Plasma orifice plate substrate for spectral analysis and manufacturing method thereof

Abstract

The invention provides a plasma orifice plate substrate for spectral analysis and a manufacturing method thereof. And a method for manufacturing the same. The present invention provides a plasma orifice substrate which is suitable for detection of a solution phase sample and has an improved signal enhancement effect and uniformity, and a method for manufacturing a plasma orifice substrate for spectral analysis which can improve the signal enhancement effect and uniformity by easily adjusting the forms of various polycrystalline structures.

Description

Technical Field

[0001] The present invention relates to a plasma aperture plate substrate for spectral analysis and a method for manufacturing the same. More specifically, the present invention relates to a plasma aperture plate substrate suitable for detecting solution-phase samples and having improved signal enhancement and uniformity, as well as a method for manufacturing a plasma aperture plate substrate for spectral analysis that can easily adjust the morphology of various polycrystalline structures to improve signal enhancement and uniformity. Background Art

[0002] Raman scattering is a type of inelastic scattering in which the energy of incident light changes. When light strikes a specific molecule, the inherent vibrational transitions of the molecule produce light with a wavelength slightly different from that of the incident light.

[0003] In fact, almost all organic molecules have an inherent Raman shift. Therefore, Raman spectroscopy, which utilizes Raman scattering, can obtain signals even for non-polar molecules whose induced polarizability changes. Furthermore, Raman spectroscopy is not affected by interference caused by water molecules, making it more suitable for detecting biomolecules such as proteins and genes. The wavelength of the Raman emission spectrum indicates the chemical composition and structural characteristics of the light-absorbing molecules within the sample, so the target substance can be directly analyzed by analyzing this Raman signal.

[0004] To detect biomolecules and other substances using Raman spectroscopy, samples are typically dried, requiring a separate sample drying step. This step increases time and cost, and can also lead to contamination and sample deterioration.

[0005] On the other hand, the orifice plate is an experimental test device composed of a plate formed by arranging a plurality of holes or slots, and is widely used in biochemical analysis or clinical testing, etc. When detecting or diagnosing a specific target substance or disease, etc. based on the orifice plate, the sample to be analyzed is usually put into the hole to induce a reaction, and then the target substance is detected or the disease is diagnosed, etc., with the substance used for the detection of the target substance or the diagnosis of the disease being impregnated on the inner surface of the hole of the orifice plate. When this orifice plate is used, it is easy to detect the target substance in multiple samples at the same time. In addition, it is suitable for the analysis of liquid samples, so body fluids such as urine, saliva, tears and sweat can be directly used as samples in a liquid state, thereby effectively reducing costs and shortening time in the detection of target substances or the diagnosis of diseases.

[0006] As background technology for this application, Korean Patent Publication No. 10-2016-0014866 describes a method and apparatus for diagnosing viral infection using teardrops. This patent publication includes a separate drying step to prepare the collected tears into a sample for measurement, thereby obtaining a Raman spectrum. Summary of the Invention

[0007] Problems to be solved by the invention

[0008] The purpose of the present application is to provide a plasma aperture plate substrate for spectral analysis, which has excellent signal enhancement effect and uniformity suitable for liquid sample analysis when used for spectral analysis.

[0009] Another object of the present application is to provide a plasma aperture plate substrate for spectral analysis, which can perform qualitative and quantitative analysis on the analyzed substance in a non-labeling manner.

[0010] Another object of the present application is to provide a plasma aperture plate substrate for spectral analysis, which is suitable for analyzing multiple samples simultaneously.

[0011] Another object of the present application is to provide a method for manufacturing a plasma aperture plate substrate for spectral analysis, which can easily adjust and form the morphology of the nanostructure of the polycrystalline structure formed on the aperture plate member, thereby having improved signal enhancement effect and uniformity.

[0012] Another object of the present application is to provide a method for effectively manufacturing a plasma aperture plate substrate for spectral analysis, wherein the plasma aperture plate substrate for spectral analysis is suitable for analyzing solution-phase samples and has excellent signal enhancement effect and uniformity.

[0013] The objects of the present application are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood from the detailed description.

[0014] Means used to solve problems

[0015] According to one aspect, a plasma aperture plate substrate for spectral analysis is provided, comprising: an aperture plate member having one or more apertures; and a polycrystalline structure formed within the aperture, wherein the polycrystalline structure is composed of an aggregate formed by a plurality of gold nanostructures and includes a plurality of pores therein.

[0016] According to one embodiment, the gold nanostructures or aggregates formed of the gold nanostructures may be connected to each other.

[0017] According to one embodiment, the polycrystalline structure may be in the form of one or more of a nano-sponge, a nano-tree, a nano-branch, and a nano-coral.

[0018] According to one embodiment, the polycrystalline structure may further include a gold nanoparticle layer on its surface.

[0019] According to one embodiment, the polycrystalline structure may further include a gold nanolayer formed by further growing a gold precursor on the gold nanoparticle layer.

[0020] According to one embodiment, the polycrystalline structure may further include a gold nanolayer further grown on the surface of the polycrystalline structure by a gold precursor.

[0021] According to one embodiment, the polycrystalline structure may further include a gold nanoparticle layer on the gold nanolayer.

[0022] According to one embodiment, the average thickness of the gold nanoparticle layer or the gold nanolayer may be 5 nm to 100 nm.

[0023] According to one embodiment, the plasma aperture plate substrate for spectral analysis can be used for surface enhanced Raman spectroscopy (SERS), plasmon-enhanced fluorescence (PEF) or fluorescence analysis.

[0024] According to another aspect, a method for manufacturing a plasma orifice plate substrate for spectral analysis is provided, the method comprising: step i) preparing an orifice plate member having one or more holes; and step ii) immersing the orifice plate member in a composition for manufacturing a substrate for spectral analysis comprising a gold precursor and a reducing agent solution, thereby forming a polycrystalline structure in the hole, wherein the polycrystalline structure formed in step ii) is composed of aggregates (formed by a plurality of gold nanostructures) and includes a plurality of pores therein, and further comprising: a step of adjusting the morphology of the polycrystalline structure by making the ratio of the gold precursor to the reducing agent in step ii) be 1:1 to 1:10.

[0025] According to one embodiment, after step ii), the method may further include attaching gold nanoparticles to the orifice plate member having the polycrystalline structure formed thereon, so as to form a gold nanoparticle layer on the surface of the polycrystalline structure.

[0026] According to one embodiment, after the step of forming a gold nanoparticle layer on the surface of the polycrystalline structure, the method may further include: immersing the aperture plate member in a composition for manufacturing a spectral analysis substrate comprising a gold precursor and a reducing agent solution to form a further grown gold nanolayer on the gold nanoparticle layer.

[0027] According to one embodiment, after step ii), the method may further include: immersing the aperture plate member formed with the polycrystalline structure in a composition for manufacturing a spectral analysis substrate comprising a gold precursor and a reducing agent solution to form a further grown gold nanolayer on the surface of the polycrystalline structure.

[0028] According to one embodiment, after the step of forming a further grown gold nanolayer on the surface of the polycrystalline structure, the method may further include: attaching gold nanoparticles to the aperture plate member to form a gold nanoparticle layer on the surface of the polycrystalline structure.

[0029] According to one embodiment, before the step of forming the gold nanoparticle layer, the method may further include the step of modifying the surface of the polycrystalline structure.

