Polymer-mediated plasmonic well plate substrate and manufacturing method thereof

Abstract

The present application provides a polymer-mediated plasmonic well plate substrate and a manufacturing method thereof. More specifically, the present application provides a polymer-mediated plasmonic well plate substrate having an excellent signal enhancement effect and uniformity suitable for analysis of a plurality of samples when used for spectroscopic analysis, and a manufacturing method thereof.

Description

Polymer-mediated plasmonic well-plate substrate and method for manufacturing the same

[0001] The present invention relates to a polymer-mediated plasmonic well-plate substrate and a method for manufacturing the same. More specifically, the present invention relates to a polymer-mediated plasmonic well-plate substrate having excellent signal enhancement effects and uniformity suitable for analyzing a large number of samples for spectroscopic analysis, and a method for manufacturing the same.

[0002]

[0003] Most organic molecules have a unique Raman shift, so Raman spectroscopy using Raman scattering can obtain signals even from nonpolar molecules with induced polarization changes.

[0004] Furthermore, Raman spectroscopy is unaffected by interference from water molecules, making it more suitable for the detection of biomolecules such as proteins and genes. The wavelength of the Raman emission spectrum indicates the chemical composition and structural characteristics of light-absorbing molecules within a sample, so analyzing these Raman signals allows for direct analysis of the target substance.

[0005] Meanwhile, a well plate is an experimental and testing device consisting of a plate formed by arranging a number of 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 to place a substance for detecting the target substance or diagnosing the disease on the inner surface of a well of the well plate, and then induce a reaction by introducing a sample to be analyzed into the well, after which the target substance is detected or the disease is diagnosed.

[0006] Utilizing these well plates facilitates the simultaneous detection of target substances in multiple samples. Furthermore, they are suitable for the analysis of liquid samples, allowing the use of bodily fluids such as urine, saliva, tears, and sweat as liquid samples. This can improve the cost and time efficiency of target substance detection and disease diagnosis.

[0007] However, spectroscopic analysis methods that analyze multiple samples using well plates have limitations in terms of sensitivity and uniformity.

[0008] As a background technology for this application, Korean Patent Publication No. 10-2016-0014866 describes a method and device for diagnosing viral infections using teardrops. This patent includes a separate step of drying the collected tears to prepare them as a sample for measurement, in order to obtain a Raman spectrum.

[0009]

[0010] The purpose of this invention is to provide a plasmonic well plate substrate having excellent signal enhancement effect and uniformity suitable for analysis of multiple samples when used for spectroscopic analysis.

[0011] Another purpose of the present invention is to provide a plasmonic well plate substrate capable of qualitative and quantitative analysis of unlabeled analytes.

[0012] Another object of the present invention is to provide a plasmonic well plate substrate suitable for simultaneous analysis of multiple samples.

[0013] Another object of the present invention is to provide a method for manufacturing a plasmonic well-plate substrate having improved signal enhancement effect and uniformity by easily controlling and forming the nanostructure form of a polycrystalline structure layer formed on a well-plate member.

[0014] Another object of the present invention is to provide a method for efficiently manufacturing a plasmonic well plate substrate having excellent signal enhancement effect and uniformity suitable for analysis of multiple samples.

[0015] The purpose of this invention is not limited to the purposes mentioned above, and other purposes not mentioned can be clearly understood from the description in detail.

[0016]

[0017] According to one aspect, a polymer-mediated plasmonic wellplate substrate is provided, comprising: a wellplate member having one or more wells; a polymer-mediated layer formed within the wells; and a polycrystalline structure layer formed on the polymer-mediated layer, wherein the polycrystalline structure layer is composed of a cluster formed of a plurality of metal nanostructures and includes a plurality of pores therein.

[0018] According to one embodiment, the polymer intermediary layer may be formed of at least one selected from catechols and polyphenols with a thickness of 10 nm to 10 μm.

[0019] According to one embodiment, the metal nanostructure or the cluster formed by the plurality of metal nanostructures may be connected to each other.

[0020] According to one embodiment, the polycrystalline structure layer may include a metal seed layer formed of metal nanoparticles; and a metal nanostructure layer additionally grown one or more times on the metal seed layer using a metal precursor and a reducing agent.

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

[0022] According to one embodiment, the average thickness of the metal seed layer, metal nanostructure layer, or metal nanoparticle layer may be 5 nm or more.

[0023] In one embodiment, the polymer-mediated plasmonic wellplate substrate of the present invention can be used for Surface Enhanced Raman Spectroscopy (SERS), Plasmon-Enhanced Fluorescence (PEF), or Fluorescence analysis.

[0024]

[0025] According to another aspect, a method for manufacturing a polymer-mediated plasmonic well-plate substrate is provided, comprising: i) preparing a well-plate member having one or more wells; ii) forming a polymer mediator layer within the wells; and iii) immersing the well-plate member having the polymer mediator layer formed thereon in a composition for forming a polycrystalline structure layer comprising a metal precursor and a reducing agent solution, thereby forming a polycrystalline structure layer within the wells; wherein the polycrystalline structure layer formed in step iii) is composed of a cluster formed of a plurality of metal nanostructures and includes a plurality of pores therein.

[0026] According to one embodiment, the polymer mediator layer in step ii) may be formed by coating with 0.1 to 20 mg / mL of polydopamine.

[0027] According to one embodiment, the polymer intermediary layer in step ii) may be formed by coating 2 to 5 times.

[0028] According to one embodiment, the method for manufacturing a polymer-mediated plasmonic well-plate substrate of the present invention may further include a step of forming a metal seed layer by immersing a well-plate member on which a polymer-mediated layer is formed in a metal precursor solution for forming a metal seed layer after step ii) and before step iii).

[0029] According to one embodiment, the pH of the metal precursor solution for forming the metal seed layer may be 2-9.

[0030] According to one embodiment, the step iii) may include a step of controlling the shape of the polycrystalline structure layer by setting the ratio of metal precursor:reducing agent to 1:0.5 to 1:10.

[0031] According to one embodiment, in step iii), the reducing agent may be at least one of hydroxylamine and derivatives thereof.

[0032] According to one embodiment, the step of forming a polycrystalline structure layer in step iii) may be performed two or more times.

[0033] According to one embodiment, the concentration of the metal ion among the metal precursors in the composition for forming a polycrystalline structure layer in step iii) may be 0.5 to 10 mg / mL.

[0034] According to one embodiment, the formation of the polycrystalline structure layer in step iii) may be performed for 5 minutes to 24 hours.

[0035] According to one embodiment, the method for manufacturing a polymer-mediated plasmonic well-plate substrate of the present invention may further include a step of forming a metal nanoparticle layer by attaching metal nanoparticles on a polycrystalline structure layer after step iii).

