Plasmonic well plate substrate for spectroscopic analysis and method for manufacturing the same

Abstract

This application provides a plasmonic well plate substrate for spectroscopic analysis and a method for manufacturing the same. More specifically, this application provides a plasmonic well plate substrate with improved signal enhancement effect and uniformity, which is suitable for detecting solution-like samples, and a method for manufacturing a plasmonic well plate substrate for spectroscopic analysis that can improve signal enhancement effect and uniformity by easily adjusting the morphology of various polycrystalline structures.

Description

Technical Field

[0001] This application relates to a plasmonic well plate substrate for spectroscopic analysis and a method for manufacturing the same. More specifically, this application relates to a plasmonic well plate substrate with improved signal enhancement effect and uniformity, which is suitable for detecting liquid samples, and a method for manufacturing a plasmonic well plate substrate for spectroscopic analysis that can improve the signal enhancement effect and uniformity by easily adjusting various polycrystalline structures.

Background Art

[0002] Raman scattering is inelastic scattering in which the energy of the incident light changes. When light is applied to a specific molecule, light with a slightly different wavelength from the irradiated light is generated due to the vibration transition peculiar to the molecular body.

[0003] In fact, almost all organic molecules have a unique Raman shift. Therefore, according to Raman spectroscopy using Raman scattering, signals can be obtained even in the case of non-polar molecules with a change in the induced polarizability of the molecule. In addition, Raman spectroscopy is not affected by the interference of water molecules, so it is more suitable for detecting biomolecules such as proteins and genes. Since the wavelength of the Raman emission spectrum indicates the chemical composition and structural characteristics of the light-absorbing molecules in the sample, the analyte can be directly analyzed by analyzing such Raman signals.

[0004] In order to detect biomolecules and the like using such Raman spectroscopy, it is common to use the sample in a dried form, so a step of drying the sample is separately required. Therefore, time and cost are added by the step of drying the sample, and problems such as contamination or sample alteration may occur in the step of drying the sample.

[0005] On the other hand, a well plate is an experimental and testing instrument consisting of a plate formed by arranging numerous wells or grooves, and is actively used in biochemical analysis and clinical testing. When detecting or diagnosing a specific target substance or disease based on a well plate, it is common practice to place the substance for detecting the target substance or diagnosing the disease on the surface inside the wells of the well plate, then introduce the sample to be analyzed into the wells to induce a reaction, and finally detect the target substance or diagnose the disease. Utilizing such a well plate makes it easy to simultaneously detect target substances in multiple samples. Furthermore, it is suitable for the analysis of liquid samples, allowing bodily fluids such as urine, saliva, tears, and sweat to be used as samples in their liquid form, which can be efficient in terms of cost and time in the process of detecting target substances or diagnosing diseases.

[0006] As background technology for this application, Korean Published Patent No. 10-2016-0014866 describes a method and apparatus for diagnosing viral infection using teardrops. The above-mentioned published patent separately includes a step of drying the collected tears to prepare them as a sample for measurement in order to obtain a Raman spectrum. [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of this application is to provide a plasmonic well plate substrate for spectroscopic analysis that has excellent signal enhancement effect and uniformity, making it suitable for the analysis of liquid samples when used for spectroscopic analysis.

[0008] Another object of this application is to provide a plasmonic well plate substrate for spectroscopic analysis that enables label-free qualitative and quantitative analysis of analytes.

[0009] Another objective of this application is to provide a plasmonic well plate substrate for spectroscopic analysis that is suitable for the simultaneous analysis of multiple samples.

[0010] Another object of this application is to provide a method for manufacturing a plasmonic well plate substrate for spectroscopic analysis that allows for easy adjustment and formation of the nanostructure morphology of the polycrystalline structure formed on the well plate member, and has improved signal enhancement effect and uniformity.

[0011] Another object of this invention is to provide a method for efficiently manufacturing a plasmonic well plate substrate for spectroscopic analysis that has excellent signal enhancement effect and uniformity, making it suitable for the analysis of solution-like samples.

[0012] The purposes of this application are not limited to those stated above, and other purposes not mentioned can be clearly understood from the detailed description. [Means for solving the problem]

[0013] According to one embodiment, a plasmonic well plate substrate for spectroscopic analysis is provided, comprising a well plate member having one or more wells and a polycrystalline structure formed in the wells, wherein the polycrystalline structure is composed of a cluster of multiple gold nanostructures and contains multiple voids inside.

[0014] According to one embodiment, the gold nanostructures or the clusters formed by the gold nanostructures may be connected to one another.

[0015] According to one embodiment, the polycrystalline structure may be one or more of the following forms: nano-sponge, nano-tree, nano-branch, and nano-coral.

[0016] According to one embodiment, the polycrystalline structure may further include a layer of gold nanoparticles on its surface.

[0017] According to one embodiment, the polycrystalline structure may further include a gold nanolayer grown on the gold nanoparticle layer with a gold precursor.

[0018] According to one embodiment, the polycrystalline structure may further include a gold nanolayer grown on its surface with a gold precursor.

[0019] According to one embodiment, the polycrystalline structure may further include a layer of gold nanoparticles on the gold nanolayer.

[0020] According to one embodiment, the average thickness of the gold nanoparticle layer or gold nanolayer may be 5 to 100 nm.

[0021] According to one embodiment, the plasmonic well plate substrate for spectroscopic analysis may be used for surface-enhanced Raman spectroscopy (SERS), plasmon-enhanced fluorescence (PEF), or fluorescence analysis.

[0022] In another embodiment, a method for manufacturing a plasmonic well plate substrate for spectroscopic analysis is provided, comprising the steps of: i) preparing a well plate member having one or more wells; and ii) supporting the well plate member on a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution to form a polycrystalline structure in the wells, wherein the polycrystalline structure formed in step ii) is composed of a cluster of a plurality of gold nanostructures and contains a plurality of voids inside, and adjusting the morphology of the polycrystalline structure by setting the gold precursor:reducing agent ratio in step ii) to 1:1 to 1:10.

[0023] According to one embodiment, after step ii), the further step may include attaching gold nanoparticles to the well plate member on which the polycrystalline structure is formed to form a gold nanoparticle layer on the surface of the polycrystalline structure.

[0024] According to one embodiment, after the step of forming a gold nanoparticle layer on the surface of the polycrystalline structure, the well plate member is supported on a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution, and a step of forming a gold nano layer formed by epitaxial growth on the gold nanoparticle layer may be further included.

[0025] According to one embodiment, after the step ii), the well plate member on which the polycrystalline structure is formed is supported on a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution, and a step of forming a gold nano layer formed by epitaxial growth on the surface of the polycrystalline structure may be further included.

[0026] According to one embodiment, after the step of forming a gold nano layer formed by epitaxial growth on the surface of the polycrystalline structure, a step of attaching gold nanoparticles to the well plate member to form a gold nanoparticle layer on the surface of the polycrystalline structure may be further included.

