Spectroscopic analysis substrate for diagnosing dementia including hydrophilized graphene layer and spectroscopic device including same

Abstract

The present application provides: a spectroscopic analysis substrate for diagnosing dementia, comprising a hydrophilized graphene layer; and a spectroscopic device comprising same. More specifically, the present application provides: a spectroscopic analysis substrate for diagnosing dementia, having improved accuracy due to increased binding force and selectivity for amyloid beta, the substrate comprising a hydrophilized graphene layer; and a spectroscopic device comprising the spectroscopic analysis substrate.

Description

A substrate for spectroscopic analysis for dementia diagnosis comprising a hydrophilized graphene layer and a spectroscopic device comprising the same

[0001] The present invention relates to a substrate for spectroscopic analysis for dementia diagnosis comprising a hydrophilized graphene layer and a spectroscopic device comprising the same. More specifically, the present invention relates to a substrate for spectroscopic analysis for dementia diagnosis comprising a hydrophilized graphene layer with improved accuracy due to increased binding affinity and selectivity with amyloid beta, and a spectroscopic device comprising the same.

[0002] Dementia refers to a clinical syndrome in which cognitive functions in various areas, such as memory, language, and judgment, decline due to acquired factors, making it difficult to perform daily activities properly. Dementia includes senile dementia, known as Alzheimer's disease, and vascular dementia caused by conditions such as stroke; in addition, there is dementia caused by various other factors.

[0003] It is known that Alzheimer's disease develops when proteins such as amyloid beta and tau tangle in the brain and damage brain cells.

[0004] In particular, Alzheimer's disease is known to be caused by the formation of plaques, which are fibrils resulting from the polymerization of amyloid beta (Aβ) proteins (Ross, CA; Poirier, MA Protein Aggregation and Neurodegenerative Disease Nature Medicine 2004, 10, S10).

[0005] Meanwhile, to diagnose dementia, levels of proteins such as amyloid beta are evaluated through cerebrospinal fluid (CSF) tests, positron emission tomography (PET), and magnetic resonance imaging (MRI) following cognitive intelligence tests, and the presence of dementia and treatment prognosis are confirmed.

[0006] Cerebrospinal fluid (CSF) testing is an invasive diagnostic method based on CSF extraction, which carries the risk of causing pain and aftereffects to the human body. Furthermore, such analysis using CSF is costly and time-consuming.

[0007] In addition, PET and MRI scans require a significant amount of time and expensive equipment, and can be accompanied by side effects such as nausea and headaches; furthermore, they may not be suitable for healthcare purposes, such as monitoring during daily life.

[0008] On the other hand, while the method of detecting biomarkers from blood (plasma) using diagnostic kits is useful for health management in daily life, existing diagnostic kits have the problem of high production costs due to being manufactured through complex processes, while having low accuracy (analytical sensitivity).

[0009] Therefore, there is a need for a dementia diagnostic device and method that are non-invasive, save time and costs, have a low production cost, are highly accurate, and are useful for health management in daily life.

[0010] As background technology of the present invention, Korean published patent No. 10-2024-0012659 describes a composition, kit, and method for diagnosing Alzheimer's disease dementia by detecting the methylation level of a gene CpG site.

[0011]

[0012] The purpose of this invention is to provide a spectroscopic analysis substrate for dementia diagnosis that is non-invasive, saves time and costs, has a low production cost, is highly accurate, and is useful in terms of health management in daily life.

[0013] Another objective of the present invention is to provide a spectroscopic analysis device for dementia diagnosis that is non-invasive, saves time and costs, has a low production cost, is highly accurate, and is useful in terms of health management in daily life.

[0014] Another objective of this institution is to provide a spectroscopic analysis method for dementia diagnosis that is non-invasive, saves time and costs, offers high diagnostic accuracy, and is useful for health management in daily life.

[0015] The objectives of the present invention are not limited to those mentioned above, and other unmentioned objectives will be clearly understood from the description in detail.

[0016]

[0017] According to one aspect, a substrate for spectroscopic analysis for dementia diagnosis is provided, comprising: a base substrate; a graphene layer formed on the base substrate; and a nucleic acid layer formed on the graphene layer.

[0018] According to one embodiment, the nucleic acid layer may hydrophilize the graphene layer.

[0019] According to one embodiment, the substrate for spectroscopic analysis for dementia diagnosis of the present invention can improve binding strength and / or selectivity for amyloid beta fibrils.

[0020]

[0021] According to another aspect, a spectroscopic device is provided comprising: a light source; a substrate for spectroscopic analysis for dementia diagnosis as described herein; and a detector for detecting spectra.

[0022] According to one embodiment, the spectroscopic device of the present invention may be able to diagnose dementia using a biological sample including blood, serum, saliva, or urine.

