KR101338291B1 - Apparatus and method for separating microparticles by controlling the dielectrophoresis and magnetophoresis - Google Patents
Apparatus and method for separating microparticles by controlling the dielectrophoresis and magnetophoresis Download PDFInfo
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Abstract
The present invention relates to a device for separating microparticles using the techniques of dielectric electrophoresis (DEP) and magnetophoresis (MAP) and a method for separating the microparticles using the same, the mutual symmetry around the centerline of the microfluidic channel A device for separating the fine particles by the difference in the path change of the fine particles by the dielectric and magnetophoretic generated by the patterned electrode and the linear magnetic microstructure to have a predetermined inclination angle with respect to the flow direction of the sample so that A method for separating fine particles.
Description
The present invention relates to an apparatus for separating fine particles using the technique of dielectric electrophoresis (DEP) and magnetophoresis (MAP) and a method for separating the fine particles using the same.
Recently, with the development of microfabrication technology, research has been actively conducted to manufacture microelectronic devices for concentrating, assembling, and separating fine particles, and to apply them to biological, chemical, and new material fields.
In such microelectronic devices, various electrokinetics such as electrophoresis, dielectrophoresis, and magnetophoresis are mainly used to precisely drive or isolate microparticles, proteins, cells, and bacteria. It is available.
Electrophoresis is a phenomenon in which fine particles with positive and negative charges are moved by coulomb force in an electric field. The movement characteristics of the particles by electrophoresis may vary depending on the amount of charge, the size of the particles, the electrical, chemical, physical properties of the medium, or the strength of the electric field.
Dielectrophoretic (DEP) methods are known as the primary technique for separating target cells from heterogeneous cell mixtures. Dielectrophoresis is a very efficient way to manipulate and isolate species. In general, Dielectrophoresis (DEP) is a method in which dielectric particles exhibit inductive dipoles in a non-uniform electric field, and use them to move the dielectric particles. The dielectrophoresis is a method of freely controlling solid particles in a fluid, and the movement of the particles by the dielectrophoresis is changed depending on the frequency of the applied alternating voltage, the type of fluid, and the type of solid particles, and the direction of the electric field is weak. It is composed of negative (-) dielectric phenomena which move to, and positive (+) dielectric drift in which particles move in the direction of strong electric field.
This technique mainly applies to cells, organelles and other particles (eg cell contents and membranes). When a particle is placed in an electric field, charge is induced due to the relative permittivity and conductivity of the particle compared to the media. This process is called polarization. If the external electric field is not uniform, the particles can move by the electrostatic force. In particular, in an alternating electric field, particle polarization is frequency dependent, so that the induced force and therefore the movement of the particle can be adjusted. This is called genetic electrophoresis (DEP). By varying the induced force, the particles can be brought close to or repelled by conventional electrophoresis (DEP), or can be moved in both directions by traveling wave electrophoresis (DEP). Dielectrophoresis (DEP) can also be used to identify or separate other particles. (E.g., different kinds of bacteria, living cells or dead cells), the main advantage of genetic electrophoresis (DEP) is that the movability and thus mode of movement can be controlled by a single electric field.
However, DEP performance is very sensitive to fluids such as buffers, in particular ionic strength. Large DEP forces can only be obtained in low ionic strength media. On the other hand, the ionic strength of real samples such as blood is much higher. Moreover, since the DEP force strength is usually proportional to the volume of the particles, it is only suitable for manipulation of large particles such as cells and is too small to manipulate small molecules. In addition, DEP of a bioanalyte does not necessarily reflect the biological properties of the analyte, but has a large physical effect. Thus, it can be difficult to manipulate analytes with specific specificities in complex environments.
Another method for bioanalyte migration is the use of magnetophoresis (MAP). Magnetic particles can be operated by magnetophoretic force. The advantage of this magnetophoresis is that the biospecificity is maintained by the biocompatible bond between the magnetic particles and the bioanalyte, and the magnetophoretic force is not affected by the media. The conventional magnetophoretic method uses a method of applying a magnetic field from the outside to the sample to be separated according to the magnetic density gradient.
