KR101721837B1 - Blood pre-treatment chip using centrifugal separation - Google Patents

Blood pre-treatment chip using centrifugal separation Download PDF

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Publication number
KR101721837B1
KR101721837B1 KR1020150060738A KR20150060738A KR101721837B1 KR 101721837 B1 KR101721837 B1 KR 101721837B1 KR 1020150060738 A KR1020150060738 A KR 1020150060738A KR 20150060738 A KR20150060738 A KR 20150060738A KR 101721837 B1 KR101721837 B1 KR 101721837B1
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South Korea
Prior art keywords
blood
valve
centrifugal
chamber
vertical
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KR1020150060738A
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Korean (ko)
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KR20160128802A (en
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김수경
위성량
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김수경
위성량
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler
    • C12M1/264—Devices involving centrifugal, centripetal or rotational forces
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Abstract

The present invention relates to a blood pretreatment chip having convenience and economy, comprising: a blood inlet; A first centrifuge channel connected to the blood inlet; A first vertical valve connected to the first centrifugal channel in the first aspect for discharging the blood component A in the second aspect and a first horizontal valve for discharging the blood components B and C in the third aspect A first chamber; A second chamber connected to the first horizontal valve in the first aspect, a second vertical valve for discharging the blood component in the second side, and a second horizontal valve for discharging the blood component in the third side; And a third chamber connected to the second horizontal valve in the first aspect and having a third vertical valve for discharging the blood component in the second aspect.

Description

[0001] Blood pre-treatment chip using centrifugal separation [0002]

The present invention relates to a pretreatment chip capable of detecting various diseases or early cancer-related biomarkers by easily centrifuging blood in the field for screening of healthy patients such as hospitals and homes or for early screening diagnosis of cancer suspect patients.

A specific protein in the blood is a biomarker that is used as a key indicator to determine various diseases and health conditions. In recent years, a biochip has been developed which can easily and quickly measure a biomarker by injecting a small amount of blood into a strip-shaped chip.

Usually, blood is composed of blood cells and plasma. The blood cells have a volume of about 40% or more such as red blood cells, white blood cells, and platelets. Plasma is composed of water, protein, fat, saccharides and other minerals.

Protein biomarkers are mostly present in trace amounts in plasma. Therefore, in order to obtain a high sensitivity and high reproducibility result by using the biochip, it is preferable that only plasma components from which blood cells have been removed from blood are used for the detection of proteins. To this end, various methods have been proposed for separating plasma from blood on a chip. For example, hemocyte filtration using chip paper, glass fiber, microstructure, etc., and blood cell deflection, separation and sedimentation using centrifugal force, electromagnetic force, gravity, etc. have been mainly used.

However, there is still a shortage of methods that can remove blood cells quickly and efficiently with a simple method with a small amount of undiluted blood.

It is an object of the present invention to provide a pretreatment chip for separating blood components using density difference and centrifugal force of blood components.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the precise form disclosed. It can be understood.

A centrifugal blood pretreatment chip for achieving the above object comprises: a blood inlet; A first centrifuge channel connected to the blood inlet; A first vertical valve connected to the first centrifugal channel in the first aspect for discharging the blood component A in the second aspect and a first horizontal valve for discharging the blood components B and C in the third aspect A first chamber; A second chamber connected to the first horizontal valve in the first aspect, a second vertical valve for discharging the blood component in the second side, and a second horizontal valve for discharging the blood component in the third side; And a third chamber having a third vertical valve connected to the second horizontal valve in a first aspect and discharging a blood component in a second aspect.

In one embodiment, the first to third vertical and horizontal valves may be a one-way valve.

In another embodiment, the valve opening / closing force of the vertical valve and the horizontal valve may be a relationship of 'first vertical valve <first horizontal valve <second vertical valve <second horizontal valve <third vertical valve'.

In another embodiment, the density of the A blood component, B blood component, and C blood component may be a relationship of 'A blood component> B blood component> C blood component'.

In another embodiment, the A blood component is erythrocyte, the B blood component is leukocyte and platelet, and the C blood component may be plasma.

In yet another embodiment, the first vertical valve is connected to a second centrifugal channel, the second vertical valve is connected to a third centrifugal channel, and the third vertical valve is connected to a fourth centrifugal channel .

In yet another embodiment, the apparatus may further include a first foreign matter storage unit for removing foreign matter between the second centrifugation channels.

In yet another embodiment, the apparatus may further include a mixing chamber and a second foreign matter storage unit for obtaining pure plasma between the fourth centrifugal channels.

In another embodiment, the second to fourth centrifugal channels may further include a discharge port at an end thereof.