[0036]

[0037] In one embodiment, the polymer-mediated plasmonic well-plate substrate of the present invention includes a polycrystalline structure layer of various shapes formed as a cluster of metal nanostructures by mediating a polymer on a well-plate member, and is suitable for analyzing a large number of samples when used for spectroscopic analysis and can exhibit excellent signal enhancement effect and uniformity.

[0038] In one embodiment, the use of the polymer-mediated plasmonic well-plate substrate of the present invention can improve signal enhancement and uniformity without a drying step when analyzing a liquid sample, thereby enabling accurate and highly sensitive on-site analysis of a target substance.

[0039] In one embodiment, the polymer-mediated plasmonic well plate substrate of the present invention enables qualitative and quantitative analysis of unlabeled analytes.

[0040] In one embodiment, by using the polymer-mediated plasmonic well plate substrate of the present invention, a large number of samples to be analyzed can be analyzed simultaneously with high sensitivity and accuracy.

[0041] According to one embodiment, the method for manufacturing a polymer-mediated plasmonic well-plate substrate of the present invention can easily form a polycrystalline structure layer formed by a cluster of metal nanostructures by mediating a polymer on a well-plate member in various shapes, thereby efficiently manufacturing a plasmonic well-plate substrate for spectroscopic analysis with improved signal enhancement effect and uniformity.

[0042] According to one embodiment, the method for manufacturing a polymer-mediated plasmonic wellplate substrate of the present invention can efficiently manufacture a plasmonic wellplate substrate for spectroscopic analysis with improved signal enhancement effect and uniformity suitable for analysis of a large number of samples.

[0043]

[0044] Figure 1 is a photograph showing a type of polymer-mediated plasmonic well plate substrate manufactured by one embodiment of the present invention.

[0045] Figure 2 is a drawing schematically showing a method for manufacturing a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0046] Figure 3 is a photograph of a plasmonic well plate substrate in each step according to a method for manufacturing a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0047] Figure 4 is an SEM photograph showing the surface of a well-plate substrate according to the concentration of a polymer when forming a polymer-mediated layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present invention.

[0048] Figure 5 is an SEM photograph showing the surface of a well-plate substrate according to the number of times the polymer is coated when forming a polymer-mediated layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present invention.

[0049] Fig. 6 (a) is a graph showing the SERS signal intensity measured at 633 nm of a well-plate substrate coated only with a polymer according to one embodiment of the present invention, and Fig. 6 (b) is a graph showing the SERS signal intensity measured at 785 nm of a well-plate substrate coated only with a polymer according to one embodiment of the present invention.

[0050] FIG. 7 is an SEM photograph showing a metal seed layer according to the concentration of a metal precursor when forming a metal seed layer among the polycrystalline structure layers of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0051] FIG. 8 (a) is a graph showing the SERS signal intensity measured at 633 nm according to the pH of the metal precursor solution when forming a metal seed layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present disclosure, and FIG. 8 (b) is a graph showing the SERS signal intensity measured at 785 nm according to the pH of the metal precursor solution when forming a metal seed layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present disclosure.

[0052] FIG. 9 (a) and (b) are SEM photographs showing a polycrystalline structure layer according to the pH of a metal precursor solution for forming a metal seed layer of a polymer-mediated plasmonic well-plate substrate according to an embodiment of the present disclosure, and FIG. 9 (c) is a photograph of a well-plate substrate according to the pH of a metal precursor solution for forming a metal seed layer of a polymer-mediated plasmonic well-plate substrate according to an embodiment of the present disclosure.

[0053] Figure 10 is a graph showing the SERS signal intensity measured at 633 nm according to the type of reducing agent and the ratio of metal precursor and reducing agent when forming a polycrystalline structure layer of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0054] Figure 11 is a graph showing the SERS signal intensity measured at 785 nm according to the type of reducing agent and the ratio of metal precursor and reducing agent when forming a polycrystalline structure layer of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0055] Figure 12 is an SEM photograph showing the change in density of a polycrystalline structure layer according to the number of additional growths of a metal nanostructure layer when forming a polycrystalline structure layer of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0056] FIG. 13 (a) is a graph showing the SERS signal intensity measured at 633 nm according to the number of additional growths of a metal nanostructure layer when forming a polycrystalline structure layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present disclosure, and FIG. 13 (b) is a graph showing the SERS signal intensity measured at 785 nm according to the number of additional growths of a metal nanostructure layer when forming a polycrystalline structure layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present disclosure.

[0057] Figure 14 is an SEM photograph showing the change in density of a polycrystalline structure according to the additional growth time of a metal nanostructure layer when forming a polycrystalline structure layer of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0058] Figure 15 is a graph showing the SERS signal intensity measured at 633 nm of a polymer-mediated plasmonic well-plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention and a commercial well-plate substrate.

[0059] FIG. 16 is a graph showing the SERS signal intensity measured at 633 nm according to the number of additional growths of a metal nanostructure layer of a polymer-mediated plasmonic well plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0060] Figure 17 is a graph of SERS signal intensity measured at 633 nm using malachite green to confirm the detection sensitivity of a polymer-mediated plasmonic well plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0061] FIG. 18 is a graph showing the SERS signal uniformity measured at 633 nm for a liquid sample in one well of a polymer-mediated plasmonic well plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0062] Fig. 19 (a) is a UV-vis absorbance graph showing that there is no SPR because a polymer intermediary layer is not formed by a comparative example of the present disclosure, and Fig. 19 (b) is a graph showing UV-vis absorbance according to the number of times the polymer is coated when a polymer intermediary layer is formed by an example of the present disclosure.

[0063] Figure 20 is a graph of SERS signal intensity measured at 785 nm using malachite green to confirm the detection sensitivity of a polymer-mediated plasmonic well plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0064] FIG. 21 is a graph showing the SERS signal uniformity measured at 785 nm for a liquid sample in one well of a polymer-mediated plasmonic well-plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0065] Figure 22 is a graph showing the SERS signal intensity measured at 633 nm of a polymer-mediated plasmonic well-plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 and pH 8 according to one embodiment of the present invention and a commercial well-plate substrate.

[0066] Figure 23 is a graph showing the absorbance according to wavelength of a polymer-mediated plasmonic wellplate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 and pH 8 according to one embodiment of the present invention, and a bare wellplate and a commercial wellplate substrate as a control group.

[0067] Figure 24 is a graph showing the detection sensitivity of a polymer-mediated plasmonic well-plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 8 according to one embodiment of the present invention, using malachite green for liquid and dry samples.

[0068]

[0069] The purpose, specific advantages and novel features of the present disclosure will become more apparent from the following detailed description and examples taken in conjunction with the accompanying drawings.

[0070] Prior to this, the terms or words used in this specification and claims should not be interpreted in their usual or dictionary meanings, but should be interpreted in their meanings and concepts that are consistent with the technical idea of ​​the present disclosure based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0071] In this specification, when a component, such as a layer, portion, or 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, and one or more other components may be interposed between the two components. Conversely, when 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.

