KR20120116245A - Method for lysing cells or viruses - Google Patents

Abstract

PURPOSE: A pulverizing method for cells or viruses is provided to efficiently pulverize cells or viruses using bead-collision. CONSTITUTION: A pulverizing method for cells or viruses in cells comprises the following steps: introducing a sample in which cells or viruses are included to a first chamber in which beads are included; and pulverizing the cells or viruses by agitating the mixture including the beads and the samples. The liquid volume fraction among the first chamber is 0.6 or less. The liquid volume fraction is a value in which the liquid volume divides the total sum of the liquid volume and the air gap volume. The entrance or the exit of the first chamber is formed for beads not to be passed. One or more of the walls of the first chamber and the bead surface combine with materials which combine with the cells or viruses. The combining material is a material which specifically or non-specifically combines with the cells or viruses.

Description

Method for lysing cells or viruses

A method of disrupting cells or viruses using bead-collision.

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

As a cell destruction method, a bead-beating method is known. Bead-collision disrupts cells by vigorously stirring the cells in the presence of small beads. A simple form is to disrupt cells by mixing cells and beads in a test tube and mixing with a vortex mixer. However, this bead-collision method has a problem mainly performed in tabletop equipment.

Even with this prior art, there is still a need for a method for efficiently disrupting cells in a microfluidic device.

Provided are methods for efficiently disrupting cells or viruses using bead-collision.

One aspect includes introducing a sample containing cells or viruses into a first chamber containing beads; And disrupting cells or viruses in the sample by agitating the mixture of beads and samples, wherein the liquid volume fraction (f _L ) in the first chamber into which the beads and samples are introduced is determined. It is 0.6 or less, wherein the liquid volume fraction is calculated by dividing a net void volume corresponding to the sum of the fluid volume (V _L) and a pore volume (V _O) of the first chamber to a liquid volume (V _L) of the first chamber Provide a method.

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

In the sample introduction step, the first chamber may be to provide a closed space. The first chamber may be, for example, a chamber formed in the microfluidic device. The first chamber is 1 to 1000 µl, 1 to 500 µl, 1 to 300 µl, 1 to 200 µl, 1 to 100 µl, 1 to 50 µl, 10 to 50 µl, 10 to 40 µl. Or may have an internal space volume of 20 μl to 40 μl. The first chamber can include an inlet and an outlet. The inlet and outlet may be at the same or different location location. At least one of the inlet and outlet may be fluidly connected to the first chamber via a channel, such as a channel. At least one of the inlet and the outlet may have a diameter smaller than the diameter of the beads so that the beads cannot pass therethrough. The flow path for fluidly connecting at least one of the inlet and the outlet with the first chamber may include a region whose diameter is smaller than the diameter of the bead such that the bead cannot be removed or introduced therethrough. At least one of the inlet, the outlet, and the passage may include a dam so that the beads may not be removed or introduced through the dam.

In addition, the first chamber may be connected to a means for stirring the mixture of the beads and the sample therein or formed to agitate the mixture of the beads and the sample therein. For example, the first chamber is connected to an ultrasonic generator so that the beads therein may be agitated via ultrasonic waves. At least a portion of the wall of the first chamber may be composed of a flexible membrane. For example, at least a part of the wall of the first chamber may be composed of a flexible membrane, and the flexible membrane may constitute a part of the second chamber. That is, at least a portion of the first chamber and the second chamber may be defined by a common flexible film. The second chamber may be connected with the pressurizing or decompressing means. The pressurizing or depressurizing means may be a pump. The pump may be a pneumatic pump. The flexible membrane can be vibrated by alternating application of pressure and pressure to the flexible membrane of the first chamber, thereby allowing the beads in the first chamber to be stirred. The second chamber may also be connected to the ultrasonic generator. When ultrasonic waves are applied to the flexible membrane through the ultrasonic generator, the beads in the first chamber may be stirred. 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 a bead collision, for example, a collision between the beads and beads, by inducing the movement of the beads by applying various kinds of energy to the mixture of the beads and the sample, Causing collisions between the beads and the liquid medium, collisions between the beads and the wall, and collisions between the beads and the cells or viruses. By such agitation, cells or viruses in the sample can be disrupted. Fracture may occur due to mechanical collision or shear force, but is not limited to a specific mechanism. The disruption of cells or viruses by agitation of the mixture of beads and samples is also referred to as bead-beating method below. Singh stirring may be performed in a closed state of the first chamber.

