KR101902923B1 - Method for lysing cells or viruses - Google Patents

Abstract

Provides a method for efficiently disrupting a cell or virus using bead-collision.

Description

Methods for lysing cells or viruses {Methods for lysing cells or viruses}

To a method for disrupting cells or viruses using bead-collision.

Cell destruction is a process normally required for biological analysis because it releases cell contents. For example, in order to analyze a nucleic acid in a cell, the cell is usually destroyed to release the nucleic acid, and the released nucleic acid is analyzed.

As a cell destruction method, a bead-beating method is known. The bead-collision method destroys cells by vigorously agitating the cells in the presence of small beads. In a simple form, cells may be disrupted by mixing cells and beads in a test tube and mixing with a vortex mixer. However, such a bead-collision method has a problem that is mainly performed in a table top apparatus.

Even with these conventional techniques, there is still a need for a method for efficiently disrupting cells in a microfluidic device.

The present invention provides a method for effectively disrupting a cell or a virus using bead-collision.

One aspect includes introducing a sample containing cells or virus into a first chamber containing beads; And a step of disrupting the cell or the virus in the sample, wherein the liquid volume fraction (f _L ) of the first chamber into which the beads and the sample are introduced is Wherein the liquid volume fraction is a value obtained by dividing the liquid volume (V _L ) of the first chamber by the net void volume corresponding to the sum of the liquid volume (V _L ) and the void volume (V ₀ ) of the first chamber ≪ / RTI >

The method of disrupting cells or viruses in the sample includes introducing a sample containing cells or virus into a first chamber containing beads.

In the sample introduction step, the first chamber may provide an enclosed space. The first chamber may be, for example, a chamber formed in the microfluidic device. The first chamber contains 1 내지 to 1000,, 1 내지 to 500,, 1 내지 to 300,, 1 내지 to 200,, 1 내지 to 100,, 1 내지 to 50,, 10 내지 to 50,, Mu] l or 20 [mu] l to 40 [mu] l. The first chamber may include an inlet and an outlet. The inlet and outlet may be at the same or different location. At least one of the inlet and the outlet may be fluidly connected to the first chamber through a channel, such as a channel. At least one of the inlet and the outlet may be such that the diameter is smaller than the diameter of the bead such that the bead can not pass. The flow path that fluidly connects one or more of the inlet and outlet to the first chamber may include a region having a diameter smaller than the diameter of the bead so that the bead can not be removed or introduced. At least one of the inlet, the outlet and the flow path may include a weir so that the bead can not be removed or introduced.

Also, the first chamber may be connected to means for agitating the mixture of beads and sample therein, or may be configured to agitate the mixture of beads and sample therein. For example, the first chamber is connected to an ultrasonic generator so that the beads therein can be agitated via ultrasonic waves. The first chamber may be composed of a flexible film at least a part of its wall surface. For example, in the first chamber, at least a part of the wall surface is composed of a flexible film, and the flexible film may constitute a part of the second chamber. That is, the first chamber and the second chamber may be defined by a part or more of a common flexible film. The second chamber may be connected to the pressurization or decompression means. The pressurizing or decompressing means may be a pump. The pump may be a pneumatic pump. By alternately applying pressurization and depressurization to the flexible membrane of the first chamber, the flexible membrane can be vibrated, thereby stirring the beads in the first chamber. The second chamber may also be connected to an ultrasonic generator. When the ultrasonic wave is applied to the flexible film through the ultrasonic generator, the beads in the first chamber can be agitated. The flexible membrane may be made of an elastic body. The flexible membrane may have a thickness of 1 mm to 5 mm, for example, 0.025 to 0.5 mm.

As used herein, the term "agitation " refers to the action of a bead in a mixture of the bead and a sample to induce movement of the bead by applying various kinds of energy, thereby causing bead collision, A collision between the bead and the liquid medium, a collision between the bead and the wall surface, and a collision between the bead and the cell or virus. By such stirring, the cells or viruses in the sample can be disrupted. Crushing may occur due to mechanical impact or shear force, but is not limited to a specific mechanism. Disruption of cells or viruses by stirring such a mixture of beads and a sample is also referred to as bead-beating method hereinafter. The agitating stirring can be performed in a state in which the first chamber is sealed.

The beads may be sufficiently hard to be used as cell disruption means. The beads may be of a solid material or coated with a solid material. The beads may be magnetic or non-magnetic beads. The beads may be beads of one or more of glass beads, metal beads, and metal oxides. The metal oxide may be any one of ZrO ₂ , SiO ₂ , Al ₂ O ₃ , Fe ₂ O ₃ , TiO _2, and mixtures thereof. The mixture may be, for example, a mixture comprising ZrO ₂ and SiO ₂ . The metal beads may be, for example, steel or stainless steel.

The beads do not necessarily have micrometer-scale dimensions, but may be of a size capable of disrupting cells by the operation of the device. For example, the beads may have a length of the shortest width of the beads of 10 nm to 1000 μm, 10 nm to 500 μm, 100 nm to 100 μm, or 1 μm to 500 μm. The beads may be spherical, plate-like, or have a plurality of faces. The beads may comprise a plurality of, for example, two or more, ten, hundred, 1000, 10,000, 100,000 or more than 10 ⁸ of the first chambers. Specifically, the beads may include the above 1-10 ⁸ , for example, 100-10 ⁶ of the chambers. The beads may be pre-introduced at the time of manufacturing the first chamber and can not be discharged through the inlet or outlet. The density of the beads may be D> 1 g / cm 3, for example, 1-20 g / cm 3.