[0072] The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the present disclosure. Singular expressions include plural expressions unless the context clearly dictates otherwise.

[0073] In this specification, terms such as “include” or “have” are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but should be understood not to exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

[0074] In this specification, when a part is said to "include" a component, this does not exclude other components, unless otherwise specifically stated, but rather implies the inclusion of other components. Furthermore, throughout this specification, the term "on" means located above or below the target part, and does not necessarily mean located above the direction of gravity.

[0075] The present disclosure may be subject to various modifications and embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present disclosure to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present disclosure. In describing the present disclosure, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present disclosure.

[0076] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the attached drawings. In describing with reference to the attached drawings, identical or corresponding components are assigned the same drawing numbers, and redundant descriptions thereof will be omitted.

[0077]

[0078] Fig. 1 is a photograph showing a type of polymer-mediated plasmonic well-plate substrate manufactured according to one embodiment of the present invention. Fig. 2 is a drawing schematically showing a method for manufacturing a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present invention.

[0079] Referring to FIGS. 1 and 2, the polymer-mediated plasmonic well-plate substrate of the present invention according to one aspect comprises: a well-plate member having one or more wells; a polymer-mediated layer (10) formed within the wells; and a polycrystalline structure layer formed on the polymer-mediated layer (10), wherein the polycrystalline structure layer is composed of a cluster formed of a plurality of metal nanostructures and includes a plurality of pores therein.

[0080] Referring to FIG. 1, the polymer-mediated plasmonic wellplate member of the present disclosure may form a polycrystalline structure layer composed of a cluster of metal nanostructures on a wellplate member having one or more wells, so that it appears as if a metal film is formed inside the well. In addition, the polymer-mediated plasmonic wellplate member of the present disclosure may change color depending on the pH of the metal precursor solution for forming the metal seed layer when forming the metal seed layer.

[0081] Although not limited thereto, it may be a multi-well plate having two or more wells, such as a known 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, and 384-well plate, with various well sizes.

[0082] Although not limited thereto, single-well petri dishes are used for the detection of unlabeled bacteria, and the polymer-mediated plasmonic well-plate substrate according to the present invention can be utilized for in-situ mapping, species classification, and detection of drug-resistant bacteria.

[0083] Although not limited thereto, the above 6-well plate is used for cancer cell culture and metabolite profiling using a label-free method, and in-situ Raman imaging, drug response, and immune response monitoring may be possible using the polymer-mediated plasmonic well plate substrate according to the present invention.

[0084] Although not limited thereto, the 96-wellplate can be utilized in the fields of high-throughput chemical screening and ultra-sensitive ELISA platform by fluorescence (PEF) and Raman spectroscopy (SERS) analysis using the polymer-mediated plasmonic wellplate substrate of the present invention.

[0085]

[0086] The above polymer intermediary layer (10) simultaneously functions as a reducing agent and a metal ion catcher, thereby promoting spontaneous formation of metal seeds (metal nuclei) without using a separate reducing agent, and the formation of the metal nanostructure layer (30) of the polycrystalline structure layer can be promoted by the formation of the metal seeds.

[0087] Although not limited thereto, the polymer intermediary layer (10) may be composed of at least one selected from catechols and polyphenols. Although not limited thereto, the catechols may be a material containing a catechol functional group and may be polycatechol. Although not limited thereto, the material containing the catechol functional group may be selected from dopamine, norepinephrine, polydopamine, poly-norepinephrine, PEG-catechol, and PEI-catechol.

[0088] The above polydopamine is produced by polymerizing dopamine and can promote the spontaneous formation of metal seeds (metal nuclei). Furthermore, the polydopamine can be used as a coating material that can modify various surfaces to have consistent chemical properties regardless of their structure or material, and it has excellent adhesive strength.

[0089] The above polyphenol is a type of aromatic alcohol compound found in plants, and has multiple hydroxyl groups as a functional group with two or more phenol groups per molecule, and can play a role in promoting the spontaneous formation of metal seeds (metal nuclei).

[0090] Although not limited thereto, the polymer intermediary layer (10) formed with a thickness of 10 nm to 10 ㎛ may be suitable for promoting spontaneous formation of metal seeds. Although not limited thereto, if the thickness of the polymer intermediary layer (10) is less than 10 nm, the promotion of spontaneous formation of metal seeds may be insufficient, and if it exceeds 10 ㎛, the effect of promoting spontaneous formation of metal seeds due to an increase in the thickness of the polymer intermediary layer (10) may be minimal, and thus may not be suitable in terms of manufacturing efficiency.

[0091]

[0092] The above polycrystalline structure layer is formed on the polymer intermediary layer (10), and is composed of a cluster formed of a plurality of metal nanostructures and includes a plurality of pores therein. Although not limited thereto, the metal may be one of Au, Ag, Al, Co, Cu, Fe, Li, Ni, Pd, Pt, Rh, Ru, and alloys thereof, and a noble metal may be suitable, and Au may be more suitable. The metal nanostructure may be directly grown on the polymer intermediary layer (10) by a solution process using a metal precursor and a reducing agent to form a cluster.

[0093] More specifically, Au precursors among precious metals form nanostructures and clusters, and in particular, these can be directly grown on the polymer intermediary layer (10) using a solution process. Specifically, the clustered nanostructures form small-sized polygonal structure grains through coalescence, and the grains then gradually grow larger through oriented attachment. Accordingly, a polycrystalline structure layer having multiple grain boundaries is formed. Although not limited thereto, the polycrystalline structure layer can grow to an average size of micrometers. As described above, the polycrystalline structure layer having multiple grain boundaries can dramatically improve signal intensity and signal uniformity when used as a substrate for spectroscopic analysis because scattering increases at the multiple grain boundaries.

[0094]

[0095] In addition, the polycrystalline structure layer may be composed of a cluster formed of a plurality of metal nanostructures and may include a plurality of pores therein. Although not limited thereto, the plurality of metal nanostructures or the cluster formed of the plurality of metal nanostructures may be connected to each other and include pores therein. Although not limited thereto, the plurality of metal nanostructures or the clusters formed of the plurality of metal nanostructures may be connected to each other in a branch structure. However, the present invention does not completely exclude a structure in which the plurality of metal nanostructures or the clusters formed of the plurality of metal nanostructures are not connected.

[0096] Although not limited thereto, the polycrystalline structure layer may have an average particle diameter of 0.1 to 100 μm. The well plate substrate of the present disclosure is grown into a polycrystalline structure layer by a solution process, and is connected in a branch structure, so that the average particle diameter may be 0.1 to 100 μm. Although not limited thereto, the average particle diameter of the polycrystalline structure layer may be suitable for improving signal intensity and signal uniformity within the above range.

[0097] Although not limited thereto, the pores included in the polycrystalline structure layer may be formed in a disordered but uniform manner between a plurality of metal nanostructures, or in a disordered but uniform manner between clusters formed by a plurality of metal nanostructures. Accordingly, the polymer-mediated plasmonic well-plate substrate of the present disclosure can enhance the SERS signal enhancement effect and uniformity for analytes.

