Method for manufacturing a target substance detection substrate based on surface enhanced Raman scattering, target substance detection substrate based thereon, and target substance detection method using the same

Abstract

The present invention relates to a method for manufacturing a target substance detection substrate based on surface enhanced Raman scattering, which utilizes centrifugal separation to facilitate coating of metal nanoparticles on the surface of a target substance, a target substance detection substrate based thereon, and a target substance detection method using the same.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing a substrate for detecting a target substance based on surface enhanced Raman scattering, a substrate for detecting a target substance based on the substrate, and a target substance detection method using the substrate. Background Art

[0002] Surface enhanced Raman scattering (SERS), as one of the most promising analytical methods, means a spectroscopic method that measures the Raman scattering of the analyzed substance when the substance to be analyzed is adsorbed on the surface of a Raman active material such as a roughened metal or is located within a distance of several hundred nanometers. The intensity of the Raman scattering is increased by more than 104 to 106 times compared to the general Raman intensity due to the surface plasmon provided by the roughness of the surface.

[0003] The wavelength of the Raman emission spectrum shows the chemical composition and structural characteristics of the light-absorbing molecules in the sample, so analyzing this Raman signal can directly analyze the object of analysis.

[0004] Furthermore, for the analysis of proteins, most of the existing research results on the detection of intrinsic SERS signals of proteins so far are examples of containing a single protein in the sample or using separated and extracted proteins as the object. However, the actual biopsy sample is mixed with complex substances, and the process of separating / extracting the target protein before detection is complicated and requires a large amount of sample. A simple method for separating / extracting the target protein in advance is to use antibodies and then use immune binding. For example, a specific antibody is bound to the target protein on the surface of a metal particle, and a composite particle in which the target protein is bound to the antibody is used.

[0005] On the one hand, the SERS signal has a characteristic of decreasing exponentially according to the distance from the nanogap, and since the composite particle has a predetermined distance between the surface of the metal particle and the target protein, there is a problem of decreased detection sensitivity.

[0006] At the same time, the preparation of highly sensitive nanostructures requires focused ion-beam lithography, E-beam lithography, ion-milling and other high-performance equipment that takes a long time and is expensive, and has not yet been commercialized.

[0007] Therefore, there is a need for a method for producing a sensor or the like that does not require a separate protein separation / extraction process and has a simple production process, and there is a need for a biosensor that can perform uniform signal detection. Summary of the invention

[0008] Technical issues

[0009] The present invention aims to solve the above-mentioned problems and provide a method for manufacturing a target substance detection substrate based on surface enhanced Raman scattering with a simple manufacturing process, a target substance detection substrate based on the same, and a target substance detection method using the same.

[0010] Technical Solution

[0011] In order to achieve the above object, the present invention provides a method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering (SERS), which comprises the following steps:

[0012] Immersing the substrate to which the bioreceptor is attached into a sample solution containing the target substance, thereby forming a primary capture structure that combines the target substance and the bioreceptor;

[0013] placing the substrate forming the primary capture structure inside a centrifuge, and placing a solution containing metal nanoparticles into the centrifuge; and

[0014] By operating the centrifuge, a secondary capture structure in which metal nanoparticles are coated on the surface of the target substance is formed.

[0015] In addition, the present invention provides a target substance detection substrate based on surface enhanced Raman scattering (SERS), comprising:

[0016] substrates for adhesion of biological receptors;

[0017] Target substances captured by biological receptors;

[0018] Metal nanoparticles capped with a target substance; and

[0019] Raman dye attached to metal nanoparticles.

[0020] The target substance is sandwiched between the bioreceptor and the metal nanoparticles to form a capture structure.

[0021] In addition, the present invention provides:

[0022] A target substance detection method based on surface enhanced Raman scattering (SERS) is characterized in that a target substance is detected using a target substance detection substrate based on surface enhanced Raman scattering (SERS).

