Surface enhanced raman spectroscopy substrate, and method and system for detecting biomarker using surface enhanced raman spectroscopy and cyclic voltammetry

Abstract

Disclosed is a surface-enhanced Raman spectroscopy substrate that includes a multi-stage nanograting structure comprising a first nanograting including silver (Ag), a second nanograting including gold (Au), and a third nanograting including platinum (Pt). The surface enhanced Raman spectroscopy substrate may be used to detect a plurality of target materials.

Description

Surface-enhanced Raman spectroscopy substrate, method for detecting biomarkers using surface-enhanced Raman spectroscopy and cyclic voltammetry, and detection system

[0001] The present invention relates to a detection system using surface-enhanced Raman spectroscopy. More specifically, the present invention relates to a surface-enhanced Raman spectroscopy substrate, a method for detecting a biomarker using surface-enhanced Raman spectroscopy and cyclic voltammetry, and a detection system.

[0002] Biomarkers are molecular data composed of DNA, RNA, metabolites, and proteins. They are biological indicators that can detect changes in the body caused by diseases or other conditions. Biomarkers can be utilized for disease diagnosis and prognosis, personalized medical technologies, and the development of intelligent healthcare systems that create patient-friendly treatment environments. For example, biomarkers currently being clinically used for cancer diagnosis include Prostate-Specific Antigen (PSA), Carcinoembryonic Antigen (CEA), and Alphafetoprotein (AFP).

[0003] However, diagnosis using a single biomarker can have low accuracy. For example, a meta-analysis found that PSA has a sensitivity of approximately 70% and a specificity of 58% for prostate cancer. Furthermore, a single biomarker is frequently associated with multiple cancer types. Therefore, diagnostics based on multi-biomarker panels, which utilize multiple biomarkers simultaneously, are currently being studied.

[0004] Multiplexing biosensors currently under development utilize a horizontal array-type sensor structure, measuring and quantifying multiple biomarkers individually. This increases the number of clinical samples required, measurement time, and costs. Accurate quantification of individual components is difficult due to false positives caused by non-specific binding of antibodies, aptamers, and other biomarker components used in the selective binding process, as well as cross-interference effects.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] 1) Republic of Korea Patent Publication No. 2021-0137215

[0008] 2) Republic of Korea Patent Publication No. 2014-0140179

[0009] 3) Republic of Korea Patent No. 1589039

[0010] One object of the present invention is to provide a surface-enhanced Raman spectroscopy substrate usable for multiple detection.

[0011] Another object of the present invention is to provide a detection method capable of improving the accuracy of detection and quantification of multiple target substances.

[0012] Another object of the present invention is to provide a detection system capable of improving the accuracy of detection and quantification of multiple target substances.

[0013] However, the problem to be solved by the present invention is not limited to the problem mentioned above, and may be expanded in various ways without departing from the spirit and scope of the present invention.

[0014] According to exemplary embodiments of the present invention for achieving the above-described object of the present invention, a surface-enhanced Raman spectroscopy substrate includes a multi-stage nano-lattice structure including a first nano-lattice including Ag (silver), a second nano-lattice including Au (gold), and a third nano-lattice including Pt (platinum).

[0015] In one embodiment, at least one of the first nanolattice, the second nanolattice, and the third nanolattice comprises a first nanowire array comprising a plurality of nanowires extending in a first direction and a second nanowire array comprising a plurality of nanowires extending in a second direction intersecting the first direction, wherein the first nanowire array and the second nanowire array are welded at a contact area.

[0016] In one embodiment, the second nanolattice is disposed on the first nanolattice, and the third nanolattice is disposed on the second nanolattice.

[0017] In one embodiment, the second nanolattice has a period greater than the first nanolattice, and the third nanolattice has a period greater than the second nanolattice.

[0018] According to one embodiment, the first to third nanolattices each include a plurality of nanowires, and the nanowires of the first nanolattice and the nanowires of the second nanolattice are exposed through the openings of the third nanolattice.

[0019] In one embodiment, the first to third nanolattices have different periods.

[0020] In one embodiment, each of the first to third nanolattices has an antibody immobilized on its surface.

[0021] In one embodiment, the antibodies immobilized on the first to third nanolattices are different from each other.

[0022] In one embodiment, the third nanolattice is heat treated at 500°C to 850°C.

[0023] A method for detecting a biomarker according to one embodiment of the present invention includes the steps of contacting a sample containing a biomarker with a surface-enhanced Raman spectroscopy substrate including a multi-stage nanolattice structure, identifying the biomarker bound to the surface-enhanced Raman spectroscopy substrate through Raman spectroscopy analysis, and quantifying the biomarker through cyclic voltammetry of the multi-stage nanolattice structure.

[0024] According to one embodiment, the method for detecting the biomarker comprises, prior to performing the Raman spectroscopy analysis, a step of binding an aptamer coupled to a Raman label to the biomarker bound to the surface-enhanced Raman spectroscopy substrate.