The beads may be hard enough to be used as cell disrupting means. The beads may be made of a solid material or coated with a solid material. The beads can be magnetic or nonmagnetic 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 dimensions at the micrometer level, but may have dimensions that can disrupt cells by operation of the device. For example, the beads may have a length 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-shaped, or in the form of a plurality of faces. The beads may include a plurality of first chambers, for example, two or more, 10, 100, 1000, 10,000, 100,000, or 10 ⁸ or more. Specifically, the beads may be included in the 1-10 ⁸ , for example, 100-10 ⁶ of the chamber. The beads may be introduced in the manufacture of the first chamber in advance so that they cannot 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 walls of the first chamber and the surface of the beads may be made of a material that binds to a cell or virus or to which the material is bound. The binding agent may be a substance specifically binding to the cell or virus. The specifically binding agent may be selected from the group consisting of antibodies to antigens, substrates or inhibitors for enzymes, enzymes for substrates, receptors for ligands, ligands for receptors, and mixtures thereof. The binding agent may be a substance that specifically binds to the cell or virus. The non-specifically binding material may be a material having a hydrophobicity of water contact angle of 70 ° to 95 ° or a material having one or more amino groups. The material having at least one amino group may be a polymer. The material having at least one amino group may be PEIM. The hydrophobic material having a water contact angle of 70 ° to 95 ° may be OTC or DFS. Thus, the wall of the first chamber containing the binding material and the surface of the beads can be used for cell or virus isolation by trapping or adsorbing the cells or viruses.

In the introducing step, the sample may contain one or more of cells and viruses. The sample may be a biological sample. "Biological sample" refers to an organism or a sample derived therefrom. The biological sample can be, for example, one or more of blood, urine, saliva and tissue. The cell may be a prokaryotic or eukaryotic cell. The prokaryotic cell may be a bacterial cell. Bacteria 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 (eg PBS buffer), physiological saline or water. The liquid medium may also include cell lysate. The cell lysate may be additionally introduced into the chamber separately after the liquid medium containing the cells is introduced, or may be mixed before entering the chamber and then introduced into the chamber. The cell lysate may include a non-specific cell lysate or a specific cell lysate. Non-specific cell lysates include, for example, one or more selected from the group consisting of surfactants, NaOH and chaotropic salts. Specific cell lysates may include, for example, lysozyme, lysostapine or penicillin and beta-lactam antibiotics.

In the introducing step, the introduction may be made by applying a positive pressure to the inlet of the chamber or a negative pressure to the outlet. Such application of negative or positive pressure may be achieved by, for example, a pump. The pump may be one or more of a peristaltic pump and a pneumatic pump. In addition, the introduction of the cell or virus may be directly injected by the user. For example, it may be injected by pipetting of the user. The amount and rate of influx may vary depending on the cells to be crushed, the purpose of cell crushing and the type of subsequent process after cell crushing, and will be appropriately adjusted by those skilled in the art.

The sample introduction step may include the step of introducing the sample from the inlet of the first chamber to discharge through the outlet of the first chamber to flow. The flow rate can be 10 μl / min to 1000 μl / min, for example 100 μl / min to 500 μl / min.

The method of disrupting cells or viruses in the sample also includes agitating the mixture of beads and samples to disrupt the cells or viruses. The stirring may be performed by ultrasonic waves or vibration of the wall of the first chamber. For example, the flexible membrane is vibrated by applying pressure in the order of pressurization and decompression or vice versa to a wall surface constituting a part of the first chamber, for example, a wall composed of the flexible membrane, and thus the beads and the sample. It may be to stir the mixture of. The flexible membrane may be vibrated 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. Pressure may be provided by means for providing power for inlet and outlet of fluid connected to one or more of the inlets and outlets. The power supply means may be one capable of inducing the movement of the fluid, for example, capable of providing a positive pressure or a negative pressure to the chamber including a pump. The pump may be a micropump that may be implemented in a microfluidic device. The micropump can be a mechanical or non-mechanical device. The mechanical micropump typically includes 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 may function by electro-hydrodynamic, electro-osmoticc or ultrasonic flow generation.

