TWI303276B - Method and apparatus for the rapid disruption of cells or viruses using micro magnetic beads and laser - Google Patents

Description

1303276 First, the 'sound wave energy 1 is unevenly distributed. The uneven distribution of ultrasonic energy leads to: inconsistent results. Second, due to the energy disparity in the ultrasonic bath, it is usually necessary to: complete the rupture of the cells by a number of 77 filaments. Finally, the ultrasonic method produces an unpleasant sound. The use of sputum for cell rupture has many advantages and is easy to apply to L0C (version (10) 4 people by 73, 4625-4631 (20013⁄4.

Reveals a laser-induced

Cell lysis system. When using only lasers, efficient cell lysis is not produced. Using the results of the experiment with the large intestine rod *_. μ in the extremely clear liquid, it was confirmed that when the radiation was only taken, a very low cleavage efficiency was obtained. The concentration of DNA measured after 150 seconds of exposure to the laser was 3.77 ng//^, which was the reason why energy was not efficiently transmitted to the cells. The concentration of DNA measured after the cells were measured by the heating method of F was 6.15 ng///1. U.S. Patent No. 6,685,730 discloses an absorbent nanoparticle for improved tissue repair. The method includes a method of joining tissue, including: delivering a nanometer-sized nanometer-sized nanoparticle to a tissue to be joined, the county paste can absorb light of one or more wavelengths; The paste is exposed to light or a plurality of wavelengths of light that can be absorbed by the nanoparticles. This method uses laser and nanoparticle, which only causes the loss of function of the cells. The filament has been ruptured by shaking the solution containing cells and particles 1303276. Therefore, the present invention is devoted to research to overcome the above problems and finds that when using micro • When magnetic beads and laser vibrations contain a solution of cells or viruses, they can quickly rupture cells or diseases. SUMMARY OF THE INVENTION The present invention provides a method for rupturing a cell or a virus, comprising: adding a plurality of magnetic beads to a solution containing cells or viruses; vibrating the magnetic beads; and irradiating the magnetic beads with the magnetic beads Laser to rupture such cells or viruses. The invention also provides a device for rupturing a cell or a virus, comprising: a cell lysis tank having an inlet for introducing a sample and a magnetic bead through the inlet; a vibrator attached to the cell lysis tank to lyse the cell The sample and magnetic beads are mixed, and the sputum generator is attached to the cell lysis tank to provide a laser.

The sample is evaporated. This month also provides a device for rupturing cells or viruses, including: a cell split = wafer, which has an inlet for introducing samples and magnetic beads through the inlet; vibration TM..., vibration transmission unit Attached to the cell lysis chip to mix the sample and the magnetic beads in the cell coating, the vibration transmission portion is attached to the cell lysis chip, a laser generator, attached to the cell lysis portion, attached to the cell lysis Chip, in case

The use of the device of 1303276 includes a top surface and a bottom surface of the opening and includes a reaction groove, an inlet, and an outlet; a wafer top cover attached to the top surface of the wafer body, Closing the upper portion of the reaction vessel, passing the laser, and having an inlet and an outlet; and a wafer substrate attached to the bottom surface of the wafer body via a wafer bonding portion to close the reaction vessel, the inlet, and the lower portion of the outlet . [Embodiment] The present invention will be described in detail below. A method of rupturing a cell or virus according to a specific embodiment of the present invention comprises: adding a plurality of magnetic beads to a solution containing cells or viruses; vibrating the magnetic beads; and irradiating a laser with the magnetic beads to make such a laser The cell or virus is broken. In this method, a laser containing a magnetic bead is irradiated with a laser, and the laser energy transmits a shock wave (sh〇ckwave), a vapor pressure, and heat to the cell surface due to the presence of the magnetic bead, thereby causing ablation (ablati〇) n). At this time, physical shocks are also applied to the cell surface. The magnetic beads heated by the laser raise the temperature of the solution and directly rupture the cells. The magnetic beads are not only used as a heat conductor in the solution, but also exert a reduction, mechanical vibration, and physical vibration on the filament surface to efficiently break the cell surface. Rapid cell lysis using magnetic beads and lasers is performed by heating and/or laser ablation in a liquid medium. The laser combines with the microbeads to convert the heat source into a high thermal magnetic bead/physical and mechanical vibration to enhance cell lysis. Recently, the small size, high power laser 1303276 diode has been rapidly developed, enabling the extremely small cell cell lysing device to be mounted on the LOC. Furthermore, the power and energy of the laser can be concentrated on a particular area by fiber optics, mirrors, or lenses. The advantage of the magnetic beads is that the DNA separation step is reduced because the protein is denatured by lysis of micromagnetic beads and laser cells. Denatured proteins and cell debris are attached to the magnetic beads and removed by gravity or magnetic force. As a result, the detection limit was lowered, and the DNA extraction time was significantly shortened due to a step in the private sequence of DNA extraction, and the analysis result was significantly improved due to the increase in the Congrui. The microbeads and the laser rupture_the required slant_ is 4 sec. The laser burning material is exposed to the laser-induced surface. Laser burning surnames quickly increase (four) surface temperature hundreds to thousands of degrees. If the surface temperature of the material is increased to or more than the evaporation of the shaft material, the surface saturated vapor pressure will increase rapidly. The saturated helium pressure is expressed by the temperature function of the Clausius-Clapeyr〇n equation = usually higher in the high power pulse laser program to increase the tens of fields (four). The pressure applied to the surface of the material by the vapor at the time of discharge is called "pushing force", and the magnitude of the repulsion pressure is 5%, and 4 is the vapor pressure. The seismic wave system uses a laser with a very high instantaneous intensity, such as a pulsed laser, which produces a short time to heat the treatment point or higher in the range of nano nanoseconds to tens of nanoseconds. The vapor generated on the surface of the material increases from a few atmospheres to several tens of atmospheres M' and forms a shock wave when the vapor expands to the surrounding air towel. Since 12 1303276 is extremely high pressure, this expansion vapor applies about 0.56 Ps to the material (where Ps represents the saturated vapor pressure of the surface). In a particular embodiment of the invention, the laser may comprise a pulsed laser or a continuous wave (CW) laser. When the laser power is too low, the laser burning surname cannot be effectively occurred. For CW lasers, the laser power is 10 mW or higher, and for pulse lasers, it is 1 mj/pulse or higher. Preferably, the pulse laser is 3 mJ/pulse or higher, and the CW laser has a power of 100 mW or higher. This is because when the CW laser is less than 1 〇mw and the pulse laser is less than 1 mj/pulse, sufficient energy cannot be transmitted to rupture the cells. In a particular embodiment of the invention, the resulting laser should be within a particular wavelength range in which the magnetic beads can absorb the laser energy. The resulting laser is preferably in the wavelength range of 4 〇〇 nm or longer, and more preferably in the wavelength range of 750 nm to 1300 nm. This is because the DNA is denatured or damaged at wavelengths shorter than 400. The resulting laser can also be in one or more wavelength ranges. That is, the laser may have one wavelength or two or more different wavelengths within the above range. In a specific embodiment of the invention, the diameter of the magnetic beads is preferably 5 〇 nm to 〗, 〇〇〇 //m, and more preferably 1/zm to 50/zm. When the diameter of the beads is less than 5〇11111, physical and mechanical vibrations are not sufficient to cause cell lysis. When the diameter of the bead is larger than Looovm •, it is not suitable for L〇C. The magnetic beads may also be mixed with beads of two or more sizes 13 1303276. That is, the magnetic beads may also have a size equal to each other or a mixture of beads having different sizes. In a particular embodiment of the invention, the magnetic beads can be any magnetized material. In particular, the magnetic bead includes at least a material selected from the group consisting of Fe, Μ, & and its oxides. φ In a specific embodiment of the invention, the magnetic beads may be a polymer coated with a ferromagnetic metal, an organic material, tantalum, or glass. In a particular embodiment of the invention, the surface of the bead is preferably negatively charged such that (10) eight: attached to the magnetic bead. Because the DNA is negatively charged, it is not attached to the V-negative magnetic beads due to the repulsive force. When the DNA is attached to the magnetic beads, it is difficult to separate the DNa from the magnetic beads after the cells are broken, making DNA purification difficult. • In a specific embodiment of the invention, the functional groups on the surface of the magnetic beads may be hydrophilic, and the solution containing the magnetic beads may have a pH of from 6 to 9. The efficiency of DNA amplification obtained from the lysed cells may depend on the functional groups on the surface of the magnetic beads and the value of the solution containing the magnetic beads. As the hydrophilicity of the functional group increases, the efficiency of DNA amplification after cell lysis increases. Preferably, the functional group is a negatively charged carboxyl group or a derivative thereof. Carboxylic derivatives include iminodiacetic acid (IDA), ethylenediaminetetraacetic acid (EDTA), citric acid, polycarboxylic acid, etc. • pH of a solution containing magnetic beads The value is preferably from 6 to 9. If the pH is outside the above range, the DNA amplification efficiency after cell lysis is reduced. In a specific embodiment of the present invention, the solution may be selected from saliva, urine, blood, The serum, and the fine listening group, may be any solution of the annihilation (such as animal cells, plant cells, bacteria, viruses, bacteriophages, and the like). According to another embodiment of the present invention The device for rapidly rupturing cells or viruses includes a cell lysis tank having an inlet through which a sample and a magnetic bead '-vibration II are introduced, attached to a cell lysis tank to lyse the sample and micromagnetic in the cell lysis tank Light-light; and - Lei Chengsheng, squatting in the cell lysis tank to provide laser. Figure 1 is the specific system of the system used for laser lysis and micro-magnetic beads for cell lysis. Via population supply. The product is completely combined with the magnetic beads i. The complete mixing of the sample with the magnetic beads 1 is achieved by the vibrator 2. When the mixture is vibrated, the laser 3 is simultaneously irradiated. The light transmission window (-) of the cell lysis tank should be laser It can be composed of materials that can be filled with a knife. The magnetic beads that are exposed to the laser 3 convert the light into heat, that is, 'the laser is burned. The heat is transmitted from the field by continuous vibration and the magnetic beads and the two cells are Collision 'so heat, vibration, shock wave, and pressure material can be transmitted efficiently. § When the temperature of the cell cracking is increased by the laser, ^ 赖 (4) affmvalve) will open 'this can be used; 5_ thickness control. After the foot touches the cells, the laser is turned off and the remaining microbeads are removed using an electromagnet. If the stone is removed by heat, the solution is red-PCR, and the PCR is used to enlarge the leg.