As described above, the present invention, which is developed to solve the problems of the prior art, can separate blood components by a simple treatment method using density difference and centrifugal force of blood components.

In addition, it is possible to separate blood components precisely by a simple facility configuration, which is convenient and economical.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing an example of a rotation driving device to which a blood pretreatment chip of the present invention is applied.
2 is a view showing an example of a circular substrate for a rotation driving device to which a blood pretreatment chip of the present invention is applied.
3 is an exemplary view showing an embodiment of a blood pretreatment chip according to the present invention.
4 is an exemplary view showing an embodiment of the chamber of the present invention.
FIG. 5 is another exemplary view showing an embodiment of the blood pretreatment chip of the present invention.
6 is a graph showing the RPM of the present invention in terms of time.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms " comprising, "" including, &quot;or" having &quot;, when used in this application, specify features, numbers, steps, operations, elements, But do not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description of the present invention, the well-known functions or constructions are not described in order to simplify the gist of the present invention.

The centrifugal blood pretreatment chip 100 of the present invention can be applied to and driven by the rotation driving device 10 illustrated in FIG. The rotation driving device 10 may be mounted on the circular substrate 20 for the rotation driving device into which the centrifugal blood pretreatment chip 100 is inserted as shown in FIG. The blood pretreatment chip 100 may be formed in a structure that can be attached to and detached from the circular substrate 20 for a rotation drive device. The specific structure and operation principle of the rotation driving device 10 and the circular substrate 20 for the rotation driving device are described in detail in the publication of the inventor's application No. 10-2007-0136837, No. 10-2011-0038235, It is based on -2011-0043366.

Hereinafter, the structure of the blood pretreatment chip 100 of the present invention will be mainly described with reference to FIGS. 3 to 5. FIG. In Fig. 3, the direction of the arrow indicates the direction of movement of the blood by the centrifugal force.

The centrifugal blood pretreatment chip 100 according to the present invention includes a blood inlet 200, first inner four centrifugal channels 300, 301, 301 and 303, and first to third chambers 400, 500, .

The blood inlet 200 is connected to the first centrifuge channel 300. The first centrifugal channel 300 may have various shapes capable of separating the blood due to the difference in density due to centrifugal force and may be formed in a zigzag shape in order to sufficiently secure a section for centrifugal separation as shown in FIG.

The first chamber 400 may have a rectangular shape, as shown in FIG. A first vertical valve 401 connected to the first centrifugal channel 300 on the first side and formed in the direction of centrifugal force indicated by an arrow on the second side of the four sides, A valve 402 may be provided. When the blood is divided into three A, B and C components according to the density difference, the blood component A having the largest density is discharged to the first vertical valve, and the blood components B and C having the density smaller than the blood component A are discharged to the first horizontal And is discharged to the valve.

The principle of separation and discharge by horizontal and vertical valves for each blood component in each chamber is due to the difference between the closing force and the centrifugal driving force of each valve and will be described in detail later. Where A blood component is erythrocyte, B blood component is white blood cell and platelet, and C blood component is plasma. Hereinafter, the terms of red blood cells, white blood cells, platelets, and plasma are used for convenience of explanation.

Platelets and plasma discharged to the first horizontal valve 402 are introduced into the second chamber 500 connected to the first horizontal valve 402 on the first side. Plasma leukocytes and platelets having higher density are introduced into the second vertical valve 501 formed on the second side of the second chamber 500 and the plasma is discharged through the second horizontal valve 502, .

The plasma discharged to the second horizontal valve 502 flows into the third chamber 600 connected to the second horizontal valve 502 on the first side. The introduced plasma is discharged to the third vertical valve 601 connected to the second side of the third chamber 600. Both the discharge and the inflow are effected by the centrifugal force by the driving of the rotation drive device.

The first to third vertical valves and the horizontal valves 401, 402, 501, 502, and 601 may be a one-way valve. A one-way valve is a valve that can flow only in one direction and not in the opposite direction after the circuit. Various types of valves can be used as well as mechanical valves, as well as one-way valves It includes everything that plays a role.

The first to third vertical valves and the horizontal valves 401, 402, 501, 502, and 601 may have different opening and closing forces. The opening / closing force is a force applied to the valve by a blood component having a fluid force, and the valve is opened by the force, so that the blood component can be discharged in one direction.

The valve is a one-way valve that is opened only when a pressure equal to or greater than a certain level of force is applied. Once the valve is closed, it can not be opened any more after the blood is discharged, and can be operated only in a direction closed by capillary force or elastic restoring force.

