WO2024090849A1

WO2024090849A1 - Cartridge for centrifugal separation device and centrifugal separation device comprising same

Info

Publication number: WO2024090849A1
Application number: PCT/KR2023/015601
Authority: WO
Inventors: 이성훈; 길민석; 배진한
Original assignee: 주식회사 클리노믹스
Priority date: 2022-10-27
Filing date: 2023-10-11
Publication date: 2024-05-02
Also published as: KR20240059058A

Abstract

The present invention relates to a cartridge structure that can centrifuge specific components from body fluids such as a large volume of blood, and can extract and separate a target substance within a single cartridge. The cartridge structure includes a chamber for cell lysis and a magnetic bead reaction chamber for extracting target components, in the cartridge, and at the same time, a target substance recovery chamber is placed within the cartridge to implement sample separation and efficient extraction of a target component in a single centrifugal separation cartridge even without any additional manual work.

Description

Cartridge for centrifugal separator and centrifugal separator including the same

The present invention relates to a cartridge structure that can perform centrifugal separation of specific components from a large volume of body fluid, such as blood, and extract and separate target substances within a single cartridge. This application is the result of the market-friendly global competitiveness product development project [Project identification number: 1711174434 (Project number RS-2020-KD000019)], a project supported by the Pan-Ministry Life Cycle Medical Device Research and Development Project, and is a hospital information system (HIS) ) The application was filed by claiming priority for Korean Patent Application No. 10-2022-0139833, which was filed as a result of the development of a real-time liquid biopsy molecular diagnosis system for diagnosing linked lung cancer.

Molecular diagnosis using nucleic acids has the advantages of high accuracy, reproducibility, and speed, and is a method that has recently become a hot issue in the fields of food hygiene and forensics. However, despite these advantages, it is necessary to have several additional measurement facilities, so recently, many studies are being conducted to implement this in the form of a lab-on-a disk.

Specifically, the rotary circular or polygonal Lab-on-a-Disk technology involves putting whole blood into a single tube container to separate it into each component (plasma, buffy coat, white blood cells, and red blood cells). This is a method that allows efficient separation without cross-contamination of samples compared to separation methods based on specific gravity by inserting them into a centrifugal separation device.

However, further manual work is required for in-depth analysis of components in whole blood using existing rotating circular or polygonal lab-on-a-discs. In particular, cell-derived nucleic acid (cfDNA), which has recently been widely used in cancer diagnosis, exists in the plasma layer, and in order to separate it, it must be separated manually using a filter or magnetic beads.

In order to analyze the genetic information contained in cell-derived nucleic acid (cfDNA), analysis methods such as RT-PCR or NGS must be used, but performing this series of processes requires a lot of time and labor. Additionally, it involves several problems such as sample contamination and crossover during the separation and analysis process.

Accordingly, there is a need for a device that can extract and separate cell-derived nucleic acids present in plasma in the blood within a single cartridge without the need for manual work.

The present invention was devised to solve the above-mentioned problem, and the purpose of the present invention is to extract a chamber for cell lysis and a target component within a cartridge for a centrifugal separator capable of separating and analyzing body fluid (e.g., whole blood) components. A cartridge structure that provides a magnetic bead reaction chamber for the reaction and at the same time places a target material recovery chamber within the cartridge, enabling sample separation and efficient extraction of target components in a single centrifugal separation cartridge without separate manual work. is to provide.

As a means for solving the above-described problem, in an embodiment of the present invention, as shown in FIGS. 1 and 2, a disk-shaped cartridge body 100 having a constant thickness that can be rotated by receiving power from a drive unit; Separated component storage module (A) including a plurality of chambers for storing separated components centrifuged by receiving body fluid through the inlet of the cartridge body (A): disposed inside the cartridge body (A) and storing the separated components An extraction module (B) that lyses the components introduced from the module (A) and then extracts the target components through a magnetic bead reaction; a cleaning fluid module (C) that communicates with a part of the chamber of the extraction module (B) and has at least one cleaning fluid supply chamber for supplying a cleaning fluid; It is possible to provide a cartridge for a centrifugal separation device, including an impurity storage module (D) that selectively communicates with the extraction module (B) through centrifugal force and a valve and stores cleaning liquid and impurities other than the target component.

According to an embodiment of the present invention, a chamber for cell lysis and a magnetic bead reaction chamber for extracting target components are provided in a cartridge for a centrifugal separator capable of separating components of body fluid (e.g., whole blood), and at the same time, target substances. By arranging the recovery chamber within the cartridge, there is an effect of enabling sample separation and efficient extraction of target components in a single centrifugal separation cartridge without any additional manual work.

In particular, by applying the cartridge of the present invention with this structure, even a skilled technician can accurately and efficiently separate target components (e.g., cell-derived nucleic acid, cfDNA) from a sample (e.g., whole blood) in one device without cross-contamination. We can provide you with the convenience to do it.

