KR20160133837A - Apparatus for separating fine endoplasmic reticulum by electrophoresis sample pH adjustment - Google Patents

Apparatus for separating fine endoplasmic reticulum by electrophoresis sample pH adjustment Download PDF

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KR20160133837A
KR20160133837A KR1020150066882A KR20150066882A KR20160133837A KR 20160133837 A KR20160133837 A KR 20160133837A KR 1020150066882 A KR1020150066882 A KR 1020150066882A KR 20150066882 A KR20150066882 A KR 20150066882A KR 20160133837 A KR20160133837 A KR 20160133837A
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solution
biological sample
sample
flow channel
separated
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KR1020150066882A
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Korean (ko)
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천홍구
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고려대학교 산학협력단
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept

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  • Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Fluid Mechanics (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The present invention relates to a microfibrillar separator, and more particularly, to a microfibrillar separator for continuously separating a sample by adjusting the pH of a biological sample and applying electrophoresis to the sample.
To this end, the present invention relates to a flow channel unit in which a biological sample mixed with an acidic solution or an alkaline solution and a buffer are separated and flow side by side along one direction; A first path part connected to one end of the flow channel part to inject the buffer; A second path part connected to one end of the flow channel part to inject the acidic solution or the alkaline solution and the biological sample; An electric field forming unit forming an electric field in a direction orthogonal to the one direction of the flow channel unit such that the biological sample flowing along the flow channel unit is separated according to electrophoretic fluidity; A third path part connected to the other end of the flow channel part so as to discharge the separated first material in accordance with the electrophoretic fluidity of the electric field forming part; And a neutralizing solution for neutralizing the acidic solution or the alkaline solution injected from the second path portion, wherein the neutralizing solution for neutralizing the acidic solution or the alkaline solution injected from the second path portion A fourth path portion to be injected; .

Description

[0001] The present invention relates to an apparatus for separating a pH-adjusted sample by an electrophoresis method. [0002] Apparatus for separating fine endoplasmic reticulum by electrophoresis [

[0001] The present invention relates to a microfibrillar separator, and more particularly, to a microfibrillar separator which comprises a biological sample to which an acidic solution or an alkaline solution is mixed to make a positive charge or a negative charge and electrophoresis is applied to the sample, To an apparatus for separating a microfibrillar body by an electrophoresis method.

In recent years, interest and research on biotechnology have been actively pursued. However, existing bioanalytical systems are difficult to rapidly process rapidly growing bio information. Therefore, the biological detection system for the identification of life phenomena and drug development and diagnosis is based on microfluidics, and a micro-comprehensive analysis system (μ-TAS : micro-Total Analysis System) and lab-on-a-chip. Most of the biochemical samples to be analyzed are present in solution, so the technique of delivering liquid samples is the most important factor. Microfluidics is a research field for controlling the flow of such microfluidics, and is a field for research and development of core technologies that are based on commercialization of the microcomputer analysis system and lab-on-a-chip.

The microcomputer analysis system is a system that comprehensively implements chemical and biological experiments and analyzes, which are subjected to a plurality of experimental steps and reactions, on a single unit existing on one laboratory. Such a micro total analysis system is composed of a sampling region, a microfluidic circuit, a detector, and a controller for controlling them.

Also, the lab-on-a-chip means that the concept and function of the micro-comprehensive analysis system are implemented on a single chip in the meaning of a 'laboratory on a chip'. Therefore, in order to develop the lab-on-a-chip, a circuit is formed with microchannels through which a solution can flow on the surface of plastic, glass, or silicon, and then pretreatment, separation, dilution, mixing, biochemical reaction, Chip and integrated on a chip of a semiconductor device.

On the other hand, in vivo micro-vesicles (micro vesicles) are small vesicles of membrane structure that are present in various kinds of cells or secreted from cells. Microvesicles secreted out of the cell are (1) exosomes: membrane vesicles of 30-100 nm in diameter originated from the origin of the bacteria, (2) shedding microvesicles (SMVs): flowing directly from the plasma membrane (3) Apoptotic blebs: vesicles having a diameter of 50 to 5000 nm, which are discharged by dying cells.

