KR100285560B1 - Multi Cell Shredding Device - Google Patents

Abstract

The present invention relates to a multi-cell crusher. More specifically, the present invention provides a sample container holding unit including a sample container holder for fixing the sample container is provided on the upper plate, the upper plate of the electrical sample plate and a lower portion of the upper plate is placed; A driving unit for transmitting power to the electrical sample container holding unit; And, it relates to a multi-cell crushing device including a control unit for adjusting the driving force transmitted from the electric drive unit to the sample container fixing unit. By using the multi-cell crushing apparatus of the present invention, the detachable rack can be equipped with various types of sample containers, and the frictional force between the beads in the sample container by converting the motion direction of the beads in the sample container into vertical vibration. It is possible to prevent the noise, vibration and the distortion of the shaft caused by the vibration movement guide of the magnetic or spring type, and the vibration absorbing spring is installed between the shaft support and the main frame so that the top plate does not tilt to one side even at low speed, Not only is it more effective in blocking the noise and vibration generated during vibration, it is equipped with a crushing speed setting RPM meter and crushing time setting timer has the advantage that the sample can be crushed more easily, quickly and reproducibly.

Description

Multi Cell Shredding Device

The present invention relates to a multi-cell crusher. More specifically, the present invention is a sample container fixing unit that can be equipped with a vibration guide and a removable sample container that can be crushed and processed at the same time in order to avoid abrasion due to noise and friction even at high speed rotation And it relates to a multi-cell crushing device including a control unit for controlling the driving force of the electric drive unit and the drive unit for providing a driving force of the up and down vibration movement of the electrical sample container holding unit.

Today, most biological experiments require the simultaneous processing of multiple small samples. In other words, in order to extract a gene or protein from a sample of cells, tissues, etc., the work of crushing the cell tissues to diffuse the intracellular material such as genes and proteins into a solution must be preceded. . In particular, in the field of genetic engineering or biotechnology that analyzes or manipulates genes or proteins, the number of samples to be analyzed gradually increases as various studies progress, and thus, an apparatus for extracting genes by crushing cell tissues more easily and quickly is needed. It became.

As such a device, a cell crushing device (BeadBeater, manufactured by BioSpec) shown in FIG. 1 is widely used together with a sample to break glass beads having a diameter of about 0.1 to 2.5 mm in the sample container 4. In this case, when the motor 1 is driven, the rotating upper plate 3 rotates and hits the glass beads by the driving force transmitted by the rotating shaft 2, and the glass beads rotate and collide with each other. As a result, the sample container ( The sample in 4) is continuously broken down little by little by friction between beads. However, despite the advantage of the large capacity of the cell crushing device, only one type of sample can be crushed at a time, and the rotating top plate and the glass beads continuously collide, so that the rotational force of the motor is reduced to the frictional force between the beads. The conversion efficiency is lowered, the high-speed rotation is difficult and takes a lot of time when cell breakage, there is a problem that the degree of breakdown is also reduced.

In addition, another prior art cell crushing device (Mini-BeadBeater, BioSpec Co., Ltd.) shown in FIG. 2 is equipped with one sample container 9 in the form of a small capsule, unlike the above-mentioned large-capacity product. Therefore, the glass sample is put together with the sample to be broken into glass beads in the capsule-shaped sample container 9, and the capsule-shaped sample container 9 is shaken from side to side to collide the beads with the beads, and the sample is broken by the frictional force. . However, although the cell crushing device has also improved efficiency and degree of crushing than the cell crushing device of FIG. 1, the motor 6 rotating at high speed, the belt 7 transmitting the rotational force, and the rotation axis 8 are unevenly centered. It consists only of) and converts the rotational movement to the left and right vibrational movement, and the vibration is severe as the actual sample weight increases due to the twisting action of the rotational shaft 8, so that the mechanical rigidity such as the shaft bending or the shaft fitting part is broken is inferior. In addition, there has been a problem that only one sample can be processed at a time.

