Information acquisition method, determination method, and information acquisition device

Abstract

This information acquisition method includes a step of preparing an analysis device having a plurality of metal nanostructures arranged apart from each other so that a plurality of nanogaps are formed on a substrate, a step of irradiating a solution containing a nucleic acid or a protein with excitation light, the solution being provided in a region of the analysis device including the plurality of nanogaps, a step of detecting Raman scattered light occurring from the solution due to the irradiation with the excitation light, and a step of acquiring information about the nucleic acid or the protein in the solution on the basis of the detected Raman scattered light. The shape of each of the plurality of metal nanostructures in a planar view is a triangle.

Description

Information acquisition method, determination method, and information acquisition apparatus

[0001] The present invention relates to an information acquisition method, a determination method, and an information acquisition apparatus.

[0002] Nucleic acids include deoxyribonucleic acid (DNA) containing genetic information, and ribonucleic acid (RNA) including messenger RNA (mRNA) that transcribes the genetic information. Then, based on the genetic information (base sequence) of DNA transcribed by mRNA, proteins are synthesized in ribosomes. Nucleic acids or proteins in body fluids are used as cancer markers, and cancer detection can be performed by distinguishing between nucleic acids or proteins in body fluids of healthy subjects and nucleic acids or proteins in body fluids of cancer patients. Therefore, acquiring information related to nucleic acids or proteins in a specimen obtained from a subject is important for determining the presence or absence of cancer cells.

[0003] Various methods are known for acquiring information related to nucleic acids. For example, next-generation sequencing (NGS method) is known for analyzing nucleic acid base sequences (Non-Patent Document 1). In the NGS method, for example, a fragment of a target nucleic acid molecule is reacted with one base at a time that forms a complementary pair to the bases constituting the nucleic acid molecule, and the fluorescence generated when the reaction occurs is detected, whereby the base sequence of the nucleic acid molecule can be analyzed.

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

[0005] Furthermore, an analysis method using a microarray method is also known (Non-Patent Document 3). In this method, using a substrate on which probes that complementary bind to bases constituting a nucleic acid molecule are arrayed, a fluorescence-labeled target nucleic acid molecule is reacted with the probes. Then, the binding position between the nucleic acid molecule and the probe is detected by fluorescence, and the base sequence of the target nucleic acid molecule can be analyzed.

[0006] “An overview of recent RNA-Seq publications featuring Illumina technology” [Retrieved on February 17, 2020] <URL: https: / / www.illumina.com / content / dam / illumina-marketing / documents / products / research_reviews / rna-sequencing-methods-review-web.pdf> SA Deepak, et al., “Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes”, Current Genomics, vol. 8, pp. 234-251. Kunihisa Nagino, et al., “Ultrasensitive DNA Chip: Gene Expression Profile Analysis without RNA Amplification”, The Journal of Biochemistry, vol. 139, pp. 697-703.

[0007] As described in Non-Patent Documents 1-3, analytical methods for obtaining information about nucleic acids are known. However, these methods take several hours to several days to perform the analysis, making it difficult to obtain information about nucleic acids in a short time.

[0008] Furthermore, these methods involve high costs of several hundred thousand yen for preparing the bases to react with nucleic acids (Non-Patent Documents 1-2), fluorescent molecules for fluorescently labeling nucleic acids (Non-Patent Documents 1-3), and substrates on which probes are arranged (Non-Patent Document 3), making it difficult to obtain information on nucleic acids at a low cost.

[0009] Furthermore, there is a need to obtain information about proteins quickly and inexpensively in order to determine the presence or absence of cancer cells.

[0010] Therefore, the inventors considered using surface-enhanced Raman spectroscopy to obtain information on nucleic acids and proteins quickly and inexpensively. However, conventional surface-enhanced Raman spectroscopy could not accurately obtain information on nucleic acids and proteins.

[0011] The object of the present invention is to provide an information acquisition method and information acquisition apparatus that can accurately acquire information about nucleic acids or proteins in a short time and at low cost, as well as a determination method using the above method.

[0012] One aspect of the present invention for achieving the above objective relates to the following information acquisition methods [1] to

[11] : [1] A method for acquiring information about nucleic acids or proteins by surface-enhanced Raman scattering spectroscopy, comprising: preparing an analytical device having a substrate and a plurality of metal nanostructures spaced apart from each other on the substrate so as to form a plurality of nanogaps; irradiating a solution containing the nucleic acids or proteins, provided to a region of the analytical device including the plurality of nanogaps, with excitation light; detecting Raman scattered light generated from the solution by irradiation with the excitation light; and acquiring information about the nucleic acids or proteins in the solution based on the detected Raman scattered light, wherein the planar shape of each of the plurality of metal nanostructures is triangular, and the plurality of metal nanostructures are arranged on the substrate such that each of the plurality of nanogaps is formed between the corners of the triangles. [2] The information acquisition method according to [1], wherein the shapes of the plurality of metal nanostructures are substantially the same. [3] The information acquisition method according to [1] or [2], wherein the width of each of the plurality of nanogaps is 1 nm or more and 20 nm or less. [4] The information acquisition method according to any one of [1] to [3], wherein the height of each of the plurality of metal nanostructures is 1 nm or more and 100 nm or less. [5] The information acquisition method according to any one of [1] to [4], wherein the length of each side of the triangle is 50 nm or more and 200 nm or less. [6] The information acquisition method according to any one of [1] to [5], wherein in the irradiation step, the excitation light is irradiated onto the solution containing a plurality of nucleic acids or a plurality of proteins, and in the acquisition step, information concerning the plurality of nucleic acids or a plurality of proteins in the solution is acquired collectively. [7] The information acquisition method according to any one of [1] to [6], wherein in the acquisition step, the information is acquired by performing statistical analysis. [8] The information acquisition method according to [7], wherein in the acquisition step, the information is acquired using principal component analysis. [9] The information acquisition method according to any one of [1] to [8], wherein the solution contains the nucleic acid, and the information is information concerning the nucleic acid.