[0098] The above polycrystalline structure layer may include a nano-sponge, a thick sponge, a nano-tree, a nano-branch, and a nano-coral shape. The nano-sponge is a sponge shape that includes a number of pores inside, and the thick sponge may refer to a sponge shape that is the same or similar in shape to the nano-sponge but is formed in a thicker cluster. The nano-tree, nano-branch, and nano-coral may refer to a cluster that has grown long and formed in a tree shape, a branch shape, or a coral shape.

[0099] Although not limited thereto, the shape of the polycrystalline structure layer formed on the plasmonic well-plate substrate can be easily controlled by adjusting the optimal ratio of the reducing agent according to the type of reducing agent for the metal precursor. In addition, the shape of the polycrystalline structure layer can be easily controlled by adjusting the time for which the well-plate member is immersed in the composition for forming the metal nanostructure layer.

[0100]

[0101] Although not limited thereto, the polycrystalline structure layer may include a metal seed layer (20) made of metal nanoparticles; and a metal nanostructure layer (30) additionally grown one or more times on the metal seed layer (20) using a metal precursor and a reducing agent. The metal seed layer (20) is spontaneously formed on the polymer intermediary layer (10) through a solution process using a metal precursor. The metal seed layer (20) can promote the formation of the metal nanostructure layer (30). In addition, the metal seed layer (20) is spontaneously formed without using a separate reducing agent, thereby increasing the efficiency of the overall manufacturing process of the well plate substrate of the present disclosure.

[0102]

[0103] Although not limited thereto, the metal nanostructure layer (30) may be in the form of a continuous layer or may be in the form of a partially discontinuous layer, but a continuous layer form may be preferable. The metal nanostructure layer (30) may be in the form of covering the metal seed layer (20) or in the form of thinly covering it (laminated).

[0104]

[0105] Although not limited thereto, the polycrystalline structure layer may further include a metal nanoparticle layer (not shown) formed on the metal nanostructure layer (30). The metal nanoparticle layer may be formed by adding metal nanoparticles synthesized in advance and attaching the metal nanoparticles on the metal nanostructure layer (30). Although not limited thereto, the metal nanoparticle layer may be in the form of a continuous layer or a discontinuous form.

[0106]

[0107] Although not limited thereto, the average thickness of each of the metal seed layer (20), the metal nanostructure layer (30), or the metal nanoparticle layer may be 5 nm or more, 5 to 100 nm, 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. Although not limited thereto, if the average thickness of each of the metal seed layer (20), metal nanostructure layer (30), or metal nanoparticle layer is less than 5 nm, the SERS signal enhancement effect and uniformity improvement effect for the analyte may be insufficient when the polymer-mediated plasmonic well plate substrate of the present invention is used for spectroscopic analysis.

[0108]

[0109] Although not limited thereto, the polymer-mediated plasmonic well-plate substrate of the present disclosure may be used for Surface Enhanced Raman Spectroscopy (SERS), Plasmon-Enhanced Fluorescence (PEF), or Fluorescence analysis. Although not limited thereto, the well-plate substrate of the present disclosure may be more suitable for Surface Enhanced Raman Spectroscopy (SERS) analysis. That is, when the polymer-mediated plasmonic well-plate substrate of the present disclosure is used for Surface Enhanced Raman Spectroscopy analysis, an analyte can be efficiently detected and quantitatively analyzed with high sensitivity without a label such as a fluorescent substance.

[0110]

[0111] As described above, by using the polymer-mediated plasmonic well plate substrate of the present invention, analytes can be efficiently detected and quantitatively analyzed without a separate label for the sample or analyte to be analyzed, i.e., without a label.

[0112]

[0113] Although not limited thereto, the analyte may be selected from one or more of cells, metabolites, proteins, nucleic acids, DNA, RNA, mRNA, lipids, hormones, metabolites, enzymes, organic molecules, viruses, bacteria, antigens, antibodies, neurotransmitters, extracellular vesicles, microvesicles, exosomes, and fats.

[0114]

[0115] Although not limited thereto, the sample may be either a dry sample or a liquid sample, with a dry sample being more suitable.

[0116] Although not limited thereto, the sample may be a sample containing the various analytes, and may be various body fluids, substrates and secretions such as urine, saliva, blood, sweat, tears, ascites, gastric juice, and cerebrospinal fluid.

[0117] The polymer-mediated plasmonic well-plate substrate of this institute can support liquid samples and can be quickly analyzed and diagnosed on-site by drying the sample as a dry sample or as a liquid sample.

[0118] Although not limited thereto, the polymer-mediated plasmonic wellplate substrate of this disclosure can be used to diagnose cancer using dried or liquid samples. Furthermore, metabolite analysis of the samples and analytes described above can provide information necessary for cancer diagnosis, enabling early cancer diagnosis through noninvasive testing, and enabling multiplex analysis with extremely high sensitivity.

[0119] The above cancer may be one or more selected from, but is not limited to, 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.

[0120]

[0121] Figure 2 is a drawing schematically showing a method for manufacturing a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0122] Referring to FIG. 2, a method for manufacturing a polymer-mediated plasmonic well-plate substrate according to another aspect of the present invention comprises the steps of: i) preparing a well-plate member having one or more wells; ii) forming a polymer-mediated layer (10) within the wells; and iii) immersing the well-plate member on which the polymer-mediated layer (10) is formed in a composition for forming a polycrystalline structure layer including a metal precursor and a reducing agent solution, thereby forming a polycrystalline structure layer within the wells; wherein the polycrystalline structure layer formed in step iii) is composed of a cluster formed of a plurality of metal nanostructures and includes a plurality of pores therein.

[0123]

[0124] Step i) is a step of preparing a wellplate member having one or more wells ((a) of FIG. 2). The wellplate member having one or more wells may be a variety of known multi-well plates such as a 6-wellplate, a 12-wellplate, a 24-wellplate, a 48-wellplate, a 96-wellplate, a 384-wellplate, etc.

[0125]

[0126] Step ii) is a step of forming a polymer intermediary layer (10) within the well (Fig. 2 (b)). The polymer intermediary layer (10) can be formed using various known methods. For example, the polymer intermediary layer (10) can be formed using dip-coating, spin-coating, drop casting, spraying, etc.

[0127] Although not limited thereto, the polymer intermediary layer (10) may be formed of one or more selected from catechols and polyphenols.

[0128] Although not limited thereto, the catechols may be substances containing a catechol functional group and may be polycatechol. Although not limited thereto, the substances containing the catechol functional group may be selected from dopamine, norepinephrine, polydopamine, poly-norepinephrine, PEG-catechol, and PEI-catechol.