(S 15 1303276; in the specific implementation of the present invention, the vibrator may include sonic vibration crying _ theory), the _ field morality of the electric field of the vibrating wire, the machine (such as (four) thief, etc.) or pressure Electrical material. The vibrator is attached to the cell lysis tank and can be any device capable of vibrating a mixed solution of cells and microbeads. In a specific embodiment of the present invention, the device for rapidly rupturing cells or viruses can be further modified to include an electromagnet attached to a cell lysis tank for removing the lysis of the cells after the cells are lysed. . The electromagnet can be attached to the cell lysis tank, and in order to perform LOC, the magnetic beads are removed by electromagnet after the cell lysis is completed, and the solution of the ruptured cells can flow directly to the PCR tank without the separation step of the magnetic beads. . The beads can be magnetized for removal using an electromagnet. In a particular embodiment of the invention, if a more complete purification is desired, the means for rapidly disrupting the cells or virus can be further stepped by including a D. purification tank connected to the cell lysis trough via a channel of the PCR channel. If the paraffin valve or a MEMS (Micro Electro Mechanical Systems) structure valve using a magnetic or electric field is turned on after cell lysis is completed, the *dna purification tank is attached to the cell lysis tank to purify the DNA. In a particular embodiment of the invention, the means for rapidly disrupting cells or viruses may further comprise a paraffin valve located in the channel connected to the cell lysis tank, the thickness of which is controlled by the cell lysis time. When the temperature of the cell lysis tank is increased by the laser, the stone valve is opened, which can be controlled by the thickness of the stone sluice valve. 1303276: In a specific embodiment of the invention, a rapid rupture of a cell or virus can be advanced. A step includes a DNA amplification channel connected to the cell lysis cell via a channel. Since the microbead purification effect can be produced as described above, the DNA amplification tank can be directly attached to the cell lysis tank.

In a particular embodiment of the invention, the means for rapidly disrupting cells or viruses may include a DNA amplification channel connected to the cell lysis cell via a channel. For the purpose of performing LOC, an amplification system for purifying 2DNA is required. The purified dna can be detected using a spectrometer, microbeads, electrochemical methods, electrochemiluminescence, emission and fluorescence labels, real-time PCR (R-PCR), and the like. The PCR method is most suitable for fully amplifying the desired DNA. Other DNA amplification methods can be used, but can also be directly detected by an instant PCR method or the like. A device for rupturing a cell or a virus according to another embodiment of the present invention includes: a cell lysis wafer having an inlet for introducing a sample and a magnetic bead through the inlet; and a vibrator transmitting through a vibration Connected to the cell lysis wafer to mix the sample and magnetic beads in the cell lysis wafer, the vibration transmission portion is attached to the cell lysis wafer to transmit vibration to the cell lysis wafer; a laser generator attached to the cell lysis wafer 'To provide a laser; and an anti-volatiles, attached to the cell lysis wafer to prevent sample evaporation. Figure 2A is a schematic diagram of a system embodiment of a cell cleavage using a laser and microbeads 9 on a microchip 8, and a system design shown in Fig. 2A. The cell lysis chip is a device for lysing cells or viruses which are introduced from the inlet and the magnetic beads. The cell lysis wafer includes a wafer top cover 11, a wafer body 12, a wafer bonding portion, and a wafer substrate 13. The components of the cell lysing wafer will be described in more detail later. The cell cleaves the wafer as a function of the reaction tank to lyse the cells or viruses therein. The vibrator 16 is connected to the cell lysis wafer via the vibration transmission portion 15, and the sample and the magnetic beads are mixed in the eutrophic cell lysis wafer. The vibrator can vibrate vertically. The vibrator may include a vibrating motion n, a vibrating using a magnetic field, a vibrator of a long electric field, a mechanical vibrator (such as a vortex finder, etc.) or a piezoelectric material. The vibrator can be any device capable of mixing the vibrating cells with the micromagnetic beads (4). The vibrator can be a vibration motor for a mobile phone. The vibration transmitting portion transmits the vibration generated by the vibrator 16 to the cell lysis tank via the cell lysis chip. The vibration transmission part can be transferred to the cell lysis chip by the metal (for example, the laser chip is generated, and the laser cell rupture anti-volatile part is provided). The thunder is to reduce the volatilization. For this purpose, the anti-volatile anti-volatile part should have (4) ride 1Gpsi or larger diligent 1303276. The anti-volatile part can include the partition 18. The optical tape can be attached to the inlet 10 And the outlet 19 then fixes the separator 18 to the cell lysis wafer. Figure 3 shows the cell lysis device. The plate ", sheet. The separator can be a valve, a polymer structure, or a metal structure, but as long as it can prevent volatilization, Particularly limited to this. In a specific embodiment of the invention, the laser may comprise a pulsed laser or a continuous wave (CW) laser. • ^ When the laser power is too low, laser ablation is not effectively produced. The laser power is 10mW or more, and the pulse laser power is imJ/pulse wave or larger. This is because when the cw laser power is less than 10mW and the pulse laser power is less than 丨mj/pulse, it cannot be fully transferred. Energy to rupture cells. Specific implementation of the invention The resulting laser should have a specific wavelength range such that the magnetic beads are capable of absorbing the energy of the laser. The resulting laser preferably has a wavelength of 4 〇〇 nm or a larger range, and more preferably 75 〇 11111 Up to 13 波 波 波 波. This is because the DNA will be denatured or damaged below the wavelength below 4 〇〇 nm. The resulting laser also has a range of wavelengths. That is, the laser can have One wavelength within the above range, or two or more different wavelengths. In a specific embodiment of the invention, the diameter of the magnetic beads is preferably 5 〇 (10) to 丨, (10) • m, and more preferably 1 to 50. When the diameter of the magnetic beads is less than 50 nm, the physical and mechanical vibrations are not enough to cause cell lysis. When the diameter of the magnetic beads is larger than l, 〇〇0//m, it is used for 1303276 LOC. The magnetic beads can also have two or more sizes. The bead mixture, that is, the magnetic beads may have the same size or a mixture of beads of different sizes. In a specific embodiment of the invention, the magnetic beads may be any magnetized material. In particular, the magnetic beads are preferably Including at least one selected from ferromagnetic Fe, Ni, Cr, and oxygen thereof The material of the group consisting of φ. In a specific embodiment of the invention, the magnetic beads may be a polymer coated with a ferromagnetic metal, an organic material, tantalum, or glass. In a specific embodiment of the invention, the magnetic The surface of the bead is preferably negatively charged, so that the DNA is not attached to the magnetic bead. Because the DNA is negatively charged, it is not attached to the π-negative magnetic bead due to the repulsive force. When the DNA is attached to the magnetic bead, the cell is broken. It is difficult to separate from the magnetic beads, making DNA purification difficult. • In a specific embodiment of the present invention, the sample may be selected from the group consisting of saliva, urine, blood, serum, and cell culture. The sample may have Thieves, such as animal cells, plant cells, bacteria, viruses, rhymes, and the like. A cell lysis wafer for use in a device for rupturing cells or viruses according to another embodiment of the present invention comprises: a wafer body having an open top surface and a bottom surface and including a reaction tank, an inlet, and an outlet - wafer top cover attached to the crystal

