Nucleic acid binding particle and method for producing nucleic acid binding particle

Abstract

Nucleic acid binding particles according to the present invention each have at least a portion of the surface covered with silica, have a sphericity of 0.86 or more as determined from formula (A), and have silanol groups adjacent to the surface. (A): [Sphericity] = particle diameter (nm) determined through image analysis / BET diameter (nm)

Description

Nucleic acid binding particles and method for producing nucleic acid binding particles

[0001] The present invention relates to particles for binding nucleic acids and a method for producing such particles. This application claims priority to Japanese Patent Application No. 2023-207662, filed December 8, 2023, the contents of which are incorporated herein by reference.

[0002] In recent years, with the improvement in the detection sensitivity of equipment, the spread of next-generation sequencers, and improvements in computer performance, methods for visualizing and quantifying gene and protein expression in individual cells in biological tissue and analyzing them together with their location within the tissue have become widely used.

[0003] Due to the above-mentioned techniques, there is an increasing demand for efficient recovery of nucleic acids from samples containing nucleic acids and genome analysis. For example, magnetic particles are known that allow nucleic acids to be adsorbed onto magnetic particles and then recovered using magnetic force to efficiently obtain nucleic acids (see, for example, Patent Document 1).

[0004] The nucleic acid recovered by the method described in Patent Document 1 may be a nucleic acid of a living organism or a synthetic nucleic acid. That is, the "sample containing nucleic acid" may be a biological sample containing nucleic acid of a living organism or a sample containing synthetic nucleic acid.

[0005] As used herein, the term "biological sample" refers to a sample derived from a living organism and containing nucleic acid from that organism. "Nucleic acid from a living organism" refers to both DNA and RNA contained in the cells of that organism. The nucleic acid may be the DNA or RNA itself contained in the cells, or may be a biopolymer in which part of the DNA or RNA is missing.

[0006] Japanese Patent Application Laid-Open No. 2023-046202

[0007] The above-mentioned analytical method is attracting attention as a technology that can lead to the early detection of diseases such as early-stage cancer by analyzing the genomes contained in minute tissues. From this perspective, when considering highly efficient extraction of nucleic acids, there is still room for improvement in the particles used to extract nucleic acids.

[0008] The present invention has been made in view of the above circumstances, and aims to provide nucleic acid binding particles that enable efficient recovery of nucleic acids from samples, and a method for easily producing such nucleic acid binding particles.

[0009] One aspect of the present invention is a particle for nucleic acid binding, at least a portion of whose surface is covered with silica, and the particle has vicinal silanol groups on the surface and a sphericity of 0.86 or more as determined by the following formula (A): [Sphericity] = particle diameter (nm) determined by image analysis ÷ BET diameter (nm) ... (A)

[0010] One aspect of the present invention is a method for producing particles for nucleic acid binding, which comprises a step of treating particles whose surfaces are covered with silica to increase the number of vicinal silanol groups, and controlling the evaluation value for the hydrophilicity of the surface, calculated from the following formula (B), to 0.35 or more: S=α Ｈ２Ｏ / α Ｎ２ ...(B) S: Evaluation value α regarding the hydrophilicity of the particle surface Ｈ２Ｏ α: BET specific surface area of ​​the combined particles determined by water vapor adsorption measurement Ｎ２ : BET specific surface area of ​​bonded particles determined by nitrogen adsorption measurement

[0011] According to the present invention, it is possible to provide particles for nucleic acid binding that enable efficient recovery of nucleic acids from biological samples, and a method for producing such particles for nucleic acid binding that enables easy production of such particles for nucleic acid binding.

[0012] Fig. 1 is an explanatory diagram illustrating a binding particle 100. Fig. 2 is a flowchart showing a method for manufacturing a particle for nucleic acid binding. Fig. 3 is a flowchart showing a method for extracting nucleic acid using a particle for nucleic acid binding. Fig. 4 is a graph showing the evaluation results. Fig. 5 is a graph showing the evaluation results. Fig. 6 is a graph showing the evaluation results.

[0013] The nucleic acid binding particles, the method for producing the nucleic acid binding particles, and the method for extracting nucleic acid according to this embodiment will be described below with reference to Figures 1 to 3. Note that in all of the following figures, the dimensions and proportions of each component have been appropriately changed to make the drawings easier to understand.

[0014] <<Nucleic Acid Binding Particles>> The nucleic acid binding particles of this embodiment are particles at least partially covered with silica and have adjacent silanol groups on the surface. In the following description, the nucleic acid binding particles may be simply referred to as "binding particles."

[0015] The binding particles are used in the "Boom method," a method for isolating nucleic acids from biological samples. The Boom method utilizes the solid-phase adsorption of nucleic acids to silica beads (binding particles) in the presence of a chaotropic substance, and isolates nucleic acids by adsorbing the nucleic acids to the silica beads in a biological sample containing nucleic acids and impurities and separating the nucleic acids together with the beads from the biological sample.

[0016] The Boom method using the binding particles of this embodiment refers to the Boom method in a broad sense, including the operation of adsorbing nucleic acids onto silica beads and recovering the nucleic acids, and is not limited to the original (narrow sense) Boom method. For example, it may be a modified version of the original Boom method, in which the method of washing the silica beads or the method of separating the nucleic acids bound to the silica beads are modified.

[0017] Many nucleic acid binding particles are commercially available as products that apply this principle. Among them, as described in Patent Document 1, nucleic acid binding particles that are manufactured from a magnetic material and thereby enable control of the behavior of the beads by magnetic force have been proposed, manufactured, and sold.

[0018] The inventors focused on the surface of binding particles in order to recover nucleic acids more efficiently than conventional nucleic acid binding particles. After extensive investigation, the inventors focused on silanol groups exposed on the particle surface, which are thought to contribute to the adsorption of nucleic acids, and discovered that binding particles having adjacent silanol groups on their surfaces are highly effective, thereby completing the invention.

[0019] As will be described in detail later, the nucleic acid binding particles of this embodiment have an appropriate surface shape, which makes it easy to form vicinal silanols, and as a result, the nucleic acid binding particles of this embodiment can easily adsorb (capture) nucleic acids by the vicinal silanol groups on the particle surface, making it possible to preferably recover nucleic acids.