At least one of the wall surface of the first chamber and the surface of the bead may be a substance that binds to a cell or a virus, or a substance to which the substance is bound. The binding substance may be a substance that specifically binds to the cell or virus. The specifically binding material may be selected from the group consisting of an antibody to an antigen, a substrate or inhibitor for an enzyme, an enzyme for a substrate, a receptor for a ligand, a ligand for a receptor, and mixtures thereof. The binding substance may be a substance that binds nonspecifically with the cell or virus. The non-specifically binding substance may be a substance having a hydrophobic property at a water contact angle of 70 DEG to 95 DEG or a substance having at least one amino group. The material having the at least one amino group may be a polymer. The material having the at least one amino group may be PEIM. The substance having a hydrophobic property with a water contact angle of 70 ° to 95 ° may be OTC or DFS. Accordingly, the wall surface of the first chamber and the surface of the bead containing the binding substance can be used for cell or virus isolation by capturing or adsorbing cells or viruses.

In the introducing step, the sample may be one containing at least one of a cell and a virus. The sample may be a biological sample. "Biological sample" refers to an organism or a sample derived therefrom. The biological sample may be, for example, one or more of blood, urine, saliva, and tissue. The cell may be a prokaryotic cell or a eukaryotic cell. The prokaryotic cell may be a bacterial cell. Bacterias can be gram negative or gram positive bacteria. The eukaryotic cell may be a plant cell, an animal cell, or a fungal cell. The cell or virus may be contained in a suitable liquid medium. The liquid medium may be, for example, a cell culture medium, a buffer (e.g. PBS buffer), physiological saline or water. The liquid medium may also include a cell lysate. The cell lysate may be added separately to the chamber after the liquid medium containing the cells is introduced, or may be mixed before being introduced into the chamber and then introduced into the chamber. The cell lysate may be a non-specific cytolytic agent or a specific cytolytic agent. Non-specific cytolytic agents include, for example, one or more selected from the group consisting of surfactants, NaOH, and chaotropic salts. Specific cytolytic agents may include, for example, lysozyme, resourcetapine or penicillin and beta-lactam antibiotics.

In the introducing step, the introduction may be effected by applying a positive pressure to the inlet of the chamber or by applying a negative pressure to the outlet. The application of such a negative pressure or a positive pressure can be effected, for example, by a pump. The pump may be at least one of a peristaltic pump and a pneumatic pump. In addition, the introduction of the cell or virus may be directly performed by a user. For example, it may be injected by the user's pipetting. The amount and rate of introduction may vary depending on the cell to be disrupted, the purpose of cell disruption and the type of subsequent process after cell disruption, and may be suitably adjusted by those skilled in the art.

In the sample introduction step, the sample may be introduced from the inlet of the first chamber and discharged through the outlet of the first chamber. The flow rate can be 10 / / min to 1000 / / min, for example, 100 / / min to 500 / / min.

The method of disrupting a cell or a virus in the sample further includes a step of shaking the mixture of the bead and the sample to destroy the cell or the virus. The stirring may be performed by ultrasonic waves or vibrations of the wall surface of the first chamber. For example, it is possible to vibrate the flexible membrane by applying pressure to the wall surface constituting a part of the first chamber, for example, a wall surface composed of a flexible film, in the order of pressurization and depressurization or vice versa, ≪ / RTI > The flexible film may be oscillated at 0.001 Hz-100 kHz, for example, 0.01 Hz-100 kHz, 0.1 Hz-100 kHz, 1 Hz-100 kHz, 5 Hz-100 kHz or 10 Hz-100 kHz. The pressure may be provided by means for providing power for the inflow and outflow of fluid connected to at least one of the inlet and the outlet. The power providing means may be one capable of inducing movement of fluid, for example, providing positive or negative pressure to the chamber including the pump. The pump may be a micropump that may be implemented in a microfluidic device. The fine pump may be a mechanical or non-mechanical device. The mechanical micropump usually comprises moving parts, which are actuators and membranes or flaps.

The driving force can be generated using piezoelectric, electrostatic, thermo-pneumatic, pneumatic or magnetic effects. Non-mechanical pumps can function by electro-hydrodynamic, electro-osmoticc or ultrasonic flow generation.

The inlet and the outlet may be in fluid communication with the chamber, for example, fluidly connected to the chamber through a microchannel. The microchannel may have a channel width of 1 μm-10 mm, for example, 1 μm-1 mm, or 1 μm-500 μm.

Wherein the liquid volume fraction (f _L ) of the first chamber into which the beads and the sample are introduced is less than or equal to 0.6, wherein the liquid volume fraction is less than or equal to the liquid volume (V _L) of the first chamber ) Divided by the net void volume corresponding to the sum of the liquid volume (V _L ) and the void volume (V _O ) in the first chamber. For example, the liquid volume fraction may be 0.25 to 0.6 or 0.3 to 0.5. Also, the liquid volume fraction may be zero. That is, the cells or viruses in the sample can be disrupted in a substantially moisture-free dry state. It was confirmed that the cell or virus breaking efficiency was significantly higher at a liquid volume fraction (f _L ) of 0.6 or less.