20 1303276 The top surface of the sheet body to close the upper portion of the reaction vessel, pass the laser, and have an inlet and an outlet, and a wafer substrate attached to the bottom surface of the wafer body via a wafer bonding portion to close the reaction The trough, the inlet, and the lower part of the outlet. Figure 4 is a schematic illustration of a specific embodiment of a microchip used in a device that uses microbeads and laser ruptured cells. Referring to Fig. 4, the wafer body 20 has an open top surface and a bottom surface and includes a reaction tank 21, an inlet 22, and an outlet 23. The wafer body 10 can be a stone wafer that can withstand temperatures of 100 ° C or higher. The wafer body may be composed of glass, a compound, or a hexanone. The glass is preferably Pyrex 7740. The top surface of the wafer body 2 is attached to the wafer top cover 24, and the wafer bottom surface is attached to the wafer bonding portion 26. The inner surface of the wafer body can be hydrophobically treated to prevent the generation of bubbles. For example, the inner surface of the wafer body can be coated with Sigmacoat. A wafer top cover 24 is attached to the top surface of the wafer body 20 to close the upper portion of the reaction vessel _21. The wafer top cover 24 allows the laser to pass through and has an inlet u and an outlet 23. The top cover of the wafer may be composed of glass, polymer, bismuth tin oxide (O.sub.2) glass, and the like. The slope is preferably the Pyrex side. The material of the chick can be warmed up and has a penetration rate of 90/〇 or more. The wafer top cover may have an anti-reflective (ar) coating to increase the penetration of the laser. The antireflective coating can be formed using methods known in the art. Thus, the wafer top cover can be prepared using AR coated pyrex 7740. The wafer substrate 25 is attached to the bottom surface of the wafer body via the wafer bonding portion 26 to close the reaction vessel 21, the inlet 22, and the lower portion of the outlet 23. The wafer backplane can be gathered

21 1303276 ^ (5) paste glass, IT glass and other components. The material of the wafer backplane is preferably tolerable. It is also door/in and flexible. The wafer substrate is preferably composed of a material (e.g., a polycarbonate film) that is capable of effectively transmitting the vibration generated by the vibrator to the body of the cymbal. The wafer bonding portion 26 attaches the wafer substrate 25 to the wafer body 20, and serves as a buffer containing the H-slot 21. This is achieved by a transition material selected from the group consisting of adhesive tapes and adhesives. Although it can be formed only by the wafer body 10 and the (9) substrate, the leakage of the reaction solution may occur. The wafer bonding portion prevents the exposure of the reaction solution. Figure 5 is a photograph of a microchip 30 in accordance with an embodiment of the present invention. The invention is described in more detail with reference to the following examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Preparation Example 1: Cell lysis system As shown in Fig. 1, bacterial cells (90//1) and micromagnetic beads (30 // Dynabeads® M-270 carboxylic acid (Carboxylic) of Dynal, Norway, were prepared as shown below. Acid)) Mixed in a vial 4 located in a vial guide 5 (vialguide, AMITECH, Korea). In individual experiments, a mixture of 8 〇 8 nm, 13·8 W high power laser beam 6 (HLU25F100-808 from LIMO, Germany) was irradiated to the mixture for a period of time to rupture the cells by vortexing the vial. (See Figure 1). , Preparation Example 2 Determination of Viability of Vibrio and Bacterial Cells

22 1303276 Culture of large intestine sputum (5·μ") sputum BL21 and transglycosmic sputum (ATCC# 35668) in a vigorously aspirated brain heart extract culture medium (BHI media) at 37 °C, To the logarithmic period (exp0nentiai phase) (〇£)_〇! 〇). Bacterial cells were collected by centrifugation and washed twice with 3 ml of phosphate-buffered saline (PBS) solution. The cells were resuspended in PBS (cell density; 1χ1〇5 cells/#1). The number of viable cells was determined by the ability of a single cell to form colonies. The large intestine rod φ 闺 cells (1χ1〇3) irradiated by the laser beam are distributed in several portions on several BHI plates. These BHI plates were incubated overnight at 37 ° C, and the number of colonies was counted. Culture of epithelial cancer (Staphylococcus epidermidis) (AJCC# \4990 > 12228) ' to logarithmic phase (〇〇6〇0=〇) in 37C vigorously aerated nutrient locus (Nutrient Agar, NA) medium ·5~1·〇). Bacterial cells were collected by centrifugation and washed twice with 3 ml of phosphate buffered saline. The cells were resuspended in pBS (cell density; 1 x 10 cells///1). The number of viable cells is determined by the ability of a single cell to form colonies. Staphylococcus epidermidis cells (lxl〇3) irradiated with a laser beam were distributed in several portions on several NA disks. Put these BHIs at 37. The underarms were cultured overnight and the number of colonies was counted. Preparation Example 3 · Extraction of bacterial genomic DNA In order to compare the release efficacies of the DNA by the laser method and other conventional methods, a method for preparing a large intestine rod due to the method of f for 5 minutes was prepared. • 〇.9xl〇5 bacteria, equivalent to the number of cells used in each laser lysis). 8 23 1303276 Preparation 4 · Measurement of DNA released from bacteria To monitor cell lysis and DNA release from lysed cells, use

An Agilent Bioanalyzer (Agiient Bi〇analyzer) was then amplified using polymerase chain reaction (PCR). Use the following pair of primers (pair 〇f primers) for pCR use · primer A (sequence 1D number: 1); primer B (sequence ID number · 2). This primer pair is complementary to the 16S ribosomal RNA, encoding each end of the gene, and amplifies the entire coding region during PCR. PCR amplification of E. coli using Taq polymerase (Korea Solgent Co., Ltd.) for 25 cycles (95 C for 1 minute for pre-denature, 95 C for 5 seconds for denaturation, 6 ° C for 13 seconds for slow cooling Pairing, and 72. 〇 effect for 15 seconds to extend (extension), and 72. 〇 effect 丨 minutes for an additional extension). For Gram-positive bacterial cells; use Taq polymerase (Korea s〇lgent Co., Ltd.) to perform pCR amplification of S. mutans and S. epidermidis cells up to 3 〇% (95 C for 1 minute to pre-denature (pre-denature), 95 ° C for 5 seconds to denature, 60 C for 13 seconds to slow the pair (anneai), and 72. The 〇 effect for 15 seconds to prolong, and 72 C for 1 minute for an additional extension). After the completion of the amplification cycle, the sample was slowly heated (O.rc/sec) from 6 Torr to 90 ° C to obtain a melting curve. PCR was carried out by LightCyder0 (Roche Diagnostics, Inc., USA) and total volume Zheng Zheng reaction mixture containing iXFastStartDNAMasterSYBR (Roche Diagnostics Inc., Indiana), 〇·25//μ. Primer (Genotech, Korea), 4mM MgCl2 (R〇che 24 d 1303276, Indiana, USA)

Diagnostics, distilled water (PCR grade, R〇che, Di^ostics, Indiana, USA). The commercially available DNA 5〇〇 assay size reagent was used to analyze the amplified DNA by the BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA). Preparation Example 5: The cell lysing wafer of the present invention was produced by using a linaloic acid, a glass 'polycarbonate film, and a double-sided tape (D〇uble c〇ated 叩 e φ (9495ΜΡ, Minnesota, USA)) For microchips with a wafer size of 7.5 mm x 15 mm for use in a sample volume of 1 (1), as shown in Fig. 4, for laser-induced sample preparation, the remainder includes two micro-shirts such as annihilation. A bonding step of a double-sided tape having a polycarbonate film. A six-inch diameter and a 500/m thick glass wafer (giass wafer) was cleaned and laminated with a BF41 tantalum photoresist. The photoresist is patterned by lithography to form a hole having a diameter of 15 mm as an inlet and an exit through which the sample mouth passes. The holes are formed on the glass disc by sand blasting.晶圆 The wafer is a double-sided polished stone substrate with a diameter of six inches and a thickness of __. Due to cost considerations, 10 reaction tanks S are formed on the chopped wafer by m technology. To optimize sample loading, Sigmac〇at8 (Sigma-aldrich, Missouri, USA) was applied to the blasted surface of the wafer. Then, a polycarbonate film having a thickness of 100 // m was bonded to the ruthenium wafer using a double-sided tape having a thickness of 15 Å/m. Further Preparation Example 6: Laser-Initiated On-Chip Sample Preparation System As shown in FIG. 2A, for bacterial cell lysis, bacterial cells (1 W) and micromagnetic beads (9/zl, approximately 9×10 6) prepared as follows were prepared. Beads //zl, 烕d(4) 8 25 1303276 The company's Dynabeads® MyOneTM Carboxylic Acid (Carboxylic Acid) is mixed in a microchip placed in a chip guide module (AMITECH, Korea) (South Korea SAIT) . For the surface charge effect of magnetic beads and bead materials, an additional preparation of australis (3·0 μιη, Bangs Laboratories, Inc., USA), amine-terminated polystyrene Beads (1·5 μιη, Bangs Laboratories, Inc., Indiana), polystyrene beads (4·16 μm, Bangs Laboratories, Inc., Indiana), and carboxylic acid-terminated Benzene magnetic beads. In the experiment, a fiber-coupled laser system (HLU25F100-808 from LIMO, Germany) was used to vibrate the microchip by a copper plate type vibration motor (DMJBRK20X, Samsung Electronics Co., Ltd., Korea) using an aluminum vibrating rod. , 0.22 NA divergence angle 'irradiation wavelength 808 nm (l W) high power laser beam to rupture the cell up to # again. With 30W wide frequency power / energy meter (Broadband