[0025] In one embodiment, the multi-stage nanolattice structure comprises at least three nanolattices comprising different materials, wherein the aptamer comprises a first aptamer coupled to rhodamine 6G (R6G), a second aptamer coupled to methylene blue (MB), and a third aptamer coupled to malachite green (MG).

[0026] In one embodiment, the concentration of the third aptamer is greater than the concentration of the first aptamer and the concentration of the second aptamer.

[0027] In one embodiment, the malachite green is bound to both the head and the tail of the third aptamer, and the rhodamine 6G and the methylene blue are bound to one of the head or the tail of the first aptamer and the second aptamer.

[0028] According to one embodiment, the multi-stage nanolattice structure includes a plurality of nanolattices containing different materials, and the step of quantifying the biomarker includes the step of measuring a cyclic voltammetry of the multi-stage nanolattice structure and the step of calculating the amount of the biomarker based on a change in a reduction voltage peak corresponding to the material of each nanolattice from a result obtained by measuring the cyclic voltammetry.

[0029] A biomarker detection system according to one embodiment of the present invention includes a surface-enhanced Raman spectroscopy substrate including a multi-stage nanolattice structure capable of being combined with a plurality of biomarkers in a sample, a light source for irradiating light onto the surface-enhanced Raman spectroscopy substrate combined with the biomarker, an identification unit for analyzing Raman spectra of light reflected from the surface-enhanced Raman spectroscopy substrate to identify the biomarker, and a quantification unit for quantifying the biomarker through cyclic voltammetry of the surface-enhanced Raman spectroscopy substrate on which the biomarker is captured.

[0030] According to the exemplary embodiments of the present invention described above, a target material can be rapidly detected using surface-enhanced Raman spectroscopy.

[0031] Additionally, multiple target substances can be detected simultaneously using multi-stage nanolattice structures that can selectively bind to different substances.

[0032] In addition, since each nanolattice within the multi-stage nanolattice structure has a different period, the surface enhancement effect of the lower nanolattice can be prevented from being reduced.

[0033] Additionally, the surface enhancement effect can be increased through heat treatment of Pt, which is known to have a low surface enhancement effect.

[0034] Additionally, identification and quantification of target materials are possible through a combination of surface-enhanced Raman spectroscopy and cyclic voltammetry.

[0035] In addition, by using label-conjugated aptamers, multiple biomarkers that are difficult to identify and distinguish, such as proteins, can be detected.

[0036] In addition, the accuracy of multiple detection can be improved by combining labels suitable for multiple detection, and the accuracy of multiple detection can be improved by varying the number of labels bound to the aptamer.

[0037] FIG. 1 is a cross-sectional view schematically illustrating a process of forming a nanowire array in a method for manufacturing a surface-enhanced Raman spectroscopy substrate according to one embodiment of the present invention.

[0038] FIG. 2 is a cross-sectional view schematically illustrating a process of transferring a nanowire array in a method for manufacturing a surface-enhanced Raman spectroscopy substrate according to one embodiment of the present invention.

[0039] FIG. 3 is a plan view illustrating a single layer of nano-grating in a method for manufacturing a surface-enhanced Raman spectroscopy substrate according to one embodiment of the present invention.

[0040] FIG. 4 is an exploded perspective view illustrating a surface-enhanced Raman spectroscopy substrate according to one embodiment of the present invention.

[0041] FIG. 5 is a schematic diagram illustrating a step of fixing an antibody to a surface-enhanced Raman spectroscopy substrate according to one embodiment of the present invention.

[0042] Figure 6 is a schematic diagram schematically illustrating a biomarker detection system according to one embodiment of the present invention.

[0043] Figure 7 is a schematic diagram illustrating a method for detecting a biomarker according to one embodiment of the present invention.

[0044] Figure 8 shows the results after dropping R6G onto the third nano-lattice of Example 1 (10 -6 M) This is a graph showing the obtained Raman spectrum.

[0045] Figure 9 is a scanning electron microscope (SEM) image (a) after transferring the second nanolattice onto the first nanolattice in Example 1, and an SEM image (b) after transferring the third nanolattice onto the second nanolattice.

[0046] Figure 10 is a graph showing the cyclic voltammetry of the multi-stage nanolattice structure of Example 1.

[0047] Hereinafter, with reference to the attached drawings, a surface-enhanced Raman spectroscopy substrate, a detection method using surface-enhanced Raman spectroscopy and cyclic voltammetry, and a detection system according to embodiments of the present invention will be described in detail. The present invention may have various modifications and may take various forms, and thus specific embodiments will be illustrated and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In the attached drawings, the dimensions of structures may be illustrated larger than actual dimensions to ensure clarity of the present invention.

[0048] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof.

[0049] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0050] Surface-enhanced Raman spectroscopy substrate and manufacturing method thereof

[0051] A surface-enhanced Raman spectroscopy substrate according to one embodiment of the present invention can be manufactured using thermally assisted nanotransfer printing (T-nTP).

