Information acquisition method, determination method, and information acquisition device

Abstract

To provide an information acquisition method in which information regarding nucleic acid or protein can be accurately acquired in short time and inexpensively.SOLUTION: A method for acquiring information regarding nucleic acid or protein by surface-enhanced Raman-scattering spectroscopy includes: a step of preparing an analysis device having a substrate and a plurality of metal nano structures which are arranged apart from each other such that a plurality of nano gaps are formed on the substrate; a step of irradiating solution containing the nucleic acid or the protein, which is provided for a region including the plurality of nano gaps of the analysis device, with excitation light; a step of detecting Raman scattered light generated from the solution by irradiation of the excitation light; and a step of acquiring information regarding the nucleic acid or the protein in the solution on the basis of the detected Raman scattered light.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an information acquiring method, a determination method, and an information acquiring device. [Background technology]

[0002] Nucleic acids include deoxyribonucleic acid (DNA) containing genetic information and ribonucleic acid (RNA) including messenger RNA (mRNA) that copies the genetic information. Based on the genetic information (base sequence) of DNA copied by mRNA, proteins are synthesized in ribosomes. Nucleic acids or proteins in body fluids are used as cancer markers, and cancer can be detected by distinguishing between nucleic acids or proteins in the body fluids of healthy subjects and those of cancer patients. Therefore, obtaining information about nucleic acids or proteins in a specimen obtained from a subject is important in determining the presence or absence of cancer cells.

[0003] There are various known methods for obtaining information about nucleic acids. For example, the next generation sequencing method (NGS method) for analyzing the base sequence of nucleic acids is known (Non-Patent Document 1). In the NGS method, for example, a fragment of a target nucleic acid molecule is reacted with a base that is a complementary pair of a base constituting the nucleic acid molecule, one base at a time, and the fluorescence generated during the reaction is detected, thereby analyzing the base sequence of the nucleic acid molecule.

[0004] Also, real-time PCR is known as a method for identifying and quantifying nucleic acid molecules (Non-Patent Document 2). In real-time PCR, for example, an intercalator that enters the structure of a nucleic acid molecule and emits fluorescence is used, and the amount of amplification of the nucleic acid molecule is monitored by detecting the fluorescence from the intercalator. This allows the identification and quantification of the nucleic acid molecule.

[0005] Furthermore, an analytical method using a microarray method is also known (Non-Patent Document 3). In this method, a substrate on which probes that bind complementarily to bases constituting a nucleic acid molecule are arranged is used, and a fluorescently labeled target nucleic acid molecule is reacted with the probes. The binding position between the nucleic acid molecule and the probe is then detected by fluorescence, and the base sequence of the target nucleic acid molecule can be analyzed. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] “An overview of recent RNA-Seq publications featuring Illumina technology” [Searched on August 1, 2020] <URL:https: / / www.illumina.com / content / dam / illumina-marketing / documents / products / research_reviews / rna-sequencing-methods-review-web.pdf> [Non-Patent Document 2] SA Deepak, et al., “Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes”, Current Genomics, Vol. 8, pp. 234-251. [Non-Patent Document 3] Kunihisa Nagino, et al., “Ultrasensitive DNA Chip: Gene Expression Profile Analysis without RNA Amplification”, The Journal of Biochemistry, Vol. 139, pp. 697-703. Summary of the Invention [Problem to be solved by the invention]

[0007] Analytical methods for obtaining information about nucleic acids are known, such as those described in Non-Patent Documents 1 to 3. However, these methods require analysis time of several hours to several days, making it difficult to obtain information about nucleic acids in a short period of time.

[0008] Furthermore, in these methods, the costs of preparing the bases to react with nucleic acids (Non-Patent Documents 1-2), the fluorescent molecules to fluorescently label the nucleic acids (Non-Patent Documents 1-3), and the substrate on which the probes are arranged (Non-Patent Document 3) are high, costing hundreds of thousands of yen, making it difficult to obtain information about nucleic acids inexpensively.

[0009] Furthermore, there is a demand for obtaining information about proteins quickly and inexpensively in determining the presence or absence of cancer cells.

[0010] Therefore, the inventors considered using surface-enhanced Raman spectroscopy to obtain information about nucleic acids and proteins quickly and inexpensively, but conventional surface-enhanced Raman spectroscopy was not able to accurately obtain information about nucleic acids and proteins.