Power/Energy Meter, USA MELLES GRIOT) measures laser power. The laser wavelength is selected by the absorption coefficient of the wavelength in water. The 808 nm laser beam with an absorption coefficient of 0.021773 (cnf1) in water mostly penetrates the water and reaches the microbead. For the purposes of the present invention, φ can produce visible light lasers, but high power lasers have not yet been developed to be portable H and cost is not beneficial. In addition, the ir wavelength is high in the reading of the water towel; most of the IR radar is absorbed in the water and is not suitable for this use. The uv laser beam is not good for cell lysis and DNA purification. Because UV light is known to be caused! The phoenix is damaged. The dna that illuminates the uv light accumulates the thymus ♦ the two nucleus as the main nuclear. Therefore, the 8Q8nm spectrum of 26 1303276 continuous laser diodes. Using a vibration motor (Molybdenum Samsung Electronics Co., Ltd. DMJBRK20X), which is mostly used for mobile phones, designs a vibration system for the (10)_chip sample preparation test module on the wafer, which has an aluminum part (AMITECH, Korea) to vibrate only The flexible polycarbonate film in the cell sample reaction cell area has a vibration rate of 12 Å. The vibration power of the vibration motor was adjusted by the power supply (E3620A, Agilent, Calif.). The temperature of the sample in the microchip reaction cell was measured by a thermocouple (Omega K type) and a data acquisition system (34970A, Agilent, Calif.). As shown in Fig. 3, after loading the sample solution and the magnetic beads, the inlet and the outlet were sealed with a translucent tape (Applied Biosystems, Calif.). To ensure that no leakage occurred during the laser exposure, two elastomers (thermogreen LB_2 'Sigma-Aldrich, Missouri, USA) on the top of the sample preparation test module on the wafer were used to compact the inlet and preparation example 7: live After irradiating the laser with photographs of cells and dead cells and magnetic beads, in order to observe the living cells and dead cells left in the sample solution, use the Live/Dead®BacLightTM Bacterial Survivability Kit (Bacterial) according to the recommended steps provided by the supplier. Viability kit, L7012, Molecular Probe, Oregon, USA) stained the cells. Images were taken with a microscope (Eclipse TE 3〇〇, 曰本 Nikon). Preparation Example 8: DNA analysis after laser irradiation 1303276 The genomic DNA and plastid DNA were separated from the BU1 cells containing the PCR® H_T〇P〇® (Invitmgen) plastid by various methods: For laser lysis, the cells are mixed with magnetic beads, and the laser is irradiated for 4 sec. After washing with sulphur/isoamyl alcohol, the ethanol precipitation is carried out with 0.3 M sodium acetate in ethanol solution. The DNA was purified. For the turpentine lysis, the cells were heated at 95 C for 5 minutes and the DNA was purified as in the case of laser cleavage. The plastids were separated using the Qiagen QIAprep® Miniprep kit. The genome of BL21 was isolated using a ton of (3) φ QIAamp® DNA Mini kit. DNA analysis was carried out with a 7% agarose gel and a 1 kb marker. Example 1: Effect of Laser Irradiation on Cell Survival in the Presence of Magnetic Beads The effect of Rena's cell survival in the presence of magnetic beads was investigated. Figure 6 shows the results of measuring cell viability after irradiation with a laser. The cells (3χ1〇3) were irradiated with lasers in the presence of microbeads (Α and Β) or in the absence of (C) for a period of time, and then distributed on the LB disk. The LB disk was cultured overnight at 37 ° C, and then the number of colonies formed was counted out (A: the cells of the suspension towel irradiated with a laser for a period of time in the presence of micromagnetic beads; B: a simple f shot After washing the cells recovered by the beads; C: cells under the same conditions as A, but the laser irradiation is carried out in the absence of micromagnetic beads, and the period during which the cells are irradiated with laser light is prolonged; D: unirradiated The positive control group of the laser). As can be seen in Figure 6, bacterial cells lose their viability due to exposure to lasers. The addition of magnetic beads has suddenly increased the loss of viability. Irradiating the laser in the presence of magnetic beads

(S 28 1303276 seconds later 5% of the starting cells (153 of 3,000 cells) survived, irradiated with lightning: after 10 seconds of exposure, cell-free survival (Figure A). Conversely, the magnetic beads are not In the presence, even after 30 seconds of exposure to the laser, about two-thirds (about 2 cells) of the starting cells survive (C in Figure 6). To identify cells that are not specifically bound to the magnetic beads. The magnetic beads were washed with a high salt concentration buffered saline (PBS + OJMNaCl), and the living cells of the washing solution were also examined (B of Fig. 6). A small amount of living cells were recovered, and their survival kinetics and self were recovered. The survival power of the cells recovered by the suspension is the same, showing that the cells recovered from the φ magnetic beads are similar to the cells in the solution between the magnetic beads. These results are not taught: because the laser can be irradiated in the presence of magnetic beads. Causes rapid cell rupture, so it can be used to release any type of DNA, such as genomic, episomal, or viral DNA present in cells. Example 2: Effect of irradiating a laser on the release of DNA from cells because of the above observation Loss of viability does not necessarily correspond to the release of DNA from cells Cell lysis is related, so 16S _ rDNA is amplified by using the above PCR to check the presence of DNA in the solution. The 7th ugly laser can only efficiently release bacterial DNA in the presence of magnetic beads. The long graph indicates amplification of DNA. Wavelength (ng/ //1). The amount of pCR product was quantified by Agilent Bi〇Analyzer 2丨〇〇. The error bar graph represents the standard deviation of the mean. As shown in Figure 7, when the solution is The use of the template DMA source B$ 'i6S rDNA is most efficiently amplified. Furthermore, the 1 of the pcr product is proportional to the irradiation time, and the magnetic beads can dramatically increase the pCR efficiency (2 times or 8 29 1303276 more. These results are consistent with cell viability. That is, these results show that the loss of cell viability is not due to the thermal inactivation of cells produced by cell-free lysis, but rather to Cellular physical rupture. Example 3: Comparison of DNA release efficiency of laser ablation cell lysis and chemical cell lysis using micromagnetic beads DNeasy was used to directly compare the DNA release efficiency of cell lysis according to the present invention. Knowing the DNA release efficiency of chemical cell lysis, DNeasy is a Qiagen kit for cell lysis and release of DNA. Figure 8 shows DNA released by laser ablation compared to DNA prepared by conventional methods. More efficiently amplified by Taq polymerase. DNA was prepared using the same number of DNA in all experiments. The bar graph indicates the concentration of amplified DNA (ng///l). The amount of PCR product was quantified by Agilent BioAnalyzer 2100. . The error bar graph represents the standard deviation of the mean.

❿ As shown in Figure 8, the DNA obtained after irradiation with a laser in the presence of magnetic beads can amplify 16S rDNA more efficiently. Considering that the Qiagen kit is not the most suitable for samples containing the number of small cells (<1x10), the DNAI recovered using the Qiagen kit may be less than expected. Despite this, the cell lysis by laser and magnetic beads can be easily integrated with the LOC, thus providing a more versatile application. In addition, it was found that the PCR amplification efficiency of the DNA released by laser ablation is larger than that of the DNA obtained by using the Qiagen. kit or boiling method. This shows that the amount of DNA released by the laser can be the same or more than the amount of DNA released by the other two conventional methods to 8 30 1303276. If the same amount of DNA is released, the PCR amplification from the laser ablated dna is more efficient, that is, the release of the laser ablation reducing inhibitor is shown. Example 4: Effect of magnetic bead size on cell lysis efficiency The effect of magnetic bead size on the release of DNA from cells was investigated. Figure 9 shows the effect of the bead size. The long bar graph indicates the concentration of amplified DNA (ng///1). The amount of pCR product was quantified by Agilent BioAnalyzer 2100. The error bar graph represents the standard deviation of the mean. As shown in Figure 9, a 2j/m diameter magnetic bead is much more effective at cell lysis than 5nm gold particles (G1402 from Sigma, Missouri, USA). Example 5 · Laser penetration test on the top cover of the wafer. Pyrex 7740 and AR-coated Pyrex 7740 (Coming) were used as the wafer top cover to check whether the laser generated by the laser generator is effective. Through the wafer top cover, the laser penetration rate was measured at a laser power of 1, 2, 3, and 4 W using a 30 W Broadband Power/Energy Meter (MELLES GRIOT, USA). As shown in Fig. 10, the 1^1> dazzle 774 coated with argon has a higher laser transmittance of about 1.75% than the Pyrex 7740 which is not coated with AR. Therefore, the AR coated Pyrex 7740 is suitable for efficient delivery of lasers to cell lysis wafers. Example 6: Anti-volatile test on a cell lysing wafer 31 13 〇 3276 produced: = test to check whether the cell lysis crystal of the present invention is caused by a laser 21 __. Using the cell lysing wafer prepared as described above, the same experiment was carried out at least 2 (10) times under the laser power of the tearing == tear. Figure n is a photograph of the cell lysing wafer of the present invention after firing. If the power is (4), or , 'the temperature of the sample solution increases, the system pressure increases, and the cell lysis film does not volatilize. Example 7: Effect of the amount of magnetic beads on cell lysis efficiency The effect of the amount of magnetic beads on the cell lysis efficiency, i.e., the amount of DNA released, was investigated. Different amounts of magnetic beads were added to the sample solution (〇 to (4) 6 beads (10) and irradiated _ thief's 丨w turn (four) shot Qing. The DNA ❺ PCR results released by the 120th bacterium of Enterobacter sphaeroides were plotted against the amount of magnetic beads. Again (cr〇ssing point,

Cp) is the number of cycles in which the detectable fluorescence is first measured in g immediately. That is, the Cp value decreases as the initial concentration of DNA increases. Cp is also involved in DNA purification.