[0030] According to one embodiment, the reducing agent may be one or more selected from ascorbic acid, hydroxylamine, hydroxylamine-O-sulfonic acid, and O-Methylhydroxylamine hydrochloride.

[0031] Effects of the Invention

[0032] According to one embodiment, the plasma aperture plate substrate for spectral analysis of the present application includes various forms of polycrystalline structures formed by aggregates of gold nanostructures on the aperture plate component. When used for spectral analysis, it is suitable for the analysis of solution phase samples and can also exhibit excellent signal enhancement effect and uniformity.

[0033] According to one embodiment, when analyzing a liquid sample using the plasma aperture plate substrate for spectral analysis of the present application, no drying step is required.

[0034] According to one embodiment, the plasma aperture plate substrate for spectral analysis of the present application can be used to perform qualitative and quantitative analysis of non-labeled analytical substances.

[0035] According to one embodiment, the plasma aperture plate substrate for spectral analysis of the present application is effectively used to simultaneously analyze a plurality of samples to be analyzed with high sensitivity.

[0036] According to one embodiment, the manufacturing method of the plasma aperture plate substrate for spectral analysis of the present application can easily adjust and form the polycrystalline structure composed of aggregates of gold nanostructures into various forms on the aperture plate component, thereby effectively manufacturing a plasma aperture plate substrate for spectral analysis with improved signal enhancement effect and uniformity.

[0037] According to one embodiment, the method for manufacturing a plasma aperture plate substrate for spectral analysis of the present application can effectively manufacture a plasma aperture plate substrate for spectral analysis that is suitable for analyzing liquid samples and has improved signal enhancement effect and uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The following is a photograph of plasma aperture plate substrates for spectral analysis manufactured according to an embodiment of the present application, classified by aperture size.

[0039] Figure 2 Parts (a) to (c) of FIG. 5 are scanning electron microscope (SEM) photographs showing various forms of polycrystalline structures formed on the aperture plate member according to one embodiment of the present application.

[0040] Figure 3 Parts (a) and (b) are scanning electron microscope (SEM) photographs showing a thick sponge-like polycrystalline structure formed on a hole plate member according to an embodiment of the present application, wherein the thick sponge-like polycrystalline structure includes a gold nanoparticle layer formed by gold nanoparticles attached to the surface with thicknesses of 25 nm and 40 nm, respectively.

[0041] Figure 3 Part (c) is a scanning electron microscope (SEM) photograph showing a thick sponge-like polycrystalline structure formed on a hole plate member according to an embodiment of the present application, wherein the thick sponge-like polycrystalline structure includes a gold nanolayer formed by further growing a gold precursor on the gold nanoparticle layer.

[0042] Figure 4 Parts (a) and (b) are scanning electron microscope (SEM) photographs showing short tree-shaped and long tree-shaped polycrystalline structures formed on a hole plate member according to an embodiment of the present application, wherein the short tree-shaped and long tree-shaped polycrystalline structures include a gold nanoparticle layer formed by gold nanoparticles attached to the surface with a thickness of 25 nm.

[0043] Figure 5 1 is a graph showing X-ray diffraction (XRD) results of a polycrystalline structure of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0044] Figure 6 FIG. 1 is a diagram briefly illustrating various methods of manufacturing a plasma aperture plate for spectral analysis according to an embodiment of the present application.

[0045] Figure 7 Part (a) is a graph showing the surface enhanced Raman spectroscopy (SERS) signal intensity of the nanostructure of the polycrystalline structure of the plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0046] Figure 7 Part (b) is a graph showing the SERS signal intensity at the main peak of the nanostructure of the polycrystalline structure of the plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0047] Figure 8 3 is a graph showing the SERS signal intensity measured at 633 nm according to the type of reducing agent and the ratio of the gold precursor to the reducing agent when a polycrystalline structure of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application is formed.

[0048] Figure 9 3 is a graph showing the SERS signal intensity measured at 785 nm according to the type of reducing agent and the ratio of the gold precursor to the reducing agent when a polycrystalline structure of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application is formed.

[0049] Figure 10 This is a transmission electron microscope (TEM) image of the nanostructure of a polycrystalline structure in a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application, in which gold nanoparticles are attached to a thick sponge-like polycrystalline structure to form a gold nanoparticle layer, and then a gold precursor is further grown to form a gold nanolayer.

[0050] Figure 11 The present invention is a diagram and an image showing the size distribution of a thick sponge-like polycrystalline structure in a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application, the nanostructure of the polycrystalline structure on which gold nanoparticles are attached to form a gold nanoparticle layer, and the nanostructure of the polycrystalline structure on which a gold precursor is further grown on the gold nanoparticle layer to form a gold nanolayer.

[0051] Figure 12Part (a) is a graph showing the SERS signal intensity of the nanostructure of the polycrystalline structure of a dried sample measured at 633 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0052] Figure 12 Parts (b) and (c) are graphs showing SERS signal intensity and detection limit according to sample concentration when measuring a dry sample at 633 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0053] Figure 13 Part (a) is a graph showing the SERS signal intensity of the nanostructure according to the polycrystalline structure of a dried sample measured at 785 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0054] Figure 13 Parts (b) and (c) are graphs showing SERS signal intensity and detection limit according to sample concentration when measuring a dry sample at 785 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0055] Figure 14 Part (a) is a graph showing the SERS signal intensity of the nanostructure of the polycrystalline structure of a liquid sample measured at 785 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0056] Figure 14 Parts (b) and (c) are graphs showing SERS signal intensity and detection limit according to sample concentration when measuring a liquid sample at 785 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0057] Figure 15 Part (a) is a diagram showing the uniformity of the SERS signal of a liquid sample in a single well of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0058] Figure 15 Part (b) is a graph showing the uniformity of SERS signals of 96 liquid samples between different wells of a plasma well plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0059] Figure 162 is a graph showing the SERS signal intensities of six metabolites (uracil, guanine, xanthine, purine, hypoxanthine, and adenine) of a liquid sample measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0060] Figure 17 Parts (a) to (d) are graphs showing the SERS signal intensities and detection limits of four metabolites (adenine, xanthine, hypoxanthine, and purine) of a liquid sample measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0061] Figure 18 is a graph showing normalized SERS signal intensities of urine samples of cancer patients measured using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0062] Figure 19 is a graph showing the SERS signal intensities of a candidate group of cancer metabolites in a urine sample of a cancer patient, measured using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0063] Figure 20 Parts (a) to (d) are graphs showing the positions of Raman shifts where differences occur between various cancer types and normal people within a confidence interval of 95% or more, measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0064] Figure 20 Part (e) is a graph showing a comparison of the relative magnitudes of Raman shifts at different positions in various cancer types measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0065] Figure 21 Parts (a) to (d) are confusion matrix result diagrams used to illustrate the accuracy of distinguishing urine samples of cancer patients from normal people according to cancer type, measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0066] Figure 21Part (e) is a receiver operating characteristics (ROC) curve diagram showing the accuracy of distinguishing urine samples of cancer patients from normal people according to cancer type, measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application. DETAILED DESCRIPTION

[0067] The objects, specific advantages and novel features of the present disclosure will become more apparent through the following detailed description and examples related to the accompanying drawings.

[0068] Before this, the terms and words used in this specification and claims should not be interpreted according to their ordinary meanings and dictionary meanings, but should be interpreted as meanings and concepts that are consistent with the technical ideas of the present disclosure based on the principle that the inventor can appropriately define the concepts of the terms to interpret his invention in the best way.