[0020] [Vicinal silanol groups] Fig. 1 is an explanatory diagram illustrating a bonded particle 100. The surface of the bonded particle 100 is covered with silica. Fig. 1 shows the bonded particle 100 as being entirely made of silica.

[0021] Figure 1 illustrates the silanol groups of bonded particles 100. As shown in Figure 1, the silica covering the surface of bonded particles 100 has silanol groups. These silanol groups can be divided into surface silanol groups (silanol groups A) present on the surface of the particles and internal silanol groups (silanol groups B) present inside the particles. The surface silanol groups can be further divided into adjacent silanol groups A1 and isolated silanol groups A2.

[0022] Here, "vicinal silanol groups" refers to hydroxyl groups present in the silica (particle surface) covering the surface of the particle, and bonded to two adjacent silicon atoms via oxygen atoms in a siloxane bond (Si-O-Si).

[0023] Furthermore, an "isolated silanol group" refers to a hydroxyl group bonded to a silicon atom present on the particle surface, and the adjacent silicon atom bonded to the silicon atom via oxygen does not have a hydroxyl group exposed on the particle surface.

[0024] At least a portion of the surface of the bonded particle 100 is covered with silica. That is, at least a portion of the surface of the bonded particle 100 is made of silica, and the silica is exposed on the surface. The silica exposed on the surface of the bonded particle 100 has vicinal silanol groups A1. The vicinal silanol groups A1 are silanol groups that are directly bonded to the silica that constitutes the surface of the bonded particle. In this sense, a configuration in which the surface of a silica particle is modified with an organic group and the organic group has a silanol group at the tip of the organic group cannot be said to have vicinal silanol groups A1, and does not fall under the category of the bonded particle of this embodiment.

[0025] Whether the silanol groups on the particle surface are vicinal silanol groups A1 or isolated silanol groups A2 can be confirmed by Raman spectroscopy. In this embodiment, "having vicinal silanol groups on the surface" means "the peaks of vicinal silanol groups can be confirmed in the Raman spectrum."

[0026] (Raman Spectroscopic Analysis) Raman spectroscopic analysis can be carried out using a known Raman spectroscopic analyzer (for example, Raman microscope inVia Reflex, manufactured by Renishaw).

[0027] When using the above Raman microscope, a laser with an excitation wavelength of 532 nm is used, and the measurement range is 690 cm －１ From 4000 cm －１ The Raman shift is 520.5 cm from single crystal Si. －１ The obtained spectrum is subjected to baseline correction and peak separation using the data processing software WiRE4.2 attached to the instrument. The peak intensity is approximately 800 cm originating from the Si-O skeleton. －１ The Raman peak intensity is normalized using the Raman peak intensity of

[0028] In the obtained Raman spectrum, the peak of the vicinal silanol group is at 3610 cm －１ , the peak of the isolated silanol group is 3750 cm －１ appears in.

[0029] [Particle Surface Condition 1: Sphericity] Assuming a model in which "nucleic acids bind to adjacent silanol groups on the surface of a bound particle" as described above, it is natural to assume that increasing the surface area is preferable to increase the amount of nucleic acid bound. Generally, to increase the surface area, particles with uneven surfaces or porous particles are considered preferable. However, in the case of the bound particles of this embodiment, contrary to this expectation, it was found that a smooth surface and a shape approaching a perfect sphere are preferable. In other words, the bound particles are not porous and have a particle shape that is close to a perfect sphere.

[0030] The shape of the combined particles is evaluated using the sphericity calculated by the following formula (A). The greater the sphericity, the closer the shape of the combined particles is to a perfect sphere. [Sphericity] = particle diameter (nm) calculated by image analysis ÷ BET diameter (nm) (A)

[0031] The BET diameter of the bonded particles is α Ｎ２ The BET specific surface area of ​​the bonded particles determined by nitrogen adsorption measurement and the density of the bonded particles can be calculated from the following formula: [BET diameter]=6 / α Ｎ２ × (density of bound particles)

[0032] The BET specific surface area is a value measured by the BET (Brunauer, Emmett, Teller) method, which is a gas adsorption method. The specific surface area by the gas adsorption method can be measured using a known gas adsorption measuring device (e.g., BELSORP mini II or BELSORP MAX II, both manufactured by Microtrac BEL Co., Ltd.).

[0033] α Ｎ２ When measuring the adsorbate, nitrogen (for example, nitrogen (G1 grade, manufactured by Taiyo Nippon Sanso Corporation)) is used as the adsorbate.

[0034] Prior to measurement, the bound particles to be measured are subjected to vacuum degassing treatment overnight at 100°C using a known pretreatment device (e.g., Belprep vac II, manufactured by Microtrac-Bell Co., Ltd.) to remove moisture physically adsorbed on the surface.

[0035] The particle diameter determined by image analysis of the bound particles is a value measured using a known measuring device such as a scanning electron microscope. As an example, the SEM image can be an image taken using a scanning electron microscope (Scanning Electron Microscope S-3400N, Hitachi High-Technologies Corporation) at an accelerating voltage of 3 kV and a magnification of 10,000 times. The particle diameter can be automatically detected using software (e.g., MacView Version 4, manufactured by Mountech Co., Ltd.) that calculates the particle diameter based on the obtained SEM image.

[0036] Therefore, when the average particle size of the bound particles is the same, it is preferable that the particles have a sphericity close to 1.

[0037] The sphericity of the combined particles is preferably 0.86 or more, more preferably 0.88 or more. The ideal upper limit of the sphericity is "1".

[0038] The average particle size of the binding particles is preferably 0.1 μm or more and 1000 μm or less. Even if the average particle size of the binding particles exceeds 1000 μm, the effect of the invention of efficiently recovering nucleic acids from biological samples is still achieved. However, when using binding particles with a large particle size, it becomes necessary to increase the amount of reaction solution required for processing. Therefore, when the goal is to efficiently recover nucleic acids from "small-scale biological samples," the average particle size of the binding particles is preferably 0.1 μm or more and 200 μm or less, more preferably 0.1 μm or more and 50 μm or less, and even more preferably 0.1 μm or more and 10 μm or less.