The method of disrupting cells or viruses in the sample may also include removing at least a portion of the liquid in the first chamber to reduce the volume of the liquid prior to the vibration step after sample introduction. The liquid volume reduction key step may include drying the interior of the first chamber. The drying may be performed by flowing air into the first chamber or by applying heat.

The method of disrupting a cell or a virus in the sample may further include washing the surface of the wall surface of the first chamber or the surface of the bead after the sample introduction step to remove the substance not bound to the surface. Washing may be a liquid medium that does not interfere with the binding of the bound cells or viruses. The liquid medium may be water, such as buffer or distilled water, such as PBS. The stirring step may be performed after the washing step.

And drying the surface after the washing step. The phase agitation step may be performed after the drying step. The stirring step may be performed after the drying step with the liquid volume fraction being substantially zero. The drying may be performed by flowing air into the first chamber or by applying heat.

The method of disrupting cells or viruses in the sample may further include introducing a cell or virus disruption solution into the first chamber. The cell solution may be selected from the group consisting of NaOH, KOH, chaotrope, and a surfactant. The crushing solution may be introduced after the drying step. The stirring step may be performed after introducing the crushing solution.

A method for disrupting cells or viruses in a sample, the first chamber comprising: a first chamber; A second chamber; And a flexible membrane between the first and second chambers, the second chamber being a first chamber disposed in a device for cell disruption which is a pressurization and decompression chamber for vibrating the membrane, It may be to vibrate the flexible membrane between the chambers.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a drawing showing one embodiment (20) of a device for cell disruption which can be used in the cell disruption method. Referring to FIG. 1, the apparatus 20 includes a top plate 21, a bottom plate 23, and a membrane 26. The membrane 26 is provided between the upper plate 21 and the lower plate 23. A space provided in a part of the upper plate 21 forms the first chamber 22 and a space provided in a part of the lower plate 23 forms the second chamber 24. [ The first and second chambers 22 and 24 are separated by a membrane 26 so that a membrane 26 is provided between the first and second chambers 22 and 24. In other words, the first chamber 22 is a space defined by the upper plate 21 and the membrane 26, and the first chamber 24 is bounded by the lower plate 23 and the membrane 26 .

The membrane 26 may be a polymer membrane that is flexible and may be, for example, a polydimethylsiloxane (PDMS) membrane. The thickness of the film 26 may be, for example, 1 占 퐉 to 5 mm. The first chamber 22 comprises a plurality of particles for cell disruption. Figure 1 shows a bead 28 with such particles. The first chamber 22 is bordered by the membrane 26 so that the bead 28 can contact the membrane 26. The beads 28 may be microbeads. The plurality of beads 28 are merely an example of micro-means for cell disruption, and other means may be provided in place of the plurality of beads 28. One size of the beads 28 may be, for example, 1-500 占 퐉. The density D of the beads 28 in the first chamber 22 may be D> 1 g / cm 3, for example the density D may be 1-20 g / cm 3. The beads may be one or more, for example, 10, 100, 1000, 10,000, or 10 ⁹ or more per 1 μl liquid medium. Specifically, the micro means may be included in the chamber in the range of 1-10 ⁸ , for example, 100-10 ⁶ per μl of liquid medium in the chamber. The bead 28 may be a glass bead. The beads 28 may also be metal oxide beads or metal beads. The metal oxide may be any one of ZrO ₂ , SiO ₂ , Al ₂ O ₃ , Fe ₂ O ₃ , TiO _2, and mixtures thereof. The mixture may be, for example, a mixture comprising ZrO ₂ and SiO ₂ . The metal beads may be, for example, steel or stainless steel. If the beads 28 have a composition of glass or metal oxide, surface modification for cell capture or adsorption can be easily performed.

The apparatus 20 includes an inlet 30 and an outlet 32. The diameter of the inlet 30 and the outlet 32 may be less than the diameter of the bead 28. A solution containing the cells to be disrupted enters the first chamber 22 through the inlet 30. The result of the crushing process including the nucleic acid due to cell wall breakage is discharged through the outlet 32. The inlet 30 passes through one wall of the upper plate 21 and is connected to one side of the first chamber 22. The outlet 32 passes through the other wall of the top plate 21 and is connected to the other side of the first chamber.

The second chamber 24 may be a pneumatic chamber containing a fluid, such as air, into which the membrane 26 is periodically or aperiodically pressurized. The high pressure fluid is introduced into the second chamber 24, so that the membrane 26 is pressurized. When the membrane 26 is pressurized, the membrane 26 is convex toward the first chamber 22 to reduce the volume of the space in the first chamber 22 and, when the membrane 26 is depressurized, ) Concave downward. The second chamber 24 has a port 34 which is an inflow passage for fluid to be pressurized inside the chamber and which is a discharge passage of the fluid. By periodically or non-periodically introducing and discharging fluid into the second chamber 24 through the port 34, the membrane 26 can be oscillated periodically or aperiodically. The vibrations of this membrane 26 exert a periodic or aperiodic pressure on the beads 28 in the first chamber 22 eventually in direct contact with the beads 28 or through the solution contained in the first chamber 22 . As a result, motion of the bead 28 is induced, and during the movement of the bead, the beads 28 collide with each other or collide with the wall surface of the chamber. By this movement of the beads 28, the cells introduced into the first chamber 22 are sheared or grinded and the cells are broken. The pressurization and the depressurization of the inside of the second chamber 24 through the supply and discharge of the fluid to the second chamber 24 can be freely controlled within a range in which the frequency of the membrane 26 is 0.001 Hz to 100 kHz.