[0023] According to one embodiment, the spectroscopic device of the present invention can diagnose dementia by analyzing Raman shift fluctuations.

[0024] According to one embodiment, the target substance of the detector in the spectroscopic device of the present invention may be amyloid beta fibrils in a biological sample.

[0025] According to one embodiment, a biological sample may be provided in a gel state in the spectroscopic device of the present invention.

[0026]

[0027] According to another aspect, a method for manufacturing a substrate for spectroscopic analysis is provided, comprising: a) a graphene layer forming step of forming a graphene layer on a base substrate; and b) a nucleic acid layer forming step of coating a nucleic acid on the graphene layer to form a nucleic acid layer.

[0028]

[0029] According to another aspect, a Raman spectroscopic analysis method is provided, comprising: 1-1) a step of adsorbing a biological sample onto a substrate for spectroscopic analysis as described herein; and 1-2) a dementia analysis step of analyzing whether there is a change in Raman shift by comparing the Raman shift of the biological sample adsorbed onto the substrate for spectroscopic analysis with the Raman shift of a control group using a Raman spectroscopic device.

[0030]

[0031] According to another aspect, a Raman spectroscopy analysis method is provided, comprising: 2-1) a step of adsorbing a solution containing a biological sample and nucleic acid onto a substrate for spectroscopic analysis on which a graphene layer is formed on a base substrate; and 2-2) a dementia analysis step of analyzing whether there is a change by comparing the Raman shift of the biological sample adsorbed on the substrate for spectroscopic analysis with the Raman shift of a control group using a Raman spectroscopy device.

[0032]

[0033] According to one embodiment, a substrate for spectroscopic analysis comprising a hydrophilized graphene layer of the present invention improves the adsorption of biological samples and increases the binding strength and selectivity with amyloid beta, thereby enabling non-invasive, time- and cost-saving, and high-accuracy diagnosis of dementia, and can be usefully utilized for health management such as monitoring in daily life.

[0034] According to one embodiment, a substrate for spectroscopic analysis comprising a hydrophilized graphene layer of the present invention has a simple structure and a reduced number of production processes, resulting in a low production cost, and is label-free yet has high accuracy in diagnosing dementia.

[0035] According to one embodiment, a spectroscopic device comprising a substrate for spectroscopic analysis including a hydrophilized graphene layer of the present invention can be used for health management, such as monitoring in daily life, with high accuracy and low production cost, as well as non-invasive and time- and cost-saving methods for diagnosing dementia, due to improved adsorption of biological samples and increased binding strength and selectivity with amyloid beta.

[0036] According to one embodiment, the method for manufacturing a substrate for spectroscopic analysis comprising a hydrophilized graphene layer of the present invention can efficiently manufacture a substrate for spectroscopic analysis with improved adsorption of biological samples, increased binding strength and selectivity with amyloid beta by forming a nucleic acid layer on the graphene layer.

[0037] According to one embodiment, the Raman spectral analysis method for diagnosing dementia according to the present invention is non-invasive, can save time and cost, has high accuracy in diagnosing dementia, and can be usefully utilized for health management, such as monitoring in daily life.

[0038]

[0039] FIG. 1 is a schematic diagram showing the preparation of a substrate for spectroscopic analysis including a hydrophilized graphene layer and a method for diagnosing dementia according to one embodiment of the present invention.

[0040] FIG. 2 is a diagram schematically showing a method for improving the adsorption of a biological liquid sample on a graphene layer according to one embodiment of the present invention.

[0041] FIG. 3 is a diagram showing the results of Raman spectral analysis performed using a substrate for spectroscopic analysis including a hydrophilized graphene layer according to one embodiment of the present invention, compared with a comparative example.

[0042] Figure 4 is a graph showing the downshifting of the G peak in the presence of amyloid beta fiprils, based on the results of Raman spectral analysis using a substrate for spectroscopic analysis comprising a hydrophilized graphene layer according to one embodiment of the present invention, compared with a comparative example.

[0043] FIG. 5a is a graph showing the result of downshifting the G peak based on the results of Raman spectroscopic analysis at different concentrations of amyloid beta fibrils using a substrate for spectroscopic analysis comprising a hydrophilized graphene layer in which the base substrate is silica (SiO2) according to one embodiment of the present invention.

[0044] FIG. 5b is a graph showing the result of downshifting the G peak based on the results of Raman spectroscopic analysis at different concentrations of amyloid beta fibrils using a substrate for spectroscopic analysis comprising a hydrophilized graphene layer in which the base substrate is glass according to one embodiment of the present invention.

[0045] The object, specific advantages, and novel features of the present disclosure will become more apparent from the following detailed description and embodiments in conjunction with the accompanying drawings.