[0011] An object of the present invention is to provide an information acquiring method and information acquiring device that can accurately acquire information about nucleic acids or proteins in a short time and at low cost, and a determination method using said method. [Means for solving the problem]

[0012] In order to achieve the above object, one aspect of the present invention relates to the following information acquisition methods [1] to

[11] . [1] A method for obtaining information about a nucleic acid or a protein by surface-enhanced Raman scattering spectroscopy, comprising: Providing an analytical device having a substrate and a plurality of metallic nanostructures spaced apart from one another on the substrate such that a plurality of nanogaps are formed; Irradiating an excitation light onto a solution containing the nucleic acid or the protein provided in a region of the analysis device that includes the plurality of nanogaps; detecting Raman scattered light generated from the solution by irradiation with the excitation light; obtaining information about the nucleic acid or the protein in the solution based on the detected Raman scattered light; (c) a method of obtaining information; [2] The information acquisition method described in [1], wherein the shapes of the multiple metal nanostructures are substantially identical. [3] The planar shape of each of the plurality of metal nanostructures is polygonal; the plurality of metallic nanostructures are disposed on the substrate such that each of the plurality of nanogaps is formed between corners of the polygon; The method of obtaining information described in [1] or [2]. [4] The width of each of the nanogaps is 1 nm or more and 20 nm or less. ]~[3] A method for obtaining information described in any one of the items. [5] The information acquisition method according to any one of [1] to [4], wherein the height of each of the metallic nanostructures is not less than 1 nm and not more than 100 nm. [6] In the irradiating step, the solution containing the plurality of types of the nucleic acid molecules or the plurality of types of the protein molecules is irradiated with excitation light; In the acquiring step, information on the multiple types of nucleic acid molecules or the multiple types of protein molecules in the solution is acquired collectively. The method for obtaining information described in any one of [1] to [5]. [7] The information acquiring method according to any one of [1] to [6], wherein in the acquiring step, the information is acquired by performing a statistical analysis. [8] The information acquisition method according to [7], wherein in the acquiring step, the information is acquired using principal component analysis. [9] The solution contains the nucleic acid, The information is information about the nucleic acid. The method for obtaining information described in any one of [1] to [8].

[10] The method for obtaining information described in [9], wherein the nucleic acid is RNA.

[11] The information acquisition method described in

[10] , wherein the RNA is microRNA.

[0013] Another aspect of the present invention for achieving the above object relates to the determination methods

[12] and

[13] below.

[12] A method for determining the presence or absence of cancer cells, comprising: [1] to

[11] , by the information acquisition method according to any one of the above. [1] to

[11] , determining whether or not the specimen contains the nucleic acid or the protein derived from a cancer cell based on the first information; A method for determining whether or not a

[13] The method further comprises the step of obtaining second information about a nucleic acid or a protein contained in a sample obtained from a healthy subject by the information obtaining method; the step of determining includes comparing the first information with the second information to determine whether or not the specimen obtained from the subject contains the nucleic acid or the protein derived from a cancer cell; The method for determining whether or not a person is a person skilled in the art has the following characteristics.

[0014] Another aspect of the present invention for achieving the above object relates to an apparatus as described below in

[14] .

[14] An apparatus for obtaining information about a nucleic acid or a protein by surface-enhanced Raman scattering spectroscopy, comprising: An analytical device having a substrate and a plurality of metal nanostructures arranged on the substrate at a distance from each other so that a plurality of nanogaps are formed; an irradiation unit provided in a region including the nanogaps of the analysis device for irradiating a solution including the nucleic acid or the protein with excitation light; a detection unit for detecting Raman scattered light generated from the solution by irradiation with the excitation light; an information acquiring unit for acquiring information about the nucleic acid or the protein in the solution based on the detected Raman scattered light; An information acquisition device comprising: Effect of the Invention

[0015] According to the present invention, there are provided an information acquiring method and an information acquiring device that can accurately acquire information about nucleic acids or proteins in a short time and at low cost, and a determination method using the above method. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a flowchart showing an information acquisition method according to an embodiment of the present invention. [Figure 2A] FIG. 2A is a partially enlarged cross-sectional view of the analytical device. [Figure 2B] FIG. 2B is an example of an electron microscope image of the surface of an analytical device. [Figure 2C] FIG. 2C is an enlarged image of portion α of FIG. 2B. [Diagram 3] FIG. 3 is a flowchart illustrating a determination method according to one embodiment of the present invention. [Figure 4] FIG. 4 is a schematic diagram showing the configuration of an information acquisition device according to one embodiment of the present invention. [Diagram 5] 5A and 5B show Raman spectra obtained from solution 1 and solution 2, respectively, in an embodiment of the present invention. [Figure 6] FIG. 6 is a dot plot obtained by principal component analysis from the Raman spectra of FIGS. 5A and 5B. [Figure 7] FIG. 7 is a dot plot obtained by principal component analysis from the Raman spectra of solutions 3 and 4 when the analytical device of the embodiment is used in the embodiment of the present invention. [Figure 8] FIG. 8 is a dot plot obtained by principal component analysis from the Raman spectra of solutions 3 and 4 when a comparative analytical device (a commercially available SERS substrate) was used in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0018] 1.How to obtain information FIG. 1 is a flowchart showing an information acquisition method according to an embodiment of the present invention.

[0019] As shown in FIG. 1, an information acquisition method according to one embodiment of the present invention is a method for acquiring information about nucleic acids or proteins by surface-enhanced Raman scattering spectroscopy, and includes the steps of (1) preparing an analytical device having a plurality of metal nanostructures (step S10), (2) irradiating a solution containing nucleic acids or proteins with excitation light (step S20), (3) detecting Raman scattered light generated from the solution (step S30), and (4) acquiring information about the nucleic acids or proteins in the solution (step S40).