On the other hand, as shown in FIG. 2, the beads 28 may have an organic film 28A capable of capturing specific or nonspecific cells on its surface. For example, when capturing only specific cells, the surface of the beads 28 can be coated with an antibody or an aptamer. In the case of non-specific cells, they can be captured by the action of hydrophobic or electrostatic forces.

The organic film 28A can be formed by modifying the surface of the beads 28 variously by using an organosilane.

Thus, when there is a means on the surface of the bead 28 that has an affinity for a specific or non-specific cell, the cells introduced into the first chamber 22 can be captured by the bead 28. When the membrane 26 is vibrated in this state, the movement of the beads 28 is caused to strike against each other or against the wall surface. In this process, the cells captured in the beads 28 can be disrupted.

Meanwhile, after supplying a solution containing cells to be crushed into the first chamber 22, a substance capable of increasing the cell crushing effect may be supplied to the first chamber 22, followed by cell crushing. At this time, the substance may be a cell lysis solution. The cell lysate may be, for example, NaOH, KOH, a chaotropic solution, a surfactant, or the like. The concentration of such material to be used can be applied to the PCR without additional purification steps after cell disruption by using a concentration that does not affect subsequent processing, for example, PCR, after cell disruption. If a concentration that affects the PCR is used, further purification steps can be performed. The cell lysate may be supplied after cell lysis to facilitate the release of the nucleic acid.

On the other hand, after the solution containing the cells is supplied to the first chamber 22, it is possible to perform cell disruption without supplying the cell lysis solution to the first chamber 22.

Figure 3 shows a variation of the device 20 of Figure 1. Only the portions different from those in Fig. 1 will be described. The same is applied to the other drawings.

3, the diameter of the inlet 30 and outlet 32 is greater than the diameter of the bead 28. As shown in FIG. A plurality of first protrusions (40) are distributed on the inner surface of the inlet (30). The plurality of first protrusions 40 can be uniformly distributed on the inner surface of the inlet 30. The plurality of first projections 40 may be provided to face each other. The substantial diameter or effective diameter of the inlet 30 is smaller than that of the bead 28 by the first projection 40. The second projection 42 is distributed on the inner surface of the outlet 32. The distribution of the second protrusions 42 may follow the distribution of the first protrusions 40. The effective or effective diameter of the outlet 32 by the second projection 42 may be less than the bead 28. [ The shapes of the first and second projections 40 and 42 may be the same, but they may be different. Instead of providing the first and second projections 40 and 42, the inner surfaces of the inlet 30 and the outlet 32 may be corrugated.

Fig. 4 shows another variation of the device 20 of Fig.

Referring to FIG. 4, the structural features of the inlet 30 may be the same as described in FIG. A filter 44 may be provided in the outlet 32. The filter 44 may be a porous material through which the contents of the disrupted cell can pass. The diameter of the inlet 30 may be less than the diameter of the bead 28, as in FIG.

Figure 5 shows another variation of the device 20 of Figure 1.

Referring to Fig. 5, there are two chambers, i.e., the third and fourth chambers 24A and 24B, in the second chamber 24 of Fig. The third and fourth chambers 24A and 24B are separated by a partition wall 48. The role of the third and fourth chambers 24A and 24B may be the same as that of the second chamber 24. The third chamber 24A has a first port 34A and the fourth chamber 24B has a second port 34B. The role of the first and second ports 34A, 34B may be the same as the port 34 of the second chamber 24. The structural features of the inlet 30 and the outlet 32 may be the same as those of FIG. 3 or 4. The application of pressure can be applied to the first and second ports 34A, 34B simultaneously or sequentially so that the membrane 26 can be vibrated simultaneously or sequentially. In addition, the application of the pressure may apply pressure of the same or different phase to the first and second ports 34A, 34B so that the membranes 26 of each chamber are vibrated in the same or different phases. For example, application of the pressure may be performed by applying a positive pressure to the first port 34A and applying a negative pressure to the second port 34B so that the film 26 and the fourth chamber 24B of the third chamber 24A, To cause the membrane 26 of the membrane 26 to vibrate in opposite phases.

Figure 6 shows another variation of the device 20 of Figure 1.