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

[11] The information acquisition method according to

[10] , wherein the RNA is microRNA.

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

[12] and

[13] below.

[12] A method for determining the presence or absence of cancer cells, comprising: a step of obtaining first information relating to nucleic acids or proteins contained in a sample obtained from a subject by an information acquisition method described in any one of [1] to

[11] ; and a step of determining, based on the first information, whether or not the sample contains nucleic acids or proteins derived from cancer cells.

[13] The determination method according to

[12] , further comprising a step of obtaining second information relating to nucleic acids or proteins contained in a sample obtained from a healthy person by an information acquisition method, wherein the determination step involves comparing the first information and the second information to determine whether or not the sample obtained from the subject contains nucleic acids or proteins derived from cancer cells.

[0014] Another aspect of the present invention for achieving the above objective relates to the apparatus described in

[14] below.

[14] An apparatus for acquiring information about nucleic acids or proteins by surface-enhanced Raman scattering spectroscopy, comprising: an analytical device having a substrate and a plurality of metal nanostructures spaced apart from each other on the substrate so as to form a plurality of nanogaps; an irradiation unit provided in a region of the analytical device including the plurality of nanogaps for irradiating a solution containing the nucleic acids or proteins with excitation light; a detection unit for detecting Raman scattered light generated from the solution by irradiation with the excitation light; and an information acquisition unit for acquiring information about the nucleic acids or proteins in the solution based on the detected Raman scattered light, wherein the planar shape of each of the plurality of metal nanostructures is triangular, and the plurality of metal nanostructures are arranged on the substrate such that each of the plurality of nanogaps is formed between the corners of the triangles.

[0015] The present invention provides an information acquisition method and information acquisition apparatus that can accurately obtain information on nucleic acids or proteins in a short time and at low cost, as well as a determination method using the above method.

[0016] Figure 1 is a flowchart of an information acquisition method according to one embodiment of the present invention. Figure 2A is a partially enlarged plan view of an analysis device according to one embodiment of the present invention, and Figure 2B is a partially enlarged cross-sectional view taken along line B-B in Figure 2A. Figures 3A to 3C show examples of plan view shapes of metal nanostructures. Figure 4 is a flowchart of a determination method according to one embodiment of the present invention. Figure 5 is a schematic diagram showing the configuration of an information acquisition device according to one embodiment of the present invention. Figure 6A is an electron microscope image of an example of the analysis device of the example, and Figure 6B is an electron microscope image of an example of the analysis device of the comparative example. Figure 7 is a graph showing the relationship between the size of the metal nanostructure and the intensity of the Raman peak for the analysis device of the example and the analysis device of the comparative example. Figure 8A is the Raman spectrum of sample solution 1 obtained using the analysis device of the example, and Figure 8B is the Raman spectrum of sample solution 2 obtained using the analysis device of the example. Figure 9A is the Raman spectrum of sample solution 1 obtained using the analysis device of the comparative example, and Figure 9B is the Raman spectrum of sample solution 2 obtained using the analysis device of the comparative example. Figure 10 is a dot plot showing the results of principal component analysis of the Raman spectrum obtained using the analysis device of the example. Figure 11 is a dot plot showing the results of principal component analysis of the Raman spectrum obtained using the analysis device of the comparative example.

[0017] The embodiments of the present invention will be described in detail below. In this specification, the lower and upper limits of a numerical range indicated by "~" are included within that range. However, the present invention is not limited to the following embodiments.

[0018] 1. Information Acquisition Method Figure 1 is a flowchart of an information acquisition method according to one embodiment of the present invention.

[0019] As shown in Figure 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: (1) a step of preparing an analytical device having a plurality of metal nanostructures (step S10); (2) a step of irradiating a solution containing nucleic acids or proteins with excitation light (step S20); (3) a step of detecting Raman scattered light generated from the solution (step S30); and (4) a step of acquiring information about nucleic acids or proteins in the solution (step S40). Each step will be described below.

[0020] (1) Step of preparing the analysis device (Step S10) In this step, an analysis device is prepared having a substrate and a plurality of metal nanostructures arranged spaced apart from each other on the substrate so as to form a plurality of nanogaps. In this embodiment, the plan view shape of each of the plurality of metal nanostructures is triangular. The plurality of metal nanostructures are arranged on the substrate so as to form each of the plurality of nanogaps between the corners of the triangles.

[0021] Figure 2A is a partially enlarged plan view of the analysis device 100 according to this embodiment, and Figure 2B is a partially enlarged cross-sectional view taken along the line B-B in Figure 2A. As shown in Figures 2A and 2B, the analysis device 100 has a substrate 110 and a plurality of metal nanostructures 120 arranged spaced apart from each other on the substrate 110. Nanogaps 120a exist between the plurality of metal nanostructures 120. In this specification, "nanogaps" refers to the shortest gap between adjacent plurality of metal nanostructures 120, and the gap having a width on the order of nanometers.