The opening and closing forces of the first to third vertical valves and the horizontal valves 401, 402, 501, 502, and 601 are determined according to the opening and closing forces of the first vertical valve <the first horizontal valve <the second vertical valve < Valve 'relationship. The operation principle based on this relationship will be described in the following section 'Operation Principle'.

The first vertical valve 401 of the first chamber 400 is connected to the second centrifugal channel 301 and the second vertical valve 501 of the second chamber 500 is connected to the third centrifugal channel 302, And the third vertical valve 601 of the third chamber 600 may be connected to the fourth centrifugal channel 303. That is, each chamber may have a centrifugal channel connected to the vertical valve.

The second to fourth centrifugal channels 301, 302, and 303 may also be zigzag like the first centrifugal channel 300 described above. The second centrifugal channel 301 may be configured to receive red blood cells, The separation channel 302 may serve as a white blood cell and the platelet, and the fourth centrifuge channel 303 may serve as a pathway of plasma.

Between the second centrifugal separation channel 301 through which red blood cells pass, a first foreign matter storage unit 403 for removing various precipitates present in red blood cells may be formed.

A mixing chamber 603 and a second foreign matter storage unit 604 may be formed between the fourth centrifugal separation channel 303 through which the plasma passes. The mixing chamber 603 is a place for reacting with plasma by injecting an antibody to remove fibrinogen, which is fibrin in the pure plasma, to obtain serum, and the fibrinogen removed from the plasma is stored in the second foreign substance storage unit 604 . In order to increase the accuracy of biomarker detection in plasma, albumin may be removed. In this case, an antibody reacting with albumin may be injected into the mixing chamber 603.

As shown in FIGS. 3 and 5, the discharge ports 700 may be formed at the ends of the second through fourth centrifugal channels 301, 302, and 303, respectively. The discharge port 700 is a place where blood components having passed through the second to fourth centrifugal channels 301, 302, and 303 are separated and finally collected.

As shown in FIG. 5, the separated blood collected at the discharge port 700 may be formed with the biomarker detection unit 800. The detection unit 800 may include a synthesis chamber 801 and a detection chamber 802 to perform a process necessary for the biomarker.

Hereinafter, the principle of operation of the blood pretreatment chip of the present invention will be described with reference to FIGS.

The blood is collected and dropped on the blood inlet 200 provided in the blood preprocessing chip 100. The blood pretreatment chip 100 into which the blood has been inserted is inserted into the circular substrate 20 for the rotation drive device and mounted on the rotation drive device 10. [

After the blood preprocessing chip 100 is inserted into the rotation driving device 10, the rotational speed of the rotation driving device 10 is increased to t1. (Fig. 6, section from 0 to t1) At this time, Is passed through the first centrifugal channel (300) by centrifugal force.

When the centrifugal force is applied to the first centrifugal channel 300 for a certain period of time, the blood component having the highest density among the components constituting the blood passes through the first centrifugal channel 300 the fastest, The blood components are arranged in a structure passing relatively slowly. In other words, the red blood cells, white blood cells, platelets, and plasma are mixed in the first centrifugal channel 300, but the red blood cells, white blood cells, and plasma are formed by the centrifugal force in the first centrifugal channel 300, Platelets, and plasma.

The centrifugal force is continuously applied to the first chamber 400 through the first centrifugal separation channel 300 at a constant rate so that the order of the blood flowing in the first chamber 400 is in the order of red blood cells, white blood cells, platelets, and plasma, (Fig. 6, t1 to t2 section).

When the rotation speed of the rotation driving device is increased when the order of the blood is sufficiently set to red blood cells, white blood cells, platelets, and plasma, the centrifugal force of red blood cells, which acts as pressure on the first vertical valve 401 of the first chamber 400, The first vertical valve 401 is opened to allow the red blood cells to flow into the second centrifugal separator 301. As shown in FIG. At this time, the rotation speed is maintained at a constant level (Fig. 6, t3 to t4)

The centrifugal force acting on the first vertical valve 401 is weakened when the red blood cells almost escape to the second centrifugal separation channel 301 and the leukocytes and the platelets escape. As a result, the first vertical valve 401 becomes capillary phenomenon or elastic recovery force It will close again.

The blood trapped in the first chamber 400 becomes blood in which erythrocytes are almost removed, and at this time, the rotation speed of the rotation driving device is increased. (Fig. 6, t4 to t5 section) The first horizontal valve 402 of the first chamber 400 is opened and the rotation speed is kept constant until the blood in the first chamber 400 moves to the second chamber 500 ~ t6 section)

When the rotation speed of the rotation driving device is increased when the order of the blood is sufficiently leucocyte, platelet, and plasma, the centrifugal force of leukocytes and platelets acting on the second vertical valve 501 of the second chamber 500 becomes more than a certain value The second vertical valve is opened (in the period from t6 to t7 in Fig. 6)

The second vertical valve 501 of the second chamber 500 opens and leukocytes and platelets start to flow into the third centrifugal channel 302. The rotational speed at this time is kept constant (Fig. 6, t7 to t8)

The centrifugal force acting on the second vertical valve 501 is weakened when the leukocyte and platelets almost escape to the third centrifugal separation channel 302 and the plasma is going to escape, and the second vertical valve 501 is closed again by the capillary phenomenon do.