The Cp value decreases as the purity of the DNA increases. Therefore, when the Cp value is low, the DNA in the solution is relatively pure. As shown in Fig. 12, the amount of DNA released increases with the addition of magnetic beads. When the magnetic bead concentration is higher than 5 x 106 beads/#1, good efficiency of cell lysis and DNA release are produced. Further, in order to accurately determine the starting target copy number, the Cp value of E. coli DNA amplification was examined by LightCycler® (Roche Diagnostics Co., Inc., Indiana, USA) called a instant pcr machine. As shown in Fig. 12, the Cp value decreases as more magnetic beads are added.

32 1303276 This, the result of the teaching, the number of initial target copies increases with the addition of more magnetic beads. • Example 8 • Effect of vibration power on cell lysis efficiency Use a 0 to 4 V vibration motor to check the effect of vibration power on cell lysis efficiency. In each reaction, Escherichia coli cells (1χ1〇5 cells/#1), which are Gram-negative bacterial cells, were used. The 10//1 sample was irradiated with a 808 nm laser with a power of w·5 w for 40 seconds. The distance between the cell lysis wafer and the fiber is a kneading surface. The sample in the middle of the magnetic beads is 5xl 〇 6 magnetic beads / μ Bu test conditions are repeated three times. Figure 13 plots the PCR results of the DNA released from E. coli cells versus the vibration motor voltage. In Figure 13, 'boiling (positive control group) means that E. coli cells were at 95. The case where the DNA released after boiling for 5 minutes was subjected to PCR; the negative control group was an example in which only PCR was used without water and PCR was performed. The long bar graph indicates the concentration of the amplified DNA (ng//z 1). The amount of PCR product was quantified by Agilent BioAnalyzer 2100. The error bar graph represents the standard deviation of the mean. As the amount of amplified DNA increases, the number of cell lysis increases, indicating an increase in cell lysis efficiency. As shown in Fig. 13, as the voltage of the vibration motor increases, the cell lysis efficiency increases. Vibration motor voltages above 3 V are sufficient to achieve higher cell lysis efficiency than using the boiling method. Further, when the voltage of the vibration motor is increased, the amount of released DNA is increased due to an increase in cell lysis efficiency, and therefore, the Cp value is decreased. - Therefore, after mixing cells or viruses with magnetic beads, cells can be increased by vibration to increase the efficiency of lysis. 33 © 1303276 Example 9 • Effect of laser cleavage efficiency on Gram-positive fine g cells using 〇·5 to 3 W laser power to check the laser cleavage efficiency of Gram-positive bacterial cells should. The same experiment as in Example 8 was carried out, but using Staphylococcus epidermidis cells of Gram-positive bacterial cells (1χ1〇5 cells (4), the distance between the cell lysis wafer and the optical fiber was 3 mm, and the sample bead concentration was 9 χι. 〇6 magnetic beads/μb Figure 14 shows the PCR results of Staphylococcus epidermidis cells (1χ1〇5 cells (4) released DNA. In the 14th towel, the control group refers to the S. epidermidis cells After centrifugation at 13,200 rpm for 5 minutes, an example was obtained in which the above solution was subjected to pCR. Boiling (positive control group) refers to an example in which PCR was carried out on DNA released after S. epidermidis cells were boiled for 5 minutes at 95% under the armpits;贞 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The standard deviation of the values. As shown in Figure 14, when the laser power increases, the cell lysis efficiency increases. In particular, the laser power above 丨w is sufficient to obtain cells that are the same or higher than the boiling method. Cracking efficiency. Therefore, when When the microchip of the present invention lyses cells or viruses, the laser power can be remarkably reduced. To examine the effect of cell concentration on cell lysis efficiency, the same experiment as described above is performed, but using E. coli cells which are Gram-positive bacterial cells (1χ1〇2 cells///1).

