WO2015098959A1 - Sample disruption method, biomolecule extraction method, and disruption solution containing nanowires - Google Patents

Abstract

　Provided are a sample disruption method and a disruption solution in which biomolecules such as nucleic acids and proteins that leak into the disruption solution after sample disruption are not denatured, and in which there is no risk of negatively affecting analysis of extracted biomolecules. A sample can be disrupted, without negatively affecting the analysis of biomolecules, by a suspension step for suspending a sample selected from cells, viruses, and bacteria in a disruption solution containing nanowires and a stirring step for stirring the disruption solution in which the sample has been suspended.

Description

Sample crushing method, biomolecule extraction method, and crushing liquid containing nanowires

The present invention relates to a sample crushing method, a biomolecule extraction method, and a crushing liquid containing nanowires. In particular, a sample of cells, viruses, bacteria, etc. (hereinafter sometimes referred to as “cells”) is suspended in a disruption solution containing nanowires (hereinafter also simply referred to as “disruption solution”). By stirring the cells, the cells and the like are disrupted without pretreatment such as lysis, and DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) (hereinafter, DNA and RNA may also be referred to as “nucleic acid”). The present invention relates to a sample crushing method, a biomolecule extraction method, and a crushing liquid containing nanowires, which can recover biomolecules such as proteins.

In recent years, due to advances in molecular biology, genetic analysis such as gene diagnosis such as gene deletion and drug susceptibility SNP (Single Nucleotide Polymorphism), diagnosis of infectious diseases due to pathogenic bacteria, and allergen diagnosis in hospitals and other medical institutions Accordingly, there is a need for a method for extracting a nucleic acid, which is a gene, from a cell or the like by an efficient and simple operation. In addition to genetic diagnosis, for example, a cancer diagnosis is performed by extracting a protein from a cell and identifying whether the extracted protein is a cancer marker protein. There is also a need for a method for extracting proteins with simple operations.

As a method of extracting biomolecules such as nucleic acids and proteins from cells and the like, a method of extracting biomolecules by crushing cells and the like using reagents and enzymes is known. Kits for this purpose are also commercially available from several companies, and biomolecules can be extracted from cells and the like relatively easily.

However, the above-mentioned kit for crushing cells and the like to extract biomolecules contains reagents such as phenol, chloroform and surfactant, and these reagents denature the biomolecules after crushing and extraction. It is known that the analysis may be adversely affected (see Non-Patent Documents 1, 2, and 3).

On the other hand, as a method for disrupting cells and the like without using a reagent, a method is known in which cells and the like are disrupted by moving the cells or the like so as to rub against a nanowire provided on a substrate (see Non-Patent Document 3). . However, all of the methods described in Non-Patent Document 3 are obtained by growing nanowires in microchannels provided on a substrate. Therefore, since the amount of cells and the like that can be crushed is limited by the size of the microchannel and the flow rate per unit time, there is a problem that the crushing efficiency is poor.

In addition, the invention described in Non-Patent Document 3 is an invention in which cells and the like are crushed when passing through the nanowire bundles grown in the microchannel at a flow rate of a predetermined level or more. The interval between them is constant. Therefore, for example, if the interval between the nanowire bundles is narrowed, clogging is likely to occur when the cells to be crushed are large, and conversely, if the interval between the nanowire bundles is widened, the nanowire bundles when the cells to be crushed are small. It passes between and becomes difficult to crush. For this reason, it is necessary to prepare chips with different intervals between nanowire bundles according to the size of cells to be crushed, which is inefficient. Furthermore, since the invention described in Non-Patent Document 3 grows nanowires in the microchannel, the surface of the grown nanowires is extracted from the target biological components after crushing cells or the like. There is a problem that it is difficult to apply.

A. Abolmaty et al. , “Effect of lysing methods and thevariables on the yield of Escherichia coli O157: H7 DNA and its PCR amplification13 (Journal of BioMole13) Dino Di Carlo et al. , “Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation”, Lab Chip, 2003, 3, 287-291 Jung Kim et al. , “Nanoire-integrated microfluidic devices for facilities and reagent-free mechanical cell lysis”, Lab Chip, 2012, 12, 2914-2921

The present invention is an invention made to solve the above-mentioned conventional problems, and as a result of extensive research, a sample such as a cell is suspended in a disruption solution containing nanowires, and then a disruption solution in which the sample is suspended is obtained. It was newly found that, by stirring, cells and the like can be efficiently disrupted without using a reagent or the like. Furthermore, it has been newly found that biomolecules such as DNA, RNA, and protein can be extracted with high purity by using nanowires in which one or two selected from proteolytic enzymes, ribonucleases, and deoxyribonucleases are immobilized. The present invention has been completed.

That is, the object of the present invention is to provide a method for crushing a sample using nanowires, a method for extracting biomolecules, and a crushing liquid containing nanowires.

The present invention relates to a sample disruption method using nanowires, a biomolecule extraction method, and a disruption solution containing nanowires, as described below.

(1) A suspension step of suspending a sample selected from cells, viruses, and fungi in a disruption solution containing nanowires,
A stirring step of stirring the crushed liquid in which the sample is suspended;
A method for crushing a sample.
(2) A suspension step of suspending a sample selected from cells, viruses, and fungi in a disruption solution containing nanowires,
A stirring step of stirring the crushed liquid in which the sample is suspended;
A residue removal step of removing the residue by centrifuging the stirred crushed liquid,
A method for extracting biomolecules.
(3) The method for extracting a biomolecule according to (2) above, wherein the nanowire is a nanowire on which one or two selected from proteolytic enzymes, ribonucleases, and deoxyribonucleases are immobilized.
(4) A sample disruption solution selected from cells, viruses, and fungi containing nanowires.
(5) The sample disruption liquid according to (4) above, wherein the nanowire is a nanowire on which one or two selected from proteolytic enzymes, ribonucleases, and deoxyribonucleases are immobilized.