In Korean Patent Laid-Open Publication No. 10-2010-0026270, as shown in FIG. 1, dielectric and magnetophoresis are simultaneously applied, and the position of the electrode and the magnetic field generator are separated, thereby inducing the movement of the particles by the force of the electrophoresis. In the case of electrophoresis, a method of separating by using a magnetic field gradient generator to specify the location of the separated particles is disclosed.
However, in such a conventional method, if the distance from the magnetic field generating material to a certain distance, the effect of the magnetic force is reduced, and the magnetic field and the electric field applied to the sample cannot be effectively controlled with each other, resulting in low separation efficiency and long separation time. There was a problem taken.
The present invention is to solve the above problems, while simultaneously applying the electrophoresis and magnetophoresis to the sample, the apparatus for separating the microparticles by adjusting the direction of action of the electrophoresis and magnetophoresis and separating the fine particles using the same It is an object to provide a method.
The present invention is an upper glass substrate including a patterned electrode structure to solve the above problems; A bottom glass substrate comprising a patterned linear magnetic structure; A microfluidic channel formed between the upper glass substrate and the lower glass substrate and having a sample including particles to be separated; And it provides a device for separating the fine particles by adjusting the direction of action of the electrophoresis and magnetophoresis including an external magnetic field source (not shown) and an external power source (not shown).
In the present invention, the microfluidic channel may include a microfluidic channel region for sample injection for injecting a sample and a buffer, respectively; A separation microfluidic channel region through which fine particles contained in the injected sample pass while being separated by dielectric and magnetophoresis; And a plurality of discharge microfluidic channel regions in which the separated fine particles and the remaining sample are separated and discharged, respectively.
In the present invention, the patterned electrode of the upper glass substrate is arranged to cross each other to have a predetermined inclination angle with respect to the flow direction of the sample in the separation microfluidic channel region, and the driving of the sample particles by applying power It is characterized by forming a potential difference for.
In the present invention, the patterned linear magnetic structure of the lower glass substrate is patterned and included in the lower glass substrate so as to have a predetermined inclination angle with respect to the flow direction of the sample in the separation microfluidic channel region. .
In the present invention, the patterned linear magnetic structure of the lower glass substrate may be patterned on the lower glass substrate to have a predetermined inclination angle with respect to the flow direction of the sample in the separation microfluidic channel region.
In the present invention, the patterned electrode of the upper glass substrate and the patterned linear magnetic structure of the lower glass substrate are patterned so as to have a predetermined inclination angle with respect to the flow direction of the sample, and center the centerline of the microfluidic channel. It characterized in that the pattern is formed so as to be mutually symmetrical.
In the present invention, the predetermined inclination angle with respect to the flow direction of the sample of the patterned electrode of the upper glass substrate and the patterned linear magnetic microstructure of the lower glass substrate is adjusted according to the characteristics of the fine particles to be separated, Preferably it is characterized in that 5 ° to 12 °.
The present invention also provides a method comprising: a first step of injecting a sample containing fine particles to be separated into a sample injecting unit of a microfluidic channel region for sample injection; A second step of injecting a buffer into a buffer injecting unit so that the injected sample flows aligned with the center of the microfluidic channel; A third step of applying a current to a plurality of electrodes of the upper glass substrate and applying an external magnetic field to the microfluidic channel part through which the sample including the microparticle passes, thereby separating the microparticles while the sample passes through the microfluidic channel part ; And a fourth step of capturing the fine particles separated in the third step in the discharge microfluidic channel by adjusting the direction of action of the dielectric and magnetophoresis to separate the fine particles. Provided are methods for separating particles.