[0052] FIG. 1 is a cross-sectional view schematically illustrating a process of forming a nanowire array in a method for manufacturing a surface-enhanced Raman spectroscopy substrate according to an embodiment of the present invention. FIG. 2 is a cross-sectional view schematically illustrating a process of transferring a nanowire array in a method for manufacturing a surface-enhanced Raman spectroscopy substrate according to an embodiment of the present invention. FIG. 3 is a plan view illustrating a single layer of nano-lattices in a method for manufacturing a surface-enhanced Raman spectroscopy substrate according to an embodiment of the present invention.

[0053] Referring to (a) of Fig. 1, a replica mold (12) is formed by coating a polymer on a master mold (10) having a pattern formed on the surface. For example, a polymer composition including the polymer and a solvent may be coated on the uneven surface (surface on which the pattern is formed) of the master mold (10) using spin coating, deep coating, or spray coating, and the solvent may be removed (dried) to form a replica mold (12). In a cross-sectional view, the uneven surface of the master mold (10) may have a shape in which protrusions and depressions are repeated in a horizontal direction. In addition, the horizontal lengths of the protrusions and the depressions may be the same or different, and may be variously adjusted depending on the shape and period of the pattern to be transferred.

[0054] For example, the replica mold (12) may include, but is not limited to, polymethyl methacrylate (PMMA), polystyrene, polyvinyl pyridine (poly-4-viyl pyridine), or a combination thereof, and may be formed of various polymeric materials. In addition, the replica mold (12) may have a single-layer structure or a multi-layer structure including different materials.

[0055] In addition, so that the replica mold (12) can be easily separated from the master mold (10) in a later step, the patterned surface of the master mold (10) may be coated in advance with a hydrophobic SAM (self-assembled monolayer) such as a PDMS brush polymer or HMDS (hexamethylene di silazane) having low surface energy.

[0056] Referring to (b) of Fig. 1, the replica mold (12) is separated from the master mold (10). According to one embodiment, an adhesive film (20) is attached to the upper surface of the replica mold (12) (the opposite surface of the surface on which the pattern of the replica mold is transferred) and an external force is applied, thereby physically separating the replica mold (12) and the master mold (10). An adhesive may be coated on the adhesive surface of the adhesive film (20).

[0057] Since the above-mentioned copy mold (12) is formed to fill the unevenness of the surface pattern of the master mold (10), it can have an unevenness pattern corresponding to the reverse of the surface pattern of the master mold (10).

[0058] Referring to (c) and (d) of FIG. 2, a functional material may be deposited on a portion of the replica mold (12) to form a nanowire array (30). According to one embodiment, when E-beam lithography, thermal evaporation, sputtering, etc. are performed while the replica mold is tilted so that the surface on which the deposition of the replica mold (12) is performed and the deposition direction have a certain angle, the functional material is deposited only on a portion of the surface pattern of the replica mold (12) centered on the protruding portion (edge). Accordingly, the pattern of the functional material can be obtained without a photolithography process. For example, when the surface pattern of the replica mold (12) has a wire array shape (when the protruding portion or the recessed portion has a shape extending in one direction), the nanowire array (30) can be formed through the inclined deposition.

[0059] The functional material may include a metal such as Au, Ag, Cu, Ni, Pt, Cr, Co or Pd, but embodiments of the present invention are not limited thereto, and various materials may be used as the functional material as needed.

[0060] Next, the above nanowire array (30) is transferred to a transfer substrate.

[0061] Referring to (a) and (b) of FIG. 2, the nanowire array (30) is brought into contact with the surface of the transfer substrate (40), and the adhesive film (20) is heated while being pressed using a pressing member (42). Accordingly, the bonding force between the adhesive film (20) and the replication mold (12) is weakened by the heat, so that the adhesive film (20) can be easily removed from the replication mold (12). The heating temperature may be 100°C or higher, for example, 100°C to 300°C, but embodiments of the present invention are not limited thereto.

[0062] The replica mold (12) remaining on the nanowire array (30) can be removed using a solvent. For example, in order to remove the replica mold (12), an organic solvent such as toluene, acetone, or isopropyl alcohol can be applied to the replica mold (12), thereby removing the replica mold (12).

[0063] As a result, as illustrated in (c) of FIG. 2, a nanowire array (30) can be transferred onto a transfer substrate (40). For example, the line width of the nanowire array (30) may be 10 nm to 100 nm, and the pitch may be 20 nm to 500 nm, but is not limited thereto.

[0064] In one embodiment, the above process can be repeated to form a nano-lattice. For example, referring to FIG. 3, a first nano-wire array (32) including a plurality of nano-wires extending in a first direction (D1) is formed on a transfer substrate. The process illustrated in FIG. 1 is repeated to form a nano-wire array on a replication mold, and heat-assisted transfer is performed on the transfer substrate (40) onto which the first nano-wire array (32) has been transferred, in the manner illustrated in FIG. 2, such that the first nano-wire array (32) and the additional nano-wire array intersect, thereby forming a nano-lattice including a second nano-wire array (34). For example, the second nano-wire array (34) can include a plurality of nano-wires extending in a second direction (D2) perpendicular to the first direction (D1).

[0065] A nanolattice opening can be defined between adjacent nanowires of the first nanowire array (32) and adjacent nanowires of the second nanowire array (34).