Therefore, due to such a problem, there is a need in the art to process a plurality of samples at the same time, and also to block the noise or vibration to double the robustness of the device and to develop an improved cell crushing device in terms of function and efficiency. It is desperately emerging.

Accordingly, the present inventors have made intensive research to solve the above-mentioned problems. As a result, the removable sample container can be crushed and processed at the same time, and the magnet type can be prevented from abrasion due to noise and friction even during high-speed rotation. It includes a tension spring type vibration guide and converts the sample container into vertical vibration to prevent vibration caused by excessive friction between the beads and the shaft. In addition, vibration is prevented between the shaft support and the main frame to prevent vibration. In addition to locating the spring for absorption, it also includes a crushing speed setting RPM meter and a crushing time setting timer, so that multiple samples can be processed at the same time. Development of cell crushing device significantly improved in terms of efficiency, completed the present invention It was good.

After all, an object of the present invention is to provide a multi-cell shredding device that can simultaneously crush and process a plurality of samples, and also doubles the robustness of the device by blocking noise or vibration, and improved in terms of its function and efficiency.

1 is a schematic view showing an embodiment of a cell disruption apparatus of the prior art.

Figure 2 is a schematic diagram showing another embodiment of the cell disruption apparatus of the prior art.

Figure 3 is a schematic diagram showing the overall configuration of a multi-cell crushing device according to an embodiment of the present invention.

Figure 4 is a schematic diagram showing the overall configuration of a multi-cell crushing device according to another embodiment of the present invention.

Figure 5a is a photograph showing the results of electrophoresis of the crushed bacterial cells and DNA extracted therefrom,

Figure 5b is a photograph showing the results of electrophoresis of the crushed yeast cells and DNA extracted therefrom,

Figure 5c is a photograph showing the results of electrophoresis of the DNA cells extracted from the crushing and animal cells,

Figure 5d is a photograph showing the results of the electrophoresis of the crushed and extracted DNA from plant cells.

Figure 6a is a photograph showing the results of electrophoresis of the disruption of bacteria cells and proteins extracted therefrom,

Figure 6b is a photograph showing the results of electrophoresis of crushed animal cells and proteins extracted therefrom.

7a and 7b are photographs showing the results of electrophoresis of bacterial cell disruption and DNA extracted from the deep well plate.

[Description of Symbols for Main Parts of Drawing]

10: drive unit 11: body frame 12: vibration absorbing spring

13: shaft support 1, 6, 14: motor 7, 15: belt

2, 8, 16: Rotating shaft 21: Coil 22: Permanent magnet support spring

23: permanent magnet 30: sample container fixing part 3, 31: top plate

32: sample vessel holder 33: captive screw 34: sample vessel cover

4, 9, 35: sample container 36: vibration movement guide 50: control unit

51: motor speed controller 52: RPM meter 53: timer

54: Amplifier 55: Voltage Controlled Oscillator (VCO)

Multi-cell crushing device of the present invention

The upper plate 31 on which the sample container is placed, the upper plate 31 is provided on the upper portion of the sample container holding means for fixing the sample 32 and the vibration movement guide 36 located in the lower portion of the upper plate 31 Sample container fixing portion 30;

A driving unit 10 for transmitting power to the electrical sample container fixing unit 30; And,

It includes a control unit 50 for adjusting the driving force transmitted from the electric drive unit 10 to the sample container fixing unit 30.

Hereinafter, with reference to the accompanying drawings will be described in more detail the multi-cell crushing apparatus of the present invention.

Figure 3 is a schematic diagram (sectional view) schematically showing a multi-cell crushing apparatus using a rotating shaft as the center of the main components as an embodiment of the present invention.