[0069] In this specification, when a component such as a layer, a part, or a substrate is described as being “on,” “connected to,” or “coupled to” another component, it may be directly “on,” “connected to,” or “coupled to” the other component, or one or more other components may be interposed between the two components. In contrast, if a component is described as being “directly on,” “directly connected to,” or “directly coupled to” another component, no other components may be interposed between the two components.

[0070] The terms used in this specification are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context clearly dictates otherwise, a singular expression includes a plural expression.

[0071] It should be understood that, in this specification, terms such as "including" or "having" are used to specify the existence of features, numbers, steps, actions, constituent elements, parts or combinations thereof recorded in the specification, and do not preclude the existence or additional possibilities of one or more other features, numbers, steps, actions, constituent elements, parts or combinations thereof.

[0072] In this specification, when a portion is described as "including" a certain component, unless otherwise stated, it means that the portion may also include the other components, not that the portion is excluded. Furthermore, throughout this specification, the term "on" refers to being above or below the target portion and does not necessarily mean being located on the upper side relative to the direction of gravity.

[0073] The present disclosure is susceptible to numerous variations and embodiments, and specific embodiments are illustrated in the accompanying drawings and described in detail in the detailed description. However, it should be understood that the present disclosure is not limited to specific embodiments, but rather encompasses all variations, equivalents, and even substitutes within the spirit and technical scope of the present disclosure. When describing the present disclosure, if a detailed description of a related known technology is determined to obscure the main purpose of the present disclosure, the detailed description will be omitted.

[0074] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings, but when describing with reference to the accompanying drawings, the same or corresponding constituent elements are given the same reference numerals and repeated description thereof will be omitted.

[0075] According to one aspect, a plasma aperture plate substrate for spectral analysis is provided, comprising: an aperture plate member having one or more apertures; and a polycrystalline structure formed within the aperture, wherein the polycrystalline structure is composed of an aggregate formed by a plurality of gold nanostructures and includes a plurality of pores therein.

[0076] Figure 1 The following is a photograph of plasma aperture plate substrates for spectral analysis manufactured according to an embodiment of the present application, classified by aperture size.

[0077] Reference Figure 1 The plasma well plate component for spectral analysis of the present application can form a polycrystalline structure composed of aggregates (formed by gold nanostructures) on the well plate component having more than one well, so that it looks like a gold film is formed inside the well.

[0078] Without limitation, the wells may have various sizes, for example, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, a 384-well plate, or other well-known multi-well plates having two or more wells.

[0079] Although not limited thereto, petri dishes are used for label-free bacterial detection, and the plasma aperture plate substrate for spectral analysis according to the present application can be applied to in-situ mapping, species classification, drug-resistant bacteria detection, etc.

[0080] Without limitation, the 6-well plate is used for cancer cell culture and metabolite analysis by a label-free method. By utilizing the plasma well plate substrate for spectral analysis according to the present application, in-situ Raman imaging and monitoring of drug responses and immune responses can be achieved.

[0081] Without limitation thereto, in the plasma well plate substrate for spectral analysis according to the present application, a 96-well plate or the like can be applied to the fields of high-speed screening of a large number of chemical substances by fluorescence (PEF) and surface-enhanced Raman spectroscopy (SERS) analysis and ultra-high sensitivity enzyme-linked immunosorbent assay (ELISA) platform.

[0082] Figure 2 Parts (a) to (c) of FIG. 5 are scanning electron microscope (SEM) images showing polycrystalline structures of various nanostructures formed on the aperture plate member according to one embodiment of the present application.

[0083] Reference Figure 2 From parts (a) to (c) of FIG. 1 , it can be confirmed that a polycrystalline structure composed of aggregates (formed by a plurality of gold nanostructures) is formed on the orifice plate member. The gold nanostructures are grown directly on the orifice plate member by a solution process using a gold precursor and a reducing agent to form aggregates.

[0084] In the embodiment of the present invention, the nanostructure of the present invention is to form an aggregate, and the nanostructure of the present invention is to form an aggregate. More specifically, the gold (Au) precursor in the noble metal forms a nanostructure to constitute an aggregate, and particularly, it is grown directly on the orifice plate member by solution process. Specifically, the nanostructure of aggregation forms a small-sized polygonal structure grain (grain) by coalescence (coalescence), and then by oriented attachment growth (oriented attachment), grain grows up gradually. Therefore, a polycrystalline structure with multiple grain boundaries (grain boundary) is formed. Not limited to this, the polycrystalline structure can be grown to micron size on average. As mentioned above, the polycrystalline structure with multiple grain boundaries can significantly improve signal intensity and signal uniformity due to the increase in scattering at multiple grain boundaries when it is used as the substrate for spectral analysis.

[0085] In addition, described polycrystalline structure is made of aggregation (being formed by a plurality of gold nanostructures), and can comprise a plurality of holes inside.Be not limited to this, described a plurality of gold nanostructures or the aggregation being formed by a plurality of gold nanostructures can be the form that is interconnected and comprises hole inside.Be not limited to this, described a plurality of gold nanostructures or the aggregation being formed by a plurality of gold nanostructures can be connected with branch (branch) structure between.

[0086] Without limitation thereto, the average particle size of the polycrystalline structure may be 0.1 μm to 100 μm. The substrate for spectral analysis of the present application is grown into a polycrystalline structure by a solution process and connected in a branch structure, and the average particle size thereof may be 0.1 μm to 100 μm. Without limitation thereto, the average particle size of the polycrystalline structure may be suitable for improving signal intensity and signal uniformity within the range.

[0087] As described above, the plurality of gold nanostructures are connected to each other and constitute an aggregate, and the polycrystalline structure may be constituted by the aggregate (formed by the plurality of gold nanostructures).

[0088] As described above, in the plasma orifice plate substrate for spectral analysis of the present application, the gold nanostructures are connected to each other, rather than spaced apart from each other, and form a morphology of aggregates including pores distributed within. Therefore, compared with the case where the gold nanostructures are spaced apart from each other, the nanogaps and hot spots may be significantly increased. Therefore, the plasma orifice plate substrate for spectral analysis of the present application can show excellent SERS signal enhancement effect and uniformity for the analyzed substance.

[0089] Without limitation, the pores contained in the polycrystalline structure can be formed by a porous structure between multiple gold nanostructures in a disordered but overall uniform manner, or can be formed by a porous structure between aggregates formed by multiple gold nanostructures in a disordered but overall uniform manner. Therefore, the plasma aperture plate substrate for spectral analysis of the present application can improve the SERS signal enhancement effect and uniformity for the analyzed substance.

[0090] Reference Figure 2 (a) part to (c) part, not limited thereto, the polycrystalline structure can be in the form of nano sponge (nanosponge), thick sponge (thick sponge), nano tree (nano-tree), nano branch (nano-branch) and nano coral (nano-coral) etc. The nano sponge is a sponge form including multiple pores inside. Compared with the nano sponge, the thick sponge can refer to the form of a sponge with the same or similar shape but thick aggregate formed. The nano tree, nano branch, nano coral can refer to the length of the aggregate grown, formed into a tree shape, a branch shape or a coral shape.

[0091] Without limitation, the shape of the polycrystalline structure formed on the substrate for spectral analysis can be easily adjusted by adjusting the optimal reducing agent ratio according to the type of reducing agent used for the gold precursor. Furthermore, the shape of the polycrystalline structure formed on the substrate for spectral analysis can be easily adjusted by adjusting the time the orifice plate member is immersed in the composition for manufacturing the substrate for spectral analysis.

