Patent Application
20220168672
This disclosure relates to a multi-layered filter for separating blood components and a disease diagnosis kit using the same, and more specifically, to a multi-layered filter for separating blood components, which includes a first filter unit for separating blood cells from a whole blood sample, a second filter unit for separating cell fragments having blood platelet and exosome, and a third filter unit for separating proteins, wherein the whole blood sample is separated into blood components through each filter unit provided to have various pore sizes, and after filtering, the multi-layered filter may be disassembled such that each component remaining in the filters is collected and used for diagnosis, and a disease diagnosis kit using the same.
Blood includes cells, such as white blood cells and red blood cells, small particles derived from cells such as blood platelets and exosomes, and trace molecules including plasma proteins, lipids, nucleic acid fragments, and metabolites.
Health status and presence of diseases may be diagnosed by measuring individual components of blood, so blood is used as a useful biological sample for diagnosis.
Since it is difficult to use blood directly in the measurement method used for diagnosis, a technique for measuring purely separated individual components is applied after a treatment procedure is performed to separate blood components.
However, the methods currently used to separate individual components from blood require complex procedures, and various instruments and reagents are required to perform each procedure, so there is a disadvantage in that the methods can be performed only in well-equipped laboratories.
Patent Literature 1 discloses a plasma separation chip having a radial filter structure, which includes an upper substrate and a lower substrate and has a blood inlet and a plasma outlet, wherein one of the substrates has a plasma transfer channel for moving plasma using capillary force and includes a first filter unit for filtering leukocytes and a second filter unit for filtering red blood cells in order along the movement path of plasma, and it reports that the plasma separation chip rapidly and efficiently removes plasma from blood without a separate driving means. However, this disclosure is limited to separating only plasma from blood.
In addition, Patent Literature 2 relates to a leukocyte removal filter device and a leukocyte removal method for removing microaggregates or leukocytes that cause side effects of transfusion from whole blood preparations for transfusion, red blood cell preparations, blood platelet preparations, plasma preparations, and the like, and it proposes a leukocyte removal filter device and a leukocyte removal method that exhibit excellent filtration performance under both use conditions of room temperature filtration and refrigerated filtration. However, this disclosure is also directed to a method for separating a specific component from blood and is limited to laboratory practice, with poor field applicability.
Accordingly, inventors of this application have made efforts to simplify conventional complex procedures such as the use of various devices and reagents for separating individual components of blood, and, by using the point that individual components of blood have different sizes and physicochemical properties, the inventors have found that individual components of blood may be efficiently separated without a separate machine by providing a multi-layered filter so that the whole blood sample is separated into blood components through various filter units with various pore sizes, and after filtering, the multi-layered filter may be disassembled such that each component remaining in the filters is collected and used for diagnosis, thereby having completed the present disclosure.
This disclosure is directed to providing a multi-layered filter that may separate blood components from whole blood.
This disclosure is also directed to providing a disease diagnosis kit using the multi-layered filter.
In one general aspect, the present disclosure provides a multi-layered filter, comprising: a first filter unit configured to separate blood cells from a whole blood sample; a second filter unit configured to separate cell fragments having blood platelet or exosome; and a third filter unit configured to separate protein, wherein the whole blood sample is separated into blood components by passing through the filter units provided to have different pore sizes.
In the multi-layered filter according to the present disclosure, the third filter unit may further include an additional filter unit according to a size of the protein.
The multi-layered filter according to the present disclosure may further comprise a fourth filter unit configured to separate small protein or nucleic acid components that have passed through the third filter unit.
In addition, in the multi-layered filter according to the present disclosure, the filter units may be combined in order, and after filtering, the combined filter units may be disassembled for diagnosis of each component.
In addition, the multi-layered filter according to the present disclosure may be configured such that the filter units are combined very close to each other and are spaced apart from each other with a space therebetween.
In the multi-layered filter according to the present disclosure, the whole blood sample may be administrated in an amount of 20-100 μL to allow separation of blood components immediately at a medical spot.