In the present invention, since the sample can be crushed with the crushing liquid containing nanowires, it is not necessary to use a reagent or the like when crushing the sample. Therefore, biomolecules such as nucleic acids and proteins that leak into the crushing liquid after crushing the sample are not denatured, and there is no possibility that the analysis of the extracted biomolecules may be adversely affected or can be reduced.

In the present invention, the sample is crushed by suspending and stirring the sample in a crushing liquid containing nanowires. Therefore, there is no limitation on the capacity and flow rate of the micro-channel as in the prior art, and all the nanowires contained in the disruption liquid contribute to the disruption of the sample. It can be crushed.

In the present invention, since the nanowires are stirred in the crushing liquid in a dispersed state, there is no risk of clogging due to the residue of the sample after crushing or it can be reduced.

In the crushing liquid of the present invention, nanowires on which one or two selected from proteolytic enzymes, ribonucleases, and deoxyribonucleases are immobilized can be dispersed. Therefore, the target biomolecule can be recovered with high purity.

FIG. 1 is a diagram showing an example of a procedure for producing nanowires 4 used in the crushing liquid of the present invention. FIG. 2 is a drawing-substituting photograph, which is a field emission scanning electron microscope (FESEM) photograph of the nanowire 4 grown on the substrate 1. FIG. 3 is a graph showing that there is a correlation between the concentration of nanowires and absorbance in phosphate buffered saline (PBS). FIG. 4 is a graph showing the relationship between the stirring time and the number of cycles in Example 2 and Comparative Examples 3 to 5. FIG. 5 is a graph showing the relationship between the stirring time and the number of cycles in Example 3 and Comparative Examples 6 and 7. FIG. 6 is a graph showing the relationship between the stirring time and the number of cycles in Example 4, Comparative Examples 8 and 9, and Comparative Example 4 in which cells and the like were chemically disrupted. FIG. 7 is a graph showing the relationship between the stirring time of Example 5 and Comparative Examples 10 to 12 and the number of cycles. FIG. 8 is a chart showing the analysis results of Example 7 and Reference Example 1. FIG. 9 is a drawing-substituting photograph, (1) is Example 10, (2) is Example 11, and (3) is a scanning electron microscope (SEM) photograph at the end of stirring in Comparative Example 13. is there. FIG. 10 is a graph showing the DNA concentration in the suspensions of Examples 10 and 11 and Comparative Example 13.

Hereinafter, a sample crushing method using nanowires, a biomolecule extraction method, and a crushing solution containing nanowires will be described in detail.

The crushing liquid containing the nanowire of the present invention can be prepared by dispersing the nanowire in a dispersion liquid. The “nanowire” in the present invention is not particularly limited in terms of production method, size, material, etc., as long as it can be crushed by damaging cells and the like when dispersed in a dispersion and stirred with cells and the like. .

FIG. 1 is a diagram showing an example of a procedure for producing nanowires used in the crushed liquid of the present invention. Nanowires can be produced by the following procedure.
(1) The substrate 1 is prepared.
(2) The nanowire-producing particles 2 or the catalyst 3 are applied on the substrate 1.
(3) The nanowire 4 is grown on the substrate 1.
(4) The nanowire 4 is peeled from the substrate 1 by running water treatment or ultrasonic treatment.
(5) The separated nanowire 4 is washed and centrifuged.
(6) The separated nanowire 4 is put in a container 5 such as an Eppendorf tube and dispersed in the dispersion liquid 6 to prepare a crushed liquid.

The material of the substrate 1 is PDMS (poly (siloxane siloxane)), PMMA (poly (methyl methacrylate)), PC (polycarbonate), plastic made of hard polyethylene, etc., as long as the nanowire 4 can be grown. There is no particular limitation.

Examples of the particles 2 for producing the nanowire 4 include ZnO. The nanowire 4 using ZnO fine particles can be produced using the hydrothermal synthesis method described in Non-Patent Document 3. Specifically, first, ZnO particles are applied on the substrate 1. Next, the heated substrate 1 is immersed in a precursor solution in which zinc nitrate hexahydrate (Zn (NO ₃ ) ₂ .6H ₂ O) and hexamethylenetetramine (C ₆ H ₁₂ N ₄ ) are dissolved in deionized water. By doing so, the ZnO nanowire 4 can be grown.

Examples of the catalyst 3 for producing the nanowire 4 include gold, platinum, aluminum, copper, iron, cobalt, silver, tin, indium, zinc, and gallium. The nanowire 4 using the catalyst 3 can be produced by the following procedure.
(A) The catalyst 3 is deposited on the substrate 1.
_{(B) SiO 2, Li 2} O, MgO, Al 2 O 3, CaO, TiO 2, Mn 2 O 3, Fe 2 O 3, CoO, NiO, CuO, ZnO, Ga 2 O 3, SrO, In 2 O _3. Using a material such as SnO ₂ , Sm ₂ O ₃ , or EuO, a core nanowire is formed by physical vapor deposition such as pulse laser deposition or VLS (Vapor-Liquid-Solid).
(C) SiO ₂ , TiO _2, etc., which are materials that are difficult to adsorb crushed / extracted nucleic acid due to electrostatic interaction, sputtering, EB (Electron Beam) deposition, PVD (Physical Vapor Deposition), ALD (Atomic Layer) A coating layer is formed around the core nanowire by a general vapor deposition method such as Deposition).
In addition, the nanowire 4 produced using the catalyst 3 may be a nanowire 4 having no branched chain, or may be a nanowire 4 having a branched chain.