[0022] The substrate 110 supports a plurality of metal nanostructures 120. The material of the substrate 110 is not particularly limited, but an insulator (dielectric) is preferred. Examples of materials for the substrate 110 include glass, silicon nitride, silicon dioxide, and silicon. Of these, silicon nitride is preferred for the substrate 110. The substrate 110 may be a gel, but it is preferable that it is not a gel from the viewpoint of precisely controlling the size (width g) of the plurality of nanogaps 120a. The shape of the substrate 110 is not particularly limited, but it is preferable that the surface of the substrate 110 on which the plurality of metal nanostructures 120 are arranged is planar.

[0023] Multiple metal nanostructures 120 are arranged spaced apart on the substrate 110 so as to form multiple nanogaps 120a. Each of the multiple metal nanostructures 120 is a triangular prism. That is, the plan view shape of each of the multiple metal nanostructures 120 is a triangle. The type of triangle is not particularly limited. Examples of triangles include equilateral triangles, isosceles triangles, right triangles, acute triangles, and obtuse triangles. It is preferable that the shapes of the multiple metal nanostructures 120 are substantially identical. By doing so, the intensity of Raman scattered light generated from the solution containing nucleic acids or proteins can be increased, making it easier to obtain more accurate information about nucleic acids or proteins by detecting the Raman scattered light.

[0024] In this specification, the concept of "triangle" includes not only the general definition of a triangle (a polygon consisting of three points that are not on the same straight line and three line segments connecting them), but also figures with shapes similar to triangles. For example, the angles of the triangle may be rounded, or the sides of the triangle (lines connecting two angles) may be curved, as long as the three angles can be identified. Specifically, the shape of the metal nanostructure 120 may be a general triangle as shown in Figure 3A, a figure similar to a triangle with rounded angles as shown in Figure 3B, or a figure similar to a triangle with curved sides (for example, a Reuleaux triangle) as shown in Figure 3C.

[0025] In the example shown in Figure 2A, the planar shapes of the multiple metal nanostructures 120 are substantially identical, and each is an equilateral triangle.

[0026] The material of the multiple metal nanostructures 120 is not particularly limited as long as it is a metal that can induce localized surface plasmon resonance, for example, gold, silver, copper, aluminum, etc. Of these, the material is preferably gold. The multiple metal nanostructures 120 may have other substances (e.g., organic molecules) fixed to them or be coated with other substances, but it is preferable that they do not have other substances fixed to them or be coated with other substances. In other words, it is preferable that the material of the metal nanostructures 120 is exposed.

[0027] Multiple metal nanostructures 120 are arranged such that nanogaps 120a are formed between the metal nanostructures 120 that are spaced apart from each other. As mentioned above, the planar shape of the multiple metal nanostructures 120 is triangular. The nanogaps 120a are formed between the corners of the triangles. That is, the multiple metal nanostructures 120 are arranged on the substrate 110 such that each of the multiple nanogaps 120a is formed between the corners of the triangles. The nanogaps 120a may be formed between two corners, three corners, or four or more corners. By doing so, the intensity of Raman scattered light generated from a solution containing nucleic acids or proteins can be increased, making it easier to obtain information about nucleic acids or proteins more accurately by detecting the Raman scattered light. The opposing sides of two adjacent triangles may be parallel, but it is preferable that they are not parallel.

[0028] The height h of the multiple metal nanostructures 120 (see Figure 2B) is preferably between 1 nm and 100 nm, more preferably between 10 nm and 50 nm, and even more preferably between 20 nm and 40 nm. Having the height h within this range makes it easier for nucleic acid or protein molecules to penetrate into the nanogap 120a. This facilitates the generation of surface-enhanced Raman scattering by irradiation with excitation light, making it easier to detect the Raman scattered light. The height h can be determined by observing the analysis device 100 with an electron microscope or atomic force microscope.

[0029] The width g of each of the multiple nanogaps 120a (see Figure 2B) is preferably 1 nm to 20 nm, more preferably 2 nm to 20 nm, even more preferably 4 nm to 16 nm, and particularly preferably 6 nm to 10 nm. A width g of 1 nm or more makes it easier for nucleic acid or protein molecules to enter the multiple nanogaps 120a. Furthermore, a width g of 20 nm or less allows for a greater increase in the intensity of Raman scattered light generated from the solution containing nucleic acid or protein. For these reasons, having a width g within the above range makes it easier to obtain information about nucleic acid or protein. The width g can be determined by observing the surface of the analysis device 100 with an electron microscope.

[0030] The size of the multiple metal nanostructures 120 is not particularly limited and can be appropriately set according to the wavelength of the excitation light. As mentioned above, the planar shape of the multiple metal nanostructures 120 is triangular. The length of one side s of this triangle (see Figure 2A) is not particularly limited, but is, for example, 50 to 200 nm, preferably 130 to 160 nm, and more preferably 140 to 150 nm. Here, if the corners of the triangle are rounded as shown in Figure 3B, the length of one side of this triangle means the length of one side of a triangle with unrounded corners (a triangle formed by extending the straight line portion of the figure shown in Figure 3B) as shown in Figure 3A.

[0031] The method for manufacturing the analysis device 100 is not particularly limited. For example, the analysis device 100 can be manufactured by (I) forming a resist pattern on the surface of a substrate 110, (II) forming a metal film on the surface of the substrate 110 on which the resist pattern is formed, and (III) removing the resist pattern together with the metal film covering the resist pattern. Steps (I) to (III) described below will be explained below.