[0046] Prior to this, terms and words used in this specification and claims should not be interpreted in their ordinary and dictionary meanings, but should be interpreted in a meaning and concept consistent with the technical spirit of this disclosure, based on the principle that the inventor can appropriately define the concept of the terms to best describe his invention.

[0047] In this specification, where a component, such as a layer, part, or substrate, is described as being "on," "connected," or "joined" to another component, it may be directly "on," "connected," or "joined" to the other component, or it may have one or more other components interposed between the two components. In contrast, where a component is described as being "directly on," "directly connected," or "directly joined" to another component, no other components may be interposed between the two components.

[0048] The terms used herein are merely for describing specific embodiments and are not intended to limit the disclosure. The singular expression includes the plural expression unless the context clearly indicates otherwise.

[0049] In this specification, terms such as “comprising” or “having” are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should not be understood as precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0050] In this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. Furthermore, throughout the specification, "on" means located above or below the subject part, and does not necessarily mean located on the upper side with respect to the direction of gravity.

[0051] The present disclosure is capable of various modifications and may have various 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, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present disclosure. In describing the present disclosure, if it is determined that a detailed description of related prior art may obscure the essence of the present disclosure, such detailed description is omitted.

[0052] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In describing with reference to the accompanying drawings, identical or corresponding components are given the same reference numerals, and redundant descriptions thereof will be omitted.

[0053] FIG. 1 is a schematic diagram showing the preparation of a substrate for spectroscopic analysis including a hydrophilized graphene layer and a method for diagnosing dementia according to one embodiment of the present invention.

[0054] Referring to FIG. 1, a substrate for spectroscopic analysis for dementia diagnosis according to one aspect of the present invention comprises: a base substrate (10); a graphene layer (20) formed on the base substrate (10); and a nucleic acid layer (30) formed on the graphene layer (20).

[0055] Although not limited thereto, the base substrate (10) may be selected from silicon, silica (SiO2), quartz, glass, ceramic, and polymer. Although not limited thereto, silica or glass may be suitable for the base substrate (10). Although not limited thereto, the base substrate (10) may be suitable for improving sensitivity during spectroscopic analysis if it has a thickness of about 10 to 500 nm.

[0056] Although not limited thereto, the graphene of the graphene layer (20) is synthesized by exfoliating layered graphite through mechanical or ultrasonic treatment, or by epitaxial growth, chemical vapor deposition (CVD), etc., and has a two-dimensional sheet shape of several layers, and has structural and electrical properties (electrical conductivity) similar to graphite. Graphene is capable of Raman signal amplification due to its inherent structural properties.

[0057] In the present invention, graphene may include all types of graphene, such as graphene oxide (GO) and reduced graphene oxide (rGO).

[0058] Although not limited thereto, the graphene layer (20) in the present invention may be suitable for being manufactured by CVD and then transferred onto the base substrate (10).

[0059] Although not limited thereto, the graphene layer (20) may have a thickness of about 1 to 500 nm to be suitable for improving sensitivity during spectroscopic analysis, and may be 10 to 300 nm, 10 to 200 nm, 10 to 100 nm, 50 to 300 nm, 50 to 200 nm, or 50 to 100 nm.

[0060]

[0061] The above nucleic acid layer (30) facilitates the adsorption of biological samples by surface-modifying the graphene layer (10) to be hydrophilic. Although not limited thereto, the above nucleic acid layer (30) may be suitable for improving sensitivity during spectroscopic analysis if it has a thickness of about 1 to 10 nm.

[0062] In addition, the nucleic acid layer (30) can improve the binding strength and / or selectivity of the graphene layer (20) to amyloid beta fibrils. Therefore, by using the spectroscopic analysis substrate of the present invention, the accuracy of dementia diagnosis can be improved non-invasively with a simple and label-free configuration.

[0063] In this institution, nucleic acids from various organisms can be used, for example, salmon polynucleic acids can be used.

[0064] Although not limited thereto, the nucleic acid layer (30) may be suitable for hydrophilization of the graphene layer (20), improvement of the adsorption of biological samples, increase in binding strength and selectivity with amyloid beta, and improvement in sensitivity during spectroscopic analysis.

[0065] Although not limited thereto, the nucleic acid layer (30) may be formed on the graphene layer (20) after dissolving nucleic acid in liquid media to form a media gel.

[0066] The above media may include, but is not limited to, any liquid capable of dissolving nucleic acids. For example, it may include physiological saline (PBS, phosphate buffered saline) and cell culture media, which are liquids similar to those found in the human body. The above cell culture media may include, but is not limited to, DMEM (Dulbecco's Modified Eagle Medium), a general-purpose culture medium used for culturing various animal cells.

[0067] A spectroscopic device according to another aspect of the present invention comprises: a light source; a substrate for spectroscopic analysis for dementia diagnosis as described herein; and a detector for detecting spectra.