[0027] According to one embodiment, before the step of forming the gold nanoparticle layer, a step of surface-modifying the surface of the polycrystalline structure may be further included.

[0028] According to one embodiment, the reducing agent may be one or more of ascorbic acid, hydroxylamine, hydroxylamine-O-sulfonic acid, and O-methylhydroxylamine hydrochloride.

Advantages of the Invention

[0029] According to one embodiment, the plasmonic well plate substrate for spectroscopic analysis of the present application includes various forms of polycrystalline structures formed by clusters of gold nanostructures on the well plate member, and is suitable for analyzing solution samples when used for spectroscopic analysis, and can exhibit excellent signal enhancement effects and uniformity.

[0030] According to one embodiment, using the plasmonic well plate substrate for spectroscopic analysis of the present invention eliminates the need for a drying step when analyzing liquid samples.

[0031] According to one embodiment, the plasmonic well plate substrate for spectroscopic analysis of the present invention enables qualitative and quantitative analysis of unlabeled analytes.

[0032] According to one embodiment, using the plasmonic well plate substrate for spectroscopic analysis of the present invention is efficient for simultaneously analyzing a large number of samples with high sensitivity.

[0033] According to one embodiment, the method for manufacturing a plasmonic well plate substrate for spectroscopic analysis of the present invention allows for the easy adjustment of various forms of polycrystalline structures formed of clusters of gold nanostructures on a well plate member, and enables the efficient production of a plasmonic well plate substrate for spectroscopic analysis with improved signal enhancement effect and uniformity.

[0034] According to one embodiment, the method for manufacturing a plasmonic well plate substrate for spectroscopic analysis of the present invention can efficiently manufacture a plasmonic well plate substrate for spectroscopic analysis that has improved signal enhancement effect and uniformity, making it suitable for the analysis of liquid samples. [Brief explanation of the drawing]

[0035] [Figure 1] Figure 1 is a photograph showing different types of plasmonic well plate substrates for spectroscopic analysis, categorized by well size, manufactured according to one embodiment of the present invention. [Figure 2] Figures 2(a) to 2(c) are SEM images showing various forms of polycrystalline structures formed on a well plate member according to one embodiment of the present invention. [Figure 3]Figures 3(a) and 3(b) are SEM images showing a thick sponge-shaped polycrystalline structure formed on a well plate member according to one embodiment of the present invention, which includes a gold nanoparticle layer formed by depositing gold nanoparticles on the surface with thicknesses of 25 nm and 40 nm, respectively. Figure 3(c) is an SEM image showing a thick sponge-shaped polycrystalline structure formed on a well plate member according to one embodiment of the present invention, which includes a gold nanolayer formed by additional growth of a gold precursor on the gold nanoparticle layer. [Figure 4] Figures 4(a) and 4(b) are SEM images showing short tree and long tree shaped polycrystalline structures formed on a well plate member according to one embodiment of the present invention, which include a gold nanoparticle layer formed by depositing gold nanoparticles to a thickness of 25 nm on the surface. [Figure 5] Figure 5 is a graph showing the XRD results of a polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 6] Figure 6 is a schematic diagram illustrating various manufacturing methods for a plasmonic well plate for spectroscopic analysis according to one embodiment of the present invention. [Figure 7] Figure 7(a) is a graph showing the SERS signal intensity due to the nanostructure of the polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 7(b) is a graph showing the SERS signal intensity at the main peak due to the nanostructure of the polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 8] Figure 8 is a graph showing the SERS signal intensity measured at 633 nm, depending on the type of reducing agent and the ratio of gold precursor to reducing agent, when forming a polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 9]Figure 9 is a graph showing the SERS signal intensity measured at 785 nm, depending on the type of reducing agent and the ratio of gold precursor to reducing agent, when forming a polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 10] Figure 10 is a TEM image of the nanostructure of a polycrystalline structure obtained by attaching gold nanoparticles to a thick sponge-shaped polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis, manufactured according to one embodiment of the present invention, to form a gold nanoparticle layer, and then further growing a gold nanolayer with a gold precursor. [Figure 11] Figure 11 shows graphs and images illustrating the size distribution of a thick sponge-shaped polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention, the nanostructure of a polycrystalline structure in which a gold nanoparticle layer is formed by depositing gold nanoparticles on the thick sponge-shaped polycrystalline structure, and the nanostructure of a polycrystalline structure in which a gold nanolayer is formed by additional growth of a gold precursor on the gold nanoparticle layer. [Figure 12] Figure 12(a) is a graph showing the SERS signal intensity due to the nanostructure of a polycrystalline structure measured at 633 nm on a dry sample using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 13] Figures 12(b) and (c) are graphs showing the SERS signal intensity and detection limit at sample concentration measured at 633 nm for a dry sample using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 13(a) is a graph showing the SERS signal intensity due to the nanostructure of a polycrystalline structure measured at 785 nm for a dry sample using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figures 13(b) and (c) are graphs showing the SERS signal intensity and detection limit at sample concentration measured at 785 nm for a dry sample using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 14]Figure 14(a) is a graph showing the SERS signal intensity due to the nanostructure of a polycrystalline structure measured at 785 nm for a liquid sample using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figures 14(b) and (c) are graphs showing the SERS signal intensity and detection limit at 785 nm for a liquid sample measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 15] Figure 15(a) is a graph showing the uniformity of the SERS signal for a liquid sample in one well of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 15(b) is a graph showing the uniformity of the SERS signal for a liquid sample between 96 different wells of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 16] Figure 16 is a graph showing the SERS signal intensity of six metabolites (Uracil, Guanine, Xanthine, Purine, Hypoxanthine, and Adenine) in a liquid sample, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 17] Figures 17(a) to (d) are graphs showing the SERS signal intensity and detection limits of four metabolites (Adenine, Xanthine, Hypoxanthine, and Purine) for a liquid sample, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 18] Figure 18 is a graph showing the normalized SERS signal intensity for urine samples from cancer patients, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 19] Figure 19 is a graph showing the SERS signal intensity of candidate cancer metabolites in urine samples from cancer patients, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 20] Figures 20(a) to (d) are graphs showing the locations of Raman shifts for each cancer type that differ from those of healthy individuals in intervals with a confidence level of 95% or higher, as measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 20(e) is a graph comparing the relative magnitudes of the Raman shifts for each cancer type at different locations, as measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Figure 21] Figures 21(a) to (d) are graphs showing the results of confusion matrices, which indicate the accuracy of distinguishing urine samples from cancer patients from healthy individuals of each cancer type, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 21(e) is a curve graph of receiver operating characteristics (ROC), which indicates the accuracy of distinguishing urine samples from cancer patients from healthy individuals of each cancer type, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. [Modes for carrying out the invention]

[0036] The purpose of this disclosure, specific advantages, and novel features will become even clearer from the accompanying drawings and the detailed description and examples below.

[0037] First, the terms and words used in this specification and claims should not be interpreted in their ordinary dictionary sense, but rather in a sense and concept consistent with the technical idea of ​​this disclosure, based on the principle that inventors can appropriately define the concepts of terms in order to best describe their invention.