Figure 15 is a graph showing the PCR results of the DNA 34 1303276 released by Staphylococcus epidermidis cells (1χ1〇2 cells/“丨) for the laser power. In Figure 15, the control group refers to the Staphylococcus epidermidis cells. An example of obtaining a supernatant solution after centrifugation at 13,200 rpm for 5 minutes; boiling (positive control group) refers to an example of performing PCR on DNA released after S. epidermidis cells are still boiled for 5 minutes; The negative control group refers to an example in which only the water in the library is used without PCR, and the long graph indicates the concentration of the amplified DNA (ng/ //1). The amount of the PCR product is quantified by the Agilent BioAnalyzer 2100. The graph represents the standard deviation of the mean. As shown in Figure 15, as the laser power increases, the cell lysis efficiency increases. In particular, laser power above 3 w is sufficient to obtain the same or more than the boiling method. High cell lysis efficiency. Therefore, regardless of the cell concentration, the Gram-positive bacterial cells can be efficiently lysed using the method of the present invention. Furthermore, it is possible to efficiently lyse another cell for examination using the cell lysis of the present invention. The Gram-positive bacterial cell S. mutans was subjected to the same experiment as above, but sputum sputum cells and transglycogen spheroid cells were used, and the lura power of iw was used for 40 seconds. Fig. 16 is the epidermis PCR results of Staphylococcus aureus cells and S. mutans cells releasing 2 DNA. In Figure 16, sample 1 refers to an example when PCR is performed on DNA released from S. epidermidis cells; sample 2 refers to Staphylococcus epidermidis The case where the DNA released by the cell after 5 minutes at 95 C is subjected to PCR; the sample • 3 refers to the case when PCr is released from the DNA released by S. mutans cells; sample 4 • refers to the transglycan Example of PCR when streptococci cells boil at 95 ° C for 5 minutes and dna 8 35 l3 〇 3276 is released; and sample 5 refers to an example in which only distilled water is used and no DNA is used for pcR. The long graph indicates amplification of DNA. Concentration (ng///l). The amount of PCR product was quantified by Agiient BioAnalyzer 2100. The error bar graph represents the standard deviation of the mean. As shown in Figure 16, the cell lysis method of the present invention is in the epidermis. Staphylococcus cells and S. mutans Both cells have better cell lysis efficiency than when using the boiling method. Example 10 · The effect of laser power on the cleavage efficiency of Gram-negative bacterial cells using 〇 to 3 Wfr projectiles to check the laser god Effect of lysis efficiency of negative bacterial cells. The same experiment as in Example 9 was carried out, but Escherichia coli cells (lxl〇5 cells///1) which are Gram-negative bacterial cells were used. Figure 17 shows Escherichia coli cells. (lxl〇5 cells//zl) Map of DNA2 PCR results released. In Fig. 17, boiling (positive control group) means that E. coli cells are in %. An example in which the released DNA was subjected to pCR after boiling for 5 minutes; and a negative control group was an example in which only steamed water was used without PCR. The long bar graph indicates the amplification of the DNA (ng/ //1). The amount of pcR product was quantified by Agilent Bi〇AnalyZer 2 j 〇〇. The error bar _ represents the standard deviation of the mean. As shown in Figure 17, as the laser power increases, the cell lysis scale increases. In particular, ^ = power above i W is sufficient to achieve higher cell lysis efficiency than using the boiling method. In addition, as the laser power increases, the Cp value decreases, indicating an increase in the amount of released DNA. However, = the firing power is above 2 w and it is saturated. This result teaches that the target copy number Ik increases with increasing laser power until all cells are lysed. 36 1303276 Thus, when using the microchip of the present invention to lyse cells or viruses, the laser power can be significantly reduced. In order to examine the effect of cell concentration on cell lysis efficiency, the same experiment as above was performed, but Escherichia coli cells (1χ1〇2 cells///1) which are Gram-negative bacterial cells were used, and Escherichia coli was shown in Fig. 18. A plot of PCR results for DNA released from cells (ιχι〇2 cells/#丨). In Fig. 18, the control group refers to an example in which E. coli cells are centrifuged at 13, 2 rpm for 5 minutes to obtain a supernatant solution for PCR; boiling (positive control group) means that E. coli cells are obtained at 95. An example in which the released DNA was subjected to PCR after boiling for 5 minutes at ° C; and a negative control group was an example in which only distilled water was used and no DNA was used for pCR. The long bar graph indicates the concentration of amplified DNA (ng^i). The amount of PCR product was quantified by AgUent BioAnalyzer 2100. The error bar graph represents the standard deviation of the mean. As shown in Figure 18, as the laser power increases, the cell lysis efficiency increases. In particular, a laser power higher than 〇·5 w is sufficient to achieve a higher cell lysis efficiency than the boiling method. The 3 W laser power provides much more cell lysis efficiency than using the boiling method. Therefore, when the microchip of the present invention is used to lyse cells or viruses, the laser power can be remarkably reduced. Therefore, regardless of the cell concentration, the Gram-negative bacterial cells can be efficiently cleaved using the method of the present invention. 37 1303276 Example 11 • The effect of the laser power on the temperature of the E. coli sample, using the laser power of 0·5, 1, and 2 w to check the laser power in the E. coli risk (6) = effect as in Example 1 The same experiment was carried out, but Escherichia coli cells (IxlG5 cells///1) which were Gram-like cells were used. » 19 Figure shows the large = the plot of the temperature of the sample sample versus the laser power. As shown in Fig. 19, the sample temperature Ik is increased by the laser power. In particular, after a few seconds of laser power with an illumination power higher than 丨w, the sample temperature rapidly increased to above 65 °C. Example 12: Effect of magnetic bead surface charge and magnetic bead material Four different types of beads were used to examine the effect of the magnetic bead surface charge and magnetic bead material. The same experiment as in Example 1 was carried out, but the bead concentration was 〇·5%. Figure 2 shows the DNA2 PCR results of E. coli cells (lxl〇5 cells//ζ1) versus the surface charge of the beads and the magnetic bead material. In the second diagram, samples 1, 2, 3, and 4 refer to the release of styrene magnetic beads using terminal amine-based polystyrene beads, argon beads, polystyrene beads, and terminal carboxylic acids. An example of DNA when PCR is performed. Sample 5 (positive control group) refers to an example of PCR for DNA released after boiling E. coli cells at 95 ° C for 5 minutes; and sample 6 (negative control group) refers to PCR using only distilled water without DNA An example of time. The bar graph indicates the concentration of amplified DNA (ng/#^. The amount of PCR product was quantified by Agilent BioAnalyzer 2100. The error bar graph indicates the standard deviation of the mean. As shown in Figure 20, only the carboxylic acid at the terminal carboxylic acid In the presence of ethylene magnetic beads, the E. coli DNA can be released by efficient irradiation of the laser. In addition, the temperature of the sample solution is studied in four types of magnetic beads (data not shown) 38 1303276 Sample solution with helium oxygen beads The temperature increase is extremely slow, because the oxygen beads do not fully absorb the laser beam with a power of 1 w. The temperature of the sample solution with the terminal amine-based polystyrene beads increases like the styrene beads of the terminal carboxylic acid, but the release The dna is bound to the microbeads due to the positive charge of the amine on the surface of the beads. Because of the heat of the beads, the temperature of the sample solution of the polyethylene beads increases at the speed between the stones and the beads.最大 The most advantageous advantage of styrene beads with terminal carboxylic acids is the reduction of the DNA separation step, as cell lysis using lasers and microbeads can lead to denaturation and removal of proteins. Denatured proteins and cells The fragments stick to the polystyrene surface of the magnetic beads due to adsorption, so that they can be easily removed by gravity or magnetic field. Because the carboxylic acid of the beads is negatively charged and electrically repelled, the DNA does not bind to the beads. Detection limits, reduced dna extraction time, and increased signal size, which significantly improved PCR yield. Example 13: Magnetic Bead Surface Functionality Based on DNA Amplification Efficiency Effect Study of Cellular Fission Explained DNA Amplification Efficiency to Check Magnetic Bead Surface Functionality Based Cells The effect of cleavage efficiency. First, various functional groups are synthesized on the surface of the magnetic beads. Figure 21 shows an example of synthesizing imine diacetic acid (IDA), hydrazine, and thiol functional groups on the surface of magnetic beads. The procedure for synthesizing each functional group on (Dynabeads®) is as follows: (1) IDA beads Take 500 // 1 magnetic beads with amine groups (Dynabeads® M-270 Amine, 30 mg/ml) and remove the solution using a magnet. 5 〇〇//1 of 丨-methylpyrrolidone 39 1303276 (l-methyl-2-pyrrolidone, NMP) was thoroughly mixed with the beads, and then the solution was removed. This procedure was repeated three times. Ethyl bromoacetate (ethylbromoacetate) ) (6# 1) with triethylamine (1 0 //1) The solution of NMP (500 //1) is thoroughly mixed with the beads and placed at 45 ° C for 1 day. After the reaction is completed, the beads are washed at 500//1 (χ3) and then Wash the beads with 500 /zl of ethanol (χ3). Remove the solution with a magnet, and then add 500//1 of a 1: 1 (v/v) 1 Ν mixture of NaOH and ethanol to the beads and place at room temperature. After 1 hour, after the reaction was completed, the beads were washed with 500//1 of ethanol (χ3), and then the beads were washed with 500//1 of tertiary distilled water (χ3). After that, the solution was removed and 500//1 of the desired buffer solution was added. The resultant was stored in a refrigerator. (2) Cu-IDA beads A solution of Cu(N03)2 (100 mg) and TEA (100//l) in NMP (500 /zl) was added to IDA beads, and the resultant was allowed to stand for 1 day. After the reaction was completed, the beads were washed with 500 //1 (ΝΜΡ3), 500 //1 ethanol (χ3), and tertiary distilled water (χ3). After that, the solution was removed and 500//1 of the desired buffer solution was added. Store the contents in the refrigerator. (3) Pyrene-beads Take 500 // 1 Amine-based magnetic beads (Dynabeads® M-270 Amine, 30 mg/ml) and remove the solution with a magnet. 500//1 of NMP was thoroughly mixed with the beads and the solution was removed. Repeat this step three times. U-Pyrenebutyric acid (15 mg), 〇-benzotriazole·1_yl-N,N,N,,N,_tetramethyl" glandular hexafluoride Acid salt 1303276

(o-benzotriazole· 1 -yl-N,N,N,,N'-tetramethy luroniumhexafluorophosp hate,HBTU) (22 mg) and triethylamine (TEA) (7 //1) in NMP (500 // 1) The solution was thoroughly mixed with the beads and left at room temperature for 1 day. After the reaction was completed, the beads were washed with 500//1 NMP (x3), 500//1 ethanol (x3), and 500//1 third grade distilled water (x3). Thereafter, the solution was removed and 500//1 of the desired buffer solution was added. The resultant was stored in a refrigerator. (4) Thiol-beads ® Take 500 //1 magnetic beads with amine groups (Dynabeads® M_270 Amine, 30 mg/ml) and remove the solution with a magnet. 500//1 of NMP was thoroughly mixed with the beads and the solution was removed. Repeat this step three times. Mix 3-methoxycaptopropionic acid (10 //1), HBTU (22 mg) and TEA (7 //1) in NMP (500//1) with the beads and place at room temperature Next day. The beads were washed at 500//1 (χ3) and 500/1 ethanol (χ3) after the reaction was completed. Use a magnet to remove the solution, and then add 500//1 of 1:1 (v/v) in NaOH and B? This mixture was poured into the beads and placed at room temperature for 1 hour. After the reaction was completed, the beads were washed with 500//1 of ethanol (X3) and 500//1 of tertiary distilled water (X3). After that, the solution was removed and 500//1 of the desired buffer solution was added. Store the resultant in a refrigerator. Magnetic beads with functional groups synthesized thereon were used to study DNA amplification efficiency. Perform • The same experiment as in Example 12, but using E. coli cells (lxlO7 cells/# • 丨). Figure 22 is a graph showing the PCR results (Cp) of DNA released from Escherichia coli cells for the surface functional groups of 41 8 1303276 magnetic beads. In Fig. 22, a carboxy carboxy bead refers to an example in which PCR is carried out after cell lysis using Dynabeads® MyOneTM Carboxylic Acid (DYNAL, Norway); boiling (positive control) means that Escherichia coli cells are The case where the released DNA was subjected to PCR after boiling at 95 ° C for 5 minutes; the control group refers to an example in which E. coli cells were centrifuged at 13,200 rpm for 5 minutes to obtain a supernatant solution for PCR; and a negative control group ( NTC) refers to an example in which only distilled water is used and no DNA is used for PCR. As shown in Fig. 22, the IDA in the functional group has the lowest Cp value, and the Cp value of the thiol is smaller than the Cp value of Cu-IDA. Furthermore, as the hydrophilicity of the functional group increases, the Cp value decreases. That is,: 0) Eight has a lower value than the thiol. From the above results, it is understood that beads having a hydrophilic carboxyl group have an optimum cell cleavage efficiency' and Cu-IDA beads having a blocked functional group have low cell lysis efficiency. Example 14: Effect of pH of a solution containing magnetic beads on DNA amplification efficiency Three types of beads, namely, a magnetic bead having a carboxyl group (Dynabeads®), a magnetic bead having IDA, and a polycarboxy magnetic bead were used for inspection. The effect of the pH of the solution containing the magnetic beads on the efficiency of DNA amplification. Since the beads having a carboxyl group have the highest cell cleavage efficiency as a result of the above experiment, polycarboxylate beads having a plurality of carboxyl groups are synthesized. Figure 23 shows the process for synthesizing polycarboxylates on the surface of the beads. The procedure for synthesizing the polycarboxylate on the magnetic beads was as follows: Take 500 // 1 magnetic beads with amine groups (Dynabeads® M 27 Amine, 3 〇 mg/ml). The solution was removed using a magnet. Mix the NMp of the lion... with the beads