When whole blood is used as a sample through the cartridge of the present invention, cell-derived nucleic acid (cfDNA) can be extracted quickly without cross-contamination, and such cell-derived nucleic acid contains various genetic information, especially cell-derived nucleic acid derived from cancer cells. (cfDNA) contains genetic information about cancer cells and can be useful in cancer diagnosis.

1 and 2 show a plan cross-sectional conceptual diagram of a cartridge for a centrifugal separator according to an embodiment of the present invention.

Figure 3 shows an image of a triple disk type used in a centrifugal separation device using three of the above-described cartridges of the present invention.

Figures 4 to 7 show the results of testing the application performance of a cartridge for a centrifugal separator according to an embodiment of the present invention.

[Explanation of symbols]

100: Cartridge body A: Separate component storage module

B: Extraction module C: Cleaning liquid module

D: Impurity storage module 110: First chamber part

120: second chamber part 130: third chamber part

140: Fourth chamber part

The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosure will be thorough and complete and so that the spirit of the invention can be sufficiently conveyed to those skilled in the art.

The terms used in this application are only used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Figures 1 and 2 show a plan cross-sectional conceptual diagram of a cartridge for a centrifugal separator (hereinafter referred to as 'the present invention') according to an embodiment of the present invention.

Referring to Figures 1 and 2, the present invention includes a disk-shaped cartridge body 100 having a constant thickness (t) that can be rotated by receiving power from a driving unit, and receiving body fluid through the injection port of the cartridge body (A). A separation component storage module (A) including a plurality of chambers for storing centrifuged components, disposed inside the cartridge body (A), and magnetically lysing the components flowing from the separation component storage module (A). An extraction module (B) that extracts the target component through a bead reaction, a cleaning fluid module (C) having at least one cleaning fluid supply chamber that communicates with a part of the chamber of the extraction module (B) and supplies a cleaning fluid, the extraction module It selectively communicates with (B) through centrifugal force and a valve, and may be configured to include an impurity storage module (D) that stores cleaning liquid and impurities other than the target ingredients.

Specifically, the cartridge body 100 is implemented as a three-dimensional structure with a plurality of chambers inside and passages between each chamber, as shown in Figure 1. Although not shown, the cartridge body 100 A flat cover structure is provided at the top and bottom of the top and bottom to seal the three-dimensional structure from top and bottom. In particular, the cartridge body 100 injects body fluid, which is an object requiring separation and analysis, into the interior, and implements chamber structures such as a chamber for cell lysis, a reaction chamber for separation, and a cleaning fluid supply chamber embedded within the cartridge body. Each chamber structure is implemented as a closed structure, and material movement between them is realized by centrifugal force and the opening and closing operation of the valve.

The cartridge structure of the present invention is applied to a device that performs centrifugation by injecting body fluid, and the various body fluids to be analyzed in the present invention may be whole human blood, sweat, or other samples, but the present invention As a preferred example, human whole blood is used as the target body fluid, the first separated material is plasma, and the target component is cell free DNA (cfDNA).

The cartridge body 100 may be implemented as an external shape having a fan-shaped flat cross-section with a constant thickness, as shown in FIGS. 1 and 2, and in the present invention, the center (P1) of this external shape is separated from the left and right sides. A structure defined as the intersection of line segments extending the outer border of and the angle a formed by the center P1 is 120° will be described as a preferred embodiment.

First, the injection port 1 is formed closest to the center P1 of the present invention, and the chamber parts that perform the separation function for body fluids can be sequentially arranged around the injection port outward.

The injection port 1 is a place where body fluid (whole blood) is injected, and is the part where the body fluid separated by the present invention by performing a centrifugation operation is first injected.

The separated component storage module (A) includes a plurality of chambers that receive body fluid through an injection port and store the separated components centrifuged, and the separated material is stored.

The separate component storage module (A) includes a first chamber portion 110, an adjacent chamber 115, an outer chamber 117, and a micro-channel 11 that communicates them.

Adjacent to the injection port 1, among the components of whole blood separated by centrifugation, plasma, which is the first separation material, is stored in the first chamber part 110, and among the components separated by centrifugation, buffy The buffy coat is moved to the adjacent chamber 115, and the red blood cells are moved to the outer chamber 117 for storage. That is, the main material containing the target ingredient is stored in the first chamber 110, and other ingredients are stored separately in the adjacent chamber 115 and the outer chamber 117. This process is separated by centrifugal force, and each chamber communicates with each other through the microchannel 111.

The extraction module (B) performs the function of lysing the components introduced from the separated component storage module (A) and then extracting the target component through a magnetic bead reaction.