[0038] In this specification, when a component such as a layer, portion, or substrate is described as being "on top of," "connected to," or "bonded" to another component, this may be a direct "on top of," "connected to," or "bonded" to the other component, and furthermore, one or more other components may be interposed between the two components. In contrast, when a component is described as being "directly on top of," "directly connected to," or "directly bonded" to another component, there should be no other components interposed between the two components.

[0039] The terms used herein are used solely to describe specific embodiments and are not intended to limit the disclosure. Singular expressions include plural expressions unless the context clearly indicates otherwise.

[0040] In this specification, terms such as “includes” or “have” are intended to indicate the presence of features, figures, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood not to preemptively exclude the possibility of the presence or addition of one or more other features or figures, steps, actions, components, parts, or combinations thereof.

[0041] In this specification, when a part is described as "including" a component, unless otherwise stated, this does not mean that other components are excluded, but rather that other components may be included. Throughout this specification, "above" means located above or below the part in question, and does not necessarily mean located above the direction of gravity.

[0042] This disclosure can be modified in various ways and may include many different embodiments. Therefore, specific embodiments are illustrated in the drawings and described in detail. However, this is not intended to limit this disclosure to any particular embodiment, but rather to be understood as including all modifications, equivalents, or substitutes that fall within the concept and technical scope of this disclosure. In describing this disclosure, if a specific description of relevant known technology is deemed to obscure the gist of this disclosure, such detailed description will be omitted.

[0043] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. In this description, identical or corresponding components will be assigned the same drawing number, and redundant descriptions thereof will be omitted.

[0044] According to one embodiment, a plasmonic well plate substrate for spectroscopic analysis is provided, comprising a well plate member having one or more wells and a polycrystalline structure formed in the wells, wherein the polycrystalline structure is composed of a cluster of multiple gold nanostructures and contains multiple voids inside.

[0045] Figure 1 is a photograph showing different types of plasmonic well plate substrates for spectroscopic analysis, categorized by well size, manufactured according to one embodiment of the present invention.

[0046] Referring to Figure 1, the plasmonic well plate member for spectroscopic analysis of the present invention may be configured such that a polycrystalline structure composed of a cluster of gold nanostructures is formed on a well plate member having one or more wells, and a gold film is formed inside the wells.

[0047] The multi-well plates may include, but are not limited to, known multi-well plates having two or more wells, such as 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, and 384-well plates, with varying well sizes.

[0048] While petri dishes are used for detecting unlabeled bacteria, the plasmonic well plate substrate for spectroscopic analysis according to this invention can be used for in-situ mapping, species classification, and detection of drug-resistant bacteria, among other applications.

[0049] While the above 6-well plates are used for cancer cell culture and metabolite profiling using label-free methods, the plasmonic well plate substrate for spectroscopic analysis according to this invention enables in-situ Raman imaging, drug response, and immune response monitoring.

[0050] While not limited to the following, plasmonic well plate substrates for spectroscopic analysis according to this application, such as 96-well plates, can be used in the fields of high-speed, high-volume chemical screening and ultra-high-sensitivity ELISA platforms by fluorescence (PEF) and Raman spectroscopy (SERS) analysis.

[0051] Figures 2(a) to (c) are SEM images showing various nanostructured polycrystalline structures formed on a well plate member according to one embodiment of the present invention.

[0052] Referring to Figures 2(a) to (c), a polycrystalline structure composed of a cluster of multiple gold nanostructures formed on the well plate member can be observed. These gold nanostructures are grown directly on the well plate member in a solution process using a gold precursor and a reducing agent, forming a cluster.

[0053] More specifically, gold (Au) precursors among the precious metals form nanostructures and aggregate, and these are grown directly on a well plate material using a solution process. Specifically, the aggregated nanostructures form small polygonal grain structures through coalescence, and then the grains gradually grow larger through oriented attachment. Thus, a polycrystalline structure with multiple grain boundaries is formed. The polycrystalline structure can grow to an average size of a micrometer, but is not limited to the following. As described above, polycrystalline structures with multiple grain boundaries have increased scattering at the multiple grain boundaries, which can dramatically improve signal intensity and signal uniformity when used as a substrate for spectroscopic analysis.

[0054] Furthermore, the polycrystalline structure may consist of a cluster formed of multiple gold nanostructures and may contain multiple voids within it. The multiple gold nanostructures or clusters formed of multiple gold nanostructures may be interconnected while containing internal voids. The multiple gold nanostructures or clusters formed of multiple gold nanostructures may be connected by a branch structure.

[0055] The average particle size of the polycrystalline structure may be 0.1 to 100 μm, although this is not limited to the above. The spectroscopic analysis substrate of this application is grown into a polycrystalline structure by a solution process, connected by a branch structure, and may have an average particle size of 0.1 to 100 μm. Although this is not limited to the above, having an average particle size of the polycrystalline structure within the above range is suitable for improving signal intensity and signal uniformity.

[0056] As described above, the multiple gold nanostructures may be formed in a manner in which they are interconnected and constitute a community, and the polycrystalline structure may be composed of a community formed of the multiple gold nanostructures.

[0057] As described above, the plasmonic well plate substrate for spectroscopic analysis of this application has a configuration in which gold nanostructures are connected to each other without separation, forming a cluster containing internally distributed voids. Therefore, compared to the case where gold nanostructures are formed separated from each other, nanogaps and hot spots can be significantly increased. Accordingly, the plasmonic well plate substrate for spectroscopic analysis of this application can exhibit excellent SERS signal enhancement effect and uniformity for the analyte.

[0058] The voids contained in the polycrystalline structure may be formed in a disordered but uniform manner between multiple gold nanostructures, or in a disordered but uniform manner between clusters formed by multiple gold nanostructures. Therefore, the plasmonic well plate substrate for spectroscopic analysis of this invention can improve the SERS signal enhancement effect and uniformity for the analyte.

[0059] Referring to Figures 2(a) to (c), the above polycrystalline structures may be nano-sponge, thick sponge, nano-tree, nano-branch, and nano-coral forms, but are not limited to these. The nano-sponge is a sponge form containing numerous voids inside, and the thick sponge is the same or similar in shape to the nano-sponge, but may refer to a sponge form in which the community is formed thickly. The nano-tree, nano-branch, and nano-coral may refer to communities that have grown long and are formed in the shape of a tree, branches, or coral.

[0060] The shape of the polycrystalline structure formed on the spectroscopic analysis substrate can be easily adjusted by adjusting the optimal ratio of reducing agent to the gold precursor according to the type of reducing agent, although this is not limited to the above. Furthermore, the shape of the polycrystalline structure formed on the spectroscopic analysis substrate can be easily adjusted by adjusting the time during which the well plate member is supported on the spectroscopic analysis substrate manufacturing composition.