[0032] (Process for forming a resist pattern) In this process, after applying a resist to the substrate 110, the substrate is exposed using a photomask so that openings are formed at the positions where multiple metal nanostructures 120 should be formed, and the resist pattern is formed using a developer.

[0033] The above resist pattern may be formed using a positive-type photosensitive resin composition or a negative-type photosensitive resin composition.

[0034] The positive-type and negative-type photosensitive resin compositions that can be used in this process are known resin compositions used as lift-off resists.

[0035] (Step to form a metal film) In this step, a metal film is formed on the surface of the substrate 110 on which the resist pattern is formed (specifically, the openings formed between the surface of the resist and the resist pattern). The metal film formed in the openings remains as a metal nanostructure 120 after the resist pattern is removed in the step to remove the resist pattern described later.

[0036] The method for forming the metal film is not particularly limited, but examples include vapor deposition and sputtering. Examples of metals used as materials for the metal film include the same metals as those used for the metal nanostructures described above.

[0037] (Step to remove the resist pattern) In this step, the resist pattern is removed along with the metal film covering the resist pattern. Specifically, this step is performed using a stripping solution for removing the resist pattern.

[0038] In this step, for example, the resist pattern can be removed by a method of immersing the substrate in a stripping solution.

[0039] Examples of the stripping solution include anisole, dimethyl sulfoxide, and the like.

[0040] (2) Step of irradiating excitation light (Step S20) In this step, excitation light is irradiated to a solution containing a nucleic acid or a protein provided in a region including a plurality of nanogaps 120a of the analysis device 100.

[0041] The nucleic acids and proteins from which information is to be obtained are not particularly limited. Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The number of nucleotide units constituting the nucleic acid is not particularly limited. Nucleic acids also include oligonucleotides and polynucleotides. RNA includes messenger RNA (mRNA) and non-coding RNA (ncRNA). ncRNA includes microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA). Further, the number of amino acid residues constituting a protein is not particularly limited. Proteins include oligopeptides and polypeptides.

[0042] In this step, in the step of obtaining information described later (Step S40), after irradiating a solution containing a plurality of types of nucleic acids or a plurality of types of proteins with excitation light, information on the plurality of types of nucleic acids or the plurality of types of proteins in the solution may be collectively obtained. This makes it possible to obtain information on a plurality of types of nucleic acids or a plurality of types of proteins in a shorter time.

[0043] When determining the presence or absence and / or type of cancer cells based on information obtained in the step of obtaining information described later (Step S40), the solution preferably contains a nucleic acid. In this case, the information obtained in the step of obtaining information described later (Step S40) is preferably information related to the nucleic acid.

[0044] Preferably, the nucleic acid is RNA, more preferably microRNA (miRNA) or circulating tumor RNA (ctRNA) in blood, and even more preferably microRNA (miRNA). In the step of acquiring information described below (step S40), acquiring information related to microRNA (miRNA) and circulating tumor RNA (ctRNA) enables easy determination of the presence or absence and / or type of cancer cells based on the acquired information.

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

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

[0047] Preferably, the excitation light is substantially light of a 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, more preferably 750 nm or more and 800 nm or less. In addition, the type of excitation light is not particularly limited, but from the viewpoint of further increasing the intensity of Raman scattered light, laser light is preferable. The excitation light can be irradiated using a known light source (for example, a laser diode).

[0048] The information acquisition method according to the present embodiment may further include, before the present step (step S20), a step of providing a solution containing a nucleic acid or a protein to a region including a plurality of nanogaps 120a of the analysis device 100.

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

[0050] When a solution containing nucleic acids or proteins is provided to a region of the analysis device 100 that includes multiple nanogaps 120a, it is believed that nucleic acid or protein molecules enter the nanogaps 120a. In this state, when the provided solution is irradiated with excitation light, localized surface plasmon resonance occurs with the multiple nanogaps 120a acting as hot spots (enhanced electric fields). As a result, the intensity of the Raman scattered light generated from the solution increases significantly (Surface-Enhanced Raman Scattering (SERS)). Thus, in this invention, by applying surface-enhanced Raman scattering spectroscopy to nucleic acids or proteins using the analysis device 100, it is possible to detect Raman scattered light with higher sensitivity than when using an analysis device that does not include multiple metal nanostructures forming multiple nanogaps, or when using an analysis device that includes multiple metal nanostructures whose planar shape is not triangular. This makes it possible to accurately obtain information about nucleic acids or proteins.

[0051] The method for detecting Raman scattered light is not particularly limited, and for example, Raman scattered light can be detected using a CCD detector. From the viewpoint of obtaining information about nucleic acids or proteins more accurately from Raman scattered light, in this step, the Raman scattered light generated from the above solution may be spectrally analyzed and the spectrally analyzed Raman scattered light may be detected.

[0052] This process allows you to obtain a Raman spectrum of nucleic acids or proteins in the above solution.

[0053] (4) Step to acquire information (Step S40) In this step, information about nucleic acids or proteins in the solution is acquired based on the Raman scattering light detected in Step S30.

[0054] Specific examples of the above information include information for determining whether or not the solution contains nucleic acids or proteins derived from cancer cells, and information for specifically identifying the nucleic acids or proteins in the solution.

[0055] The method for obtaining information about nucleic acids or proteins is not particularly limited; for example, the above information can be obtained by performing statistical analysis. The above statistical analysis may be performed using machine learning.