[0092] Figure 3 Parts (a) and (b) are scanning electron microscope (SEM) photographs showing a thick sponge-like polycrystalline structure formed on a hole plate member according to an embodiment of the present application, wherein the thick sponge-like polycrystalline structure includes a gold nanoparticle layer formed by gold nanoparticles attached to the surface with thicknesses of 25 nm and 40 nm, respectively.

[0093] Figure 4 Parts (a) and (b) are scanning electron microscope (SEM) photographs showing short tree-shaped and long tree-shaped polycrystalline structures formed on a hole plate member according to an embodiment of the present application, wherein the short tree-shaped and long tree-shaped polycrystalline structures include a metal nanoparticle layer formed by gold nanoparticles attached to its surface with a thickness of 25 nm.

[0094] Reference Figure 3 Part (a), Part (b), Figure 4 The polycrystalline structure of the present application may further include a gold nanoparticle layer on the surface. The gold nanoparticle layer may be formed by adding pre-synthesized gold nanoparticles so that the gold nanoparticles are attached to the surface of the polycrystalline structure.

[0095] Figure 3 Part (c) is a scanning electron microscope (SEM) photograph showing a thick sponge-like polycrystalline structure formed on a hole plate member according to an embodiment of the present application, wherein the thick sponge-like polycrystalline structure includes a gold nanolayer formed by further growth of a gold precursor on the gold nanoparticle layer.

[0096] Reference Figure 3 In part (c), the polycrystalline structure of the present application may further include a gold nanolayer formed by further growing a gold precursor on the gold nanoparticle layer. The gold nanolayer may be formed by further growing gold nanoparticles on the gold nanoparticle layer using a composition that is the same as or different from the composition containing the gold precursor and reducing agent solution used when the polycrystalline structure is initially formed.

[0097] Figure 5 : is a graph showing the X-ray diffraction (XRD) results of a polycrystalline structure of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application. Specifically, Figure 5 The XRD results of the polycrystalline structure formed by further growing a gold nanolayer on the gold nanoparticle layer confirm the polycrystalline nature of the polycrystalline structure of the plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0098] Without limitation, the polycrystalline structure of the present application may further include a gold nanolayer formed by further growing a gold precursor on its surface. The gold nanolayer may be formed by further growing a composition that is the same as or different from the composition comprising the gold precursor and reducing agent solution used when the polycrystalline structure is initially formed.

[0099] Without limitation thereto, the polycrystalline structure of the present application may further include a gold nanoparticle layer on the gold nanolayer. The gold nanoparticle layer may be formed by attaching synthesized gold nanoparticles to the gold nanolayer.

[0100] Without being limited thereto, the gold nanoparticle layer can be a continuous layer morphology or a discontinuous layer morphology. Without being limited thereto, the gold nanolayer can be a continuous layer morphology or a partially discontinuous morphology, but it may be more appropriate to use a continuous layer morphology. The gold nanolayer can be in the form of a covering polycrystalline structure or in the form of a laminate in a thin layer form.

[0101] Without being limited thereto, the average thickness of the gold nanolayer or gold nanoparticle layer may be 5 nm to 100 nm, and the case having the said average thickness range may be suitable for the improvement of signal enhancement effect and uniformity caused by further growth, and may be 10 nm to 95 nm, may be 15 nm to 90 nm, may be 20 nm to 85 nm, may be 20 nm to 80 nm, may be 20 nm to 75 nm, may be 20 nm to 70 nm, may be 20 nm to 65 nm, may be 20 nm to 60 nm, may be 20 nm to 55 nm, may be 20 nm to 50 nm, may be 20 nm to 45 nm, may be 25 nm to 40 nm.

[0102] The plasma aperture plate substrate for spectral analysis of the present application can be used for surface enhanced Raman spectroscopy (SERS), plasmon-enhanced fluorescence (PEF) or fluorescence (Flurorescence) analysis. Not limited to this, the plasma aperture plate substrate for spectral analysis of the present application may be more suitable for surface enhanced Raman spectroscopy (SERS) analysis. When it is used for the surface enhanced Raman spectroscopy analysis, the analyte can be effectively detected and analyzed with high sensitivity without the need for labels such as fluorescent substances.

[0103] By using the plasma aperture plate substrate for spectral analysis of the present application, it is not necessary to separately label the sample to be analyzed or the analyzed substance, that is, the analyzed substance can be effectively detected and analyzed in a non-labeling manner.

[0104] Without limitation, the analyte may be one or more selected from cells, metabolites, proteins, nucleic acids, DNA, RNA, mRNA, lipids, hormones, metabolites, enzymes, organic molecules, viruses, bacteria, antigens, antibodies, neurotransmitters, extracellular vesicles, microvesicles, exosomes, and fat.

[0105] Without limitation, the sample may be a liquid sample, various solutions containing the various analytes, or various body fluids, substrates, and secretions such as urine, saliva, blood, sweat, tears, ascites, gastric juice, and cerebrospinal fluid.

[0106] Conventionally, due to the low hygroscopicity of substrates used for spectral analysis, the sensitivity when measuring liquid samples was very low. However, the plasma aperture plate substrate for spectral analysis according to the present application can be loaded with liquid samples, allowing even liquid samples to be rapidly analyzed and diagnosed on-site.

[0107] Without limitation, the plasma aperture plate substrate for spectral analysis of the present application can be used to diagnose cancer using liquid samples. Without limitation, metabolite analysis of the sample and analyte described above can provide information necessary for cancer diagnosis, enabling early diagnosis of cancer through non-invasive testing, and enabling multiple analyses with extremely high sensitivity.

[0108] The cancer may be one or more selected from pancreatic cancer, prostate cancer, lung cancer, colon cancer, bronchial cancer, colorectal cancer, breast cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, thyroid cancer, esophageal cancer, uterine cancer, liver cancer, kidney cancer and bile duct cancer, but is not limited thereto.

[0109] Figure 18 is a graph showing normalized SERS signal intensities of urine samples of cancer patients measured using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application. Figure 19 is a graph showing the SERS signal intensities of a candidate group of cancer metabolites in a urine sample of a cancer patient, measured using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0110] Refer to the Figure 18 and Figure 19 It has been confirmed that the plasma aperture plate substrate for spectral analysis of the present application is suitable for cancer diagnosis using liquid samples such as patient urine that may contain cancer metabolites.

[0111] Figure 6FIG. 1 is a diagram briefly illustrating various methods of manufacturing a plasma aperture plate substrate for spectral analysis according to an embodiment of the present application.

[0112] Reference Figure 6 According to another aspect of the present application, a method for manufacturing a plasma orifice plate substrate for spectral analysis is provided, the method comprising: step i) preparing an orifice plate member having one or more holes; and step ii) immersing the orifice plate member in a composition for manufacturing a substrate for spectral analysis comprising a gold precursor and a reducing agent solution, thereby forming a polycrystalline structure in the hole, wherein the polycrystalline structure formed in step ii) is composed of aggregates (formed by a plurality of gold nanostructures) and includes a plurality of pores therein, and comprises a step of adjusting the morphology of the polycrystalline structure by making the ratio of the gold precursor to the reducing agent in step ii) be 1:1 to 1:10.

[0113] Step i) is a step of preparing a well plate member having one or more wells. The well plate member having one or more wells can be various well-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.