[0066] Since heat is provided in the process of combining the first nanowire array (32) and the second nanowire array (34), the contact area (JT, intersection area) of the first nanowire array (32) and the second nanowire array (34) is welded. Accordingly, the stability of the nanolattice can be improved, and deformation or damage in subsequent processes can be prevented.

[0067] Fig. 4 is an exploded perspective view illustrating a surface-enhanced Raman spectroscopy substrate according to one embodiment of the present invention. Fig. 5 is a schematic diagram illustrating a step of fixing an antibody to a surface-enhanced Raman spectroscopy substrate according to one embodiment of the present invention.

[0068] Referring to FIG. 4, the surface-enhanced Raman spectroscopy substrate (100) may include a multi-stage nanolattice structure.

[0069] For example, the multi-stage nano-lattice structure may include a first nano-lattice (110), a second nano-lattice (120) disposed on the first nano-lattice (110), and a third nano-lattice (130) disposed on the second nano-lattice (120). The multi-stage nano-lattice structure may be disposed on a base substrate (101). The base substrate (101) may be conductive or may include an electrode (conductive terminal) electrically connected to the multi-stage nano-lattice structure.

[0070] In one embodiment, the nanolattices of the multi-stage nanolattice structure may comprise different materials. For example, the first nanolattice (110) may comprise Ag, the second nanolattice (120) may comprise Au, and the third nanolattice (130) may comprise Pt.

[0071] In one embodiment, the third nanolattice (130) containing Pt can be heat-treated. Heat-treated Pt can have increased crystallinity, thereby improving its surface enhancement effect. Accordingly, the surface enhancement effect of Pt, which is low compared to Ag and Au, can be improved, and thus it can be utilized as a material for a surface-enhanced Raman spectroscopy substrate.

[0072] For example, the heat treatment temperature of Pt may be 500°C to 850°C, preferably 650°C to 800°C. If the heat treatment temperature is excessively high, crystallinity is reduced due to melting, and the surface enhancement effect is not increased.

[0073] In addition, the nanolattices may have different periodicities. According to one embodiment, the first nanolattice (110) may have a first period (hereinafter, the period may refer to the pitch of adjacent nanowires), the second nanolattice (120) may have a second period greater than the first period, and the third nanolattice (130) may have a third period greater than the second period. In addition, the size (area on a plan view) of the opening (unit opening) of the second nanolattice (120) may be larger than the opening of the first nanolattice (110), and the size of the opening of the third nanolattice (130) may be larger than the opening of the second nanolattice (120). According to one embodiment, the nanowires of the second nanolattice (120) and the nanowires of the first nanolattice (110) may be exposed through the opening of the third nanolattice (130). That is, on the plan view, the opening of the third nano-lattice (130) can overlap with the nano-wire of the second nano-lattice (120) and the nano-wire of the first nano-lattice (110).

[0074] In one embodiment, in a multi-stage nanolattice structure, since the aperture size of the upper nanolattice is larger than that of the lower nanolattice, the lower nanolattice can be easily exposed to the target material entering from above. Furthermore, the area variation of each nanolattice exposed to the sample can be reduced, thereby improving detection reliability.

[0075] In addition, considering the difference in surface enhancement effect of each nano-lattice material (Ag > Au > Pt), the nano-lattice formed with a material having a relatively large surface enhancement effect is placed at the bottom (the area where the surface enhancement effect is reduced by the upper layer), and the nano-lattice formed with a material having a relatively small surface enhancement effect is placed at the top, thereby preventing a decrease in detection reliability due to the difference in surface enhancement effect.

[0076] Each nano-lattice of the above multi-stage nano-lattice structure can be formed by the method described with reference to FIGS. 1 to 3. When the transfer substrate of the nano-lattice is removed using an etchant or the like and brought into contact with another nano-lattice, the nano-lattices are bonded to each other by van der Waals forces or the like without a separate adhesive, thereby forming the above multi-stage nano-lattice structure.

[0077] The above surface-enhanced Raman spectroscopy substrate can be used for the detection of biomarkers, and specifically, can be effectively used for multiple detection of biomarkers. For multiple detection, each nanolattice can be designed to selectively bind to a different biomarker.

[0078] In one embodiment, the first nanolattice (110) may include a first antibody (114), the second nanolattice (120) may include a second antibody (124), and the third nanolattice (130) may include a third antibody (134). Each antibody may bind to a different target substance (biomarker) through antigen-antibody binding. In addition, after the antibody captures the target substance, the target substance may be bound to an aptamer linked to a different Raman label depending on the type of the target substance.

[0079] In one embodiment, a linker such as dithiobis-N-succinimidyl propionate (DTSP) may be used to bind an antibody to the nanolattice.

[0080] For example, as illustrated in FIG. 5, DTSP can be provided to a nanolattice (110), a linker (112) can be formed on the surface of the nanolattice, and then an antibody (114) can be bound to the linker.

[0081] The step of immobilizing antibodies onto each nanolattice can be performed after forming each nanolattice, and then the nanolattices can be combined to form a multi-stage nanolattice structure. Additionally, the heat treatment of the Pt nanolattice can be performed after forming the nanolattice containing Pt, and then the antibodies can be immobilized onto the Pt nanolattice.