Referring to Fig. 6, there are three chambers, i.e., third to fifth chambers 24A, 24B, and 24C, at the position of the second chamber 24 in Fig. The role of the third to fifth chambers 24A, 24B, 24C may be the same as the second chamber 24 of Fig. The third and fifth chambers 24A and 24C are separated by a first partition 48A. The fourth and fifth chambers 34B and 34C are separated by a second partition wall 48B. The third to fifth chambers 24A, 24B and 24C have first to third ports 34A, 34B and 34C, respectively. The role of the first to third ports 34A, 34B, 34C may be the same as the port 34 of the second chamber 24. FIG. 6 shows a case where the second chamber 24 of FIG. 1 is divided into three chambers, but the second chamber 24 of FIG. 1 may be divided into three or more chambers. The application of pressure may be applied to the first to third ports 34A, 34B, 34C simultaneously or sequentially so that the membranes 26 of each chamber are vibrated simultaneously or sequentially. In addition, application of the pressure may apply pressure of the same or different phases to the first to third ports 34A, 34B, 34C so that the membranes 26 of each chamber are vibrated in the same or different phases. For example, application of pressure may be performed by applying positive pressure to the first port 34A and the third port 34C and applying negative pressure to the second port 34B to apply pressure to the third chamber and the fifth chamber 24A, To cause the membrane 26 of the second chamber 24 and the membrane 26 of the fourth chamber 24B to vibrate in opposite phases to each other.

1 and 3 to 6, the roles of the first and second chambers 22 and 24 may be changed. The bead 28 may be provided in any one of the first and second chambers 22 and 24 and the other may be used as a chamber into which the fluid for pressurization flows. In addition, when the second chamber 24 is a chamber into which fluid for pressurization flows, the port 34 of the second chamber 24 is connected to a pressure regulator (not shown) which can pressurize or depressurize the interior of the second chamber 24. [ Lt; / RTI > The third to fifth chambers 24A, 24B and 24C are also the same.

According to one embodiment, there is provided a method of efficiently disrupting a cell or virus using bead-collision.

Figures 1 and 3-6 are cross-sectional views of one embodiment of an apparatus that may be used in a cell disruption method.
2 is a cross-sectional view showing a case where an organic film enabling cell capture is present on the bead surface.
7 and 8 are views showing an example of a cell disruption apparatus used in a cell disruption method.
FIG. 9 is a graph showing the cell destruction efficiency according to the change in the liquid volume fraction f _L. FIG.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: liquid volume In fraction  Cell destruction effect

1. Production of device for cell or virus disruption

An apparatus for disrupting cells or viruses is produced by placing a common elastic membrane between two glass chips, each of which has a chamber and a part or all of a channel formed therein, And joining the first glass chip and the second glass chip, thereby completing a cell or virus destruction apparatus using bead-beating.

Specifically, the first and second glass chips were fabricated by forming channels and chambers in a glass wafer through conventional photolithography, etching, and wet etching processes. The amorphous polysilicon layer was subjected to low pressure chemical vapor deposition (LPCVD) to a thickness of 500 nm on the cleaned glass wafer after the 6-inch glass wafer (borosilicate, 700 μm thick) was washed with Piranha solution. Next, a patterning process was performed to expose a part of the deposited polysilicon layer using a photoresist film. The exposed portions of the polysilicon layer were then removed by dry etching. Thereafter, the photoresist film was stripped, and the exposed glass wafer was wet-etched with a hydrofluoric acid solution (HF 49%) to form a channel having a depth and a width of about 100 μm and about 200 μm, respectively. In order to isolate the beads in the etching process for forming the channel, a dam (protrusion: weir) protruding from the inner surface of the channel to the center of the channel by about 20 μm was formed. This dam makes it possible to simultaneously perform the functions of a valve seat and a bead trapping dam.

Next, the polysilicon layer is removed, and a dry film resist (DFT) is coated and patterned. Thereafter, a chamber having a volume of about 15.5 mu l in which the beads are located and holes for the inflow or outflow of fluid were formed by a sand-blasting method. Subsequently, the glass wafer was diced into chips and then cleaned using plasma. Next, using a plasma-activated 254 μm thick PDMS film (Rogers) as an intermediate layer, a fluidic chip (hereinafter also referred to as a first glass chip) and a bead containing a chamber to contain the bead prepared above A pneumatic chip (hereinafter also referred to as a second glass chip) including a chamber to serve as a pneumatic pump is permanently coupled. The PDMS membrane was monolithic and was used as an atuator for bead collision through pneumatic vibration as well as as a tool for fluid flow control, .

Approximately 15-16 mg (about 2x10 ⁵ ) surface-modified glass beads were placed in the resulting bead chamber and the chamber was sealed by placing a PCR-compatible adhesive tape (Applied biosystems). A polycarbonate plate was covered on the glued tape to prevent movement of the tape during the DNA extraction process, for example.

The PDMS membrane operation was controlled by applying positive or negative pressure to the array of solenoid valves (S070-5DC, SMC) in the pneumatic chamber. The valves were connected to an electro-pneumatic-regulator (ITV0030-3BL, SMC) and LabVIEW software (National Instruments). The valve manipulation coupled with the fluid transfer was visualized at each step with the LabVIEW interface to monitor the nucleic acid extraction process.

Figs. 7 and 8 are views showing a cell disruption apparatus used in this embodiment. Fig. 7 is a cross-sectional view of a bead-impact device for inducing bead collision through vibration of a PDMS film. 7, protrusions 40 and 42 are formed in the inlet 40 and the outlet 42, respectively, and the inlet 40 and the outlet 42 are connected to the inlet port and the outlet port through the liquid channels 48 and 50, respectively. And valve seats 44 and 46 are formed on the upper plate of the inlet port 40, the outlet port 42 and the outlet port 42. Pneumatic chambers 34A and 34D are formed on the lower plate corresponding thereto .

8 is an enlarged view of a monolithic glass-PDMS-glass device composed of three layers and its fluid and pneumatic components.