The diameter of the nanowire 4 is not particularly limited as long as it is a size that can damage and crush cells by stirring as described above. However, the size of the cell, virus, fungus, etc. that is the purpose of crushing, cell membrane or You may change according to the intensity | strength of a cell wall, etc. For example, since prokaryotic cells are 1 μm to 10 μm, eukaryotic cells are 5 μm to 10 μm, viruses are several tens to several hundreds of nm, and blood cells are 7 μm to 15 μm, the diameter of the nanowire 4 is 10 nm or more, 15 nm or more. , 20 nm or more, or 25 nm or more. Further, the diameter of the nanowire 4 needs to be smaller than that of the cell or the like that is to be crushed, and is, for example, 200 nm or less, 150 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, or 50 nm or less. May be.

The diameter of the nanowire 4 can be appropriately adjusted by changing the size of the particle 2 or forming a coating layer on the produced nanowire 4 when the particle is formed using the particle 2. The coating layer may be formed with the same material and coating method as the coating layer when the catalyst 3 is used, and the deposition time may be adjusted as appropriate. Moreover, also when growing using the catalyst 3, a diameter can be suitably adjusted by changing the vapor deposition time at the time of forming a coating layer.

In the present invention, when the nanowires 4 and the cells that are released are agitated and the nanowires 4 come into contact with the cells or the like, the nanowires 4 damage the cells or the like due to the inertial force in the length direction due to the difference in aspect ratio. I think that. The length of the nanowire 4 is not particularly limited as long as it is a length capable of damaging cells and the like by crushing, but is preferably 300 nm or more. If it is smaller than 300 nm, it is not substantially different from the particle 2 and it is not preferable because it is difficult to damage because of its short length even when it comes into contact with a cell or the like. On the other hand, if the length of the nanowire 4 is longer than the size of a cell or the like to be crushed, the probability of contact is reduced and the nanowires 4 are easily entangled with each other. The length of the nanowire 4 can be adjusted by changing the immersion time in the precursor solution when formed using the particles 2. Moreover, when forming using the catalyst 3, the length of the nanowire 4 can be suitably adjusted by changing the growth time by VLS after forming a core nanowire.

The nanowires 4 grown on the substrate 1 can be peeled off from the substrate 1 by running water treatment, but if necessary, the nanowires 4 are peeled off from the substrate 1 by immersing the substrate 1 in the dispersion 6 and performing ultrasonic treatment. May be. The ultrasonic treatment may be performed for about several minutes using a commercially available general ultrasonic treatment apparatus.

The crushing liquid of the present invention can be produced by adding nanowires 4 peeled from the substrate 1 to the dispersion liquid 6 and dispersing the aggregated nanowires 4 by ultrasonic treatment. The concentration of the nanowire 4 in the crushed liquid may be adjusted as appropriate according to the concentration of cells and the like, and the concentration may be adjusted so that the absorbance of the spectrophotometer becomes a desired value.

The dispersion 6 of the nanowire 4 has no particular problem as long as it is a liquid that does not denature biomolecules such as nucleic acids and proteins or does not affect the subsequent analysis. For example, phosphate buffered saline (PBS) or the like can be used. A buffer solution may be mentioned.

In the present invention, the collected nanowires 4 may be dispersed in the dispersion 6 as they are, but may be dispersed in the dispersion 6 after the nanowires 4 are treated. For example, when nucleic acids are recovered using the disruption solution of the present invention, proteolytic enzymes such as Protease K, endopeptidase, and exopeptidase are immobilized around the recovered nanowires 4 so that the disruption solution and cells, etc. You may enable it to decompose | disassemble the protein which leaks in a crushing liquid during stirring. The proteolytic enzyme can be fixed to the nanowire 4 by suspending the nanowire 4 collected in a solution containing the proteolytic enzyme and leaving it for a predetermined time, followed by washing with distilled water or the like.

In addition, when proteins are recovered using the disrupted liquid of the present invention, nucleic acid-degrading enzymes (RNase A, RNase L, etc., which degrade RNA, deoxygenases, such as DNase I, DNase II, etc., which degrade DNA, are collected around the collected nanowires 4. By fixing the ribonuclease), the nucleic acid leaking into the disruption solution while stirring the disruption solution and the cells may be decomposed. The nucleolytic enzyme can be fixed to the nanowire 4 by suspending the collected nanowire 4 in a solution containing the nucleolytic enzyme and leaving it for a predetermined time, and then washing with distilled water or the like.

Furthermore, in the present invention, the above proteolytic enzyme and nucleolytic enzyme may be used in combination. For example, when DNA is extracted with high purity using the disruption solution of the present invention, a proteolytic enzyme and ribonuclease are immobilized on the nanowire 4, and when RNA is extracted with high purity, a proteolytic enzyme and Deoxyribonuclease may be immobilized. For proteolytic enzymes and nucleolytic enzymes, the recovered nanowire 4 is suspended in a mixed solution of proteolytic enzyme and ribonuclease, or a mixed solution of proteolytic enzyme and deoxyribonuclease, and left for a predetermined time, and then washed with distilled water or the like. Can be fixed to the nanowire 4.