[0068] Although not limited thereto, the light source may use a laser capable of providing high-output incident light, such as that used in general spectroscopic devices.

[0069] The spectroscopic device of the present invention utilizes the aforementioned substrate for spectroscopic analysis for dementia diagnosis to facilitate the adsorption of biological samples and improve the binding strength and / or selectivity of the graphene layer toward amyloid beta fibrils. Accordingly, the spectroscopic device of the present invention can accurately diagnose dementia non-invasively with a simple and label-free configuration.

[0070] Although not limited thereto, the spectroscopic device of the present invention may be capable of diagnosing dementia using biological samples including blood, serum, saliva, or urine. The spectroscopic device of the present invention can diagnose dementia using easily obtainable biological samples. Therefore, according to the above configuration, health management, such as early diagnosis of dementia, can be facilitated non-invasively in daily life. Furthermore, dementia is a disease that requires monitoring of its progression, and using the spectroscopic device of the present invention can facilitate such monitoring.

[0071] The spectroscopic device of the present invention is a device that separates and analyzes light by wavelength and may include various known spectroscopic devices. Although not limited thereto, suitable devices may include a Raman spectroscopic device that measures the intrinsic molecular vibration of a substance using Raman scattering, a fluorescence spectroscopic device that measures a fluorescence signal emitted after absorbing light at a specific wavelength, etc.

[0072] Although not limited to this, the spectroscopic device of the present invention can diagnose dementia by analyzing Raman shift fluctuations.

[0073] The graphene in the graphene layer (20) of the spectroscopic analysis substrate of the present invention can amplify Raman signals due to its inherent structural characteristics. In addition, dementia can be accurately diagnosed by converting minute electrochemical changes resulting from the binding of amyloid beta, a target substance, to the graphene layer (20) into data. More specifically, when amyloid beta fibrils are present, the Raman signal is amplified and the G peak is downshifted compared to when amyloid beta fibrils are not present, making it easy to determine whether dementia is present (see FIGS. 3 to 5b).

[0074] The target substance of the detector may be amyloid beta fibrils in a biological sample, although this is not limited thereto.

[0075] Furthermore, the electrical properties of amyloid beta change depending on the fibrillation stage. Therefore, when amyloid beta fibrils bind, the G peak is downshifted, which can be identified as dementia.

[0076]

[0077] Although not limited to this, biological samples may be provided in a gel state to the spectroscopic device of the present invention. According to the above configuration, the hydrophilicity of the biological sample with respect to the graphene layer (20) is increased so that it is adsorbed well onto the graphene layer (20), thereby improving the sensitivity of spectroscopic analysis.

[0078]

[0079] Referring to FIG. 1, a method for manufacturing a substrate for spectroscopic analysis according to another aspect of the present invention comprises: a) a graphene layer (20) forming step of forming a graphene layer (20) on a base substrate (10); and b) a nucleic acid layer (30) forming step of coating a nucleic acid on the graphene layer (20) to form a nucleic acid layer (30).

[0080] Referring to FIG. 1 (a), step a) is a step of forming a graphene layer (20) on a base substrate (10).

[0081] Although not limited thereto, the graphene layer (20) in the present invention can be formed by known processes such as CVD (Chemical Vapor Deposition), ALD (Atomic Layer Deposition), Thermal Evaporation, E-beam Evaporation, Sputtering, and ICP (Inductively Coupled Plasma).

[0082] The graphene layer (20) may be formed on the base substrate (10) by a non-transfer method, or may be formed on a copper substrate, etc. and then transferred to the base substrate (10). Although not limited thereto, in an embodiment of the present invention, a graphene layer was prepared on a copper substrate by CVD, and then the graphene layer was transferred onto the base substrate (10) to form the graphene layer (20). Although not limited thereto, the graphene layer (20) may be formed with a thickness of about 1 to 500 nm to be suitable for improving sensitivity during spectroscopic analysis, and may be formed with a thickness of 10 to 300 nm, 10 to 200 nm, 10 to 100 nm, 50 to 300 nm, 50 to 200 nm, or 50 to 100 nm.

[0083]

[0084] Referring to FIG. 1(b), step b) is a step of forming a nucleic acid layer (30) by coating a nucleic acid on the graphene layer (20). The nucleic acid layer (30) facilitates the adsorption of biological samples by surface-modifying the graphene layer (20) to be hydrophilic. Although not limited thereto, the nucleic acid layer (30) may be formed with a thickness of about 1 to 10 nm to be suitable for improving sensitivity during spectroscopic analysis.

[0085] In addition, the nucleic acid layer (30) can improve the binding strength and / or selectivity of the amyloid beta fibrils of the graphene layer (20). Therefore, by using a spectroscopic analysis substrate manufactured according to the method for manufacturing a spectroscopic analysis substrate of the present invention, the accuracy of dementia diagnosis can be improved non-invasively with a simple and label-free configuration.