In the present invention, the separation or capture of the fine particles of the third step or the fourth step has a predetermined inclination angle with respect to the flow direction of the sample, and the pattern-formed electrode to be symmetrical with respect to the center line of the microfluidic channel and By using the device for separating the microparticles by adjusting the direction of action of the electrophoresis and magnetophoresis, characterized in that it is carried out by the difference in the path change of each microparticle due to the electrophoresis and magnetic domain generated by the linear magnetic body structure To provide a method for separating fine particles.
Hereinafter, with reference to the drawings will be described in detail the apparatus for separating the fine particles by adjusting the direction of action of the electrophoresis and magnetophoresis of the present invention and a method for separating the fine particles using the same.
2 and 3 are cross-sectional views and plan views of a device for separating nucleated cells from blood by genophoresis and magnetophoresis according to an embodiment of the present invention. An
In addition, the
4 shows a top view of an
The plurality of electrodes of the upper glass substrate may be patterned into two or more spaced apart electrodes, and may be patterned so as to intersect with each other with a predetermined inclination angle θd with respect to the flow direction of the sample so as to exist for all possible driving areas. . In addition, in the present invention, the predetermined inclination angle (θd) of the electrode with respect to the flow direction of the sample increases the electrophoretic force acting on the particles as the value increases, but the number of electrodes present per unit area is reduced. It is preferable that the predetermined inclination angle θd is 5 ° to 12 °.
5 illustrates a patterned electrode included in the upper glass substrate and a patterned linear magnetic structure included in the lower glass substrate at the same time according to an embodiment of the present invention. As shown in FIG. 5, the
The linear magnetic structure is made of Co and Fe-based amorphous metals, such as Ni, Fe, Co, Ti, W, such as magnetic, permalloy, supermalloy, Invar, etc. have excellent soft magnetic properties to use the material as a magnetic material, magnetic material The shape of is formed by sputtering or vacuum evaporation using a linear magnetic body such as a ribbon, a wire, a thin film, or the like. Preferably, it is formed in 50-200 micrometers by electroplating using the magnetic alloy film which consists of NiFe.
The predetermined inclination angle θm of the linear
In addition, the predetermined inclination angle θm of the linear magnetic structure of the lower glass substrate with respect to the flow direction of the sample is centered on the predetermined inclination angle θd of the electrode with respect to the flow direction of the sample and the centerline of the microfluidic channel. The pattern is formed so as to be mutually symmetrical. That is, in the present invention, as the external magnetic field and current are applied, the fine particles are separated by magnetophoresis and dielectric electrophoresis symmetrically formed in a predetermined pattern with respect to the flow of the sample. In the present invention, a predetermined inclination angle θm of the linear magnetic structure of the lower glass substrate with respect to the flow direction of the sample and a predetermined inclination angle θd of the electrode with respect to the flow direction of the sample are to be separated. Can be adjusted according to the nature of the.
The electrophoretic and magnetophoretic forces acting on the particles located between the patterned electrode included in the upper glass substrate and the patterned linear magnetic body structure included in the lower glass substrate are illustrated in FIGS. 7 is shown. As shown in FIG. 6, although both the dielectric and magnetic domains act on the particles by the patterned electrodes included in the upper glass substrate and the patterned linear magnetic structures included in the lower glass substrate, the patterned electrodes And the direction of the patterned linear magnetic structure can be controlled by the inclination angle with respect to the flow direction of the sample, and the difference in the degree of reaction by the dielectric and magnetic domains due to the magnetic properties and the weight of the particles Thereby, the direction of travel is changed and separated.
As shown in FIG. 7, the magnetophoretic force acts at right angles to the patterned linear magnetic body microstructure, and the electrophoretic force also acts at right angles to the patterned planar electrode, thereby being symmetrical with respect to the flow direction of the sample. As the direction of action of the electrophoretic force and the magnetophoretic force is changed by the electrode having the inclination angle and the linear magnetic body structure, the particles having a large influence of the magnetophoretic force and the particles having a large influence of the electrophoretic force are separated.