[0129]

[0130] Although not limited thereto, in the step ii), the polymer intermediate layer (10) may include immersing the polymer intermediate layer in a dopamine solution of 0.1 to 20 mg / mL to polymerize polydopamine to form the polymer intermediate layer. Although not limited thereto, if the concentration of the dopamine solution is less than 0.1 mg / mL, spontaneous formation of the metal seed layer (20) may not be easy, and if it exceeds 20 mg / mL, spontaneous growth of the metal seed layer (20) may be inhibited as the concentration increases.

[0131] Although not limited thereto, the polymer intermediary layer (10) in step ii) may include forming the layer by coating 2 to 5 times. Forming the layer by coating 2 or more times can improve the SERS signal enhancement effect and uniformity for the analyte.

[0132]

[0133] Although not limited thereto, the method for manufacturing a polymer-mediated plasmonic well-plate substrate of the present invention may further include a step ((c) of FIG. 2) of forming a metal seed layer (20) on the polymer-mediated layer (10) after step ii) and before step iii). The metal seed layer (20) may be formed by various known methods. Although not limited thereto, the well-plate member on which the polymer-mediated layer (10) is formed may be immersed in a metal precursor solution for forming a metal seed layer to form the metal seed layer. The metal seed layer (20) may be spontaneously formed on the polymer-mediated layer (10) without a reducing agent.

[0134] There is no particular limitation on the metal precursor solution for forming the metal seed layer as long as it contains a metal ion that can react with the polymer intermediary layer (10). For example, the metal precursor solution for forming the metal seed layer may be at least one of HAuCl4, NaAuCl4, and AgNO3 aqueous solutions, and it is not excluded that the metal precursor solutions for forming the metal seed layer may be mixed or used sequentially. In the example of the present application, the well plate substrate on which the polymer intermediary layer (10) is formed is immersed in an HAuCl4 solution to form the metal seed layer (20).

[0135] Although not limited thereto, the metal ion concentration of the metal precursor solution for forming the metal seed layer may be 0.5 mg / mL to 50 mg / mL. Although not limited thereto, if the metal ion concentration of the metal precursor solution for forming the metal seed layer is less than 0.5 mg / mL, spontaneous growth of the metal seed layer (20) may not be easy, and if it exceeds 50 mg / mL, spontaneous growth of the metal seed layer (20) may be inhibited or nanogap formation may be suppressed as the concentration increases.

[0136] In the step of forming a metal seed layer (20) on the polymer intermediate layer (10), the pH of the metal precursor solution for forming the metal seed layer may be 2-9 or 3-8. Although not limited thereto, when the pH of the metal precursor solution for forming the metal seed layer is 2-9, the SERS signal enhancement effect and uniformity for the analyte may be improved. In addition, depending on the pH of the metal precursor solution for forming the metal seed layer, the color of the polymer-mediated plasmonic well plate substrate of the present disclosure may change, and when the metal is Au, the color may change to a color more similar to gold as the pH increases.

[0137]

[0138] Step iii) is a step of forming a polycrystalline structure layer by immersing the well plate member on which the polymer intermediate layer (10) is formed in a composition for forming a polycrystalline structure layer including a metal precursor and a reducing agent solution, thereby forming a polycrystalline structure layer through additional growth within the well (Fig. 2 (d)). The formation of the polycrystalline structure in step iii) means forming a metal nanostructure layer by growing a metal nanostructure on the polymer intermediate layer (10) of the well plate member or the metal seed layer (20).

[0139] The polycrystalline structure layer formed in the above step iii) is composed of a cluster formed of a plurality of metal nanostructures and may include a plurality of pores therein. Although not limited thereto, the plurality of metal nanostructures or the cluster formed of the plurality of metal nanostructures may be connected to each other and include pores therein. Although not limited thereto, the plurality of metal nanostructures or the cluster formed of the plurality of metal nanostructures may be connected to each other in a branch structure.

[0140] Although not limited thereto, the polycrystalline structure layer may be grown by a solution process and may be connected in a branch structure, and may have an average particle diameter of 0.1 to 100 μm. Although not limited thereto, the average particle diameter of the polycrystalline structure layer may be suitable for improving signal intensity and signal uniformity within the above range.

[0141] As described above, the plurality of metal nanostructures are formed in a form in which they are connected to each other and constitute a cluster, and the polycrystalline structure layer may be formed of a cluster formed by the plurality of metal nanostructures. In addition, the polycrystalline structure layer may be formed of a plurality of connected clusters and may have a plurality of grain boundaries.

[0142] The polymer-mediated plasmonic well-plate substrate of the present invention, which includes a polycrystalline structure layer formed as described above, has a form in which the metal nanostructures are not spaced apart from each other but are connected and form a cluster including pores distributed inside, so that the nano-gaps and hot spots can be significantly increased compared to the case in which the metal nanostructures are formed spaced apart from each other. Therefore, the polymer-mediated plasmonic well-plate substrate of the present invention can exhibit excellent SERS signal enhancement effect and uniformity for analytes.

[0143] Although not limited thereto, the pores included in the polycrystalline structure layer may be formed in a disordered but uniform manner between a plurality of metal nanostructures, or may be formed in a disordered but uniform manner between clusters formed by a plurality of metal nanostructures. Accordingly, the polymer-mediated plasmonic well-plate substrate of the present disclosure can enhance the SERS signal enhancement effect and uniformity for analytes.

[0144] Although not limited thereto, the step iii) may include a step of controlling the shape of the polycrystalline structure layer by setting the ratio of the metal precursor:reducing agent to 1:0.5 to 1:10. By controlling the optimal ratio of the reducing agent depending on the type of reducing agent, the shape of the polycrystalline structure layer formed on the polymer-mediated plasmonic well-plate substrate of the present disclosure can be easily controlled. In addition, by controlling the time for which the well-plate member is immersed in the composition for forming a polycrystalline structure layer, the shape of the polycrystalline structure layer formed on the substrate for spectroscopic analysis can be easily controlled. The method for manufacturing the polymer-mediated plasmonic well-plate substrate of the present disclosure can simultaneously improve signal enhancement and signal uniformity by a simple process of immersion once or twice. Furthermore, performing the step of forming the polycrystalline structure layer in the step iii) twice or more may be suitable for simultaneously improving signal enhancement and signal uniformity.

[0145]

[0146] Although not limited thereto, the composition for forming the polycrystalline structure layer of the present invention in step iii) may include a gold precursor and a reducing agent.

[0147] Although not limited thereto, the gold sphere may be selected from the group consisting of HAuCl4, AuCl, AuCl2, AuCl3, Na2Au2Cl8, and NaAuCl2, but HAuCl4 and NaAuCl2 One or more of these may be more suitable.

[0148] Although not limited thereto, in the step iii), the reducing agent may be at least one of hydroxylamine and its derivatives. Although not limited thereto, the hydroxylamine derivative may be at least one of hydroxylamine hydrochloride, hydroxylamine sulfate, and O-methylhydroxylamine.