[0039] [Particle Surface State 2: Hydrophilicity of Particle Surface] The bound particles of this embodiment have high surface hydrophilicity due to the presence of adjacent silanol groups as described above. The hydrophilicity of such particle surfaces is evaluated by an evaluation value calculated according to the following formula (B): S = α Ｈ２Ｏ / α Ｎ２ ...(B) S: Evaluation value α regarding the hydrophilicity of the particle surface Ｈ２Ｏ : BET specific surface area (m) of the combined particles obtained by water vapor adsorption measurement ２ / g) α Ｎ２ : BET specific surface area (m) of the bonded particles determined by nitrogen adsorption measurement ２ / g)

[0040] α Ｈ２Ｏ can be determined by the method for measuring the BET specific surface area described in [Particle surface condition 1: sphericity]. Ｈ２Ｏ When measuring the adsorbate, water vapor (e.g., pure water that has been degassed three or more times) is used as the adsorbate. The pure water can be produced using a water purifier (e.g., Elix Advantage 5, manufactured by Merck Ltd.).

[0041] The combined particles of this embodiment more preferably have an evaluation value calculated by the above formula (B) of 0.35 or more.

[0042] In addition, since the BET specific surface area is used in calculating the evaluation value of hydrophilicity, it is thought that the calculation is strongly influenced by the sample amount (total surface area) of the bonded particles used to measure the specific surface area. As a result of the inventors' studies, it was found that even if the bonded particles are made of the same material and differ only in particle size, that is, even if the distribution state of adjacent silanol groups on the surface is similar, the evaluation value of hydrophilicity tends to increase as the particle size of the bonded particles increases.

[0043] From the above formula (B), the theoretical upper limit of the evaluation value is 1. On the other hand, experimentally, it was found that the evaluation value calculated from the above formula (B) greatly exceeds 1 for bonded particles having an average particle size large enough to exceed 10 μm, for example, and having many adjacent silanol groups on the surface.

[0044] For bound particles with a large average particle size and many vicinal silanol groups, the results of the evaluation value exceeding 1 are reproducible, and it has been confirmed that nucleic acids can be suitably recovered even from particles with an evaluation value exceeding 1 (see Examples below). Therefore, the inventors have determined that bound particles with a required evaluation value of 0.35 or greater are "highly hydrophilic."

[0045] From the above findings and experimental confirmation results by the inventors, it is preferable that the combined particles of this embodiment have an evaluation value calculated by the above formula (B) of 0.35 or more and 130 or less.

[0046] [Particle Surface State 3: Abundance Ratio of Vicinal Silanol Groups] The bound particles preferably have an abundance ratio of vicinal silanol groups of 0.93 or more and 1.00 or less, as calculated by the following formula (C): [Abundance Ratio of Vicinal Silanol Groups]=Rv / Rt (C) (Rv: peak area of ​​vicinal silanol groups in the Raman spectrum; Rt: sum of peak areas of vicinal silanol groups and isolated silanol groups in the Raman spectrum)

[0047] In formula (C), Rv and Rt can be determined from the Raman spectrum obtained by performing Raman spectroscopic analysis of the bound particles based on the description in (Raman spectroscopic analysis) above.

[0048] The ratio of vicinal silanol groups to the amount of silanol groups present on the surface is preferably as high as possible, and is preferably 0.93 or more. The upper limit of the ratio of vicinal silanol groups is preferably "1".

[0049] [Particle Structure] The bound particles may have at least a portion of their surface made of silica. That is, the bound particles may have silica exposed on at least a portion of their surface, and for example, a portion of the particle surface may be coated with a resin. It is preferable that the entire surface of the bound particles be made of silica.

[0050] Furthermore, as long as at least a portion of the surface of the bonded particles is made of silica, the interior of the particles can be made of various materials. The interior of the bonded particles may be made of a material other than silica. Examples of "materials other than silica" include resin materials with a lower specific gravity than silica and metals with a higher specific gravity than silica.

[0051] The binding particles preferably contain a magnetic material inside, which allows the behavior of the particles to be controlled by magnetic force, facilitating nucleic acid separation by the Boom method.

[0052] Such bonded particles can be produced, for example, by coating the surface of a particle with iron and then coating the surface of the iron-coated particle with silica.

[0053] The method for coating the particle surfaces with iron can be, for example, the method described in “Engineered Magnetic Core-Shell SiO2 / Fe Microspheres and “Medusa-like” Microspheres of SiO2 / Iron Oxide / Carbon Nanofibers or Nanotubes” Langmuir 2014, 30, 32, 9850-9858.

[0054] Furthermore, the surface of iron-coated particles can be further coated with silica by the method described in J. Am. Chem. Soc. 2008, 130, 28-29.

[0055] The binding particles may be silica particles whose interiors are also composed of silica.

[0056] Silica particles are commercially available as gap fillers for electric and electronic devices such as displays, and as fillers for semiconductor encapsulation. The inventors investigated the adjacent silanol groups on the particle surfaces of these electronic materials, which are in completely different technical fields from the particles for nucleic acid binding, and found that there are materials that can be used as the binding particles of this embodiment that can effectively adsorb nucleic acids.

[0057] The electronic material is much cheaper than commercially available particles for adsorbing nucleic acids used in the Boom method. Therefore, by converting silica particles, which are electronic materials, into the binding particles of this embodiment, i.e., by using silica particles, at least a portion of whose surface is covered with silica and which have adjacent silanol groups on the surface, for adsorbing nucleic acids by the Boom method, it is possible to obtain particles for binding nucleic acids that are cheaper than conventional particles.

[0058] <<Method for Manufacturing Nucleic Acid-Binding Particles>> Fig. 2 is a flowchart showing a method for manufacturing nucleic acid-binding particles. The method for manufacturing nucleic acid-binding particles includes the following steps 1 to 4.