Next, a sample crushing method and a biomolecule extraction method using the crushing liquid of the present invention will be described. In the present invention, the “sample” means one that can recover internal biomolecules by crushing using a crushing solution, and examples thereof include cells, viruses, and fungi. More specifically, the cells include those having a cell membrane structure, such as staphylococci, Bacillus subtilis, Escherichia coli, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae, Shigella, Bacillus anthracis, tuberculosis, Clostridium botulinum, tetanus, Examples include bacteria such as streptococci, granulocytes, lymphocytes, reticulocytes, erythrocytes, leukocytes, blood cells such as platelets, and the like. Examples of viruses include norovirus, rotavirus, influenza virus, adenovirus, coronavirus, measles virus, rubella virus, hepatitis virus, herpes virus, HIV and the like. Examples of the fungi include mushrooms, molds, and yeasts, and specifically include ringworm fungi, Candida, Aspergillus, and budding yeast. Mitochondria, exosomes and extracellular vesicles can also be mentioned as samples. Note that the sample is not limited to the above example as long as the material inside the sample can be recovered by crushing.

The crushing of a sample such as a cell can be performed according to the procedure described below. First, a step of suspending cells collected by centrifugation or the like in a disruption solution to prepare a suspension is performed. The suspension may be prepared by putting it in a known container 5 such as an Eppendorf tube that does not easily affect biomolecules. It should be noted that the concentration of nanowire 4 and the concentration of cells, etc. in the suspension are sufficiently agitated in the suspension in the agitation step, and the tip of nanowire 4 comes into contact with the cells, etc. If it is the density | concentration which crushes, there will be no restriction | limiting in particular.

After the step of preparing the suspension, a step of stirring the suspension is performed. The stirring step is not particularly limited as long as the nanowire 4 and the cells can be stirred. For example, the container 5 may be stirred using a vortex mixer, or a stirring bar of a magnetic stirrer may be placed in the container 5 and stirred. The stirring speed and time may be appropriately adjusted according to the concentration of the nanowire 4 and the type and concentration of the sample.

Through the above-described steps, it is possible to break up cells and leak biomolecules without pretreatment of cells and the like and without using reagents that affect biomolecules such as denaturation.

After the agitation step, the suspension residue removal step such as centrifugation is performed to precipitate and remove residues such as nanowires 4, unbroken cells, and cell walls in the suspension, and extract biomolecules. can do.

In addition, as described above, when the nanowire 4 to which one or two selected from proteolytic enzymes, ribonucleases, and deoxyribonucleases are used, the biomolecules leaked into the suspension during the stirring step are nanowires. Therefore, the desired biomolecule can be extracted with high purity. Furthermore, when the enzyme is immobilized on the nanowire 4, the enzyme can be simultaneously removed from the suspension by removing the nanowire 4 by centrifugation after the stirring step. Compared with the case of adding the enzyme, the enzyme that degrades the biomolecule can be easily removed.

Of course, instead of immobilizing one or two kinds of enzymes selected from proteolytic enzymes, ribonucleases, or deoxyribonucleases to the nanowire 4, the enzymes may be added during the stirring step or after the stirring step. In that case, after degrading the biomolecule with the enzyme, the added enzyme may be removed from the suspension according to a conventional method.

Hereinafter, the present invention will be specifically described with reference to examples. However, these examples are provided merely for the purpose of explaining the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

[Preparation of crushing liquid]
<Example 1>
A mixed solution in which NANOBYK (registered trademark) -3820 (produced by Big Chemie Japan Co., Ltd .; ZnO diameter: 20 nm) as ZnO particles was mixed with 5 mL and 1.35 mg (1 mmol / L) of 10-CDPA (Dojindo) as a crosslinker was mixed. And left at room temperature for 3 days. Thereafter, the mixed solution was diluted 10,000 times, and uniformly applied onto the substrate 1 (PMMA (poly (methymethacrylate), 50 mm × 50 mm) using a pipette, and fixed at 85 ° C. for 1 hour. 0.5608 g of zinc nitrate hexahydrate ((Zn (NO ₃ ) ₂ .6H ₂ O, 98%, manufactured by Sigma Adrich), hexamethylenetetramine ((C ₆ H ₁₂ N ₄ ), 99 ⁺ %, A precursor solution was prepared by adding 1.16 g of Sigma Adrich Co.) to 200 mL of deionized water, and then the substrate 1 was placed in the precursor solution and immersed in an oven at 85 ° C. for 24 hours. Is a field emission scanning electron microscope (FESEM) of nanowires 4 grown on a substrate 1. Next, the substrate 1 is treated with a phosphate buffered saline solution (PBS; 5 mL), and then the substrate 1 is immersed in the PBS, and an ultrasonic device (US-1, manufactured by Asone) is used. Was sonicated at 38 kHz (80 W) for about 1 and a half minutes, and 5 mL of PBS was placed in a 15 mL centrifuge tube (Iwaki), and an ultrasonic device (UT-250S, manufactured by Sharp) was installed. Then, ultrasonic treatment was performed at 35 kHz (200 W) for about 2 hours to highly disperse the nanowires 4 in PBS, and then the crushed liquid in which the nanowires 4 were dispersed was put into 5424R manufactured by Eppendorf, and centrifuged and / or Alternatively, by adding PBS, the absorbance (measured at 405 nm) of the crushed liquid when using POLARstar OPTIMA manufactured by BMG Labtech is 0.630. The concentration of the nanowire 4 was adjusted, and the concentration of the nanowire 4 in the crushed liquid is defined by the absorbance, but it is difficult to accurately measure the weight of the minute amount of the nanowire 4, but FIG. As shown, the concentration of the nanowire 4 in the PBS solution is correlated with the absorbance, and the diameter of the prepared nanowire 4 was about 100 nm and the average length was 2 μm. 700 μL of the crushed liquid was collected in a tube to prepare the crushed liquid of Example 1.

<Comparative Example 1>
In place of the nanowire 4 of Example 1, ZnO particles (by Big Chemie Japan Co., Ltd .; ZnO diameter: 20 nm) were used, and the crushing was performed in the same manner as in Example 1 except that the absorbance was 0.629 as in Example 1. A liquid was prepared.