[0114] Step ii) is a step of immersing the aperture plate member in a composition for manufacturing a spectroscopic analysis substrate comprising a gold precursor and a reducing agent solution to form a polycrystalline structure in the aperture. Formation of the polycrystalline structure in step ii) means growing gold nanostructures on the aperture surfaces of the aperture plate member.

[0115] The polycrystalline structure formed in the step ii) is made of aggregation (being formed by a plurality of gold nanostructures), and can comprise a plurality of holes inside it.Be not limited to this, the aggregation that described a plurality of gold nanostructures or are formed by a plurality of gold nanostructures can be the form that is interconnected and comprises hole inside.Be not limited to this, can be connected with branch (branch) structure between the aggregation that described a plurality of gold nanostructures or are formed by described a plurality of gold nanostructures.

[0116] Without limitation, the polycrystalline structure is grown by a solution process and connected in a branch structure, and its average particle size may be 0.1 μm to 100 μm. Without limitation, the average particle size of the polycrystalline structure may be within the above range to improve signal intensity and signal uniformity.

[0117] As described above, the multiple gold nanostructures are in a form connected to each other and constituted as aggregates, and the polycrystalline structure can be composed of the aggregates (formed by the multiple gold nanostructures). In addition, the polycrystalline structure is formed by multiple aggregates connected to each other and can have multiple grain boundaries.

[0118] In the plasma aperture plate substrate for spectral analysis of the present application, which includes the polycrystalline structure formed as described above, the gold nanostructures are connected to each other, rather than spaced apart from each other, and form a morphology of aggregates including pores distributed within. Therefore, compared with the case where the gold nanostructures are spaced apart from each other, the nanogaps and hot spots may be significantly increased. Therefore, the plasma aperture plate substrate for spectral analysis of the present application can exhibit excellent SERS signal enhancement effect and uniformity for the analyzed substance.

[0119] Without limitation, the pores contained in the polycrystalline structure can be formed by a porous structure between multiple gold nanostructures in a disordered but overall uniform manner, or can be formed by a porous structure between aggregates formed by multiple gold nanostructures in a disordered but overall uniform manner. Therefore, the plasma aperture plate substrate for spectral analysis of the present application can improve the SERS signal enhancement effect and uniformity for the analyzed substance.

[0120] A shape adjustment step may be included to adjust the shape of the polycrystalline structure by adjusting the ratio of the gold precursor to the reducing agent in the step ii) to 1:1 to 1:10. Depending on the type of reducing agent, the optimal ratio of the reducing agent is adjusted, so that the shape of the polycrystalline structure formed on the plasma aperture plate substrate for spectral analysis can be easily adjusted. In addition, the shape of the polycrystalline structure formed on the substrate for spectral analysis can be easily adjusted by adjusting the time for immersing the aperture plate member in the composition for manufacturing the spectral analysis substrate. The manufacturing method of the substrate for spectral analysis of the present application can simultaneously improve signal enhancement and signal uniformity through a simple process of immersing once to twice.

[0121] Without being limited thereto, after step ii), the method may further include attaching gold nanoparticles to a well plate member having a polycrystalline structure formed thereon, so as to form a gold nanoparticle layer on the surface of the polycrystalline structure.

[0122] As mentioned above, it can be to utilize gold nanoparticles manufactured through independent synthesis, and the gold nanoparticles are attached to the surface of the polycrystalline structure formed in the hole of the orifice plate member to form a gold nanoparticle layer. Not limited to this, the gold nanoparticles can be manufactured by various known gold nanoparticle synthesis methods. Not limited to this, the attachment of the gold nanoparticles in the step of forming the gold nanoparticle layer can utilize conventional known technology. For example, the gold nanoparticles can be attached by physical adsorption, or the gold nanoparticles can be attached after surface modification using a chemical linker, so that the gold nanoparticles are fixed on the surface of the polycrystalline structure. Through the above-mentioned formation, the plasma orifice plate substrate for spectral analysis of the present application can improve the SERS signal enhancement effect and uniformity for analyzing substances.

[0123] Without limitation thereto, after the step of forming the gold nanoparticle layer on the surface of the polycrystalline structure, the method may further include: immersing the orifice plate member in a composition for manufacturing a spectral analysis substrate comprising a gold precursor and a reducing agent solution, so as to further grow gold nanoparticles on the gold nanoparticle layer to form a gold nanolayer. Without limitation thereto, the gold nanolayer may be in a form covering the polycrystalline structure on which the gold nanoparticle layer is formed, or in a form laminated in the form of a thin layer.

[0124] Without limitation, the composition for manufacturing the spectral analysis substrate comprising a gold precursor and a 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 such a configuration, the plasma aperture plate substrate for spectral analysis of the present application can improve the SERS signal enhancement effect and uniformity for the analyte.

[0125] After step ii), the method may further include: immersing the orifice plate member formed with a polycrystalline structure in a composition for manufacturing a spectral analysis substrate comprising a gold precursor and a reducing agent solution, so as to further grow gold nanoparticles on the surface of the polycrystalline structure to form a gold nanolayer. Not limited to this, the gold nanolayer may be in the form of covering the polycrystalline structure, or in the form of a laminate in the form of a thin layer. Through the above-mentioned configuration, the plasma orifice plate substrate for spectral analysis of the present application can improve the SERS signal enhancement effect and uniformity for the analyzed substance.

[0126] Without limitation, after forming the further grown gold nanolayer on the surface of the polycrystalline structure, the method may further include attaching gold nanoparticles to the aperture plate member having the polycrystalline structure to form the gold nanoparticle layer on the surface of the polycrystalline structure. With this configuration, the plasma aperture plate substrate for spectral analysis of the present application can improve the SERS signal enhancement effect and uniformity for the analyzed substance.

[0127] Without being limited thereto, the thickness of the gold nanolayer formed by further growth of the gold precursor or the gold nanoparticle layer formed by attachment of the gold nanoparticles may be 5 nm to 100 nm. The case having the said average thickness range may be suitable for improving the signal enhancement effect and uniformity by further growth, and may be 10 nm to 95 nm, 15 nm to 90 nm, 20 nm to 85 nm, 20 nm to 80 nm, 20 nm to 75 nm, 20 nm to 70 nm, 20 nm to 65 nm, 20 nm to 60 nm, 20 nm to 55 nm, 20 nm to 50 nm, 20 nm to 45 nm, or 25 nm to 40 nm.

[0128] Without limitation thereto, the method for manufacturing a plasma aperture plate substrate for spectral analysis of the present application may further comprise, before the step of forming the gold nanoparticle layer, a step of surface-modifying the surface of the polycrystalline structure. The surface modification step is a step for effectively attaching the gold nanoparticles to the surface of the polycrystalline structure when forming the gold nanoparticle layer. Without limitation thereto, the surface modification step may utilize a chemical linker for fixing the gold nanoparticles, and a substance containing a thiol group may be used as the chemical linker.

[0129] The composition for manufacturing a spectroscopic analysis substrate of the present application comprises a gold precursor and a reducing agent.

[0130] Without being limited thereto, the gold precursor may be selected from the group consisting of HAuCl 4 , AuCl, AuCl 2 , AuCl 3 , Na 2 Au 2 Cl 8 , and NaAuCl 2 , but one or more of HAuCl 4 and NaAuCl 2 may be more suitable.