[0149]

[0150] Although not limited thereto, in the composition for forming a polycrystalline structure layer in step iii), the concentration of the metal ion in the metal precursor may be 0.5 to 10 mg / mL. Although not limited thereto, if the concentration of the metal ion in the metal precursor solution in the composition for forming a metal nanostructure layer is less than 0.5 mg / mL, the formation of a polycrystalline structure layer may not be easy, and if it exceeds 10 mg / mL, the formation of a polycrystalline structure layer may be inhibited or the formation of nano-gap may be suppressed as the concentration increases.

[0151]

[0152] Although not limited thereto, the formation of the polycrystalline structure layer in the step iii) may be performed for 5 minutes to 24 hours, for 5 minutes to 15 hours, for 5 minutes to 10 hours, for 5 minutes to 5 hours, for 5 minutes to 3 hours, for 5 minutes to 1 hour, or for 10 minutes to 1 hour. Although not limited thereto, if the polycrystalline structure layer formation time is less than 5 minutes, the formation of the polycrystalline structure layer may not be easy, and if it exceeds 24 hours, the formation of the polycrystalline structure layer may be inhibited or the formation of nanogap may be suppressed depending on the increase in concentration.

[0153]

[0154] Although not limited thereto, the method for manufacturing a polymer-mediated plasmonic well-plate substrate of the present invention may further include a step of forming a metal nanoparticle layer by attaching metal nanoparticles on a polycrystalline structure layer after step iii).

[0155] As described above, the metal nanoparticles may be separately synthesized and manufactured to form a metal nanoparticle layer by attaching the metal nanoparticles to the surface of the polycrystalline structure layer formed in the well of the well plate member. Although not limited thereto, the metal nanoparticles may be manufactured by various known metal nanoparticle synthesis methods. Although not limited thereto, the attachment of the metal nanoparticles in the step of forming the metal nanoparticle layer may utilize a conventionally known technique. For example, the metal nanoparticles may be attached by physical adsorption, or the metal nanoparticles may be attached after surface modification using a chemical linker to fix the metal nanoparticles on the surface of the polycrystalline structure layer. By the above configuration, the polymer-mediated plasmonic well plate substrate of the present disclosure can improve the SERS signal enhancement effect and uniformity for the analyte.

[0156]

[0157] Example

[0158] Example 1. Fabrication of a polymer-mediated plasmonic well plate substrate

[0159] A well plate member including one or more wells was prepared as a base member.

[0160] A 1 mg / mL polydopamine solution was coated twice on the well plate member for 5 hours and 24 hours, respectively.

[0161] Next, the well plate member on which the polydopamine layer was formed was washed twice with DI, and a 0.5 mg / mL HAuCl4 gold precursor solution at pH 8 was coated to grow a gold seed layer on the polydopamine layer.

[0162] The well-plate member on which the gold seed layer was formed was washed twice with DI after 24 hours, and a composition for forming a polycrystalline structure layer containing a 10 mM / mL HAuCl4 gold precursor and a 20 mM hydroxylamine hydrochloride (HH) reducing agent was added three times to induce additional growth on the gold seed layer, thereby completing the formation of the polycrystalline structure layer. After the formation of the polycrystalline structure layer was completed, the remaining precursor and reducing agent were removed by washing twice with ethanol and three or more times with water. Thereafter, the well-plate member was dried at room temperature for more than 1 hour to complete the manufacture of a polymer-mediated plasmonic well-plate substrate.

[0163] FIG. 3 is a photograph of a plasmonic well plate substrate manufactured step by step according to a method for manufacturing a polymer-mediated plasmonic well plate substrate according to an embodiment of the present invention as described above.

[0164]

[0165] Experimental Example 1. Effects according to polymer concentration

[0166] Figure 4 is an SEM photograph showing the surface of a well-plate substrate according to the concentration of a polymer when forming a polymer-mediated layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present invention.

[0167] As shown in Figure 4, the coating results for different concentrations of the polymer polydopamine showed no significant difference. Considering process efficiency, it may be appropriate to coat polydopamine twice at a concentration of 1 mg / mL over 24 hours.

[0168]

[0169] Experimental Example 2. Effects of Repeated Polymer Coating Processes

[0170] Figure 5 is an SEM photograph showing the surface of a well-plate substrate according to the number of times the polymer is coated when forming a polymer-mediated layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present invention.

[0171] As shown in Fig. 5, when the concentration of the polymer polydopamine is 1 mg / mL and the treatment is performed for 24 hours and only the number of coatings is changed, it may be appropriate to coat more than twice.

[0172]

[0173] Experimental Example 3. SERS activity according to polymer coating

[0174] Fig. 6 (a) is a graph showing the SERS signal intensity measured at 633 nm of a well-plate substrate coated only with a polymer according to one embodiment of the present disclosure, and Fig. 6 (b) is a graph showing the SERS signal intensity measured at 785 nm of a well-plate substrate coated only with a polymer according to one embodiment of the present disclosure. At this time, 100 μL of 10 μM malachite green was used as the analyte.

[0175] As shown in Fig. 6, when only the polymer polydopamine was coated on the well plate substrate, no SERS signal appeared.

[0176]

[0177] Experimental Example 4. Effect of metal precursor concentration on metal seed layer formation

[0178] Figure 7 is an SEM photograph showing a metal seed layer according to the concentration of a metal precursor when forming a metal seed layer among the polycrystalline structure layers of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention. In this case, the reaction time was set to 24 hours.

[0179] As shown in Fig. 7, in the solution for forming a gold seed layer, the gold seed layer begins to form when the concentration of gold ions in the gold precursor spheres is 0.5 mg / mL, and as the concentration increases, the formation of the gold seed layer is promoted, and when it exceeds 10 mg / mL, the gold seed layer may be excessively formed.

[0180]

[0181] Experimental Example 5. Effect of pH on metal precursor solution for forming metal seed layer

[0182] FIG. 8 (a) is a graph showing the SERS signal intensity measured at 633 nm according to the pH of the metal precursor solution when forming a metal seed layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present disclosure, and FIG. 8 (b) is a graph showing the SERS signal intensity measured at 785 nm according to the pH of the metal precursor solution when forming a metal seed layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present disclosure.

[0183] At this time, 100 μL of 10 μM malachite green was used as the analyte, and measurement was performed under the following conditions.

[0184] (a) in Figure 8: Laser wavelength 633nm, laser power 1mW, exposure time 0.1s

[0185] (b) in Figure 8: Laser wavelength 785nm, laser power 1mW, exposure time 1s

[0186]

[0187] FIG. 9 (a) and (b) are SEM photographs showing a polycrystalline structure layer according to the pH of a metal precursor solution for forming a metal seed layer of a polymer-mediated plasmonic well-plate substrate according to an embodiment of the present disclosure, and FIG. 9 (c) is a photograph of a well-plate substrate according to the pH of a metal precursor solution for forming a metal seed layer of a polymer-mediated plasmonic well-plate substrate according to an embodiment of the present disclosure.