The in vivo micro-vesicles (microvesicles), such as exosomes, are vesicles of the size of a few tens of nanometers secreted from the cells, and are produced in the cytoplasm or cells inside the lipid bilayer or lipid monolayer It is a structure containing protein and RNA. Exosomes are a means of intercellular communication through the exchange of proteins and RNA. In addition, exosomes are also responsible for the release of unnecessary substances in the cells, and they contain microRNAs (microRNAs and miRNAs), which can be used as useful markers in molecular diagnostics such as early diagnosis of diseases such as cancer. Although the importance and the value of the in vivo micro-endoplasmic reticulum as described above are revealed, it is difficult to obtain the micro-endoplasmic reticulum.

The method of isolating the existing microbejicle is a method of immune-capturing and isolating the microbequicle by combining the microbezyme and the antibody. Such a method may cause a bias depending on the separation or detection target due to masking of antibody recognition sites due to changes in the protein structure, microbial heterogeneity, protein interaction, and the like. Complex processes or expensive equipment may be required for separation or detection, and sample consumption may be high. Therefore, it is necessary to efficiently separate microbeads from a small amount of sample, independent of the target.

In addition, in order to separate microbicules or exosomes from each other, generally, the centrifugal separation method was widely used. A solution of Ficoll solution or OptiPrep (Nycomed Pharma, Norway) or the like was added to the cell or tissue sample solution and centrifuged to obtain microbicule. However, this method not only requires pretreatment of the cell or tissue sample solution, but also requires a large volume of sample. In addition, this method requires several centrifugation steps and requires a special reagent and device for centrifugation, which is time consuming and expensive. As a result, the pellet containing the microbeques obtained through the centrifugation contains a lot of impurities such as fine protein molecules and cell debris similar in density and mass to the microbequicle. In addition, since the impurities do not differ greatly in density from the microvacles, they are not easily separated and thus are not applicable to on-site diagnostics that require immediate response. In addition, microvessels to be obtained may be damaged due to high inertial force for a long time, which may cause fundamental problems in studying biological reactions.

Therefore, it is necessary to develop a new system capable of continuously separating the micro-vesicles from biological samples without damaging the micro-vesicles.

In addition, there is also a demand for a technique of effectively separating the sample efficiently by electrophoresis, by mixing the biological sample with an acidic solution or an alkaline solution, and adjusting the pH to charge the biological sample arbitrarily.

Korean Patent Publication No. 2005-0119128

SUMMARY OF THE INVENTION The present invention has been conceived to solve the above-mentioned problems, and an object of the present invention is to provide a method of selectively removing a micro-vesicle of a desired size without damaging the micro-vesicle from a biological sample, The present invention also provides a device for separating fine microorganisms from each other by electrophoresis to adjust the pH so that the microorganisms can be separated more efficiently.

To this end, the apparatus for separating micro-fibrils according to the present invention for continuously separating a pH-adjusted sample by an electrophoresis method comprises: a flow channel part in which a biological sample and an buffer mixed with an acidic solution or an alkaline solution are flowed in parallel along one direction; A first path part connected to one end of the flow channel part to inject the buffer; A second path part connected to one end of the flow channel part to inject the acidic solution or the alkaline solution and the biological sample; An electric field forming unit forming an electric field in a direction orthogonal to the one direction of the flow channel unit such that the biological sample flowing along the flow channel unit is separated according to electrophoretic fluidity; A third path part connected to the other end of the flow channel part so as to discharge the separated first material in accordance with the electrophoretic fluidity of the electric field forming part; And a neutralizing solution for neutralizing the acidic solution or the alkaline solution injected from the second path portion, wherein the neutralizing solution for neutralizing the acidic solution or the alkaline solution injected from the second path portion A fourth path portion to be injected; .

According to an embodiment of the present invention, the second path portion includes a sample injection port into which the biological sample is injected; And a solution injection port into which the acidic solution or the alkaline solution is injected; .

In addition, the second path portion according to the embodiment of the present invention may include a first mixing portion formed in a wave form (wave form) for mixing the biological sample with the acidic solution or the alkaline solution, ; .

Meanwhile, the fourth path portion according to the embodiment of the present invention may include a sample outlet through which the second material is discharged; And a neutralization solution inlet for injecting a neutralization solution for neutralizing the acid solution or the alkaline solution injected from the second path portion; .

The fourth path portion according to an embodiment of the present invention may include a second mixing portion formed between the neutralizing solution inlet and the sample outlet in wave form for mixing the second material and the neutralizing solution; .

Further, the biological sample according to an embodiment of the present invention is a protein, a micro-vesicle or a mixture thereof.