[0059] (STEP 1: Preparation of raw material particles) First, particles whose surfaces are covered with silica are prepared as raw material particles (raw material particles) for the nucleic acid binding particles. The raw material particles only need to have at least a portion of their surfaces made of silica. That is, the raw material particles only need to have silica exposed on at least a portion of their surfaces, and for example, a portion of the particle surface may be coated with a resin. It is preferable that the entire surface of the raw material particles is made of silica. The interior of the raw material particles can have a structure that is common to the internal structure of the binding particles described above in [Particle structure].

[0060] (STEP 2: Adjustment of Hydrophilicity of Particle Surface) Next, the prepared raw material particles are subjected to a hydrothermal treatment to adjust the hydrophilicity evaluation value S of the particle surface of the raw material particles (evaluation value calculated from the following formula (B)) to 0.35 or more. Ｈ２Ｏ / α Ｎ２ ...(B) S: Evaluation value α regarding the hydrophilicity of the particle surface Ｈ２Ｏα: BET specific surface area of ​​the combined particles determined by water vapor adsorption measurement Ｎ２ : BET specific surface area of ​​bonded particles determined by nitrogen adsorption measurement

[0061] In this embodiment, "hydrothermal treatment" refers to heat treatment of raw material particles in the presence of high-temperature, high-pressure water. The pressure during treatment can be set appropriately as long as the desired heating temperature can be achieved. Furthermore, the hydrothermal treatment may be replaced by a superheated steam irradiation treatment.

[0062] Step 2 will be described in detail. Silanol groups in silica are dehydrated by heat treatment in a dry environment, forming siloxane bonds and decreasing in number. On the other hand, siloxane bonds are cleaved (depolymerized) by hydrothermal treatment, generating silanol groups.

[0063] Therefore, in the method for producing particles for nucleic acid binding of this embodiment, raw material particles whose surfaces are covered with silica can be subjected to a process that generates and increases vicinal silanol groups, such as hydrothermal treatment or superheated steam treatment, to increase the amount of vicinal silanol groups on the particle surface, thereby increasing the density of vicinal silanol groups. The amount of vicinal silanol groups on the particle surface can be adjusted by changing the conditions of the hydrothermal treatment. For example, if the amount of vicinal silanol groups after the hydrothermal treatment is lower than expected, the amount of vicinal silanol groups can be increased by changing the conditions, such as extending the hydrothermal treatment time or increasing the treatment temperature. In this embodiment, particles for nucleic acid binding can be produced by changing the treatment conditions to set the evaluation value to 0.35 or more.

[0064] The hydrothermal treatment is carried out, for example, by heating the raw material particles overnight at a temperature in the range of 100° C. to 180° C. As an example, the hydrothermal treatment is carried out by heating the raw material particles at 130° C. overnight.

[0065] When commercially available silica particles are used as raw material particles to produce bonded particles, the silica particles may be heat-treated (calcined) in a dry environment prior to the hydrothermal treatment to remove surface silanol groups. That is, STEP 2 may be performed in two stages: drying the raw material particles and hydrothermal treatment of the raw material particles. This allows differences in the amount of surface silanol groups due to differences in the manufacturer or lot of silica particles to be ignored, and by performing hydrothermal treatment under constant treatment conditions, it becomes possible to stably produce silica particles (bonded particles) with a desired density of vicinal silanol groups.

[0066] The heat treatment in a dry environment is carried out by, for example, firing the raw material particles at a temperature in the range of 500° C. to 1000° C.

[0067] The conditions for the hydrothermal treatment can be determined by conducting a preliminary experiment. That is, the ratio of vicinal silanol groups in the raw material particles treated in Step 2 is measured, and the conditions under which the ratio of vicinal silanol groups is 0.93 to 1.00 are determined. The hydrothermal treatment in Step 2 is performed under the conditions thus determined.

[0068] (STEP 3: Drying) After the hydrothermal treatment, the supernatant is discarded and the resulting particles are dried overnight. This results in particles (particles for nucleic acid binding) obtained by hydrothermally treating the raw material particles. The drying treatment can be carried out by a commonly known method. For example, the drying treatment can be carried out by heating, reducing pressure, blowing air, or a combination of these. When drying treatment is carried out by heating, it can be carried out at a temperature of, for example, room temperature or higher and 250°C or lower.

[0069] (STEP 4: Dispersion in Dispersion Medium) Next, the obtained nucleic acid binding particles may be dispersed in sterile distilled water to prepare a dispersion liquid with a predetermined particle concentration. The concentration of the dispersion liquid may be 20 mg / mL or more, or 40 mg / mL or more. The concentration of the dispersion liquid may be 200 mg / mL or less, or 160 mg / mL or less. The upper and lower limit values ​​of the dispersion liquid concentration can be arbitrarily combined. For example, the concentration of the dispersion liquid may be 20 mg / mL or more and 200 mg / mL or less, or 40 mg / mL or more and 160 mg / mL or less. In the examples described below, the concentration of the dispersion liquid is 40 mg / mL. The produced nucleic acid binding particles are finally supplied in the form of a dispersion liquid.

[0070] <<Nucleic Acid Extraction Method>> Figure 3 is a flowchart showing a method for extracting nucleic acids using nucleic acid-binding particles. The method for extracting nucleic acids using the nucleic acid-binding particles described above comprises the steps of mixing the nucleic acid-binding particles with a biological sample containing nucleic acids to form a dispersion (STEP 11), adsorbing the nucleic acids to the surfaces of the nucleic acid-binding particles (STEP 12), washing the nucleic acid-binding particles separated from the dispersion with a washing solution (STEP 13), and separating and recovering the nucleic acids from the washed nucleic acid-binding particles (STEP 14). Furthermore, the amount of recovered nucleic acid is measured and quantified (STEP 15), and the amount of nucleic acid is calculated (STEP 16).

[0071] The inventors' investigations revealed that, depending on the type of nucleic acid binding particles used, if the washing solution used in the washing step contains alcohol, nucleic acid may be detached from the nucleic acid binding particles in the washing step, resulting in a reduced amount of nucleic acid recovered. Details are explained below.