[0131] Without limitation thereto, the reducing agent may be one or more selected from ascorbic acid, FeSO4, hydroxyquinone, hydroxylamine, hydroxylamine-O-sulfonic acid, and O-Methylhydroxylamine hydrochloride. Without limitation thereto, when the reducing agent is one or more selected from hydroxylamine, hydroxylamine-O-sulfonic acid, and O-Methylhydroxylamine hydrochloride, it may be suitable for manufacturing a plasma aperture plate substrate for spectral analysis having excellent signal enhancement and uniformity.

[0132] Without limitation, the ratio of the gold precursor to the reducing agent may be 1:1 to 1:10. When the ratio is 1:2 to 1:10, it may be suitable for the manufacture of a plasma aperture plate substrate for spectral analysis with excellent signal enhancement effect and uniformity, and may be more suitable when the ratio is 1:5 to 1:10.

[0133] Example

[0134] Example 1: Fabrication of a Plasma Aperture Plate Substrate for Spectroscopic Analysis Comprising a Polycrystalline Structure

[0135] A well plate member including one or more holes is prepared as a base member. A gold precursor solution and a reducing agent solution are mixed at ratios of 1:0.5, 1:1, 1:2, 1:5 and 1:10 to produce a composition for producing a spectral analysis substrate. Distilled water, ethanol and methanol can be used as manufacturing solvents for producing the composition, but ethanol may be more suitable for the uniform formation of a polycrystalline structure, so ethanol is used. The composition is loaded into each hole of the well plate for 1 minute to 24 hours to form a polycrystalline structure on the well plate member, wherein the polycrystalline structure is composed of aggregates (formed by a plurality of gold nanostructures) and includes a plurality of pores therein.

[0136] HAuCl4 was used as the gold precursor solution for the composition used to manufacture the spectral analysis substrate, and hydroxylamine, hydroxylamine-O-sulfonic acid, and O-methylhydroxylamine hydrochloride were used as reducing agents. After the polycrystalline structure was formed, it was washed twice with ethanol and three or more times with water to remove any residual precursor and reducing agent. The composition was then dried at room temperature for at least one hour, completing the manufacture of the plasma aperture plate substrate for spectral analysis.

[0137] Example 2: Plasmonic aperture plate for spectral analysis comprising a polycrystalline structure forming a gold nanoparticle layer Substrate manufacturing

[0138] The surface of the plasma aperture plate substrate for spectral analysis is formed with a polycrystalline structure inside the hole manufactured in the embodiment 1, and the surface of the polycrystalline structure is modified by using a substance containing a thiol group as a chemical linker. At this time, cysteine ​​is used as a substance containing a thiol group, and the concentration range is 1uM to 1000uM. Afterwards, a gold nanoparticle solution synthesized separately in advance is added inside each hole to attach gold nanoparticles and form a gold nanoparticle layer with a thickness of 25nm to 40nm on the surface of the polycrystalline structure. The substance containing the thiol group can be selectively used, and even when not in use, gold nanoparticles can be attached by physical adsorption. The reaction is carried out at a temperature of 40°C until the gold nanoparticle solution is completely dry. After the formation of the gold nanoparticle layer is completed, it is washed with water more than twice to complete the manufacture of the plasma aperture plate substrate for spectral analysis.

[0139] Example 3: Use of a polycrystalline structure comprising a gold nanoparticle layer formed on a gold nanoparticle layer for spectral analysis Fabrication of the Plasma Orifice Plate Substrate

[0140] For the plasma orifice plate substrate for spectral analysis, which had a gold nanoparticle layer formed on the surface of the polycrystalline structure, manufactured in Example 2, the composition for manufacturing a spectral analysis substrate, described in Example 1, was loaded into each well of the orifice plate for 1 minute to 24 hours to further grow gold nanoparticles on the gold nanoparticle layer to form a gold nanolayer. After the further grown gold nanolayer was formed, the supernatant liquid was removed and the substrate was completely dried. Thereafter, the substrate was rinsed with water two or more times to remove any residual precursor and reducing agent. The substrate was then dried at room temperature for at least 1 hour, completing the manufacture of the plasma orifice plate substrate for spectral analysis.

[0141] Example 4: Plasma aperture plate substrate for spectral analysis comprising a polycrystalline structure having a gold nanolayer formed thereon Board manufacturing

[0142] For the plasma orifice plate substrate for spectral analysis manufactured in Example 1, which has a polycrystalline structure formed inside the hole, the composition for manufacturing the spectral analysis substrate in Example 1 is loaded inside each hole of the orifice plate for 30 seconds to 24 hours, so that further growth is allowed to form a gold nanolayer on the surface of the polycrystalline structure.

[0143] After the gold nanolayer is further grown, it is washed twice or more with water or ethanol to remove any remaining precursors and reducing agents. It is then dried at room temperature for at least one hour to complete the fabrication of the plasmonic aperture plate substrate for spectral analysis.

[0144] Example 5: A polycrystalline structure comprising a gold nanoparticle layer formed on a gold nanolayer for spectral analysis Fabrication of plasma aperture plate substrate

[0145] In each hole of the plasma aperture plate substrate for spectral analysis manufactured in Example 4, which further grows a gold nanolayer on the surface of the polycrystalline structure, gold nanoparticles that have been synthesized separately in advance are added so that the gold nanoparticles adhere to the gold nanolayer, thereby forming a gold nanoparticle layer with a thickness of 25nm to 40nm.

[0146] After the gold nanoparticles are attached to complete the formation of the gold nanoparticle layer, the substrate is dried at room temperature to complete the manufacture of the plasma aperture plate substrate for spectral analysis.

[0147] Experimental example

[0148] 1. Comparison of signal intensity and size distribution based on polycrystalline structure morphology

[0149] Figure 7 Part (a) is a graph showing the surface enhanced Raman spectroscopy (SERS) signal intensity of the nanostructure of the polycrystalline structure of the plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0150] Figure 7 Part (b) is a graph showing the SERS signal intensity at the main peak of the nanostructure of the polycrystalline structure of the plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0151] Reference Figure 7Can confirm, due to the polycrystalline structure on the plasma orifice plate substrate for spectral analysis of the application, formed peak (peak).Particularly, can confirm, comprise the polycrystalline structure (AuNP25nm / short tree (AuNP25nm / Short Tree) and AuNP25nm / long tree (AuNP25nm / Long Tree) that are formed with gold nano-particle layer), the polycrystalline structure (laminaration / AuNP40nm / thick sponge (Laminate / AuNP40nm / Thick Sponge) that are formed with gold nano-particle layer), be formed with gold nano-particle layer or on gold nano-particle layer, be formed with the polycrystalline structure (AuNP40nm / thick sponge (AuNP40nm / Thick Sponge)) of the plasma orifice plate substrate for spectral analysis higher, present obvious main peak (main peak) .

[0152] On the other hand, when SERS signal measurement was performed using a commercially available gold coated microplate from Nirmidas Biotech as a comparative example, no clear peak was observed.

[0153] Figure 10 This is a transmission electron microscope (TEM) image of the nanostructure of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application, in which gold nanoparticles are attached to a thick sponge-like polycrystalline structure to form a gold nanoparticle layer, and then a gold precursor is further grown to form a polycrystalline structure with a gold nanolayer.

[0154] Figure 11 The present invention is a diagram and an image showing the size distribution of the nanostructures of a thick sponge-like polycrystalline structure in a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application, a polycrystalline structure on which gold nanoparticles are attached to form a gold nanoparticle layer, and a polycrystalline structure on which a gold precursor is further grown on the gold nanoparticle layer to form a gold nanolayer.