[0188]

[0189] As shown in FIGS. 8 and 9, when the metal precursor solution for forming the metal seed layer was at pH 3, the polycrystalline structure layer was formed better than when the solution was at pH 8, the SERS signal intensity increased, and the color of the plasmonic well plate substrate was found to be reddish.

[0190]

[0191] Experimental Example 6. Effects of the type of reducing agent and the ratio of metal precursor and reducing agent in the formation of a polycrystalline structure layer.

[0192] Figure 10 is a graph showing the SERS signal intensity measured at 633 nm according to the type of reducing agent and the ratio of metal precursor and reducing agent when forming a polycrystalline structure layer of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0193] Figure 11 is a graph showing the SERS signal intensity measured at 785 nm according to the type of reducing agent and the ratio of metal precursor and reducing agent when forming a polycrystalline structure layer of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0194] At this time, 100 μL of 10 μM malachite green was used as the analyte, and measurement was performed under the following conditions.

[0195] Figure 10: Laser wavelength 633nm, laser power 1mW, exposure time 0.2s

[0196] Figure 11: Laser wavelength 785nm, laser power 1mW, exposure time 1s

[0197] As shown in Figures 10 and 11, there may be some differences depending on the type of reducing agent, but the ratio of the metal precursor to the reducing agent may be 1:0.5 to 1:10.

[0198]

[0199] Experimental Example 7. Density change according to the number of additional growths of metal nanosphere layers during polycrystalline structure layer formation.

[0200] Figure 12 is an SEM photograph showing the change in density of a polycrystalline structure layer according to the number of additional growths of a metal nanostructure layer when forming a polycrystalline structure layer of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0201] FIG. 13 (a) is a graph showing the SERS signal intensity measured at 633 nm according to the number of additional growths of a metal nanostructure layer when forming a polycrystalline structure layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present disclosure, and FIG. 13 (b) is a graph showing the SERS signal intensity measured at 785 nm according to the number of additional growths of a metal nanostructure layer when forming a polycrystalline structure layer of a polymer-mediated plasmonic well-plate substrate according to one embodiment of the present disclosure.

[0202] At this time, the concentration of polydopamine was 1 mg / mL, and the treatment was performed twice for 24 hours each time, and the pH of the gold precursor solution for forming the gold seed layer was fixed at 3 when forming the gold seed layer. The gold ion concentration was 0.5 mg / mL, and the metal seed layer was formed for 24 hours. The treatment time of HH, a reducing agent, was fixed at 1 hour, and the number of treatment repetitions varied from 1 to 3 times.

[0203] As shown in Figures 12 and 13, as the number of HH treatment repetitions increased, the density of the polycrystalline structure layer increased and the SERS signal was enhanced.

[0204]

[0205] Experimental Example 8. Density change according to additional growth time of metal nanostructure layer during polycrystalline structure layer formation.

[0206] Figure 14 is an SEM photograph showing the change in density of a polycrystalline structure layer according to the additional growth time of a metal nanostructure layer when forming a polycrystalline structure layer of a polymer-mediated plasmonic well plate substrate according to one embodiment of the present invention.

[0207] At this time, the concentration of polydopamine was 1 mg / mL, and the treatment was performed twice for 24 hours each time, and the pH of the gold precursor solution for forming the gold seed layer was fixed at 3 when forming the gold seed layer. The concentration of gold ions was 0.5 mg / mL, and the metal seed layer was formed for 24 hours. The number of treatments with HH, a reducing agent, was fixed at 1 time, and the treatment time was varied from 0 to 60 minutes.

[0208] As shown in Fig. 14, a polycrystalline structure layer was formed starting from 5 minutes of HH processing time, and as the processing time increased, the density of the polycrystalline structure layer increased.

[0209]

[0210] Experimental Example 9. Comparison of SERS signal intensities between our polymer-mediated plasmonic well-plate substrate and a commercial well-plate substrate.

[0211] Figure 15 is a graph showing the SERS signal intensity measured at 633 nm of a polymer-mediated plasmonic well-plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention and a commercial well-plate substrate.

[0212] As shown in Fig. 15, the SERS signal enhancement effect of the polymer-mediated plasmonic well-plate substrate (PD Well-Plate) of the present invention was significantly greater than that of a commercial well-plate substrate (Nirmidas).

[0213]

[0214] Experimental Example 10. Comparison of SERS signal intensity according to the number of additional growths of the metal nanostructure layer on the polymer-mediated plasmonic well plate substrate of our center.

[0215] FIG. 16 is a graph showing the SERS signal intensity measured at 633 nm according to the number of additional growths of a metal nanostructure layer of a polymer-mediated plasmonic well plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0216] As shown in Fig. 16, the SERS signal intensity increased as the number of additional growths of the metal nanostructure layer increased.

[0217]

[0218] Experimental Example 11. Detection Sensitivity of Our Polymer-Mediated Plasmonic Well Plate Substrate

[0219] Figure 17 is a graph of SERS signal intensity measured at 633 nm using malachite green to confirm the detection sensitivity of a polymer-mediated plasmonic well plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0220] As shown in Fig. 17, the polymer-mediated plasmonic well plate substrate of the present invention showed that the SERS signal was proportionally enhanced as the concentration of the analyte increased, and thus the analyte detection sensitivity was excellent and quantitative evaluation was possible.

[0221]

[0222] Experimental Example 12. SERS signal uniformity of our polymer-mediated plasmonic well plate substrate.

[0223] FIG. 18 is a graph showing the SERS signal uniformity measured at 633 nm for a liquid sample in one well of a polymer-mediated plasmonic well plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0224] As shown in Fig. 18, the polymer-mediated plasmonic well-plate substrate of the present invention showed high reliability with excellent SERS signal uniformity.

[0225]

[0226] Experimental Example 13. Effects according to the presence or absence of the polymer intermediary layer of the present invention.

[0227] Figure 19 (a) is a UV-vis absorbance graph showing that there is no SPR because a polymer intermediary layer is not formed by a comparative example of the present disclosure (Without PD Layer), and Figure 19 (b) is a graph showing UV-vis absorbance according to the number of polymer coatings when a polymer intermediary layer is formed by an example of the present disclosure (With PD Layer).

[0228] As shown in Fig. 19, it was confirmed that if the polymer intermediary layer of the present invention is not formed, it is difficult to form a nanocrystal structure layer even if a metal seed layer is formed and further grown.

[0229]

[0230] Experimental Example 14. Detection sensitivity and uniformity of our polymer-mediated plasmonic well plate substrate.

[0231] Figure 20 is a graph of SERS signal intensity measured at 785 nm using malachite green to confirm the detection sensitivity of a polymer-mediated plasmonic well plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0232] As shown in Fig. 20, using the polymer-mediated plasmonic well plate substrate of the present invention, it is possible to accurately detect and quantitatively analyze analytes at concentrations of several tens of nM.