[0086] In the method for manufacturing a substrate for spectroscopic analysis of the present invention, nucleic acids from various organisms may be used, for example, polynucleic acids from salmon may be used.

[0087] Although not limited thereto, the nucleic acid layer (30) may be suitable for hydrophilization of the graphene layer (20), improvement of the adsorption of biological samples, increase in binding strength and selectivity with amyloid beta, and improvement in sensitivity during spectroscopic analysis.

[0088] Although not limited thereto, the nucleic acid layer (30) can be formed on the graphene layer (20) after dissolving the nucleic acid in a liquid media to form a media gel.

[0089] The above media may include, but is not limited to, any liquid capable of dissolving nucleic acids. For example, it may include physiological saline (PBS, phosphate buffered saline) and cell culture media, which are liquids similar to those found in the human body. The above cell culture media may include, but is not limited to, DMEM (Dulbecco's Modified Eagle Medium), a general-purpose culture medium used for culturing various animal cells.

[0090] The media gel produced in this way has abundant electrons, which can impart very high wettability to the hydrophobic graphene layer (20).

[0091] Although not limited thereto, the step of drying after forming the nucleic acid layer (30) may be further included.

[0092]

[0093] A Raman spectroscopic analysis method A according to another aspect comprises: 1-1) a step of adsorbing a biological sample onto a substrate for spectroscopic analysis as described herein; and 1-2) a dementia analysis step of analyzing whether there is a change in Raman shift by comparing the Raman shift of the biological sample adsorbed onto the substrate for spectroscopic analysis with the Raman shift of a control group using a Raman spectroscopic device.

[0094] Referring to FIG. 1 (c), step 1-1) is a step of adsorbing a biological sample onto a substrate for spectroscopic analysis as described herein. At this time, the substrate for spectroscopic analysis described herein may have a hydrophobic graphene layer surface modified and hydrophilized with nucleic acid as described above to improve the adsorption of the biological sample. In addition, the nucleic acid layer (30) may improve the binding strength and / or selectivity of the graphene layer (20) for amyloid beta fibrils. Therefore, by using the Raman spectroscopic analysis method A of the present invention, the accuracy of dementia diagnosis during spectroscopic analysis can be improved non-invasively with a simple and label-free configuration.

[0095] Although not limited thereto, the method may further include a step of drying the biological sample after adsorption. With the above-described configuration, sensitivity during Raman spectroscopic analysis can be improved through the concentration of target substances, etc.

[0096] Referring to Fig. 1(d), step 1-2) is a dementia analysis step for analyzing whether there is a change in Raman shift using a Raman spectroscopic device. The change in Raman shift can be determined by comparing the Raman shift of a biological sample adsorbed on the substrate for spectroscopic analysis with the Raman shift of a control group. The control group may be a biological sample in which amyloid beta protein is not present, or a biological sample containing amyloid beta monomers.

[0097] The graphene in the graphene layer (20) of the spectroscopic analysis substrate of the present invention is capable of amplifying Raman signals due to its inherent structural characteristics. In addition, dementia can be accurately diagnosed by converting minute electrochemical changes resulting from the binding of amyloid beta, a target substance, to the graphene layer (20) into data. Therefore, when amyloid beta fibrils are present, the G peak is downshifted compared to when amyloid beta fibrils are not present, allowing for the determination of dementia (see FIGS. 3 to 5b).

[0098] Furthermore, the electrical properties of amyloid beta change depending on the fibrillation stage. Therefore, when amyloid beta fibrils bind, the G peak is downshifted, which can be used to identify dementia. Additionally, the progression of dementia can be diagnosed based on the degree of G peak downshift according to the amount of amyloid beta fibrils.

[0099] A Raman spectroscopic analysis method B according to another aspect comprises: 2-1) a step of adsorbing a solution containing a biological sample and nucleic acid onto a spectroscopic analysis substrate having a graphene layer formed on a base substrate; and 2-2) a dementia analysis step of analyzing whether there is a change by comparing the Raman shift of the biological sample adsorbed on the spectroscopic analysis substrate with the Raman shift of a control group using a Raman spectroscopic device.

[0100] The Raman spectroscopic analysis method B of the present invention differs from the Raman spectroscopic analysis method A of the present invention only in that it does not form a separate nucleic acid layer but mixes the nucleic acid with the biological sample to increase the adsorption of the biological sample onto the hydrophobic graphene layer.

[0101] In the Raman spectroscopic analysis method B of the present invention, nucleic acids from various organisms can be used, for example, salmon polynucleic acid can be used.