[0020] In this specification, the nucleic acids and proteins are not particularly limited. The nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The number of nucleotide units constituting the nucleic acid is not particularly limited. The nucleic acids also include oligonucleotides and polynucleotides. The RNA includes messenger RNA (mRNA) and non-coding RNA (ncRNA). The ncRNA includes microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA). The number of amino acid residues constituting the protein is not particularly limited. The protein includes oligopeptides and polypeptides.

[0021] 1-1. Step of preparing an analytical device (step S10) Fig. 2A is a partially enlarged cross-sectional view of the analytical device 100, Fig. 2B is an example of an electron microscope image of the surface of the analytical device 100, and Fig. 2C is an enlarged image of a portion α of Fig. 2B. In this step, an analytical device 100 is prepared, which has a substrate 110 and a plurality of metallic nanostructures 120 arranged on the substrate at a distance from each other so that a plurality of nanogaps 120a are formed.

[0022] When a solution containing nucleic acid or protein is provided to a region including a plurality of nanogaps 120a of the analytical device 100, it is believed that molecules of the nucleic acid or protein enter the nanogaps 120a. When excitation light is irradiated to the provided solution in this state, localized surface plasmon resonance occurs with the nanogaps 120a as hot spots (enhanced electric field). This significantly increases the intensity of Raman scattered light generated from the solution (Surface-Enhanced Raman Scattering (SERS)). Thus, in the present invention, by applying surface-enhanced Raman scattering spectroscopy to nucleic acid or protein using the analytical device 100, Raman scattered light can be detected with sufficiently higher sensitivity than when an analytical device not including a metal nanostructure 120 forming a plurality of nanogaps 120a is used. This allows accurate acquisition of information about nucleic acid or protein.

[0023] In addition, since there is no need to react the target nucleic acid or protein with other molecules in the process of obtaining information about the nucleic acid or protein, the information about the nucleic acid or protein can be obtained in a shorter time than before. In addition, since there is no need to prepare a nucleic acid for capturing the nucleic acid and a fluorescent molecule for fluorescently labeling the nucleic acid, the cost of obtaining information about the nucleic acid or protein can be reduced compared to before.

[0024] The substrate 110 supports a plurality of metal nanostructures 120 arranged on the substrate 110. The material of the substrate 110 is not particularly limited, but may be, for example, glass, silicon nitride, silicon dioxide, silicon, etc. Of these, the material of the substrate 110 is preferably silicon nitride. The shape of the substrate 110 is not particularly limited.

[0025] The multiple metallic nanostructures 120 are arranged spaced apart from each other so that multiple nanogaps 120a are formed on the substrate 110. The shapes of the multiple metallic nanostructures 120 are not particularly limited, but are preferably substantially identical. This can increase the intensity of the Raman scattered light generated from a solution containing nucleic acids or proteins, making it easier to obtain information about nucleic acids or proteins more accurately by detecting the Raman scattered light.

[0026] Also, for example, the shape of the plurality of metal nanostructures 120 is a polygonal column, a circular column, or an elliptical column. That is, the planar shape of the plurality of metal nanostructures 120 is a polygon, a circle, or an ellipse. Of these, from the viewpoint of further increasing the intensity of Raman scattering light generated from a solution containing nucleic acid or protein, the planar shape of the plurality of metal nanostructures 120 is preferably a polygon, and more preferably a rectangle. From the same viewpoint, it is preferable that the polygon includes an acute angle. As shown in FIG. 2B, in this embodiment, the planar shapes of the plurality of metal nanostructures 120 are substantially the same, and each is approximately a rectangle. In this specification, the "nanogap" refers to the gap between the adjacent plurality of metal nanostructures 120 that has the shortest width and has a width on the order of nanometers.

[0027] The material of the multiple metallic nanostructures 120 is not particularly limited as long as it is a metal that can generate localized surface plasmon resonance, and examples of the material include gold, silver, copper, aluminum, etc. Among these, the material is preferably gold.

[0028] The multiple metallic nanostructures 120 are arranged such that nanogaps 120a are formed between the metallic nanostructures 120 spaced apart from one another. When the multiple metallic nanostructures 120 each have a polygonal shape in plan view, the multiple metallic nanostructures 120 are preferably arranged on the substrate 110 such that each of the multiple nanogaps 120a is formed between the corners of the polygon. This can further increase the intensity of the Raman scattered light generated from a solution containing nucleic acids or proteins, making it easier to more accurately obtain information about nucleic acids or proteins by detecting the Raman scattered light.

[0029] The height of the plurality of metallic nanostructures 120 (the length of the arrow A in FIG. 2A) is preferably 1 nm or more and 100 nm or less, more preferably 10 nm or more and 50 nm or less, and even more preferably 20 nm or more and 40 nm or less. When the height is within the above range, the nucleic acid or protein molecules can more easily enter the nanogap 120a. This makes it easier to generate surface-enhanced Raman scattering by irradiation with excitation light, and makes it easier to detect the Raman scattered light. The height can be determined by observing the analytical device 100 with an electron microscope or atomic force microscope.