42 1303276 Close, then remove the solution. Repeat this step three times. Mix the (ethylene/alt-maleic anhydride) (100 mg) and triethylamine (10//1) solution in NMP (500//1) with the beads. And placed at room temperature for 1 day. After the reaction was completed, the beads were washed with 500//1 NMP (x3) and 500#1 ethanol (χ3). The solution was removed using a magnet, and then 500//1 of 100 mM Tris-HCl buffer solution (pH 9·0) was added to the beads and allowed to stand at room temperature for 1 hour. After the reaction was completed, the beads were washed with 500//1 ethanol (χ3) and 500//1 tertiary distilled water (χ3). After that, the solution was removed and 500//1 of the desired buffer solution was added. Store the resultant in a refrigerator. The same experiment as in Example 13 was carried out, but the three types of beads were adjusted to ρ Η 5, 7, and 9. Figure 24 is a graph showing the PCR results (Cp) of DNa released from Escherichia coli cells for the surface functional groups of the magnetic beads and the pH of the magnetic bead solution. In Fig. 24, the control group is an example in which PCR is carried out after cell lysis using Dynabeads® My OneTM Carboxylic Acid (dynal company, Norway). As shown in Fig. 24, the Cp value p is now decreased as the pH value is increased. Therefore, it is understood that the cell lysis efficiency increases as the pH of the solution containing the magnetic beads increases. However, at the same pH, the cell lysis efficiency did not change significantly due to the uterine base. Example 15: Comparison of PCR efficiencies in the presence of inhibitors In κ cases 13 and 14, when pure E. coli cells were used, the cell lysis rate was determined by the pH of the solution containing the magnetic beads. When the surface functional group is a carboxyl group, the cell cleavage efficiency changes finely due to its structure. This side is #only when pure 43 8 1303276 E. coli cells are used, the inhibitory effect of cell debris on PCR is not significant. Therefore, comparing the cell lysis efficiency according to the magnetic bead pattern and pH to examine the surface of the bead is based on the effect of removing the PCR inhibitor. First, the same experiment as in Example 14 was carried out, except that 1% by weight of serum was added as an inhibitor, and the pH of the solution containing the magnetic beads was adjusted to 7 and 9. Figure 25 is a graph showing the PCR results of DNA released from E. coli cells in the presence of ίο% serum (4). In Figure 25, the control group refers to the use of Dynabeads8.

MyOn# CarboxylicAdd (Norway's DYNAL) is a case of PCR after cell lysis in the presence of 1% serum; PTC (positive control) refers to the addition of 1% serum to the DNA released by E. coli An example of a pcR. As shown in Fig. 25, the Cp value of the magnetic beads having the same functional group varies depending on the pH. That is, the carboxyl group and IDA have a lower Cp value at pH 7 than pH 9, and the polycarboxy group has a lower Cp value at pH 9 than pH 7. Therefore, it is understood that when the carboxyl group is appropriately combined with the pH of the solution containing the magnetic beads, the effect of the inhibitor on pCR2 can be remarkably reduced.

Next, the same experiment as described above was carried out, but Escherichia coli was replaced with hepatitis B virus (HBV), and the pH of the solution containing the magnetic beads was adjusted to 7. The following primer pairs were used for PCR: positive stock primer (sequence ID number: 3); anti-strand primer (sequence ID number: 4). This pair of primers is the site corresponding to the core region of the HBv genome. Figure 26 is a graph showing the pCR results (concentration of pCR • product) of the DNA released by HBv in the presence of 1 〇〇 / 0 serum for the functional groups on the surface of the magnetic beads. In Figure 26, the control group • refers to the case of using pcRR after HBV lysis in the presence of serum in the presence of Dynabeads® MyOneTM Carboxylic Acid (DYNAL, Norway); pTc (positive, control) refers to In the presence of 1% by weight of serum, PCR is performed on the DNA isolated from HBV. As shown in Fig. 26, the PCR product can be produced even in the presence of 1 G% of serum. Because &, when the carboxyl group is properly combined with the solution containing the magnetic beads, the effect of the inhibitor on PCR can be significantly reduced. Example 16 • Study on cell viability according to laser irradiation φ Using E. coli cells, observe the presence of live and dead cells in the sample solution according to laser irradiation. Figure 27 is a photograph showing the viability of large intestine rod S cells according to laser irradiation. In Figure 27, Panel A (PandA) is an image of E. coli cells before laser irradiation without microbeads; Plate B is E. coli cells irradiated with a laser power of 0.5 W at microbeads. _ nm laser image after 4 sec.; c-plate E. coli cells in the presence of micro-magnetic beads with 丨w laser power illuminating 8 〇 8 laser 40 seconds after the image. The green stained cells are living cells, and the red stained cells are dead cells. As shown in Fig. 27, most of the cells are alive before laser irradiation, and the proportion of dead cells in the dead increases with the increase in laser power. Example 17: Effect of laser irradiation on genomic DNA damage Check whether the isolated DNA is deformed and sheared after 4 hours of exposure to the laser to check whether the laser irradiation damages the genome. Figure 28 is a DNA analysis photograph of the E. coli BL21 cells obtained by harboring pCR®II_T〇P〇® (Invitr〇gen) plastids. In Fig. 28, the first channel (1^^ i) - refers to the case when the DNA is isolated by the method of the present invention; the second track refers to the DNA separation after 5 minutes of the sputum 45 8 1303276 Example; the third refers to the case when the Qiagen QIAprep8-miniprep kit is used to isolate the plastid DNA; the fourth refers to the case when the Qiagen, QIAamp® DNA minikit is used to isolate the BL21 genome DNA; And the fifth finger is an example when the genome DNA of BL21 is isolated from Escherichia coli BL21 cells using the Qiagen QiAamp8 DNA small set without plastid. Use lane 5 to identify the correct band of the genomic DNA. As shown in Figure 28, DNA is not damaged. As expected, plastids were prepared using the QIAprep Mini Kit (Qiagen), and φ had little contamination of the genomic DNA (lane 3). Interestingly, the use of the method of the present invention can preferentially isolate the genomic DNA, which is rarely contaminated by plastid DNA. On the other hand, when the genomic DNA was isolated by the QIAamp subgroup, a large amount of plastid contamination was observed because of the use of the Shihe oxygen-gel-membrane technique after the protein s#k treatment of the bacteria (Track 4). This may explain why the DNA isolated by the method of the present invention is subjected to PCR amplification, and a better yield can be obtained than the DNA isolated using the Qiagen kit. In the present invention, an efficient cell splitting solution is developed by combining a laser and a micromagnetic bead; when a Weiguang New product is applied, the microbeads in the suspension of the fine cypress float can cause rapid Cell lysis allows the bacterial cells to rupture in just a few seconds. Most importantly, the DNA released by cells ruptured in this way is more efficient than PCR-expressed DNA released by cells lysed by two other conventional methods, showing release during cell lysis. Out of the interference dna magnified material, with other methods she is less. This simple and efficient cell lysis and release of the ship is very suitable for integration into the LOC lysis process. 1303276 As described above, according to the method of the present invention, rapid cell lysis is possible within 40 seconds, and a laser or a rupture device of a virus can be miniaturized by using a laser diode, and DNA purification can be directly performed after rupturing a cell or a virus. In the step, and after removing the cell debris and the microbeads (which will adhere to the inhibitor that inhibits the subsequent reaction) using a magnet, the DNA-containing solution is transferred to the subsequent step. In addition, by the cell lysis of the cell of the invention, the volatilization problem can be solved; the magnetic beads can be efficiently transmitted to the cells via the magnetic beads; the micro-jet problem on the rough surface can be solved by hydrophobic treatment of the inner surface of the wafer; The cell lysis wafer was applied to the LOC. While the invention has been particularly shown and described with reference to the embodiments of the present invention, it is to be understood that category. Illustrated in Figure 8 is a system used in cell lysis using microbeads and lasers. Figure 2A shows the use of lasers and microbeads in accordance with a specific embodiment of the present invention. Cell lysis system on microchips. Figure 2B shows the system design of Figure 2A. Figure 3 is a photograph of the separator member of the cell lysis apparatus. Figure 11 is a schematic diagram of the micro-months and body shells used in the micro-magnetic beads and laser rupture cell devices. 'Back is a microchip photograph in accordance with a specific embodiment of the present invention. 47 1303276 Figure 6 shows the results of measuring cell viability after irradiation with a laser. Figure 7 shows that laser irradiation can effectively release bacterial DNA only in the presence of magnetic beads. Figure 8 shows DNA released by laser ablation, compared to DNA prepared by conventional methods. It can be amplified more efficiently by Taq polymerase (called polymerase). • Figure 9 shows the effect of the bead size. Figure 10 is a graph showing the penetration rate (transmi gance) of pyrex 7740 and pyrex 7740 treated by anti-reflection (AR) coating. Figure 11 is a photograph of a cell lysing wafer irradiated with a laser in accordance with a specific embodiment of the present invention. Figure 12 is a plot of PCR results for magnetic beads released from E. coli cells. Figure 13 is a graph showing the pCR results of DNA released from E. coli cells versus vibrator voltage. Figure 14 is a plot of the PCR results of Staphylococcus epidermidis (and the DNA released from the 哗 / / 卿 ^ 谈 细胞 cell (jχ 105 cells / / / 1) on the laser power. Figure 15 is the S. epidermidis cells ( 1χ1〇2 cell 仏丨) The PCR result of the released DNA is plotted against the laser power. Figure 16 is the DNA released by the S. epidermidis cells and the PCR of the S. mutans (and the DNA released by the cells) Results map.