[0102] Although not limited thereto, including 0.1 to 10 mass% of nucleic acid in the total solution containing the above biological sample and nucleic acid may be suitable for hydrophilization of the graphene layer, improvement of the adsorption of the biological sample, increase in binding strength and selectivity with amyloid beta, and improvement in sensitivity during spectroscopic analysis.

[0103] Although not limited to this, nucleic acids can be dissolved in media to form a media gel, and then a biological sample can be mixed and adsorbed onto the graphene layer.

[0104]

[0105] The above media may include, but is not limited to, any liquid capable of dissolving nucleic acids and proteins. For example, it may include physiological saline (PBS, phosphate buffered saline) and cell culture media, which are liquids similar to those found in the human body. The cell culture media may include DMEM (Dulbecco's Modified Eagle Medium), a general-purpose culture medium used for culturing various animal cells.

[0106] The media gel produced in this way has abundant electrons, which can impart very high wettability to the hydrophobic graphene layer.

[0107]

[0108] For the reasons stated above, the substrate for spectroscopic analysis, the spectroscopic device including the same, and the Raman spectroscopic analysis method of the present invention can be usefully utilized for screening dementia treatments and monitoring dementia prognosis.

[0109] Furthermore, by utilizing the Raman spectroscopy analysis method of this institute to database the fibril levels of amyloid beta protein, a platform for the early diagnosis of dementia, where early diagnosis is crucial, can be established. Moreover, since management following detection in the early to mid-stages of dementia is also important, a platform can be built to monitor the progression of the disease through periodic examinations using the institute's Raman spectroscopy analysis method after diagnosis. Based on the accumulated database, this platform can contribute to the promotion of public health in a rapidly aging society by developing related apps to provide services that transmit regular dementia diagnosis and monitoring results to patients via smartphones.

[0110]

[0111] [Example]

[0112] Example 1 Preparation of a substrate for spectroscopic analysis

[0113] Example 1-1 Graphene layer formation

[0114] (1) Graphene synthesis

[0115] Graphene was synthesized on a 35 μm Cu foil (99.99%) using a synthesis gas of CH4 (10–100 sccm), H2 (10–100 sccm), Ar, or N2 (10,000 sccm) via a CVD process at a temperature of 900–1060°C.

[0116] (2) Copper etching and doping

[0117] The copper substrate on which the graphene was formed was etched with H2SO4 and H2O2 and doped with benzotriazole as a dopant.

[0118] (3) Warrior

[0119] A graphene layer was formed by transferring it onto a wafer substrate, which serves as the base substrate, using the thermal release tape technique (TRT).

[0120]

[0121] Example 1-2 Formation of nucleic acid layer

[0122] The graphene layer formed in Example 1-1 above was hydrophilized by coating a nucleic acid dissolved in media onto the graphene layer to form a nucleic acid layer.

[0123] DMEM (Dulbecco's Modified Eagle Medium) was used for the media; more specifically, the media was prepared by adding 10 mM HCl to an F12 / DMEM (Giboco, Thermo Fisher Scientific) solution (without phenol red) to achieve a pH of 1.5. Nucleic acid was added to gel the media. The nucleic acid was prepared by dissolving 0.01 g of nucleic acid extracted from salmon in 1 ml of media to achieve a concentration of 1 mass%. As the nucleic acid dissolved, the liquid state changed into a gel state. The media gel containing the nucleic acid prepared in this way possesses abundant electrons, which can impart very high wettability to hydrophobic graphene.

[0124] The media gel containing the nucleic acid was coated onto the graphene layer. Subsequently, the substrate for spectroscopic analysis was prepared by drying at room temperature for more than one hour.

[0125]

[0126] Experimental Example

[0127] Experimental Example 1

[0128] A sample (300) containing amyloid fibrils dissolved in a media gel was loaded onto the graphene layer prepared in Example 1-1.

[0129] At this time, DMEM (Dulbecco's Modified Eagle Medium) was used as the media; more specifically, the media was prepared by adding 10 mM HCl to an F12 / DMEM (Giboco, Thermo Fisher Scientific) solution (without Phenol Red) to achieve a pH of 1.5. Nucleic acid was added to gel the media. The nucleic acid was prepared by dissolving 0.01 g of nucleic acid extracted from salmon in 1 ml of media to achieve a concentration of 1 mass%. As the nucleic acid dissolved, the liquid state changed into a gel state. The media gel containing the nucleic acid prepared in this way possesses abundant electrons, which can impart very high wettability to hydrophobic graphene.

[0130] 10 μM of Aβ42 peptide (Catalog num AS-20276, AnanSpec, San Jose, USA) was used for amyloid beta. The Aβ42 peptide was dissolved in the above-mentioned media gel. In addition, to form amyloid beta fibrils, the mixture was incubated at 37°C for at least 24 hours. The stock solution was diluted so that the amyloid beta concentration was 10 μM. 10 to 40 μL of the media gel containing dissolved amyloid beta fibrils was loaded onto a graphene layer.