The inlet and outlet may be in fluid communication with the chamber, for example fluidly connected to the chamber via 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.

In a method of disrupting cells or viruses in the sample, the liquid volume fraction f _L in the first chamber into which the beads and the sample are introduced is 0.6 or less, wherein the liquid volume fraction is the liquid volume V _L in the first chamber. ) Is 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. In addition, the liquid volume fraction may be zero. That is, the cells or viruses in the sample can be broken down in a dry state substantially free of moisture. The liquid volume fraction (f _L ) was found to be significantly higher in the efficiency of cell or virus disruption at 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 before the vibrating step after sample introduction. The reducing of the liquid volume may include drying the inside of the first chamber. The drying may be made by flowing air in the first chamber or by applying heat.

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

After the washing step may further comprise the step of drying the surface. The phase stirring step may be performed after the drying step. The stirring step may be performed in a state where the liquid volume fraction is substantially zero after the drying step. The drying may be made by flowing air in the first chamber or by applying heat.

The method for 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, chaotropes and surfactants. The crushing solution may be introduced after the drying step. The stirring step may be performed after introducing the shredding solution.

A method of disrupting cells or viruses in a sample, the method 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 an apparatus for cell disruption that is a pressurized and reduced pressure chamber for vibrating the membrane. It may be to vibrate the flexible membrane between the chambers.

1 is a diagram showing one embodiment 20 of a cell disruption apparatus that can be used in the cell disruption method. Referring to FIG. 1, the apparatus 20 includes an upper plate 21, a lower plate 23, and a membrane 26. The film 26 is provided between the upper plate 21 and the lower plate 23. The space provided in a portion of the upper plate 21 forms the first chamber 22, and the space provided in a portion of the lower plate 23 forms the second chamber 24. The first and second chambers 22, 24 are separated by a membrane 26, so it can be seen that the membrane 26 is provided between the first and second chambers 22, 24. In detail, the first chamber 22 is a space defined by the upper plate 21 and the film 26, and the first chamber 24 is defined by the lower plate 23 and the film 26. It is a space.

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 μm-5 mm. The first chamber 22 includes a plurality of particles for cell disruption. 1 shows beads 28 with these particles. Since the first chamber 22 is bounded by the membrane 26, the beads 28 may contact the membrane 26. Bead 28 may be a microbead. The plurality of beads 28 is only one example of micro means for cell disruption, and other means may be provided instead of the plurality of beads 28. The size of one bead 28 may be, for example, 1-500 μm. 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, 10000, or 10 ⁹ or more per 1 μl of the liquid medium. Specifically, the micro means may be included in the chamber of 1-10 ⁸ , for example 100-10 ⁶ per 1 μl of the liquid medium in the chamber. Bead 28 may be a glass bead. In addition, the beads 28 may 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 beads 28 have a composition of glass or metal oxide, surface modification for cell capture or adsorption can be readily performed.

The apparatus 20 includes an inlet 30 and an outlet 32. The diameter of the inlet 30 and the outlet 32 may be smaller than the diameter of the beads 28. The solution containing the cells to be crushed is introduced into the first chamber 22 through the inlet port 30. Through the outlet 32, a crushed product including nucleic acid due to cell wall rupture flows out. The inlet 30 penetrates one wall of the upper plate 21 and is connected to one side of the first chamber 22. The outlet 32 penetrates 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 including a space into which fluid, such as air, that presses the membrane 26 periodically or aperiodically is introduced. When the high pressure fluid flows into the second chamber 24, the membrane 26 is pressurized. When the membrane 26 is pressurized, the membrane 26 is convex toward the first chamber 22, so that the volume of the space in the first chamber 22 is reduced, and when the pressure is reduced, the membrane 26 becomes the second chamber 24. ) Will be concave downwards. The second chamber 24 has a port 34 which is an inflow passage of the fluid to pressurize the inside of the chamber and a discharge passage of the fluid. The membrane 26 may be vibrated periodically or aperiodically by introducing and discharging fluid into the second chamber 24 through the port 34 periodically or aperiodically. This vibration of the membrane 26 results in periodic or non-periodic pressure on the beads 28 in the first chamber 22 through direct contact with the beads 28 or through a solution contained in the first chamber 22. . As a result, the motion of the beads 28 is induced, and during the movement of the beads, 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 ground so that the cells are crushed. Pressurization and depressurization in the second chamber 24 through supply and discharge of the fluid to the second chamber 24 can be freely controlled in a range in which the frequency of the membrane 26 becomes 0.001 Hz to 100 kHz.