[0061] Figures 3(a) and 3(b) are SEM images showing a thick sponge-shaped polycrystalline structure formed on a well plate member according to one embodiment of the present invention, which includes a gold nanoparticle layer formed by depositing gold nanoparticles on its surface with thicknesses of 25 nm and 40 nm, respectively.

[0062] Figures 4(a) and 4(b) are SEM images showing short tree and long tree shaped polycrystalline structures formed on a well plate member according to one embodiment of the present invention, which include a metal nanoparticle layer formed by depositing gold nanoparticles to a thickness of 25 nm on the surface.

[0063] Referring to Figures 3(a) and 3(b) and Figures 4(a) and 4(b), the polycrystalline structure of the present invention may further include a layer of gold nanoparticles on its surface. The gold nanoparticle layer may be formed by adding gold nanoparticles that have been synthesized and manufactured in advance, causing the gold nanoparticles to adhere to the surface of the polycrystalline structure.

[0064] Figure 3(c) is an SEM image showing a thick sponge-shaped polycrystalline structure formed on a well plate member according to one embodiment of the present invention, which includes a gold nanolayer formed by additional growth of a gold precursor on a gold nanoparticle layer.

[0065] Referring to Figure 3(c), the polycrystalline structure of the present invention may further include a gold nanolayer formed by additional growth of a gold precursor on the gold nanoparticle layer. The gold nanolayer may be formed by additional growth of gold nanoparticles on the gold nanoparticle layer using the same or different compositions containing the gold precursor and reducing agent solution used in the formation of the initial polycrystalline structure.

[0066] Figure 5 is a graph showing the XRD results of a polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Specifically, Figure 5 shows the XRD results for a polycrystalline structure formed by additionally growing a gold nanolayer on a gold nanoparticle layer, and the polycrystalline nature of the polycrystalline structure of the plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention can be confirmed.

[0067] The polycrystalline structure of the present invention may further include a gold nanolayer grown on the surface with a gold precursor. The gold nanolayer may be formed by growing it with the same or different compositions containing the gold precursor and reducing agent solution used in the formation of the initial polycrystalline structure.

[0068] The polycrystalline structure of the present invention may further include a gold nanoparticle layer on the gold nanolayer. The gold nanoparticle layer may be formed by the attachment of gold nanoparticles to the gold nanolayer using synthesized gold nanoparticles.

[0069] The gold nanoparticle layer may be in the form of a continuous layer or a discontinuous layer, although it is not limited to the following. The gold nanoparticle layer may be in the form of a continuous layer or a partially discontinuous layer, but it is preferable that it be in the form of a continuous layer. The gold nanoparticle layer may be in the form of a cover over the polycrystalline structure or in the form of a thin lamination.

[0070] The average thickness of the gold nanolayer or gold nanoparticle layer may be 5 to 100 nm, and when it is within the above average thickness range, it is suitable for signal enhancement and uniformity improvement by additional growth, and may be 10 to 95 nm, 15 to 90 nm, 20 to 85 nm, 20 to 80 nm, 20 to 75 nm, 20 to 70 nm, 20 to 65 nm, 20 to 60 nm, 20 to 55 nm, 20 to 50 nm, 20 to 45 nm, or 25 to 40 nm.

[0071] The plasmonic well plate substrate for spectroscopic analysis of this application may be used for surface-enhanced Raman spectroscopy (SERS), plasmon-enhanced fluorescence (PEF), or fluorescence analysis. While not limited to the above, the plasmonic well plate substrate for spectroscopic analysis of this application is more preferably used for surface-enhanced Raman spectroscopy (SERS) analysis. When used for surface-enhanced Raman spectroscopy, the analyte can be efficiently detected and analyzed with high sensitivity without labeling such as fluorescent substances.

[0072] By using the plasmonic well plate substrate for spectroscopic analysis of this invention, it is possible to efficiently detect and analyze the analyte without any additional labeling of the sample or analyte to be analyzed, i.e., without labeling.

[0073] The analyte may be one or more selected from, but is not limited to, cells, metabolites, proteins, nucleic acids, DNA, RNA, mRNA, lipids, hormones, metabolites, enzymes, organic molecules, viruses, bacteria, antigens, antibodies, neurotransmitters, extracellular vesicles, microvesicles, exosomes, and lipids.

[0074] The above sample is preferably a liquid sample, and may be a variety of solutions containing the various analytes mentioned above, and may be a variety of bodily fluids such as urine, saliva, blood, sweat, tears, ascites, gastric juice, cerebrospinal fluid, substrates, and secretions. Conventionally, spectroscopic analysis substrates have low hygroscopicity, resulting in very low sensitivity when measuring liquid samples. However, the plasmonic well plate substrate for spectroscopic analysis according to this invention can support liquid samples, enabling rapid analysis and diagnosis on-site even with liquid samples.

[0075] Without limiting the following, the plasmonic well plate substrate for spectroscopic analysis of this invention enables cancer diagnosis using liquid samples. Without limiting the following, metabolite analysis of the samples and analytes described above can provide information necessary for cancer diagnosis, enabling early diagnosis of cancer through non-invasive testing, and allowing for multiplex analysis with an extremely high level of sensitivity.

[0076] The cancers mentioned above may be, but are not limited to, one or more selected from pancreatic cancer, prostate cancer, lung cancer, colorectal cancer, bronchial cancer, colon cancer, breast cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, thyroid cancer, esophageal cancer, uterine cancer, liver cancer, kidney cancer, and biliary tract cancer.

[0077] Figure 18 is a graph showing the normalized SERS signal intensity for a urine sample from a cancer patient, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 19 is a graph showing the SERS signal intensity of a candidate group of cancer metabolites for a urine sample from a cancer patient, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0078] Referring to Figures 18 and 19 above, it can be confirmed that the plasmonic well plate substrate for spectroscopic analysis of this application is suitable for use in cancer diagnosis using liquid samples such as patient urine that may contain cancer metabolites.

[0079] Figure 6 is a schematic diagram illustrating various manufacturing methods for a plasmonic well plate substrate for spectroscopic analysis according to one embodiment of the present invention.

[0080] Referring to Figure 6, another aspect of the present invention provides a method for manufacturing a plasmonic well plate substrate for spectroscopic analysis, comprising the steps of: i) preparing a well plate member having one or more wells; ii) supporting the well plate member on a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution to form a polycrystalline structure in the wells, wherein the polycrystalline structure formed in step ii) is composed of a cluster of a plurality of gold nanostructures and contains a plurality of voids inside, and adjusting the morphology of the polycrystalline structure by setting the gold precursor:reducing agent ratio in step ii) to 1:1 to 1:10.

[0081] Step i) is the step of preparing a well plate member having one or more wells. The well plate member having one or more wells may be a variety of known multi-well plates such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, and a 384-well plate.

[0082] Step ii) is the step of supporting a well plate member on a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution, and forming a polycrystalline structure in the wells. The formation of the polycrystalline structure in step ii) means growing gold nanostructures on the well surface of the well plate member.