[0072] (STEP 11: Preparation of Biological Sample) First, a biological sample from which nucleic acids are extracted is prepared. For example, the biological sample is diluted with a known buffer to adjust the concentration. As the buffer, for example, 10 mmol / L trishydroxymethylaminomethane hydrochloride (pH 8.5) can be used. Binding particles are added to the obtained aqueous solution of the biological sample, and the mixture is shaken at room temperature for, for example, 10 minutes. The binding particles added may be the dispersion liquid described above.

[0073] (STEP 12: DNA adsorption) Next, a lysis buffer containing 8 mol / L guanidine salt is added to the dispersion obtained in STEP 11 to adsorb the DNA to the bound particles. The dispersion containing the dispersed bound particles is centrifuged, for example, at 700 rpm for 5 minutes to precipitate the bound particles. The separated lysis buffer is discarded.

[0074] (STEP 13: Washing of DNA-bound particles) Next, a buffer solution (washing solution) containing 10 mmol / L tris(hydroxymethylaminomethane) hydrochloride and ethanol is added to the obtained bound particles. The washing solution containing the dispersed bound particles is then centrifuged at 700 rpm for 5 minutes to precipitate the bound particles. The separated washing solution is discarded, and the bound particles are washed.

[0075] When the above-described steps from adding the buffer solution to the bound particles to discarding the separated washing solution are considered as one washing step, the washing step of the bound particles may be performed multiple times, for example, twice.

[0076] (STEP 14: Desorption and Recovery of DNA) The bound particles are re-dispersed in a buffer solution (extraction solution) (e.g., 60 μL) containing 10 mmol / L trishydroxymethylaminomethane hydrochloride, and heated at 70° C. for 10 minutes. The extract solution in which the bound particles are dispersed is then centrifuged at 700 rpm for 10 minutes to precipitate the particles, and the supernatant (extraction solution containing DNA, e.g., 60 μL) is recovered.

[0077] (STEP 15: Measurement and Quantification of DNA Amount) A portion of the obtained supernatant (e.g., 2 μL) is used to quantify the DNA concentration of the supernatant using a DNA concentration measurement device and a detection reagent. The DNA concentration measurement device may be, for example, a Qubit4 Fluorometer (manufactured by Thermofisher). The detection reagent may be, for example, a Qubit 1X dsDNA HS Assay Kit (manufactured by Thermofisher).

[0078] (STEP 16: Calculation of DNA Amount) The amount of extracted DNA is calculated by multiplying the measured concentration by the volume of the supernatant (60 μL).

[0079] The nucleic acid binding particles having the above-described configuration can efficiently recover nucleic acids from biological samples. Furthermore, the nucleic acid binding particles can be easily produced by the method for producing such particles.

[0080] While the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to these examples. The shapes and combinations of the components shown in the above examples are merely examples, and various modifications can be made based on the design, specifications, etc., without departing from the spirit of the present invention.

[0081] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0082] [Hydrophilicity of particle surface] In this example, the nitrogen adsorption amount, water vapor adsorption amount, and BET specific surface area of ​​the bonded particles were determined according to the descriptions in [Particle surface condition 1: sphericity] and [Particle surface condition 2: hydrophilicity of particle surface] above, and an evaluation value for the hydrophilicity of the surface of the bonded particles was calculated.

[0083] [Particle Sphericity] In this example, the sphericity of the combined particles was calculated according to the description in [Particle surface condition 1: sphericity] above.

[0084] [Raman Spectroscopic Analysis] In this example, Raman spectroscopic analysis of the bound particles was carried out in accordance with the description above (Raman Spectroscopic Analysis).

[0085] [Biological Samples] Biological Sample 1: Dispersion containing λDNA (Nippon Gene, concentration 550 ng / μL) Biological Sample 2: Dispersion prepared by the following method. (Preparation Method) After culturing HeLa cells, a cell line established from human cervical cancer cells, the cell number was counted using a Thoma hemocytometer and the cells were collected into tubes so that the cell counts in each sample were 1,000, 2,000, and 10,000. The cells in each tube were lysed using Proteinase K to prepare Biological Sample 2 containing DNA.

[0086] (Common Procedure: DNA Extraction) DNA was extracted based on the description in the above "Nucleic Acid Extraction Method." The biological sample was diluted with 10 mmol / L trishydroxymethylaminomethane hydrochloride (pH 8.5) to prepare the various concentrations used in the experiment. 23.1 μL of the bound particle dispersion was added to a tube containing the aqueous solution of each biological sample, and the tube was shaken at room temperature for 10 minutes (STEP 11 in Figure 3).

[0087] A lysis buffer containing 8 mol / L guanidine salt was added to the resulting dispersion, allowing the DNA to be adsorbed onto the bound particles. The dispersion containing the dispersed bound particles was centrifuged at 700 rpm for 5 minutes to precipitate the bound particles, and the separated lysis buffer was discarded (STEP 12 in Figure 3).

[0088] A buffer solution containing 10 mmol / L tris(hydroxymethyl)aminomethane hydrochloride and ethanol (washing solution) was added to the bound particles, and the washing solution containing the dispersed bound particles was centrifuged at 700 rpm for 5 minutes to precipitate the bound particles. The separated washing solution was discarded, and the bound particles were washed. This washing procedure was repeated twice (STEP 13 in Figure 3).

[0089] The resulting bound particles were re-dispersed in 60 μL of a buffer solution (extract solution) containing 10 mmol / L trishydroxymethylaminomethane hydrochloride, and then heated at 70° C. for 10 minutes. The extract solution with the dispersed bound particles was then centrifuged at 700 rpm for 10 minutes to precipitate the particles, and the supernatant (60 μL of extract solution containing DNA) was collected (STEP 14 in FIG. 3 ).

[0090] A portion (2 μL) of the resulting supernatant was used to quantify the DNA concentration of the supernatant using a DNA concentration measuring device (Qubit4 Fluorometer, Thermofisher) and a detection reagent (Qubit 1X dsDNA HS Assay Kit, Thermofisher) (STEP 15 in Figure 3).

[0091] The amount of extracted DNA was calculated by multiplying the measured concentration by the volume of the supernatant (60 μL) (STEP 16 in FIG. 3).

[0092] DNA was extracted from each type of bound particle according to the common procedure described above. The experimental results (Evaluation 1 to Evaluation 3) are explained below.