[0082] In addition, according to embodiments of the present invention, since each nanolattice is made of a different material, when measuring cyclic voltammetry, reduction voltage peaks of different values ​​can be observed for each nanolattice. The characteristics (current value, area, etc.) of the reduction voltage peak can vary depending on the amount of target materials bound to each nanolattice. Accordingly, after reacting the substrate with a sample, the target materials bound to each substrate or included in the sample can be quantified based on the change in the reduction voltage peak obtained from the cyclic voltammetry measurement (before / after reaction).

[0083] Method and system for detecting biomarkers

[0084] A method for detecting a biomarker according to one embodiment of the present invention may include a step of contacting a sample including a biomarker with a surface-enhanced Raman spectroscopy substrate including a multi-stage nanolattice structure, a step of identifying the biomarker bound to the surface-enhanced Raman spectroscopy substrate through Raman spectroscopy analysis, and a step of quantifying the biomarker through cyclic voltammetry of the multi-stage nanolattice structure.

[0085] Figure 6 is a schematic diagram schematically illustrating a biomarker detection system according to one embodiment of the present invention.

[0086] Referring to FIG. 6, a biomarker detection system according to one embodiment of the present invention may include a surface-enhanced Raman spectroscopy substrate (100) that can be combined with a biomarker in a sample (500), a light source (210) for irradiating light to the surface-enhanced Raman spectroscopy substrate combined with the biomarker, an identification unit (220) for analyzing Raman spectroscopy of light reflected from the surface-enhanced Raman spectroscopy substrate to identify the biomarker, and a quantification unit (300) for quantifying the biomarker through cyclic voltammetry of the surface-enhanced Raman spectroscopy substrate on which the biomarker is captured. In addition, the system may further include a stage (400) for supporting the surface-enhanced Raman spectroscopy substrate (100).

[0087] The above surface-enhanced Raman spectroscopy substrate may be the same as that described above.

[0088] Figure 7 is a schematic diagram illustrating a method for detecting a biomarker according to one embodiment of the present invention.

[0089] Referring to Fig. 7, a sample is brought into contact with a nanolattice (110) of a multi-stage nanolattice structure having an antibody bound to the surface, thereby binding a biomarker (target) in the sample to the antibody.

[0090] For example, an appropriate amount of sample can be dropped onto the surface-enhanced Raman spectroscopy substrate, and contact can be maintained for a period of time sufficient for the biomarker within the sample to sufficiently bind to the antibody. In addition, the substrate can be moved or rotated so that the antibody and the biomarker can easily bind.

[0091] The biomarker may be derived from a biological organism. For example, the sample may include a biological fluid. The biological fluid may include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, etc. In addition, the sample may include biological tissues, cells, or intracellular materials of humans, animals, plants, bacteria, fungi, and viruses. In addition, the sample may include an environmental sample, such as drinking water or food.

[0092] In one embodiment, after the antibody and the biomarker are combined, an aptamer coupled with a Raman label is combined with the biomarker. If the biomarker is a protein, peak detection through Raman spectroscopy may not be easy. According to one embodiment of the present invention, by combining different Raman labels depending on the type of biomarker and identifying the peaks corresponding to the corresponding Raman labels, the reliability of multiple detection can be improved.

[0093] When there are multiple biomarkers to be detected, different aptamers that can selectively bind to each biomarker are used, and each aptamer can be conjugated to a different Raman label.

[0094] For example, Table 1 below presents biomarkers used in the diagnosis of lung cancer. Studies have shown that a combination of Carcinoembryonic Antigen (CEA), CYFRA 21-1, and neuron-specific enolase (NSE) among the biomarkers listed below has high diagnostic accuracy (75% sensitivity and 82% specificity).

[0095] Table 1

[0096]

[0097] The aptamer structure corresponding to (bindable to) the above biomarker is known and is as follows.

[0098]

[0099] For example, when simultaneously detecting the three biomarkers from a sample (such as blood of a test subject), a first aptamer that can selectively bind to CEA, a second aptamer that can selectively bind to CYFRA 21-1, and a third aptamer that can selectively bind to NSE can be provided on the substrate to bind to the corresponding biomarkers.

[0100] The first to third aptamers may be conjugated to different labels. For example, the labels may be conjugated to the heads and / or tails of the aptamers.

[0101] Since the above labels are for distinguishing the above biomarkers, it is desirable that the Raman peaks by the labels are easily distinguishable from each other, and in order to confirm each Raman peak, it is desirable that the Raman cross-sections are similar.

[0102] In one embodiment, the first to third aptamers may each comprise a combination of multiple labels (dyes) to which they bind, such as rhodamine 6G (R6G), methylene blue (MB), and malachite green (MG). The label-aptamer combinations need not be specific, but the same labels must bind to the same aptamers. For example, the first aptamer may bind to R6G, the second aptamer may bind to MB, and the third aptamer may bind to MG.

