filter, a filter having a hydrophilic surface may be used. In addition, since the saliva sample passes through the filter, it is preferable to be made of a material that has durability capable of withstanding the flow rate of the saliva sample and does not damage passing bioparticles. To meet these conditions, the filter is selected from the group consisting of polytetrafluoroethylene (PTFE), polycarbonate (PC), polyester, and polyethersulfone (PES) It may consist of one or more polymers. However, the filter does not necessarily have to be made of the above polymer, and a filter made of a different polymer or other durable material and having a hydrophilic film formed on its surface may be manufactured and used.

The bioparticle separation step (S300) is a process of separating bioparticles from the saliva sample that has passed through the filter. Here, bioparticles may be separated and extracted by centrifugation. However, the separation method is not necessarily limited to centrifugation, and other separation methods may be used depending on the type of bioparticles.

Overall, according to the present invention, saliva-derived bioparticles can be separated in high yield without damage by performing stabilization and filtration processes of saliva samples, and the separated bioparticles can be used for diagnosing diseases in the oral cavity. Specific biomarkers can be provided with

2 is a flowchart of a saliva-derived bioparticle separation method according to another embodiment of the present invention.

Referring to FIG. 2 , the saliva-derived bioparticle separation method according to another embodiment of the present invention may further include storing the bioparticles separated through the bioparticle separation step (S300) (S400). Here, the bioparticles are stored in an appropriate environment, where the storage temperature may be -20 to -90 °C. However, the storage temperature may be selectively adopted depending on the bioparticles, and for example, in the case of exosomes, approximately -75 to -85 ° C is preferable.

3 is an exploded perspective view of a saliva-derived bioparticle separation device according to an embodiment of the present invention, and FIG. 4 is a combined cross-sectional view of the saliva-derived bioparticle separation device according to an embodiment of the present invention.

As shown in FIGS. 3 and 4, the saliva-derived bioparticle separation device according to an embodiment of the present invention includes a collection sample accommodating portion 11 accommodating a saliva sample 1 therein, and a saliva sample ( 1) a sample collection container 10 having a collection sample outlet 13 communicating with the collection sample accommodating portion 11 so that 1) flows in and out; A filter 20 for filtering the saliva sample 1 discharged through the collected sample outlet 13; and a separated sample accommodating part 31 having a filter 20 disposed therein and accommodating the separated sample 3 passing through the filter 20, and a separated sample accommodating part 31 so that the separated sample 3 flows in and out at one end. ) and a sample separation container 30 having a separated sample outlet 33 communicating with the sample separation container 30.

Specifically, the device for separating bioparticles derived from saliva according to an embodiment of the present invention includes a sample collection container 10, a filter 20, and a sample separation container 30.

The sample collection container 10 is a container for temporarily storing the collected saliva sample 1, and includes a collection sample accommodating part 11 and a collection sample outlet 13. Here, the collection sample accommodation unit 11 provides an accommodation space formed to accommodate the saliva sample 1 therein. The collection sample outlet 13 is an opening provided at one end of the sample collection container 10 and is formed to communicate with the collection sample accommodating part 11 . Therefore, the saliva sample 1 flows into the collected sample receiving unit 11 through the collected sample outlet 13, is temporarily stored, and then discharged through the collected sample outlet 13. Here, one end of the sample collection container 10 in which the collected sample outlet 13 is formed is preferably an upper surface portion so that the saliva sample 1 can be dropped and injected from the top to the bottom, but may also be a side portion. In addition, the sample collection container 10 may be formed of a transparent material so as to check the amount of the saliva sample 1 received or discharged. Furthermore, scales and numerical values for accurately measuring the amount of the saliva sample 1 may be displayed on the outer circumferential surface of the waste collection container 10 . At this time, not only a scale and a value for measuring the amount of the saliva sample 1 flowing in, but also a scale and a value for measuring the amount of the discharged saliva sample 1 may be displayed together. For example, values and scales that decrease from one end where the collection sample outlet 13 is formed to the other end direction are displayed (for measuring the amount of saliva sample stored), and separately, numbers and scales that increase from the one end toward the other end It can be displayed (for measuring the amount of saliva sample discharged).