[0030] The width of the nanogaps 120a (the length of the arrow B in FIG. 2C) is preferably 1 nm or more and 20 nm or less, more preferably 2 nm or more and 20 nm or less, even more preferably 4 nm or more and 16 nm or less, and particularly preferably 6 nm or more and 10 nm or less. When the width is 1 nm or more, the nucleic acid or protein molecules can easily enter the nanogaps 120a. When the width is 20 nm or less, the intensity of the Raman scattered light generated from the solution containing the nucleic acid or protein can be further increased. For these reasons, when the width is within the above range, it is possible to easily obtain information about the nucleic acid or protein. The width can be determined by observing the surface of the analysis device 100 with an electron microscope.

[0031] The size of the multiple metallic nanostructures 120 in a plan view (the length of the arrows C and D in FIG. 2C) is not particularly limited, and can be set appropriately depending on the wavelength of the excitation light.

[0032] The manufacturing method of the analytical device 100 is not particularly limited, but may include, for example, the steps of (1) forming a resist pattern on the surface of the substrate 110, (2) forming a metal film on the surface of the substrate 110 on which the resist pattern is formed, and (3) removing the resist pattern together with the metal film covering the resist pattern. The steps (1) to (3) are described below.

[0033] (Process for forming a resist pattern) In this process, after applying a resist onto the substrate 110, the resist is exposed using a photomask so that openings are formed in the positions where the metallic nanostructures 120 are to be applied, and the resist pattern is formed using a developer.

[0034] The resist pattern may be formed using a positive-type photosensitive resin composition, or may be formed using a negative-type photosensitive resin composition.

[0035] The positive-type photosensitive resin composition and negative-type photosensitive resin composition that can be used in this step may be any known resin composition used as a lift-off resist.

[0036] (Metal film forming process) In this step, a metal film is formed on the surface of the substrate 110 on which the resist pattern is formed (specifically, on the surface of the resist and in the openings formed between the resist patterns). The metal film formed in the openings functions as metal nanostructures 120 after the resist pattern is removed in a step of removing the resist pattern described below.

[0037] The method for forming the metal film is not particularly limited, but may be, for example, a vapor deposition method, a sputtering method, etc. Examples of metals that can be used as the material for the metal film include the same metals as the materials for the metal nanostructures described above.

[0038] (Step of removing the resist pattern) In this step, the resist pattern is removed together with the metal film covering the resist pattern. Specifically, this step is performed using a stripping solution for removing the resist pattern.

[0039] In this step, the resist pattern can be removed, for example, by a method of immersing the substrate in a stripping solution.

[0040] Examples of the stripping solution include anisole, dimethyl sulfoxide, and the like.

[0041] 1-2. Step of irradiating excitation light (step S20) In this step, a solution containing nucleic acid or protein, which is provided in a region of the analytical device 100 that includes the plurality of nanogaps 120a, is irradiated with excitation light.

[0042] In this step, a solution containing multiple types of nucleic acid molecules or multiple types of protein molecules may be irradiated with excitation light, and information on the multiple types of nucleic acid molecules or multiple types of protein molecules in the solution may be acquired collectively in the step of acquiring information described below (step S40). This allows information on multiple types of nucleic acid molecules or multiple types of protein molecules to be acquired in a shorter time.

[0043] When the presence or absence of cancer cells is determined based on information acquired in the step of acquiring information (step S40) described below, the solution preferably contains nucleic acid. In this case, the information acquired in the step of acquiring information (step S40) described below is preferably information related to nucleic acid.

[0044] The nucleic acid is preferably RNA, more preferably microRNA (miRNA) or tumor circulating RNA (ctRNA), and even more preferably microRNA (miRNA). In the step of acquiring information (step S40) described later, by acquiring information on microRNA (miRNA) or tumor circulating RNA (ctRNA), it is possible to easily determine the presence or absence of cancer cells based on the acquired information.

[0045] The solvent of the above solution is not particularly limited, but includes TE buffer and Tris-HCl.

[0046] The concentration of nucleic acid or protein in the above solution is 1.0 x 10 -6 mol / L or more 1.0×10 -1 It is preferable that the concentration is less than mol / L, and 1.0×10 -4 mol / L or more 1.0×10 -3 It is more preferable that it is less than mol / L.

[0047] The excitation light is preferably a light of a substantially single wavelength. The wavelength of the excitation light is not particularly limited, and is, for example, 300 nm or more and 1000 nm or less. From the viewpoint of preventing autofluorescence from nucleic acid molecules or protein molecules, the wavelength of the excitation light is preferably 700 nm or more and 1000 nm or less, and more preferably 750 nm or more and 800 nm or less. In addition, the type of excitation light is not particularly limited, but is preferably a laser light from the viewpoint of further increasing the intensity of Raman scattering light. The excitation light is irradiated by a known light source (for example, a laser diode).