[0093] [Evaluation 1: Vicinal silanol groups] In the following evaluations, commercially available silica particles used as electronic materials (gap materials) were used as bonding particles. Note that, because the particle diameters of gap materials are uniform with extremely high precision, there is no significant difference between the particle diameters of individual particles and the average particle diameter.

[0094] The particle diameter of the combined particles 1 used in Evaluation 1 determined by image analysis is as follows. The presence of vicinal silanol groups on the surfaces of the combined particles 1 was confirmed by Raman spectroscopic analysis, and the hydrophilicity evaluation value of the particle surfaces was measured. Combined particles 1: particle diameter 1 μm, hydrophilicity evaluation value 0.51, sphericity 0.89

[0095] Each of the bound particles was suspended in sterile distilled water to prepare a dispersion with a particle concentration of 40 mg / mL.

[0096] Evaluation was performed using untreated bound particles 1 and bound particles 1 that had been subjected to each of the baking treatments (heat treatment in a dry environment) shown in Table 1. Using biological sample 1 and an input DNA amount of 60 ng, DNA was adsorbed and extracted according to the common procedure described above. The evaluation results are shown in Table 1.

[0097]

[0098] The evaluation results showed that baking at 250°C showed DNA adsorption capacity similar to that of untreated samples, whereas treatments at 500°C, 750°C, and 1000°C all lost the ability to adsorb DNA. It is believed that baking reduces the number of vicinal silanol groups on the surface, causing the loss of adsorption capacity.

[0099] As will be described later, for the bound particles No. 1-3, DNA can be recovered by adjusting the composition of the washing solution.

[0100] The bound particles No. 1-3 in Table 1 were subjected to hydrothermal treatment overnight at 130°C. Using biological sample 1 and an input DNA amount of 51 ng, DNA was adsorbed and extracted using untreated bound particles 1 (No. 1-1 in Table 1) and the bound particles after hydrothermal treatment, following the same procedure as above. The evaluation results are shown in Table 2. The hydrophilicity evaluation value of the bound particles after hydrothermal treatment (No. 1-4 in Table 2) was measured and found to be 0.63.

[0101]

[0102] As a result of the evaluation, it was found that the particles that had been subjected to hydrothermal treatment after calcination treatment had regained their DNA adsorption ability, similar to that of untreated particles, strongly suggesting that the hydrothermal treatment of the particles results in the formation of vicinal silanols on the particles, thereby acquiring DNA adsorption ability.

[0103] [Evaluation 2: Influence of surface shape of combined particles] In Evaluation 2, the influence of surface shape was confirmed using the above-mentioned combined particles 1 and combined particles with different sphericity. First, the influence of shape was confirmed using combined particles 1 and combined particles 2-1, which are porous particles. The particle diameter of combined particles 2-1 determined by image analysis is as follows. The presence of vicinal silanol groups on the surface of combined particles 2-1 was confirmed by Raman spectroscopy, and the hydrophilicity evaluation value of the particle surface was measured. Combined particles 2-1: particle diameter 1 μm, hydrophilicity evaluation value 0.38, sphericity 0.01

[0104] Using biological sample 1 and an input DNA amount of 55 ng, DNA was adsorbed and extracted according to the above-mentioned common procedure.

[0105] The evaluation results are shown in Figure 4 and the following Table 3. Figure 4 is a graph showing the evaluation results.

[0106]

[0107] As shown in Figure 4, it was found that binding particle 1, which has a sphericity close to 1 and a relatively smooth surface, was able to recover more DNA than binding particle 2-1, which has a low sphericity and a rough surface. The above results make it clear that, with the nucleic acid binding particles of the present invention, a surface structure that increases the surface area, such as a porous structure, is detrimental to the DNA yield, and that particles with a smooth surface are advantageous.

[0108] Additionally, the influence of shape was confirmed using combined particles 2-2, which were formed from the same material as combined particles 1 but had slightly smaller sphericity than combined particles 1. The particle diameter of combined particles 2-2 determined by image analysis was as follows. The presence of vicinal silanol groups on the surface of combined particles 2-2 was confirmed by Raman spectroscopic analysis, and the hydrophilicity evaluation value of the particle surface was measured. Combined particles 2-2: particle diameter 1 μm, hydrophilicity evaluation value 44.25, sphericity 0.86

[0109] The combined particle 2-2 exhibits a high evaluation value of hydrophilicity of 44.25, which is thought to be due to the shape of the particle surface of the combined particle 2-2. Since the combined particle 2-2 has low sphericity, it is thought that the particle surface has gentle irregularities or undulations compared to other combined particles. It can be inferred that due to this surface shape, the combined particle 2-2 exhibits a tendency for moisture to easily adsorb and condense on the hydroxyl groups. For this reason, when measuring the hydrophilicity of the combined particle 2-2, the BET specific surface area (α Ｈ２Ｏ ) tend to show large values, which can be inferred to result in a high evaluation value.

[0110] Using biological sample 2 containing 10,000 cells, DNA was adsorbed and extracted according to the common procedure described above. The evaluation results are shown in Table 4 below.

[0111]

[0112] As shown in Table 4, compared with bound particles 1 with a sphericity of 0.89, bound particles 2-2 with a sphericity of 0.86 yielded a significantly reduced amount of extracted DNA, but it was found that DNA could still be recovered.

[0113] In the above examples, the effect was confirmed by adsorbing DNA to the binding particles, but it is expected that the same behavior will be observed with RNA due to the similarity in structure.

[0114] [Evaluation 3: Hydrophilicity of Particle Surface] It was found that bound particles 1 retained their ability to adsorb DNA when untreated or baked at 250°C, but lost their DNA adsorption ability at 500°C. Meanwhile, in addition to the adsorption process, the washing process (STEP 13 in Figure 3), which is typically performed to increase DNA purity, must be considered when considering the effect on DNA yield. Therefore, the following experiment was conducted. The sphericity of bound particles No. 3-1 to No. 3-3 used in Evaluation 3 was not affected by the baking treatment and was 0.89, the same as bound particles 1.