Meanwhile, since the above-described saliva sample 1 is stabilized in the sample collection container 10, the buffer solution may be accommodated in the sample collection container 11. Here, the buffer is to adjust the pH of the saliva sample 1, and before the saliva sample 1 flows in, the collection sample receiving part 11 is first filled, and then the saliva sample 1 flows in and can be mixed with each other. there is. Due to the addition of such a buffer, the saliva sample 1 can be stabilized under an acidic condition of pH 3-6. However, the pH range may be determined differently depending on the bioparticles, and in the case of exosome separation, a pH of 4 to 5 is preferable. In addition, the sample collection container 10 may be adjusted so that the saliva sample 1 maintains a temperature of 2 to 6 °C in order to stabilize the saliva sample 1 . Accordingly, although not shown, a temperature control unit may be mounted on the sample collection container 10 . As an example of the temperature control means, it may be a water jacket disposed on the outer surface of the sample collection container 10 and through which a cooling fluid flows along the inner hollow. However, the temperature control means is not necessarily limited to the water jacket, and is not particularly limited as long as it is a means capable of controlling the temperature of the saliva sample 1 in the sample collection container 10.

In addition, the open sample collection outlet 13 of the sample collection container 10 may be opened and closed by the sample collection container cap 15 . Here, the sample collection container cap 15 may be screwed to the sample collection container 10, and for this purpose, one end of the sample collection container 10 may be formed in a bottleneck shape, and a screw thread may be provided on the outer circumferential surface thereof. there is. However, the shape of the sample collection container 10 is sufficient as long as the saliva sample 1 is stored therein and the saliva sample 1 is formed so that the saliva sample 1 flows in and out, and must be screwed together with the sample collection container cap 10. It doesn't have to be.

The filter 20 is a porous member that filters the saliva sample 1 discharged through the collected sample outlet 13 . In one embodiment of the filter 20, the filter 20 may have a hollow tube shape with one end open so that the saliva sample 1 flows into the inside, and may have a plurality of pores 23 on the outer circumferential surface. Here, the size of the pores 23 may be 0.8 to 1.2 μm suitable for exosome separation.

Such a filter 20 may be formed in a "U" shape in cross section cut along the longitudinal direction. In addition, the outer circumferential surface of the filter 20 refers to all or a part of the outer wall forming the hollow tube except for one end opened with respect to the body portion 21 of the hollow tube shape. Accordingly, the pores 23 may be formed on all or part of the outer surface forming the other end opposite to one end of the body portion 21 and the side surface connecting the one end and the other end. Here, the pores 23 have a size through which bioparticles can pass. As a result, the saliva sample 1 introduced into the inside from one end of the body portion 21 passes through the pores 23, and in the process, some of the components in the saliva sample 1 are filtered out and saliva containing bioparticles. Sample 1 is discharged. Hereinafter, the saliva sample 1 that has passed through the filter 20 is defined as the separated sample 3.

On the other hand, according to the position of the pores 23, the saliva sample 1 is not only discharged to the other end of the filter 20 but also to the side, so that the pressure is not concentrated in one direction but dispersed and the flow rate is increased. Since it slows down, damage to bioparticles due to contact with the filter 20 can be reduced.

In addition, in order to prevent non-specific binding to bioparticles from occurring during the separation process by the filter 20, and thereby reducing yield, the filter 20 may have a hydrophilic surface. In addition, since the saliva sample 1 passes through the filter 20, it is preferable to be made of a material that has durability capable of withstanding the flow rate of the saliva sample 1 and does not damage passing bioparticles. Accordingly, the filter 20 is made of one or more polymers selected from the group consisting of polytetrafluoroethylene (PTFE), polycarbonate (PC), polyester, and polyethersulfone (PES). It can be done. However, the filter 20 does not necessarily have to be made of the above polymer, and may be made of a different polymer or other durable material and have a hydrophilic film formed on its surface.

The sample separation container 30 is a container for accommodating the separated sample 3 passing through the filter 20 and separating bioparticles from the separated sample 3 . The sample separation container 30 includes a separated sample accommodating part 31 and a separated sample outlet 33 . Here, the separated sample accommodating part 31 is an accommodation space formed therein, where the filter 20 is disposed and the separated sample 3 is accommodated. The separated sample outlet 33 is an opening formed at one end of the sample separation container 30 and communicates with the separated sample receiving portion 31, so that the saliva sample 1 temporarily stored in the sample collection container 10 moves through it. while passing through the filter 20. The separated sample outlet 33 can be opened and closed by the sample separation container cap 35, and at this time, a thread is formed on the outer circumferential surface of one end of the sample separation container 30 so as to be detachable from the sample separation container cap 35. can be screwed on.

As a method of separating bioparticles from the separation sample 3 in the sample separation container 30, a centrifugation method may be used, and the bioparticles separated thereby may be exosomes. In the process of centrifugation, the filter 20 may be separated from the sample separation vessel 30, sealed by the sample separation vessel cap 35, and provided alone to the centrifuge. However, if stability is ensured, the filter 20 may be installed inside, or provided with the sample collection container 10 attached as will be described later.