[0131]

[0132] Additionally, for comparison, a media gel sample (100) containing nucleic acid, a media sample in solution state (200) not containing nucleic acid, and a sample (300) containing amyloid fibrils dissolved in the media gel were loaded together on a wafer, which is a base substrate according to the present invention, by forming a graphene layer on the graphene layer, and the results are shown in FIG. 2.

[0133] As shown in FIG. 2, the media sample (200) in solution state is shown to have a high contact angle on the graphene layer, which is a hydrophobic surface, and maintains a round shape, so the media sample (200) in solution state is not sufficiently adsorbed onto the graphene surface.

[0134] In contrast, the media gel sample (100) containing nucleic acid was found to have high wettability and was uniformly spread and adsorbed on the graphene layer.

[0135] In addition, it was confirmed that the sample (300) containing amyloid fibrils dissolved in the media gel also spread evenly and well on the graphene layer, improving the adsorption capacity.

[0136] Using a gel-state solvent containing the above nucleic acid improves hydrophilicity, thereby improving the adsorption capacity of the liquid sample onto the hydrophobic graphene layer, which can facilitate subsequent Raman spectroscopy measurements.

[0137]

[0138] Experimental Example 2

[0139] A sample (Graphene+Gel(fibril)(Example)) containing amyloid beta fibril (10 μM) dissolved in a media gel was loaded onto a spectroscopic analysis substrate prepared in Examples 1-2 and then dried.

[0140] In addition, as a comparative example, a media gel sample (Graphene (comparative example)) was loaded onto a spectroscopic analysis substrate having only a graphene layer formed thereon, and the results are shown in FIGS. 3 and 4.

[0141] Raman spectroscopic analysis was performed by measuring the graphene Raman spectrum under the following conditions.

[0142] - Excitation laser wavelength: 532 nm

[0143] - Laser power: 0.5 ~ 10 mW

[0144] - Laser exposure time: 1 ~ 15 s

[0145] - Laser spot size: 30 ~ 150 μm

[0146]

[0147] In FIG. 3(a), the left portion (the portion with circle I) represents the dried portion after loading a sample (Graphene+Gel(fibril) (Example)) containing amyloid beta fibrils (10 μM) dissolved in a media gel onto the spectroscopic substrate prepared in Example 1-2. In FIG. 3(a), the right portion (the portion with circle II) represents the dried portion after loading a media gel sample (Graphene (Comparative Example)) onto a spectroscopic substrate formed only with a graphene layer. It represents the dried portion after loading a sample (Graphene+Gel(fibril) (Example)) containing amyloid beta fibrils (10 μM) dissolved in a media gel onto the spectroscopic substrate prepared in Example 1-2. Figure 3(b) is a graph showing the results of the Raman analysis for circle I, and Figure 3(c) is a graph showing the results of the Raman analysis for circle II.

[0148] As shown in FIG. 3, it can be confirmed that the Raman signal intensity is improved in the Raman analysis results using the spectroscopic analysis substrate of the present invention (Fig. 3(b) - Example) compared to the Raman analysis results using a spectroscopic analysis substrate formed only with a graphene layer (Fig. 3(c) - Comparative Example). In addition, regarding the example, the G peak at 1586 cm⁻¹ in FIG. 3(b) -1 It can be confirmed that the signal strength of the part is detected more uniformly than in Fig. 3 (c) regarding the comparative example.

[0149] In addition, referring to FIG. 4, when comparing the positional distribution of the G peak based on 600 Raman signal detection data, the Raman analysis result using the spectroscopic analysis substrate of the present invention (Fig. 3(b) - Example) shows that the G peak is at 1586 to 1588 cm⁻¹ compared to the Raman analysis result using a spectroscopic analysis substrate formed only with a graphene layer (Fig. 3(c) - Comparative Example). -1 From 1581 to 1583 cm -1 You can clearly see that it has been downshifted.

[0150] Referring to FIGS. 3 and 4, only when a sample (Example) containing amyloid beta fibrils dissolved in a media gel is loaded onto a spectroscopic analysis substrate of the present invention and Raman spectroscopic analysis is performed, the Raman G peak is approximately 3 to 7 cm⁻¹ on average. -1 It was confirmed that shifting was taking place.

[0151]

[0152] Experimental Example 3

[0153] The following experiment was conducted to analyze the effects of changes in the type of base substrate and the concentration of amyloid beta fibrils on graphene G-peak shifting.

[0154] Except for using silica or glass as the base substrate, samples containing amyloid beta fibrils at various concentrations of 1 pM to 1 μM dissolved in media gel (Graphene+Gel(fibril)(Example)) were loaded onto a spectroscopic analysis substrate prepared as described in Examples 1-2 and then dried.