Specifically, the extraction module (B) receives the first isolate by centrifugal force from the first chamber unit 110, which separates and stores the first isolate containing the target ingredient, and collects the first isolate (plasma). ) A second chamber unit 120 that dissolves the inner cells and separates them into a target component and a second separation material. In the second chamber unit, the dissolved target component is introduced, and the magnetic beads and the target component react to form the target component. It may be configured to include a third chamber part 130 that separates and a fourth chamber part 140 that stores the target material flowing from the third chamber part.

The extraction module (B) extends from the center (P1) of the cartridge body 100 to the outside (P2), and includes the first chamber part 110, the second chamber part 120, and the third chamber part 130. ) and the fourth chamber portion 140 are arranged sequentially. That is, in FIG. 2, considering an arbitrary straight line P2 extending from the center P1 to the outside, each chamber portion includes a first chamber portion 110, a second chamber portion 120, and a third chamber portion 130. ) and the fourth chamber part 140 are arranged in that order, and this arrangement is to ensure smooth transfer of the separated material according to centrifugal force.

That is, the movement of materials between the first chamber part 110, the second chamber part 120, the third chamber part 130, and the fourth chamber part 140 in communication with each other occurs in the flow path according to the opening and closing of the valve. It opens and moves selectively, and is implemented by centrifugal force resulting from the rotation of the cartridge body (A).

To explain the operation of the configuration of these chamber parts, first, the second chamber 120 disposed outside the first chamber 110 is selectively communicated with the first chamber 110 by the valve 14, During the centrifugation process, the plasma can be moved to the second chamber 120 through the fine connection passage 121 by controlling the opening and closing of the valve 14 and centrifugal force.

The second chamber 120 performs the function of lysing cells in plasma by receiving lysing substances necessary for lysing the injected cells. The dissolved material injected into the second chamber 120 may be, for example, a Protease K solution. The lysed material and the plasma reflect each other to perform cell lysis. For example, to completely achieve cell lysis, centrifugation at 180 degrees can be performed in a centrifugal separator.

Thereafter, the third chamber 130 is disposed adjacent to the outside of the second chamber 120. When the plasma dissolved in the second chamber 120 moves to the third chamber 130 by the opening and closing operation of the valve 15 and centrifugal force, the magnetic bead provided in the third chamber 130 Cell-derived nucleic acid (cf DNA) reacts through a reaction with beads. That is, in the third chamber, magnetic beads and cell-derived nucleic acid (cfDNA) in dissolved plasma are combined. For example, in order to efficiently implement this binding, it is desirable to perform incomplete centrifugation at 180 degrees in a centrifugal separation device to increase the binding reaction.

In this case, the present invention allows a washing process to be performed to remove impurities present in the plasma present in the third chamber.

The cleaning fluid module (C) for performing this cleaning process includes at least one cleaning fluid chamber disposed on one side of the extraction module (B), and the cleaning fluid chamber 160 includes the third chamber portion 130 and The cleaning solution is supplied through communication to remove impurities in the first separating component.

To this end, the cleaning fluid module (C) may be configured to further include at least one cleaning fluid supply chamber 160 that communicates with the third chamber portion 130 and supplies cleaning fluid.

That is, as shown in FIG. 2, the cleaning liquid supply chamber 160 of the present invention is used to remove impurities present in plasma before and after the reaction of plasma flowing into the magnetic beads performed in the third chamber 130. Provides a supply of cleaning liquid to perform cleaning. As shown in Figure 2, the cleaning fluid supply chamber 160 is provided with a total of four cleaning fluid chambers (161, 162, 163, 164), and can supply a total of four cleaning fluids to perform four cleaning processes. Let it happen. At this time, the necessary cleaning solution is injected and filled in advance before centrifugation, and when centrifugation begins, the cleaning solution is sequentially supplied to the third chamber 130 by centrifugal force, the opening and closing operation of the

valves

16, 17, 18, and 19, and centrifugal force. This movement removes impurities. The removed impurities are moved to the impurity storage chamber (D) by the opening and closing operation of the valve 23 and centrifugal force.

Thereafter, the cell-derived nucleic acid (cfDNA) bound to the magnetic beads in the third chamber 130 is combined with the extraction buffer solution and dissolved. This extraction buffer solution is injected through the extraction buffer solution inlet 12, and is injected into the third chamber 130 by the opening and closing operation of the valve 21 and centrifugal force, and the cells combined with the magnetic beads are injected. The derived nucleic acid is dissolved.

Cell-derived nucleic acid (cfDNA) dissolved in the elution buffer flows into the fourth chamber 140 disposed outside the third chamber 130 by centrifugal force and the opening and closing operation of the valve 29.