In addition, the bioparticles can be accommodated and stored in the sample separation container 30, and in this case, it is preferable to store them at a temperature of -20 to -90 ° C. Similarly, a temperature control means (which may be a water jacket for example) may be mounted. However, each temperature control unit does not necessarily have to be the same.

The sample collection container 10 and the sample separation container 30 may be detachably coupled so that the saliva sample 1 in the sample collection container 10 may be injected into the sample separation container 30 . As the coupling method, a method in which threads are formed at one end of the sample collection container 10 and one end of the sample separation container 30 to be screwed together may be employed. At this time, one end of the sample collection container 10 may be screwed while being inserted into one end of the sample separation container 30 . To this end, a thread may be formed on the outer circumferential surface of one end of the sample collection container 10 and the inner circumferential surface of one end of the sample separation container 30, respectively. Here, the screw on the outer circumferential surface of one end of the sample collection container 10 may be a screw thread that is screwed into the sample collection container cap 15 . In addition, although not shown, one end of the sample separation container 30 may be inserted into one end of the sample collection container 10 in the same manner as above and screwed together.

Meanwhile, the filter 20 is disposed inside the sample separation container 30, and at this time, the filter 20 may be detachably fixedly disposed. As a detachable means, a protruding portion 25 protruding outward is formed on the outer circumferential surface of the body portion 21 of the filter 20, and the protruding portion 25 of the filter 20 is caught and fixed on the inner surface of the sample separation container 30. A locking jaw 37 may be formed. Here, the protruding portion 25 may be formed in a ring shape surrounding the outer circumferential surface of the body portion 21 or in a column shape. Therefore, the filter 20 inserted through the separated sample outlet 33 of the sample separation container 30 is fixed by the protruding portion 25 being caught on the locking jaw 37, and if necessary, the separated sample outlet inlet ( 33) can be removed. However, detachable means of the filter 20 and the sample separation container 30 do not necessarily have to be provided by the protruding portion 25 and the locking jaw 37, and as long as the filter 20 can be detachably attached, other It may be formed by the configuration.

Overall, according to the saliva-derived bioparticle separation device according to the present invention, saliva collection, pretreatment, and separation and storage of bioparticles are integrally performed at the clinical site, so saliva samples are transferred to the laboratory, separated, and stored for a certain period of time And it is possible to prevent a decrease in the recovery rate and loss of characteristics of saliva-derived bioparticles that may occur during the maintenance process.

Hereinafter, the present invention will be described in more detail through experimental examples.

1. Experiment purpose and method

The purpose of this experiment is to confirm the effect of the pore size of the filter according to the present invention on the separation of exosomes, and to derive the optimal pore size of the filter capable of separating exosomes from samples in a stable and high yield.

Total Exosome Isolation Reagent (from cell culture media) - After collecting sample groups under the same pretreatment conditions using Invitrogen products and filtering the sample groups with PTFE (Polytetra fluoro ethylene) Teflon syringe filters with different pore sizes, Nanoparticle tracking analysis (NTA) analysis and western blot analysis were performed on each filtered sample group, and the results were compared.

Specifically, 22 ml of osteoblasts (MG63 cells) cultured for 48 hours were collected, divided into 11 ml each in a 50 ml tube, and centrifuged at 2,000 rpm for 10 minutes. Since cell debris settles after centrifugation, 10 ml of the supernatant was transferred to a new tube, 5 ml of Total Exosome Isolation Reagent (from cell culture media) was added thereto, stirred, and overnight at 4°C. . The overnight sample was centrifuged at 4° C. at 10,000 g for 1 hour. After centrifugation, exosomes were present in the form of pellets on the wall and bottom of the tube. After removing the liquid in the tube, 5 ml of 1X phosphate buffer saline (PBS) was added to resuspend the exosomes in the form of pellets. After addition and mixing, the PBS-mixed sample was divided into tubes by 1 ml each. Except for the control group, each sample was injected into three syringes equipped with filters having different pore sizes and filtered. Here, polytetra fluoro ethylene (PTFE) Teflon filters having pore sizes of 1.0, 0.45, and 0.2 μm were used. Since exosomes have a size of 30 to 200 nm, a filter with a pore size through which all exosomes can pass was selected.

2. NTA assay

NTA (nanoparticle tracking analysis) is a method of measuring the size and concentration of nanoparticles from light scattered by nanoparticles in a solution by receiving a laser beam.

