WO2016084945A1 - Particles containing graphene oxide and/or graphite oxide as well as cellulose, composition for nucleic acid extraction, method for extracting nucleic acid, and method for recycling particles or composition for nucleic acid extraction - Google Patents

Particles containing graphene oxide and/or graphite oxide as well as cellulose, composition for nucleic acid extraction, method for extracting nucleic acid, and method for recycling particles or composition for nucleic acid extraction Download PDF

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WO2016084945A1
WO2016084945A1 PCT/JP2015/083438 JP2015083438W WO2016084945A1 WO 2016084945 A1 WO2016084945 A1 WO 2016084945A1 JP 2015083438 W JP2015083438 W JP 2015083438W WO 2016084945 A1 WO2016084945 A1 WO 2016084945A1
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nucleic acid
composition
acid extraction
particles
particle
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PCT/JP2015/083438
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French (fr)
Japanese (ja)
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永宏 齋藤
オイ ルン ヘレナ リー
ガルビス アクセオグル
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国立大学法人名古屋大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/34—Regenerating or reactivating
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/05—Preparation or purification of carbon not covered by groups C01B32/15, C01B32/20, C01B32/25, C01B32/30
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/10—Devices for withdrawing samples in the liquid or fluent state

Definitions

  • the present invention relates to particles containing graphene oxide and / or graphite oxide and cellulose, a composition for nucleic acid extraction, a method for nucleic acid extraction, and a method for recycling particles or a composition for nucleic acid extraction.
  • the present invention relates to a particle and a composition for nucleic acid extraction that are easy to extract nucleic acid from various samples containing nucleic acid and that can extract nucleic acid with high purity and efficiency.
  • the present invention relates to a nucleic acid extraction method using the particles and the nucleic acid extraction composition, and a recycling method of the particles or nucleic acid extraction composition after the nucleic acid extraction.
  • the analysis of a small amount of nucleic acid contained in a sample is performed in many fields such as biochemistry, medical treatment, food, and crime investigation.
  • Friedrich Mischer's method has been used for a long time, and then the phenol / chloroform method was developed in 1967.
  • the nucleic acid extraction method performs nucleic acid extraction by denaturing a component to be removed, such as protein, and then removing it from a solution in which a biological component is dissolved by centrifugation or the like. Therefore, since the nucleic acid to be extracted is in a state dissolved in a liquid, there is a problem that the nucleic acid is easily decomposed due to the influence of an enzyme or the like. Further, since the extraction operation repeats mixing of liquids, there is a problem that the extraction operation tends to spill.
  • a nucleic acid extraction method using solid silica particles was developed instead of the conventional nucleic acid extraction method using a liquid.
  • a laboratory in a university or the like sometimes recycles equipment and materials used for nucleic acid extraction for budget reasons.
  • As a method for recycling silica particles it is known to treat silica particles after nucleic acid extraction with HCl (see Non-Patent Document 1).
  • As a method for extracting nucleic acid in a sample a method using magnetic particles is also known.
  • Non-Patent Document 1 has a problem that when silica particles are surface-treated with HCl, the surface is changed and the recycling ability is lowered, and the recycling takes about 2 to 3 days.
  • the nucleic acid extraction method using magnetic particles requires a large-scale apparatus and the like, and also requires a long extraction time, resulting in complicated nucleic acid extraction.
  • a particle or nucleic acid extraction composition in which graphene oxide and / or graphite oxide and cellulose are mixed can extract nucleic acid from a solution in which a biological component is dissolved,
  • nucleic acids in the sample can be extracted with higher efficiency than conventional silica particles,
  • the silica particles once used could not be recycled, but the used particles or nucleic acid extraction composition of the present invention should be treated with a nucleic acid cleaning solution.
  • the nucleic acid on the surface of the particle or nucleic acid extraction composition can be removed and can be recycled, (4)
  • a magnetic substance is added to the particle or nucleic acid extraction composition
  • the particle or nucleic acid extraction composition on which the nucleic acid is adsorbed by the magnet can be recovered, so that the work efficiency can be further improved.
  • nucleic acid can be extracted without using a chaotropic salt or a binding buffer containing Na + . Newly found.
  • an object of the present invention is to provide particles containing graphene oxide and / or graphite oxide and cellulose, a nucleic acid extraction composition, a nucleic acid extraction method, and a recycling method of the particles or nucleic acid extraction composition.
  • the present invention relates to the following particles containing graphene oxide and / or graphite oxide and cellulose, a composition for nucleic acid extraction, a method for nucleic acid extraction, and a method for recycling the particles or the composition for nucleic acid extraction.
  • Particles containing graphene oxide and / or graphite oxide and cellulose (2) The particle according to (1), wherein the ratio of graphene oxide and / or graphite oxide to cellulose in the particle is 47% to 64%. (3) The particle according to (1) or (2), further including a magnetic substance and / or a substance that is positively charged in the particle. (4) A solid nucleic acid extraction composition containing graphene oxide and / or graphite oxide and cellulose. (5) The nucleic acid extraction composition according to (4), wherein the ratio of graphene oxide and / or graphite oxide to cellulose in the nucleic acid extraction composition is 47% to 64%.
  • nucleic acid extraction composition according to (4) or (5), further comprising a magnetic substance and / or a positively charged substance in the composition.
  • a nucleic acid extraction method comprising: (8) The particles or nucleic acid extraction composition after the nucleic acid extraction step according to (7) is treated with a nucleic acid cleaning solution to remove nucleic acids remaining on the surface of the particles or nucleic acid extraction composition.
  • Surface recovery process A method for recycling the particle or nucleic acid extraction composition comprising:
  • the graphene oxide and / or graphite oxide (hereinafter, sometimes referred to as “GO”) and cellulose-containing particles of the present invention, and the composition for nucleic acid extraction are nucleic acids from a solution in which biological components are dissolved. Can be selectively extracted.
  • GO graphite oxide
  • cellulose-containing particles of the present invention By adjusting the ratio of graphene oxide to cellulose, nucleic acids in various samples can be extracted with higher efficiency than conventional silica particles. Therefore, even a very small amount of nucleic acid contained in saliva can be extracted.
  • silica particles once used could not be recycled, but the particles or the composition for nucleic acid extraction of the present invention should be treated with a nucleic acid cleaning solution.
  • the nucleic acid on the surface of the particle or nucleic acid extraction composition can be removed and can be recycled. Therefore, since it can be used repeatedly for nucleic acid extraction, the cost of laboratories and the like can be reduced.
  • a magnetic substance is added to the particle or nucleic acid extraction composition, the particle or nucleic acid extraction composition to which the nucleic acid is adsorbed by the magnet can be recovered, so that the working efficiency is improved.
  • FIG. 1 is a drawing-substituting photograph
  • FIG. 1 (A) is a photograph of particles produced in Example 1
  • FIG. 1 (B) is a photograph of particles produced in Comparative Example 1.
  • FIG. 2-1 is a photograph substituted for a drawing, and is an SEM photograph of particles with various GO ratios.
  • FIG. 2-2 is a photograph substituted for a drawing, and is an SEM photograph of particles with various GO ratios.
  • FIG. 3 is a drawing-substituting photograph showing a photograph in which particles having GO ratios of 53%, 56%, 60%, and 64% are pressed with the tip of a pipette, respectively.
  • 4A is a graph of 260 nm / 280 nm values in Table 2, and FIG.
  • FIG. 4B is a graph of 260 nm / 230 nm values in Table 2.
  • FIG. 5 (A) is a graph showing the amount of nucleic acid (ng) contained in 1 ⁇ l of the nucleic acid eluate in Table 2.
  • FIG. 5 (B) is a graph of Total Yield in Table 2.
  • FIG. 6 is a graph showing the results of Example 4 and Comparative Example 2, and showing the Total Yield value of DNA extraction from various samples.
  • FIG. 7A is a drawing-substituting photograph, the left is a photograph of CGM0 produced in Example 5, and the right is a photograph of CGM45 produced in Example 5.
  • 7B and 7C are photographs substituted for drawings, FIG.
  • FIG. 7B is a photograph in which a magnet is brought close to a container in which CGM0 is dispersed in pure water
  • FIG. 7C is a photograph in which CGM45 is dispersed in pure water. It is the photograph which brought the magnet close to the container.
  • FIG. 8A is a drawing-substituting photograph and an SEM photograph of CGM45 of Example 5.
  • FIGS. 8B to 8D are photographs substituted for drawings, and are mappings obtained by analyzing the surface of CGM45 particles with an energy dispersive X-ray analyzer (EDX).
  • FIG. 8B is a carbon element
  • FIG. 8D represents dispersion of Fe element.
  • FIG. 9 is a graph of the amount of nucleic acid (ng / ⁇ l) of each sample of Example 5 and Total Yield.
  • FIG. 10 is a graph of the amount of nucleic acid (ng / ⁇ l) and the total yield of each sample of Example 6.
  • the particles containing GO and cellulose of the present invention a composition for nucleic acid extraction, a method for nucleic acid extraction, and a method for recycling the particle or composition for nucleic acid extraction will be described in detail.
  • a substance for breaking hydrogen bonds between cellulose and cellulose molecules is added to and mixed with water containing GO (hereinafter sometimes referred to as “mixed solution”). It can be produced by dropping the mixed solution into the coagulation solution and coagulating the mixed solution.
  • graphene oxide Graphene Oxide; a carbon atom sheet is a single layer
  • graphite oxide Graphite Oxide; a carbon atom sheet is a multilayer
  • GO may be produced by a known method such as Marcano's method, or a commercially available one may be used. What is necessary is just to use the cellulose generally marketed.
  • the coagulation solution is not particularly limited as long as the mixed solution can be coagulated, and examples thereof include HNO 3 , H 2 SO 4 , acetone, ethanol, saline, and other acidic solutions.
  • HNO 3 and H 2 SO 4 are preferable from the viewpoints of stability and coagulation ability.
  • the coagulation solution needs to be adjusted to 5 ° C. to 50 ° C. as necessary, but HNO 3 is preferable from the viewpoint of operability because it can be used at room temperature.
  • the size of the particles may be adjusted by adjusting the diameter of an instrument (such as a dropper) when dropping the mixed solution and adjusting the amount of the mixed solution dropped into the coagulation solution.
  • the “particle” of the present invention means a particle formed by dripping the mixed solution into the coagulation solution as described above, and varies depending on the viscosity of the mixed solution. Can be mentioned
  • the substance for breaking hydrogen bonds between cellulose molecules is not particularly limited as long as hydrogen bonds between cellulose molecules are broken.
  • NaOH, urea, etc. are mentioned, and they may be used alone or in combination.
  • the ratio of GO to cellulose is not particularly limited as long as the nucleic acid in the sample can be extracted by adsorbing the nucleic acid in the sample and releasing the adsorbed nucleic acid with the nucleic acid eluate.
  • the “ratio of GO to cellulose” means (GO / (cellulose + GO)) ⁇ 100 (%).
  • the ratio of GO to cellulose (hereinafter sometimes simply referred to as “GO ratio”) is preferably 5% or more, and more preferably 31% or more. Furthermore, when nucleic acids are extracted with high efficiency comparable to conventional silica particles, the GO ratio is particularly preferably 47% or more.
  • the upper limit of the GO ratio is not particularly limited as long as the produced particles or composition can maintain a solid state.
  • the solid state can be maintained even if the GO ratio is 64%, it may be set to 64% or less, and considering the convenience of handling, 60% or less, 56% or less, 53% or less, etc. Adjust it.
  • GO and cellulose are made particulate. Shape. Furthermore, it has been newly found in the present invention that a nucleic acid can be extracted and recycled using a composition containing GO and cellulose.
  • the shape of the composition is not particularly limited, but the specific surface area is increased, and the composition is efficiently applied to an Eppendorf tube or the like generally used for nucleic acid extraction. Since it can be filled, a particulate form is preferable. Since the particles of the present invention can selectively adsorb nucleic acids in a sample, for example, after adding and mixing the particles of the present invention in a sample, the particles are removed from the sample to remove nucleic acids from the sample. Can be used for the purpose of concentrating proteins and the like. Therefore, the use of the particles of the present invention is not limited to nucleic acid extraction.
  • the composition containing GO and cellulose of the present invention can provide a new application for nucleic acid extraction.
  • the shape of the composition is preferably particulate, but is not limited to particles.
  • the composition is formed into a tube shape, a sample containing nucleic acid is flowed into the tube, and then a nucleic acid eluate is flowed to perform continuous processing. You may be able to do it.
  • nucleic acid can be adsorbed by contacting with a sample solution containing nucleic acid.
  • nucleic acid extraction method of the present invention first, a sample solution containing a nucleic acid, a binding buffer, and the particle or the composition for nucleic acid extraction are mixed to adsorb the nucleic acid to the particle or the composition for nucleic acid extraction, and then washed with ethanol. Thereafter, the nucleic acid adsorbed on the particle or nucleic acid extraction composition is separated by mixing the nucleic acid-adsorbed particles or nucleic acid extraction composition and the nucleic acid eluate, and the nucleic acid in the sample is recovered in the nucleic acid eluate. be able to.