2. Glass Bead  Modification

Glass beads (Polysciences, Inc.) having a diameter of 30 to 50 μm were washed with a Piranha solution, thoroughly washed with distilled water, filtered and vacuum dried.

Next, 5% (v / v) trimethoxysilylpropyl modified polyethyleneimine (Gelest, Inc.) solution in ethanol was prepared as a bead surface modification solution. The beads were immersed in the prepared solution and allowed to react for 2 hours while gently mixing. The beads were then filtered and washed three times with fresh ethanol. The final recovered glass beads were incubated in an oven at 110 캜 for 50 minutes. As a result, a glass bead having a surface coated with polyethyleneimine (PEIM) was obtained. Since the PEIM has non-specific binding properties to cells, the glass beads can be used to nonspecifically separate cells.

3. Nucleic Acid Extraction Experiment

1 ml of sodium acetate buffer (50 mM, pH 4, Sigma-Aldrich) containing 10 ⁶ CFU / ml of S. aureus or MRSA as a sample, 0.5 ml TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0, Ambion ) And 10 [mu] l or 20 [mu] l NaOH (0.02 N, Sigma-Aldrich) for disruption were pre-dispensed in a liquid reservoir. The liquid solution was conveyed in a pressure-driven manner and its working liquid pressure was determined in a preliminary experiment. First, the sample solution was passed through a chamber filled with beads at a flow rate of about 200 l / min at 30 kPa while applying pressure to the PDMS membrane at 150 kPa. The flown-through solution was recovered to evaluate cell capture efficiency. After loading the initial sample, the chambers filled with the beads were washed by flowing the wash solution at a flow rate of about 500 l / min (80 kPa) and air dried at 100 kPa for 30 seconds. To disrupt the captured cells, 6 μl of the lysis solution (0.02 N NaOH) was injected and the valves on both sides of the chamber filled with the beads were closed. Next, the solenoid valve was adjusted through the LabVIEW program to adjust the pressure of the two pneumatic chambers to 80 kPa and -80 kPa, respectively, to cause the PDMS membrane to vibrate for 5 minutes at a frequency of 10 Hz, Respectively. After the crushing process, the inlet and outlet were opened and an oil pressure of 100 kPa was applied, and further 4 또는 or 14 의 NaOH solution was injected to recover cell lysate through the outlet. Thus, a cell lysate containing a total of 10 또는 or 20 의 of nucleic acid was obtained. The whole process took less than 20 minutes. No additional DNA purification steps were followed.

Positive lysis control (PLC) and negative lysis control (NLC) experiments were prepared as follows.

PLC experiments were carried out by two types of bench top fracturing methods, an enzymatic method and a bead - impact method. 1 ml S. aureus samples having two different concentrations, i.e., 10 ⁴ CFU / ml and 10 ⁶ CFU / ml were centrifuged at 13,200 rpm for 20 minutes in a microcentrifuge tube to precipitate the cells, . The precipitated pellets were each treated in the above manner. For the enzymatic method, the cell pellet was incubated with lysostaphin solution (200 mg / mL, Sigma) for 30 min at 37 ° C and applied to a Qiagen DNA extraction kit (Cat 51304, QIAamp DNA Mini Kit) , And 20 μl of purified DNA solution was obtained as described in its protocol. In the case of the bench top bead-collision method, 30 mg of bare glass beads and 20 mL of disruption solution (ie, 0.02 N NaOH solution or distilled water) were added to the cell pellet, Fisher Scientic) was vigorously vortexed for 5 min at full speed. After simple centrifugation, the extracted DNA solution (cell lysate) was recovered. As an NLC experiment, the cell pellet was vortexed vigorously for 5 minutes at full speed (3,200 rpm) using only distilled water under the condition of no glass beads and using a universal probe (GENIE 2, Fisher Scientic). The target sequence was amplified using the resulting control sample, PLC and NLC, and the amplified result was compared with the result of amplifying the target sequence using the DNA solution obtained by the bead-impact device as a template. For accurate comparison, the total number of S. aureus injected into the chamber and the final DNA elution volume were adjusted to be always equal to that of the sample.

4. Real time PCR  Amplification

Real-PCR was performed on a GenSpector ^R TMC-1000 instrument (Samsung Electronics) to perform cell disruption and the resulting DNA quality as a result. 3 (SEQ ID NO: 1) and reverse: 5'-ATG ACC AGC TTC GGT ACT ACT AAA GAT-3 'specific for the SA442 region of the S. aureus genome (forward: 5'-GTT GCA TCG GAA ACA TTG TGT- (SEQ ID NO: 2), GeneBank accession number AF033191) and a primer set specific for the mecA fragment of the MRSA genome (forward: 5'-ACG AGT AGA TGC TCA ATA- ATG ACG CTA TGA T-3 '(SEQ ID NO: 4) and GeneBank accession number EF190335.1) were designed using Primer3 software (Whitehead Institute / MT Center for Genome Research).