The sample to be separated by a device for separating fine particles by adjusting the direction of action of the electrophoretic and magnetophoretic of the present invention can be selected from the group consisting of biological samples, biological samples, environmental samples, and environmental samples.
As an example, considering blood containing white blood cells and red blood cells as an example of a sample including the fine particles, red blood cells are ferromagnetic / paramagnetic, so they move in the direction of magnetophoretic force by magnetic force. In comparison, leukocytes, which are nucleated cells, are subjected to genetic activity only. Therefore, although red blood cells have a property of moving to the region where the magnetic flux is concentrated by the magnetic flux density gradient, the leukocytes are not affected by the magnetic flux density gradient and are only influenced by the electrophoresis. Purified erythrocytes can be obtained without causing surface area deformation and maintaining their original properties.
The apparatus for separating fine particles by controlling the direction of dielectric and magnetophoresis according to the present invention generates dielectric and magnetophoretic symmetrically with a predetermined angle with respect to the sample flow by a patterned electrode and a patterned linear magnetic structure. As a result, the influence on the dielectric and magnetophoretic changes according to the characteristics of the fine particles, thereby increasing the separation efficiency and reducing the separation time.
1 is a view showing a fine particle separator by magnetophoresis and dielectric electrophoresis.
2 and 3 show a cross-sectional view and a plan view, respectively, of an apparatus for separating nucleated cells from blood by electrophoresis and magnetophoresis according to an embodiment of the present invention.
4 shows a top view of an
5 illustrates a patterned electrode included in the upper glass substrate and a patterned linear magnetic structure included in the lower glass substrate at the same time according to an embodiment of the present invention.
6 and 7 illustrate dielectric and magnetophoretic forces acting on particles positioned between the patterned electrode included in the upper glass substrate and the patterned linear magnetic structure included in the lower glass substrate.
8 shows a process of manufacturing an apparatus for separating fine particles by controlling the direction of dielectric and magnetophoresis according to an embodiment of the present invention.
9 shows an apparatus for separating fine particles by adjusting the direction of dielectric and magnetophoresis prepared according to an embodiment of the present invention.
10 is a red blood cell and white blood cell including a fluorescent probe (or fluorescent material) passing through the fine particle channel for separation of the device for separating the microparticles by adjusting the direction of the electrophoresis and magnetophoresis prepared according to an embodiment of the present invention Represents an image of.
11 is a red blood cell and nucleus when the flow rate of the sample is changed to 4, 8, 12 μl / h in the apparatus for separating microparticles by controlling the direction of dielectrophoresis and magnetophoresis prepared according to an embodiment of the present invention The separation efficiency of the cells is shown.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention, the content of the present invention is not limited by the examples.
Preparation Example 1 Preparation of a Device for Separating Fine Particles by Adjusting the Directions of Dielectrophoresis and Magnetophoresis
An apparatus for separating fine particles by adjusting the direction of dielectric and magnetophoresis of the present invention was prepared by the method shown in FIG.
First, a 0.7 mm thick bottom glass substrate (Borofloat ™, Howard Glass Co., Worchester, Mass.) Was etched to a depth of 30 μm using 25% hydrofluoric acid. A Ti / Cu / Cr seed layer was electron beam deposited on the etched substrate (FIG. 8A), and a nickel wire of permalloy Ni 0.8 Fe 0.2 film was formed to a thickness of 40 μm as a linear magnetic structure using electroplating (FIG. 8B). ). Then, planarized by a chemical mechanical polishing (CMP) process (FIG. 8C), and the epoxy-based photocurable material SU-8 2050 (Microchem, USA) was applied (FIG. 8D), and then 30 μm thick using a photoresist. The fluid channel structure was formed (FIG. 8E).
The patterned electrode structure of the upper glass substrate was patterned and evaporated Cr / Au (200Å / 2000Å) on a glass substrate 602 using AZ1512 photoresist (AZ Electronic Materials, USA). Was prepared (FIG. 8F). Thereafter, holes for sample injection and discharge of the separator were formed in the prepared upper glass substrate (FIG. 8G).