  • the nucleic acid of the present invention includes DNA and RNA. Further, the DNA may be single-stranded or double-stranded.
  • guanidinium chloride (GuHCl), chaotropic salts (GITC), NaCl, PB buffer, or the like may be used.
  • the nucleic acid eluate is not particularly limited as long as the nucleic acid can be eluted by breaking the ion bond between the particle or the surface of the nucleic acid extraction composition and the nucleic acid.
  • examples include AE buffer (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0, manufactured by Qiagen).
  • the nucleic acid remaining on the surface of the particle or nucleic acid extraction composition may be damaged, and the surface treatment may be performed with a nucleic acid cleaning solution that can be removed from the surface.
  • the nucleic acid cleaning solution is not particularly limited as long as it damages the nucleic acid as described above and can remove the nucleic acid from the surface of the particle or the composition for nucleic acid extraction.
  • an acidic solution such as HCl or an alkaline solution such as NaOH Or Urea or the like.
  • the particle or nucleic acid extraction composition of the present invention may contain a magnetic substance. Since the particles or the composition for nucleic acid extraction containing a magnetic material can be collected with a magnet, the working efficiency is improved.
  • the magnetic material is not particularly limited as long as it is attracted by a magnet.
  • magnetic iron oxide such as magnetite, maghemite, and ferrite, and iron oxide containing other metal oxides
  • iron Metals such as cobalt, nickel, or these metals and metals such as aluminum, cobalt, copper, lead, magnesium, tin, zinc, antimony, beryllium, bismuth, cadmium, calcium, manganese, selenium, titanium, tungsten, vanadium And alloys thereof, (3) and mixtures thereof.
  • the extraction efficiency of the nucleic acid is improved, and further, the nucleic acid can be extracted without using a binding buffer containing a chaotropic salt or Na + .
  • the positively charged substance is not particularly limited as long as a negatively charged nucleic acid can be attracted, and examples thereof include iron oxides such as magnetite, maghemite, and ferrite, aluminum oxide, and magnesium oxide.
  • the magnetic substance and the positively charged substance may be added separately to the particles or the nucleic acid extraction composition, or may be added in combination.
  • a substance having both magnetic properties and positively charged properties such as iron oxide (eg, magnetite, maghemite, ferrite) is preferable because it has two different effects independently.
  • the amount of iron oxide contained in the particle or nucleic acid extraction composition has no particular lower limit because an effect can be obtained if it is added even in a trace amount. On the other hand, if the content of iron oxide is too large, solid particles or a composition for nucleic acid extraction cannot be formed.
  • Example 1 The particles of the present invention were produced by the steps shown in the following (1) to (5).
  • GO was manufactured by the following Marcano's method.
  • a mixture of 360 ml of H 2 SO 4 and 40 ml of H 3 PO 4 was mixed with 3.0 g of flake graphite (graphite flakes; manufactured by Sigma-Aldrich) and 18.0 g of KMnO 4 (Wako Pure Chemical Industries, Ltd.) The resulting mixture was heated to 30-40 ° C.
  • B Next, the mixture obtained in the above (a) was heated to 50 ° C. and stirred for 12 hours.
  • FIG. 1 (A) is a photograph of the particles produced in Example 1, which was an ellipse with a black major axis of about 3 mm mixed with GO and cellulose.
  • Example 1 shows the added amount (g) of GO in the above step (2) and the ratio of GO when the added amount is added.
  • FIG. 1B is a photograph of the particles produced in Comparative Example 1. Since GO was not included, elliptical particles having a white major axis of about 3 mm were obtained.
  • FIGS. 2-1 and 2-2 are SEM photographs of particles having various GO ratios shown in Table 1 and particles of Comparative Example 1.
  • FIG. As apparent from the SEM photograph, when the GO ratio was increased, fine irregularities increased on the particle surface, but when the ratio exceeded 60%, a slight smoothing was observed.
  • FIG. 3 shows a photograph in which particles having a GO ratio of 53%, 56%, 60%, and 64% were pressed with the tip of a pipette, respectively. It was confirmed that particles were formed at any GO ratio. However, when the GO ratio was 64%, the particles were soft and needed to be handled with care.
  • Example 2 Thirteen types of particles having different GO ratios shown in Table 2 below were prepared, and 5 particles each were placed in a 1.5 ml Eppendorf tube. Next, 20 ⁇ l of a DNA sample (Sigma D7290, Sigma-Aldrich)) was added to an Eppendorf tube, and 180 ⁇ l of GuHCl (Sigma G4505, Sigma-Aldrich) was added as a binding buffer and stirred. Next, after washing twice with 70% ethanol, 500 ⁇ l of nucleic acid eluate (AE buffer; 10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0) is added, heated to 70 ° C. and stirred for 5 minutes.
  • AE buffer 10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0
  • the DNA adsorbed on the particles prepared in Example 1 was eluted.
  • the absorbance at 230 nm, 260 nm and 280 nm of the nucleic acid eluate (1 ⁇ l) was manually measured using NanoDrop ND-2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). And 260nm / 280nm and 260nm / 230nm values were calculated.
  • the total yield was determined by multiplying the amount of nucleic acid (ng / ⁇ l) contained in the 1 ⁇ l nucleic acid eluate displayed on the NanoDrop ND-2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) by 500 times.
  • the DNA sample was extracted according to the attached manual using Qiaamp DNA mini kit (Qiagen Co., Ltd.), a commercially available nucleic acid purification kit, and the absorbance was measured by the same procedure as above. 260/280 nm and 260/230 nm values were calculated.
  • the total yield was determined by multiplying the value of the amount of nucleic acid (ng / ⁇ l) by 200. By comparing the Total Yield value, the nucleic acid extraction efficiency can be compared.
  • Table 2 includes particles with the GO ratio changed from 0% to 53%, 260 nm / 280 nm and 260 nm / 230 nm values when DNA extraction was performed using Qiaamp DNA mini kit, and 1 ⁇ l of nucleic acid eluate. It summarizes the amount of nucleic acid (ng) and Total Yield.
  • 4A is a graph of 260 nm / 280 nm values in Table 2
  • FIG. 4B is a graph of 260 nm / 230 nm values in Table 2.
  • FIG. 5 (A) is a graph showing the amount of nucleic acid (ng) contained in 1 ⁇ l of the nucleic acid eluate shown in Table 2.
  • FIG. 5 (B) is a graph showing Total Yield in Table 2. is there.
  • 260/230 nm when the value of 260/230 nm is in the range of 2.0 to 2.2, it is considered to be a beautiful DNA with few contaminants such as buffer salts.
  • the GO ratio is 31% or more, better results are obtained when using a commercially available nucleic acid purification kit Qiaamp DNA mini kit, and when the GO ratio is 47% or more, 260 nm / 280 nm and It has been found that any value of 260 nm / 230 nm falls within the desired range.
  • Example 2 260 nm / 280 nm and 260 nm / 230 nm values, and the nucleic acid amount (ng) and Total Yield contained in 1 ⁇ l of nucleic acid eluate were measured only up to a GO ratio of 53%. As shown, since it has been confirmed that particles can be formed even when the GO ratio is 64%, the same effect can be expected.
  • the GO ratio is preferably 5% to 64% and more preferably 31% to 64%. It was.
  • the Total Yield did not increase until the GO ratio was 20%, but thereafter, the GO ratio increased. Total Yield increased. Moreover, the GO ratio was 47%, which was close to the total yield of the commercially available Qiaamp DNA mini kit, and by further increasing the GO ratio, a value greater than the Total Yield value of the Qiaamp DNA mini kit was obtained.
  • the ratio of GO to cellulose changes the total yield after DNA extraction in the sample, and the GO ratio is preferably 20% to 64%, more preferably 47% to 64%. preferable.
  • Example 3 [Extraction of DNA from various samples] ⁇ Example 3> Except that the sample DNA of Example 2 was replaced by 1 cm ⁇ 1 cm of the filter part of the smoked tobacco filter, 15 mg of chewing gum after chewing, 20 mg of dry yeast, and 15 mg of chicken breast were used as samples. Total Yield was determined in the same procedure as in Example 2.
  • FIG. 6 is a graph showing the results of Example 3 and Comparative Example 2.
  • QIAamp DNA Investigator Kit-QIAamp MinElute columns is a highly sensitive DNA detection kit used in criminal investigations.
  • the sample DNA used in Example 2 is single-stranded, but the DNA of Example 3 is double-stranded. Therefore, it was confirmed that the particles of the present invention can extract both single-stranded and double-stranded nucleic acids.
  • Example 4 the recyclability of the particles (GO ratio 53%) subjected to the DNA extraction experiment in Example 2 was confirmed.
  • the particle recycling ability was prepared by preparing a large number of particles subjected to DNA extraction experiments under the same conditions as in Example 2, washing once with 500 ⁇ l AE buffer, washing twice with AE buffer, 1 hour, 2 hours with 2M HCl. Particles treated for 4, 8 and 12 hours were obtained.
  • 500 ⁇ l of AE buffer was added to each particle, heated to 70 ° C. and stirred for 5 minutes to elute the DNA remaining on the particle surface, and the amount of DNA contained in the eluate was determined to be Quick 2.0 (life Technology).
  • Table 3 shows the detection value (ng / ml) of Quick 2.0.
  • nucleic acid extraction ability of HCl-treated particles was confirmed by the following procedure.
  • Particles having a GO ratio of 53% in the mixed solution were prepared, DNA was extracted from the DNA sample in the same procedure as in Example 2 above, and 260 nm / 280 nm and 260 nm / 230 nm values were calculated. Further, the total yield was determined by multiplying the amount of nucleic acid (ng / ⁇ l) contained in 1 ⁇ l of nucleic acid eluate by 500 times.
  • the amount of DNA remaining on the particle surface after 2 hours of treatment with 2M HCl was measured by the same procedure as described above.
  • 260 nm / 280 nm and 260 nm / 230 nm values were calculated in the same procedure as in (1) except that the particles treated with HCl in (2) were used. Further, the total yield was determined by multiplying the amount of nucleic acid (ng / ⁇ l) contained in 1 ⁇ l of nucleic acid eluate by 500 times. The values of 260 nm / 280 nm and 260 nm / 230 nm of the above (3) and the value of Total Yield were all about 99% of the value of (1). From the above results, it became clear that the particles of the present invention can be recycled.
  • Example 5 Particles containing Fe 3 O 4 were prepared in the same procedure as in Example 1 except that 0.3 g to 4.5 g of Fe 3 O 4 was added in the step (3) of Example 1 above. . Table 4 shows the mixing ratio of the particles produced in Example 5.
  • FIG. 7A The left side of FIG. 7A is a photograph of CGM0 and the right side is CGM45, which is a substantially spherical shape having a diameter of about 2.6 mm.
  • FIG. 7B is a photograph of a magnet brought close to a container in which CGM0 is dispersed in pure water
  • FIG. 7C is a photograph of a magnet brought close to a container in which CGM45 is dispersed in pure water.
  • the particles (CGM45) containing Fe 3 O 4 were attracted to the magnet when approaching the magnet. Therefore, when the particles contain Fe 3 O 4 , the particles adsorbed with the nucleic acid can be collected using a magnet, so that the convenience of operation is improved.
  • FIGS. 8B to 8D are mappings obtained by analyzing the CGM45 particle surface with an energy dispersive X-ray analyzer (EDX)
  • FIG. 8B is a carbon element
  • 8C shows the oxygen element
  • FIG. 8D shows the dispersion of the Fe element.
  • FIG. 9 is a graph of the amount of nucleic acid (ng / ⁇ l) and Total Yield of each sample.
  • the nucleic acid extraction efficiency was improved as the amount added was increased. This is thought to be because Fe 3 O 4 is positively charged in the particle, attracting the negatively charged nucleic acid, and the aromatic ring of GO and the aromatic ring of nucleic acid interact by ⁇ - ⁇ stacking. It is done.
  • Example 5 since pure water was used instead of the binding buffer (GuHCl), the value was lower than the nucleic acid extraction efficiency shown in Table 2.
  • binding buffers contain chaotropic salts such as guanidinium ions, urea, iodide ions, and Na + may be added to increase the extraction efficiency of nucleic acids.
  • the extracted nucleic acid solution contains Na + or a chaotropic salt, it becomes an inhibiting factor when the nucleic acid is amplified by PCR. Therefore, when the extracted nucleic acid is amplified by PCR, the use of particles obtained by adding Fe 3 O 4 to GO + cellulose shown in Example 5 eliminates the need for a step of removing Na + or chaotropic salt. It is very useful as a particle.
  • Example 6 Preparation of particles containing Fe 2 O 3 ] ⁇ Example 6> Particles were prepared in the same procedure as in Example 5 except that Fe 2 O 3 was used instead of Fe 3 O 4 and the mixing ratio was as shown in Table 5, and the amount of nucleic acid (ng / ⁇ l) and total yield were determined. .
  • FIG. 10 is a graph of the amount of nucleic acid (ng / ⁇ l) and Total Yield of each sample.