[0056] Examples of methods for performing the above statistical analysis include multivariate analysis, correlation analysis, regression analysis, and factor analysis. Of 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). Furthermore, when the above solution contains multiple types of nucleic acids or multiple types of proteins, using multivariate analysis makes it easier to accurately obtain information about multiple types of nucleic acids or multiple types of proteins together.

[0057] Examples of multivariate analysis methods include principal component analysis, sparse principal component analysis, non-negative matrix factorization, cluster analysis, independent component analysis, linear discriminant analysis, logistic regression analysis, or Gaussian mixture models. Of these, principal component analysis is preferred from the viewpoint of obtaining more accurate information about nucleic acids or proteins.

[0058] As described above, when irradiating a solution containing multiple types of nucleic acids or multiple types of proteins with excitation light in step S20, it is preferable to obtain information about the multiple types of nucleic acids or multiple types of proteins in the solution collectively in this step.

[0059] Furthermore, as described above, when the solution contains nucleic acids, it is preferable to obtain information about the nucleic acids in this step. The information about the nucleic acids is preferably about RNA, more preferably about microRNA (miRNA), more preferably about circulating tumor RNA (ctRNA), and even more preferably about microRNA (miRNA).

[0060] 2. Determination Method Figure 4 is a flowchart of a determination method according to one embodiment of the present invention. As shown in Figure 4, a determination method according to one embodiment of the present invention is a method for determining the presence or absence of cancer cells, and includes (1) a step of obtaining first information (step S110) concerning nucleic acids or proteins contained in a sample obtained from a subject by the information acquisition method described above, and (2) a step of determining whether or not the sample contains nucleic acids or proteins derived from cancer cells 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 (step S120) concerning nucleic acids or proteins contained in a sample obtained from a healthy person by the information acquisition method described above. Each step will be described below.

[0061] (1) Step to acquire first information (Step S110) In this step, first information concerning nucleic acids or proteins contained in the sample obtained from the subject is acquired by the information acquisition method described above.

[0062] The subjects described above are not particularly limited. Examples of subjects include humans, monkeys, cattle, and pigs. Human subjects are preferred. The types of specimens are also not particularly limited. Examples of specimens include blood (serum, plasma), urine, nasal fluid, saliva, feces, body fluids (cerebrospinal fluid, ascites, pleural fluid, etc.), and various biological tissues.

[0063] From the viewpoint of making it easier to determine the presence or absence of cancer cells, the first information described above is preferably information about nucleic acids, more preferably information about RNA, even more preferably information about microRNA (miRNA) or circulating tumor RNA (ctRNA) in the blood, and particularly preferably information about microRNA (miRNA).

[0064] The types of cancer to be assessed are not particularly limited. Examples of cancers include stomach cancer, penile cancer, various bone and soft tissue tumors, liver cancer, thyroid cancer, retroperitoneal tumors, bone metastases, uterine cancer, esophageal cancer, kidney cancer, renal pelvis and ureteral cancer, pancreatic cancer, testicular cancer, prostate cancer, colorectal cancer, multiple myeloma, biliary tract cancer, head and neck cancer, breast cancer, lung cancer, leukemia, skin cancer, adrenal cancer, bladder cancer, ovarian cancer, and lymphoma.

[0065] (2) Step to acquire second information (Step S120) The determination method according to this embodiment preferably includes a step to acquire second information concerning nucleic acids or proteins contained in a sample obtained from a healthy person by the information acquisition method described above.

[0066] By obtaining the second information in this process, it becomes possible to compare it with the first information obtained in step S110. This makes it possible to determine, for example, in the determination process described later (step S130), that the sample contains nucleic acids or proteins derived from cancer cells when the first and second information are not similar, thus facilitating the determination.

[0067] In this specification, "healthy subjects" refers to subjects who are not found to have cancer. The subjects are the same as those described in step S110. The samples are also the same as those described in step S110.

[0068] From the viewpoint of making it easier to determine the presence or absence of cancer cells, the second information described above is preferably information about nucleic acids, more preferably information about RNA, even more preferably information about microRNA (miRNA) or circulating tumor RNA (ctRNA) in the blood, and particularly preferably information about microRNA (miRNA).

[0069] Furthermore, from the viewpoint of making it easier to determine the presence or absence of cancer cells, the above information is preferably obtained by statistical analysis, more preferably by multivariate analysis, and even more preferably by principal component analysis.

[0070] (3) Determination step (step S130) In this step, based on the information in step 1 above, it is determined whether or not the sample contains the nucleic acid or protein derived from cancer cells.

[0071] The method for making the determination described above is not particularly limited. For example, when the sample prepared in advance contains nucleic acids or proteins derived from cancer cells, the determination can be made by comparing information about the nucleic acids or proteins with the first information. Furthermore, if the determination method according to this embodiment includes a step of acquiring second information (step S120), the determination can be made by comparing the first information with the second information.

[0072] 3. Information Acquisition Device Figure 5 is a schematic diagram showing the configuration of an information acquisition device 200 according to one embodiment of the present invention. As shown in Figure 5, the information acquisition device 200 is an apparatus for acquiring information on nucleic acids or proteins by surface-enhanced Raman scattering spectroscopy, and includes an analysis device 100 having a substrate 110 and a plurality of metal nanostructures 120 arranged spaced apart from each other on the substrate 110 so as to form a plurality of nanogaps 120a; an irradiation unit 210 for irradiating a solution L containing the nucleic acid or protein with excitation light E, provided in a region of the analysis device 100 including the plurality of nanogaps 120a; a detection unit 250 for detecting Raman scattered light R generated from the solution L by irradiation with excitation light E; and an information acquisition unit 260 for acquiring information on the nucleic acid or protein in the solution L based on the detected Raman scattered light R. The above-described information acquisition method can be carried out using the information acquisition device 200. The analysis device 100 is the same as that described in the information acquisition method above.