[0103] In one embodiment, the first nanolattice comprising Ag can be coupled with a CYFRA 21-1 specific antibody, and an aptamer coupled with MB can be used to label CYFRA 21-1, the second nanolattice comprising Au can be coupled with an NSE specific antibody, and an aptamer coupled with R6G can be used to label NSE, and the third nanolattice comprising Pt can be coupled with a CEA specific antibody, and an aptamer coupled with MG can be used to label CEA.

[0104] To increase the accuracy of Raman spectroscopy, it is desirable that the difference in intensity of the Raman peaks of each marker be small. Therefore, the following methods may be considered to ensure that the intensity of the Raman peaks of each marker remains within a certain range.

[0105] In one embodiment, when providing aptamers to the substrate, the concentration of each aptamer may be varied. For example, if the Raman peak of MG is weak, the concentration of the third aptamer bound to MG may be increased compared to the concentrations of the first and second aptamers.

[0106] In another embodiment, the number of labels conjugated to the aptamers can be varied. For example, if the Raman peak of MG is weak, MG can be conjugated to both the head and tail of the third aptamer, and the first and second aptamers can have labels conjugated to either the head or the tail.

[0107] The above quantification unit (300) measures the cyclic voltammetry of a surface-enhanced Raman spectroscopy substrate combined with a biomarker and quantifies the biomarker based on this.

[0108] According to embodiments of the present invention, the surface-enhanced Raman spectroscopy substrate includes a plurality of nanolattices comprising different materials. Therefore, when the cyclic voltammetry of the surface-enhanced Raman spectroscopy substrate is measured, a reduction voltage peak having a different value may be observed by each nanolattice. The characteristics (current value, area, etc.) of the reduction voltage peak may vary depending on the amount of biomarkers bound to each nanolattice. Accordingly, after reacting the substrate with a sample to bind the substrate and the biomarkers, the amount of the biomarkers may be calculated based on the change in the reduction voltage peak (before / after reaction) obtained from the cyclic voltammetry measurement. Accordingly, the biomarkers bound to the substrate or the biomarkers contained in the sample may be distinguished and quantified. In addition, various known calibrations may be utilized for quantification based on the cyclic voltammetry measurement.

[0109] According to embodiments of the present invention, a target material can be rapidly detected using surface-enhanced Raman spectroscopy.

[0110] Additionally, multiple target substances can be detected simultaneously using multi-stage nanolattice structures that can selectively bind to different substances.

[0111] In addition, since each nanolattice within the multi-stage nanolattice structure has a different period, the surface enhancement effect of the lower nanolattice can be prevented from being reduced.

[0112] Additionally, the surface enhancement effect can be increased through heat treatment of Pt, which is known to have a low surface enhancement effect.

[0113] Additionally, identification and quantification of target materials are possible through a combination of surface-enhanced Raman spectroscopy and cyclic voltammetry.

[0114] In addition, by using label-conjugated aptamers, multiple biomarkers that are difficult to identify and distinguish, such as proteins, can be detected.

[0115] In addition, the accuracy of multiple detection can be improved by combining labels suitable for multiple detection, and the accuracy of multiple detection can be improved by varying the number of labels bound to the aptamer.

[0116] Below, the effects of the present invention will be examined through specific examples and experiments.

[0117] Example 1

[0118] Fabrication of the first nanolattice

[0119] PMMA was coated on the uneven surface of a master mold with a period of 80 nm (protrusion width: 40 nm, depression width: 40 nm), and then dried to form a replica mold. An adhesive film was attached to the upper surface of the replica mold, and then the replica mold was separated from the master mold.

[0120] The first Ag nanowire array was formed by depositing the replica mold while tilting it. The first Ag nanowire array was placed on a SiO2 wafer substrate, heated (150°C) and pressurized, and then the adhesive film was removed. After the adhesive film was removed, isopropyl alcohol was added to remove the replica mold, and the first Ag nanowire array was transferred to the SiO2 wafer substrate.

[0121] After forming a second Ag nanowire array in the same manner as the first Ag nanowire array, it was brought into contact with the first Ag nanowire array so as to intersect it on a plan view. Next, in the same manner as above, heating and pressing were performed, the adhesive film was removed, and the replication mold was removed to prepare a first nanolattice including the first Ag nanowire array and the second Ag nanowire array welded to each other at the contact area.

[0122] In order to fix the antibody to the first nanolattice, a dimethyl sulfoxide (DMSO) solution (NaBH475 mM) containing DTSP (3 mg / mL) was incubated for 30 minutes, and the first nanolattice was immersed in the solution for 30 minutes and washed with DMSO and deionized water.

[0123] Next, the first nanolattice was immersed in a PBS solution (1-2 g / ml) containing a CYFRA 21-1 specific antibody (monoclonal antibody) for 4 hours, then immersed in a PBS solution containing 10% ethanolamine for 4 hours, and washed with 0.1 M Tris-HCl (pH 8.0).

[0124] Fabrication of the second nanolattice

[0125] PMMA was coated on the uneven surface of a master mold with a period of 100 nm (protrusion width: 50 nm, depression width: 50 nm), and then dried to form a replica mold. An adhesive film was attached to the upper surface of the replica mold, and then the replica mold was separated from the master mold.