As shown in Figure 3, the multi-cell crushing device of the present invention

It is provided on the upper plate 31, the upper plate 31 on which the sample container is placed, the sample container holder 32 for fixing the sample container therein, and the sample container inside the sample container holder 32 to cover and cover the sample container. Vibration movement guide located under the sample container cover 34, the fastening screw 33 for fixing the sample container cover 34 to the electric sample container holder 32, the removable sample container 35 and the upper plate 31. Sample container holding portion 30 comprising a 36;

A motor that provides a driving force for the up and down vibration motion of the sample container fixing part 30 and is located in the body frame 11 and the body frame 11 to provide the driving force for the up and down vibration motion of the sample container fixing part 30. 14, the shaft support 13 for supporting the belt 15 and the rotating shaft 16, the electric rotating shaft 16 for transmitting the rotational motion of the motor 14 to the sample container fixing portion 30 in the form of vertical vibration movement. A driving unit 10 including a vibration absorbing spring 12 for absorbing vibration between the body frame 11 and the shaft support 13; And,

The controller 50 includes a motor speed controller 51, a crushing speed setting RPM meter 52, and a crushing time setting timer 53.

Looking at the operation principle of the present invention, first mixing the sample to be crushed and beads in the desired sample container 35, put on the sample container holder 32 on the top plate 31, and then the sample container cover 34 Covering, fixing with a fastening screw 33, and performing a crushing operation, while adjusting the motor speed regulator 51 while checking the RPM meter 52 according to the sample to be crushed. Then, the rotational force of the motor 14 is transmitted to the rotating shaft 16 through the belt 15, and the center of the bearing (not shown) coupled with the upper plate 31 and the center of the rotating shaft 16 do not coincide, so that the belt When the rotating shaft 16 rotates by the rotational force transmitted by the (15), the upper plate 31 moves up and down by the amount which shifted the center, and the rotational movement of the motor 14 is converted into the up-down vibration movement. However, at this time, since the rotating shaft 16 rotates and pulls down the upper plate 31, since the actual force applied to the upper plate 31 is not a vertical force directed downward, the upper plate 31 tilts left and right at a certain speed or less. The problem with the shaft support 13 arises. Therefore, in order to solve this problem, the present invention includes a vibration guide 36 in the form of a permanent magnet between the shaft support 13 and the top plate 31, thereby converting any kinetic energy into vibration In the end, the beads collide with each other and rub against each other in the sample container 35, and the cells rapidly begin to be broken. At this time, when the upper plate 31 and the sample container 35 vibrates up and down, it is accompanied by a considerable noise and vibration, so as to include a sound absorbing material (not shown) in the body frame 11, or the shaft support ( Several vibration absorbing springs 12 may be included between the 13 and the body frame 11. In addition, the appropriate crushing time of the sample can be variously determined by adjusting the timer 53 according to the type of the sample.

Figure 4 is a schematic diagram (sectional view) schematically showing a multi-cell crushing apparatus using electron induction as a main embodiment of the multi-cell crushing apparatus according to the present invention with the main components.

As shown in Figure 4, the multi-cell crushing device of the present invention

Place and cover the sample container inside the upper plate 31, the upper plate 31, the sample container holder 32 for fixing the sample container therein, and the sample container holder 32 in which the sample container is placed. Vibration movement located under the sample container cover 34, the fastening screw 33 for fixing the sample container cover 34 to the electric sample container holder 32, the removable sample container 35 and the upper plate 31. Sample container holding portion 30 including a guide 36;

The permanent magnet 23, which is located below the main body frame 11 and the main body frame 11 and provides a driving force for the up and down vibration movement of the sample container fixing part 30, and the coil 21 and the coil 21 wound thereon And a driving member 10 including a shaft support 13 supporting the permanent magnet support spring 22 and a vibration absorbing spring 12 positioned between the main frame 11 and the shaft support 13 to absorb vibration. ); And,

The controller 50 includes a motor speed controller 51, a crushing speed setting RPM meter 52, an amplifier 54, and a voltage controlled oscillator (VCO) 55.