[0155] Reference Figure 10 and Figure 11 It was confirmed that the gold nanoparticles adhered to the surface of a thick, spongy polycrystalline structure (AuNS) formed by aggregates (formed by multiple gold nanostructures) to form a gold nanoparticle layer (AuNP), and that the polycrystalline structure further grew on the gold nanoparticle layer to form a gold nanolayer. In addition, it was confirmed that the size distribution of the polycrystalline structure shifted toward larger values ​​as the gold nanoparticle layer and the gold nanolayer formed.

[0156] 2. Comparison of signal intensity according to reducing agent type and gold precursor to reducing agent ratio

[0157] Figure 8 3 is a graph showing the SERS signal intensity measured at 633 nm according to the type of reducing agent and the ratio of the gold precursor to the reducing agent when a polycrystalline structure of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application is formed.

[0158] Figure 9 3 is a graph showing the SERS signal intensity measured at 785 nm according to the type of reducing agent and the ratio of the gold precursor to the reducing agent when a polycrystalline structure of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application is formed.

[0159] For a plasmonic aperture plate substrate for spectral analysis, where a polycrystalline structure is formed by direct growth of gold nanostructures on the aperture plate member without forming a gold nanolayer or gold nanoparticle layer, SERS signal intensity was measured based on the type of reducing agent and the ratio of gold precursor to reducing agent. Signals were measured at 633 nm and 785 nm, using Malachite Green as the target molecule.

[0160] Reference Figure 8 and Figure 9 Hydroxylamine (HA), hydroxylamine-O-sulfonic acid (HOS), and O-methylhydroxylamine hydrochloride (OMH) were confirmed to be suitable reducing agents for manufacturing the plasmonic aperture plate substrate for spectral analysis of the present application. Furthermore, for each reducing agent, as the gold precursor:reducing agent ratio increased from 1:0.5 to 1:10, the SERS signal intensity further increased, and the ratio of 1:10, where the strongest signal was observed, was set as the optimal ratio, with 1:0.5 to 1:10 being set as the optimal ratio range.

[0161] 3. Signal Detection and Comparison of Dry and Liquid Samples

[0162] Figure 12 Part (a) is a graph showing the SERS signal intensity of the nanostructure of the polycrystalline structure of a dried sample measured at 633 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0163] Figure 12Parts (b) and (c) are graphs showing the SERS signal intensity and limit of detection (LOD) according to the sample concentration of a dry sample measured at 633 nm using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0164] Figure 13 Part (a) is a graph showing the SERS signal intensity of the nanostructure according to the polycrystalline structure of a dried sample measured at 785 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0165] Figure 13 Parts (b) and (c) are graphs showing SERS signal intensity and detection limit according to sample concentration when measuring a dry sample at 785 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0166] Figure 14 Part (a) is a graph showing the SERS signal intensity of the nanostructure of the polycrystalline structure of a liquid sample measured at 785 nm using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0167] Figure 14 Parts (b) and (c) are graphs showing the SERS signal intensity and limit of detection (LOD) according to the sample concentration when measuring a liquid sample at 785 nm using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0168] Figure 15 Part (a) is a diagram showing the uniformity of the SERS signal of a liquid sample in a single well of a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application. Figure 15 Part (b) is a graph showing the uniformity of SERS signals of 96 liquid samples between different wells of a plasma well plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0169] Using 4-ATP and malachite green as analytes, signal detection of a solution sample of a plasma orifice plate substrate for spectral analysis of the present application and a dried sample obtained by drying the solution were compared. Signal detection of 4-ATP and signal detection of malachite green were performed using a substrate comprising a polycrystalline structure, wherein the polycrystalline structure was formed by attaching gold nanoparticles to a polycrystalline structure formed by directly growing a gold nanostructure on an orifice plate member to form a gold nanoparticle layer, and further growing a gold nanolayer on the gold nanoparticle layer to form a gold nanolayer.

[0170] Reference Figures 12 to 14 After gold nanoparticles were attached to the polycrystalline structure formed by directly growing the gold nanostructure on the orifice plate member to form a gold nanoparticle layer, and then a gold nanolayer was further grown on the gold nanoparticle layer to form a polycrystalline structure, both the dry sample and the solution sample showed the highest signal intensity.

[0171] On the other hand, the signal intensity of the solution sample may be higher than that of the dried sample, referring to Figure 15 It can be confirmed that the signal uniformity of the solution sample is also excellent. Therefore, the plasma aperture plate substrate for spectral analysis of the present application can effectively detect the solution sample and can also ensure the reliability of the measurement results.

[0172] 4. Solution-phase Metabolite Detection and Sensitivity Assessment

[0173] Figure 16 2 is a graph showing the SERS signal intensities of six metabolites (uracil, guanine, xanthine, purine, hypoxanthine, and adenine) of a liquid sample measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0174] Figure 17 Parts (a) to (d) are graphs showing the SERS signal intensities and detection limits of four metabolites (adenine, xanthine, hypoxanthine, and purine) of a liquid sample measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0175] The metabolites are four types: xanthine, purine, hypoxanthine, and adenine, and are provided to a substrate in the form of a solution-phase sample. The SERS signal is confirmed using a substrate comprising a polycrystalline structure, wherein the polycrystalline structure is formed by attaching gold nanoparticles to the polycrystalline structure formed by directly growing a gold nanostructure on an orifice plate member to form a gold nanoparticle layer, and further growing a gold nanolayer on the gold nanoparticle layer.

[0176] Reference Figure 16 It was confirmed that xanthine, purine, hypoxanthine, and adenine were significantly detected among the six metabolites by confirming the Raman spectroscopy signal intensity using the plasma aperture plate substrate for spectral analysis of the present application.

[0177] Reference Figure 17 It can be confirmed that the plasma aperture plate substrate for spectral analysis of the present application has excellent signal sensitivity to the four metabolites xanthine, purine, hypoxanthine and adenine, which show obvious Raman spectral signal intensity.

[0178] 5. Signal Detection and Sensitivity Assessment in Urine Samples from Cancer Patients

[0179] Figure 18 is a graph showing normalized SERS signal intensities of urine samples of cancer patients measured using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0180] Figure 19 is a graph showing the SERS signal intensities of a candidate group of cancer metabolites in a urine sample of a cancer patient, measured using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0181] The SERS signal of a liquid sample is confirmed using a substrate comprising a polycrystalline structure, wherein the polycrystalline structure is formed by attaching gold nanoparticles to the polycrystalline structure formed by directly growing a gold nanostructure on a well plate member to form a gold nanoparticle layer, and further growing a gold nanolayer on the gold nanoparticle layer.

[0182] Reference Figure 18 and Figure 19 It has been confirmed that the plasma aperture plate substrate for spectral analysis of the present application is suitable for cancer diagnosis using liquid samples such as patient urine that may contain cancer metabolites.

[0183] 6. Raman shift analysis of samples from cancer patients and healthy subjects according to cancer type

[0184] Figure 20 Parts (a) to (d) are graphs showing the positions of Raman shifts where differences occur between various cancer types and normal people within a confidence interval of 95% or more, measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application. Figure 20 Part (e) is a comparative diagram showing the relative magnitudes of Raman shifts at different positions for various cancer types measured using a plasma aperture plate substrate for spectral analysis manufactured according to one embodiment of the present application.

[0185] The SERS signal of a liquid sample is confirmed using a substrate comprising a polycrystalline structure, wherein the polycrystalline structure is formed by attaching gold nanoparticles to the polycrystalline structure formed by directly growing a gold nanostructure on a well plate member to form a gold nanoparticle layer, and further growing a gold nanolayer on the gold nanoparticle layer.