The predetermined inclination angle θm of the linear magnetic structure of the lower glass substrate with respect to the flow direction of the sample was 7 °, and the predetermined inclination angle θd with respect to the flow direction of the sample was 11 °. .
The prepared lower glass substrate and the upper glass substrate are bonded with a UV adhesive (1187-M, DYMAX Co., Torrington, CT), and a plastic microfluidic interface for forming a sample inlet in the hole formed in the upper glass substrate. The device for separating fine particles by controlling the direction of dielectric and magnetophoresis by bonding the interface) was finally completed as shown in FIG.
Example 1 Isolation of Nucleated Cells from Blood Sample
Blood samples were prepared from anti-coagulated (Heparin-Agarose, H-1027, Sigma Diagnostics, USA) human whole blood diluted at a 1 to 5 ratio including PBS solution (10010, Invitrogen, USA).
The sample thus prepared was injected into the sample inlet at a rate of 8 μl / h. Images of erythrocytes and leukocytes containing fluorescent probes (or fluorescent materials) passing through the microparticle channel for separation of the microparticle separator of the present invention at a volumetric flow rate with a sinusoidal voltage of 4 Vp-p of 2 MHz, respectively, are shown in FIG. 10. Shown in
10A is an external magnetic field and when no external power is applied, FIG. 10B is a case where only an external magnetic field of 0.3T is applied, and FIG. 10C is an external power comparison example when only 2MHz and 4Vp-p power are applied. 10D shows a case where both an external magnetic field of 0.3T and an external power source of 2MHz and 4Vp-p are applied.
When no external voltage and magnetic field is applied, there is no directivity. When only external magnetic field is applied, white blood cells are not affected by magnetic field but are discharged together with red blood cells. In addition, when only external power is applied, both red blood cells and white blood cells are affected by electrophoretic force, but in the case of electrophoresis, red blood cells having a smaller particle size have the same direction as the white blood cells but have a smaller moving distance. Therefore, in this case, additional work is required to separate the white blood cells and red blood cells. In contrast, in the case of FIG. 10D to which an external voltage and a magnetic field are applied, red blood cells and white blood cells are separated and flow along the channel.
<Example 2> Separation efficiency measurement according to the flow rate of the sample
When the flow rate of the sample was changed to 4, 8, and 12 μl / h, the efficiency of separation of red blood cells and nucleated cells in the blood samples of the respective microfluidic channels was measured. Indicated.
As shown in FIG. 11, as the flow rate of the sample is increased, the separation efficiency is lowered. Therefore, it is understood that the flow rate of the sample is 4 to 8 μl / h in view of the separation efficiency and the separation speed.
As shown in FIG. 11, when the flow rate is 8 μl / h or less, the ratio of erythrocytes and leukocytes in the discharged sample is 95.3 to 96.7%. The ratio of erythrocytes and leukocytes in natural blood is generally 1: 1000. In consideration of this, as a result of concentration of 20,000 times or more, it can be seen that the effect of the apparatus for separating microparticles by adjusting the action direction of the electrophoresis and magnetophoresis according to the present invention and the method for separating the microparticles using the same are very excellent. .
100: upper glass substrate
200: lower glass substrate
300, 310, 320: patterned electrode
400: upper glass substrate
500: microfluidic channel
510: microfluidic channel region for sample injection
520: microfluidic channel region for separation
530: microfluidic channel area for discharge
540: sample injection unit
550: buffer injection unit
560: discharge part
Claims (9)
A bottom glass substrate comprising a patterned linear magnetic structure;
A microfluidic channel formed between the upper glass substrate and the lower glass substrate and having a sample including particles to be separated; And
In the device for separating fine particles by adjusting the direction of action of the electrophoresis and magnetophoresis including an external magnetic field source and an external power source,
The patterned electrodes of the upper glass substrate are arranged to cross each other to have an inclination angle θd with respect to the flow direction of the sample in the microfluidic channel region, and form a potential difference for driving the sample particles when an external power source is applied.