[0048] The information acquiring method according to this embodiment may include a step of providing a solution containing nucleic acid or protein to a region of the analytical device 100 including the nanogaps 120a before this step (step S20).

[0049] 1-3. Step of detecting Raman scattered light (step S30) In this step, Raman scattered light generated from the solution by irradiation with excitation light in step S20 is detected.

[0050] The method for detecting the Raman scattered light is not particularly limited, and for example, the Raman scattered light can be detected using a CCD detector, etc. From the viewpoint of more accurately obtaining information about the nucleic acid or protein from the Raman scattered light, in this step, the Raman scattered light generated from the above-mentioned solution may be spectrally separated and the spectrally separated Raman scattered light may be detected.

[0051] This step makes it possible to obtain a Raman spectrum for the nucleic acid or protein in the solution.

[0052] 1-4. Information acquisition process (process S40) In this step, information about the nucleic acid or protein in the solution is obtained based on the Raman scattered light detected in step S30.

[0053] Specific examples of the information include information for determining whether the solution contains nucleic acid or protein derived from cancer cells, and information for specifically identifying the nucleic acid molecules or protein molecules in the solution.

[0054] The method for obtaining information about nucleic acids or proteins is not particularly limited, and the information can be obtained, for example, by performing statistical analysis. The statistical analysis may be performed using machine learning.

[0055] Examples of the method for performing the statistical analysis include multivariate analysis, correlation analysis, regression analysis, factor analysis, etc. Among these, multivariate analysis is preferred. By using multivariate analysis, information about nucleic acids or proteins can be obtained more accurately based on the detected Raman scattered light (more specifically, the obtained Raman spectrum). In addition, when the solution contains multiple types of nucleic acid molecules or multiple types of protein molecules, by using multivariate analysis, it is possible to easily and accurately obtain information about multiple types of nucleic acid molecules or multiple types of protein molecules collectively.

[0056] Examples of multivariate analysis methods include principal component analysis, sparse principal component analysis, nonnegative matrix factorization, cluster analysis, independent component analysis, linear discriminant analysis, logistic regression analysis, Gaussian mixture model, etc. Among these, principal component analysis is preferred from the viewpoint of obtaining information on nucleic acids or proteins more accurately.

[0057] As described above, when excitation light is irradiated onto a solution containing multiple types of nucleic acid molecules or multiple types of protein molecules in step S20, it is preferable to collectively obtain information about the multiple types of nucleic acid molecules or multiple types of protein molecules in the solution in this step.

[0058] In addition, as described above, when the solution contains nucleic acid, it is preferable to obtain information about nucleic acid in this step. The information about nucleic acid is preferably information about RNA, more preferably information about microRNA (miRNA) or tumor circulating RNA (ctRNA), and even more preferably information about microRNA (miRNA).

[0059] 2.Judgment method Fig. 3 is a flow chart showing a determination method according to one embodiment of the present invention. As shown in Fig. 3, the determination method according to one embodiment of the present invention is a method for determining the presence or absence of cancer cells, and includes the steps of (1) obtaining first information on a nucleic acid or a protein contained in a specimen obtained from a subject by the above-mentioned information obtaining method (step S110), and (2) determining whether or not the specimen contains the nucleic acid or the protein derived from a cancer cell based on the first information (step S130). The determination method according to one embodiment of the present invention may further include, as an optional step, a step of obtaining second information on a nucleic acid or a protein contained in a specimen obtained from a healthy subject by the above-mentioned information obtaining method (step S120).

[0060] 2-1. Step of acquiring first information (step S110) In this step, first information on nucleic acids or proteins contained in a sample obtained from a subject is obtained by the above-mentioned information obtaining method.

[0061] The subject is, for example, a human, a monkey, a cow, a pig, etc., and is preferably a human. The types of specimens are, for example, blood (serum, plasma), urine, nasal fluid, saliva, feces, body cavity fluid (cerebrospinal fluid, ascites, pleural effusion, etc.), and various biological tissues.

[0062] From the viewpoint of making it easier to determine the presence or absence of cancer cells, the first information is preferably information relating to nucleic acids, more preferably information relating to RNA, even more preferably information relating to microRNA (miRNA) or blood tumor circulating RNA (ctRNA), and particularly preferably information relating to microRNA (miRNA).

[0063] 2-2. Step of acquiring second information (step S120) The determination method according to this embodiment preferably includes a step of acquiring second information on a nucleic acid or a protein contained in a sample obtained from a healthy subject by the above-mentioned information acquiring method.

[0064] Obtaining the second information in this step enables comparison with the first information obtained in step S110. This makes it possible to determine in the determination step (step S130) described below that the specimen contains a nucleic acid or protein derived from a cancer cell, for example, when the first information and the second information are not similar, facilitating the determination.

[0065] In this specification, the term "healthy subject" refers to a subject who is not found to have cancer. The subject is the same as that described in step S110. The specimen is the same as that described in step S110.