[0073] In this embodiment, the information acquisition device 200 further includes optional components: a mounting section 220, an optical system 230, and a spectroscopic section 240.

[0074] The irradiation unit 210 irradiates the solution L containing the nucleic acid or protein, which is provided to a region of the analysis device 100 including a plurality of nanogaps 120a, with excitation light E. The excitation light irradiated from the irradiation unit 210 is preferably substantially single-wavelength light. The type of excitation light is not particularly limited, but from the viewpoint of further increasing the intensity of Raman scattered light R, it is preferably laser light. The type of light source for the irradiation unit 210 is not particularly limited. The light source for the irradiation unit 210 is, for example, a laser diode.

[0075] The mounting section 220 supports the analysis device 100 in a predetermined position. The configuration of the mounting section 220 is not particularly limited. For example, the mounting section 220 is a stage for mounting the analysis device 100.

[0076] The optical system 230 guides the excitation light E emitted from the irradiation unit 210 to the analysis device 100, and also 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 functions. Figure 5 shows a beam splitter 231 that transmits the excitation light E and reflects the Raman scattered light R, and a lens (focusing lens) 232, but the configuration of the optical system 230 is not limited to this. For example, the optical system 230 may further include various filters such as an excitation light cut filter for removing Rayleigh scattered light, and various lenses such as a collimator lens.

[0077] 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 about nucleic acids or proteins more accurately.

[0078] The spectroscopic unit 240 spectrally analyzes the Raman scattered light R from the analysis device 100. From the viewpoint of obtaining information about the molecule to be measured more accurately from the Raman scattered light R, it is preferable to decompose the Raman scattered light R into wavenumbers. Therefore, the spectroscopic unit 240 spectrally analyzes the Raman scattered light R. The type of spectroscopic unit 240 is not particularly limited. The spectroscopic unit 240 is, for example, a polychromator including a diffraction grating.

[0079] The detection unit 250 detects the Raman scattered light R spectrally separated by the spectroscopic unit 240. The type of detection unit 250 is not particularly limited as long as it can appropriately detect the Raman scattered light, and can be appropriately selected according to the wavelength (wavenumber) range to be detected. The detection unit 250 is, for example, a CCD detector.

[0080] The information acquisition unit 260 acquires information about nucleic acids or proteins in 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 above information is not particularly limited, and for example, the information can be acquired by performing statistical analysis. The above statistical analysis may be performed using machine learning. Among the above statistical analyses, multivariate analysis is preferred. By performing multivariate analysis, information about nucleic acids or proteins can be acquired more accurately based on the detected Raman scattered light (more specifically, the obtained Raman spectrum). Furthermore, when the solution contains multiple types of nucleic acids or multiple types of proteins, using the multivariate analysis method makes it easier to accurately acquire information about multiple types of nucleic acids or multiple types of proteins together.

[0081] Examples of multivariate analysis methods include principal component analysis, sparse principal component analysis, non-negative matrix factorization, cluster analysis, independent component analysis, linear discriminant analysis, logistic regression analysis, or Gaussian mixture models. Of these, principal component analysis is preferred from the viewpoint of obtaining more accurate information about nucleic acids or proteins.

[0082] The configuration of the information acquisition unit 260 is not particularly limited. For example, the information acquisition unit 260 is a computer connected to the detection unit 250.

[0083] In this embodiment, the determination device may be an additional determination unit added to the configuration of the information acquisition device 200. Specifically, the determination device acquires first information regarding nucleic acids or proteins contained in a sample obtained from a subject using the information acquisition device 200, and based on the acquired first information, the determination unit determines whether or not the sample contains nucleic acids or proteins derived from cancer cells.

[0084] 4. Effects The information acquisition method, determination method, and information acquisition device according to this embodiment do not require the reaction of the target nucleic acid or protein with other molecules during the process of acquiring information about nucleic acids or proteins, thus enabling the acquisition of information about nucleic acids or proteins in a shorter time than conventional methods. Furthermore, since there is no need to prepare nucleic acids for capturing nucleic acids and fluorescent molecules for fluorescently labeling nucleic acids, the cost of acquiring information about nucleic acids or proteins can be reduced compared to conventional methods. In other words, the information acquisition method, determination method, and information acquisition device according to this embodiment enable the acquisition of accurate information about nucleic acids or proteins in a short time and at low cost.

[0085] [Example 1] (1) Fabrication of the analysis device A resist (ZEP520A, Zeon Corporation) was applied to the surface of a silicon nitride substrate using a spin coater (Mikasa Corporation). Next, an electron beam was irradiated using a photomask only on the parts of the applied resist where metal nanostructures were to be formed. After that, the substrate coated with the resist was immersed in a developer solution (ZED-N50, Zeon Corporation) to remove the parts of the resist that had been irradiated with the electron beam, thereby forming a resist pattern on the surface of the substrate.

[0086] A 50 nm thick gold film was formed by sputtering on the surface of the substrate on which the above resist pattern was formed (the resist surface and the areas where the resist had been removed). Then, the resist pattern was removed by immersing the substrate in an anisole solution. This formed multiple metal nanostructures on the substrate, and an analytical device was fabricated.