[0186] Reference Figure 20 From part (a) to part (d), urine samples of cancer patients with colorectal cancer, lung cancer, prostate cancer, and pancreatic cancer and urine samples of normal subjects were used to measure Raman spectra. Figure 18 ) were used to identify Raman shift positions within each cancer type that differed from those in healthy controls within a 95% confidence interval. The screening method used a t-test to analyze the positions of the entire Raman spectrum, excluding positions with non-corresponding peaks.

[0187] Reference Figure 20 Part (e) confirms that for four types of cancer, by selecting locations where the signal intensity of two or more cancers is higher or lower than that of normal people and expressing them in polygonal form, the patterns of different Raman shift positions are compared according to the cancer type, thereby distinguishing normal people from cancer patients.

[0188] 7. Analysis of the accuracy of signal differentiation between samples from cancer patients and normal subjects according to cancer type

[0189] Figure 21 Parts (a) to (d) are used to illustrate confusion matrix results of the accuracy of distinguishing urine samples of cancer patients from normal people according to each cancer type measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application. Figure 21Part (e) is a receiver operating characteristics (ROC) curve diagram showing the accuracy of distinguishing urine samples of cancer patients from normal people according to each cancer type, measured using a plasma aperture plate substrate for spectral analysis manufactured according to an embodiment of the present application.

[0190] The SERS signal of a liquid sample is confirmed using a substrate comprising a polycrystalline structure, wherein the polycrystalline structure is formed by attaching gold nanoparticles to the polycrystalline structure formed by directly growing a gold nanostructure on a well plate member to form a gold nanoparticle layer, and further growing a gold nanolayer on the gold nanoparticle layer.

[0191] After measuring Raman spectra using urine samples from cancer patients with colorectal cancer, lung cancer, prostate cancer, and pancreatic cancer and urine samples from normal people ( Figure 18 ), using the logistic regression method, one of the machine learning methods, to confirm the accuracy of distinguishing each cancer type from normal people. For model learning, the number of cancer patients and normal people of each cancer type is set to 70% of the total population of the cluster, and the number of cancer patients and normal people used for testing is set to 30% of the total population of the cluster. This ratio sets the general training and testing ratio, and the ratio of the training set and the test set can be set within 1 to 99% respectively. In addition, in order to verify the model formed by the training set, a validation set can be additionally set within 1 to 99%, but the sum of the training set, test set, and validation set must meet 100%.

[0192] As a method for distinguishing Raman spectra, well-known machine learning methods such as decision tree classification, random forest, support vector machine, K-nearest neighbor, Naive Bayes algorithm, partial least squares discriminant analysis (PLS-DA) can be commonly applied. However, in this experimental example, the test set of the model learned by the logistic regression method was verified for each cancer type, and the confusion matrix ( Figure 21 (a) to (d) of the ) and the receiver operating characteristics ( Figure 21 (e) of the 2012 / 13 / EC meeting).

[0193] While specific portions of the present application have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and are not intended to limit the scope of the present application. Therefore, the substantial scope of the present application should be defined by the appended claims and their equivalents.

Claims

1. A plasma aperture plate substrate for spectral analysis, characterized in that: include: An orifice member having more than one orifice, and a polycrystalline structure formed in the pore; The polycrystalline structure is composed of an aggregate formed of a plurality of gold nanostructures and includes a plurality of pores therein.

2. The plasma aperture plate substrate for spectral analysis according to claim 1, characterized in that: The gold nanostructures or aggregates formed by the gold nanostructures are in a connected state.

3. The plasma aperture plate substrate for spectral analysis according to claim 1, characterized in that: The polycrystalline structure is in one or more forms of nanosponge, nanotree, nanobranch and nanocoral.

4. The plasma aperture plate substrate for spectral analysis according to claim 1, characterized in that: The polycrystalline structure further includes a layer of gold nanoparticles on its surface.

5. The plasma aperture plate substrate for spectral analysis according to claim 4, characterized in that: The polycrystalline structure further includes a gold nanolayer formed by further growing a gold precursor on the gold nanoparticle layer.

6. The plasma aperture plate substrate for spectral analysis according to claim 1, characterized in that: The polycrystalline structure further includes a gold nanolayer formed by further growing a gold precursor on the surface of the polycrystalline structure.

7. The plasma aperture plate substrate for spectral analysis according to claim 6, characterized in that: The polycrystalline structure further includes a gold nanoparticle layer on the gold nanolayer.

8. The plasma aperture plate substrate for spectral analysis according to any one of claims 4 to 7, characterized in that: The average thickness of the gold nanoparticle layer or gold nanolayer is 5 nm to 100 nm.

9. The plasma aperture plate substrate for spectral analysis according to claim 1, characterized in that: The plasma aperture plate substrate for spectral analysis is used for surface enhanced Raman spectroscopy, plasmon enhanced fluorescence or fluorescence analysis.

10. A method for manufacturing a plasma aperture plate substrate for spectral analysis, characterized in that: include: Step i), preparing an orifice plate member having more than one hole, and Step ii), immersing the well plate member in a composition for manufacturing a spectroscopic analysis substrate comprising a gold precursor and a reducing agent solution to form a polycrystalline structure in the well; The polycrystalline structure formed in step ii) is composed of aggregates formed by multiple gold nanostructures, and includes multiple pores inside the polycrystalline structure. The method comprises the step of adjusting the morphology of the polycrystalline structure by making the ratio of the gold precursor to the reducing agent in the step ii) be 1:1 to 1:

10.

11. The method for manufacturing a plasma aperture plate substrate for spectral analysis according to claim 10, wherein: After step ii), the method further includes the step of attaching gold nanoparticles to the orifice plate member having the polycrystalline structure formed thereon, so as to form a gold nanoparticle layer on the surface of the polycrystalline structure.

12. The method for manufacturing a plasma aperture plate substrate for spectral analysis according to claim 11, characterized in that: After the step of forming a gold nanoparticle layer on the surface of the polycrystalline structure, the method further includes: immersing the aperture plate member in a composition for manufacturing a spectral analysis substrate comprising a gold precursor and a reducing agent solution to form a further grown gold nanolayer on the gold nanoparticle layer.

13. The method for manufacturing a plasma aperture plate substrate for spectral analysis according to claim 10, characterized in that: After step ii), the method further includes immersing the orifice plate member having the polycrystalline structure in a composition for manufacturing a spectral analysis substrate comprising a gold precursor and a reducing agent solution to form a further grown gold nanolayer on the surface of the polycrystalline structure.

14. The method for manufacturing a plasma aperture plate substrate for spectral analysis according to claim 13, wherein: After the step of forming a further grown gold nanolayer on the surface of the polycrystalline structure, the method further includes the step of attaching gold nanoparticles to the aperture plate member to form a gold nanoparticle layer on the surface of the polycrystalline structure.

15. The method for manufacturing a plasma aperture plate substrate for spectral analysis according to claim 11 or 14, characterized in that: Before the step of forming the gold nanoparticle layer, the method further includes the step of modifying the surface of the polycrystalline structure.

16. The method for manufacturing a plasma aperture plate substrate for spectral analysis according to claim 10, wherein: The reducing agent is one or more selected from ascorbic acid, hydroxylamine, hydroxylamine-O-sulfonic acid and O-methylhydroxylamine hydrochloride.

Images