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

The organic layer 28A may be formed by modifying the surface of the beads 28 in various ways using an organosilane.

As such, when there is a means having affinity for specific or non-specific cells on the surface of the beads 28, the cells introduced into the first chamber 22 may be captured in the beads 28. When the membrane 26 vibrates in this state, it causes the movement of the beads 28 to hit each other or the wall. In this process, the cells captured in the beads 28 may be broken.

Meanwhile, after supplying a solution including cells to be crushed to the first chamber 22, and then supplying a substance capable of increasing cell crushing effect to the first chamber 22, cell crushing may be performed. In this case, the substance may be a cell lysis solution. The cell lysate may be, for example, NaOH, KOH, chaotrop solution, surfactant, or the like. Concentrations of these substances can be applied to PCR without further purification after cell disruption by using concentrations that will not affect subsequent processes after cell disruption, such as PCR. If concentrations affecting PCR are used, further purification can be performed. The cell lysate may be supplied after cell disruption and used to easily release the nucleic acid.

On the other hand, the solution containing the cells may be supplied to the first chamber 22, and then cell disruption may be performed without additionally supplying the cell lysate to the first chamber 22.

3 shows a variant of the apparatus 20 of FIG. 1. Only parts different from FIG. 1 will be described. The same applies to other drawings.

Referring to FIG. 3, the diameters of the inlet 30 and the outlet 32 are larger than the diameter of the beads 28. The plurality of first protrusions 40 are distributed on the inner surface of the inlet port 30. The plurality of first protrusions 40 may be uniformly distributed on the inner surface of the inlet 30. The plurality of first protrusions 40 may be provided to face each other. By the first projection 40, the substantial diameter or the effective diameter of the inlet 30 is smaller than that of the beads 28. The second protrusion 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. By the second protrusion 42 the actual or effective diameter of the outlet 32 can be smaller than the beads 28. The shapes of the first and second protrusions 40 and 42 may be the same, but may be different. Instead of having the first and second protrusions 40 and 42, the inner surfaces of the inlet 30 and the outlet 32 themselves may be convexly corrugated.

4 shows another variant of the apparatus 20 of FIG. 1.

Referring to FIG. 4, the structural features of the inlet 30 may be the same as described with reference to FIG. 3. The filter 44 may be provided at the outlet 32. The filter 44 may be a porous material through which the contents of the crushed cells can pass. The diameter of the inlet 30 may be smaller than the diameter of the beads 28 as in FIG. 1.

5 shows another variant of the apparatus 20 of FIG. 1.

Referring to FIG. 5, there are two chambers, namely third and fourth chambers 24A and 24B, in the second chamber 24 position of FIG. 1. The third and fourth chambers 24A and 24B are separated by the partition wall 48. The role of the third and fourth chambers 24A and 24B may be the same as 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 in the case of FIG. 3 or 4. The application of pressure may be applied simultaneously or sequentially to the first and second ports 34A, 34B to cause the membrane 26 to vibrate simultaneously or sequentially. In addition, the application of pressure may cause pressure of the same or different phases to be applied to the first and second ports 34A and 34B to cause the membrane 26 of each chamber to vibrate in the same or different phases. For example, the application of pressure applies a positive pressure to the first port 34A, and a negative pressure is applied to the second port 34B so that the film 26 and the fourth chamber 24B of the third chamber 24A are applied. May cause the membrane 26 to vibrate in opposite phases to each other.

FIG. 6 shows another variant of the apparatus 20 of FIG. 1.