[0087] The planar shape of the metal nanostructure was either a rounded equilateral triangle (example) or a rounded square (comparative example). The side lengths of the equilateral triangles and squares were 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, or 160 nm. Here, the side length of the rounded triangle refers to the side length of the triangle formed by extending the straight portion of each side. Similarly, the side length of the rounded quadrilateral refers to the side length of the quadrilateral formed by extending the straight portion of each side. The height (thickness) of the metal nanostructure was 50 nm. The width of the nanogap between the metal nanostructures was 3 nm.

[0088] Figure 6A is an electron microscope image of an example of the analysis device in the embodiment, and Figure 6B is an electron microscope image of an example of the analysis device in the comparative example. In both the analysis devices in Figure 6A and Figure 6B, the length of one side of the metal nanostructure when viewed from above is 150 nm.

[0089] (2) Evaluation of signal intensity A 4-aminothiophenol monolayer was formed on the analysis device of the example and the analysis device of the comparative example. Using an apparatus with the same configuration as in Figure 5, the monolayer was irradiated with excitation light (laser light, wavelength 785 nm, intensity 12.0 mW) for 1 second, and the Raman scattered light generated from the monolayer was detected.

[0090] Figure 7 shows the size (length of one side when viewed from above) and Raman peak (1080 cm) of the metal nanostructures for the analysis device of the example and the analysis device of the comparative example. －１ This graph shows the relationship with the intensity of the ). Triangular plots represent the results of the analysis device in the example, and square plots represent the results of the analysis device in the comparative example.

[0091] As shown in the graph in Figure 7, when the side length of the metal nanostructure was greater than 100 nm (110 nm or more), the Raman peak intensity was greater when the plan view shape of the metal nanostructure was triangular (analysis device of the example) than when it was rectangular (analysis device of the comparative example). When the side length of the metal nanostructure was between 130 and 160 nm (especially between 140 and 150 nm), the Raman peak intensity was more than twice as greater when the plan view shape of the metal nanostructure was triangular (analysis device of the example) than when it was rectangular (analysis device of the comparative example).

[0092] [Example 2] (1) Fabrication of the analysis device An analysis device was fabricated using the same procedure as in Example 1. The planar shape of the metal nanostructure was an equilateral triangle with rounded corners (Example) or a square with rounded corners (Comparative Example). The length of one side of the equilateral triangle and square was 150 nm. The height (thickness) of the metal nanostructure was 50 nm. The width of the nanogap between the metal nanostructures was 3 nm.

[0093] Figure 6A is an electron microscope image of the analysis device of the example, and Figure 6B is an electron microscope image of the analysis device of the comparative example.

[0094] (2) Sample preparation Extracellular vesicles were isolated from the plasma of lung cancer patients using an exosome isolation kit (MagCapture Exosome Isolation Kit PS Ver.2, Fujifilm Wako Pure Chemical Corporation). MicroRNA (miRNA) was extracted from the obtained extracellular vesicles using an RNA extraction kit (Purified EV MicroRNA Extractor Kit, Fujifilm Wako Pure Chemical Corporation). The obtained microRNA was dissolved in TE buffer and prepared as sample solution 1 (concentration 5.0 × 10⁻⁶). －５ (mol / L)

[0095] Using a similar procedure, extracellular vesicles were isolated from the plasma of colorectal cancer patients, and microRNA was extracted from the extracellular vesicles. The obtained microRNA was dissolved in TE buffer to create sample solution 2 (concentration 5.0 × 10⁻⁶). －５ (mol / L)

[0096] (3) Obtaining information on microRNA Sample solution 1 was provided to a region of the analysis device containing multiple nanogaps. Using an apparatus with the same configuration as in Figure 5, the sample solution 1 was irradiated with excitation light (laser light, wavelength 785 nm, intensity 12.0 mW) for 1 second, and the Raman scattered light generated from the sample solution 1 was detected. Raman scattered light detection was performed 1443 times.

[0097] Using a similar procedure, Raman scattered light generated from sample solution 2 was also detected. Raman scattered light detection was performed 1443 times for sample solution 2 as well.

[0098] Figures 8A and 8B show the Raman spectra of the sample solutions obtained using the analytical device of the example (see Figure 6A). Figures 9A and 9B show the Raman spectra of the sample solutions obtained using the analytical device of the comparative example (see Figure 6B). Figures 8A and 9A show the Raman spectra of sample solution 1 (derived from a lung cancer patient), and Figures 8B and 9B show the Raman spectra of sample solution 2 (derived from a colorectal cancer patient).

[0099] Principal component analysis was performed on the Raman spectra obtained using the analytical device shown in Figures 8A and 8B, and dot plots were obtained with principal component 1 (PC1) and principal component 2 (PC2) as axes. The data set consisted of a total of 2886 spectra, and each spectrum had 1600 data points. Therefore, eigenvalues ​​and eigenvectors were obtained from a 2886 × 1600 variance-covariance matrix. This allowed us to obtain information about the microRNAs contained in sample solution 1 and sample solution 2.

[0100] Similarly, principal component analysis was performed on the Raman spectra obtained using the comparative analysis device shown in Figures 9A and 9B, 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 microRNAs contained in sample solution 1 and sample solution 2.

