KR20170135800A - A method and a device for cell lysis using piezoelectric energy - Google Patents

Abstract

The present invention relates to a cytolysis method using piezoelectric energy, and a device therefor. More specifically, the present invention relates to a cytolysis method, and a device therefor. To this end, a cell-containing fluid is injected into a microchannel having a piezoelectric layer at an outer wall or a lower portion, and then is allowed to pass through the microchannel while vibration is generated through application of alternating current to the piezoelectric layer. Since vibration energy in the piezoelectric layer is evenly delivered to the fluid in the microchannel, it is possible to efficiently break cell walls.

Description

TECHNICAL FIELD [0001] The present invention relates to a method and a device for cell lysis using piezoelectric energy,

The present invention relates to a cell dissolving method and apparatus using piezoelectric energy.

The term "piezoelectricity" refers to the phenomenon of causing dielectric polarization by applying mechanical strain. The first piezoelectric effect is also referred to as a reverse piezoelectric effect, or a phenomenon in which an electric field is applied to cause distortion, is called a second piezoelectric effect. The latter is electrical distortion, but the inverse piezoelectric effect is a linear function of electric field E, sometimes distinguishing it from pure electrical distortion proportional to E².

The principle of the piezoelectric is divided into ferroelectric due to the crystal structure and a case where the symmetry of the crystal structure is lost. First, the ferroelectric principle due to the crystal structure is as follows. There is also a ferroelectric material in the electric property, just as there is a ferromagnetic material in relation to the magnetic properties of the material. This is a property that polarization is applied when a strong external electric field is applied and polarization is maintained even if an external electric field is removed. That is, it maintains its polarized state because of the specificity of the crystal structure. The ferroelectric material is also a piezoelectric material. This is because the electrical polarity changes when the shape of the material changes due to mechanical pressure. For example, when compressed, the number of molecules having a dipole moment per unit volume is increased, so that the polarity increases. Next, when the symmetry of the crystal structure disappears, the principle is as follows. Quartz (SiO ₂ ), a piezoelectric material that exists naturally, is a case in which there is a ferroelectric substance that is polarized when a pressure is applied even if it is not a ferroelectric substance. The crystal structure in the normal state is symmetrical and electrically neutral, but when pressure is applied, the shape of the crystal structure changes and dipole moments are generated and thus polarized.

On the other hand, various biological treatment processes are required to analyze actual cells. First, the cells are cultured, chemically and mechanically stimulated and cultured for a certain period of time, and then trypsin or the like is used to separate the cells. Then, cells suitable for the purpose of the study are selected, and cell lysis is then performed to remove the cell membrane, thereby exposing the intracellular substance to the outside of the cell. Then, the above-mentioned intracellular substances are separated, and the DNA, RNA and protein necessary for the study are obtained, and then analyzed at the molecular level using various measuring equipment.

Under these circumstances, the inventors of the present invention confirmed that the vibrating energy of the piezoelectric material can be used to vibrate the fluid, thereby effectively breaking the cell wall contained in the fluid, and the present invention has been accomplished.

Accordingly, it is an object of the present invention to provide an efficient cell lysis method and an apparatus therefor.

In a first aspect of the present invention, there is provided a method of manufacturing a microelectronic device, comprising the steps of: (1) injecting a fluid containing cells into a microchannel having a piezoelectric layer on an outer wall or a lower portion thereof; And a step (step 2) of passing a fluid containing the cell through a microchannel while generating vibration by applying an alternating current to the piezoelectric layer.

According to a second aspect of the present invention, there is provided a method of producing a cell, comprising: an injection port for injecting a fluid containing cells; A microchannel connected to the injection port and having a piezoelectric layer on an outer wall or a lower portion thereof; An electrode for applying an alternating current to the piezoelectric layer; And an outlet connected to the microchannel and through which an intracellular substance-containing fluid having passed through the microchannel is recovered.

A third aspect of the present invention is the cytolytic device according to the second aspect; And means for analyzing the fluid contained in the cell, which is recovered from the cell dissolving apparatus.

Hereinafter, the present invention will be described in detail.

Conventional cell dissolution methods include optical, mechanical, acoustic, electrical, and chemical methods. Double acoustic cell lysis is a method in which a cell solution or suspension is placed in a chamber placed in an ultrasonic bath and ultrasound is applied to destroy cells. Cell destruction using ultrasound is difficult to uniformly form an energy distribution of ultrasonic waves, so it is difficult to obtain consistent results, and there is a disadvantage that it takes much time to destroy cells.

In the present invention, a fluid containing a cell is injected into a microchannel having a piezoelectric layer on an outer wall or a lower portion thereof, an alternating current is applied to the piezoelectric layer to generate vibration, and a fluid containing the cell is passed through the microchannel, It is possible to provide a cell dissolving method capable of efficiently breaking the cell wall by uniformly transferring the vibration energy of the layer to the fluid inside the microchannel. The present invention is based on this.