Referring to FIG. 6, three chambers, that is, third to fifth chambers 24A, 24B, and 24C, are present at the position of the second chamber 24 of FIG. 1. The role of the third to fifth chambers 24A, 24B, and 24C may be the same as the second chamber 24 of FIG. 1. The third and fifth chambers 24A and 24C are separated by the first partition wall 48A. The fourth and fifth chambers 34B and 34C are separated by the 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, and 34C may be the same as the port 34 of the second chamber 24. 6 illustrates 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 simultaneously or sequentially to the first to third ports 34A, 34B, 34C to cause the membrane 26 of each chamber to vibrate simultaneously or sequentially. In addition, the application of pressure may apply pressure of the same or different phases to the first to third ports 34A, 34B, 34C to cause the membrane 26 of each chamber to vibrate in the same or different phase. For example, the application of pressure applies positive pressure to the first port 34A and the third port 34C, and negative pressure to the second port 34B so that the third and fifth chambers 24A and 24C are applied. The membrane 26 and the membrane 26 of the fourth chamber 24B may be caused to vibrate in opposite phases.

1 and 3 to 6 the role of the first and second chambers 22, 24 may be reversed. That is, the bead 28 may be provided in one of the first and second chambers 22 and 24, and the other may be used as a chamber into which fluid for pressurization is introduced. In addition, when the second chamber 24 is a chamber into which the fluid for pressurization is introduced, the port 34 of the second chamber 24 may pressurize or depressurize the inside of the second chamber 24 (not shown). May be connected). The same applies to the third to fifth chambers 24A, 24B, and 24C.

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

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

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 fractions  Cell disruption effect

1. Manufacture of device for cell or virus disruption

An apparatus for cell or virus disruption is produced by placing a common elastic membrane between two glass chips. The glass chips are each formed with a part or all of a chamber and a channel, and the chamber and the channel between the elastic membranes. The first glass chip and the second glass chip are combined with each other, thereby completing a cell or virus crushing device using bead-beating.

Specifically, the first and second glass chips were manufactured by forming channels and chambers on the glass wafer through conventional photolithography, etching processes and wet etching processes. A 6 inch glass wafer (borosilicate, 700 μm thick) was washed with a Pirana solution, followed by low pressure chemical vapor deposition (LPCVD) of an amorphous polysilicon layer at 500 nm thickness on the cleaned glass wafer. Next, a patterning process was performed in which a portion of the deposited polysilicon layer was exposed using a photosensitive film. Next, the exposed portions of the polysilicon layer were removed by dry etching. Thereafter, the photoresist was stripped and the exposed glass wafer was wet etched with hydrofluoric acid solution (49% HF) to form a channel having a depth and a width of about 100 μm and about 200 μm, respectively. In the etching process for forming the channel, a dam (protrusion: weir) protruding from the inner surface of the channel toward the center of the channel was formed to isolate the beads. This dam allows the valve seat and bead trapping dams to perform simultaneously.

Next, the polysilicon layer is removed, and a dry film resist (DFT) is coated and patterned. Thereafter, a volume of about 15.5 μl of the volume in which the beads were placed and holes for inflow or outflow of the fluid were formed by sand blasting. Subsequently, the glass wafer was diced in chip form and then washed using plasma. Next, a plasma-activated 254 μm thick PDMS film (Rogers) was used as the intermediate layer, and contained a fluidic chip (hereinafter referred to as a first glass chip) and a bead containing a chamber which would contain the beads fabricated above. And a pneumatic chip (hereinafter also referred to as a second glass chip) containing a chamber which will serve as a pneumatic pump instead. PDMS membranes are monolithic flexible and have been used as tools for fluid flow control, i.e. pumping and valves, as well as atuators for bead collisions through pneumatic vibrations. .

About 15-16 mg (about 2 × 10 ⁵ ) surface modified glass beads were placed in the resulting bead chamber and the chamber was sealed by placing PCR-compatible adhesive tapes (Applied biosystems). A polycarbonate plate was covered on the bonded tape to prevent the tape from bending during operation, for example during DNA extraction.

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

7 and 8 show the cell crushing apparatus used in this example. 7 is a cross-sectional view of the bead-collision apparatus that induces bead collisions through vibration of the PDMS membrane. In FIG. 7, inlets 40 and outlets 42 are formed with protrusions 40 and 42, respectively, and inlets 40 and outlets 42 are connected to inlet and outlet ports through liquid channels 48 and 50. The valve seats 44 and 46 are formed on the upper plate of the inlet port and the inlet 40 and the outlet port and the outlet 42, and pneumatic chambers 34A and 34D are formed on the lower plate corresponding thereto. .