[0101] Figure 10 shows the results of principal component analysis (PC1-PC2 plot) of the Raman spectrum obtained using the analytical device of the example (see Figure 6A). Figure 11 shows the results of principal component analysis (PC1-PC2 plot) of the Raman spectrum obtained using the analytical device of the comparative example (see Figure 6B). In Figures 10 and 11, the circled plots represent the results for sample solution 1 (derived from a lung cancer patient), and the crossed plots represent the results for sample solution 2 (derived from a colorectal cancer patient).

[0102] Comparing Figure 10 and Figure 11, it can be seen that when using the analysis device of the example, where the plan view shape of the metal nanostructure is triangular (see Figure 6A), it was possible to more clearly distinguish between the distribution of microRNAs derived from lung cancer patients and those derived from colorectal cancer patients than when using the analysis device of the comparative example, where the plan view shape of the metal nanostructure is square (see Figure 6B). Thus, by using the analysis device of the example, it was possible to obtain information that allows for the differentiation between microRNAs derived from lung cancer patients and microRNAs derived from colorectal cancer patients.

[0103] These results demonstrate that cancer types can be identified from microRNAs contained within extracellular vesicles in the blood. Furthermore, by using this PC1-PC2 plot as a database or training data, it is possible to determine which data group a microRNA belongs to with just one measurement.

[0104] Furthermore, regardless of whether the analysis device in the example or the comparative example was used, the time required to obtain information on the microRNA contained in each sample solution was less than 60 minutes, which is shorter than the time required to obtain the above information using conventional analysis methods (see Non-Patent Documents 1-3). In addition, the cost required to obtain information on the microRNA contained in each sample solution was less than 20,000 yen, which is cheaper than the time required to obtain the above information using conventional analysis methods (see Non-Patent Documents 1-3).

[0105] The information acquisition method, determination method, and information acquisition apparatus according to the present invention are useful, for example, for determining the presence or type of cancer cells.

[0106] 100 Analysis device 110 Substrate 120 Metal nanostructure 200 Information acquisition device 210 Irradiation unit 220 Mounting unit 230 Optical system 240 Spectroscopy unit 250 Detection unit 260 Information acquisition unit

Claims

A method for obtaining information about nucleic acids or proteins by surface-enhanced Raman scattering spectroscopy, A step of preparing an analytical device having a substrate and a plurality of metal nanostructures arranged spaced apart from each other on the substrate so as to form a plurality of nanogaps, A step of irradiating a solution containing nucleic acid or protein with excitation light, provided to a region of the analysis device including the plurality of nanogaps, A step of detecting Raman scattered light generated from the solution by irradiation with the excitation light, A step of obtaining information about the nucleic acid or protein in the solution based on the detected Raman scattering light, Includes, The planar shape of each of the aforementioned plurality of metal nanostructures is triangular. The plurality of metal nanostructures are arranged on the substrate such that each of the plurality of nanogaps is formed between the corners of the triangles. How to obtain information. 　 The information acquisition method according to claim 1, wherein the shapes of the plurality of metal nanostructures are substantially identical. 　 The information acquisition method according to claim 1, wherein the width of each of the plurality of nanogaps is 1 nm or more and 20 nm or less. 　 The information acquisition method according to claim 1, wherein the height of each of the plurality of metal nanostructures is 1 nm or more and 100 nm or less. 　 The information acquisition method according to claim 1, wherein the length of each side of the aforementioned triangle is 50 nm or more and 200 nm or less. 　 In the irradiation step, the excitation light is irradiated onto the solution containing multiple types of nucleic acids or multiple types of proteins. In the acquisition step, information on multiple types of nucleic acids or multiple types of proteins in the solution is acquired collectively. The method for obtaining information as described in claim 1. 　 The information acquisition method according to claim 1, wherein the acquisition step involves acquiring the information by performing statistical analysis. 　 The information acquisition method according to claim 7, wherein the acquisition step involves acquiring the information using a principal component analysis method. 　 The solution comprises the nucleic acid, The aforementioned information is information relating to the nucleic acid. The method for obtaining information as described in claim 1. 　 The information acquisition method according to claim 9, wherein the nucleic acid is RNA. 　 The information acquisition method according to claim 10, wherein the RNA is a microRNA. 　 A method for determining the presence or absence of cancer cells, A step of obtaining first information concerning nucleic acids or proteins contained in a sample obtained from a subject by an information acquisition method according to any one of claims 1 to 11, A step of determining whether the sample contains the nucleic acid or protein derived from cancer cells based on the first information, A determination method that includes this. 　 The above-mentioned information acquisition method further includes a step of acquiring second information concerning nucleic acids or proteins contained in a sample obtained from a healthy person, The determination step involves comparing the first information with the second information to determine whether the sample obtained from the subject contains the nucleic acid or protein derived from cancer cells. The determination method according to claim 12. 　 A device for acquiring information about nucleic acids or proteins by surface-enhanced Raman scattering spectroscopy, An analytical device having a substrate and a plurality of metal nanostructures arranged spaced apart from each other on the substrate so as to form a plurality of nanogaps, An irradiation unit provided in a region of the analysis device including the plurality of nanogaps for irradiating a solution containing nucleic acid or protein with excitation light, A detection unit for detecting Raman scattered light generated from the solution by irradiation with the excitation light, An information acquisition unit for obtaining information about the nucleic acid or protein in the solution based on the detected Raman scattering light, Includes, The planar shape of each of the aforementioned plurality of metal nanostructures is triangular. The plurality of metal nanostructures are arranged on the substrate such that each of the plurality of nanogaps is formed between the corners of the triangles. Information acquisition device.

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