[0115] DNA was adsorbed and extracted using bound particles 1 or bound particles 1 baked at 500°C according to the common procedure shown in Figure 3. In this case, either cleaning solution 1 or 2 below was used as the cleaning solution in STEP 13. Cleaning solution 2 is the cleaning solution used in the common procedure. Cleaning solution 1: 10 mmol / L aqueous solution of trishydroxymethylaminomethane hydrochloride containing 70% ethanol Cleaning solution 2: 10 mmol / L aqueous solution of trishydroxymethylaminomethane hydrochloride containing 70% ethanol

[0116] The evaluation results are shown in Table 5.

[0117]

[0118] The results of No. 3-1 and 3-2 show that although the particles baked at 500°C can adsorb DNA, the amount of DNA that can be adsorbed is less than that of the untreated particles (No. 3-1 in Table 5). The particles baked at 500°C had a lower hydrophilicity rating than the untreated particles, suggesting that the silanol groups on the particle surface had decreased.

[0119] On the surface of the bound particles, adjacent silanol groups and water molecules are hydrogen-bonded, and it is believed that DNA is bound to adjacent silanol groups via these water molecules. When the cleaning solution does not contain ethanol, the salt in the cleaning solution shields electrostatic repulsion, allowing DNA and bound particles to bond via water molecules. Comparing the results of No. 3-1 and No. 3-2, which do not contain ethanol in the cleaning solution, it is clear that the hydrophilicity evaluation value changes depending on whether or not baking is performed, and the amount of extracted DNA decreases as the hydrophilicity evaluation value decreases. Therefore, it is believed that the amount of adjacent silanol groups decreases due to baking.

[0120] Furthermore, the results of No. 3-2 and 3-3 show that when particles baked at 500°C are used, the amount of DNA that can be extracted is significantly reduced when the particles are washed with a washing solution containing ethanol, as in the above Evaluation 1. The reason for this phenomenon is thought to be as follows.

[0121] First, when the cleaning solution contains ethanol, some of the vicinal silanol groups present on the surface of the bound particles are thought to be esterified, making them unable to form hydrogen bonds with water molecules, resulting in a decrease in the amount of vicinal silanol groups that can bond with DNA via water molecules.

[0122] Furthermore, in the bound particles with adsorbed DNA, the DNA is thought to be in an equilibrium state where it repeatedly adsorbs and desorbs from adjacent silanol groups. The polar groups of DNA that are not adsorbed to adjacent silanol groups are hydrogen-bonded to water, which is thought to stabilize the DNA in the supernatant where it is dispersed.

[0123] Here, when the cleaning solution contains ethanol, it is thought that hydrogen-bonded water in the DNA in the supernatant is dehydrated by the ethanol, making it easier for polar groups in the DNA to bond with each other within or between DNA molecules. In such bonded DNA, the sites (polar groups) to which vicinal silanol groups bond are consumed by bonds within or between DNA molecules, making it difficult for them to bond with vicinal silanol groups. In other words, when the cleaning solution contains ethanol, it is thought that the amount of DNA that easily bonds with vicinal silanol groups decreases.

[0124] When the washing solution contains ethanol, (i) the amount of vicinal silanol groups capable of binding to DNA is reduced, and (ii) the amount of DNA that readily binds to vicinal silanol groups is reduced. This is thought to result in the binding particles being less able to retain DNA during washing. As a result, when the washing solution contains ethanol, DNA is washed away by washing, and the amount of extracted DNA is thought to be reduced.

[0125] When the amount of vicinal silanol groups in the bonded particles is small, the total amount of vicinal silanol groups that are not esterified and can adsorb DNA is also small. Therefore, DNA is less likely to be adsorbed by the vicinal silanol groups, and even if DNA is adsorbed by the vicinal silanol groups, the adsorption force is weaker than that of bonded particles with a relatively large total amount of vicinal silanol groups. As a result, bonded particles with a small number of vicinal silanol groups are more susceptible to the influence of ethanol in the wash water than bonded particles with a large number of vicinal silanol groups, making DNA extraction more difficult.

[0126] As described above, it is believed that the ethanol contained in the washing solution affects the amount of DNA extracted by the binding particles. As shown in Table 5, when the binding particles were washed with washing solution 2 containing ethanol (No. 3-3), the binding particles had a weaker ability to adsorb DNA than when the binding particles were washed with washing solution 1 not containing ethanol (No. 3-2), which is thought to result in a lower amount of extracted DNA.

[0127] On the other hand, it was confirmed that if the evaluation value of hydrophilicity is 0.35, DNA can be extracted by using an aqueous solution that does not contain alcohol as a washing liquid for the binding particles.

[0128] [Evaluation 4: Consideration of particle size of combined particles] In this evaluation, the influence of particle size of combined particles was studied using combined particles 3 and 4, which were formed from the same material as combined particles 1 but differed only in particle size. The particle sizes of combined particles 3 and 4 determined by image analysis were as follows. The presence of vicinal silanol groups on the surfaces of combined particles 3 and 4 was confirmed by Raman spectroscopic analysis, and the hydrophilicity evaluation value of the particle surface was measured. Combined particle 3: particle size 0.16 μm, hydrophilicity evaluation value 0.41, sphericity 0.97 Combined particle 4: particle size 50 μm, hydrophilicity evaluation value 8.26, sphericity 1

[0129] Because combined particles 3 and 4 are made of the same material as combined particles 1 but differ only in particle diameter, it is believed that the distribution of adjacent silanol groups on the surface is similar to that of combined particles 1. However, despite being made of the same material as combined particles 1 and 3, the evaluation value of the hydrophilicity of combined particles 4 is significantly different from the evaluation values ​​of combined particles 1 and 3. As a result of investigations into this point by the inventors, it was found that as the particle diameter of the combined particles increases, the evaluation value of the hydrophilicity tends to fluctuate upward.

[0130] Because the BET specific surface area is used in calculating the evaluation value of hydrophilicity, it is thought that the evaluation value is strongly influenced by the sample amount (total surface area) of the bound particles used to measure the specific surface area. As described above, the evaluation value of hydrophilicity calculated for bound particle 4 is significantly different from that of bound particles 1 and 3. However, based on the above findings, the actual hydrophilicity of bound particle 4 is considered to be similar to that of bound particles 1 and 3, and the following evaluation is performed.