8 is an enlarged view of a monolit glass-PDMS-glass device consisting of three layers and its fluid component and pneumatic component.

2. Glass Bead  Modification

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

A 5% (v / v) trimethoxysilylpropyl modified polyethyleneimine (Gelest, Inc.) solution in ethanol was then prepared as a bead surface modification solution. The beads were soaked in the solution prepared above and reacted for 2 hours with gentle 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 ° C. for 50 minutes. The result was glass beads whose surface was coated with polyethyleneimine (PEIM). The PEIM has the property of nonspecifically binding to cells, so the glass beads can be used to nonspecifically separate cells.

3. Nucleic Acid Extraction Experiment

1 ml sodium acetate buffer (50 mM, pH 4, Sigma-Aldrich) containing 10 ⁶ CFU / mL S.aureus or MRSA as sample, 0.5 ml TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0, Ambion) for washing ) And 10 μl or 20 μl NaOH (0.02 N, Sigma-Aldrich) for crushing were pre-distributed to a liquid reservoir. The liquid solution was transferred in a pressure driven manner and the working liquid pressure was determined in preliminary experiments. First, the sample solution was allowed to flow through a chamber filled with beads at a flow rate of about 200 μl / min at 30 kPa while pressure up the PDMS membrane at 150 kPa. The cell capture efficiency was evaluated by recovering the flown-through solution. After initial sample loading, the beads filled chamber was washed with flowing the wash solution at a flow rate of about 500 μl / min (80 kPa) and air dried at 100 kPa for 30 seconds. To crush the captured cells, 6 μl of the crushing solution (0.02N NaOH) was injected and the valves on both sides of the chamber filled with the beads were closed. Next, by adjusting the solenoid valve through the LabVIEW program, the pressure in the two pneumatic chambers are adjusted to 80 kPa and -80 kPa, respectively, so that the PDMS membrane vibrates for 5 minutes at a frequency of 10 Hz to perform a cell disruption process. It was. After the crushing process, the inlet and the outlet were opened, and then 4 μl or 14 μl of NaOH solution was injected by applying a hydraulic pressure of 100 kPa to recover the cell lysate through the outlet. In this way, cell lysates containing 10 μl or 20 μl of nucleic acid in total were obtained. The whole process took less than 20 minutes. No additional DNA purification step was performed.

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

PLC experiments were performed with two types of benchtop shredding techniques: enzyme method and bead-collision method. 1 ml S.aureus samples with two different concentrations, namely 10 ⁴ CFU / ml and 10 ⁶ CFU / ml, were centrifuged at 13,200 rpm for 20 minutes in a microcentrifuge tube to precipitate cells, and then the supernatant Was removed. The precipitated pellets were each treated in the above manner. For the enzymatic method, the cell pellet is incubated with lysostaphin solution (200 mg / mL, Sigma) at 37 ° C. for 30 minutes and then applied to a Qiagen DNA extraction kit (Cat 51304, QIAamp DNA Mini Kit). 20 μl of purified DNA solution was obtained as described in its protocol. For the bench top bead-collision method, 30 mg of bare glass beads and 20 mL of crushed solution (ie 0.02 N NaOH solution or distilled water) were added to the cell pellet, followed by vortexer (GENIE 2, Fisher Scientic) was vigorously vortexed for 5 minutes at full speed. After brief centrifugation, the extracted DNA solution (cell debris) was recovered. As an NLC experiment, the cell pellet was vortexed vigorously for 5 minutes at full speed (3,200 rpm) using vortexer (GENIE 2, Fisher Scientic) in the absence of free beads. The target sequence was amplified using the resulting control lysate sample, PLC and NLC as a template, and the amplification result was compared with the result of amplifying the target sequence using the DNA solution obtained by the bead-collision apparatus as a template. For accurate comparisons, the total number of S. aureus injected into the chamber and the final DNA elution volume were adjusted to always be the same as that of the sample.