Figure 17 is a plot of PCR 8 1303276 results for laser power released from E. coli cells (lxio5 cells ///1). Figure 8 is a plot of PCR results versus laser power for DNA released from E. coli cells (lxl〇2 cells ///1). Figure 19 shows a plot of E. coli sample temperature versus laser power. Figure 20 is a diagram showing the results of PCR of DNA released from Escherichia coli cells (lxl〇5 cells///1) versus magnetic bead surface charge and magnetic bead material. Figure 21 illustrates a method of synthesizing imine φ diacetic acid, Cu_IDA '芘, and thiol functional groups on the surface of a magnetic bead in accordance with an embodiment of the present invention. Figure 22 shows the PCR results of the DNA released from E. coli cells (crossing poim (Cp)) versus the functional groups on the surface of the magnetic beads. Figure 23 shows a specific example of the synthesis of polycarboxyl groups on the surface of the magnetic beads. Figure 24 is a graph showing the PCR results (Cp) of DNA released from Escherichia coli cells on the surface of the magnetic beads and the pH of the magnetic bead solution. Figure 25 is a graph showing the PCR results (Cp) of DNA released from Escherichia coli cells for the functional groups on the surface of the magnetic beads and the magnetic bead solution in the presence of 10% serum. Lu Figure 26 shows the PCR results of the DNA released by Hepatitis B virus (HBV) (concentration of PCR product) for the functional groups on the surface of the magnetic beads with 10% serum. Figure 27 shows a photograph of the viability of E. coli cells versus laser irradiation. Figure 28 shows a DNA analysis photograph of the E. coli BL21 cells obtained by harvesting ρ€Κ®ΙΙ-Τ0Ρ0® (Invitrogen) after irradiation with a laser. [Main component symbol description] Micromagnetic bead 2 Vibrator laser 4 Vial guide tube 6 Laser beam fiber 8 Microchip microbead 10 Inlet wafer top cover 12 Wafer body wafer bottom plate 14 Cell vibration transmission unit 16 vibrator laser 18 separator outlet 20 wafer body reaction tank 22 inlet outlet 24 wafer top cover wafer substrate 26 wafer bonding portion fiber cable 28 fiber laser beam channel 30 microchip vibrator/n 50

Claims (1)

1303276 X. Patent Application Scope 1. A method for rupturing a cell or a virus, comprising: adding a plurality of magnetic beads to a solution containing cells or viruses; vibrating the magnetic beads; and irradiating the magnetic beads with a thunder Shoot to rupture the cells or viruses. 2. If applying for the method described in item 1 of the “Nina”, the laser includes a pulse wave or continuous wave (CW) laser. The method of claim 2, wherein the pulse laser is 1 plus/pulse or more, and the CW laser has a power of 1 〇mw or more. 4. The method of claim 3, wherein the pulsed laser is 3 mJ/pulse or more, and the CW laser has a power of 100 mW or more. The method of claim 1, wherein the laser has a wavelength in the range of 400 nm or more. 6. The method of claim 5, wherein the laser has a wavelength in the range of 750 nm to 1300 nm. 7. The method of claim 5, wherein the laser has one or more wavelength ranges. 51 1303276 8. The method of claim 1, wherein the magnetic beads have a size of 50 nm to 1,000^. 9. The method of claim 8, wherein the magnetic beads have a size of i to 50//m. 10. The method of claim 8, wherein the magnetic beads are a mixture of beads of two or more sizes. 11. The method of claim 1, wherein the magnetic beads comprise at least one material selected from the group consisting of ferromagnetic Fe, Ni, Cr, and oxides thereof. 12. The method of claim 1, wherein the magnetic beads are a polymer coated with a ferromagnetic metal, an organic material, a stone, or a glass. Φ1. The method of claim 1, wherein the functional groups on the surface of the magnetic beads are hydrophilic and negatively charged. The method of claim 13, wherein the functional group on the surface of the magnetic beads is a carboxyl group or a derivative thereof. 15. The method of claim 1, wherein the magnetic bead-containing solution has a pH of from 6 to 9. 52 1303276 16. The method of claim 1, wherein the solution is selected from the group consisting of saliva, silk, blood, serum, and cell culture fluid. By. 17. A skirt for rupturing a cell or virus, comprising introducing a sample and a magnetic bead through the inlet; 'to lyse the cell in the cell, a sample cell-cracking tank having an inlet, a vibrator Attached to the cell lysis tank and magnetic beads mixed; and sin- (iv) produced, attached to the cell lysis tank to provide a laser. 18. The device according to claim 17, wherein the actuator is selected from the group consisting of a vibration of a sound wave, a vibrator using a magnetic field, a vibrator using an electric field, a mechanical vibrator, and a piezoelectric material. By. The device of claim 17, further comprising an electromagnet attached to the cell lysis tank to remove the magnetic beads in the cell lysis tank after cell lysis. The device of claim 17, further comprising a DNA purification tank connected to the cell lysis tank via a channel. 21. The device of claim 17, further comprising a DNA amplification channel coupled to the cell lysis cell via a channel. The apparatus of claim 20, further comprising a dna amplification tank connected to the DNA purification tank via a channel. 23. A device for rupturing a cell or a virus, comprising: - a cell lysis wafer having a population to introduce a sample and a magnetic bead via the human σ; a vibrator coupled to the vibration transmission portion The cell cleaves the wafer to mix the sample in the cell cleavage wafer with the magnetic bead, the vibration transmission portion is attached to the cell lysis wafer to transmit vibration to the cell lysis wafer; a laser generator attached to The cell lyses the wafer to provide a laser; and an anti-volatile portion attached to the cell lysis wafer to prevent evaporation of the sample. 24. The skirt according to claim 23, wherein the vibrator is selected from the group consisting of a magnetic vibrator, an electric field vibrator, a mechanical vibrator, and a piezoelectric material. The device of claim 24, wherein the vibrator transmits vibration to the bottom of the wafer via the vibration transmission portion. 26. The device of claim 23, wherein the anti-volatile portion comprises a septa. 27. The rupture of claim 26, wherein the separator is selected from the group consisting of a valve, a polymer structure, and a metal structure. 54 1303276 A cell-cleaving wafer for use in a device for rupturing a cell or a virus as described in any one of claims 23 to 27, comprising a top surface and a bottom surface of the opening and including a reaction tank, an inlet, and an outlet; a wafer top cover attached to the top surface of the wafer body to close the upper portion of the reaction tank, pass the laser, and have An inlet and an outlet; and φ a wafer substrate attached to a bottom surface of the wafer body via a wafer bonding portion to close the reaction vessel, the inlet, and the lower portion of the outlet. The wafer of claim 28, wherein the wafer top cover comprises a material selected from the group consisting of glass, polymer, and indium tin oxide (ITO) glass. 30. The wafer of claim 29, wherein the wafer top cover is treated by an anti-reflective (AR) coating. The wafer of claim 3, wherein the wafer top cover is an anti-reflective (AR) coated Pyrex 7740 glass. 32. The wafer of claim 28, wherein the wafer substrate comprises a material selected from the group consisting of glass, polymer, silicone, and double-sided tape. 33. The wafer of claim 28, wherein the wafer substrate comprises a group of 1303276 self-polymer, linaloic, glass, and indium tin oxide-glass. 34. The wafer of claim 33, wherein the wafer joint connects the crystal body to the wafer substrate via an adhesive material selected from the tape assembly. 35. The device of claim 23, wherein the laser comprises a pulsed laser or a continuous wave (CW) laser. 36. The device of claim 35, wherein the pulsed laser is 3 (four) pulses or more, and the cw laser has a power of just mW mosquitoes. 37. The apparatus of claim 23, wherein the laser has a wavelength in the range of 400 nm or greater than 4 〇〇 nm. 38. The device of claim 3, wherein the laser has one or more wavelength ranges. The size of the magnetic beads is set, wherein the magnetic beads are two or 39. 50 nm to ΐ, 〇00//ηι as described in claim 23 of the patent application. 4. A mixture of beads of various sizes as described in claim 39 of the patent application. The apparatus of claim 23, wherein the magnetic beads comprise at least one material selected from the group consisting of ferromagnetic Fe, Ni, Cr, and oxides thereof. 42. The device of claim 23, wherein the magnetic beads are a polymer coated with a ferromagnetic metal, an organic material, tantalum, or glass. 43. The device of claim 23, wherein the sample is selected from the group consisting of saliva, urine, blood, gold, and cell culture fluid. XI. Schema: 57