[0083] The polycrystalline structure formed in step ii) above is composed of a cluster of multiple gold nanostructures and may contain multiple voids internally. The multiple gold nanostructures or clusters of multiple gold nanostructures may be interconnected and contain voids internally. The multiple gold nanostructures or clusters of multiple gold nanostructures may be connected by a branch structure.

[0084] The polycrystalline structure may be grown by a solution process, linked by a branch structure, and have an average particle size of 0.1 to 100 μm, although this is not limited to the above. However, having an average particle size within the above range for the polycrystalline structure is suitable for improving signal intensity and signal uniformity.

[0085] As described above, the multiple gold nanostructures may be formed in a manner in which they are interconnected and constitute a cluster, and the polycrystalline structure may be composed of a cluster formed of the multiple gold nanostructures. Furthermore, the polycrystalline structure may be formed of a large number of interconnected clusters and may have multiple grain boundaries.

[0086] As described above, the plasmonic well plate substrate for spectroscopic analysis of this application, which includes a polycrystalline structure, has a form in which gold nanostructures are connected to each other without separation, forming a cluster containing internally distributed voids. Therefore, compared to the case where gold nanostructures are formed separated from each other, nanogaps and hot spots can be significantly increased. Accordingly, the plasmonic well plate substrate for spectroscopic analysis of this application can exhibit excellent SERS signal enhancement effect and uniformity for the analyte.

[0087] The voids contained in the polycrystalline structure may be formed in a disordered but uniform manner between multiple gold nanostructures, or in a disordered but uniform manner between clusters formed by multiple gold nanostructures. Therefore, the plasmonic well plate substrate for spectroscopic analysis of this invention can improve the SERS signal enhancement effect and uniformity for the analyte.

[0088] The method may include a shape adjustment step in step ii) above, in which the ratio of gold precursor to reducing agent is set to 1:1 to 1:10 to adjust the shape of the polycrystalline structure. By adjusting the ratio of reducing agent to the optimal ratio according to the type of reducing agent, the shape of the polycrystalline structure formed on the plasmonic well plate substrate for spectroscopic analysis can be easily adjusted. Furthermore, by adjusting the time during which the well plate member is supported on the composition for manufacturing the spectroscopic analysis substrate, the shape of the polycrystalline structure formed on the spectroscopic analysis substrate can be easily adjusted. The method for manufacturing a spectroscopic analysis substrate according to the present invention can simultaneously improve signal enhancement and signal uniformity through a simple process of one to two loading steps.

[0089] The method may further include, but is not limited to, a step of attaching gold nanoparticles to the well plate member on which the polycrystalline structure is formed, thereby forming a gold nanoparticle layer on the surface of the polycrystalline structure, after step ii) above.

[0090] As described above, a gold nanoparticle layer may be formed by attaching gold nanoparticles, which have been synthesized separately, to the surface of a polycrystalline structure formed in the wells of the well plate member. The gold nanoparticles can be produced by various known methods for synthesizing gold nanoparticles, although this is not limited to the following. The attachment of the gold nanoparticles in the step of forming the gold nanoparticle layer can utilize conventionally known techniques, although this is not limited to the following. For example, the gold nanoparticles may be attached by physical adsorption, or a chemical linker may be used to modify the surface of the polycrystalline structure to fix the gold nanoparticles before attaching them. With the above configuration, the plasmonic well plate substrate for spectroscopic analysis of this application can improve the SERS signal enhancement effect and uniformity for the analyte.

[0091] The procedure may further include, but is not limited to, the step of forming a gold nanoparticle layer on the surface of the polycrystalline structure, supporting a well plate member on a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution, and growing additional gold nanoparticles on the gold nanoparticle layer to form a gold nanolayer. The procedure may also further include, but is not limited to, the gold nanolayer may be formed in a form that covers the polycrystalline structure on which the gold nanoparticle layer is formed, or in a form that thinly covers it.

[0092] The composition for manufacturing a spectroscopic analysis substrate, including the gold precursor and reducing agent solution in the step of forming the gold nanolayer, may be the same as or different from the composition in step ii). With the above configuration, the spectroscopic analysis plasmonic well plate substrate of the present invention can improve the SERS signal enhancement effect and uniformity for the analyte.

[0093] After step ii) above, the procedure may further include supporting the well plate member on which the polycrystalline structure is formed on a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution, and growing additional gold nanoparticles on the surface of the polycrystalline structure to form a gold nanolayer. The gold nanolayer may be formed in a form that covers the polycrystalline structure or in a form that thinly covers it. With the above configuration, the spectroscopic analysis plasmonic well plate substrate of the present invention can improve the SERS signal enhancement effect and uniformity for the analyte.

[0094] The invention may further include, but is not limited to, a step of forming a gold nanoparticle layer on the surface of the polycrystalline structure by attaching gold nanoparticles to the well plate member on which the polycrystalline structure is formed, after the step of forming an additional gold nanoparticle layer on the surface of the polycrystalline structure. With the above configuration, the plasmonic well plate substrate for spectroscopic analysis of the present invention can improve the SERS signal enhancement effect and uniformity for the analyte.

[0095] The thickness of the gold nanolayer grown with a gold precursor or the gold nanoparticle layer formed by the attachment of gold nanoparticles may be 5 to 100 nm, and when it is within the above average thickness range, it is suitable for signal enhancement effect and improvement of uniformity due to additional growth, and may be 10 to 95 nm, 15 to 90 nm, 20 to 85 nm, 20 to 80 nm, 20 to 75 nm, 20 to 70 nm, 20 to 65 nm, 20 to 60 nm, 20 to 55 nm, 20 to 50 nm, 20 to 45 nm, or 25 to 40 nm.

[0096] The present invention's method for manufacturing a plasmonic well plate substrate for spectroscopic analysis may further include a step of surface modification of the surface of a polycrystalline structure prior to the step of forming the gold nanoparticle layer. The surface modification step is a step to enable the gold nanoparticles to adhere effectively to the surface of the polycrystalline structure when forming the gold nanoparticle layer. The surface modification step may utilize a chemical linker to fix the gold nanoparticles, and a substance containing thiol groups may be used as the chemical linker.

[0097] The above-described composition for manufacturing a spectroscopic analysis substrate comprises a gold precursor and a reducing agent.

[0098] Although not limited thereto, the gold precursor may be selected from the group consisting of HAuCl4, AuCl, AuCl2, AuCl3, Na2Au2Cl8, and NaAuCl2, but one or more of HAuCl2 and NaAuCl4 are more preferred.

[0099] In one embodiment, the reducing agent may be one or more of ascorbic acid, FeSO4, hydroxyquinone, hydroxylamine, hydroxyl-O-sulfonic acid, and O-methylhydroxylamine hydrochloride. In one embodiment, the reducing agent may be one or more of hydroxylamine, hydroxyl-O-sulfonic acid, and O-methylhydroxylamine hydrochloride, which is preferable for producing plasmonic well plate substrates for spectroscopic analysis that have excellent signal enhancement effect and uniformity.