4. Real time PCR  Amplification

Real-time PCR was performed on a GenSpector ^R TMC-1000 instrument (Samsung Electronics) to perform cell disruption and to evaluate the extracted DNA quality as a result. Primer set specific for SA442 region of S.aureus genome (forward: 5'-GTT GCA TCG GAA ACA TTG TGT-3 (SEQ ID NO: 1)) and reverse: 5'-ATG ACC AGC TTC GGT ACT ACT AAA GAT-3 ' (SEQ ID NO: 2), GeneBank accession number AF033191) and primer sets specific for mecA fragments of the MRSA genome (Forward: 5'-ACG AGT AGA TGC TCA ATA-3 '(SEQ ID NO: 3) and reverse: 5'-GGA ATA) ATG ACG CTA TGA T-3 '(SEQ ID NO: 4) and GeneBank accession number EF190335.1) were each 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 MRSA FAM-5'-CCA ATC TAA CTT CCA CAT ACC ATC T-3'-BHQ1 (SEQ ID NO: 6), 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 onto the PCR chip, thermocycling was performed by denaturation step at 95 ° C. for 1 minute and extension step at 60 ° C. for 4 seconds. PCR amplicons were designed to be 72bp and 98bp 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 for Control Samples

Cell disruption and / or DNA purification effects were assessed by threshold cycle (Ct). The Ct values for the positive control sample are shown in Table 1. The values in Table 1 are repeated three times for each cell number.

Number of S.aureus Cells Applied (CFU)

Benchtop Bead-Crashing Method

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

Benchtop bead-collision methods are typically carried out with a crushing solution comprising a surfactant or chemical to increase crushing efficiency. In this example, a NaOH solution (0.02 N) was selected that did not interfere with PCR amplification without further purification. The bead-collision effect is further distinguished by NaOH solution and distilled water, indicating that NaOH contributes to DNA extraction efficiency by chemically destroying the cell membrane. For enzymatic disruption of S. aureus, lysostapine was chosen because it specifically cleaves a crosslinking pentaglycine bridge in the cell wall of staphylococci. The benchtop bead-collision method using NaOH performed equally or better than the enzyme-based DNA extraction method. Therefore, the cell or virus disruption apparatus used was evaluated by comparing it with a benchtop vortexing machine. Since optical density measurement is a rough cell quantification method, the Ct value of the PLC varied daily with a standard deviation of about 1.5 even at the same optical density. NLC samples vortexed only with distilled water showed a Ct value of about 31.5 for 10 ⁶ CFU / ml samples.

(2) cell capture results

The basic manipulation procedure in this example is as follows: (1) cell capture on glass beads, (2) washing and drying, (3) cell disruption using in situ-bead-collision, (4) extracted DNA solution Elution. Bacterial cells can be captured on solid substrates in a specific or nonspecific manner.

In addition to cell specific immunoaffinity methods, nonspecific cell capture methods using surface thermodynamics or electrostatic interaction can capture pathogenic bacterial cells. In this example, long-range Coulomb electrostatic interactions were tested by electrically modifying glass beads electrically. The surface of the glass beads was derivatized to have a positive amine group in reaction with the organosilane compound comprising 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 interaction was evaluated by comparing the modified glass beads with the modified glass beads. As a result, the Ct value of the DNA eluted from the modified glass beads was 2 smaller than the Ct value of the unmodified glass beads. This result indicates that the cell capture efficiency was increased by 4 times by surface modification (ie, 1 Ct difference represents 2 times difference of initial template copy number).

In order to obtain more quantitative data on the cell capture capacity of the cell disruption apparatus used in this example, the passed sample solution was recovered, centrifuged, and bench-top beams with min glass beads and NaOH solution as in PLC. Crushing on a scouring machine (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 proper manipulation of the bead-collision apparatus. The difference between them was used as a tool to assess cell capture efficiency and dose.

When 1 mL of various concentrations of S. aureus (ie, 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ and 10 ⁸ CFU / mL) were applied, the difference in Ct values between AC and AE was from 10 ³ CFU / mL to It remained above about 5 in the range of 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 10-fold difference in initial template copy number led to a change in Ct value of about 3.3. In addition, the prepared bead-filled device has a capacity of at least 10 ⁷ CFU S. aureus. After cell capture, the bead filled microchambers were washed and air dried to allow complete replacement with the crushing solution. These results indicate that the applied cell capture method is suitable for demonstrating in-situ bead-collision disruption of captured cells instead of releasing the captured cells. Other cell capture methods, including immunoaffinity, can be incorporated into the device of this example by employing appropriate solid surface modification chemistry.