Meanwhile, the flow channel unit according to an embodiment of the present invention adjusts the flow rate of the buffer to be injected so that the biological sample mixed with the acidic solution or the alkaline solution is separated from the buffer along one side wall of the outlet channel, Flow.

And the flow rate of the injected buffer according to the embodiment of the present invention is 1 to 20 times the flow rate of the biological sample into which the acidic solution or the alkaline solution is injected.

In addition, the biological sample mixed with the acidic solution according to an embodiment of the present invention has a positive charge, and the particles contained in the biological sample having a positive charge while passing through the electric field forming unit are mixed with the one- And is separated according to the electrophoretic fluidity.

Meanwhile, the biological sample mixed with the alkaline solution according to an embodiment of the present invention has a negative charge, and the particles contained in the biological sample having a negative charge while passing through the electric field forming unit, And is separated according to the electrophoretic fluidity.

The features and advantages of the present invention will become more apparent from the following detailed description based on the accompanying drawings.

Prior to that, terms and words used in the present specification and claims should not be construed in a conventional and dictionary sense, and the inventor may properly define the concept of the term in order to best explain its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

According to various embodiments of the present invention, the apparatus for separating micro-fibrils has the effect of continuously separating a large amount of samples according to electrophoresis fluidity without damaging the micro-endoplasmic reticulum from the biological sample.

In addition, according to various embodiments of the present invention, the apparatus for separating micro-fibrils provides an effect of efficiently separating the biological sample efficiently by electrophoresis by mixing the biological sample with an acidic solution or an alkaline solution to adjust the pH do.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an exemplary view showing the principle of a microfibrillator separation apparatus according to the present invention. FIG.
FIG. 2 is a schematic view showing a device for separating a microfibrilate according to a first embodiment of the present invention. FIG.
3 is a schematic view showing a device for separating a microfibrillar body according to a second embodiment of the present invention.
FIG. 4 is a schematic view showing a microfibrillator separation apparatus according to a third embodiment of the present invention; FIG.
FIG. 5 is a schematic view showing a device for separating a microfibrillar body according to a fourth embodiment of the present invention. FIG.
FIG. 6 is a schematic view showing a microfibrillator separation apparatus according to a fifth embodiment of the present invention; FIG.
FIG. 7 is a schematic view showing a microfibrillator separation apparatus according to a sixth embodiment of the present invention. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The objectives, specific advantages and novel features of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. It should be noted that, in the present specification, the reference numerals are added to the constituent elements of the drawings, and the same constituent elements are assigned the same number as much as possible even if they are displayed on different drawings. Also, the terms "first "," second ", and the like are used to distinguish one element from another element, and the element is not limited thereto. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following description of the present invention, detailed description of related arts which may unnecessarily obscure the gist 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 an exemplary view showing the principle of a microfibrillator separation apparatus according to the present invention.

As shown in FIG. 1, the present invention provides an apparatus for separating micro-endoplasmic reticulum or protein according to electrophoretic fluidity using an electrophoresis phenomenon.

The protein or microembossed particles contained in the biological sample are charged according to pH. Specifically, a positive charge is generated at a pH lower than the pI (Isoelectric point) at which the charge becomes zero, and a negative charge is generated at a high pH.

In addition, electrophoresis is a phenomenon in which charged particles move in an area where an electric field is applied. In the electrophoresis, there is a difference in flow rate due to factors such as size of particles, amount of charge, buffer composition, and the like. This is referred to as electrophoretic mobility. The present invention realizes continuous separation of the fine vesicle using the above-described characteristic, that is, the characteristic that charged particles are separated and flow according to the electrophoretic fluidity.

Herein, the term 'micro-vesicle' refers to a small vesicle of membrane structure existing in various kinds of cells or secreted from cells, and includes extracellular vesicles. The micro-vesicles secreted extracellularly are composed of (1) exosomes: membrane vesicles of 30-100 nm in diameter from the origin of the bacteria, (2) shedding microvesicles (SMVs) (3) Apoptotic blebs: including, but not limited to, vesicles 50-5000 nm in diameter, which are drained by dying cells. The microfilament to be obtained by the present invention may be preferably an exosome.