[0100] The ratio of the gold precursor to the reducing agent may be 1:1 to 1:10, and a ratio of 1:2 to 1:10 is suitable for producing plasmonic well plate substrates for spectroscopic analysis that have excellent signal enhancement effect and uniformity, while a ratio of 1:5 to 1:10 is more suitable.

[0101] Examples Example 1. Fabrication of plasmonic well plate substrates for spectroscopic analysis containing polycrystalline structures. A well plate member containing one or more wells was prepared as a base material. Compositions for manufacturing spectroscopic analysis substrates were prepared by mixing gold precursor solution and reducing agent solution in ratios of 1:0.5, 1:1, 1:2, 1:5, and 1:10. Distilled water, ethanol, and methanol can be used as the manufacturing solvent for producing the above compositions, but ethanol was used because it is more suitable for the uniform formation of polycrystalline structures. The above compositions were supported inside each well of the well plate for 1 minute to 24 hours to form a polycrystalline structure on the well plate member consisting of a cluster of multiple gold nanostructures and containing multiple voids inside.

[0102] HAuCl4 was used as the gold precursor solution in the above-mentioned composition for manufacturing substrates for spectroscopic analysis. Hydroxylamine, hydroxyl-O-sulfonic acid, and O-methylhydroxylamine hydrochloride were used as reducing agents. After the formation of the polycrystalline structure was complete, the remaining precursor and reducing agent were removed by washing twice with ethanol and three or more times with water. Then, the substrate was dried at room temperature for more than one hour to complete the production of plasmonic well plate substrates for spectroscopic analysis.

[0103] Example 2. Fabrication of a plasmonic well plate substrate for spectroscopic analysis containing a polycrystalline structure with a gold nanoparticle layer formed thereon. The surface of the plasmonic well plate substrate for spectroscopic analysis, in which a polycrystalline structure was formed inside the wells manufactured in Example 1 above, was surface-modified using a substance containing a thiol group as a chemical linker. At this time, cysteine ​​was used as the substance containing the thiol group, at a concentration in the range of 1 to 1000 uM. Subsequently, a gold nanoparticle solution, which had been separately synthesized and manufactured in advance, was added to the inside of each well to adhere the gold nanoparticles, forming a gold nanoparticle layer with a thickness of 25 to 40 nm on the surface of the polycrystalline structure. The substance containing the thiol group can be used selectively, and even if it is not used, gold nanoparticles can be attached by physical adsorption. The reaction was carried out at a temperature of 40°C until the gold nanoparticle solution dried, and after the formation of the gold nanoparticle layer was completed, the substrate was washed with water at least twice to complete the manufacture of the plasmonic well plate substrate for spectroscopic analysis.

[0104] Example 3. Fabrication of a plasmonic well plate substrate for spectroscopic analysis containing a polycrystalline structure in which a gold nanolayer is formed on a gold nanoparticle layer. In the plasmonic well plate substrate for spectroscopic analysis, manufactured in Example 2 above, a layer of gold nanoparticles was formed on the surface of a polycrystalline structure. The spectroscopic analysis substrate manufacturing composition from Example 1 above was supported in each well of the well plate for 1 minute to 24 hours to grow additional gold nanoparticles on the gold nanoparticle layer and form a gold nanolayer. After the formation of the additionally grown gold nanolayer was complete, the upper layer liquid was removed and the substrate was completely dried. Then, the substrate was washed with water at least twice to remove any remaining precursors and reducing agents. Finally, it was dried at room temperature for at least 1 hour to complete the manufacturing of the plasmonic well plate substrate for spectroscopic analysis.

[0105] Example 4. Fabrication of a plasmonic well plate substrate for spectroscopic analysis containing a polycrystalline structure with a gold nanolayer formed on it. In the plasmonic well plate substrate for spectroscopic analysis, which was manufactured in Example 1 above and had a polycrystalline structure formed inside the wells, the spectroscopic analysis substrate manufacturing composition of Example 1 was supported inside each well of the well plate for 30 seconds to 24 hours, and a gold nanolayer was formed by additional growth on the surface of the polycrystalline structure.

[0106] After the formation of the additionally grown gold nanolayer was complete, any remaining precursors and reducing agents were removed by washing with water or ethanol at least twice. Then, the substrate was dried at room temperature for at least one hour to complete the fabrication of the plasmonic well plate substrate for spectroscopic analysis.

[0107] Example 5. Fabrication of a plasmonic well plate substrate for spectroscopic analysis containing a polycrystalline structure in which a gold nanoparticle layer is formed on a gold nanolayer. In the plasmonic well plate substrate for spectroscopic analysis, which was manufactured in Example 4 above, a gold nanoparticle layer with a thickness of 25-40 nm was formed by adding gold nanoparticles, which were previously synthesized and manufactured separately, to each well of the substrate, thereby depositing the gold nanoparticles onto the gold nanoparticle layer.

[0108] After the gold nanoparticles were attached and the formation of the gold nanoparticle layer was completed, the substrate was dried at room temperature to complete the manufacturing of the plasmonic well plate substrate for spectroscopic analysis.

[0109] Experimental example 1. Comparison of signal intensity and magnitude distribution based on the morphology of polycrystalline structures. Figure 7(a) is a graph showing the SERS signal intensity due to the nanostructure of the polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0110] Figure 7(b) is a graph showing the SERS signal intensity at the main peak due to the nanostructure of the polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0111] Referring to Figure 7, it can be confirmed that peaks are formed by polycrystalline structures formed on the plasmonic well plate substrate for spectroscopic analysis of the present invention. In particular, it can be confirmed that the SERS signal intensity of plasmonic well plate substrates for spectroscopic analysis, including polycrystalline structures with gold nanoparticle layers (AuNP 25nm / Short Tree and AuNP 25nm / Long Tree), polycrystalline structures with gold nanoparticle layers (Laminate / AuNP 40nm / Thick Sponge), and polycrystalline structures with gold nanoparticle layers or gold nanoparticle layers formed on a gold nanoparticle layer (AuNP 40nm / Thick Sponge), shows a significant main peak and appears high.

[0112] On the other hand, when we used Nirmidas's Gold-coated microplate, a commercially available product, as a comparative example to measure the SERS signal, no significant peaks appeared in this case.

[0113] Figure 10 is a TEM image of the nanostructure of a polycrystalline structure formed by attaching gold nanoparticles to a thick sponge-shaped polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis, manufactured according to one embodiment of the present invention, to form a gold nanoparticle layer, and then further growing a gold nanolayer with a gold precursor.

[0114] Figure 11 shows graphs and images illustrating the size distribution of a thick sponge-shaped polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention, the nanostructure of a polycrystalline structure in which a gold nanoparticle layer is formed by depositing gold nanoparticles on the thick sponge-shaped polycrystalline structure, and the nanostructure of a polycrystalline structure in which a gold nanolayer is formed by additional growth of a gold precursor on the gold nanoparticle layer.