  • nucleic acid extraction efficiency was improved by adding Fe 2 O 3 to the particles. From the results of Examples 5 and 6 above, it was revealed that the nucleic acid extraction efficiency was improved by adding iron oxide to GO + cellulose particles, and the nucleic acid could be extracted without using a binding buffer.
  • the negatively charged nucleic acid is attracted when the iron oxide dispersed in the particles is positively charged, it is not limited to iron oxide, and is a substance that is positively charged in the composition for extracting particles or nucleic acids. If there is, it is thought that there is an equivalent effect.
  • nucleic acids can be extracted from a variety of samples with higher efficiency than when using a conventional nucleic acid extraction kit.
  • the particle or nucleic acid extraction composition of the present invention can be recycled. Therefore, it is useful for extracting nucleic acids from various samples in universities, companies, research institutions and the like.

Abstract

To provide a solid composition with which it is possible to extract nucleic acid highly efficiently from a solution in which biological components are dissolved, the composition being capable of being recycled after use. This problem is solved by particles containing graphene oxide and/or graphite oxide as well as cellulose.

Description

グラフェンオキサイド及び／又はグラファイトオキサイド並びにセルロースを含む粒子、核酸抽出用組成物、核酸抽出方法、及び粒子又は核酸抽出用組成物のリサイクル方法Particles containing graphene oxide and / or graphite oxide and cellulose, composition for nucleic acid extraction, method for nucleic acid extraction, and method for recycling particles or composition for nucleic acid extraction
　本発明は、グラフェンオキサイド及び／又はグラファイトオキサイド並びにセルロースを含む粒子、核酸抽出用組成物、核酸抽出方法、及び粒子又は核酸抽出用組成物のリサイクル方法に関する。特に、核酸を含む多様なサンプルからの核酸の抽出操作が容易であり、また、高純度且つ効率的に核酸を抽出することができる粒子及び核酸抽出用組成物に関する。更に、前記粒子及び核酸抽出用組成物を用いた核酸抽出方法、並びに核酸抽出後の粒子又は核酸抽出用組成物のリサイクル方法に関する。 The present invention relates to particles containing graphene oxide and / or graphite oxide and cellulose, a composition for nucleic acid extraction, a method for nucleic acid extraction, and a method for recycling particles or a composition for nucleic acid extraction. In particular, the present invention relates to a particle and a composition for nucleic acid extraction that are easy to extract nucleic acid from various samples containing nucleic acid and that can extract nucleic acid with high purity and efficiency. Furthermore, the present invention relates to a nucleic acid extraction method using the particles and the nucleic acid extraction composition, and a recycling method of the particles or nucleic acid extraction composition after the nucleic acid extraction.
　サンプル中に含まれる微量の核酸の分析は、生物化学、医療、食品、犯罪捜査等の多くの分野で行われている。サンプル中の核酸の抽出方法としては、古くは、Ｆｒｉｅｄｅｒｉｃｈ　Ｍｉｓｃｈｅｒ’ｓ　ｍｅｔｈｏｄが用いられ、次いで、１９６７年にフェノール・クロロホルム法が開発されている。前記の核酸抽出方法は、タンパク質等の除去したい成分を変性し、次いで、遠心分離等により生体成分が溶解している溶液から除去することで核酸抽出を行っている。したがって、抽出したい核酸は液体に溶解した状態であることから、酵素等の影響により核酸が分解されやすいという問題がある。また、抽出操作は液体の混合を繰り返すことから、操作中にこぼれ易い等の問題がある。 The analysis of a small amount of nucleic acid contained in a sample is performed in many fields such as biochemistry, medical treatment, food, and crime investigation. As a method for extracting nucleic acids from samples, Friedrich Mischer's method has been used for a long time, and then the phenol / chloroform method was developed in 1967. The nucleic acid extraction method performs nucleic acid extraction by denaturing a component to be removed, such as protein, and then removing it from a solution in which a biological component is dissolved by centrifugation or the like. Therefore, since the nucleic acid to be extracted is in a state dissolved in a liquid, there is a problem that the nucleic acid is easily decomposed due to the influence of an enzyme or the like. Further, since the extraction operation repeats mixing of liquids, there is a problem that the extraction operation tends to spill.
　上記問題点を解決するため、１９７９年には、従来の液体を用いた核酸の抽出方法に代え、固体のシリカ粒子を用いた核酸の抽出方法が開発された。とろこで、大学等の研究室では、予算の都合上、核酸等の抽出に用いる器具・材料等をリサイクルする場合がある。シリカ粒子をリサイクルする方法としては、核酸抽出後のシリカ粒子をＨＣｌで処理することが知られている（非特許文献１参照）。また、サンプル中の核酸を抽出する方法としては、磁性粒子を用いた方法も知られている。 In order to solve the above problems, in 1979, a nucleic acid extraction method using solid silica particles was developed instead of the conventional nucleic acid extraction method using a liquid. In a lab, a laboratory in a university or the like sometimes recycles equipment and materials used for nucleic acid extraction for budget reasons. As a method for recycling silica particles, it is known to treat silica particles after nucleic acid extraction with HCl (see Non-Patent Document 1). As a method for extracting nucleic acid in a sample, a method using magnetic particles is also known.
　しかしながら、非特許文献１に記載されている方法は、ＨＣｌでシリカ粒子を表面処理すると表面が変化しリサイクル能が低下することと、リサイクルには２～３日程度要するという問題がある。また、磁性粒子を用いた核酸の抽出方法は、大型の装置等が必要であり、又抽出時間もかかり、核酸抽出が煩雑であるという問題がある。
　現状では、サンプル中の核酸を抽出することができ且つリサイクル可能な固体状の組成物は知られていない。 However, the method described in Non-Patent Document 1 has a problem that when silica particles are surface-treated with HCl, the surface is changed and the recycling ability is lowered, and the recycling takes about 2 to 3 days. In addition, the nucleic acid extraction method using magnetic particles requires a large-scale apparatus and the like, and also requires a long extraction time, resulting in complicated nucleic acid extraction.
At present, there is no known solid composition that can extract nucleic acid in a sample and can be recycled.
　本発明は、上記の問題点を解決するためになされた発明で、鋭意研究を行ったところ、
（１）グラフェンオキサイド及び／又はグラファイトオキサイドとセルロースを混合した粒子又は核酸抽出用組成物は、生体成分が溶解した溶液中から核酸を抽出できること、
（２）セルロースに対するグラフェンオキサイド及び／又はグラファイトオキサイドの比を調整することで、サンプル中の核酸を、従来のシリカ粒子より高効率で抽出できること、
（３）従来のシリカ粒子を用いた核酸の抽出方法では、一度使用したシリカ粒子はリサイクルできなかったが、本発明の使用済みの粒子又は核酸抽出用組成物は、核酸クリーニング液で処理することで粒子又は核酸抽出用組成物表面の核酸を除去することができ、リサイクルが可能であること、
（４）粒子又は核酸抽出用組成物に磁性体を加えると、磁石により核酸が吸着した粒子又は核酸抽出組成物を回収できるので作業効率を更に向上できること、
（５）粒子又は核酸抽出用組成物にプラスに帯電する物質を加えると、カオトロピック塩やＮａ+を含むバインディングバッファーを用いなくても核酸を抽出できること、
を新たに見出した。 The present invention has been made in order to solve the above-mentioned problems.
(1) A particle or nucleic acid extraction composition in which graphene oxide and / or graphite oxide and cellulose are mixed can extract nucleic acid from a solution in which a biological component is dissolved,
(2) By adjusting the ratio of graphene oxide and / or graphite oxide to cellulose, nucleic acids in the sample can be extracted with higher efficiency than conventional silica particles,
(3) In the conventional nucleic acid extraction method using silica particles, the silica particles once used could not be recycled, but the used particles or nucleic acid extraction composition of the present invention should be treated with a nucleic acid cleaning solution. The nucleic acid on the surface of the particle or nucleic acid extraction composition can be removed and can be recycled,
(4) When a magnetic substance is added to the particle or nucleic acid extraction composition, the particle or nucleic acid extraction composition on which the nucleic acid is adsorbed by the magnet can be recovered, so that the work efficiency can be further improved.
(5) By adding a positively charged substance to the particle or nucleic acid extraction composition, nucleic acid can be extracted without using a chaotropic salt or a binding buffer containing Na + .
Newly found.
　すなわち、本発明の目的は、グラフェンオキサイド及び／又はグラファイトオキサイド並びにセルロースを含む粒子、核酸抽出用組成物、核酸抽出方法、及び粒子又は核酸抽出用組成物のリサイクル方法を提供することである。 That is, an object of the present invention is to provide particles containing graphene oxide and / or graphite oxide and cellulose, a nucleic acid extraction composition, a nucleic acid extraction method, and a recycling method of the particles or nucleic acid extraction composition.
　本発明は、以下に示す、グラフェンオキサイド及び／又はグラファイトオキサイド並びにセルロースを含む粒子、核酸抽出用組成物、核酸抽出方法、及び粒子又は核酸抽出用組成物のリサイクル方法に関する。 The present invention relates to the following particles containing graphene oxide and / or graphite oxide and cellulose, a composition for nucleic acid extraction, a method for nucleic acid extraction, and a method for recycling the particles or the composition for nucleic acid extraction.
（１）グラフェンオキサイド及び／又はグラファイトオキサイド、並びにセルロースを含む粒子。
（２）前記粒子における、セルロースに対するグラフェンオキサイド及び／又はグラファイトオキサイドの比が、４７％～６４％である上記（１）に記載の粒子。
（３）磁性体及び／又は粒子中でプラスに帯電する物質を更に含む上記（１）又は（２）に記載の粒子。
（４）グラフェンオキサイド及び／又はグラファイトオキサイド、並びにセルロースを含む固体状の核酸抽出用組成物。
（５）前記核酸抽出用組成物における、セルロースに対するグラフェンオキサイド及び／又はグラファイトオキサイドの比が、４７％～６４％である上記（４）に記載の核酸抽出用組成物。
（６）磁性体及び／又は組成物中でプラスに帯電する物質を更に含む上記（４）又は（５）に記載の核酸抽出用組成物。
（７）上記（１）～（３）の何れか一に記載の粒子、上記（４）～（６）の何れか一に記載の核酸抽出用組成物の何れかに、核酸を含む溶液を混合して核酸を前記粒子又は核酸抽出用組成物に吸着する工程、
　前記核酸が吸着した前記粒子又は核酸抽出用組成物に核酸溶出液を混合することで、前記粒子又は核酸抽出用組成物に吸着した核酸を分離する核酸抽出工程、
を含む核酸抽出方法。
（８）上記（７）に記載の核酸抽出工程後の前記粒子又は核酸抽出用組成物を、核酸クリーニング液で処理して前記粒子又は核酸抽出用組成物の表面に残留している核酸を除去する表面回復工程、
を含む前記粒子又は核酸抽出用組成物のリサイクル方法。 (1) Particles containing graphene oxide and / or graphite oxide and cellulose.
(2) The particle according to (1), wherein the ratio of graphene oxide and / or graphite oxide to cellulose in the particle is 47% to 64%.
(3) The particle according to (1) or (2), further including a magnetic substance and / or a substance that is positively charged in the particle.
(4) A solid nucleic acid extraction composition containing graphene oxide and / or graphite oxide and cellulose.
(5) The nucleic acid extraction composition according to (4), wherein the ratio of graphene oxide and / or graphite oxide to cellulose in the nucleic acid extraction composition is 47% to 64%.
(6) The nucleic acid extraction composition according to (4) or (5), further comprising a magnetic substance and / or a positively charged substance in the composition.
(7) A solution containing a nucleic acid in any one of the particles according to any one of (1) to (3) and the nucleic acid extraction composition according to any one of (4) to (6). Mixing and adsorbing the nucleic acid to the particle or the composition for nucleic acid extraction,
A nucleic acid extraction step of separating nucleic acid adsorbed on the particles or nucleic acid extraction composition by mixing a nucleic acid eluate with the particles or nucleic acid extraction composition on which the nucleic acids have been adsorbed;
A nucleic acid extraction method comprising:
(8) The particles or nucleic acid extraction composition after the nucleic acid extraction step according to (7) is treated with a nucleic acid cleaning solution to remove nucleic acids remaining on the surface of the particles or nucleic acid extraction composition. Surface recovery process,
A method for recycling the particle or nucleic acid extraction composition comprising:
（１）本発明のグラフェンオキサイド及び／又はグラファイトオキサイド（以下、「ＧＯ」と記載することがある。）並びにセルロースを含む粒子、及び核酸抽出用組成物は、生体成分が溶解した溶液中から核酸を選択的に抽出できる。
（２）セルロースに対するグラフェンオキサイドの比を調整することで、多様なサンプル中の核酸を、従来のシリカ粒子より高効率で抽出できる。したがって、唾液等に含まれる極微量の核酸であっても抽出することができる。
（３）従来のシリカ粒子を用いた核酸の抽出方法では、一度使用したシリカ粒子はリサイクルすることができなかったが、本発明の粒子又は核酸抽出用組成物は、核酸クリーニング液で処理することで粒子又は核酸抽出用組成物表面の核酸を除去することができ、リサイクルが可能である。したがって、繰り返し核酸の抽出に用いることができるので、研究室等の経費を節減することができる。
（４）粒子又は核酸抽出用組成物に磁性体を加えた場合、磁石により核酸が吸着した粒子又は核酸抽出組成物を回収できるので作業効率が向上する。
（５）粒子又は核酸抽出用組成物にプラスに帯電する物質を加えると、カオトロピック塩やＮａ+を含むバインディングバッファーを用いなくても核酸を抽出できるので、核酸抽出後にＰＣＲで増幅する場合に必要であった、Ｎａ+やカオトロピック塩を除去する工程が不要となる。 (1) The graphene oxide and / or graphite oxide (hereinafter, sometimes referred to as “GO”) and cellulose-containing particles of the present invention, and the composition for nucleic acid extraction are nucleic acids from a solution in which biological components are dissolved. Can be selectively extracted.