Looking at the operation principle of the present invention, first mixing the sample to be crushed and beads in the desired sample container 35, put on the sample container holder 32 on the top plate 31, and then the sample container cover 34 Put on, and fixed with a fastening screw 33 to perform a crushing operation, while adjusting the motor speed regulator 51 while checking the crushing speed setting RPM meter 52 according to the sample to be crushed. At this time, the RPM meter 52 displays the frequency per minute by counting the output of the voltage adjusting oscillator 55, and the output voltage can be varied by adjusting the knob of the motor speed controller 51, the output voltage As the oscillation frequency of the voltage regulating oscillator (VCO) 55 changes, the frequency output from the amplifier 54 is changed, so that the direction of the current flowing through the coil 21 in the shaft support 13 can be changed according to the frequency. Will be. That is, when it is described in more detail based on the attached FIG. 4, ⓧ indicates the direction of the current in the cross-sectional view of the coil, ⊙ indicates the direction of the current coming out, and when the current flows in this way, the force by the law of the right hand of Fleming The permanent magnet (23) is moved upward to the upper side, and if the current direction is reversed, it is lowered to enable the vertical vibration movement of the sample container, by changing the current direction can be made the desired frequency, the As a result, the up and down moving vibration speed of the permanent magnet 23 can be adjusted. However, at this time, too, as in the multi-cell crushing device by the electric rotary shaft, since the problem occurs that the upper plate 31 is inclined to the left and right to hit the shaft support 13 below a certain speed, in order to solve the present invention, the shaft support A vibration guide 36 in the form of a spring is provided between the top plate 31 and the top plate 31 to convert any kinetic energy into vertical vibration and eventually collide with each other within the sample container 35. Friction with the cells causes the cells to break down rapidly. In addition, when the upper plate 31 and the sample container 35 vibrates up and down, they are accompanied by considerable noise and vibration, so that a sound absorbing material (not shown) is included in the main frame 11, or the shaft support ( Several vibration absorbing springs 12 may be included between the 13 and the body frame 11.

As described above, the vibrating motion guide 36 used in the present invention is the same pole (N or S) so that the top plate 31 can maintain the equilibrium without hitting the shaft support 13 even during the initial operation of the motor 14. Use a permanent magnet with strong magnetic force attached to face) or use a tension spring that can adjust the vibration width as the vibration width can be increased as it is attached to the upper side. In addition, in the present invention, the sample container 35 placed on the top plate 31 may use all of the detachable sample containers that are currently widely available in the field of genetic engineering and biotechnology, for example, 96 deep wells. A plate, a 1 ml volume or a 1.5 ml volume microtube, or the like is used.

Hereinafter, the present invention will be described in more detail with respect to the effect thereof.

Experimental Example 1 Crushing of Various Cells

The bacteria were collected from the bacterial culture cultured overnight in 1.5 ml ampicillin-containing LB medium, and then 300 µl of bacterial lysate (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 0.1 N NaOH, 0.5% SDS). Containing). Yeast was collected in yeast broth cultured overnight in 1.5 ml of YPD medium, and then suspended in 300 μl of yeast lysate (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA). . In addition, animal cells were collected from the culture medium incubated in 5 ml of MEM medium for 3 days, and then 300 µl of animal cell lysate (100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0), 0.5%). Suspension containing SDS). Finally, plant cells were cut into 0.1 × 0.1 cm in size and suspended in 300 μl of lysate (containing 100 mM Tris-HCl (pH 8.0), 100 mM EDTA, 250 mM NaCl) to prepare a sample. Various cell lysates prepared above were added to a 1.5 ml tube containing 0.3 g of various glass beads. The glass beads used at this time were three types of diameter 0.12-0.18 mm, diameter 0.45-0.52 mm, and diameter 0.90-1.25 mm. It was mounted on a cell crusher and reacted at 5,000 rpm for 5 minutes. Thereafter, the mixture was centrifuged at 12,000 rpm for 3 minutes, and then the supernatant and the same amount of isopropanol were added to precipitate DNA according to a general method. It was washed with 80% ethanol and then suspended in 30 μl of TE, 4-10 μl of which was electrophoresed on 0.8% agarose gel.