The 'exosome' is a small vesicle of membrane structure secreted from various kinds of cells. The diameter of the exosome can be as large as 30-1000 nm. Exosomes originate from specific compartments within the cell called multivesicular bodies (MVBs) and are released and secreted out of the cell, rather than being removed directly from the plasma membrane in electron microscopic studies. That is, when fusion of the polycation and the plasma membrane occurs, such vesicles are released into the extracellular environment, which is called exosomes. It is unclear how these exosomes are produced by molecular mechanisms, but it is possible that not only red blood cells but also various types of immune cells and tumor cells, including B-lymphocytes, T-lymphocytes, dendritic cells, platelets and macrophages, It is known to produce and secrete exosome in the state of being. Exosomes are known to be released from many different cell types under normal, pathological, or both conditions.

Biological samples contain protein, particles of similar size to the micro-endoplasmic reticulum, and vesicles of various sizes including micro-endoplasmic reticulum. As the follicle, especially the micro-endoplasmic reticulum, differs in the cell-producing region, the lipid layer constituting the follicle varies in monolayer or bilayer, and the size of the follicle varies.

The biological sample containing the protein and the microfibrillar of the present invention refers to a biological sample from which a desired type of microfibrillar body can be obtained and includes, for example, a body fluid or a cell culture. The body fluid may be at least one selected from the group consisting of urine, mucus, saliva, tears, plasma, serum, urine, sputum, spinal fluid, pleural fluid, aspiration nipple, lymphatic fluid, airway fluid, intestinal fluid, urinary reproductive fluid, , Ascites, cystic tumor body fluids, positive sap or combinations thereof. The cell culture medium means a culture medium from which cells have been removed after cell culture. The composition of the medium may be optionally changed by a person skilled in the art so as to secrete a large amount of micro-endoplasmic reticulum from the cells, but it is preferably a conditioned medium (serum-free medium) or serum .

In addition, the filtration and concentration processes may be arbitrarily added to the sample according to the experimental efficiency desired by the ordinary skilled in the art. The filtration process may be performed by a known filtration method. For example, centrifugation or filtration using a microfilter may be used. The concentration process may be performed by a known concentration process, but is not limited thereto. For example, the process can be performed using a centrifugation method.

The biological sample containing the protein and the microfibrillar of the present invention may preferably be a culture medium after the cell culture or a serum concentrate.

Next, referring to Figs. 2 to 7, the apparatus 100, 100A, 100B, 100C, 100D, and 100E for separating micro-fibrils according to various embodiments of the present invention will be described below.

FIG. 2 is a schematic view showing a microfibrillator separation apparatus according to a first embodiment of the present invention, FIG. 3 is a schematic view showing a microfibrillator separation apparatus according to a second embodiment of the present invention, and FIG. 5 is a schematic view showing a device for separating a microfibrilate according to a fourth embodiment of the present invention, and FIG. 6 is a schematic view of a microfibril separator according to a fifth embodiment of the present invention. FIG. 7 is a schematic view showing a microfibrillar separation apparatus according to a sixth embodiment of the present invention. FIG.

2, the apparatus 100 for separating a microfibrilate according to the first embodiment of the present invention includes a flow channel unit 110, a first path unit 120, a second path unit 130, A second path portion 140, a third path portion 150, and a fourth path portion 160.

The flow channel portion 110 flows along the biological sample and the buffer side by side along one direction. Here, the buffer is injected into the first path portion 120 to flow into the flow channel portion 110, and the biological sample is injected into the second path portion 130 and flows to the flow channel portion 110. It is preferred that an acidic or alkaline solution is injected into the second pathway 130 with the biological sample or a pH controlled biological sample is injected to adjust the pH of the biological sample.

When the biological sample mixed with the acidic solution or the alkaline solution and the buffer are injected from the first path part 120 and the second path part 130 as described above, the electric field forming part 140 forms the flow path along the flow channel part 110 An electric field is formed in a direction orthogonal to one direction of the flow channel portion 110 so that the biological sample is separated according to the electrophoretic fluidity.

The third channel part 150 and the fourth channel part 160 are connected to the other end of the flow channel part 110 so that separated samples are separated and flowed according to the electrophoretic fluidity of the flow channel part 110 . That is, the first material separated according to the electrophoretic fluidity is discharged through the third path portion 150, and the second material is discharged through the fourth path portion 160. Since the second material that the fourth path unit 160 separates and discharges is mixed with the acidic solution or the alkaline solution in the second path unit 130, it is preferable to mix the neutralization solution in the second material. The biological sample is preferably a protein, a micro-vesicle or a mixture thereof. It is also assumed that the separated samples are discharged through the third path part 150 and the fourth path part 160 assuming the first material and the second material for convenience of explanation, It is preferable that a discharge path can be further provided depending on the kind of the sample.