[0115] Referring to Figures 10 and 11, we can see the morphology of a polycrystalline structure in which gold nanoparticles adhere to the surface of a thick, sponge-shaped polycrystalline structure (AuNS) formed from a cluster of multiple gold nanostructures, forming a gold nanoparticle layer (AuNP), and then additional growth occurs on the gold nanoparticle layer to form a gold nanolayer. Furthermore, we confirmed that as the gold nanoparticle layer and then the gold nanolayer are formed, the size distribution of the polycrystalline structure shifts to larger values.

[0116] 2. Comparison of signal intensity based on the type of reducing agent and the ratio of gold precursor to reducing agent. Figure 8 is a graph showing the SERS signal intensity measured at 633 nm, depending on the type of reducing agent and the ratio of gold precursor to reducing agent, when forming a polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0117] Figure 9 is a graph showing the SERS signal intensity measured at 785 nm, depending on the type of reducing agent and the ratio of gold precursor to reducing agent, when forming a polycrystalline structure of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0118] We measured the SERS signal intensity for plasmonic well plate substrates for spectroscopic analysis, where gold nanostructures were directly grown on a well plate member without a gold nanolayer or gold nanoparticle layer to form a polycrystalline structure, depending on the type of reducing agent and the ratio of gold precursor to reducing agent. Signals were measured at 633 nm and 785 nm, and malachite green was used as the target molecule.

[0119] Referring to Figures 8 and 9, it was confirmed that hydroxylamine (HA), hydroxyl-O-sulfonic acid (HOS), and O-methylhydroxylamine hydrochloride (OMH) are suitable reducing agents for manufacturing the plasmonic well plate substrate for spectroscopic analysis according to the present invention. Furthermore, as the gold precursor:reducing agent ratio for each reducing agent increases from 1:0.5 to 1:10, the SERS signal intensity increases further, and the 1:10 ratio at which the strongest signal is observed can be set as the optimal ratio, with 1:0.5 to 1:10 being the range of the optimal ratio.

[0120] 3. Signal detection and comparison for dry and liquid samples Figure 12(a) is a graph showing the SERS signal intensity due to the nanostructure of a polycrystalline structure measured at 633 nm on a dry sample using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0121] Figures 12(b) and (c) are graphs showing the SERS signal intensity and detection limit at 633 nm for a dry sample, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0122] Figure 13(a) is a graph showing the SERS signal intensity due to the nanostructure of a polycrystalline structure measured at 785 nm on a dry sample using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0123] Figures 13(b) and (c) are graphs showing the SERS signal intensity and detection limit at 785 nm for a dry sample, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0124] Figure 14(a) is a graph showing the SERS signal intensity due to the nanostructure of a polycrystalline structure measured at 785 nm for a liquid sample using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0125] Figures 14(b) and (c) are graphs showing the SERS signal intensity and detection limit at 785 nm for a liquid sample, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0126] Figure 15(a) is a graph showing the uniformity of the SERS signal for a liquid sample in one well of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 15(b) is a graph showing the uniformity of the SERS signal for a liquid sample between 96 different wells of a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0127] The signal detection for solution samples and dried samples (after the solution has been dried) on the plasmonic well plate substrate for spectroscopic analysis of this invention was compared using 4-ATP and malachite green as the analytes. For signal detection for 4-ATP and malachite green, the SERS signal was confirmed using a substrate containing a polycrystalline structure in which gold nanoparticles were attached to a polycrystalline structure formed by directly growing gold nanostructures on the well plate member, and a gold nanoparticle layer was formed on the gold nanoparticle layer by additional growth.

[0128] Referring to Figures 12 to 14, both the dry and solution samples showed the highest signal intensity when a polycrystalline structure was formed by directly growing gold nanostructures on a well plate member, with gold nanoparticles adhering to the polycrystalline structure, forming a gold nanoparticle layer, and then further growth created a polycrystalline structure with a gold nanolayer on top of the gold nanoparticle layer.

[0129] On the other hand, the signal intensity for solution samples is even higher than that for dry samples, and as can be seen in Figure 15, the signal uniformity for solution samples is also excellent. Thus, the plasmonic well plate substrate for spectroscopic analysis of this invention is effective for detecting solution samples and ensures the reliability of the measurement results.

[0130] 4. Detection and sensitivity evaluation of metabolites in solution Figure 16 is a graph showing the SERS signal intensity of six metabolites (Uracil, Guanine, Xanthine, Purine, Hypoxanthine, and Adenine) in a liquid sample, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0131] Figures 17(a) to (d) are graphs showing the SERS signal intensity and detection limits of four metabolites (Adenine, Xanthine, Hypoxanthine, and Purine) for a liquid sample, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0132] The above metabolites were xanthine, purine, hypoxanthine, and adenine, and were provided to the substrate in solution form. The substrate used was a polycrystalline structure in which gold nanoparticles were attached to a polycrystalline structure formed by directly growing gold nanostructures on a well plate member to form a gold nanoparticle layer, and further growth formed a gold nanolayer on top of the gold nanoparticle layer. The SERS signal was then confirmed using this substrate.

[0133] Referring to Figure 16, by examining the Raman spectroscopy signal intensity using the plasmonic well plate substrate for spectroscopic analysis of this application, it can be confirmed that xanthine, purine, hypoxanthine, and adenine are significantly detectable among the six metabolites mentioned above.

[0134] Referring to Figure 17, it can be confirmed that the plasmonic well plate substrate for spectroscopic analysis of this application exhibits excellent signal sensitivity for the four metabolites, xanthine, purine, hypoxanthine, and adenine, for which the Raman spectroscopy signal intensity was clearly shown.

[0135] 5. Signal detection and sensitivity evaluation of urine samples from cancer patients Figure 18 is a graph showing the normalized SERS signal intensity for urine samples from cancer patients, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0136] Figure 19 is a graph showing the SERS signal intensity of candidate cancer metabolites in urine samples from cancer patients, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0137] As a substrate, we used a substrate containing a polycrystalline structure in which gold nanoparticles were attached to a well plate member to form a gold nanoparticle layer, and further growth formed a gold nanolayer on top of the gold nanoparticle layer to confirm the SERS signal for a liquid sample.

[0138] Referring to Figures 18 and 19, it can be seen that the plasmonic well plate substrate for spectroscopic analysis of this application is suitable for use in cancer diagnosis using liquid samples such as urine from patients that may contain cancer metabolites.

[0139] 6. Raman shift analysis of cancer patient samples compared to healthy controls, categorized by cancer type. Figures 20(a) to (d) are graphs showing the locations of Raman shifts for each cancer type that differ from those of healthy individuals in intervals with a confidence level of 95% or higher, as measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 20(e) is a graph comparing the relative magnitudes of the Raman shifts for each cancer type at different locations, as measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0140] As a substrate, we used a substrate containing a polycrystalline structure in which gold nanoparticles were attached to a well plate member to form a gold nanoparticle layer, and further growth formed a gold nanolayer on top of the gold nanoparticle layer to confirm the SERS signal for a liquid sample.