Comparative Experimental Example 1 : Disintegration of Various Cells and Extraction of DNA from the Cells

Centrifugation and electrophoresis were performed in the same manner as in Experimental Example 1, except that the culture medium of each cell prepared in Experimental Example 1 was heat treated at 95 ° C. for 5 minutes.

Comparative Experimental Example 2 Fracture of Various Cells and Extraction of DNA from the Cells

Centrifugation and electrophoresis were performed in the same manner as in Experimental Example 1, except that the culture solution of each cell prepared in Experimental Example 1 was vortexed for 5 minutes.

The results in Experimental Example 1 and Comparative Experimental Examples 1 and 2 above are summarized in FIGS. 5A to 5D. As a result, it was confirmed that bacteria, yeast, plant cells and animal cells can be efficiently crushed by the multi-cell crushing apparatus of the present invention, and DNA can be efficiently separated therefrom. In particular, yeasts and plants that are known to be difficult to crush the cells, compared to the heat treatment or vortexing performed in the Comparative Experimental Examples 1 and 2, the cells are more efficiently crushed by the multi-cell crushing apparatus of the present invention. Able to know. 5A to 5D show the results of electrophoresis by crushing bacteria, yeast, animal cells and plant cells, respectively, the first lane is heat treatment (Comparative Experiment 1), and the second lane is vortexing (Comparative Experiment 2). , Lanes 3 to 5 show the results of electrophoresis with glass beads having a diameter of 0.12 to 0.18 mm, a diameter of 0.45 to 0.52 mm, and a diameter of 0.90 to 1.025 mm (Experimental Example 1).

Experimental Example 2 Fracture of Various Cells and Extraction of Proteins Therefrom

In the same manner as described in Experiment 1, bacterial and animal cell cultures (50 mM Tris-HCl; pH 8.0; containing 0.1% Triton X-100, 0.5% SDS) were prepared in 1.5 μl tubes, and 0.45 in diameter. 0.3 g of glass beads of ˜0.52 mm were added thereto. Then, it was mounted on the multi-cell crusher of the present invention and reacted for 5 minutes at 5,000 rpm. Sampling buffer was added to 10-20 μl of the centrifuged supernatant and reacted for 3 minutes at 95 ° C., followed by electrophoresis with 10% SDS-PAGE.

Comparative Experimental Example 3 Fracture of Various Cells and Extraction of Proteins from the Cells

Centrifugation and electrophoresis were performed in the same manner as in Experimental Example 2, except that the culture medium of each cell prepared in Experimental Example 2 was heat treated at 95 ° C for 5 minutes.

Comparative Experimental Example 4 Fracture of Various Cells and Extraction of Proteins from the Cells

Centrifugation and electrophoresis were performed in the same manner as in Experimental Example 2, except that the culture solution of each cell prepared in Experimental Example 2 was vortexed for 5 minutes.

The results in Experimental Example 2 and Comparative Experimental Examples 3 and 4 above are summarized in FIGS. 6A and 6B. As shown in Figure 6a and 6b, it was confirmed that by the multi-cell crushing apparatus of the present invention, bacterial cells and animal cells can be efficiently disrupted, and proteins can be efficiently separated therefrom. Figures 6a and 6b shows the results of electrophoresis by crushing bacteria and animal cells, respectively, the first lane is heat treatment (Comparative Experiment Example 3), the second lane is vortexing (Comparative Experiment Example 4), the third and fourth Lanes were glass beads having a diameter of 0.45 to 0.52 mm, and samples crushed by the multi-cell crushing apparatus of the present invention (Experimental Example 2), and lanes 5 represent protein markers, respectively.