Stokes-Einstein equation

(Dt: diffusion constant, K _b : Boltzmann constant, T: absolute temperature, η: viscosity, d: diameter of spherical particles)

The scattered light that appears as the laser passes through the solution is recorded as an image, and the behavior of the nanoparticles is traced from the image. The temperature (T) is measured separately, and the diffusion constant (Dt) and the Boltzmann constant (Kb) are fixed values, so the viscosity (η) and diameter (d) can be calculated. In general, solvents with little viscosity, such as water and buffers, are used, so if treated as a constant or set separately, the particle size can be measured. At the same time, it is also possible to quantify the concentration through the number of individual particles per frame.

NTA analysis was performed on the samples filtered with the filters having different pore sizes. 5 is a nanoparticle tracking analysis (NTA) analysis result according to an experimental example.

Referring to FIG. 5, when the filtered samples were compared with the control group, the concentration of exosomes decreased as the pore size of the filter decreased, especially in the case of a 0.45 μm pore filter compared to the control group. That amount was reduced by about a third. In addition, the amount of protein tended to decrease as the pore size of the filter decreased.

3. Western blot analysis

Western blotting is a technique for separating proteins by size, and a specific protein can be detected from all proteins using an antigen-antibody reaction.

Western blot analysis was performed on the samples filtered with the filters having different pore sizes. 6 is a Western blot analysis result according to an experimental example.

Referring to FIG. 6 , when the expression levels of CD9, CD63, and CD81 bands, which are specific exosome markers, were graphed, the amount of exosome specific markers decreased as the pore size of the filter decreased. This indicates that, as the pore size of the filter becomes smaller, it is lost due to being caught in the small pores of a specific exosome and damaged.

4. Conclusion

Nano endoplasmic reticulum has substances that exchange information between cells, such as exosomes, microvesicles, and apoptotic bodies. Since materials other than exosomes exist in the control group before filtering, a filter having a specific pore size capable of filtering out materials other than exosomes was selected and this experiment was conducted to leave only information exchange materials including pure exosomes. When filtering through a filter after pretreatment and separation by the precipitation method, considering that the amount of protein decreases as the pore size of the filter decreases, it can be seen that various substances other than exosomes are present in the sample. In addition, even within the pore size range through which exosomes can pass, the smaller the pore size, the more the exosomes become clogged or adhere to the filter and disappear. In conclusion, when the pore size is 1.0 μm, impurities in the sample can be filtered out and exosomes can be isolated in high yield without damage.

Although the present invention has been described in detail through specific examples, this is for explaining the present invention in detail, the present invention is not limited thereto, and within the technical spirit of the present invention, by those skilled in the art It is clear that modifications and improvements are possible.

All simple modifications or changes of the present invention belong to the scope of the present invention, and the specific protection scope of the present invention will be clarified by the appended claims.

1: Saliva sample 3: Separation sample
10: sample collection container 11: collection sample receiving unit
13: collected sample outlet 15: sample collection container cap
20: filter 21: body part
23: pores 25: projections
30: sample separation container 31: separation sample receiving unit
33: separation sample outlet 35: sample separation container cap
37: snag

Claims (17)

Using a filter, including the step of filtering the sample,
The filter is made of one or more polymers selected from the group consisting of polytetrafluoroethylene (PTFE), polycarbonate (PC), and polyester, and polyethersulfone (PES). lose,
The filter has a pore size of 0.8 to 1.2 μm,
The sample is an exosome separation method comprising the exosome obtained by centrifuging after reacting the cell culture medium containing the exosome with an exosome separation reagent.
The method of claim 1,
The sample is saliva exosome separation method.
delete delete delete delete delete delete delete a sample collection container having a collection sample accommodating portion accommodating a sample therein, and a sample collection outlet communicating with the collected sample accommodating portion so that the sample flows in and out at one end;
a filter for filtering the sample discharged through the collected sample outlet; and
A sample separation container having a separated sample accommodating part having the filter disposed therein and accommodating the separated sample passing through the filter, and a separated sample outlet communicating with the separated sample accommodating part at one end so that the separated sample flows in and out; including,
The filter is made of one or more polymers selected from the group consisting of polytetrafluoroethylene (PTFE), polycarbonate (PC), polyester, and polyethersulfone (PES), and ,
The filter has a pore size of 0.8 to 1.2 μm,
The sample is an exosome separation device comprising the exosome obtained by reacting the cell culture medium containing the exosome with an exosome separation reagent and then centrifuging.
The method of claim 10,
One end of the sample collection container and one end of the sample separation container are detachably screwed together.
The method of claim 10,
The filter is an exosome separation device having a hollow tube shape with one end open so that the sample flows in, and a plurality of pores through which the separated sample is discharged on an outer circumferential surface.
The method of claim 10,
The sample is an exosome separation device that is saliva.
delete delete The method of claim 10,
The collection sample accommodating part The exosome separation device further comprising a buffer solution accommodated within and in which the sample is mixed.
delete