[0233] FIG. 21 is a graph showing the SERS signal uniformity measured at 785 nm for a liquid sample in one well of a polymer-mediated plasmonic well-plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 according to one embodiment of the present invention.

[0234] As shown in Fig. 21, the polymer-mediated plasmonic well-plate substrate of the present invention showed high reliability with excellent SERS signal uniformity.

[0235]

[0236] Experimental Example 15. Comparison of SERS signal intensity and absorbance between various polymer-mediated plasmonic well-plate substrates of our laboratory and commercial well-plate substrates.

[0237] Figure 22 is a graph showing the SERS signal intensity measured at 633 nm of a polymer-mediated plasmonic well-plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 and pH 8 according to one embodiment of the present invention and a commercial well-plate substrate.

[0238] Figure 23 is a graph showing the absorbance according to wavelength of a polymer-mediated plasmonic wellplate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 3 and pH 8 according to one embodiment of the present invention, and a bare wellplate and a commercial wellplate substrate as a control group.

[0239] As shown in FIGS. 22 and 23, the polymer-mediated plasmonic well-plate substrates of the present invention (PD Well-Plate (pH 3) and PD Well-Plate (pH 8)) were found to be significantly superior to commercial products (Nirmidas), and in particular, the polymer-mediated plasmonic well-plate substrates of the present invention manufactured using a metal precursor solution for forming a metal seed layer at pH 3 were found to have a more superior SERS signal enhancement effect.

[0240]

[0241] Experimental Example 16. Comparison of Signal Detection for Liquid and Dry Samples

[0242] Figure 24 is a graph showing the detection sensitivity of a polymer-mediated plasmonic well-plate substrate manufactured with a metal precursor solution for forming a metal seed layer at pH 8 according to one embodiment of the present invention, using malachite green for liquid and dry samples.

[0243] As shown in Fig. 24, the various polymer-mediated plasmonic well-plate substrates of the present invention exhibit excellent detection sensitivity for both liquid-state measurement and dry-state measurement, and the SERS signal enhancement effect is shown to be more excellent for dry samples.

[0244]

[0245] While specific portions of the present application have been described in detail above, it will be clear to those skilled in the art that these specific descriptions merely represent preferred embodiments and do not limit the scope of the present application. Therefore, the substantial scope of the present application is defined by the appended claims and their equivalents.

[0246]

[0247] [Explanation of symbols]

[0248] 10: Polymer mediator layer

[0249] 20: Metal seed layer

[0250] 30: Metal nanostructure layer

Claims

1. A well plate member having one or more wells; a polymer mediating layer formed within the well; and A polycrystalline structure layer formed on the polymer intermediary layer; A polymer-mediated plasmonic well plate substrate, wherein the polycrystalline structure layer is composed of a cluster formed of a plurality of metal nanostructures and includes a plurality of pores therein.

2. In paragraph 1, A polymer-mediated plasmonic well plate substrate, wherein the polymer-mediated layer is formed with a thickness of 10 nm to 10 μm of at least one selected from catechols and polyphenols.

3. In paragraph 1, A polymer-mediated plasmonic well plate substrate, wherein the metal nanostructure or a cluster formed of a plurality of metal nanostructures is connected to each other.

4. In paragraph 1, The above polycrystalline structure layer A metal seed layer composed of metal nanoparticles; and A polymer-mediated plasmonic well plate substrate comprising a metal nanostructure layer grown one or more times with a metal precursor and a reducing agent on a metal seed layer.

5. In paragraph 4, The above polycrystalline structure layer A polymer-mediated plasmonic well plate substrate further comprising a metal nanoparticle layer formed on the metal nanostructure layer.

6. In paragraph 5, A polymer-mediated plasmonic well plate substrate, wherein the average thickness of the metal seed layer, the metal nanostructure layer, or the metal nanoparticle layer is each 5 nm or more.

7. In paragraph 1, The above polymer-mediated plasmonic wellplate substrate is a polymer-mediated plasmonic wellplate substrate for surface-enhanced Raman spectroscopy (SERS), plasmon-enhanced fluorescence (PEF), or fluorescence analysis.

8. A method for manufacturing a polymer-mediated plasmonic well plate substrate described in Article 1, i) a step of preparing a well plate member having one or more wells; ii) forming a polymer mediating layer within the well; and iii) a step of forming a polycrystalline structure layer within the well by immersing the well plate member on which the polymer intermediary layer is formed in a composition for forming a polycrystalline structure layer including a metal precursor and a reducing agent solution; A method for manufacturing a polymer-mediated plasmonic well plate substrate, wherein the polycrystalline structure layer formed in the above step iii) is composed of a cluster formed of a plurality of metal nanostructures and includes a plurality of pores therein.

9. In paragraph 8, A method for manufacturing a polymer-mediated plasmonic well plate substrate, comprising forming the polymer-mediated layer in step ii) by coating it with 0.1 to 20 mg / mL of polydopamine.

10. In paragraph 8, A method for manufacturing a polymer-mediated plasmonic well plate substrate, comprising forming the polymer-mediated layer by coating it 2 to 5 times in the above step ii).

11. In paragraph 8, A method for manufacturing a polymer-mediated plasmonic well-plate substrate, further comprising a step of forming a metal seed layer by immersing a well-plate member on which a polymer-mediated layer has been formed prior to step iii) in a metal precursor solution for forming a metal seed layer after step ii) above.

12. In paragraph 11, A method for manufacturing a polymer-mediated plasmonic well plate substrate, wherein the pH of the metal precursor solution for forming the metal seed layer is 2-9.

13. In paragraph 8, A method for manufacturing a polymer-mediated plasmonic well-plate substrate, comprising a step of controlling the shape of the polycrystalline structure layer by adjusting the ratio of metal precursor:reducing agent to 1:0.5 to 1:10 in step iii).

14. In paragraph 8, A method for manufacturing a plasmonic well plate substrate for spectroscopic analysis, wherein in the above step iii), the reducing agent is at least one of hydroxylamine and a hydroxylamine derivative.

15. In paragraph 8, A method for manufacturing a polymer-mediated plasmonic well plate substrate, wherein the step of forming a polycrystalline structure layer in step iii) above is performed two or more times.

16. In paragraph 8, A method for manufacturing a polymer-mediated plasmonic well plate substrate, wherein the concentration of metal ions in the metal precursor in the composition for forming a polycrystalline structure layer in step iii) above is 0.5 to 10 mg / mL.

17. In paragraph 8, A method for manufacturing a polymer-mediated plasmonic well plate substrate, wherein the formation of a polycrystalline structure layer in the above step iii) is performed for 5 minutes to 24 hours.

18. In paragraph 8, A method for manufacturing a polymer-mediated plasmonic well plate substrate, further comprising a step of forming a metal nanoparticle layer by attaching metal nanoparticles on a polycrystalline structure layer after the above step iii).

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