(2) By adjusting the ratio of graphene oxide to cellulose, nucleic acids in various samples can be extracted with higher efficiency than conventional silica particles. Therefore, even a very small amount of nucleic acid contained in saliva can be extracted.
(3) In the conventional nucleic acid extraction method using silica particles, silica particles once used could not be recycled, but the particles or the composition for nucleic acid extraction of the present invention should be treated with a nucleic acid cleaning solution. Thus, the nucleic acid on the surface of the particle or nucleic acid extraction composition can be removed and can be recycled. Therefore, since it can be used repeatedly for nucleic acid extraction, the cost of laboratories and the like can be reduced.
(4) When a magnetic substance is added to the particle or nucleic acid extraction composition, the particle or nucleic acid extraction composition to which the nucleic acid is adsorbed by the magnet can be recovered, so that the working efficiency is improved.
(5) Necessary when PCR is amplified after nucleic acid extraction because nucleic acid can be extracted without using chaotropic salt or binding buffer containing Na + by adding a positively charged substance to the particle or nucleic acid extraction composition The step of removing Na + and chaotropic salt, which has been described above, becomes unnecessary.
図１は、図面代用写真で、図１（Ａ）は実施例１で作製した粒子の写真、図１（Ｂ）は比較例１で作製した粒子の写真である。FIG. 1 is a drawing-substituting photograph, FIG. 1 (A) is a photograph of particles produced in Example 1, and FIG. 1 (B) is a photograph of particles produced in Comparative Example 1. 図２－１は、図面代用写真で、ＧＯの比を種々変えた粒子のＳＥＭ写真である。FIG. 2-1 is a photograph substituted for a drawing, and is an SEM photograph of particles with various GO ratios. 図２－２は、図面代用写真で、ＧＯの比を種々変えた粒子のＳＥＭ写真である。FIG. 2-2 is a photograph substituted for a drawing, and is an SEM photograph of particles with various GO ratios. 図３は、図面代用写真で、ＧＯの比が、夫々５３％、５６％、６０％、６４％の粒子をピペットの先端で押した写真を示している。FIG. 3 is a drawing-substituting photograph showing a photograph in which particles having GO ratios of 53%, 56%, 60%, and 64% are pressed with the tip of a pipette, respectively. 図４（Ａ）は、表２の２６０ｎｍ／２８０ｎｍ値をグラフ化したもので、図４（Ｂ）は、表２の２６０ｎｍ／２３０ｎｍ値をグラフ化したものである。4A is a graph of 260 nm / 280 nm values in Table 2, and FIG. 4B is a graph of 260 nm / 230 nm values in Table 2. 図５（Ａ）は、表２の核酸溶出液１μｌに含まれる核酸量（ｎｇ）値をグラフ化したもので、図５（Ｂ）は、表２のＴｏｔａｌ　Ｙｉｅｌｄをグラフ化したものである。FIG. 5 (A) is a graph showing the amount of nucleic acid (ng) contained in 1 μl of the nucleic acid eluate in Table 2. FIG. 5 (B) is a graph of Total Yield in Table 2. 図６は、実施例４及び比較例２の結果を表すグラフで、多様なサンプルからのＤＮＡ抽出のＴｏｔａｌ　Ｙｉｅｌｄ値を示すグラフである。FIG. 6 is a graph showing the results of Example 4 and Comparative Example 2, and showing the Total Yield value of DNA extraction from various samples. 図７（Ａ）は、図面代用写真で、左側は実施例５で作製したＣＧＭ０、右側は実施例５で作製したＣＧＭ４５の写真である。図７（Ｂ）及び（Ｃ）は、図面代用写真で、図７（Ｂ）はＣＧＭ０を純水に分散した容器に磁石を近付けた写真、図７（Ｃ）はＣＧＭ４５を純水に分散した容器に磁石を近付けた写真である。FIG. 7A is a drawing-substituting photograph, the left is a photograph of CGM0 produced in Example 5, and the right is a photograph of CGM45 produced in Example 5. 7B and 7C are photographs substituted for drawings, FIG. 7B is a photograph in which a magnet is brought close to a container in which CGM0 is dispersed in pure water, and FIG. 7C is a photograph in which CGM45 is dispersed in pure water. It is the photograph which brought the magnet close to the container. 図８（Ａ）は、図面代用写真で、実施例５のＣＧＭ４５のＳＥＭ写真である。図８（Ｂ）～（Ｄ）は、図面代用写真で、ＣＧＭ４５粒子表面をエネルギー分散型Ｘ線分析装置（ＥＤＸ）で解析したマッピングで、図８（Ｂ）は炭素元素、図８（Ｃ）は酸素元素、図８（Ｄ）はＦｅ元素の分散を表している。FIG. 8A is a drawing-substituting photograph and an SEM photograph of CGM45 of Example 5. FIGS. 8B to 8D are photographs substituted for drawings, and are mappings obtained by analyzing the surface of CGM45 particles with an energy dispersive X-ray analyzer (EDX). FIG. 8B is a carbon element, and FIG. Represents oxygen element, and FIG. 8D represents dispersion of Fe element. 図９は、実施例５の各サンプルの核酸量（ｎｇ／μｌ）及びＴｏｔａｌ　Ｙｉｅｌｄのグラフである。FIG. 9 is a graph of the amount of nucleic acid (ng / μl) of each sample of Example 5 and Total Yield. 図１０は、実施例６の各サンプルの核酸量（ｎｇ／μｌ）及びＴｏｔａｌ　Ｙｉｅｌｄのグラフである。FIG. 10 is a graph of the amount of nucleic acid (ng / μl) and the total yield of each sample of Example 6.
　以下に、本発明のＧＯ及びセルロースを含む粒子、核酸抽出用組成物、核酸抽出方法、及び粒子又は核酸抽出用組成物のリサイクル方法ついて詳しく説明する。 Hereinafter, the particles containing GO and cellulose of the present invention, a composition for nucleic acid extraction, a method for nucleic acid extraction, and a method for recycling the particle or composition for nucleic acid extraction will be described in detail.
　本発明のＧＯ及びセルロースを含む粒子は、ＧＯを含む水に、セルロース及びセルロース分子間の水素結合を壊すための物質を添加・混合（以下、「混合溶液」と記載することがある。）し、混合溶液を凝固用溶液に滴下し、混合溶液を凝固することで製造することができる。 In the particles containing GO and cellulose of the present invention, a substance for breaking hydrogen bonds between cellulose and cellulose molecules is added to and mixed with water containing GO (hereinafter sometimes referred to as “mixed solution”). It can be produced by dropping the mixed solution into the coagulation solution and coagulating the mixed solution.
　本発明に用いるＧＯは、グラフェンオキサイド（Ｇｒａｐｈｅｎｅ　Ｏｘｉｄｅ；炭素原子シートが単層）、グラファイトオキサイド（Ｇｒａｐｈｉｔｅ　Ｏｘｉｄｅ；炭素原子シートが多層）を夫々用いてもよいし、両者を混合して用いてもよい。ＧＯはＭａｒｃａｎｏ’ｓ　ｍｅｔｈｏｄ等の公知の法で製造してもよいし、市販されているものを用いてもよい。セルロースも一般的に市販されているものを用いればよい。 As the GO used in the present invention, graphene oxide (Graphene Oxide; a carbon atom sheet is a single layer), graphite oxide (Graphite Oxide; a carbon atom sheet is a multilayer) may be used, or a mixture of both may be used. . GO may be produced by a known method such as Marcano's method, or a commercially available one may be used. What is necessary is just to use the cellulose generally marketed.
　凝固用溶液としては、混合溶液を凝固できれば特に制限は無く、例えば、ＨＮＯ3、Ｈ2ＳＯ4、アセトン、エタノール、食塩水、その他、酸性溶液等が挙げられる。前記凝固用溶液の中では、安定性や凝固能力等の面から、ＨＮＯ3、Ｈ2ＳＯ4が好ましい。また、凝固用溶液は、必要に応じて５℃～５０℃に調整する必要があるが、ＨＮＯ3は室温で使用可能であることから、操作性の観点から好ましい。粒子の大きさは、混合溶液を滴下する際の器具（スポイト等）の口径を調整し、凝固用溶液中に滴下する混合溶液の量を調整すればよい。なお、本発明の「粒子」とは、上記のとおり、凝固用溶液中に混合溶液を滴下して形成された粒子を意味し、混合溶液の粘度により異なるものの、球形、楕円形、涙形等が挙げられる The coagulation solution is not particularly limited as long as the mixed solution can be coagulated, and examples thereof include HNO 3 , H 2 SO 4 , acetone, ethanol, saline, and other acidic solutions. In the coagulation solution, HNO 3 and H 2 SO 4 are preferable from the viewpoints of stability and coagulation ability. The coagulation solution needs to be adjusted to 5 ° C. to 50 ° C. as necessary, but HNO 3 is preferable from the viewpoint of operability because it can be used at room temperature. The size of the particles may be adjusted by adjusting the diameter of an instrument (such as a dropper) when dropping the mixed solution and adjusting the amount of the mixed solution dropped into the coagulation solution. The “particle” of the present invention means a particle formed by dripping the mixed solution into the coagulation solution as described above, and varies depending on the viscosity of the mixed solution. Can be mentioned
　セルロース分子間の水素結合を壊すための物質としては、セルロース分子間の水素結合が壊れれば特に制限は無い。例えば、ＮａＯＨ、尿素等が挙げられ、単独でも組み合わせて用いてもよい。 The substance for breaking hydrogen bonds between cellulose molecules is not particularly limited as long as hydrogen bonds between cellulose molecules are broken. For example, NaOH, urea, etc. are mentioned, and they may be used alone or in combination.
　セルロースに対するＧＯの比は、サンプル中の核酸を吸着し、核酸溶出液により吸着した核酸を離脱することでサンプル中の核酸を抽出できれば特に制限はない。なお、本発明において、「セルロースに対するＧＯの比」とは、（ＧＯ／（セルロース＋ＧＯ））×１００（％）を意味する。混合溶液を凝固用溶液中に滴下すると混合溶液が固化し、セルロースとＧＯは凝固用溶液に溶解しないので、混合溶液中のセルロースに対するＧＯの比と粒子中のセルロースに対するＧＯの比は同じと推測される。 The ratio of GO to cellulose is not particularly limited as long as the nucleic acid in the sample can be extracted by adsorbing the nucleic acid in the sample and releasing the adsorbed nucleic acid with the nucleic acid eluate. In the present invention, the “ratio of GO to cellulose” means (GO / (cellulose + GO)) × 100 (%). When the mixed solution is dropped into the coagulating solution, the mixed solution is solidified, and cellulose and GO are not dissolved in the coagulating solution. Therefore, it is assumed that the ratio of GO to cellulose in the mixed solution is the same as the ratio of GO to cellulose in the particles. Is done.
　セルロースに対するＧＯの比（以下、単に「ＧＯの比」と記載することがある。）としては、５％以上が好ましく、３１％以上がより好ましい。更に、従来のシリカ粒子と遜色ない高効率で核酸を抽出する場合は、ＧＯの比を４７％以上とすることが特に好ましい。 The ratio of GO to cellulose (hereinafter sometimes simply referred to as “GO ratio”) is preferably 5% or more, and more preferably 31% or more. Furthermore, when nucleic acids are extracted with high efficiency comparable to conventional silica particles, the GO ratio is particularly preferably 47% or more.
　一方、ＧＯの比の上限は、製造した粒子又は組成物が固体状態を維持できる比であれば特に制限はない。例えば、ＧＯの比が６４％でも固体状態を維持することができるので、６４％以下とすればよく、取り扱いの利便性等を考慮し、６０％以下、５６％以下、５３％以下等、適宜調整すればよい。 On the other hand, the upper limit of the GO ratio is not particularly limited as long as the produced particles or composition can maintain a solid state. For example, since the solid state can be maintained even if the GO ratio is 64%, it may be set to 64% or less, and considering the convenience of handling, 60% or less, 56% or less, 53% or less, etc. Adjust it.