The flow channel unit 110 is formed to be long in one direction, i.e., a buffer, a biological sample mixed with an acidic solution or an alkaline solution to be separated, is separated and flows side by side. At this time, the buffer (buffer) flows through the channel and carries and serves as a carrier buffer and a partitioning fluid because it transports and separates the micro-vesicles in the sample.

The buffer is injected into a first path part 120 connected to one end of the flow channel part 110 and the biological sample is introduced into a second path part 130 connected to one end of the flow channel part 110 . As described above, the injected buffer and the biological sample flow at one end of the flow channel part 140 and flow in one direction. As described above, the buffer serves as a carrier buffer and a separation liquid, Separate from the biological sample and flow side by side. At this time, it is preferable that the biological sample flows along one side wall of the flow channel part 110 in order to increase the separation effect. This is achieved by controlling the flow rate of the buffer injected through the first path part 120 can do.

Meanwhile, the flow rate of the injected buffer is preferably 1 to 20 times the flow rate of the biological sample to be injected. If the upper limit value is exceeded, the flow along the one side wall of the sample becomes unstable. If the lower limit is exceeded, the sample may flow into the third and fourth path portions 150 and 160.

The buffer is not limited in its kind as long as it does not affect the lipid membrane structure of the micro-vesicles contained in the biological sample, which is known in the art. For example, a phosphate buffered saline (PBS), a PBS solution containing sucrose, a PBS solution containing glycine, etc. may be used, and preferably, a PBS solution containing sucrose may be used. no.

It is preferable that the biological sample is mixed with an acid solution or an alkaline solution so that the pH of the biological sample is adjusted before passing through the electric field forming part 140 along the flow channel part 110, +) Charge or negative (-) charge. Therefore, mixing the acidic or alkaline solution with the biological sample is optional and the two solutions can be selectively injected.

Next, the apparatus 100A for separating a microfibrillar body according to the second embodiment of the present invention will be described with reference to FIG.

Referring to FIG. 3, the apparatus 100A for separating a microfibrilate according to the second embodiment of the present invention is different from the apparatus 100 for separating microfibrillate according to the first embodiment of the present invention shown in FIG. 130A may further include a solution injection port 131 and a sample injection port 132.

In the description of FIG. 3, the description overlapping with the description of FIG. 2 will be omitted, while the same elements as in FIG. 2 will be described with the same reference numerals.

First, the sample inlet 132 shown in FIG. 3 injects a biological sample into the second path portion 130A, and the solution inlet 131 injects an acidic solution or an alkaline solution to adjust the pH of the biological sample. Thereafter, the second material discharged to the fourth path portion 160 is subjected to a post-treatment process in which the neutralizing solution is mixed to neutralize the acid solution or the alkaline solution injected from the solution injection port 131.

Therefore, the apparatus 100 for separating a microfibrilate according to the first embodiment of the present invention shown in FIG. 2 injects a biological sample mixed with an acidic solution or an alkaline solution into the second path part 130, The apparatus 100A for separating a microfibrillar body according to the second embodiment of the present invention may selectively mix an acidic solution or an alkaline solution into a biological sample.

Next, the apparatus 100B for separating a microfibrillar body according to the third embodiment of the present invention will be described with reference to FIG.

Referring to FIG. 4, the apparatus 100B for separating a microfibrilate according to the third embodiment of the present invention is different from the apparatus 100A for separating microfibrillate according to the second embodiment of the present invention shown in FIG. 3, 130B have a first mixing portion 133 instead of the solution injection port 131 and the sample injection port 132. [

In the description of FIG. 4, the description overlapping with the description of FIG. 3 will be omitted, while the same elements as in FIG. 3 will be described with the same reference numerals.

Referring to FIG. 4, in the apparatus 100B for separating a microfibrilate according to the third embodiment of the present invention, the second path part 130B separates the acidic solution or the alkaline solution for adjusting the pH of the biological sample, And a first mixing portion 133 formed in a shape (wave form). Therefore, the biological sample and the acidic solution or the alkaline solution can increase the mixing efficiency while flowing the first mixing part 133 through the zigzag.