[0023] Technical Effects

[0024] According to the method for manufacturing a substrate for detecting a target substance based on surface enhanced Raman scattering (SERS) of the present invention, centrifugal separation can be used to quickly and uniformly coat the surface of the target substance.

[0025] Furthermore, by directly coating the target substance with metal nanoparticles, the distance between the surface of the metal ions and the target substance can be shortened, thereby improving the detection sensitivity of the target substance and facilitating detection of the target substance based on surface enhanced Raman scattering (SERS). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 FIG. 1 is a diagram schematically showing a process of manufacturing a substrate for detecting a target substance based on surface enhanced Raman scattering (SERS) according to an embodiment of the present invention.

[0027] Figure 2 FIG. 1 is a flow chart showing a process of manufacturing a substrate for detecting a target substance based on surface enhanced Raman scattering (SERS) according to an embodiment of the present invention.

[0028] Figure 3 FIG. 1 is a diagram showing a schematic diagram of a substrate for detecting a target substance based on surface enhanced Raman scattering (SERS) according to an embodiment of the present invention.

[0029] Figure 4 is a schematic diagram showing the process of target protein adhesion on a substrate.

[0030] Figure 5 1 is a diagram showing the nanoparticle adhesion process based on centrifugal separation in an embodiment of the present invention (the left side is before centrifugal separation, and the right side is after centrifugal separation).

[0031] Figure 6 The left side is a scanning electron microscope (SEM) photograph of the substrate after centrifugation. Figure 6 The right side is a dark field microscope photograph of the substrate after centrifugation.

[0032] Figure 7 A is a schematic diagram showing the centrifugal separation process. Figure 7 B is the color change of the substrate according to the number of centrifugal separation repetitions (cycles) and SEM images, Figure 7 C is a graph showing the SERS signal of the target protein according to the repetition (cycle number) of centrifugation.

[0033] Figure 8 (a) is a graph showing the results of analyzing the spectrum when the detection point is moved by 2 μm to a total of 120 μm. Figure 8 (b) is a graph showing the results of analyzing spectra at randomly set detection points.

[0034] Fig. 9 is a graph showing the results of detecting target protein signals using the protein sensors prepared in Examples and Comparative Examples ( Fig. 9 (a) is an embodiment, Fig. 9(b) is a comparative example).

[0035] Reference numerals

[0036] 100: Substrate for target substance detection based on surface enhanced Raman scattering (SERS)

[0037] 110: Substrate

[0038] 120: Biological receptors

[0039] 121: Blocking molecules

[0040] 130: Target substance

[0041] 140: Metal Nanoparticles DETAILED DESCRIPTION

[0042] In the present invention, various modifications may be made and various embodiments may be provided, and thus specific embodiments will be shown in the drawings and described in detail in the detailed description.

[0043] However, this does not limit the present invention to a specific embodiment, and should be understood to include all changes, equivalents and substitutes within the technical spirit and scope of the present invention. When the specific description of the function of the related known technology is judged to make the subject of the present invention unclear, its detailed description will be omitted.

[0044] The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the present invention. Unless the context clearly indicates otherwise, an expression in the singular includes an expression in the plural.

[0045] In the present invention, terms such as including or having are intended to indicate the existence of features, numbers, steps, actions, constituent elements, parts or their combinations recorded in the specification, and should be understood as not excluding the existence or additional possibilities of one or more other features, numbers, steps, actions, constituent elements, parts or their combinations in advance.

[0046] The present invention relates to a method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering (SERS), a substrate for target substance detection based on the substrate and a target substance detection method using the same, and more specifically to a method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering (SERS) using a centrifugal separation process for a target substance of immunoadhesive metal nanoparticles, a substrate for target substance detection based on the substrate and a target substance detection method using the same.

[0047] In particular, according to the method for manufacturing a target substance detection substrate based on surface enhanced Raman scattering (SERS) of the present invention, centrifugal separation can be used to quickly and uniformly coat the surface of the target substance.