Following the PCR reaction mixture (about 2㎕) having a final concentration was prepared as: 0.4μM Taqman probe ((SEQ ID NO: FAM-5'-TGT ATG TAA AAG CCG TCT TG-3'-MGB-NFQ against S. aureus No. 5) and FAM-5'-CCA ATC TAA CTT CCA CAT ACC ATC T-3'-BHQ1 (SEQ ID NO: 6) for MRSA), 1X Z- Tag buffer (Takara Bio), 1 μM each primer (Applied Biosystems Or Sigma), 0.05 U Z- Taq polymerase (Takara Bio), 0.2 mM dNTP (Takara Bio), 0.5 μl PCR feed (Ambion), and 1 μl extracted DNA solution. After loading the mixture into the PCR chip, thermal cycling was performed by a denaturation step at 95 DEG C for 1 minute and an extension step at 4O < 0 > C for 4 seconds. The size of PCR amplicon was designed to be 72 bp and 98 bp for S. aureus and MRSA, respectively. The size of the PCR amplicon was further confirmed by gel electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies).

5. Experimental results

(1) Experimental results on control samples

The cell crushing and / or DNA purification effect was evaluated by the threshold cycle (Ct). The Ct values for the positive control samples are shown in Table 1. The values in Table 1 are repeated three times for each cell number.

Number of applied S. aureus cells (CFU)

Bench Top Bead - How to crash

Enzyme method
(Resource tapin)
NaOH (0.02N) Distilled water About 10 ⁴ 30.5 ± 0.35 34.5 ± 0.26 31.6 ± 0.56 About 10 ⁶ 23.7 ± 0.29 26.7 ± 0.15 25.7 ± 1.43

Typically, a bench top bead-impact method is performed with a disruption solution comprising a surfactant or chemical agent to increase the fracture efficiency. In this example, a NaOH solution (0.02 N) which was found not to interfere with PCR amplification without further purification was selected. The bead-impact effect is further divided by NaOH solution and distilled water, indicating that NaOH contributes to DNA extraction efficiency by chemically destroying the membrane. For enzymatic disruption of S. aureus, resource tapin was chosen because it specifically cleaves the crosslinking pentaglycine bridge in the cell wall of staphylococci. The bench top bead-collision method using NaOH showed the same or better results for the enzyme-based DNA extraction method. Therefore, the cell or viral shredding device used was evaluated by comparison with a benchtop vortexing machine. Since the optical density measurement is an approximate cell quantification method, the Ct values of the PLC changed daily with a standard deviation of about 1.5 at the same optical density. The Nt sample vortexed with distilled water showed a Ct value of about 31.5 for 10 ⁶ CFU / ml sample.

(2) Results of cell capture

The basic operating procedure in this example is as follows: (1) cell capture on glass beads, (2) washing and drying, (3) cell disruption using situ-bead-collision, (4) Lt; / RTI > Bacterial cells can be captured on a solid substrate in a specific or non-specific manner.

Nonspecific cell capture methods using surface thermodynamics or electrostatic interaction, along with cell specific immunoaffinity methods, can capture pathogenic bacterial cells. In this example, long-range Coulomb electrostatic interactions were tested by modifying the glass beads electrically positively. The surface of the glass beads was derivatized to have a positive amine group in the reaction with organosilane compounds containing PEIM.

After filling the interior of the microchamber with surface modified glass beads, a 1 ml sample solution containing S. aureus cells (10 ⁶ CFU / mL) was passed through the chamber. First, the role of electrostatic interactions was evaluated by comparing unmodified glass beads with modified glass beads. As a result, the Ct value of the DNA eluted from the modified glass beads was smaller by 2 than the Ct value of the unmodified glass beads. This result indicates that the cell capture efficiency is increased by a factor of four by surface modification (ie, the 1 Ct difference represents a 2-fold difference from the initial template copy number).

In order to obtain more quantitative data on the cell trapping ability of the device for cell disruption used in this example, the passed sample solution was recovered, centrifuged, and transferred to a bench top with a glass bead and NaOH solution as in PLC (Hereinafter referred to as "AC"). The Ct value of AC was compared to the Ct value (hereinafter referred to as "AE ") of the eluted DNA solution obtained from appropriate manipulation of the bead-impact apparatus. The difference between them was used as a tool to evaluate cell capture efficiency and capacity.

With various concentrations of 1 mL S. aureus (i.e., 10 ^4, 10 ^5, 10 ^6, 10 ⁷ and 10 ⁸ CFU / mL) when applied to, Ct values between AC and AE difference is 10 ³ CFU / mL to Maintained at about 5 over 10 ⁷ CFU / mL and dropped to about 2.5 for 10 ⁸ CFU / mL. Therefore, the capture efficiency was about 90% in that a difference of ten times the number of initial template copies leads to a change in Ct value of about 3.3. In addition, the bead-filled device produced has a capacity of 10 ⁷ CFU S. aureus or greater. After cell harvesting, the bead-filled microchambers were washed and air-dried to complete exchange with the rinsing solution. These results indicate that the applied cell capture method is suitable for demonstrating in-situ bead-collisional breakdown of the captured cells instead of releasing the captured cells. Other cell capture methods, including immunoaffinity, can be incorporated into the apparatus of this embodiment by employing suitable solid surface modification chemistries.