[0066] From the viewpoint of making it easier to determine the presence or absence of cancer cells, the second information is preferably information regarding nucleic acids, more preferably information regarding RNA, even more preferably information regarding microRNA (miRNA) or blood tumor circulating RNA (ctRNA), and particularly preferably information regarding microRNA (miRNA).

[0067] Furthermore, from the viewpoint of making it easier to determine the presence or absence of cancer cells, the above information is preferably information obtained by statistical analysis, more preferably information obtained by multivariate analysis, and even more preferably information obtained by principal component analysis.

[0068] 2-3. Judging step (step S130) In this step, it is determined whether or not the specimen contains the nucleic acid or the protein derived from cancer cells, based on the first information.

[0069] The method of the determination is not particularly limited, and for example, when the sample prepared in advance contains a nucleic acid or a protein derived from a cancer cell, the determination can be made by comparing information on the nucleic acid or the protein with the first information. Also, when the determination method according to the present embodiment includes a step of acquiring second information (step S120), the determination can be made by comparing the first information with the second information.

[0070] 3. Information acquisition device FIG. 4 is a schematic diagram showing the configuration of an information acquisition device 200 according to one embodiment of the present invention. As shown in FIG. 4, the information acquisition device 200 is a device for acquiring information about a nucleic acid or a protein by surface-enhanced Raman scattering spectroscopy, and includes an analysis device 100 having a substrate 110 and a plurality of metal nanostructures 120 arranged at a distance from each other so that a plurality of nanogaps 120a are formed on the substrate 110, an irradiation unit 210 for irradiating an excitation light E onto a solution L containing the nucleic acid or the protein provided in a region including the plurality of nanogaps 120a of the analysis device 100, a detection unit 250 for detecting Raman scattered light R generated from the solution L by irradiation with the excitation light E, and an information acquisition unit 260 for acquiring information about the nucleic acid or the protein in the solution L based on the detected Raman scattered light R. The above-mentioned information acquisition method can be implemented using the information acquisition device 200. The analysis device 100 is the same as that described in the above-mentioned information acquisition method.

[0071] In this embodiment, the information acquisition device 200 includes a mounting unit 220, an optical system 230, and a spectroscopic unit 240, which are optional components.

[0072] The irradiation unit 210 irradiates the solution L containing the nucleic acid or the protein, which is provided in a region including the plurality of nanogaps 120a of the analysis device 100, with excitation light E. The excitation light irradiated from the irradiation unit 210 is preferably light of a substantially single wavelength. The type of excitation light is not particularly limited, but is preferably laser light from the viewpoint of further increasing the intensity of the Raman scattering light R. The type of the light source of the irradiation unit 210 is not particularly limited. The light source of the irradiation unit 210 is, for example, a laser diode.

[0073] The mounting part 220 supports the analytical device 100 at a predetermined position. There is no particular limitation on the configuration of the mounting part 220. The mounting part 220 is, for example, a stage on which the analytical device 100 is placed.

[0074] The optical system 230 guides the excitation light E emitted from the irradiation unit 210 to the analysis device 100, and guides the Raman scattered light R from the analysis device 100 to the spectroscopic unit 240. The configuration of the optical system 230 is not particularly limited as long as it can perform the above-mentioned functions. Although FIG. 4 shows a beam splitter 231 that transmits the excitation light E and reflects the Raman scattered light R, and a lens (condenser lens) 232, the configuration of the optical system 230 is not limited thereto. For example, the optical system 230 may further include various filters such as an excitation light cut filter for removing Rayleigh scattered light, various lenses such as a collimator lens, and the like.

[0075] Furthermore, the lens 232 may be an objective lens. In this case, it is preferable that the objective lens is in contact with the solution L from the viewpoint of obtaining information related to nucleic acids or proteins more accurately.

[0076] The spectroscopic section 240 disperses the Raman scattered light R from the analytical device 100. From the viewpoint of obtaining information on the measured molecules from the Raman scattered light R more accurately, it is preferable to resolve the Raman scattered light R into wave numbers. Therefore, the spectroscopic section 240 disperses the Raman scattered light R. The type of the spectroscopic section 240 is not particularly limited. The spectroscopic section 240 is, for example, a polychromator including a diffraction grating.

[0077] The detection unit 250 detects the Raman scattered light R dispersed by the spectroscopic unit 240. The type of the detection unit 250 is not particularly limited as long as it can appropriately detect the Raman scattered light, and can be appropriately selected depending on the wavelength (wave number) range to be detected. The detection unit 250 is, for example, a CCD detector.

[0078] The information acquisition unit 260 acquires information about the nucleic acid or protein in the solution L based on the Raman scattered light R detected by the detection unit 250. The method by which the information acquisition unit 260 acquires the information is not particularly limited, and the information can be acquired by performing statistical analysis, for example. The statistical analysis may be performed using machine learning. Of the statistical analyses, it is preferable to perform multivariate analysis. By using the multivariate analysis method, it is possible to acquire information about the nucleic acid or protein more accurately based on the detected Raman scattered light (more specifically, the obtained Raman spectrum). In addition, when the solution contains multiple types of nucleic acid molecules or multiple types of protein molecules, it is possible to easily acquire information about multiple types of nucleic acid molecules or multiple types of protein molecules collectively and accurately by using the multivariate analysis method.