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

The effect of various factors on bead-collision cell disruption was identified. First, experiments were conducted on membrane vibration frequency (5 Hz to 10 Hz), membrane drive pressure (20 kPa to 80 kPa) and depth of pneumatic displacement chamber (100 μm to 200 μm). There was no statistically significant effect on. Next, the liquid volume fraction (f _L ) defined as the volume of the shredding solution relative to the volume of the net void microchamber filled with beads during the bead-collision induction during the bead-collision induction Was tested by changing. The final eluted DNA volume was adjusted to the same 20 μl by adjusting additional NaOH solution when eluting the extracted DNA from the chamber. 9 is a graph showing cell disruption efficiency according to the change of the liquid volume fraction (f _L ). As shown in FIG. 9, the liquid volume fraction (f _L ) was the key factor in determining cell disruption efficiency. The liquid volume fraction (f _L ) was excellent in cell disruption 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, cells could be efficiently crushed. This is believed to be because the relative amount of liquid solution (NaOH) greatly affects the viscosity of the mixed liquid (gas and liquid) since the viscosity of the liquid (NaOH) is at least 100 greater than that of air.

20: Cell or virus disruption apparatus 21, 23: upper and lower plates
22, 24, 24A, 24B, 24C: first to fifth chambers
26: membrane 28: microbead
28A: Organic membrane 30: Inlet
32: outlet 34: port
34A, 34B, 34C: first to third ports
40, 42: First and second protrusions 48: Bulkhead
48A, 48B: first and second bulkheads

<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 viruses into a first chamber containing beads; And
A method of disrupting cells or viruses in a sample comprising agitating the mixture of beads and samples to disrupt cells or viruses, wherein the liquid volume fraction (f _L ) in the first chamber into which the beads and samples are introduced is 0.6. Wherein the liquid volume fraction is the liquid volume (V _L ) in the first chamber divided by the net pore volume corresponding to the sum of the liquid volume (V _L ) and the pore volume (V _O ) in the first chamber. . The method of claim 1, wherein the liquid volume fraction is 0 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 beads so that the beads cannot pass through. The method of claim 1, wherein at least one of the walls of the first chamber and the surface of the beads is bound to a substance that binds to cells or viruses. The method of claim 4, wherein the binding agent is a substance that specifically binds to the cell or virus or a substance that binds nonspecifically. The method of claim 5, wherein the specifically binding agent is selected from the group consisting of an antibody to an antigen, a substrate to an enzyme, an enzyme to a substrate, a receptor to a ligand, a ligand to a receptor, and a mixture thereof. . The method of claim 5, wherein the non-specifically binding material is a material having a hydrophobicity of water contact angle of 70 ° to 95 ° or a material having at least one amino group. 5. The method of claim 4, wherein the step of introducing the sample comprises introducing a sample from an inlet of the first chamber and discharging it through an outlet of the first chamber. The method of claim 1, comprising removing at least a portion of the liquid in the first chamber to reduce the volume of the liquid after the sample introduction and before the stirring step. 10. The method of claim 9, wherein reducing the liquid volume comprises drying the interior of the first chamber. The method of claim 10, wherein said drying is accomplished by flowing air into said first chamber or by applying heat. The method of claim 4, further comprising, after the sample introduction step, cleaning the surface of the wall of the first chamber or the surface of the beads to remove material not bound to the surface. The method of claim 1, further comprising introducing a cell or virus disruption solution into the first chamber. The method of claim 13, wherein said stirring step is performed after introducing said shredding solution. The method of claim 1, wherein the agitation is performed by ultrasonic waves or vibration of the wall of the first chamber. The apparatus of claim 1, wherein the first chamber comprises: 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 an apparatus for cell disruption that is a pressurized and reduced pressure chamber for vibrating the membrane,
Said vibration vibrating the flexible membrane between the first and second chambers. The method of claim 16, wherein the vibration vibrates the flexible membrane at 0.001 Hz-100 kHz.

Images