Further, the present disclosure may provide a disease diagnosis kit using the multi-layered filter, and in an embodiment of the present disclosure, there is provided a cerebral stroke diagnosis kit capable of measuring blood P-selectin.
The multi-layered filter for separating blood components according to the present disclosure may conveniently extract and separate individual components of blood on the spot without the help of special equipment and use the individual components for diagnosis.
In addition, even though various instruments and reagents are generally used to separate individual components of blood, in the present disclosure, it is possible to effectively separate individual components of blood by using a multi-layered filter without a separate machine.
The multi-layered filter for separating blood components according to the present disclosure simplifies the procedure for separating individual components from blood, so blood tests may be performed even in medical sites that are not equipped with many equipment or facilities, thereby securing field applicability.
Further, in the multi-layered filter of the present disclosure, the filter units are combined in order, and after filtering, the combined filter units are disassembled to diagnose each component, so it may be applied as a disease diagnosis kit.
Hereinafter, the present disclosure will be described in detail.
In the present disclosure, by using the fact that all components contained in whole blood have different sizes and physicochemical properties, a multi-layered filter according to the present disclosure is configured by combining a plurality of filter units with different pore sizes and materials suitable for each component to be separated and obtained, so that different components may remain in or pass through each filter unit while passing through the filter units using capillary action.
Specifically, the present disclosure provides a multi-layered filter, which includes a first filter unit for separating blood cells from a whole blood sample, a second filter unit for separating cell fragments having blood platelets and exosomes, and a third filter unit for separating proteins, wherein the whole blood sample is separated into blood components by passing through the filter units with different pore sizes.
In addition, in the multi-layered filter, the third filter unit may further include an additional filter unit according to a size of the protein to be separated.
In addition, small protein or nucleic acid component passing through the third filter unit may be separated using a separate filter unit (a fourth filter unit).
In addition, in the multi-layered filter of the present disclosure, the filter units may be combined in order, and after filtering, the multi-layered filter may be disassembled so that each component remaining in each filter unit may be used as a diagnostic or experimental material.
1. First Filter Unit (Filter1)
The first filter unit (Filter1) is used for separating blood cells from the administered whole blood sample, and it must have a pore size and structure that does not allow blood cells to pass therethrough and simultaneously must be made of a material that is not adsorbed to other components such that other components may easily pass therethrough. Specifically, the material may be selected from the group of natural fibers, and it is preferable as long as the material allows the filter to have a large pore size.
In addition, in order to separate blood cells without damage, the filter must satisfy standard requirements, preferably pore size of 5-15 μm, thickness of 500-1500 μm, weight of 100-400 gram/m2, micron rating of 2-5 μm, and hole-up-volume of 20-100 μL.
The most preferred filter standards employed in the embodiment of the present disclosure are pore size of 10 μm, thickness of 1000 μm, weight of 250 gram/m2, micron rating of 3 μm, and hole-up-volume of 50 μL.
In the multi-layered filter of the present disclosure, blood cells remain in the first filter unit, and the blood cells separated through the first filter unit and remaining therein may be used as an experimental sample.
2. Second Filter Unit (Filter2)
The second filter unit (Filter2) is used for separating particles other than the blood cells according to their size.
That is, the second filter unit used for separating fragments smaller than cells, such as blood platelet, cell fragments and exosomes, must have a pore size and structure that does not allow the cell fragments to pass, and at the same time, the second filter unit must be made of a material that allows other components such as plasma protein to pass through the filter easily and is not adsorbed to the filter.
Preferred filter standards are pore size of 0.05-1 μm, thickness of 100-1000 μm, weight 100-300 gram/m2, micron rating of 1-5 μm, and hole-up-volume of 2-10 μL, and more preferably, it satisfies the filter standards of pore size of 0.1 μm, thickness of 300 μm, weight of 200 gram/m2, micron rating of 2 μm, and hole-up-volume of 5 μL.