Experimental Example 3: Bacterial Cell Fracture and Extraction of DNA from 96 Deep Well Plates

In the same manner as described in Experiment 1, bacterial cell lysates containing 0.3 g of 0.45 to 0.52 mm diameter glass beads were prepared in 96 deep well plates, and then mounted in a cell crusher and reacted at 5,000 rpm for 5 minutes. I was. According to the method of Experimental Example 1, DNA was extracted from the cell lysate and electrophoresed on a 0.8% agarose gel, and the results are shown in FIG. It was confirmed that the bacterial cell was crushed at any point by the crusher, and DNA could be efficiently extracted therefrom. Each number in FIG. 7B represents a number on the 96 deep well plate shown in FIG. 7A.

As described and demonstrated in detail above, the present invention is equipped with a sample that can be equipped with a removable sample container capable of simultaneously crushing and processing a vibration guide and a plurality of samples in order to avoid abrasion due to noise and friction even during high-speed rotation Provided is a multi-cell crushing device including a control unit for controlling the driving force of the electric drive unit and the drive unit for providing the driving force of the up and down vibration movement of the container holding unit and the electrical sample container holding unit. By using the multi-cell crushing apparatus of the present invention, the detachable rack can be equipped with various types of sample containers, and the frictional force between the beads in the sample container by converting the motion direction of the beads in the sample container into vertical vibration. It is possible to prevent the noise, vibration and the distortion of the shaft caused by the vibration movement guide of the magnetic or spring type, and the vibration absorbing spring is installed between the shaft support and the main frame so that the top plate does not tilt to one side even at low speed, Not only is it more effective in blocking the noise and vibration generated during vibration, it is equipped with a crushing speed setting RPM meter and crushing time setting timer has the advantage that the sample can be crushed more easily, quickly and reproducibly.

Claims (4)

The upper plate 31 on which the sample container is placed, the upper plate 31 is provided on the upper portion of the sample container holding means for fixing the sample 32 and the vibration movement guide 36 located in the lower portion of the upper plate 31 Sample container fixing portion 30; A driving unit 10 for transmitting power to the electrical sample container fixing unit 30; And, Multi-cell crushing device comprising a control unit 50 for adjusting the driving force transmitted from the electric drive unit 10 to the sample container holding unit (30). The method of claim 1, The driving unit 10 provides a driving force for the up and down vibration movement of the sample container fixing part 30, and is located inside the main body frame 11 and the main body frame 11 so as to provide the driving force of the up and down vibration motion of the sample container fixing part 30. Supporting the belt (14), the rotating shaft (16) and the electric rotating shaft (16) for transferring the rotational motion of the motor (14) and the motor (14) providing the driving force to the sample container fixing part (30) in the form of vertical vibration. And a vibration absorbing spring 12 for absorbing vibrations between the shaft support 13, the body frame 11, and the shaft support 13, and the control unit 50 includes a motor speed controller 51, Characterized in that it comprises a crushing speed setting RPM meter 52 and a crushing time setting timer 53 Multi-cell crusher. The method of claim 1, The driving unit 10 is located in the main frame 11 and the main frame 11 to provide a driving force for the up and down vibration movement of the sample container fixing part 30, the permanent magnet 23 and the coil 21 wound around the coil 21, the coil (21) and a shaft support 13 for supporting the permanent magnet support spring 22 and a vibration absorbing spring 12 positioned between the main frame 11 and the shaft support 13 to absorb vibrations, In addition, the controller 50 may include a motor speed controller 51, a crushing speed setting RPM meter 52, an amplifier 54, and a voltage controlled oscillator (VCO) 55. Multi-cell crusher. The method of claim 1, Electric vibrating motion guide 36 is characterized in that the permanent magnet or a tension spring facing the same pole Multi-cell crusher.