<Comparative example 2>
The nanowire 4 of Example 1 was not added, and a PBS buffer solution only in an Eppendorf tube was prepared as a crushed liquid of Comparative Example 2.

[RNA extraction / quantification experiment]
Next, using the disrupted liquids of Example 1 and Comparative Examples 1 and 2, RNA extraction and quantification experiments were performed from Hela cells.
[Preparation of cells and medium]
Hela cells purchased from ECACC were used. Fetal Bovine Serum (FBS, Invitrogen) is warmed at 55 ° C., inactivated sufficiently for 30 minutes, added to Minimum Essential Medium Eagle (Sigma-Aldrich) so that the FBS capacity becomes 10%, and Penicillin− Streptomycin (manufactured by SIGMA) is 1%, L-GLUTAMINE (manufactured by SIGMA) is 1%, and 100X Non-Essential Amino Acids for MEM Eagle (manufactured by MP Biomedicals) is 1%. A culture medium was prepared.

[Culture method]
The medium and cells were placed in a culture flask (Iwaki) and cultured until confluent. Next, the medium, PBS (GIBCO, manufactured by Life Technologies), and trypsin / EDTA (GIBCO, manufactured by Life Technologies) were warmed to 37 ° C. in advance. After confirming the state of the cells with a microscope, the medium in the culture vessel was aspirated and discarded using an aspirator and Pasteur pipette. After washing once with about half the amount of PBS used for the culture, ² mL of trypsin per 25 cm ² was added to the culture container, and the culture container was allowed to stand in a CO ₂ incubator at 37 ° C. for 2 minutes. It was confirmed whether or not the state of the cells was peeled off visually, and if necessary, the side in the culture vessel was gently tapped to peel off the stuck cells. Fresh medium 3mL added per 25 cm ² in a culture vessel, and resuspended to inactivate the trypsin. The suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1,000 rpm at 25 ° C. for 3 minutes. After centrifugation, the supernatant was aspirated and discarded with a Pasteur pipette. A fresh medium was added to the remaining pellet-like cells and lightly suspended by pipetting. This cell suspension was prepared and used for experiments.

[Sample adjustment]
10 μL of the cell suspension was injected into C-Chip (Digital Bio) and the number of cells was counted using a microscope. Samples were prepared by diluting with a new medium so as to obtain the desired number of cells.

<Example 2>
[Extraction of RNA]
About 8.0 × 10 ⁵ Hela cells obtained in the above [Preparation of sample] are suspended in 700 μL of the disrupted solution prepared in Example 1 above, using a VWR vortex jenny at 2,560 rpm. Stir. The suspensions stirred for 0 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes, and 120 minutes were sampled in order to quantify the total amount of mRNA. For each sampled suspension, RNA extraction / purification was performed using miRNeasy Mini from Qiagen according to the attached manual. First, 140 μL of chloroform (Infinity Pure, manufactured by Wako) was added to each suspension, stirred for 15 seconds, and allowed to stand at room temperature for 3 minutes. Centrifugation was performed for 15 minutes at 4 ° C. and 13,000 rpm using 5424R manufactured by Eppendorf, and then 350 μL of the upper layer was transferred to a 1.5 mL tube. 525 μL of ethanol (99.5%, Infinity Pure, manufactured by Wako) was added, and pipetting was performed several times. Next, 700 μL was transferred to a spin column, and centrifuged at 25 ° C., 10,300 rpm, 15 seconds. The filtrate was discarded, 700 μL of RWT was transferred to a spin column, centrifuged at room temperature, 10,300 rpm, 15 seconds, and the filtrate was similarly discarded. Next, 500 μL of RPE was added, centrifuged at room temperature, 10,300 rpm, 15 seconds, and the filtrate was discarded. RPE was added again, and centrifugation was performed at room temperature, 10,300 rpm, and 1 minute. The spin column was transferred to a new collection tube and centrifuged at 13,000 rpm for 1 minute. The spin column was again transferred to a new 1.5 mL tube, 50 μL of RNase free water was added, and centrifugation was performed at 10,300 rpm for 1 minute. 50 μL of total RNA was extracted by the above procedure.

[Quantification of RNA]
RT-PCR was performed on each extracted RNA using a Transscriptor High Fidelity cDNA Synthesis Kit manufactured by Roche Applied Science. To 2 μL of RNA, 1 μL of anchor oligo primer and 8.4 μL of PCR grade water were added, left at 65 ° C. for 10 minutes, and stored at 4 ° C. Transscriptor High Fidelity Reverse Transscriptase Reaction Buffer 5X, 4 μL of Protector RNase Inhibitor, 0.5 μL of Protector RNase Inhibitor, 2 μL of deoxynucleotide Mix, 1 μL of DTT CDNA was prepared by treatment at 5 ° C. for 5 minutes and stored at 4 ° C. For qPCR, Light Cycler Fast Start DNA Master SYBR Green I manufactured by Roche Applied Science was used. According to the instructions for use of the kit, the PCR cycle was heated at 95 ° C. for 10 minutes, and then subjected to 40-45 cycles of 3 steps of 95 ° C. for 10 seconds, 55 ° C. for 10 seconds, and 72 ° C. for 10 seconds. The solution was treated at 0 ° C. for 15 seconds at 55 ° C., a melting curve was drawn from 60 ° C. to 95 ° C., and then kept at 40 ° C. for 30 seconds to quantify RNA. RNA was quantified by counting the number of PCR cycles until the fluorescence intensity reached 7000 using the expression level of GAPDH mRNA as an index. The smaller the number of cycles, the larger the amount of RNA at the start of RT-PCR, that is, more RNA was extracted from Hela cells.