[0048] Furthermore, by directly coating the target substance with metal nanoparticles, the distance between the surface of the metal ions and the target substance can be shortened, thereby improving the detection sensitivity of the target substance and facilitating detection of the target substance based on surface enhanced Raman scattering (SERS).

[0049] Next, the present invention will be described in detail. Figure 1 is a diagram schematically showing a manufacturing process of a substrate for detecting a target substance based on surface enhanced Raman scattering (SERS) according to an embodiment of the present invention, Figure 2 FIG. 1 is a flow chart showing a process of manufacturing a substrate for detecting a target substance based on surface enhanced Raman scattering (SERS) according to an embodiment of the present invention.

[0050] refer to Figure 1 and Figure 2 According to the present invention, a method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering (SERS) comprises the following steps:

[0051] Step S110, immersing the substrate with the bioreceptor attached thereto into a sample solution containing the target substance, thereby forming a primary capture structure combining the target substance and the bioreceptor;

[0052] Step S120, placing the substrate forming the primary capture structure inside a centrifuge, and placing a solution containing metal nanoparticles into the centrifuge; and

[0053] In step 130, a secondary capture structure is formed by operating a centrifuge to coat the surface of the target substance with metal nanoparticles.

[0054] The term "bioreceptor-adhered substrate" used in the present invention may mean a substrate to which the bioreceptor is immobilized by surface treatment of the substrate.

[0055] The substrate to which the bioreceptors are attached can be obtained by a preparation method comprising the following steps: immersing the substrate in a solution containing a surface modification substance and the bioreceptors, thereby attaching the bioreceptors to the substrate; and attaching blocking molecules to a portion of the surface of the substrate to which the bioreceptors are not attached.

[0056] Here, the surface modification material is intended to facilitate the adhesion of the bioreceptor to the substrate, and is at least one selected from the group consisting of APTES ((3-aminopropyl)triethoxysilane), APTMS ((3-aminopropyl)trimethoxysilane), cysteamine, AEAPTMS (N-[3-(trimethoxysilyl)propyl]ethylenediamine), MPTMS ((3-mercaptopropyl)trimethoxysilane), MPTES ((3-mercaptopropyl)triethoxysilane), MPA (mercaptopropionic acid), MHA (6-mercaptohexanoic acid), BDT (1,4-benzenedithiol), MBA (4-mercaptobenzoic acid), and MBIA (2-mercaptobenzimidazole-5-carboxylic acid), but is not limited to this. As a specific example, it can be APTES.

[0057] For example, the substrate may be prepared as a substrate modified with an amine group (-NH2), and as another example, a thiol group (-SH) may be used as a binder for bioreceptor immobilization through an additional treatment.

[0058] Furthermore, a "bioreceptor" is specific to a target substance, is fixed on a substrate so as to capture the target substance, and may include a substance having a binding affinity to the target substance. For example, a bioreceptor may be an "antibody," which means a protein composed of amino acids and sugar chains that can bind to a target substance.

[0059] The bioreceptor is not limited to this type. As a specific example, it can be at least one selected from the group consisting of protein antibodies, aptamers, enzymes, nucleic acids, DNA, RNA, cells, cell mimics (biometic), proteins, organic compounds and polymers. A suitable bioreceptor can be selected according to the type of target substance. For example, when the target substance is cyt C, the bioreceptor can be a protein antibody.

[0060] As a specific example, the substrate to which the bioreceptor is adhered may be prepared by immersing the substrate in a solution containing a substance for modifying the surface of the substrate and the bioreceptor.

[0061] The substrate may be at least one non-metallic material selected from the group consisting of silicon (Si), gallium arsenide (GaAs), glass, quartz, and polymer. The substrate may be glass as a specific example.

[0062] On the one hand, the substrate to which the bioreceptor is not attached may be bound with a bioreceptor. The blocking molecule may be at least one selected from the group consisting of bovine serum albumin (BSA), casein, and skim milk. As a specific example, the blocking molecule may be BSA (Bovine Serum Albumin).