Through the second filter unit (Filter2), the cell fragments remain, and the blood platelet, cell fragments, exosomes, etc. separated through the second filter unit (Filter2) may be used as experimental samples.
3. Third Filter Unit (Filter3)
The third filter unit (Filter3) is a filter used for separating proteins and must have a pore size and structure that does not allow protein to pass, and at the same time use a material with very low affinity and adsorption for nucleic acids or proteins.
In addition to the second filter unit (Filter2) and the third filter unit (Filter3) presented in
Therefore, by using two or more filters, the third filter unit (Filter3) may allow various particle components of various sizes such as proteins present in blood to remain in each filter unit so that the components are separated into each other.
The protein components remain through the third filter unit (Filter3), and the remaining protein separated through the third filter unit (Filter3) may be used as an experimental sample.
4. Fourth Filter Unit (Filter4)
If it is necessary to separate only nucleic acid fragments from the liquid sample that has passed through the third filter unit (Filter3), a fourth filter unit (Filter4) may be added.
The fourth filter unit (Filter4) may use a material to which nucleic acids are easily adsorbed. Nucleic acid remains in the fourth filter unit (Filter4), and the remaining nucleic acid components may be used as experimental samples.
In addition, in the liquid sample that has passed through the third filter unit (Filter3), very small-sized particles such as amino acids, bases and various metabolites or components dissolved in liquids exist, so they may be used for diagnostic tests.
At this time, the sample may be used directly as it is in the liquid state that has passed through the third filter unit (Filter3), or may be used by remaining in the fourth filter unit (Filter4) for the purpose of preservation or transportation. If necessary, the sample may be used by adding other sample preservation methods such as drying and freezing in a state of remaining in the fourth filter unit (Filter4).
A preferred material of the fourth filter unit (Filter4) is a silica material to help adsorption of nucleic acids, and a commercially available filter material made of silica for separating nucleic acids may be used.
The multi-layered filter of the present disclosure as described above is designed such that the filter units are combined very close to each other and are spaced apart from each other to form a space between the filter units.
Meanwhile,
From the above, in the structure of the multi-layered filter of the present disclosure, the filter units should be as closer to each other as possible, but there should be no physical interference, such as external pressure, that damages the basic structure of the filters.
In addition, in using the multi-layered filter of the present disclosure, damage to blood cells should be minimized when the whole blood is injected into the multi-layered filter.
At this time, damage to blood cells causes contamination of plasma components by cell components, leading to incorrect analysis results and hindering the separation of components of the whole blood.
20-100 μL of whole blood sample may be injected into the multi-layered filter of the present disclosure to separate blood components, so it is possible to collect blood from a finger or the like using a blood glucose meter and immediately use the blood at home as well as in a hospital or laboratory.
Therefore, the multi-layered filter of the present disclosure may simply extract and separate individual components without the help of special equipment so that the individual components are used for diagnosis.
Furthermore, the present disclosure may provide a disease diagnosis kit using the multi-layered filter described above.
As a preferred example, the present disclosure provides a cerebral stroke diagnosis kit capable of measuring blood P-selectin using the multi-layered filter described above.
P-selectin is a protein expressed on the surface of activated endothelial cells and is activated in blood platelets to express its functions. Under normal circumstances, the P-selectin is located on the surface of endothelial cells and blood platelet, but when exposed to diseases such as stroke, it is converted to sP-selectin (soluble platelet selectin).
That is, sP-selectin changed to a soluble form by disease is leaked into the plasma. Therefore, measuring sP-selectin leaked into plasma may be utilized for disease diagnosis.
If the whole blood passes through the multi-layered filter of the present disclosure, blood cells are separated in the first filter unit (Filter1) and the blood platelets are separated in the second filter unit (Filter2-1), so it is possible to check P-selectin in an inactive state.
In the second filter unit (Filter2-2), which has a smaller pore size than the second filter unit (Filter2-1), proteins in plasma may be separated. Here, if proteins are separated from the blood of stroke patients, activated forms such as sP-selectin may be found.