　なお、酵素によるＤＮＡの分解を抑えるためにグラフェンオキサイドを使用することは知られている（Ｈａｏｚｈｉ　Ｌｅｉ　ｅｔ．　ａｌ．，　“Ａｄｓｏｒｐｔｉｏｎ　ｏｆ　ｄｏｕｂｌｅ－ｓｔｒａｎｄｅｄ　ＤＮＡ　ｔｏ　ｇｒａｐｈｅｎｅ　ｏｘｉｄｅ　ｐｒｅｖｅｎｔｉｎｇ　ｅｎｚｙｍａｔｉｃ　ｄｉｇｅｓｔｉｏｎ”，Ｎａｎｏｓｃａｌｅ，２０１１，３，ｐ３８８８－３８９２、参照）。また、グラフェンオキサイドとセルロースを含む組成物をフィルム状にしてバイオセンサーに用いることも知られている（Ｑｉａｎｇ　Ｙａｎｇ　ｅｔ．　Ａｌ．，　“Ｆａｂｒｉｃａｔｉｏｎ　ｏｆ　Ｈｉｇｈ－Ｃｏｎｃｅｎｔｒａｔｉｏｎ　ａｎｄ　Ｓｔａｂｌｅ　Ａｑｕｅｏｕｓ　Ｓｕｓｐｅｎｓｉｏｎｓ　ｏｆ　Ｇｒａｐｈｅｎｅ　Ｎａｎｏｓｈｅｅｔｓ　ｂｙ　Ｎｏｎｃｏｖａｌｅｎｔ　Ｆｕｎｃｔｉｏｎａｌｉｚａｔｉｏｎ　ｗｉｔｈ　Ｌｉｇｎｉｎ　ａｎｄ　Ｃｅｌｌｕｌｏｓｅ　Ｄｅｒｉｖａｔｉｖｅｓ”，Ｊ．　Ｐｈｙｓ．　Ｃｈｅｍ．　Ｃ，２０１０，Ｖｏｌ．１１４，Ｎｏ．９，ｐ３８１１－３８１６、参照。）しかしながら、ＧＯ及びセルロースを粒子状にすることは本発明で新たに見出した形状である。更に、ＧＯ及びセルロースを含む組成物を用いて核酸を抽出し且つリサイクルできることも、本発明で新たに見出したことである。 In addition, it is known to use graphene oxide to suppress the degradation of DNA by enzymes (Haozhi Lei et. Al., “Adsorption of double-stranded DNA to grafting preventing enzymetic 1, 201”). , P 3888-3892). It is also known to use a composition containing graphene oxide and cellulose in the form of a film for use in biosensors (Qiang Yang et. Al., “Fabrication of High-Concentration and Stable Aqueous Sustainations of GraffitiNowenseNows from the World of Science. Lignin and Cellulose Derivatives ", J. Phys. Chem. C, 2010, Vol. 114, No. 9, p3811-3816, however, it was newly found in the present invention that GO and cellulose are made particulate. Shape. Furthermore, it has been newly found in the present invention that a nucleic acid can be extracted and recycled using a composition containing GO and cellulose.
　したがって、組成物を用いて核酸を抽出する場合、組成物の形状としては特に制限はないが、比表面積が大きくなり、また、核酸抽出に一般的に用いられているエッペンドルフチューブ等に効率的に充填できることから、粒子状が好ましい。なお、本発明の粒子はサンプル中の核酸を選択的に吸着することができるので、例えば、サンプル中に本発明の粒子を添加・混合した後、粒子をサンプルから除去することで、サンプルから核酸を除去しタンパク質等の濃縮用の用途にも使用することができる。したがって、本発明の粒子の用途としては、核酸抽出用に限定されない。 Therefore, when nucleic acid is extracted using the composition, the shape of the composition is not particularly limited, but the specific surface area is increased, and the composition is efficiently applied to an Eppendorf tube or the like generally used for nucleic acid extraction. Since it can be filled, a particulate form is preferable. Since the particles of the present invention can selectively adsorb nucleic acids in a sample, for example, after adding and mixing the particles of the present invention in a sample, the particles are removed from the sample to remove nucleic acids from the sample. Can be used for the purpose of concentrating proteins and the like. Therefore, the use of the particles of the present invention is not limited to nucleic acid extraction.
　また、上記のとおり、ＧＯ及びセルロースを含む組成物をフィルム状に形成することは知られているが、ＧＯ及びセルロースを含む組成物を、核酸抽出に用いることは知られていない。したがって、本発明のＧＯ及びセルロースを含む組成物は、核酸抽出用として新たな用途を提供することができる。上記のとおり、エッペンドルフチューブを用いて核酸を抽出する場合、組成物の形状は粒子状が好ましいが、粒子に限定されるものではない。例えば、混合溶液を凝固用溶液中に中空となるように押し出すことで、組成物をチューブ状に形成し、核酸を含むサンプルをチューブに流し、次いで、核酸溶出液を流すことで、連続処理ができるようにしても良い。また、ガラス板等にキャストした後に凝固用溶液中に浸漬する、又は、凝固用溶液中に薄膜状に押し出すことでフィルム状としても良い。何れの形態であっても、核酸を含むサンプル液と接触させることで、核酸を吸着させることができる。 Also, as described above, it is known to form a composition containing GO and cellulose into a film, but it is not known to use a composition containing GO and cellulose for nucleic acid extraction. Therefore, the composition containing GO and cellulose of the present invention can provide a new application for nucleic acid extraction. As described above, when nucleic acid is extracted using an Eppendorf tube, the shape of the composition is preferably particulate, but is not limited to particles. For example, by pressing the mixed solution into a coagulation solution so as to be hollow, the composition is formed into a tube shape, a sample containing nucleic acid is flowed into the tube, and then a nucleic acid eluate is flowed to perform continuous processing. You may be able to do it. Moreover, after casting to a glass plate etc., it is good also as a film form by immersing in the solution for coagulation, or extruding in a thin film form in the solution for coagulation. In any form, nucleic acid can be adsorbed by contacting with a sample solution containing nucleic acid.
　本発明の核酸抽出方法は、先ず、核酸を含むサンプル溶液及びバインディングバッファー、並びに粒子又は核酸抽出用組成物を混合することで粒子又は核酸抽出用組成物に核酸を吸着させ、次いで、エタノールで洗浄後、核酸が吸着した粒子又は核酸抽出用組成物と核酸溶出液を混合することで前記粒子又は核酸抽出用組成物に吸着した核酸を離脱させ、サンプル中の核酸を核酸溶出液中に回収することができる。なお、本発明の核酸には、ＤＮＡ及びＲＮＡが含まれる。また、ＤＮＡは一本鎖であっても２本鎖であっても良い。 In the nucleic acid extraction method of the present invention, first, a sample solution containing a nucleic acid, a binding buffer, and the particle or the composition for nucleic acid extraction are mixed to adsorb the nucleic acid to the particle or the composition for nucleic acid extraction, and then washed with ethanol. Thereafter, the nucleic acid adsorbed on the particle or nucleic acid extraction composition is separated by mixing the nucleic acid-adsorbed particles or nucleic acid extraction composition and the nucleic acid eluate, and the nucleic acid in the sample is recovered in the nucleic acid eluate. be able to. The nucleic acid of the present invention includes DNA and RNA. Further, the DNA may be single-stranded or double-stranded.
　バインディングバッファーとしては、塩化グアニジニウム（ＧｕＨＣｌ）、ｃｈａｏｔｒｏｐｉｃ　ｓａｌｔｓ（ＧＩＴＣ）、ＮａＣｌ、ＰＢ　ｂｕｆｆｅｒ等を用いればよい。 As the binding buffer, guanidinium chloride (GuHCl), chaotropic salts (GITC), NaCl, PB buffer, or the like may be used.
　核酸溶出液としては、粒子又は核酸抽出用組成物の表面と核酸のイオン結合を壊し、核酸が溶出できれば特に制限はない。例えば、ＡＥ　ｂｕｆｆｅｒ（１０ｍＭ　Ｔｒｉｓ－ＨＣｌ、０．５ｍＭ　ＥＤＴＡ、ｐＨ９．０、キアゲン社製）等が挙げられる。 The nucleic acid eluate is not particularly limited as long as the nucleic acid can be eluted by breaking the ion bond between the particle or the surface of the nucleic acid extraction composition and the nucleic acid. Examples include AE buffer (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0, manufactured by Qiagen).
　粒子又は核酸抽出用組成物のリサイクル方法は、粒子又は核酸抽出用組成物の表面に残留している核酸にダメージを与え、表面から除去できる核酸クリーニング液で表面処理を行えばよい。核酸クリーニング液は、上記のとおり核酸にダメージを与え、粒子又は核酸抽出用組成物の表面から核酸を除去できるものであれば特に制限は無く、例えば、ＨＣｌ等の酸性溶液、ＮａＯＨ等のアルカリ溶液、又はＵｒｅａ等が挙げられる。核酸クリーニング液で、粒子又は核酸抽出用組成物の表面に残留している核酸を除去して表面回復することで、粒子又は核酸抽出用組成物をリサイクルすることができる。したがって、予算が限られている研究室等で有用である。 As a method for recycling the particle or nucleic acid extraction composition, the nucleic acid remaining on the surface of the particle or nucleic acid extraction composition may be damaged, and the surface treatment may be performed with a nucleic acid cleaning solution that can be removed from the surface. The nucleic acid cleaning solution is not particularly limited as long as it damages the nucleic acid as described above and can remove the nucleic acid from the surface of the particle or the composition for nucleic acid extraction. For example, an acidic solution such as HCl or an alkaline solution such as NaOH Or Urea or the like. By removing the nucleic acid remaining on the surface of the particle or nucleic acid extraction composition with the nucleic acid cleaning liquid and recovering the surface, the particle or nucleic acid extraction composition can be recycled. Therefore, it is useful in laboratories where budgets are limited.
　また、本発明の粒子又は核酸抽出用組成物は、磁性体を含んでいてもよい。磁性体を含んだ粒子又は核酸抽出用組成物は、磁石で回収することができるので作業効率が向上する。磁性体としては、磁石により引き寄せられるものであれば特に制限はないが、例えば、（１）マグネタイト、マグヘマイト、フェライト等の磁性酸化鉄、及び他の金属酸化物を含む酸化鉄、（２）鉄、コバルト、ニッケル等の金属、又は、これらの金属とアルミニウム、コバルト、銅、鉛、マグネシウム、錫、亜鉛、アンチモン、ベリリウム、ビスマス、カドミウム、カルシウム、マンガン、セレン、チタン、タングステン、バナジウム等の金属との合金、（３）及びこれらの混合物、などが挙げられる。 Further, the particle or nucleic acid extraction composition of the present invention may contain a magnetic substance. Since the particles or the composition for nucleic acid extraction containing a magnetic material can be collected with a magnet, the working efficiency is improved. The magnetic material is not particularly limited as long as it is attracted by a magnet. For example, (1) magnetic iron oxide such as magnetite, maghemite, and ferrite, and iron oxide containing other metal oxides, (2) iron Metals such as cobalt, nickel, or these metals and metals such as aluminum, cobalt, copper, lead, magnesium, tin, zinc, antimony, beryllium, bismuth, cadmium, calcium, manganese, selenium, titanium, tungsten, vanadium And alloys thereof, (3) and mixtures thereof.
　また、粒子又は核酸抽出用組成物にプラスに帯電する物質を加えると、核酸の抽出効率が向上し、更に、カオトロピック塩やＮａ+を含むバインディングバッファーを用いなくても核酸を抽出できる。プラスに帯電する物質としては、マイナスの電荷を持つ核酸を引き寄せることができれば特に制限はなく、マグネタイト、マグヘマイト、フェライト等の酸化鉄、酸化アルミ、酸化マグネシウム等が挙げられる。 Moreover, when a positively charged substance is added to the particle or nucleic acid extraction composition, the extraction efficiency of the nucleic acid is improved, and further, the nucleic acid can be extracted without using a binding buffer containing a chaotropic salt or Na + . The positively charged substance is not particularly limited as long as a negatively charged nucleic acid can be attracted, and examples thereof include iron oxides such as magnetite, maghemite, and ferrite, aluminum oxide, and magnesium oxide.
　上記磁性体、プラスに帯電する物質は、粒子又は核酸抽出用組成物に別々に加えてもよいし、両者を組み合わせて加えてもよい。また、酸化鉄（例えば、マグネタイト、マグヘマイト、フェライト）等の磁性及びプラスに帯電の両方の性質を有する物質の場合は、単独で２つの異なる効果を奏するので好ましい。 The magnetic substance and the positively charged substance may be added separately to the particles or the nucleic acid extraction composition, or may be added in combination. In addition, a substance having both magnetic properties and positively charged properties such as iron oxide (eg, magnetite, maghemite, ferrite) is preferable because it has two different effects independently.