<Comparative Example 3>
RNA was extracted and the number of PCR cycles was counted by the same procedure as in Example 2 except that the disrupted solution of Comparative Example 1 was used instead of the disrupted solution prepared in Example 1.

<Comparative example 4>
RNA was extracted and the number of PCR cycles was counted by the same procedure as in Example 2 except that the disrupted solution of Comparative Example 2 was used instead of the disrupted solution prepared in Example 1.

<Comparative Example 5>
Instead of the disruption solution prepared in Example 1, 700 μL of QIAzol Lysis Reagent attached to miRNeasy Mini of Qiagen was added, and the procedure was the same as in Example 2 except that Hela cells were chemically disrupted. Was extracted and the number of PCR cycles was counted.

FIG. 4 is a graph showing the relationship between the stirring time of Example 2 and Comparative Examples 3 to 5 and the number of cycles. As is clear from FIG. 4, in Comparative Example 5 in which Hela cells were chemically disrupted, the number of cycles was almost the same regardless of the stirring time. On the other hand, in Example 2 using the crushed liquid in which the nanowires 4 were dispersed, a great difference was not seen from Comparative Examples 3 and 4 until the stirring time was about 60 minutes, but as the stirring time was increased, the number of cycles was Obviously it became smaller. From the above results, it became clear that by using the disruption solution containing the nanowire 4 of the present invention, cells and the like can be disrupted and RNA can be extracted without using a chemical disruption solution. Moreover, it became clear that more RNA can be extracted from a cell etc. by lengthening stirring time.

[RNA adsorption / decomposition experiment]
Next, it was confirmed whether or not RNA was adsorbed on the nanowires 4 leaked into the suspension.
<Example 3>
10 μL of GAPDH mRNA solution (concentration: 1,000 ng / μL) was added to 700 μL of the disrupted solution prepared in Example 1, and the mixture was stirred at 2560 rpm using a VWR vortex jenny. The suspensions stirred for 0 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes, and 120 minutes were sampled in whole volume (710 μL). 50 μL of RNA was extracted from each sampled suspension using Qiagen miRNeasy Mini. Thereafter, the number of PCR cycles until the fluorescence intensity reached 7,000 was counted in the same procedure as in Example 2.

<Comparative Example 6>
The number of cycles was counted in the same procedure as in Example 3 except that the crushed liquid of Comparative Example 1 was used instead of the crushed liquid produced in Example 1.

<Comparative Example 7>
The number of cycles was counted in the same procedure as in Example 3 except that the crushed liquid of Comparative Example 2 was used instead of the crushed liquid produced in Example 1.

FIG. 5 is a graph showing the relationship between the stirring time and the number of cycles in Example 3 and Comparative Examples 6 and 7. As is clear from FIG. 5, in the case of the crushed liquid containing the ZnO nanoparticles of Comparative Example 6, the number of cycles of mRNA increased, that is, the amount of RNA decreased as the stirring time increased. From this, it was found that mRNA was adsorbed to ZnO particles during the stirring step. On the other hand, in the case of only the disruption solution containing the nanowire 4 of Example 3 and the PBS buffer solution of Comparative Example 7, the number of cycles hardly changed, that is, there was no increase or decrease in the amount of mRNA. From the above results, it was found that when the crushing liquid containing the nanowire 4 was used, RNA leaked into the suspension by the stirring process was not adsorbed to the nanowire 4 or decomposed.

[Effect of adding chloroform]
When biomolecules are extracted from cells or the like by a chemical disruption method, chloroform is generally added to disrupt cell walls and cell membranes, and to separate them from layers containing biological components after disruption. When the disruption liquid of the present invention was used, it was confirmed whether biomolecules could be disrupted and extracted from cells or the like without adding chloroform.

<Example 4>
In the procedure of Example 2, except that 140 μL of chloroform was not added, the total RNA of the 0 minute and 120 minute samples was extracted in the same procedure as in Example 2, and the number of cycles was counted.

<Comparative Example 8>
Samples of 0 minutes and 120 minutes of total RNA were extracted in the same procedure as Comparative Example 3 except that 140 μ of chloroform was not added, and the number of cycles was counted.

<Comparative Example 9>
Samples of 0 minutes and 120 minutes of total RNA were extracted in the same procedure as in Comparative Example 4 except that 140 µm of chloroform was not added, and the number of cycles was counted.

FIG. 6 is a graph showing the relationship between the stirring time and the number of cycles in Example 4, Comparative Examples 8 and 9, and Comparative Example 4 in which cells were chemically disrupted. As is clear from FIG. 6, even when the crushing liquid containing the nanowire 4 of Example 4 to which no chloroform was added was used, the number of cycles after stirring for 120 minutes was the chemical crushing of Comparative Example 4. The value was close to the number of cycles of the law. In the conventional chemical disruption method, chloroform was required to damage the cell wall and cell membrane. However, since chloroform is unnecessary for the subsequent analysis, it was separated from the aqueous phase fraction containing biomolecules. It was necessary to remove. On the other hand, when the disruption liquid of the present invention is used, after disrupting cells and the like with the nanowire 4, the nanowire 4 can be removed by centrifugation, so that biomolecules can be efficiently extracted without performing phase separation. be able to. From the above results, it was revealed that the biomolecules can be extracted from the cells and the like without using any chloroform that may affect the biomolecules when the disrupted liquid of the present invention is used.