Thereafter, the second material discharged to the fourth path portion 160 is subjected to a post-treatment process in which the neutralizing solution for neutralizing the acidic solution or the alkaline solution mixed with the biological sample is mixed in the first mixing portion 133 desirable.

Next, the apparatus 100C for separating a microfibrillar body according to the fourth embodiment of the present invention will be described with reference to FIG.

Referring to FIG. 5, the apparatus 100C for separating a microfibrilate according to the fourth embodiment of the present invention is different from the apparatus 100A for separating microfibrillate according to the second embodiment of the present invention shown in FIG. 160A have a neutralization solution inlet 161A and a sample outlet 162A.

In the description of FIG. 5, the description overlapping with the description of FIG. 3 will be omitted, while the same elements as in FIG. 3 will be described with the same reference numerals.

5, when a biological sample mixed with an acidic solution or an alkaline solution is injected into the second path part 130, the biological sample is mixed with the first material and the second material according to the electrophoretic fluidity of the electric field forming part 140 Lt; / RTI > Then, the second material discharged to the sample outlet 162A of the fourth path portion 160A is neutralized by the acid solution or the alkaline solution injected from the neutralization solution inlet 161A.

Therefore, the apparatus 110C for separating a microfibrilate according to the fourth embodiment of the present invention shown in FIG. 5 can separate neutralized solution (acidic solution or alkaline solution) through the neutralization solution injection port 161A The post-treatment step of FIG.

Next, referring to FIG. 6, a microfibrillar separation device 100D according to a fifth embodiment of the present invention will be described below.

Referring to FIG. 6, the apparatus 100D for separating a microfibrilate according to the fifth embodiment of the present invention is similar to the apparatus for separating microfibrillators 100C according to the fourth embodiment of the present invention shown in FIG. 5, 160B are provided between the neutralization solution inlet 161A and the sample outlet 161B.

In the description of FIG. 6, description overlapping with the description of FIG. 5 will be omitted, while the same elements as in FIG. 5 will be described with the same reference numerals.

Referring to FIG. 6, a pH-adjusted biological sample mixed with an acidic solution or an alkaline solution is separated into a first substance and a second substance by an electric field forming unit 140. The neutralized solution (acidic solution or alkaline solution) is injected into the neutralized solution injection port 161A and mixed with the second material. Then, the second material and the neutralizing solution are mixed with each other while passing through the second mixing portion 163, so that the second material is neutralized.

Finally, referring to Fig. 7, the apparatus 100E for separating a microfibrillar body according to the sixth embodiment of the present invention will be described below.

The microfibrillator separation apparatus 100E according to the sixth embodiment of the present invention includes a flow channel unit 110, a first path unit 120, a second path unit 130A, an electric field forming unit 140, A first portion 150, and a fourth path portion 160B.

Referring to FIG. 7, a microfibrillator separation apparatus 100E according to a sixth embodiment of the present invention includes microfibrillator separation apparatuses 100 and 100A according to the first to fifth embodiments of the present invention shown in FIGS. , 100B, 100C, and 100D are all combined.

Therefore, the second path portion 130A of the apparatus 100E for separating a microfibrilate according to the sixth embodiment of the present invention includes both the sample injection port 132, the solution injection port 131 and the first mixing portion 133, A biological sample is injected into the injection port 132, and an acidic solution or an alkaline solution is injected into the solution injection port 131 and mixed with the first mixing part 133 while flowing.

The thus-mixed biological sample is separated into a first material and a second material at the electric field forming portion 140. The separated second material flows into the fourth path portion 160B and is mixed with the neutralization solution injected from the neutralization solution inlet 161A. Further, it is preferable that a second mixing portion 163 is further provided at the rear end of the neutralization solution inlet 161A to increase the mixing efficiency of the second material and the neutralization solution. The second mixing unit 163 is provided in a shape similar to that of the first mixing unit 133, and may be formed in wave form, for example, to improve mixing efficiency.

On the other hand, the biological sample mixed with the buffer and the acidic solution joining to one end of the flow channel unit 110 and flowing separately in one direction is charged with a positive charge, passes through the electric field forming unit 140, It is separated according to the fluidity. The electric field forming unit 140 forms an electric field in a direction orthogonal to one direction of the flow channel unit 110. As a result, particles having a positive charge included in the biological sample are electrophoretically moved And the moving distance varies depending on the electrophoresis fluidity. The electrophoretic fluidity may vary depending on the particle size, charge amount, buffer composition, and the like.