In another case, P-selectin is sometimes elevated due to an inflammatory response, but it may also be elevated in relation to stroke. In order to distinguish two cases from each other, the gene and protein expression levels may be checked together to make a clearer diagnosis. That is, in case of inflammation, the gene expression of inflammation-related protein increases together with P-selectin, but in case of stroke, only P-selectin protein increases.
Hereinafter, the present disclosure will be described in more detail through examples. The following examples are intended to describe the present disclosure in more detail, and the scope of the present disclosure is not limited to these examples.
A multi-layered filter was prepared by stacking the fourth filter unit, the third filter unit, the second filter unit and the first filter unit on an acrylic plate in order. The filter units were combined very close to each other, but they were fixed with a little space being formed therebetween.
The first filter unit was prepared with filter standards of pore size of 10 μm, thickness 1000 of μm, weight of 250 gram/m2, micron rating of 3 μm, and hole-up-volume of 50 μL, and was made of natural fiber to separate blood cells without damage. In addition, the second filter unit was prepared with filter standards of pore size of 0.1 μm, thickness of 300 μm, weight of 200 gram/m2, micron rating of 2 μm, and hole-up-volume of 5 μL. Each filter unit is round with diameter of 13 mm, and 100 μL of whole blood was absorbed onto the first filter unit, and after 2 minutes, it was checked whether the blood passed by using an end of the capillary or tip, and the filter units were disassembled to check front and rear sides thereof.
As shown in
A multi-layered filter was prepared in the same manner as in Example 1, except that the amount of whole blood used in Example 1 was changed to 20 μL. At this time, according to the change of blood volume, the size of all filters used in the multi-layered filter was reduced to 4 mm.
The administration amount of the whole blood was determined by the amount obtained when an individual collects blood from a finger or the like with a collection needle using a blood glucose meter at home, rather than using a syringe at a hospital to collect blood from blood vessels.
As a result, as shown in
The first filter unit (Filter1) prepared in a circular shape with a diameter of 13 mm was inserted into a 1 mL syringe, the third filter unit (Filter3) was coupled thereto in the form of a syringe filter, the fourth filter unit (Filter4) was connected to a lower position thereof, and 100 μL of whole blood was injected therein so that the whole blood is separated by passing the filter while applying pressure using the piston of the syringe.
As a result, as shown in
A multi-layered filter was prepared by coupling two acrylic plates and two O-rings and installing a multi-layered filter between the O-rings to be very close to each other. The multi-layered filter was designed so that liquid moves by capillary force even in the absence of pressure. In the multi-layered filter, the first filter unit (Filled), the third filter unit (Filter3) and the fourth filter unit (Filter4) manufactured in a circular shape with a diameter of 13 mm were stacked in order, and at this time, 100 μL of whole blood was moved by capillary force through the sample injection hole. After 2 minutes passed from injection of 100 μL of blood, the multi-layered filter was disassembled to check the status of each filter unit.
As a result, as shown in
In the case where the filter itself has the same capacity in the multi-layered filter prepared according to the Comparative Example 2, the blood separation experiment of the multi-layered filter was performed by adjusting the spatial volume generated by the O-ring and the administered whole blood volume.
Specifically, the multi-layered filter was manufactured in a circular shape with a diameter of 13 mm, the inner diameter of the O-ring was fixed to 10 mm smaller than that, and the thickness was changed to 1.5 mm, 1 mm and 0.6 mm. At this time, the administration volume of whole blood was 20 μL, which is the amount of blood when blood was collected from a finger or the like with a collection needle. After 2 minutes from the administration, the filter units were disassembled to check front and back sides thereof.
As the results are shown in
Therefore, it is expected that the blood is absorbed into the junction portion of the filter and the O-ring by the pressure of a region where the O-rings positioned at the front and rear sides of the acryl plate are engaged with each other, so that the blood cells cannot be separated and but pass therethrough as they are. Accordingly, the result caused by the structural deformation of the filter unit due to the pressure between the filter unit and the O-ring was checked.