[DNA extraction and quantification experiments]
<Example 5>
[DNA extraction]
In place of miRNeasy Mini of Qiagen in Example 2, Thermo GeneJET Genomic DNA Purification Kit was used, and instead of GAPDH mRNA, GAPDH expression level was used as an index, DNA was purified by the following procedure, and PCR was performed. The number of cycles was counted.
First, 1 × 10 ⁶ Hela cells were centrifuged (250 × g, 25 ° C., 5 minutes), the supernatant was discarded, and the pellets were washed once with PBS and processed in the same manner as in Example 1. The suspension was resuspended in 400 μL of the prepared disrupted solution, and vortexed (2,560 rpm using a VWR vortex geneny) for 0 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes, and 120 minutes.
Next, after stirring at 20 rpm and 56 ° C. (hybridization inc.) For 10 minutes, 20 μL of RNase A was added, allowed to stand at room temperature for 10 minutes, centrifuged at 250 × g, 25 ° C. for 5 minutes, and the supernatant was 400 μL. Was taken in a 1.5 mL tube, 50% ethanol (diluted by Wako) was added and vortexed. Next, the entire amount was transferred to an attached spin column, and centrifuged at 6,000 × g, 25 ° C. for 1 minute. Thereafter, the column was changed to a new collection tube, 500 μL of Wash Buffer I was added, and centrifugation was performed at 8,000 × g, 25 ° C. for 1 minute. The filtrate was discarded, 500 μL of Wash Buffer II was added, and centrifuged at 20,000 × g, 25 ° C. for 3 minutes. Change the column to a 1.5 mL tube, add 200 μL of Elution Buffer (10 mM Tris-HCl, pH.9, 0.5 mM EDTA), let stand at room temperature for 2 minutes, then 8,000 × g, 25 ° C., 1 minute. Centrifugation was performed. 200 μL of the obtained liquid was used as a DNA extraction sample.

[Quantification of DNA]
For qPCR, Light Cycler Fast Start DNA Master SYBR Green I manufactured by Roche Applied Science was used. According to the instructions for use of the kit, the PCR cycle was heated at 95 ° C. for 10 minutes, followed by 70 cycles of 3 steps of 95 ° C. for 10 seconds, 55 ° C. for 10 seconds, and 72 ° C. for 10 seconds. Second, it was treated at 55 ° C. for 15 seconds and a melting curve was drawn from 60 ° C. to 95 ° C., and then kept at 40 ° C. for 30 seconds to quantify DNA. DNA was quantified by counting the number of PCR cycles until the fluorescence intensity reached 5000 using the expression level of GAPDH as an index. The smaller the number of cycles, the greater the amount of DNA at the start of qPCR, that is, more DNA was extracted from Hela cells.

<Comparative Example 10>
The number of cycles was counted in the same procedure as in Example 5, except that 400 μL of the crushed liquid of Comparative Example 1 was used instead of the crushed liquid produced in Example 1.

<Comparative Example 11>
The number of cycles was counted in the same procedure as in Example 5 except that 400 μL of the crushed liquid of Comparative Example 2 was used instead of the crushed liquid prepared in Example 1.

<Comparative Example 12>
The number of cycles was counted in the same procedure as in Example 5 except that Hela cells were chemically disrupted using the GeneJET Genomic DNA Purification Kit manufactured by Thermo instead of the disruption solution prepared in Example 1.

FIG. 7 is a graph showing the relationship between the stirring time of Example 5 and Comparative Examples 10 to 12 and the number of cycles. As is clear from FIG. 7, in Example 5 using the crushing liquid in which nanowires 4 are dispersed, the amount of DNA extracted increases with stirring time, and when stirred for 120 minutes, Comparative Example 10 using a chemical crushing liquid is used. More DNA could be extracted. From the above results, it became clear that DNA can be extracted from cells and the like without using a chemical disruption solution by using the disruption solution containing the nanowire 4 of the present invention.

Next, a crushed liquid containing nanowire 4 immobilized with Protease K was prepared, and it was confirmed whether or not the protein in the suspension could be decomposed during the stirring step.

<Example 6>
[Preparation of crushing liquid containing nanowire 4 with Protease K immobilized]
The crushing liquid containing nanowire 4 having an absorbance of 0.630 in Example 1 was mixed with Protease K solution (GeneJET Genomic DNA Purification Kit manufactured by Thermo) and allowed to stand for 10 minutes, and then 5424R manufactured by Eppendorf was used. The mixture was centrifuged at 1,000 rpm for 3 minutes, 400 μL of the supernatant was taken, and 400 μL of PBS buffer solution was added to prepare a crushed solution containing nanowires 4 to which Protease K was fixed.

<Example 7>
[Fracture experiment using nanowire 4 with immobilized Protease K]
The suspension stirred for 120 minutes was sampled in the same procedure as in Example 2 except that the crushed liquid produced in Example 6 was used instead of the crushed liquid produced in Example 1. The sampled suspension was purified using a Thermo GeneJET Genomic DNA Purification Kit, and the DNA concentration was measured using a spectrophotometer (NanoDrop (registered trademark, manufactured by LMS Co., Ltd.)). It was measured.

<Reference Example 1>
The suspension stirred for 120 minutes in Example 2 was measured in the same manner as in Example 7.

FIG. 8 is an absorbance chart showing the analysis results of Example 7 and Reference Example 1. This chart shows that 260/280, which is the ratio of absorbance at 260 nm, which is the peak of nucleic acid, and 280 nm, which is the peak of protein, is 1.8 to 2.0, and no protein is mixed (pure). . In addition, if 260/230, which is the ratio of 230 nm to 260 nm, which is the peak of impurities, is larger than 1.8, it indicates that there are few impurities (pure). As is clear from the chart shown in FIG. 8, in Reference Example 1 in which Protease K was not immobilized, protein mixing in the suspension was conspicuous, but in Example 6 in which Protease K was immobilized, the protein was decomposed. As a result, the purity of the nucleic acid increased. From the above results, by immobilizing the enzyme on the nanowire 4, biomolecules leaked from the cells and the like can be decomposed during the stirring step, and after crushing the cells and the like, for example, the protein removal step or the nucleic acid removal The process can be omitted.