Conversely, when the alkaline solution is mixed with the biological sample, the sample is charged with a negative charge, and the first substance and the second substance can be controlled to discharge through different paths according to the direction of the electric field forming unit 140.

A third path part 150 connected to the other end of the flow channel part 110 or a fourth path part 150 connected to the other end of the flow channel part 110 so that the biological sample particles separated according to the electrophoretic fluidity passing through the electric field forming part 140 are isolated from each other, And flows through the portion 160B to be finally separated.

As described above, the electric field forming unit 140 may include a pair of electrodes for applying power to form an electric field in a direction orthogonal to one direction of the flow channel unit 110, And may be provided on both side walls of the flow channel portion.

In addition, according to the sixth embodiment of the present invention, the second path portion 130A connected to one end of the flow channel portion 110 may be provided with a second path portion 130A so that the injected acidic solution or the alkaline solution, 1 mixing unit 133. [0034] As described above, the acidic solution or the alkaline solution is used for adjusting the pH of the particles contained in the biological sample to make positive (+) or negative (-) electric charge. Most of the particles contained in the sample have a positive (+) or negative (-) charge, thereby further improving the separation effect. At this time, as shown in FIG. 7, it is preferable that the first mixer 133 is connected in a serpentine shape instead of a straight line in order to further enhance the mixing effect.

In addition, according to the sixth embodiment of the present invention, the fourth path portion 160B includes a neutralization solution inlet 161A into which a solution for adjusting the pH of the separated protein or micro- As shown in FIG. The biological sample may be injected through the sample outlet 162A in a state where the biological sample is mixed with the acidic solution at a very low pH or mixed with the alkaline solution to a very high pH. Therefore, the pH of the finally separated separated particles can be adjusted to obtain particles having a desired pH.

Hereinafter, a process of separating a biological sample using the microfibrillator separation apparatus 100 configured as described above will be described.

First, a buffer is injected through a first path portion 120 connected to one end of the flow channel portion 110, and a biological sample mixed with an acid solution or an alkaline solution through the second path portion 130A, that is, Biological samples containing negatively charged particles are injected. At this time, the flow rate of the buffer is adjusted so that the biological sample bearing the positive charges is separated from the buffer along the one side wall of the flow channel part 110 and flows side by side.

When electric power is supplied between a pair of electrodes provided in the electric field forming part 140, an electric field is formed in a direction orthogonal to one direction of the flow channel part 110.

In this state, the biological sample flowing along one side wall of the flow channel unit 110 flows in the direction of the formed electric field in the process of passing through the electric field forming unit 140. At this time, The separated particles are separated from each other according to the electrophoretic fluidity depending on the amount of charge, the buffer composition, etc., and the separated particles passing through the electric field forming part 140 are separated from each other by the electrophoretic fluid, 3 path portion 150 or the fourth path portion 160B to be finally separated.

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 evident that it is possible to modify or modify it by the owner.

100, 100A, 100B, 100C, 100D, and 100E: a microfibril separation apparatus 110: a flow channel unit 120, 120A:
130, 130A, 130B: second path portion 140: electric field forming portion
150: third path portion 160, 160A, 160B: fourth path portion
131: solution inlet 132: sample inlet
133: first mixer 161A: neutralizing solution inlet
162A and 161B: sample outlet port 163: second mixing section

Claims (10)