The patterned linear magnetic structure of the lower glass substrate is patterned and included in the lower glass substrate to have an inclination angle θm with respect to the flow direction of the sample in the microfluidic channel region.
The θd and the θm is 5 ° to 12 °,
The patterned electrode of the upper glass substrate has an inclination angle θd with respect to the flow direction of the sample in the microfluidic channel region, and the patterned linear magnetic structure of the lower glass substrate has an inclination angle with respect to the flow direction of the sample in the microfluidic channel region. Device for separating fine particles by adjusting the direction of action of the electrophoresis and magnetophoresis, characterized in that the size larger than θm.
The microfluidic channel may include a sample fluid microfluidic channel region for injecting a sample and a buffer, respectively;
A separation microfluidic channel region through which fine particles contained in the injected sample pass while being separated by dielectric and magnetophoresis; And
Apparatus for separating the fine particles by adjusting the direction of action of the electrophoresis and magnetophoresis, characterized in that the separated microparticles and the remaining sample is divided into a plurality of discharge microfluidic channel region, each discharged separately.
The predetermined inclination angle with respect to the flow direction of the sample of the patterned electrode of the upper glass substrate and the patterned linear magnetic microstructure of the lower glass substrate is controlled according to the characteristics of the fine particles to be separated And device for separating fine particles by adjusting the direction of action of magnetophoresis.
A second step of injecting a buffer into a buffer injecting unit so that the injected sample flows aligned with the center of the microfluidic channel;
Applying a current to a plurality of electrodes of the upper glass substrate, and separating the fine particles by applying an external magnetic field to the microfluidic channel through which the sample including the fine particles passes; And
The fourth step of capturing the microparticles separated through the microfluidic channel of the third step in the discharge microfluidic channel to adjust the direction of action of the electrophoresis and magnetophoresis according to claim 1 A method of separating fine particles using a device for separating fine particles.
Separation or capture of the fine particles of the third or fourth stage is caused by the patterned linear magnetic structure of the patterned electrode of the upper glass substrate and the lower glass substrate to have a predetermined inclination angle with respect to the flow direction of the sample. The fine particles using a device for separating the fine particles by adjusting the direction of action of the electrophoresis and magnetophoresis according to claim 1, characterized in that is performed by the difference of the path change of the fine particles by the electrophoresis and the electrophoresis How to separate it.
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| KR101996847B1 (en) * | 2013-01-12 | 2019-10-01 | 인제대학교 산학협력단 | Single cell trap, rotating device, and single cell trap, rotating process using the same |
| KR102140036B1 (en) * | 2013-11-21 | 2020-07-31 | 인제대학교 산학협력단 | Device for separation and capturing of micro particle |
| CN103869072B (en) * | 2014-01-22 | 2015-08-05 | 东南大学 | Dielectrophoresis enrichment method is assisted in magnetic field for nanoparticle label immune detection |
| KR101794218B1 (en) * | 2015-08-19 | 2017-11-07 | 인제대학교 산학협력단 | Method of concentrating and isolating nucleated red blood cells in maternal blood for non-invasive prenatal diagnosis |
| CN106215984B (en) * | 2016-05-04 | 2018-07-13 | 大连理工大学 | Micro-fluidic chip based on dielectrophoresis effect |
| KR101888636B1 (en) * | 2017-06-02 | 2018-08-14 | 지트로닉스 주식회사 | Magnetophoresis biochip |
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| US20220341836A1 (en) * | 2019-09-03 | 2022-10-27 | Singapore University Of Technology And Design | Microfluidic device for single cell processing and method and system for single cell processing using the microfluidic device |
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| WO2001096857A2 (en) | 2000-06-14 | 2001-12-20 | Board Of Regents, The University Of Texas System | Method and apparatus for combined magnetophoretic and dielectrophoretic manipulation of analyte mixtures |
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