(3) liquid Volume fraction  ( f _L )this Bead - Effect on collision cell disruption

The effect of various factors on bead-collision cell disruption was confirmed. First, experiments were conducted on the membrane vibration frequency (5 Hz to 10 Hz), membrane driving pressure (20 kPa to 80 kPa) and pneumatic displacement chamber (100 μm-200 μm) , But not statistically significant. Next, the effect of liquid viscosity on bead-collision cell disruption was evaluated by measuring the liquid volume fraction (f _L ) defined as the volume of the fracture solution versus the volume of the bead-filled microchambers during the bead- . The final eluted DNA volume was adjusted to the same 20 μl by adjusting the additional NaOH solution when eluting the extracted DNA from the chamber. FIG. 9 is a graph showing the cell destruction efficiency according to the change in the liquid volume fraction f _L. FIG. As shown in FIG. 9, the liquid volume fraction (f _L ) was the most important factor in determining cell crushing efficiency. The liquid volume fraction (f _L ) was excellent in cell crushing efficiency at 0.6 or less, for example, 0.5 or less, or 0.3 to 0.5. Even when the liquid volume fraction (f _L ) was 0, the cells could be efficiently broken. This is believed to be due to the fact that the viscosity of the liquid (NaOH) is greater than the viscosity of the air by more than 100, so that the relative amount of liquid solution (NaOH) greatly affects the viscosity of the mixed liquids (gas and liquid).

20: Apparatus for crushing cells or viruses 21, 23: upper and lower plates
22, 24, 24A, 24B, 24C: first to fifth chambers
26: Membrane 28: Microbead (bead)
28A: organic film 30: inlet
32: outlet 34: port
34A, 34B and 34C: first to third ports
40, 42: first and second projections 48: partition wall
48A and 48B: first and second partition walls

<110> Samsung Electronics Co. Ltd. <120> Method for lysing cells or viruses <130> PN092648 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> S.aureus <400> 1 gttgcatcgg aaacattgtg t 21 <210> 2 <211> 27 <212> DNA <213> S. aureus <400> 2 atgaccagct tcggtactac taaagat 27 <210> 3 <211> 18 <212> DNA <213> MRSA <400> 3 acgagtagat gctcaata 18 <210> 4 <211> 19 <212> DNA <213> MRSA <400> 4 ggaataatga cgctatgat 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Taqman probe for S. aureus <220> <221> misc_feature <222> (1) <223> 5 'terminal is labeled with FAM <220> <221> misc_feature <20> <223> 3'-terminal is labeled with MGB-NFQ <400> 5 tgtatgtaaa agccgtcttg 20 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Taqman probe for MRSA <220> <221> misc_feature <222> (1) <223> 5'-terminal is labeled with FAM <220> <221> misc_feature <222> (25) <223> 3'-terminal is labeled with BHQ1 <400> 6 ccaatctaac ttccacatac catct 25

Claims (17)

Introducing a sample containing cells or virus into a first chamber containing beads; And
A method for disrupting cells or viruses in a sample comprising the steps of stirring a mixture of the beads and a sample to destroy cells or viruses, wherein the liquid volume fraction (f _L ) of the first chamber into which the beads and the sample are introduced is 0.6 , Wherein the liquid volume fraction is a value obtained by dividing the liquid volume (V _L ) of the first chamber by the net void volume corresponding to the sum of the liquid volume (V _L ) and the void volume (V _O ) of the first chamber Wherein the stirring comprises: a first chamber; And a second chamber including a flexible membrane constituting a wall surface of the first chamber and constituting a wall surface of the second chamber, wherein in the apparatus for cell disruption, by alternately applying pressurization and depressurization to the flexible membrane, And vibrating the flexible membrane. The method of claim 1, wherein the liquid volume fraction is zero or 0.3 to 0.5. The method of claim 1, wherein the inlet or outlet of the first chamber is such that the diameter is less than the diameter of the bead such that the bead can not pass. The method of claim 1, wherein at least one of the wall surface of the first chamber and the surface of the bead is bonded to a material that binds to the cell or virus. 5. The method of claim 4, wherein the binding substance is a substance that binds specifically or non-specifically binds to the cell or virus. 6. The method of claim 5, wherein the specifically binding agent is selected from the group consisting of an antibody to an antigen, a substrate for an enzyme, an enzyme for a substrate, a receptor for a ligand, a ligand for a receptor, . 6. The method of claim 5, wherein the nonspecifically binding material is a material having a water contact angle of 70 ° to 95 ° or a material having at least one amino group. 5. The method of claim 4, wherein the sample introduction step includes flowing the sample from the inlet of the first chamber and discharging the sample through the outlet of the first chamber. 2. The method of claim 1, comprising removing at least a portion of the liquid in the first chamber to reduce the volume of liquid prior to stirring after sample introduction. 10. The method of claim 9, wherein reducing the liquid volume comprises drying the interior of the first chamber. 11. The method of claim 10, wherein the drying is accomplished by flowing air through the first chamber or by applying heat. 5. The method of claim 4, further comprising, after the sample introduction step, cleaning the surface of the wall surface of the first chamber or the surface of the bead to remove material unbound to the surface. The method of claim 1, further comprising introducing a solution for disrupting the cell or virus into the first chamber. 14. The method according to claim 13, wherein, in the step of shaking the cell or the virus by stirring the mixture of the bead and the sample, the stirring is performed after introduction of the solution for crushing. delete delete The method of claim 1, wherein the vibration vibrates the flexible film at 0.001 Hz to 100 kHz.

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