[0126] The first Au nanowire array was formed by depositing the replica mold while tilting it. The first Au nanowire array was placed on a SiO2 wafer substrate, heated (150°C) and pressurized, and then the adhesive film was removed. After the adhesive film was removed, isopropyl alcohol was added to remove the replica mold, and the first Au nanowire array was transferred to the SiO2 wafer substrate.

[0127] After forming a second Au nanowire array in the same manner as the first Au nanowire array, it was brought into contact with the first Au nanowire array so as to intersect it on a plan view. Next, in the same manner as above, a second nanolattice was prepared including the first Au nanowire array and the second Au nanowire array welded to each other at the contact area by heating and pressing, removing the adhesive film, and removing the replication mold.

[0128] In order to fix the antibody to the second nanolattice, a DMSO solution (NaBH475 mM) containing DTSP (3 mg / mL) was incubated for 30 minutes, and the second nanolattice was immersed in the solution for 30 minutes and washed with DMSO and deionized water.

[0129] Next, the second nanolattice was immersed in a PBS solution (1-2 g / ml) containing NSE-specific antibodies for 4 hours, then immersed in a PBS solution containing 10% ethanolamine for 4 hours, and washed with 0.1 M Tris-HCl (pH 8.0).

[0130] Fabrication of the third nanolattice

[0131] PMMA was coated on the uneven surface of a master mold with a period of 200 nm (protrusion width: 50 nm, depression width: 150 nm), and then dried to form a replica mold. An adhesive film was attached to the upper surface of the replica mold, and then the replica mold was separated from the master mold.

[0132] The first Pt nanowire array was formed by depositing the replica mold while tilting it. The first Pt nanowire array was placed on a SiO2 wafer substrate, heated (150°C) and pressurized, and then the adhesive film was removed. After the adhesive film was removed, isopropyl alcohol was added to remove the replica mold, and the first Pt nanowire array was transferred to the SiO2 wafer substrate.

[0133] After forming a second Pt nanowire array in the same manner as the first Pt nanowire array, it was brought into contact with the first Pt nanowire array so as to intersect it on a plan view. Next, in the same manner as above, a third nanolattice was prepared, including the first Pt nanowire array and the second Pt nanowire array welded to each other at the contact area by heating and pressing, removing the adhesive film, and removing the replication mold.

[0134] Next, the third nano-lattice was heat-treated by heating in an Ar atmosphere for more than 1 minute.

[0135] Next, in order to fix the antibody to the third nanolattice, a DMSO solution (NaBH475 mM) containing DTSP (3 mg / mL) was incubated for 30 minutes, and the third nanolattice was immersed in the solution for 30 minutes and washed with DMSO and deionized water.

[0136] Next, the third nanolattice was immersed in a PBS solution (1-2 g / ml) containing a CEA-specific antibody for 4 hours, then immersed in a PBS solution containing 10% ethanolamine for 4 hours, and washed with 0.1 M Tris-HCl (pH 8.0).

[0137] After the antibody fixation reaction of the first to third nanolattices, anti-IgG labeled with a fluorescent substance, FITC, was provided, and fluorescence was confirmed to confirm that the antibody was fixed to each nanolattice.

[0138] Fabrication of multi-stage nanolattice structures

[0139] After removing the SiO2 wafer substrate from the second nanolattice using an etchant (BOE), the separated second nanolattice was floated on purified water, and water transfer was performed on the first nanolattice.

[0140] After removing the SiO2 wafer substrate from the third nano-lattice using an etchant (BOE), the separated third nano-lattice was floated on purified water, and water transfer was performed on the second nano-lattice placed on the first nano-lattice.

[0141] Figure 8 shows the results after dropping R6G onto the third nano-lattice of Example 1 (10 -6 M) This is a graph showing the obtained Raman spectrum.

[0142] Referring to Fig. 8, it was confirmed that the surface enhancement effect increased when the heat treatment temperature was 500°C or higher, and that the surface enhancement effect increased the most at 700°C, but it was confirmed that the surface enhancement effect decreased again at 900°C.

[0143] Figure 9 is a scanning electron microscope (SEM) image (a) after transferring the second nanolattice onto the first nanolattice in Example 1, and an SEM image (b) after transferring the third nanolattice onto the second nanolattice.

[0144] Referring to Figure 9, it was confirmed that the lower nanolattice can be exposed by making the period of each nanolattice different.

[0145] Figure 10 is a graph showing the cyclic voltammetry of the multi-stage nanolattice structure of Example 1.

[0146] Referring to Figure 10, it was confirmed that reduction voltage peaks corresponding to each nanolattice (Ag, Au, Pt) were observed separately. Therefore, it can be seen that multiple biomarkers detected in multiple stages can be quantified by measuring the cyclic voltammetry of the multi-stage nanolattice structure.

[0147] Although the present invention has been described with reference to exemplary embodiments thereof as described above, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

[0148] Exemplary embodiments of the present invention can be used to detect a variety of substances detectable by surface-enhanced Raman spectroscopy, including biomarkers.