　粒子又は核酸抽出用組成物中に含む酸化鉄の量は、微量でも添加すれば効果が得られるので下限値は特にない。一方、酸化鉄の含有量が多く成りすぎると固体形の粒子又は核酸抽出用組成物を形成できなくなることから、固体形状を保つ範囲を上限とすればよい。 The amount of iron oxide contained in the particle or nucleic acid extraction composition has no particular lower limit because an effect can be obtained if it is added even in a trace amount. On the other hand, if the content of iron oxide is too large, solid particles or a composition for nucleic acid extraction cannot be formed.
　以下に実施例を掲げ、本発明を具体的に説明するが、この実施例は単に本発明の説明のため、その具体的な態様の参考のために提供されているものである。これらの例示は本発明の特定の具体的な態様を説明するためのものであるが、本願で開示する発明の範囲を限定したり、あるいは制限することを表すものではない。 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.
〔種々のＧＯの比の粒子の作製〕
＜実施例１＞
　以下の（１）～（５）に示す工程により、本発明の粒子を作製した。
（１）先ず、以下に示すＭａｒｃａｎｏ’ｓ　ｍｅｔｈｏｄにより、ＧＯを作製した。
（ａ）３６０ｍｌのＨ2ＳＯ4と４０ｍｌのＨ3ＰＯ4の混合液を、３．０ｇの片状黒鉛（ｇｒａｐｈｉｔｅ　ｆｌａｋｅｓ；Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）及び１８．０ｇのＫＭｎＯ4（和光純薬工業株式会社製）に添加して得られた混合物を３０－４０℃に発熱させた。
（ｂ）次いで、上記（ａ）で得られた混合物を５０℃に加熱し、１２時間撹拌した。撹拌後は、室温まで冷却し、３０％Ｈ2Ｏ2を含む氷に投入した。次いで、混合溶液のｐＨが７．０になるまで、Ｆｉｓｈｅｒｂｒａｎｄ（登録商標）透析管　ＭＷＣＯ　６０００を用いて透析を行った。
（ｃ）精製後、Ｏｍｎｉｐｏｒｅ（登録商標）　ｍｅｍｂｒａｎｅ　ｆｉｌｔｅｒｓ（０．１μｍ）を用いて混合溶液をろ過し、グラファイトオキサイドパウダーを得るために室温で乾燥した。
（ｄ）３０－４０ｍｇのグラファイトオキサイドパウダーを、吸引条件化でガラスアンプル中に密封し、炎源を用いて急速加熱した。当該熱剥離プロセス（ｔｈｅｒｍａｌ　ｅｘｆｏｌｉａｔｉｏｎ　ｐｒｏｃｅｓｓ）により、グラファイトオキサイドパウダーとグラフェンオキサイドパウダーの混合パウダーであるＧＯを得た。
（ｅ）混合パウダーを蒸留水で希釈し、６０分間超音波処理を行った。次いで、４０００ｒｐｍで２０分間、遠心分離を行って沈殿物を除去した。そして、得られた溶液を乾燥させることでＧＯを得た。（ｅ）で得られたＧＯは、（ｄ）で得られた混合パウダーより、グラフェンオキサイドの含有比が高い。
（２）２５０ｍｌビーカーに、水を９０ｍｌ及び上記（１）で合成したＧＯを４．５ｇ入れて攪拌した。
（３）ビーカーを氷中に入れ、冷却した上記（２）の溶液中に、ＮａＯＨ（和光純薬工業株式会社製）を６ｇ及びＵｒｅａ（和光純薬工業株式会社製）を４ｇ添加して攪拌した。約１０分程度の静置後、セルロース（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）を４ｇ添加して攪拌後、約１０分程度静置した。
（４）次いで、上記（３）の溶液を－２０℃の冷凍庫で２時間冷却し、常温に戻した後、５％ＨＮＯ3の中にピペットを用いて滴下し、約１日静置した。
（５）５％ＨＮＯ3を取り除き、純水で洗浄することで、実施例１のＧＯとセルロースを含む粒子を作製した。実施例１で作製した粒子のＧＯの比は、５３％であった。 [Production of particles with various GO ratios]
<Example 1>
The particles of the present invention were produced by the steps shown in the following (1) to (5).
(1) First, GO was manufactured by the following Marcano's method.
(A) A mixture of 360 ml of H 2 SO 4 and 40 ml of H 3 PO 4 was mixed with 3.0 g of flake graphite (graphite flakes; manufactured by Sigma-Aldrich) and 18.0 g of KMnO 4 (Wako Pure Chemical Industries, Ltd.) The resulting mixture was heated to 30-40 ° C.
(B) Next, the mixture obtained in the above (a) was heated to 50 ° C. and stirred for 12 hours. After stirring, the mixture was cooled to room temperature and poured into ice containing 30% H 2 O 2 . Next, dialysis was performed using a Fisherbrand (registered trademark) dialysis tube MWCO 6000 until the pH of the mixed solution reached 7.0.
(C) After purification, the mixed solution was filtered using Omnipore (registered trademark) membrane filters (0.1 μm) and dried at room temperature to obtain graphite oxide powder.
(D) 30-40 mg of graphite oxide powder was sealed in a glass ampoule under suction conditions and rapidly heated using a flame source. Through the thermal exfoliation process, GO, which is a mixed powder of graphite oxide powder and graphene oxide powder, was obtained.
(E) The mixed powder was diluted with distilled water and sonicated for 60 minutes. Subsequently, the precipitate was removed by centrifugation at 4000 rpm for 20 minutes. And GO was obtained by drying the obtained solution. The GO obtained in (e) has a higher graphene oxide content ratio than the mixed powder obtained in (d).
(2) In a 250 ml beaker, 90 ml of water and 4.5 g of GO synthesized in the above (1) were added and stirred.
(3) Put a beaker in ice and add 6 g of NaOH (manufactured by Wako Pure Chemical Industries, Ltd.) and 4 g of Urea (manufactured by Wako Pure Chemical Industries, Ltd.) to the cooled solution of (2) above and stir. did. After standing for about 10 minutes, 4 g of cellulose (manufactured by Sigma-Aldrich) was added and stirred, and then allowed to stand for about 10 minutes.
(4) Next, the solution of (3) was cooled in a freezer at −20 ° C. for 2 hours, returned to room temperature, dropped into 5% HNO 3 using a pipette, and allowed to stand for about 1 day.
(5) Particles containing GO and cellulose of Example 1 were prepared by removing 5% HNO 3 and washing with pure water. The GO ratio of the particles produced in Example 1 was 53%.
　図１（Ａ）は、実施例１で作製した粒子の写真で、ＧＯとセルロースが混合した黒色の長径が約３ｍｍの楕円であった。 FIG. 1 (A) is a photograph of the particles produced in Example 1, which was an ellipse with a black major axis of about 3 mm mixed with GO and cellulose.
　また、実施例１と同様の手順で、ＧＯの比を変えた種々の粒子を作製した。表１は、実施例１も含め、上記工程（２）におけるＧＯの添加量（ｇ）、当該添加量を添加した際のＧＯの比を示す。 In addition, various particles having different GO ratios were prepared in the same procedure as in Example 1. Table 1, including Example 1, shows the added amount (g) of GO in the above step (2) and the ratio of GO when the added amount is added.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
＜比較例１＞
　ＧＯを添加しなかった以外は、実施例１と同様の手順で粒子を作製した。図１（Ｂ）は、比較例１で作製した粒子の写真で、ＧＯが含まれていないので白色の長径が約３ｍｍの楕円の粒子が得られた。 <Comparative Example 1>
Particles were produced in the same procedure as in Example 1 except that GO was not added. FIG. 1B is a photograph of the particles produced in Comparative Example 1. Since GO was not included, elliptical particles having a white major axis of about 3 mm were obtained.
　図２－１及び図２－２は、表１に示す種々のＧＯの比の粒子及び比較例１の粒子のＳＥＭ写真である。ＳＥＭ写真から明らかなように、ＧＯの比を増加すると粒子表面は微細な凹凸が増加したが、６０％を超えるとやや平滑化が観察された。 FIGS. 2-1 and 2-2 are SEM photographs of particles having various GO ratios shown in Table 1 and particles of Comparative Example 1. FIG. As apparent from the SEM photograph, when the GO ratio was increased, fine irregularities increased on the particle surface, but when the ratio exceeded 60%, a slight smoothing was observed.
　図３は、ＧＯの比が、夫々５３％、５６％、６０％、６４％の粒子をピペットの先端で押した写真を示している。何れのＧＯの比でも粒子が形成されることを確認したが、ＧＯの比が６４％の場合、粒子が柔らかく慎重に取り扱いをする必要があった。 FIG. 3 shows a photograph in which particles having a GO ratio of 53%, 56%, 60%, and 64% were pressed with the tip of a pipette, respectively. It was confirmed that particles were formed at any GO ratio. However, when the GO ratio was 64%, the particles were soft and needed to be handled with care.
〔ＤＮＡの抽出実験〕
＜実施例２＞
　下記表２に示すＧＯの比が異なる１３種類の粒子を作製し、各々５個の粒子を１．５ｍｌのエッペンドルフチューブに入れた。次に、エッペンドルフチューブに、ＤＮＡサンプル（Ｓｉｇｍａ　Ｄ７２９０、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製））を２０μｌ、バインディングバッファーとしてＧｕＨＣｌ（Ｓｉｇｍａ　Ｇ４５０５、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）を１８０μｌ添加して攪拌した。次に、７０％エタノールで２回洗浄した後、核酸溶出液（ＡＥ　ｂｕｆｆｅｒ；１０ｍＭ　Ｔｒｉｓ－ＨＣｌ、０．５ｍＭ　ＥＤＴＡ、ｐＨ９．０）を５００μｌ添加し、７０℃に加熱して５分間攪拌することで、実施例１で作製した粒子に吸着したＤＮＡを溶出した。次に、ＤＮＡを含む核酸溶出液の純度を調べる為、ＮａｎｏＤｒｏｐ　ＮＤ－２０００ｃ　ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｅｒ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ，Ｗｉｌｍｉｎｇｔｏｎ，ＤＥ，ＵＳＡ）を用いて、核酸溶出液（１μｌ）の２３０ｎｍ、２６０ｎｍ及び２８０ｎｍの吸光度をマニュアルに従って測定し、２６０ｎｍ／２８０ｎｍ及び２６０ｎｍ／２３０ｎｍ値を計算した。
　また、ＮａｎｏＤｒｏｐ　ＮＤ－２０００ｃ　ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｅｒ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ，Ｗｉｌｍｉｎｇｔｏｎ，ＤＥ，ＵＳＡ）に表示される前記１μｌの核酸溶出液に含まれる核酸量（ｎｇ／μｌ）を５００倍することで、Ｔｏｔａｌ　Ｙｉｅｌｄを求めた。
　また、比較のため、市販の核酸精製用キットであるＱｉａａｍｐ　ＤＮＡ　ｍｉｎｉ　ｋｉｔ（株式会社キアゲン社製）を用い、添付マニュアルにしたがって上記ＤＮＡサンプルの抽出を行い、上記と同様の手順により吸光度を測定し、２６０／２８０ｎｍ及び２６０／２３０ｎｍ値を計算した。
　なお、Ｑｉａａｍｐ　ＤＮＡ　ｍｉｎｉ　ｋｉｔの標準的な核酸溶出液量は２００μｌであることから、核酸量（ｎｇ／μｌ）の値を２００倍することで、Ｔｏｔａｌ　Ｙｉｅｌｄを求めた。Ｔｏｔａｌ　Ｙｉｅｌｄ値を比較することで、核酸の抽出効率を対比することができる。 [DNA extraction experiment]
<Example 2>
Thirteen types of particles having different GO ratios shown in Table 2 below were prepared, and 5 particles each were placed in a 1.5 ml Eppendorf tube. Next, 20 μl of a DNA sample (Sigma D7290, Sigma-Aldrich)) was added to an Eppendorf tube, and 180 μl of GuHCl (Sigma G4505, Sigma-Aldrich) was added as a binding buffer and stirred. Next, after washing twice with 70% ethanol, 500 μl of nucleic acid eluate (AE buffer; 10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0) is added, heated to 70 ° C. and stirred for 5 minutes. Thus, the DNA adsorbed on the particles prepared in Example 1 was eluted. Next, in order to examine the purity of the nucleic acid eluate containing DNA, the absorbance at 230 nm, 260 nm and 280 nm of the nucleic acid eluate (1 μl) was manually measured using NanoDrop ND-2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). And 260nm / 280nm and 260nm / 230nm values were calculated.
The total yield was determined by multiplying the amount of nucleic acid (ng / μl) contained in the 1 μl nucleic acid eluate displayed on the NanoDrop ND-2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) by 500 times.
For comparison, the DNA sample was extracted according to the attached manual using Qiaamp DNA mini kit (Qiagen Co., Ltd.), a commercially available nucleic acid purification kit, and the absorbance was measured by the same procedure as above. 260/280 nm and 260/230 nm values were calculated.
Since the standard nucleic acid eluate volume of Qiaamp DNA mini kit is 200 μl, the total yield was determined by multiplying the value of the amount of nucleic acid (ng / μl) by 200. By comparing the Total Yield value, the nucleic acid extraction efficiency can be compared.