The cell dissolving method of the present invention includes the steps of injecting a fluid containing cells into a microchannel having a piezoelectric layer on an outer wall or a lower portion thereof (step 1); And passing the fluid containing the cells through a microchannel while generating vibration by applying an alternating current to the piezoelectric layer (step 2).

Further, the cell lysis method of the present invention may further include a step (step 3) of recovering the fluid containing the intracellular substance that has passed through the microchannel after the step 2).

According to another aspect of the present invention, A microchannel connected to the injection port and having a piezoelectric layer on an outer wall or a lower portion thereof; An electrode for applying an alternating current to the piezoelectric layer; And an outlet connected to the microchannel and through which the fluid containing the intracellular substance that has passed through the microchannel is recovered.

As used herein, the term "microchannel" refers to a channel that has a micro-level width of 1 mm or less and provides a passageway through which fluid can flow.

In the present invention, the width of the microchannel may preferably be 0.1 μm to 10 mm, more preferably 1 μm to 1 mm, and still more preferably 50 μm to 500 μm. If the width of the microchannel is less than 0.1 탆, the fluid may be difficult to flow. If the width of the microchannel is more than 10 탆, it may be difficult to control the flow rate of the fluid or uniformly generate the vibration energy.

In the present invention, the length of the microchannel may preferably be 10 mm to 1000 mm, more preferably 100 mm to 700 mm. Efficient cell lysis in the length range of the microchannel may be possible.

The term "piezoelectric layer" used in the present invention means a layer exhibiting piezoelectric characteristics. The piezoelectric layer may be made of a single crystal, a polycrystalline ceramic, a polymeric material, a thin film material, or a composite material obtained by compounding a polycrystalline material and a polymeric material.

Specifically, as the single crystal piezoelectric material, α-AlPO ₄ , α-SiO ₂ , LiTaO ₃ , LiNbO ₃ , SrxBayNb ₂ O ₈ , Pb ₅ -Ge ₃ O ₁₁ , Tb ₂ (MoO ₄ ) ₃ , Li ₂ B ₄ O ₇ , CdS, ZnO, Bi ₁₂ SiO ₂₀ , Bi ₁₂ GeO ₂₀ , PZN-PT [Pb (Zn _2/3 Nb _1/3 ) O ₃ -PbTiO ₃ ], PMN-PT [Pb (Mg _2/3 Nb _{1 / 3) O 3 -PbTiO 3]} , tree glycine sulfate (triglycine sulfate, TGS) and the like, with poly-crystalline piezoelectric material is _{PZT (lead zirconate titanate, PbZrO 3} -PbTiO 3) based, PT (PbTiO ₃₎ based, PZT - complex perovskite (PZT-complex perovskite) system, and BaTiO ₃ . Examples of the thin film piezoelectric material include ZnO, CdS, and AlN. Examples of the piezoelectric material include polyvinylidene fluoride (PVDF), poly (vinylidene fluoride-co-trifluroethylene [ poly (vinylidene fluoride-co-tetrafluoroethylene), P (VDF-TeFE)], and the like, Examples of the composite piezoelectric material include PZT-PVDF, PZT-silicone rubber (PZT-silicone rubber), PZT-epoxy (PZT-Epoxy), and PZT-foamed urethane. These materials may be used alone or as a mixture thereof.

The quartz crystal (α-SiO ₂ ) widely used as a single crystal piezoelectric material is used as an oscillator element that generates a signal frequency because the bandwidth of the resonant frequency is narrow and the temperature coefficient is very small. It is disadvantageous in that the price is high and the electromechanical coupling coefficient is small Which limits the application area. LiNbO ₃ and LiTaO ₃ single crystal piezoelectric materials and ZnO thin film piezoelectric materials have been applied as SAW (Surface Acoustic Wave) filters. Thin film piezoelectric materials such as AlN and ZnO have been studied in the newly developed FBAR (Film Bulk Acoustic wave Resonator) bandpass filter material. PZT ceramics, which is a polycrystalline piezoelectric material, is most widely applied to ultrasonic vibrators, filters, resonators, ignition devices and sensors because of its excellent processability and various piezoelectric characteristics and low cost. Polymer piezoelectric materials and PZT composite piezoelectric materials are mainly applied to various keyboards, underwater acoustic parts, and medical transducers because they are easy to process into sheet form due to the characteristics of materials.