[0141] Referring to Figures 20(a) to (c), Raman spectra were measured using urine samples from cancer patients with colorectal cancer, lung cancer, prostate cancer, and pancreatic cancer, as well as urine samples from healthy individuals (Figure 18). Intervals showing a difference in Raman shift positions for each cancer type with a confidence level of 95% or higher compared to healthy individuals were selected. For the selection method, a t-test was performed on all positions on the Raman spectrum, and positions that did not correspond to peaks were excluded from the selection.

[0142] Referring to Figure 20(e), we confirmed that it is possible to select locations where two or more of the four types of cancer have higher or lower signal intensity compared to healthy individuals, represent them in the form of polygons, and compare the Raman shift location patterns for each type of cancer, thereby enabling differentiation between healthy individuals and cancer patients.

[0143] 7. Signal segmentation accuracy analysis of cancer patient samples compared to healthy controls, categorized by cancer type. Figures 21(a) to (d) are graphs showing the results of confusion matrices, which indicate the accuracy of distinguishing urine samples from cancer patients from healthy individuals of each cancer type, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention. Figure 21(e) is a curve graph of receiver operating characteristics (ROC), which indicates the accuracy of distinguishing urine samples from cancer patients from healthy individuals of each cancer type, measured using a plasmonic well plate substrate for spectroscopic analysis manufactured according to one embodiment of the present invention.

[0144] As a substrate, we used a substrate containing a polycrystalline structure in which gold nanoparticles were attached to a well plate member to form a gold nanoparticle layer, and further growth formed a gold nanolayer on top of the gold nanoparticle layer to confirm the SERS signal for a liquid sample.

[0145] Raman spectra were measured using urine samples from cancer patients with colorectal cancer, lung cancer, prostate cancer, and pancreatic cancer, as well as from healthy individuals (Figure 18). The accuracy of distinguishing each cancer type from healthy individuals was then confirmed using logistic regression, a machine learning technique. For model training, the number of cancer patients and healthy individuals for each cancer type was set to 70% of the total population, and the number of cancer patients and healthy individuals for testing was set to 30% of the population. This ratio is a general training and test ratio, and the ratios of the training set and test set can be set within 1-99%. Furthermore, a validation set can be added within 1-99% to validate the model formed by the training set. However, the sum of the training, test, and validation sets must satisfy 100%.

[0146] While several well-known machine learning methods can be used to classify Raman spectra, including decision tree classification, random forest, support vector machine, K-nearest neighbor, naive Bayes algorithm, and partial least squares discriminant analysis (PLS-DA), in this experiment, we validated a test group against a model trained using logistic regression for each type of cancer to show the confusion matrix for each cancer type (Figure 21(a)-(d)) and the receiver operation characteristics (Figure 21(e)).

[0147] Although specific parts of the present application have been described in detail above, it will be clear to those with ordinary knowledge of the art that such specific descriptions are merely preferred modes of implementation and do not limit the scope of the present application. Therefore, the substantial scope of the present application will be defined by the attached claims and their equivalents.

Claims

1. A well plate member having one or more wells; and A polycrystalline structure formed in the well; The polycrystalline structure is composed of a cluster of multiple gold nanostructures and contains multiple voids within, comprising a plasmonic well plate substrate for spectroscopic analysis.

2. The plasmonic well plate substrate for spectroscopic analysis according to claim 1, wherein the gold nanostructures or the clusters formed of gold nanostructures are interconnected.

3. The plasmonic well plate substrate for spectroscopic analysis according to claim 1, wherein the polycrystalline structure is one or more of the nanosponge, nanotree, nanobranch, and nanocoral forms.

4. The plasmonic well plate substrate for spectroscopic analysis according to claim 1, wherein the polycrystalline structure further comprises a layer of gold nanoparticles on its surface.

5. The plasmonic well plate substrate for spectroscopic analysis according to claim 4, wherein the polycrystalline structure further comprises a gold nanolayer grown on the gold nanoparticle layer with a gold precursor.

6. The plasmonic well plate substrate for spectroscopic analysis according to claim 1, wherein the polycrystalline structure further comprises a gold nanolayer grown on the surface with a gold precursor.

7. The plasmonic well plate substrate for spectroscopic analysis according to claim 6, wherein the polycrystalline structure further comprises a layer of gold nanoparticles on the gold nanolayer.

8. The plasmonic well plate substrate for spectroscopic analysis according to any one of claims 4 to 7, wherein the average thickness of the gold nanoparticle layer or the gold nanolayer is 5 to 100 nm.

9. The plasmonic well plate substrate for spectroscopic analysis according to claim 1, wherein the plasmonic well plate substrate for spectroscopic analysis is for surface-enhanced Raman spectroscopy (SERS), plasmon-enhanced fluorescence (PEF), or fluorescence analysis.

10. i) the step of preparing a well plate member having one or more wells; and ii) The step of supporting a well plate member on a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution to form a polycrystalline structure in the wells; The polycrystalline structure formed in step ii) is composed of a group of multiple gold nanostructures and contains multiple voids inside, A method for manufacturing a plasmonic well plate substrate for spectroscopic analysis, comprising the step of adjusting the morphology of the polycrystalline structure by setting the ratio of gold precursor to reducing agent in step ii) to 1:1 to 1:

10.

11. A method for manufacturing a plasmonic well plate substrate for spectroscopic analysis according to claim 10, further comprising the step of attaching gold nanoparticles to the well plate member on which the polycrystalline structure is formed after step ii) to form a gold nanoparticle layer on the surface of the polycrystalline structure.

12. A method for manufacturing a plasmonic well plate substrate for spectroscopic analysis according to claim 11, further comprising the step of forming a gold nanoparticle layer on the surface of the polycrystalline structure, and then supporting the well plate member in a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution to form an additional gold nanolayer grown on the gold nanoparticle layer.

13. A method for manufacturing a plasmonic well plate substrate for spectroscopic analysis according to claim 10, further comprising the step of supporting the well plate member on which the polycrystalline structure is formed in a composition for manufacturing a spectroscopic analysis substrate containing a gold precursor and a reducing agent solution, thereby forming an additional gold nanolayer grown on the surface of the polycrystalline structure.

14. A method for manufacturing a plasmonic well plate substrate for spectroscopic analysis according to claim 13, further comprising the step of forming an additional gold nanolayer on the surface of the polycrystalline structure, followed by the step of attaching gold nanoparticles to the well plate member to form a gold nanoparticle layer on the surface of the polycrystalline structure.

15. A method for manufacturing a plasmonic well plate substrate for spectroscopic analysis according to claim 11 or 14, further comprising the step of surface modifying the surface of a polycrystalline structure before the step of forming the gold nanoparticle layer.

16. The method for producing a plasmonic well plate substrate for spectroscopic analysis according to claim 10, wherein the reducing agent is one or more of ascorbic acid, hydroxylamine, hydroxyl-O-sulfonic acid, and O-methylhydroxylamine hydrochloride.

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