[0155] Raman mapping was performed by measuring the Raman spectrum of a sample on a substrate for spectroscopic analysis of the present invention under the following conditions.

[0156] - Scan area: Graphene 5x5 mm area

[0157] - Resolution: 100 μm interval

[0158] - Laser power: 1.3 mW

[0159] - Laser exposure time: 1 s

[0160] - Grating value (groove density): 1200 grooves / mm

[0161] - Raman signal strength: 1000x amplification

[0162] - Sample count: 100

[0163] FIG. 5a is a graph showing the result of downshifting the G peak based on the results of Raman spectroscopic analysis at different concentrations of amyloid beta fibrils using a substrate for spectroscopic analysis comprising a hydrophilized graphene layer whose base substrate is silica (SiO2) according to one embodiment of the present invention. In addition, FIG. 5b is a graph showing the result of downshifting the G peak based on the results of Raman spectroscopic analysis at different concentrations of amyloid beta fibrils using a substrate for spectroscopic analysis comprising a hydrophilized graphene layer whose base substrate is glass according to one embodiment of the present invention.

[0164] As shown in Fig. 5a, when silica is used as the base substrate, the Raman G peak averages approximately 2.5–4 cm depending on the concentration of amyloid beta fibrils dissolved in the media gel on the spectroscopic analysis substrate of the present invention. -1 , especially even at extremely low concentrations of 1 pM, the Raman G peak averages about 2.5 cm -1 It was confirmed that shifting occurs. Therefore, using the substrate for spectroscopic analysis of this institution, it may be possible to diagnose the early stages of dementia.

[0165] As shown in Fig. 5b, when glass is used as the base substrate, the Raman G peak averages about 2–3.5 cm depending on the concentration of amyloid beta fibrils dissolved in the media gel on the spectroscopic analysis substrate of the present invention. -1 , particularly at a concentration of 100 pM, the Raman G peak averages about 2 cm -1 It was confirmed that shifting occurs, and within a certain range, downshifting occurs in a concentration-dependent manner, which may enable quantitative analysis.

[0166] Therefore, when silica is used as the base substrate, even low concentrations of amyloid beta fibrils can be detected, and when glass is used as the base substrate, the progression of dementia can be monitored through quantitative analysis of amyloid beta fibrils.

[0167]

[0168] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0169]

[0170] [Explanation of the symbol]

[0171] 10: Base board

[0172] 20: Graphene layer

[0173] 30: Nucleic acid layer

Claims

1. Base board; A graphene layer formed on the base substrate; and A substrate for spectroscopic analysis for dementia diagnosis comprising: a nucleic acid layer formed on the graphene layer.

2. In Paragraph 1, The above nucleic acid layer is a substrate for spectroscopic analysis for dementia diagnosis that hydrophilizes the graphene layer.

3. In Paragraph 1, A substrate for spectroscopic analysis for dementia diagnosis that improves the binding strength and / or selectivity of a graphene layer to amyloid beta fibrils.

4. Light source; A substrate for spectroscopic analysis for dementia diagnosis described in claim 1; and A spectroscopic device comprising a detector for detecting spectra.

5. In Paragraph 4, A spectroscopic device capable of diagnosing dementia using biological samples including blood, serum, saliva, or urine.

6. In Paragraph 4, Spectroscopic device that diagnoses dementia by analyzing Raman shift fluctuations.

7. In Paragraph 4, A spectroscopic device in which the target substance of the above detector is amyloid beta fibrils in a biological sample.

8. In Paragraph 4, A spectroscopic device in which biological samples are provided in a gel state. 9.a) A graphene layer formation step of forming a graphene layer on a base substrate; and b) a nucleic acid layer formation step of forming a nucleic acid layer by coating a nucleic acid on the graphene layer; comprising a method for manufacturing a substrate for spectroscopic analysis. 10.1-1) A step of adsorbing a biological sample onto a substrate for spectroscopic analysis described in claim 1; and 1-2) A dementia analysis step of analyzing whether there is a change in Raman shift by comparing the Raman shift of a biological sample adsorbed on the substrate for spectroscopic analysis with the Raman shift of a control group using a Raman spectroscopic device; comprising a Raman spectroscopic analysis method. 11.2-1) A step of adsorbing a biological sample and a solution containing nucleic acids onto a spectroscopic analysis substrate having a graphene layer formed on a base substrate; and 2-2) A dementia analysis step of analyzing whether there is a change by comparing the Raman shift of a biological sample adsorbed on the substrate for spectroscopic analysis with the Raman shift of a control group using a Raman spectroscopic device; comprising a Raman spectroscopic analysis method.

Images