In one embodiment of the present invention, PVDF was used as the material of the piezoelectric layer. PVDF, one of the polymer piezoelectric materials, exhibits piezoelectric effects several times larger than quartz. Unlike ceramics, which exhibit piezoelectric effects due to the crystal structure of the material, PVDF exhibits piezoelectric effects when elongated chains of molecules interact with each other by attracting and repelling each other when an electric field is applied. In addition, PVDF is not only resistant to corrosion, but also has superior material properties such as thermal stability and chemical resistance.

In the present invention, the thickness of the piezoelectric layer may preferably be 1 nm to 10 占 퐉, and more preferably 10 nm to 5 占 퐉. If the thickness of the piezoelectric layer is less than 1 nm, the piezoelectric characteristics may deteriorate and the width of the microchannel may be limited, so that the thickness of the piezoelectric layer may not be more than 10 탆.

In the present invention, the piezoelectric layer can generate vibration when an alternating current is applied. That is, the electrical stimulation applied to the piezoelectric layer by the inverse piezoelectric effect can cause vibration, which is a phenomenon that causes mechanical distortion. If an alternating current is applied to the piezoelectric layer to give resonance, the piezoelectric layer starts vibrating as a whole. The fluid in the microchannel vibrates due to the vibration of the piezoelectric layer, so that the cell walls of the cells contained in the fluid are broken and the intracellular substances in the cells can be exposed to the outside. That is, in the present invention, it is possible to efficiently prepare a fluid type specimen containing the intracellular substance by using the vibration energy of the piezoelectric layer, minimize the damage of the intracellular substance, and improve the specimen analysis efficiency.

As used herein, the term "alternating current (AC)" refers to a current whose magnitude and direction change periodically with time.

In the present invention, the frequency of the alternating current may preferably be 100 Hz to 900 kHz. It is possible to efficiently break the cell wall without damaging intracellular material in the frequency range.

In the present invention, the intracellular substance may be DNA, RNA, protein, or a mixture thereof.

In the cell dissolving apparatus of the present invention, the electrode may be connected to an external AC power source to generate an AC current.

As a specific embodiment, the structure of the cell lysis apparatus of the present invention is as shown in Fig. 1 (not shown in the drawings).

The cell dissolving apparatus of the present invention can be designed to have a piezoelectric structure that maximizes the piezoelectric acoustic wave energy density. For example, the arrangement of the electrodes can be adjusted to maximize the piezoelectric acoustic wave energy density.

The present invention also relates to the aforementioned cell lysis device; And means for analyzing the cell-containing substance-containing fluid recovered from the cell dissolving apparatus.

In the present invention, the means for analyzing the intracellular substance-containing fluid recovered from the cytolytic device may be a means for analyzing ordinary intracellular substances.

As a specific embodiment, the structure of the cell lysis and cell analysis integrated device is as shown in Fig. In FIG. 2, the specimen is introduced into a cell dissolving apparatus located at the upper part of the cell dissolution and cell analysis integrated device, and after the cell dissolution process, the nucleic acid is introduced into the means for analyzing the intracellular substance immobilized with the nucleic acid, A substance to be analyzed among the intracellular substances is allowed to react with the corresponding nucleic acid, and finally, an analytical sample is extracted from the well to which the nucleic acid is immobilized and analyzed by PCR or the like.

In the present invention, by using the integrated device, the fluid containing the intracellular substance recovered by using the cell lysis apparatus is used as a specimen, and the specimen is introduced into a means for analyzing a normal intracellular substance, By analyzing, cell lysis and analysis can be performed in one device.

The present invention relates to a method for manufacturing a piezoelectric element, which comprises injecting a fluid containing a cell into a microchannel having a piezoelectric layer on an outer wall or a lower portion thereof and applying an alternating current to the piezoelectric layer to generate vibration, The present invention can provide a cell dissolution method and apparatus capable of effectively breaking the cell wall by uniformly transferring the vibration energy of the layer to the fluid inside the microchannel. The cell dissolution method and apparatus of the present invention can reduce the damage of intracellular substances and improve sample analysis efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 schematically shows the structure of a cell dissolving device as one embodiment of the present invention. FIG.
2 schematically shows the structure of a cell lysis and cell analysis integrated device as one aspect of the present invention.
FIG. 3 shows a cell dissolving apparatus actually manufactured according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  1: Production of cell dissolving apparatus of the present invention

An injection port for injecting a fluid containing cells as shown in Fig. 1; A microchannel connected to the injection port and having a piezoelectric layer on an outer wall or a lower portion thereof; An electrode for applying an alternating current to the piezoelectric layer; And an outlet connected to the microchannel and through which the fluid containing the intracellular substance that has passed through the microchannel is recovered (not shown in the drawings). At this time, the width of the microchannel was 100 μm and the length was 480 mm. Further, PVDF was used as a material of the piezoelectric layer, and the thickness of the piezoelectric layer was 1 占 퐉. At this time, polycarbonate was used as the material of the cell dissolving apparatus except for the piezoelectric layer. Chromium was used as an electrode.