　表２は、ＧＯの比を０％～５３％まで変化した粒子、及びＱｉａａｍｐ　ＤＮＡ　ｍｉｎｉ　ｋｉｔを用いてＤＮＡ抽出を行った時の２６０ｎｍ／２８０ｎｍ及び２６０ｎｍ／２３０ｎｍ値、並びに、核酸溶出液１μｌに含まれる核酸量（ｎｇ）及びＴｏｔａｌ　Ｙｉｅｌｄをまとめたものである。図４（Ａ）は、表２の２６０ｎｍ／２８０ｎｍ値をグラフ化したもので、図４（Ｂ）は、表２の２６０ｎｍ／２３０ｎｍ値をグラフ化したものである。また、図５（Ａ）は、表２の核酸溶出液１μｌに含まれる核酸量（ｎｇ）値をグラフ化したもので、図５（Ｂ）は、表２のＴｏｔａｌ　Ｙｉｅｌｄをグラフ化したものである。 Table 2 includes particles with the GO ratio changed from 0% to 53%, 260 nm / 280 nm and 260 nm / 230 nm values when DNA extraction was performed using Qiaamp DNA mini kit, and 1 μl of nucleic acid eluate. It summarizes the amount of nucleic acid (ng) and Total Yield. 4A is a graph of 260 nm / 280 nm values in Table 2, and FIG. 4B is a graph of 260 nm / 230 nm values in Table 2. FIG. 5 (A) is a graph showing the amount of nucleic acid (ng) contained in 1 μl of the nucleic acid eluate shown in Table 2. FIG. 5 (B) is a graph showing Total Yield in Table 2. is there.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　一般的に、２６０／２８０ｎｍの値が１．８～２．０の範囲内にある場合は、タンパク質の混入が少なくＤＮＡの純度が高いとされている。したがって、ＧＯの比が５％以上であると、ＤＮＡを純度よく抽出できることが明らかとなった。 Generally, when the value of 260/280 nm is in the range of 1.8 to 2.0, it is considered that the purity of DNA is high with little protein contamination. Therefore, it was revealed that DNA can be extracted with high purity when the GO ratio is 5% or more.
　また、一般的に、２６０／２３０ｎｍの値が２．０～２．２の範囲内にある場合は、緩衝塩類等の共雑物が少ない奇麗なＤＮＡとされている。ＧＯの比が３１％以上であると、市販の核酸精製用キットであるＱｉａａｍｐ　ＤＮＡ　ｍｉｎｉ　ｋｉｔを使用した場合より良い結果が得られ、更に、ＧＯの比が４７％以上になると、２６０ｎｍ／２８０ｎｍ及び２６０ｎｍ／２３０ｎｍの何れの値も、望ましいとされている範囲内になることが分かった。実施例２では、ＧＯの比が５３％までしか２６０ｎｍ／２８０ｎｍ及び２６０ｎｍ／２３０ｎｍ値、並びに、核酸溶出液１μｌに含まれる核酸量（ｎｇ）及びＴｏｔａｌ　Ｙｉｅｌｄの測定を行っていないが、図３に示すとおり、ＧＯの比が６４％の時にも粒子が形成できることは確認しているので、同様の効果が期待できる。 Further, generally, when the value of 260/230 nm is in the range of 2.0 to 2.2, it is considered to be a beautiful DNA with few contaminants such as buffer salts. When the GO ratio is 31% or more, better results are obtained when using a commercially available nucleic acid purification kit Qiaamp DNA mini kit, and when the GO ratio is 47% or more, 260 nm / 280 nm and It has been found that any value of 260 nm / 230 nm falls within the desired range. In Example 2, 260 nm / 280 nm and 260 nm / 230 nm values, and the nucleic acid amount (ng) and Total Yield contained in 1 μl of nucleic acid eluate were measured only up to a GO ratio of 53%. As shown, since it has been confirmed that particles can be formed even when the GO ratio is 64%, the same effect can be expected.
　以上の結果より、ＧＯの比を変えることで、抽出後のＤＮＡ純度が変わることが明らかとなり、ＧＯの比が５％～６４％が好ましく、３１％～６４％がより好ましいことが明らかとなった。 From the above results, it is clear that changing the GO ratio changes the DNA purity after extraction, and it is clear that the GO ratio is preferably 5% to 64% and more preferably 31% to 64%. It was.
　また、表２及び図５（Ａ）及び（Ｂ）から明らかなように、ＧＯの比が２０％までは、Ｔｏｔａｌ　Ｙｉｅｌｄの増加は見られなかったが、それ以降は、ＧＯの比の増加とともに、Ｔｏｔａｌ　Ｙｉｅｌｄが増加した。また、ＧＯの比が４７％で、市販のＱｉａａｍｐ　ＤＮＡ　ｍｉｎｉ　ｋｉｔのＴｏｔａｌ　Ｙｉｅｌｄに近い値となり、更にＧＯの比を大きくすることで、Ｑｉａａｍｐ　ＤＮＡ　ｍｉｎｉ　ｋｉｔのＴｏｔａｌ　Ｙｉｅｌｄ値より大きな値が得られた。 As is clear from Table 2 and FIGS. 5 (A) and (B), the Total Yield did not increase until the GO ratio was 20%, but thereafter, the GO ratio increased. Total Yield increased. Moreover, the GO ratio was 47%, which was close to the total yield of the commercially available Qiaamp DNA mini kit, and by further increasing the GO ratio, a value greater than the Total Yield value of the Qiaamp DNA mini kit was obtained.
　以上の結果より、セルロースに対するＧＯの比を変えることで、サンプル中のＤＮＡ抽出後のＴｏｔａｌ　Ｙｉｅｌｄが変わることが明らかとなり、ＧＯの比は２０％～６４％が好ましく、４７％～６４％がより好ましい。 From the above results, it becomes clear that changing the ratio of GO to cellulose changes the total yield after DNA extraction in the sample, and the GO ratio is preferably 20% to 64%, more preferably 47% to 64%. preferable.
〔多様なサンプルからのＤＮＡの抽出〕
＜実施例３＞
　実施例２のサンプルＤＮＡに代え、サンプルとして、喫煙済みタバコのフィルター部分の紙を１ｃｍ×１ｃｍ、噛んだ後のチューイングガムを１５ｍｇ、ドライイーストを２０ｍｇ、鳥の胸肉１５ｍｇを用いた以外は、実施例２と同様の手順でＴｏｔａｌ　Ｙｉｅｌｄを求めた。 [Extraction of DNA from various samples]
<Example 3>
Except that the sample DNA of Example 2 was replaced by 1 cm × 1 cm of the filter part of the smoked tobacco filter, 15 mg of chewing gum after chewing, 20 mg of dry yeast, and 15 mg of chicken breast were used as samples. Total Yield was determined in the same procedure as in Example 2.
＜比較例２＞
　主に犯罪捜査用に用いられ、サンプル中に含まれるＤＮＡを高感度で検出できるＱＩＡａｍｐ　ＤＮＡ　Ｉｎｖｅｓｔｉｇａｔｏｒ　Ｋｉｔ－ＱＩＡａｍｐ　ＭｉｎＥｌｕｔｅ　ｃｏｌｕｍｎｓ（キアゲン社製）を用い、マニュアルに従って実施例３と同様のサンプルのＴｏｔａｌ　Ｙｉｅｌｄを求めた。 <Comparative example 2>
Using QIAamp DNA Investigator Kit-QIAamp MinElute columns (Qiagen), which is mainly used for criminal investigations and can detect DNA contained in a sample with high sensitivity, a total yield of the same sample as in Example 3 was obtained according to the manual. Asked.
　図６は、実施例３及び比較例２の結果を表すグラフである。図６に示すように、本発明の粒子（ＧＯの比は５３％）を用いると、犯罪捜査に用いられる高感度のＤＮＡ検出キットであるＱＩＡａｍｐ　ＤＮＡ　Ｉｎｖｅｓｔｉｇａｔｏｒ　Ｋｉｔ－ＱＩＡａｍｐ　ＭｉｎＥｌｕｔｅ　ｃｏｌｕｍｎｓより、多様なサンプル中のＤＮＡを高感度で抽出することができた。また、実施例２で用いたサンプルＤＮＡは一本鎖であるが、実施例３のＤＮＡは２本鎖である。したがって、本発明の粒子は、一本鎖及び２本鎖の何れの核酸も抽出できることが確認できた。 FIG. 6 is a graph showing the results of Example 3 and Comparative Example 2. As shown in FIG. 6, when using the particles of the present invention (GO ratio is 53%), QIAamp DNA Investigator Kit-QIAamp MinElute columns is a highly sensitive DNA detection kit used in criminal investigations. Was extracted with high sensitivity. The sample DNA used in Example 2 is single-stranded, but the DNA of Example 3 is double-stranded. Therefore, it was confirmed that the particles of the present invention can extract both single-stranded and double-stranded nucleic acids.
〔粒子のリサイクル能の確認〕
＜実施例４＞
　次に、実施例２でＤＮＡ抽出実験を行った粒子（ＧＯの比は５３％）のリサイクル能の確認を行った。
　粒子のリサイクル能は、実施例２と同様の条件でＤＮＡ抽出実験を行った粒子を多数準備し、５００μｌのＡＥ　ｂｕｆｆｅｒで1回洗浄、ＡＥ　ｂｕｆｆｅｒで２回洗浄、２Ｍ　ＨＣｌで１時間、２時間、４時間、８時間、１２時間処理した粒子を得た。次いで、各粒子にＡＥ　ｂｕｆｆｅｒを５００μｌ添加し、７０℃に加熱して５分間攪拌することで、粒子表面に残留していたＤＮＡを溶出し、溶出液に含まれるＤＮＡ量をＱｕｂｉｃ２．０（ライフテクノロジー社製）で検出した。表３は、Ｑｕｂｉｃ２．０の検出値（ｎｇ／ｍｌ）を示している。 [Confirmation of particle recycling ability]
<Example 4>
Next, the recyclability of the particles (GO ratio 53%) subjected to the DNA extraction experiment in Example 2 was confirmed.
The particle recycling ability was prepared by preparing a large number of particles subjected to DNA extraction experiments under the same conditions as in Example 2, washing once with 500 μl AE buffer, washing twice with AE buffer, 1 hour, 2 hours with 2M HCl. Particles treated for 4, 8 and 12 hours were obtained. Next, 500 μl of AE buffer was added to each particle, heated to 70 ° C. and stirred for 5 minutes to elute the DNA remaining on the particle surface, and the amount of DNA contained in the eluate was determined to be Quick 2.0 (life Technology). Table 3 shows the detection value (ng / ml) of Quick 2.0.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表３から明らかなように、粒子表面に吸着したＤＮＡをＡＥ　ｂｕｆｆｅｒで溶出した後に、再度粒子をＡＥ　ｂｕｆｆｅｒで洗浄しても、粒子表面からＤＮＡが検出された。一方、ＨＣｌで１時間以上処理すると、粒子表面の残留ＤＮＡの量がＱｕｂｉｃ２．０の検出限界値より低いことを表す「＜０．１」となった。 As apparent from Table 3, DNA was detected from the particle surface even when the DNA adsorbed on the particle surface was eluted with AE buffer and then the particle was washed again with AE buffer. On the other hand, when treated with HCl for 1 hour or longer, the amount of residual DNA on the particle surface was “<0.1” indicating that it was lower than the detection limit value of Quick 2.0.
　次に、ＨＣｌ処理した粒子の核酸抽出能について、以下の手順で確認を行った。
（１）混合溶液中のＧＯの比が５３％の粒子を準備し、上記実施例２と同様の手順でＤＮＡサンプルからＤＮＡの抽出を行い、２６０ｎｍ／２８０ｎｍ及び２６０ｎｍ／２３０ｎｍ値を計算した。また、１μｌの核酸溶出液に含まれる核酸量（ｎｇ／μｌ）を５００倍することで、Ｔｏｔａｌ　Ｙｉｅｌｄを求めた。
（２）２Ｍ　ＨＣｌで２時間処理した後の粒子表面に残留しているＤＮＡの量を上記と同様の手順で測定した。
（３）上記（２）でＨＣｌ処理した粒子を用いた以外は、上記（１）と同様の手順で２６０ｎｍ／２８０ｎｍ及び２６０ｎｍ／２３０ｎｍ値を計算した。また、１μｌの核酸溶出液に含まれる核酸量（ｎｇ／μｌ）を５００倍することで、Ｔｏｔａｌ　Ｙｉｅｌｄを求めた。
　上記（３）の２６０ｎｍ／２８０ｎｍ及び２６０ｎｍ／２３０ｎｍ値、並びにＴｏｔａｌ　Ｙｉｅｌｄの値は、何れも（１）の値の約９９％であった。以上の結果より、本発明の粒子は、リサイクルが可能であることが明らかとなった。 Next, the nucleic acid extraction ability of HCl-treated particles was confirmed by the following procedure.
(1) Particles having a GO ratio of 53% in the mixed solution were prepared, DNA was extracted from the DNA sample in the same procedure as in Example 2 above, and 260 nm / 280 nm and 260 nm / 230 nm values were calculated. Further, the total yield was determined by multiplying the amount of nucleic acid (ng / μl) contained in 1 μl of nucleic acid eluate by 500 times.