A flow channel part in which a biological sample mixed with an acidic solution or an alkaline solution and a buffer are separated and flow side by side along one direction;
A first path part connected to one end of the flow channel part to inject the buffer;
A second path part connected to one end of the flow channel part to inject the acidic solution or the alkaline solution and the biological sample;
An electric field forming unit forming an electric field in a direction orthogonal to the one direction of the flow channel unit such that the biological sample flowing along the flow channel unit is separated according to electrophoretic fluidity;
A third path part connected to the other end of the flow channel part so as to discharge the separated first material in accordance with the electrophoretic fluidity of the electric field forming part; And
A fourth path part connected to the other end of the flow channel part so as to discharge the separated second material according to the electrophoretic fluidity of the electric field forming part; Wherein the pH regulating sample is continuously separated by electrophoresis.
The method according to claim 1,
The second path portion
A sample inlet through which the biological sample is injected; And
A solution injection port into which the acidic solution or the alkaline solution is injected; Wherein the pH regulating sample is continuously separated by an electrophoresis method.
The method of claim 2,
The second path portion
A first mixing portion formed in wave form for mixing the biological sample with the acidic solution or the alkaline solution at the sample inlet and the rear end of the solution inlet; Further comprising an electrophoresis method for continuously separating the pH control sample by the electrophoresis method.
The method according to claim 1,
The fourth path portion
A sample outlet through which the second material is discharged; And
A neutralization solution inlet for injecting a neutralization solution for neutralizing the acidic solution or the alkaline solution injected from the second path portion; Wherein the pH regulating sample is continuously separated by an electrophoresis method.
The method of claim 4,
The fourth path portion
A second mixing portion formed between the neutralizing solution inlet and the sample outlet in wave form for mixing the second material and the neutralizing solution; Further comprising an electrophoresis method for continuously separating the pH control sample by the electrophoresis method.
The method according to claim 1,
The biological sample
Characterized in that the microorganism is a protein, a micro-vesicle or a mixture thereof, and the micro-vesicle separation apparatus for continuously separating the pH-adjusted sample by electrophoresis.
The method according to claim 1,
The flow channel portion
Wherein the biological sample mixed with the acidic solution or the alkaline solution flows separately from the buffer along one side wall of the outlet channel by controlling the flow rate of the buffer to be injected, A device for separating microfibrillar body for continuous separation.
The method according to claim 1,
The flow rate of the injected buffer
Wherein the acidic solution or the alkaline solution is 1 to 20 times the flow rate of the biological sample mixed with the microbial agent.
The method according to claim 1,
The biological sample mixed with the acidic solution
Positive (+) charges,
Characterized in that particles contained in the biological sample having a positive electrical charge while passing through the electric field forming part are separated by electrophoretic fluidity while flowing by an electric field formed in a direction orthogonal to the one direction, A device for separating microfibrillar bodies for continuous separation by centrifugation method.
The method according to claim 1,
The biological sample mixed with the alkaline solution
Negative (-) charge,
Characterized in that particles contained in the biological sample having a negative charge while passing through the electric field forming part are separated by electrophoresis fluidity while flowing by an electric field formed in a direction orthogonal to the one direction, A device for separating microfibrillar bodies for continuous separation by centrifugation method.
KR1020150066882A 2015-05-13 2015-05-13 Apparatus for separating fine endoplasmic reticulum by electrophoresis sample pH adjustment KR20160133837A (en)

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KR20190024942A (en) 2019-02-28 2019-03-08 경희대학교 산학협력단 Apparatus for extraction and separation of intracellular substance
KR20190116053A (en) * 2018-04-04 2019-10-14 광주과학기술원 Microbial concentration device
WO2020046027A1 (en) * 2018-08-30 2020-03-05 주식회사 엘지화학 Device comprising microbeads capable of adjusting ph of sample
KR20200025390A (en) * 2018-08-30 2020-03-10 주식회사 엘지화학 A disc type device incluing microbeads capable of controlling pH of a sample
KR20200025389A (en) * 2018-08-30 2020-03-10 주식회사 엘지화학 A device incluing microbeads capable of controlling pH of a sample
WO2023146067A1 (en) * 2022-01-28 2023-08-03 인천재능대학교산학협력단 Microfluidic device-based biofluid sample preprocessing apparatus and method

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190116053A (en) * 2018-04-04 2019-10-14 광주과학기술원 Microbial concentration device
WO2020046027A1 (en) * 2018-08-30 2020-03-05 주식회사 엘지화학 Device comprising microbeads capable of adjusting ph of sample
KR20200025390A (en) * 2018-08-30 2020-03-10 주식회사 엘지화학 A disc type device incluing microbeads capable of controlling pH of a sample
KR20200025389A (en) * 2018-08-30 2020-03-10 주식회사 엘지화학 A device incluing microbeads capable of controlling pH of a sample
US11440005B2 (en) 2018-08-30 2022-09-13 Lg Chem, Ltd. Device comprising microbeads capable of adjusting pH of sample
KR20190024942A (en) 2019-02-28 2019-03-08 경희대학교 산학협력단 Apparatus for extraction and separation of intracellular substance
WO2023146067A1 (en) * 2022-01-28 2023-08-03 인천재능대학교산학협력단 Microfluidic device-based biofluid sample preprocessing apparatus and method

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