[0079] Examples of multivariate analysis methods include principal component analysis, sparse principal component analysis, nonnegative matrix factorization, cluster analysis, independent component analysis, linear discriminant analysis, logistic regression analysis, Gaussian mixture model, etc. Among these, principal component analysis is preferred from the viewpoint of obtaining information on nucleic acids or proteins more accurately.

[0080] There is no particular limitation on the configuration of the information acquisition unit 260. The information acquisition unit 260 is, for example, a computer connected to the detection unit 250.

[0081] In this embodiment, the determination device may be a device further including a determination unit in addition to the configuration of the information acquiring device 200. Specifically, the determination device acquires first information on nucleic acid or protein contained in a specimen obtained from a subject using the information acquiring device 200, and determines whether or not the specimen contains nucleic acid or protein derived from cancer cells using the determination unit based on the acquired first information.

[0082] (effect) The information acquisition method, determination method, and information acquisition device of this embodiment enable accurate acquisition of information about nucleic acids or proteins in a short time and at low cost by surface-enhanced Raman spectroscopy using an analytical device including a metal nanostructure arranged to form multiple nanogaps. EXAMPLES

[0083] 1. Preparation of analysis device A silicon nitride substrate was prepared, and a resist (ZEP520A, Zeon Corporation) was applied to the surface of the substrate using a spin coater (Mikasa Corporation). Next, using a photomask, only the positions of the applied photosensitive resin composition where the metal nanostructure was to be formed were irradiated with an electron beam. Thereafter, the substrate coated with the resist was immersed in a developer (ZED-N50, Zeon Corporation), and the portions of the resist irradiated with the electron beam were removed to form a resist pattern on the surface of the substrate.

[0084] A gold film with a thickness of 30 nm was formed by sputtering on the surface of the substrate on which the resist pattern was formed (the surface of the resist and the area where the resist was removed).Then, the substrate was immersed in an anisole solution to remove the resist pattern.As a result, a metal nanostructure was formed on the substrate, and an analytical device was produced.

[0085] The shape and arrangement of the formed metallic nanostructures were similar to those shown in the microscopic image of FIG. 2B, with the metallic nanostructures having a height of 30 nm and multiple nanogaps having a width of 3 nm.

[0086] 2. Obtaining information about microRNA (miRNA) (For solution 1 and solution 2) Solution 1 containing microRNA1 (24mer-miRNA (base sequence: all A)) (solvent: TE buffer, concentration 2.0 × 10 -4 mol / L) and solution 2 containing microRNA2 (24mer-miRNA (base sequence: all C)) (solvent: TE buffer, concentration 2.0 × 10 -4mol / L) were prepared.

[0087] The prepared solution 1 was provided to an area including multiple nanogaps of the analytical device fabricated above. Using an apparatus with a configuration similar to that of Figure 4, solution 1 was irradiated with excitation light (laser light, wavelength 785 nm, intensity 10.0 mW) for 10 seconds, and the Raman scattered light generated from solution 1 was detected. For solution 1, detection of Raman scattered light was performed 367 times.

[0088] The same procedure was carried out for solution 2. For solution 2, Raman scattering light was detected 381 times.

[0089] FIG. 5A shows the Raman spectrum of 367 obtained from solution 1, and FIG. 5B shows the Raman spectrum of 381 obtained from solution 2.

[0090] Principal component analysis was performed on the obtained Raman spectra, and dot plots were obtained with principal component 1 (PC1) and principal component 2 (PC2) as axes. This allowed information about the microRNA contained in each solution to be obtained. The obtained dot plots are shown in Figure 6. As shown in Figure 6, the distributions are clearly different between solution 1 and solution 2, indicating that the microRNA contained in each solution is different.

[0091] The time required to obtain information about the microRNA contained in each solution was within 60 minutes, which was shorter than the time required for obtaining the information using conventional analysis methods (see Non-Patent Documents 1 to 3). The cost of obtaining information about the microRNA contained in each solution was less than 20,000 yen, which was cheaper than the time required for obtaining the information using conventional analysis methods (see Non-Patent Documents 1 to 3).

[0092] (For solutions 3 and 4) Solution 3 containing microRNA3 (24mer-miRNA (base sequence: A × 12-C × 12)) (solvent: TE buffer, concentration 2.0 × 10 -4Solution 4 (solvent: TE buffer, total concentration 2.0 × 10 mol / L) containing equal amounts of microRNA4-1 (24mer-miRNA (base sequence: all A)) and microRNA4-2 (24mer-miRNA (base sequence: all C)). -4 mol / L) were prepared.

[0093] The prepared solution 3 was provided to an area including multiple nanogaps of the analytical device fabricated above. Using an apparatus with a configuration similar to that of Fig. 4, solution 3 was irradiated with excitation light (laser light, wavelength 785 nm, intensity 10.0 mW) for 10 seconds, and Raman scattered light generated from solution 3 was detected. For solution 3, detection of Raman scattered light was performed 400 times.