Claims

A surface-enhanced Raman spectroscopy substrate comprising a multi-stage nano-grating structure comprising a first nano-grating comprising 1.Ag, a second nano-grating comprising Au, and a third nano-grating comprising Pt.

2. In the first paragraph, at least one of the first nano-lattice, the second nano-lattice and the third nano-lattice, A first nanowire array comprising a plurality of nanowires extending in a first direction; and A second nanowire array comprising a plurality of nanowires extending in a second direction intersecting the first direction, A surface-enhanced Raman spectroscopy substrate, characterized in that the first nanowire array and the second nanowire array are welded at a contact area.

3. A surface-enhanced Raman spectroscopy substrate, characterized in that the second nano-grating is arranged on the first nano-grating, and the third nano-grating is arranged on the second nano-grating.

4. A surface-enhanced Raman spectroscopy substrate, characterized in that in the third paragraph, the second nano-grating has a period greater than that of the first nano-grating, and the third nano-grating has a period greater than that of the second nano-grating.

5. A surface-enhanced Raman spectroscopy substrate according to claim 3, wherein the first to third nano-lattices each include a plurality of nano-wires, and the nano-wires of the first nano-lattice and the nano-wires of the second nano-lattice are exposed through the opening of the third nano-lattice.

6. A surface-enhanced Raman spectroscopy substrate, characterized in that the first to third nano-gratings in the first paragraph have different periods.

7. A surface-enhanced Raman spectroscopy substrate according to claim 1, wherein each of the first to third nanogrids has an antibody fixed to its surface.

8. A surface-enhanced Raman spectroscopy substrate according to claim 7, wherein the antibodies fixed to the first to third nanolattices are different from each other.

9. A surface-enhanced Raman spectroscopy substrate, characterized in that the third nano-lattice in the first paragraph is heat-treated at 500°C to 850°C.

10. A step of contacting a sample containing a biomarker with a surface-enhanced Raman spectroscopy substrate containing a multi-stage nanolattice structure; A step of identifying a biomarker bound to the surface-enhanced Raman spectroscopy substrate through Raman spectroscopy analysis; and A method for detecting a biomarker, comprising a step of quantifying the biomarker through cyclic voltammetry of the multi-stage nanolattice structure.

11. A method for detecting a biomarker, characterized in that in the 10th paragraph, the multi-stage nanolattice structure comprises a first nanolattice containing Ag (silver), a second nanolattice containing Au (gold), and a third nanolattice containing Pt (platinum).

12. A method for detecting a biomarker, characterized in that the first to third nanogrids in claim 11 each have different antibodies fixed to their surfaces.

13. A method for detecting a biomarker, characterized in that, in claim 10, before performing the Raman spectroscopy analysis, the method comprises a step of binding an aptamer bound to a Raman label to the biomarker bound to the surface-enhanced Raman spectroscopy substrate.

14. A method for detecting a biomarker, characterized in that in claim 13, the multi-stage nanolattice structure comprises at least three nanolattices containing different materials, and the aptamer comprises a first aptamer coupled with rhodamine 6G (R6G), a second aptamer coupled with methylene blue (MB), and a third aptamer coupled with malachite green (MG).

15. A method for detecting a biomarker, characterized in that in claim 14, the concentration of the third aptamer is greater than the concentration of the first aptamer and the concentration of the second aptamer.

16. A method for detecting a biomarker, characterized in that in paragraph 14, the malachite green is bound to both the head and the tail of the third aptamer, and the rhodamine 6G and the methylene blue are bound to one of the head or the tail of the first aptamer and the second aptamer.

17. A method for detecting a biomarker in claim 10, wherein the multi-stage nanolattice structure includes a plurality of nanolattices containing different materials, and the step of quantifying the biomarker includes a step of measuring the cyclic voltammetry of the multi-stage nanolattice structure and a step of calculating the amount of the biomarker based on a change in a reduction voltage peak corresponding to the material of each nanolattice from the result obtained by measuring the cyclic voltammetry.

18. A surface-enhanced Raman spectroscopy substrate comprising a multi-stage nanolattice structure capable of binding to multiple biomarkers in a sample; A light source for irradiating light onto a surface-enhanced Raman spectroscopy substrate combined with the above biomarker; An identification unit for identifying the biomarker by analyzing the Raman spectrum of light reflected from the surface-enhanced Raman spectroscopy substrate; and A biomarker detection system including a quantification unit that quantifies the biomarker through cyclic voltammetry of a surface-enhanced Raman spectroscopy substrate on which the biomarker is captured.

19. A biomarker detection system according to claim 18, wherein the multi-stage nanolattice structure comprises a first nanolattice containing Ag, a second nanolattice containing Au, and a third nanolattice containing Pt.

20. A biomarker detection system, characterized in that in claim 19, the second nanolattice is arranged on the first nanolattice, the third nanolattice is arranged on the second nanolattice, the second nanolattice has a period greater than that of the first nanolattice, and the third nanolattice has a period greater than that of the second nanolattice.

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