[Brushing experiment of Bacillus subtilis]
Next, crushing experiments were performed when Bacillus subtilis was used as a sample and the diameter of the nanowire 4 was changed.

[Preparation of crushing liquid]
<Example 8>
On the quartz glass substrate 1 (Crystal Base Co.), the gold catalyst 3 was deposited to a thickness of 3 nm by sputtering. Next, a core nanowire was manufactured by performing pulse laser deposition for 10 minutes at room temperature with SnO ₂ as a material. Next, a coating layer was formed around the core nanowire by sputtering with SiO ₂ as a material at room temperature for 3 to 4 minutes. Next, the substrate 1 is immersed in 1 mL of ethanol and subjected to ultrasonic treatment at 25 ° C. using an ultrasonic device (US-1, manufactured by Asone) at 38 kHz (80 W) for about 2 hours. The nanowire 4 was peeled from the substrate 1. The ethanol was collected by centrifugation, then washed twice with 500 μL of ethanol, and the ethanol was centrifuged at 21,130 g for 1 hour to precipitate the nanowires 4. After removing the supernatant, it was resuspended in ethanol and centrifuged at 21,130 g for 1 hour to precipitate the nanowires 4. The supernatant was removed and dried in a vacuum box. The nanowire 4 after drying is mixed with water so that the measured value of optical density (OD) at 405 nm is 0.1, and the nanowire 4 is suspended by sonication for 20 minutes, Produced. The diameter of the nanowire 4 contained in the produced crushed liquid was 30 nm, and the average length was 2 μm.

<Example 9>
A crushed liquid was prepared in the same procedure as in Example 8 except that the sputtering for forming the coating layer was performed for 10 minutes. The nanowire 4 contained in the prepared crushed liquid had a diameter of 110 nm and an average length of 2 μm.

[Sample adjustment]
Bacillus subtilis (ATCC6633 spore solution, Eiken Chemical Co., Ltd.) was introduced into 2 mL of LB medium and cultured at 37 ° C. and 125 rpm for 5 hours. 1 mL of the culture solution was sampled and centrifuged at 25 ° C. and 5,000 g for 10 minutes to sediment the bacteria, and the supernatant was removed. Next, the bacteria were resuspended with 1 mL of 10 mM phosphate buffer, centrifuged under the same conditions as described above, and washed again by reprecipitation of the bacteria. This washing operation was performed twice. The washed cells were suspended again in 10 mM phosphate buffer so that the OD (584 nm) was 0.4, and used as a sample.

<Example 10>
5 μL each of the disrupted liquid prepared in Example 8 and the Bacillus subtilis suspension prepared in the above [Preparation of Sample] were mixed and stirred at 2,560 rpm for 1 hour using a VWR vortex geneny. FIG. 9 (1) is an SEM photograph at the end of stirring. After stirring, the residue was removed by centrifugation at 5,000 g for 10 minutes, and then the absorbance at 260 nm was measured using a spectrophotometer (NanoDrop (registered trademark, manufactured by LMS Co., Ltd.)). Quantification of DNA was performed.

<Example 11>
The Bacillus subtilis was crushed by the same procedure as in Example 10 except that the crushing solution prepared in Example 9 was used, and DNA was quantified. FIG. 9 (2) is an SEM photograph at the end of stirring.

<Comparative Example 13>
DNA was quantified by the same procedure as in Example 10 except that the nanowire 4 was not included. FIG. 9 (3) is an SEM photograph at the end of stirring.

As is clear from the SEM photographs of FIGS. 9 (1) to 9 (3), it was confirmed that the thinner the nanowire 4 is, the easier it is to cleave Bacillus subtilis.

FIG. 10 shows the DNA concentrations in the suspensions obtained in Examples 10 and 11 and Comparative Example 13. As is clear from FIG. 10, it was confirmed that the nanowire 4 having a smaller diameter is more likely to crush Bacillus subtilis, and as a result, more DNA can be extracted.

By using the crushing liquid in which the nanowires 4 are dispersed according to the present invention, it is possible to crush cells and the like without using a chemical reagent that affects biomolecules. Therefore, the sample disruption method, biomolecule extraction method, and nanowire-containing disruption liquid of the present invention are used for sample preparation for more accurate analysis of biomolecules in medical institutions, universities, companies, research institutions, etc. Useful.

Claims (5)

A suspension step of suspending a sample selected from cells, viruses, and fungi in a disruption solution containing nanowires;
A stirring step of stirring the crushed liquid in which the sample is suspended;
A method for crushing a sample. A suspension step of suspending a sample selected from cells, viruses, and fungi in a disruption solution containing nanowires;
A stirring step of stirring the crushed liquid in which the sample is suspended;
A residue removal step of removing the residue by centrifuging the stirred crushed liquid,
A method for extracting biomolecules. The method for extracting a biomolecule according to claim 2, wherein the nanowire is a nanowire on which one or two selected from proteolytic enzymes, ribonucleases, and deoxyribonucleases are immobilized. ∙ Sample disruption fluid selected from cells, viruses, and fungi, including nanowires. The sample disruption liquid according to claim 4, wherein the nanowire is a nanowire on which one or two selected from a proteolytic enzyme, a ribonuclease, and a deoxyribonuclease are immobilized.

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