[0094] The same procedure was carried out for solution 4. For solution 4, detection of Raman scattered light was carried out 400 times.

[0095] Principal component analysis was performed on the obtained Raman spectra, and dot plots were obtained with principal component 1 (PC1) and principal component 2 (PC2) as axes. This allowed us to obtain information about the microRNA contained in each solution. The obtained dot plots are shown in Figure 7.

[0096] The same operations as described above were carried out for solutions 3 and 4, except that a commercially available SERS substrate (Wavelet, manufactured by NIDEK, not including Au nanogap periodic structure (metal nanostructure)) was used instead of the analytical device prepared above. Figure 8 shows a dot plot by principal component analysis obtained using the commercially available SERS substrate.

[0097] As shown in Figure 8, when a commercially available SERS substrate was used, overlapping regions occurred in the distribution of the microDNA contained in solutions 3 and 4, and information that could distinguish between these types could not be obtained. In contrast, as shown in Figure 7, when the analytical device prepared above was used, the types of microDNA contained in solutions 3 and 4 could be distinguished, and information on the microRNA contained in each solution could be accurately obtained. [Industrial Applicability]

[0098] INDUSTRIAL APPLICABILITY The information acquiring method, determination method and information acquiring device according to the present invention are useful, for example, for determining the presence or absence of cancer cells. [Explanation of symbols]

[0099] 100 Analysis Devices 110 Substrate 120 Metal Nanostructures 200 Information acquisition device 210 Irradiation unit 220 Placement section 230 Optical system 240 Spectroscopic section 250 Detector 260 Information Acquisition Department

Claims

1. 1. A method for obtaining information about a nucleic acid or a protein by surface-enhanced Raman scattering spectroscopy, comprising: Providing an analytical device having a substrate and a plurality of metallic nanostructures spaced apart from one another on the substrate such that a plurality of nanogaps are formed; Irradiating an excitation light onto a solution containing the nucleic acid or the protein provided in a region of the analysis device that includes the plurality of nanogaps; detecting Raman scattered light generated from the solution by irradiation with the excitation light; obtaining information about the nucleic acid or the protein in the solution based on the detected Raman scattered light; (c) a method of obtaining information;

2. The information acquisition method according to claim 1 , wherein the shapes of the plurality of metallic nanostructures are substantially identical.

3. The planar shape of each of the plurality of metallic nanostructures is a polygon; the plurality of metallic nanostructures are disposed on the substrate such that each of the plurality of nanogaps is formed between corners of the polygon; The information acquisition method according to claim 1 .

4. The information acquisition method according to claim 1 , wherein the width of each of the nanogaps is not less than 1 nm and not more than 20 nm.

5. The information acquisition method according to claim 1 , wherein the height of each of the metallic nanostructures is not less than 1 nm and not more than 100 nm.

6. In the irradiating step, the solution containing the plurality of types of the nucleic acid molecules or the plurality of types of the protein molecules is irradiated with excitation light, In the acquiring step, information on the multiple types of nucleic acid molecules or the multiple types of protein molecules in the solution is acquired collectively. The information acquisition method according to claim 1 .

7. The information acquiring method according to claim 1 , wherein in the acquiring step, the information is acquired by performing a statistical analysis.

8. The information acquiring method according to claim 7 , wherein in the acquiring step, the information is acquired using a principal component analysis method.

9. The solution contains the nucleic acid, The information is information about the nucleic acid. The information acquisition method according to claim 1 .

10. The information acquisition method according to claim 9 , wherein the nucleic acid is RNA.

11. The information acquisition method according to claim 10 , wherein the RNA is a microRNA.

12. A method for determining the presence or absence of cancer cells, comprising: A step of acquiring first information on a nucleic acid or a protein contained in a sample obtained from a subject by the information acquiring method according to any one of claims 1 to 11; determining whether or not the specimen contains the nucleic acid or the protein derived from a cancer cell based on the first information; A method for determining whether or not a

13. The method further comprises a step of obtaining second information regarding a nucleic acid or a protein contained in a sample obtained from a healthy subject by the information obtaining method, the step of determining includes comparing the first information with the second information to determine whether or not the specimen obtained from the subject contains the nucleic acid or the protein derived from a cancer cell; The method of claim 12.

14. An apparatus for obtaining information about a nucleic acid or a protein by surface-enhanced Raman scattering spectroscopy, comprising: An analytical device having a substrate and a plurality of metal nanostructures arranged on the substrate at a distance from each other so that a plurality of nanogaps are formed; an irradiation unit provided in a region including the plurality of nanogaps of the analysis device for irradiating a solution including the nucleic acid or the protein with excitation light; a detection unit for detecting Raman scattered light generated from the solution by irradiation with the excitation light; an information acquiring unit for acquiring information about the nucleic acid or the protein in the solution based on the detected Raman scattered light; An information acquisition device comprising:

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