(2) The amount of DNA remaining on the particle surface after 2 hours of treatment with 2M HCl was measured by the same procedure as described above.
(3) 260 nm / 280 nm and 260 nm / 230 nm values were calculated in the same procedure as in (1) except that the particles treated with HCl in (2) were used. Further, the total yield was determined by multiplying the amount of nucleic acid (ng / μl) contained in 1 μl of nucleic acid eluate by 500 times.
The values of 260 nm / 280 nm and 260 nm / 230 nm of the above (3) and the value of Total Yield were all about 99% of the value of (1). From the above results, it became clear that the particles of the present invention can be recycled.
〔Ｆｅ3Ｏ4を含んだ粒子の作製〕
＜実施例５＞
　上記実施例１の（３）の工程の際に、Ｆｅ3Ｏ4を０．３ｇ～４．５ｇを加えた以外は、実施例１と同様の手順でＦｅ3Ｏ4を含む粒子を作製した。実施例５で作製した粒子の配合比を表４に示す。 [Preparation of particles containing Fe 3 O 4 ]
<Example 5>
Particles containing Fe 3 O 4 were prepared in the same procedure as in Example 1 except that 0.3 g to 4.5 g of Fe 3 O 4 was added in the step (3) of Example 1 above. . Table 4 shows the mixing ratio of the particles produced in Example 5.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　図７（Ａ）の左側はＣＧＭ０、右側はＣＧＭ４５の写真で、直径約２．６ｍｍの略球形であった。また、図７（Ｂ）はＣＧＭ０を純水に分散した容器に磁石を近付けた写真、図７（Ｃ）はＣＧＭ４５を純水に分散した容器に磁石を近付けた写真である。図７（Ｂ）及び（Ｃ）から明らかなように、Ｆｅ3Ｏ4を含む粒子（ＣＧＭ４５）は磁石を近付けると磁石に引き寄せられた。したがって、粒子がＦｅ3Ｏ4を含む場合、磁石を用いて核酸を吸着した粒子を回収できるので、操作の利便性が向上する。 The left side of FIG. 7A is a photograph of CGM0 and the right side is CGM45, which is a substantially spherical shape having a diameter of about 2.6 mm. FIG. 7B is a photograph of a magnet brought close to a container in which CGM0 is dispersed in pure water, and FIG. 7C is a photograph of a magnet brought close to a container in which CGM45 is dispersed in pure water. As apparent from FIGS. 7B and 7C, the particles (CGM45) containing Fe 3 O 4 were attracted to the magnet when approaching the magnet. Therefore, when the particles contain Fe 3 O 4 , the particles adsorbed with the nucleic acid can be collected using a magnet, so that the convenience of operation is improved.
　図８（Ａ）はＣＧＭ４５のＳＥＭ写真、図８（Ｂ）～（Ｄ）はＣＧＭ４５粒子表面をエネルギー分散型Ｘ線分析装置（ＥＤＸ）で解析したマッピングで、図８（Ｂ）は炭素元素、図８（Ｃ）は酸素元素、図８（Ｄ）はＦｅ元素の分散を表している。図８（Ｂ）～（Ｄ）の写真から明らかなように、実施例５で作製した粒子の中では、ＧＯ、Ｆｅ3Ｏ4が均一に分散していることを確認した。 8A is a SEM photograph of CGM45, FIGS. 8B to 8D are mappings obtained by analyzing the CGM45 particle surface with an energy dispersive X-ray analyzer (EDX), FIG. 8B is a carbon element, 8C shows the oxygen element, and FIG. 8D shows the dispersion of the Fe element. As is clear from the photographs in FIGS. 8B to 8D, it was confirmed that GO and Fe 3 O 4 were uniformly dispersed in the particles produced in Example 5.
　次に、実施例５で作製した粒子を用い、実施例２のバインディングバッファー（ＧｕＨＣｌ）に代え純水を用いた以外は、実施例２と同様の手順でＤＮＡの抽出実験を行い、核酸量（ｎｇ／μｌ）及びＴｏｔａｌ　Ｙｉｅｌｄを求めた。図９は、各サンプルの核酸量（ｎｇ／μｌ）及びＴｏｔａｌ　Ｙｉｅｌｄのグラフである。図９から明らかなように、ＧＯとセルロースの粒子にＦｅ3Ｏ4を添加すると、添加量の増加に伴い核酸抽出効率が向上した。これは、Ｆｅ3Ｏ4が粒子内でプラスに帯電するため、マイナスに帯電している核酸を引き寄せ、そして、ＧＯの芳香環と核酸の芳香環とがπ－πスタキングにより相互作用したためと考えられる。 Next, a DNA extraction experiment was performed in the same procedure as in Example 2 except that pure water was used instead of the binding buffer (GuHCl) in Example 2 using the particles prepared in Example 5, and the amount of nucleic acid ( ng / μl) and Total Yield. FIG. 9 is a graph of the amount of nucleic acid (ng / μl) and Total Yield of each sample. As is apparent from FIG. 9, when Fe 3 O 4 was added to GO and cellulose particles, the nucleic acid extraction efficiency was improved as the amount added was increased. This is thought to be because Fe 3 O 4 is positively charged in the particle, attracting the negatively charged nucleic acid, and the aromatic ring of GO and the aromatic ring of nucleic acid interact by π-π stacking. It is done.
　なお、実施例５では、バインディングバッファー（ＧｕＨＣｌ）に代え純水を用いたため、表２に示す核酸抽出効率より低い値となっている。ところで、一般的に用いられるバインディングバッファーは、グアニジニウムイオン、尿素、ヨウ化物イオン等のカオトロピック塩を含み、更に、核酸の抽出効率を上げるためにＮａ+を添加する場合がある。しかしながら、抽出した核酸溶液にＮａ+やカオトロピック塩を含まれていると、ＰＣＲで核酸を増幅する際の阻害要因となる。したがって、抽出した核酸をＰＣＲにより増幅する場合、実施例５に示すＧＯ＋セルロースにＦｅ3Ｏ4を添加した粒子を用いると、Ｎａ+やカオトロピック塩を除去する工程が不要であることから、核酸抽出用の粒子として非常に有用である。 In Example 5, since pure water was used instead of the binding buffer (GuHCl), the value was lower than the nucleic acid extraction efficiency shown in Table 2. By the way, generally used binding buffers contain chaotropic salts such as guanidinium ions, urea, iodide ions, and Na + may be added to increase the extraction efficiency of nucleic acids. However, if the extracted nucleic acid solution contains Na + or a chaotropic salt, it becomes an inhibiting factor when the nucleic acid is amplified by PCR. Therefore, when the extracted nucleic acid is amplified by PCR, the use of particles obtained by adding Fe 3 O 4 to GO + cellulose shown in Example 5 eliminates the need for a step of removing Na + or chaotropic salt. It is very useful as a particle.
〔Ｆｅ2Ｏ3を含んだ粒子の作製〕
＜実施例６＞
　Ｆｅ3Ｏ4に代えＦｅ2Ｏ3を用い、表５に示す配合比にした以外は、実施例５と同様の手順で粒子を作製し、核酸量（ｎｇ／μｌ）及びＴｏｔａｌ　Ｙｉｅｌｄを求めた。 [Preparation of particles containing Fe 2 O 3 ]
<Example 6>
Particles were prepared in the same procedure as in Example 5 except that Fe 2 O 3 was used instead of Fe 3 O 4 and the mixing ratio was as shown in Table 5, and the amount of nucleic acid (ng / μl) and total yield were determined. .
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　図１０は、各サンプルの核酸量（ｎｇ／μｌ）及びＴｏｔａｌ　Ｙｉｅｌｄのグラフである。図１０から明らかなように、Ｆｅ3Ｏ4よりはやや劣るものの、Ｆｅ2Ｏ3を粒子中に添加することで、核酸の抽出効率は向上した。上記実施例５及び６の結果から、ＧＯ＋セルロース粒子に酸化鉄を加えることで核酸の抽出効率が向上し、また、バインディングバッファーを用いなくても核酸を抽出できることが明らかとなった。なお、粒子中に分散した酸化鉄がプラスに帯電することでマイナスに帯電する核酸を引き寄せると考えられることから、酸化鉄に限らず、粒子又は核酸抽出用組成物中でプラスに帯電する物質であれば同等の効果を奏すると考えられる。 FIG. 10 is a graph of the amount of nucleic acid (ng / μl) and Total Yield of each sample. As is clear from FIG. 10, although slightly inferior to Fe 3 O 4 , nucleic acid extraction efficiency was improved by adding Fe 2 O 3 to the particles. From the results of Examples 5 and 6 above, it was revealed that the nucleic acid extraction efficiency was improved by adding iron oxide to GO + cellulose particles, and the nucleic acid could be extracted without using a binding buffer. In addition, since it is thought that the negatively charged nucleic acid is attracted when the iron oxide dispersed in the particles is positively charged, it is not limited to iron oxide, and is a substance that is positively charged in the composition for extracting particles or nucleic acids. If there is, it is thought that there is an equivalent effect.
　本発明の粒子又は核酸抽出用組成物を用いることで、従来の核酸抽出用キットを用いた場合と比較して、多様なサンプルから核酸を高効率で抽出することができる。また、本発明の粒子又は核酸抽出用組成物は、リサイクルが可能である。
　したがって、大学、企業、研究機関等において、多様なサンプルから核酸の抽出に有用である。 By using the particle or nucleic acid extraction composition of the present invention, nucleic acids can be extracted from a variety of samples with higher efficiency than when using a conventional nucleic acid extraction kit. In addition, the particle or nucleic acid extraction composition of the present invention can be recycled.
Therefore, it is useful for extracting nucleic acids from various samples in universities, companies, research institutions and the like.

Claims (8)

  1. 　グラフェンオキサイド及び／又はグラファイトオキサイド、並びにセルロースを含む粒子。 Particles containing graphene oxide and / or graphite oxide and cellulose.
  2. 　前記粒子における、セルロースに対するグラフェンオキサイド及び／又はグラファイトオキサイドの比が、４７％～６４％である請求項１に記載の粒子。 The particle according to claim 1, wherein a ratio of graphene oxide and / or graphite oxide to cellulose in the particle is 47% to 64%.
  3. 　磁性体及び／又は粒子中でプラスに帯電する物質を更に含む請求項１又は２に記載の粒子。 The particle according to claim 1 or 2, further comprising a magnetic substance and / or a substance that is positively charged in the particle.
  4. 　グラフェンオキサイド及び／又はグラファイトオキサイド、並びにセルロースを含む固体状の核酸抽出用組成物。 A solid nucleic acid extraction composition containing graphene oxide and / or graphite oxide and cellulose.
  5. 　前記核酸抽出用組成物における、セルロースに対するグラフェンオキサイド及び／又はグラファイトオキサイドの比が、４７％～６４％である請求項４に記載の核酸抽出用組成物。 The composition for nucleic acid extraction according to claim 4, wherein the ratio of graphene oxide and / or graphite oxide to cellulose in the nucleic acid extraction composition is 47% to 64%.
  6. 　磁性体及び／又は組成物中でプラスに帯電する物質を更に含む請求項４又は５に記載の核酸抽出用組成物。 The nucleic acid extraction composition according to claim 4 or 5, further comprising a magnetic substance and / or a substance that is positively charged in the composition.
  7. 　請求項１～３の何れか一項に記載の粒子、請求項４～６の何れか一項に記載の核酸抽出用組成物の何れかに、核酸を含む溶液を混合して核酸を前記粒子又は核酸抽出用組成物に吸着する工程、
    　前記核酸が吸着した前記粒子又は核酸抽出用組成物に核酸溶出液を混合することで、前記粒子又は核酸抽出用組成物に吸着した核酸を分離する核酸抽出工程、
    を含む核酸抽出方法。     A particle containing the nucleic acid is mixed with the particle according to any one of claims 1 to 3 or the composition for nucleic acid extraction according to any one of claims 4 to 6 to mix the nucleic acid with the particle. Or a step of adsorbing to a composition for nucleic acid extraction,
    A nucleic acid extraction step of separating nucleic acid adsorbed on the particles or nucleic acid extraction composition by mixing a nucleic acid eluate with the particles or nucleic acid extraction composition on which the nucleic acids have been adsorbed;
    A nucleic acid extraction method comprising:
  8. 　請求項７に記載の核酸抽出工程後の前記粒子又は核酸抽出用組成物を、核酸クリーニング液で処理して前記粒子又は核酸抽出用組成物の表面に残留している核酸を除去する表面回復工程、
    を含む前記粒子又は核酸抽出用組成物のリサイクル方法。
          A surface recovery step of removing the nucleic acid remaining on the surface of the particle or nucleic acid extraction composition by treating the particle or nucleic acid extraction composition after the nucleic acid extraction step according to claim 7 with a nucleic acid cleaning solution. ,
    A method for recycling the particle or nucleic acid extraction composition comprising:
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