[0131] (Evaluation 4-1) The effect of particle size was investigated using bound particles 1 and bound particles 3. For bound particles 3, the concentration of the dispersion was set to 6.4 mg / mL, and the total surface area of ​​the particles in the dispersion was prepared to be the same as that of bound particles 1.

[0132] Using biological sample 1, DNA was adsorbed and extracted according to the above-mentioned common procedure, with the input DNA amount set to 5.5 ng or 11.0 ng.

[0133] The evaluation results are shown in a graph in Figure 5. As shown in Figure 5, no difference due to particle size was observed between bound particles 3 with a particle size of 0.16 µm and bound particles 1 with a particle size of 1 µm (both * and ** in Figure 5 have p values ​​of p > 0.05, indicating no significant difference).

[0134] (Evaluation 4-2) The effect of particle size on DNA extraction was investigated using binding particles 1 and binding particles 4. For binding particles 4, the concentration of the dispersion was set to 2000 mg / mL, and the dispersion was prepared so that the total surface area of ​​the particles in the dispersion was the same as that of binding particles 1. In this evaluation, biological sample 2 containing 1000 or 2000 cells was used, and DNA was adsorbed and extracted according to the common procedure described above.

[0135] The evaluation results are shown in a graph in Figure 6. As shown in Figure 6, no difference due to particle size was observed between bound particles 1 with a particle size of 1 µm and bound particles 4 with a particle size of 50 µm (the p values ​​for both *** and # in Figure 6 are p>0.05, indicating no significant difference).

[0136] When the hydrophilicity evaluation values ​​of various binding particles were confirmed, the highest evaluation value within the confirmed range was 125.54 for binding particles made of the same material as binding particle 1, with a sphericity of 0.86 or greater and a particle diameter of 10 μm determined by image analysis. It was also confirmed that these binding particles could adsorb and extract DNA using the same procedures as described above.

[0137] [Evaluation 5: Influence of contaminants in biological samples] As shown in Table 6 below, samples containing and not containing λDNA and the protease Proteinase K were prepared. When the sample contained the protease Proteinase K, the concentration of the protease Proteinase K was set to 10 mg / mL (final concentration).

[0138] Using the prepared sample, DNA was adsorbed and extracted according to the common procedure described above.

[0139] The results are shown in Table 6. In the table, (+) means that the target substance is contained in the sample, and (-) means that the target substance is not contained in the sample.

[0140]

[0141] As a result of the evaluation, it was confirmed that the protease proteinase K contained in the sample did not affect the adsorption of DNA to the binding particles.

[0142] From the above results, it was confirmed that the present invention is useful.

[0143] A... surface silanol group, A1... adjacent silanol group, A2... isolated silanol group, B... internal silanol group

Claims

1. A particle for nucleic acid binding, at least a part of which is covered with silica, and the sphericity calculated from the following formula (A) is 0.86 or more, and the particle has adjacent silanol groups on the surface. [Sphericity] = Particle diameter (nm) calculated by image analysis ÷ BET diameter (nm) ... (A) 2. The particle for nucleic acid binding according to claim 1, wherein the evaluation value of the hydrophilicity of the surface calculated from the following formula (B) is 0.35 or more. Ｈ２Ｏ / α Ｎ２ ...(B) S: Evaluation value α regarding hydrophilicity of the particle surface Ｈ２Ｏ α: BET specific surface area of ​​combined particles determined by water vapor adsorption measurement Ｎ２ : BET specific surface area of ​​bonded particles determined by nitrogen adsorption measurement 3. The particle for binding nucleic acid according to claim 2, wherein the abundance ratio of adjacent silanol groups on the surface calculated by the following formula (C) is 0.93 or more and 1.00 or less: [Abundance ratio of adjacent silanol groups]=Rv / Rt (C) (Rv: peak area of ​​adjacent silanol groups in Raman spectrum, Rt: total peak area of ​​adjacent silanol groups and isolated silanol groups in Raman spectrum) 4. Particles for nucleic acid binding according to any one of claims 1 to 3, having an average particle size of 0.1 µm or more and 1000 µm or less.

5. The nucleic acid binding particles according to claim 4, having an average particle size of 0.1 μm or more and 200 μm or less.

6. A particle for binding nucleic acid according to any one of claims 1 to 3, wherein the particle contains a magnetic material therein.

7. A method for producing particles for binding nucleic acid, comprising a step of subjecting particles having surfaces covered with silica to a treatment for increasing adjacent silanol groups, and controlling the evaluation value for the hydrophilicity of the surface calculated from the following formula (B) to 0.35 or more: S=α Ｈ２Ｏ / α Ｎ２ ...(B) S: Evaluation value α regarding hydrophilicity of the particle surface Ｈ２Ｏ α: BET specific surface area of ​​combined particles determined by water vapor adsorption measurement Ｎ２ : BET specific surface area of ​​bonded particles determined by nitrogen adsorption measurement 8. The method for producing particles for binding nucleic acid according to claim 7, wherein the treatment for increasing the number of adjacent silanol groups is a hydrothermal treatment of the particles.

9. The method for producing particles for binding nucleic acids according to claim 8, wherein the abundance ratio of adjacent silanol groups on the surface of the particles for binding nucleic acids, calculated from the following formula (C), is 0.93 or more and 1.00 or less: [Abundance ratio of adjacent silanol groups]=Rv / Rt (C) (Rv: peak area of ​​adjacent silanol groups in Raman spectrum, Rt: total peak area of ​​adjacent silanol groups and isolated silanol groups in Raman spectrum) 10. A method for producing particles for binding nucleic acid according to any one of claims 7 to 9, wherein the particles are heat-treated in a dry environment prior to the hydrothermal treatment of the particles.

11. The method for producing particles for nucleic acid binding according to any one of claims 7 to 9, wherein the particles have a sphericity of 0.86 or more calculated from the following formula (A): [sphericity] = particle diameter (nm) calculated by image analysis ÷ BET diameter (nm) ... (A)

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