The actual cell lysing apparatus is shown in Fig.

Example  2: Cell lysis using the cell lysis apparatus of the present invention

Cell lysis was performed using the cell lysis apparatus prepared in Example 1 above.

Specifically, a fluid containing cells (SiHa, Korean Cell Line Bank) is injected into an inlet of a cell dissolving apparatus, and an electrode is connected to an external AC power source to generate an alternating current. As a result, vibration of the piezoelectric layer occurs, Lt; RTI ID = 0.0 > cells. ≪ / RTI > The fluid was withdrawn from the outlet of the cell lysis device. The frequency of the alternating current applied to the piezoelectric layer was 10 kHz.

Example  3: Cell analysis using the cell lysis apparatus of the present invention

2, a fluid containing cells (SiHa, Korean Cell Line Bank) was put into a cell dissolving apparatus located at the upper part of the cell dissolution and cell analysis integrated device as shown in FIG. 2, and the electrode was connected to an external AC power source, Generating an alternating current having a frequency through which the fluid containing the cells is passed through the microchannel while causing the vibrations of the piezoelectric layer to pass therethrough and then flowing into the means for analyzing the intracellular material to which the nucleic acid is immobilized . Subsequently, through the well-to-be fluid transfer structure, the substance to be analyzed among the intracellular substances was allowed to react with the corresponding nucleic acid, and finally the analytical sample was extracted from the well to which the nucleic acid was immobilized and analyzed by PCR.

Claims (9)

As a cell lysis method,
And a piezoelectric layer having a thickness of 1 nm to 10 占 퐉 only on the outer wall and / or the lower portion and made of only piezoelectric material, wherein vibration energy generated when alternating current having a frequency of 100 Hz to 900 kHz is applied to the piezoelectric layer, Injecting a fluid containing cells into a microchannel having a width of 1 탆 to 1 탆 so as to break the cell walls of the cells in the fluid (step 1); And
And a step (2) of passing a fluid containing the cell through a microchannel and causing cell lysis by applying an alternating current having a frequency of 100 Hz to 900 kHz to the piezoelectric layer to generate vibration, Way. 2. The method of claim 1, further comprising the step of recovering an intracellular material-containing fluid (step 3) that has passed through the microchannel. The method of claim 1, wherein the microchannel has a length of 10 mm to 1000 mm. The method of claim 1, wherein the piezoelectric material constituting the piezoelectric layer is _{α-AlPO 4, α-SiO} 2, LiTaO 3, LiNbO 3, SrxBayNb 2 O 8, Pb 5 -Ge 3 O 11, Tb 2 (MoO 4) _{3, Li 2 B 4 O 7} , CdS, ZnO, Bi 12 SiO 20, Bi 12 GeO 20, PZN-PT [Pb (Zn 2/3 Nb 1/3) O 3 -PbTiO 3], PMN-PT [Pb (Mg _2/3 Nb _1/3 ) O ₃ -PbTiO ₃ ], triglycine sulfate (TGS), lead zirconate titanate, PbZrO ₃ -PbTiO ₃ , PT (PbTiO ₃ ) (PZT-Complex Perovskite), BaTiO ₃ , ZnO, CdS, AlN, polyvinylidene fluoride (PVDF), polyvinylidene fluoride- (VDF-TeFE), PZT-PVDF, PZT-PVDF, PZT-PVDF, - It is formed of silicone rubber (PZT-Silicon Rubber), PZT-Epoxy (PZT-Epoxy), PZT-foamed urethane or mixture thereof Way. 3. The method of claim 2, wherein the intracellular material is DNA, RNA, protein, or a mixture thereof. As a cell dissolving apparatus,
An injection port for injecting a fluid containing cells;
And a control unit connected to the injection port,
And a piezoelectric layer having a thickness of 1 nm to 10 占 퐉 only on the outer wall or the lower portion and made of piezoelectric material only and the vibration energy generated when an alternating current having a frequency of 100 Hz to 900 kHz is applied to the piezoelectric layer uniformly A microchannel having a width of 1 占 퐉 to 1 mm so as to break the cell walls of the cells in the fluid;
An electrode for applying an alternating current having a frequency of 100 Hz to 900 kHz to the piezoelectric layer so as to generate vibration; And
And an outlet connected to the microchannel and through which the intracellular substance-containing fluid having passed through the microchannel is withdrawn. 7. The apparatus of claim 6, wherein the microchannel has a length of 10 mm to 1000 mm. 7. The apparatus of claim 6, wherein the electrode is connected to an external AC power source to generate an AC current. 9. A cell lysing apparatus according to any one of claims 6 to 8; And means for analyzing an intracellular substance-containing fluid recovered from the cell dissolving apparatus.

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