In the experimental results using the multi-layered filter prepared according to the Example 2, it was found that blood cells were reliably separated in the first filter unit (Filled).
In the next step, in the liquid sample remaining in the fourth filter unit (Filter4), in order to determine whether plasma proteins are present, plasma proteins were checked by western blot or dot blot method. After the antigen-antibody reaction was completed, a color change was induced using 3,3′,5,5′-tetramethylbenzidine (TMB) dye, which developed blue, and observed with the naked eye.
An antibody capable of detecting IgM, which is the largest type of antibody among plasma proteins, was used to react with the blood components remaining in the fourth filter unit (Filter4) and was checked by blot analysis. To test the filter performance, three samples were used to be compared, and there were used {circle around (1)} serum separated from blood by centrifugation, {circle around (2)} components that passed directly through the first filter unit (Filter1), and {circle around (3)} components that passed the first filter unit (Filter1) and the third filter unit (Filter3) and separated in the fourth filter unit (Filter4).
As a result, as shown in
In the experimental results using the multi-layered filter prepared according to the Example 2, an experiment was performed to check the change of proteins capable of passing according to the pore size of the third filter unit (Filter3) when blood passed through the multi-layered filter.
The pore size of the third filter unit (Filter3) used for the experiment was selected as 003 μm, 0.005 μm, 0.01 μm and 0.03 μm. In order to identify proteins remaining in the fourth filter unit (Filter4) and each protein not passing therethrough after passing through the third filter unit (Filter3), electrophoresis on SDS-Page gel was used, and Coomassie blue staining method was used.
An electrophoresis experiment was performed to compare and check whether proteins in the blood passed through the third filter unit (Filter3) or did not pass therethrough but remained.
No. 1: Protein that passed only the first filter unit (Filter1)
No. 2: Protein remaining after not passing through the third filter unit (Filter3) of 0.003 μm
No. 3: Protein that passed through the third filter unit (Filter3) of 0.003 μm and remained in the fourth filter unit (Filter4)
No. 4: Protein that did not pass through the third filter unit (Filter3) of 0.005 μm and remained
No. 5: The protein that passed through the third filter unit (Filter3) of 0.005 μm and remained in the fourth filter unit (Filter4)
No. 6: Protein that did not pass through the third filter unit (Filter3) of 0.01 μm and remained
No. 7: Protein that passed through the third filter unit (Filter3) of 0.01 μm and remained in the fourth filter unit (Filter4)
No. 8: Protein that did not pass through the third filter unit (Filter3) of 0.03 μm and remained
No. 9: Protein that passed through the third filter unit (Filter3) of 0.03 μm and remained in the fourth filter unit (Filter4)
As a result, as shown in
Meanwhile, if the pore size of the third filter unit (Filter3) was 0.03 μm, it was found that a part of the proteins passed therethrough to the fourth filter unit (Filter4). Therefore, in the case of using the third filter unit (Filter3) for separation, it is possible to control the separation range according to the size by adjusting the pore size to around 0.03 μm.
From the experimental results using the multi-layered filter prepared according to the Example 2, it was found that the first filter unit reliably separated blood cells. Then, in order to check whether the nucleic acid passed through the first filter unit (Filter1) and the third filter unit (Filter3) and remained in the fourth filter unit (Filter4), DNA was extracted from the nucleic acid remaining in the fourth filter unit (Filter4) and PCR was performed with human GAPDH primer to confirm the expression of the gene.
In
As a result, in the fourth filter unit (Filter4), when PCR was checked with the human GAPDH primer, it was found that the nucleic acid passed through and separated by checking the band. Also, in the second filter unit (Filter2) and third filter unit (Filter3), since the band was out checked, it was found that all nucleic acids passed therethrough and did not remain.
In the above, the present disclosure has been described in detail only with respect to the embodiments, but it is obvious to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present disclosure, and it is natural that such changes and modifications belong to the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0148466 | Nov 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/013957 | 10/14/2020 | WO | 00 |