In the second chamber 500, white blood cells and platelets are almost completely removed and blood remains in a state in which plasma is mostly present. 6), the second horizontal valve 502 of the second chamber 500 is opened as the rotational speed increases, and the second horizontal valve 502 of the second chamber 500 is opened as the rotational speed increases. The rotation speed is kept constant until all the plasma in the chamber 500 moves to the third chamber 600 (Fig. 6, t9 to t10)

Most of the blood that has moved to the third chamber 600 is plasma, and when the rotation speed of the rotation driving device is increased, the third vertical valve 601 is opened and the plasma is discharged to the fourth centrifugal channel 303. 6, t10 to t11)

The rotation speed of the rotation driving device 10 is maintained so that plasma is completely discharged to the fourth centrifugal channel 303. (Fig. 6, t11 to t12 section) The plasma is completely discharged and the fourth centrifugal channel 303 The rotation driving device 10 is stopped after being discharged to the discharge port 700 (Fig. 6, t12 to t13)

The preprocessing process is performed by the blood preprocessing chip 100 by the above process, and further, the process necessary for the biomarker can be performed in the detecting unit 800.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It is obvious to those who have.

10: rotation drive device 20: circular substrate for rotation drive device
100: blood pretreatment chip 200: blood inlet
300: first centrifuge channel 301: second centrifuge channel
302: third centrifugal channel 403: fourth centrifugal channel
400: first chamber 401: first vertical valve
402: first horizontal valve 403: first foreign matter storage part
500: Second chamber 501: Second vertical valve
502: second horizontal valve 600: third chamber
601: Third vertical valve 603: Mixing chamber
604: second foreign matter storage part 700: discharge port
800: Detector 801: Synthesis chamber
802: Detection chamber

Claims (9)

Blood inlet;
A first centrifuge channel connected to the blood inlet;
A first vertical valve connected to the first centrifugal channel in the first aspect for discharging the blood component A in the second aspect and a first horizontal valve for discharging the blood components B and C in the third aspect A first chamber;
A second chamber connected to the first horizontal valve in the first aspect, a second vertical valve for discharging the blood component in the second side, and a second horizontal valve for discharging the blood component in the third side;
And a third chamber having a third vertical valve connected to the second horizontal valve in a first aspect and discharging a blood component in a second aspect,
The first vertical valve is connected to a second centrifugal channel,
The second vertical valve is connected to the third centrifugal channel,
And the third vertical valve is connected to a fourth centrifugal channel.
The method according to claim 1,
Wherein the first to third vertical valves and the horizontal valves are one-way valves.
The method according to claim 1,
The valve opening / closing forces of the vertical valve and the horizontal valve
The centrifugal blood pretreatment chip according to claim 1, wherein the first vertical valve, the first horizontal valve, the second vertical valve, the second horizontal valve,
The method according to claim 1,
The density of the blood components A, B, and C,
A centrifugal blood pretreatment chip characterized by the relationship of 'A blood component> B blood component> C blood component'
The method according to claim 1,
The blood component A is red blood cells,
The B blood component is leukocyte and platelet,
Wherein the C blood component is plasma. The centrifugal blood pretreatment chip
delete The method according to claim 1,
Further comprising a first foreign matter storage part for removing foreign matter between the second centrifugal separation channels.
The method according to claim 1,
Further comprising a mixing chamber and a second foreign matter storage unit for obtaining pure plasma between the fourth centrifugal channels.
The method according to claim 1,
Wherein the distal end of each of the second to fourth centrifugal channels further comprises a discharge port.
KR1020150060738A 2015-04-29 2015-04-29 Blood pre-treatment chip using centrifugal separation KR101721837B1 (en)

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JP3803078B2 (en) * 2002-09-20 2006-08-02 独立行政法人科学技術振興機構 Hematology analyzer and plasma separation method
KR100647320B1 (en) * 2005-02-05 2006-11-23 삼성전자주식회사 Microsystem for separating serum from blood
KR101922128B1 (en) * 2012-12-04 2019-02-13 삼성전자주식회사 Microfluidic apparatus and method of enriching target in boilogical sample

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