In this case, in order to prevent a small amount of cell-derived nucleic acid (cfDNA) solution from remaining in the third chamber 130, the bottom surface of the third chamber 130 of the present invention is disposed adjacent to the fourth chamber portion. It is made to have a first inclination (θ1) inclined toward the outside where (140) is located. In this case, the first inclination θ3 is preferably in the range of 3° to 5° based on the horizontal plane of the floor. If the angle exceeds the above-mentioned range, the force with which the target material flows along the slope increases due to centrifugal force, making it difficult to achieve stability, and if the slope is less than 3°, residual target ingredients remain, making it difficult to achieve the desired effect. do.

Cell-derived nucleic acid (cfDNA) collected in the fourth chamber 140 can be recovered using pellets.

Figure 3 shows an actual use image of a triple disk type in which three of the above-described cartridges of the present invention are applied to a centrifugal separation device. As in this application example, when the cartridge of the present invention with this structure is applied, even a skilled technician can accurately and efficiently remove target components (e.g., cell-derived nucleic acid, cfDNA) from a sample (e.g., whole blood) without cross-contamination. It can be separated from one piece of equipment, and when implemented as a triple type, work efficiency can be improved by more than 3 times.

[Reference experiment example]

Figures 4 to 7 show the extraction concentration of intracellular nucleic acid (cfDNA) from 120 lung cancer patients at Asan Medical Center in Seoul, where clinical blood was collected from the patients and whole blood samples from randomly selected patients were collected. This is compared to the one released using the existing manual method.

The concentrations of intracellular nucleic acid (cfDNA) separated using the cartridge according to the embodiment of the present invention were measured on average at 596 pg/ul and 691 pg/ul, respectively, and the concentration of intracellular nucleic acid (cfDNA) using the existing manual method was It was measured at 439pg/ul.

- The above results confirmed that the cartridge developed by the present invention had better purification efficiency for intracellular nucleic acid (cfDNA).

As described above, the technical idea of the present invention has been described in detail in the preferred embodiments, but the preferred embodiments described above are for illustration and not limitation. As such, a person skilled in the art will understand that various embodiments are possible through combination of embodiments of the present invention within the scope of the technical idea of the present invention.

Claims

A disk-shaped cartridge body 100 having a constant thickness that can be rotated by receiving power from a driving unit;

Separated component storage module (A) comprising a plurality of chambers for storing separated components centrifuged by receiving body fluid through the inlet of the cartridge body (A):

An extraction module (B) disposed inside the cartridge body (A), which lyses the components introduced from the separated component storage module (A) and then extracts the target component through a magnetic bead reaction;

a cleaning fluid module (C) that communicates with a part of the chamber of the extraction module (B) and has at least one cleaning fluid supply chamber for supplying a cleaning fluid;

An impurity storage module (D) that selectively communicates with the extraction module (B) through centrifugal force and a valve and stores cleaning liquid and impurities other than the target ingredients.

Cartridges for centrifuge devices.
In claim 1,

The extraction module (B) is,

In the first chamber unit 110, which separates and stores the first isolate containing the target ingredient, the first isolate is introduced by centrifugal force, and the cells in the first isolate are dissolved to form the target ingredient and the second isolate. A second chamber part 120 separated into;

A third chamber unit 130 that receives the dissolved target components from the second chamber unit and separates the target components by causing the magnetic beads to react with the target components;

Including a fourth chamber unit 140 that stores the target material flowing from the third chamber unit.

Cartridges for centrifuge devices.
In claim 2,

The extraction module (B) is,

From the center (P1) of the cartridge body 100 to the outer direction (P2), the first chamber portion 110, the second chamber portion 120, the third chamber portion 130, and the fourth chamber portion 140. ) are placed sequentially,

Material movement between chamber parts communicating with each other is selectively moved by opening the flow path according to the opening and closing of the valve, and is implemented by centrifugal force due to the rotation of the cartridge body (A),

Cartridges for centrifuge devices.
In claim 2,

The third chamber portion 130,

The bottom surface is formed to have a first inclination (θ1) inclined in the external direction (P3) where the fourth chamber portion 140 is located,

Cartridges for centrifuge devices.
In claim 4,

The first slope (θ1) is,

The bottom surface of the third chamber part 130 has an inclination angle in the range of 3 degrees to 5 degrees relative to the horizontal plane of the lower bottom surface of the cartridge body 100 in the direction of the fourth chamber part 140,

Cartridges for centrifuge devices.
In claim 4,

The cleaning fluid module (C) is,

It has at least one cleaning liquid supply chamber 160 disposed on one side of the extraction module (B),

The cleaning liquid supply chamber 160 communicates with the third chamber part 130 to supply cleaning liquid to remove impurities in the first separation component.

Cartridges for centrifuge devices.
In claim 6,

The body fluid is whole blood, and the target ingredient is cell-derived nucleic acid (cell free DNA; cfDNA).

Cartridges for centrifuge devices.
A centrifugal separator using at least one cartridge for a centrifugal separator according to claim 1.