[0063] The blocking molecules adhere to the substrate surface and can play a role in maintaining the orientation of the bioreceptor more stably. As a result, the binding site of the bioreceptor is easily exposed and the binding with the target substance can be promoted.

[0064] In addition, the blocking molecules bind to the remaining sites of the substrate where the bioreceptors are located, thereby preventing the metal nanoparticles from directly binding to the substrate or the bioreceptors.

[0065] Next, in order to carry out the centrifugal separation coating according to the present invention, the substrate or the substrate having formed a primary capture structure is arranged inside the centrifuge. Here, the centrifuge in which the substrate is arranged may mean a centrifugal separation tube, and preferably, the substrate is arranged at a position away from the bottom of the centrifuge. In more detail, in order to make the bottom surface of the centrifugal separation tube flat, a support made of a material such as PDMS can be located on the bottom surface of the tube, and the substrate can be placed flatly.

[0066] Furthermore, in order to coat the surface of the protein that has formed the primary capture structure with metal nanoparticles, a solution containing metal nanoparticles may be placed in the centrifuge (or centrifugal separation tube).

[0067] The metal nanoparticles contained in the solution may be at least one selected from the group consisting of gold (Au), silver (Ag), platinum (Pt) and aluminum (Al), but are not limited thereto. As a specific example, the metal nanoparticles may be gold (Au) nanoparticles. When prepared from the metals, the substrate according to the present invention has excellent signal amplification and signal uniformity.

[0068] On the one hand, the diameter of the metal nanoparticles can be within the range of an average of 10 to 100 nm, and the diameter of the metal nanoparticles can be 20 to 95 nm, 40 to 90 nm, 60 to 95 nm or an average of 80 nm. For example, when the diameter of the metal nanoparticles is less than 10 nm, the process of precipitating the nanoparticles by centrifugation may not be performed, and when the diameter of the metal nanoparticles is greater than 100 nm, it may be difficult to consider the range of the nanoparticles that can be synthesized in general.

[0069] The concentration of the solution containing the metal nanoparticles may be on average 1×10 9 Up to 1×10 12 particles / ml. When the concentration of the solution containing metal nanoparticles is less than 1×10 9 particles / ml, the target substance may not be coated with enough nanoparticles. 12 In the case of 1000 nanometers per ml, due to the high concentration of nanoparticles in the solution, the problem of excessive particle accumulation may occur on the surface of the target substance. In other words, when the surface of the target substance is coated with metal nanoparticles using a centrifuge, the thickness of the metal layer formed on the substrate can be adjusted by controlling the concentration of the metal nanoparticle solution.

[0070] After the substrate is set in the centrifuge and the metal nanoparticle solution is placed, the metal nanoparticles can be coated on the surface of the target material by operating the centrifuge.

[0071] More specifically, the step of forming the secondary capture structure can be performed by centrifuging at a range of 1000×g to 6000×g for 2 to 5 minutes. As a specific example, gold nanoparticles with an average size of 80 nm can be centrifuged at 1000×g for 3 minutes.

[0072] Through this centrifugal separation process, in the step of forming a secondary capture structure, the target substance is sandwiched between the bioreceptor and the metal nanoparticles, so that a capture structure can be formed. That is, a "sandwich immune complex" can be formed. In the present invention, the "sandwich immune complex" is an immune complex intended to be combined through an antibody-antigen-antibody reaction, and is named because the antigen is sandwiched between the antibodies to give it a sandwich shape. In the present invention, the sandwich immune complex may include a structure of bioreceptor-target substance-metal nanoparticles. At this time, the metal nanoparticles are centrifuged to approach the target substance on the bottom surface of the tube, and then the target substance is coated by adhering the nanoparticles to the target substance through electrostatic, hydrophobic interactions, hydrogen bonds, van der Waals forces, and steric hindrance between the target substance and the metal nanoparticles.

[0073] In particular, in most of the prior art, the target substance and the metal nanoparticles are kept tens of nanometers apart by antibodies, but in the present invention, the metal nanoparticles are directly coated on the target substance, so that a distance of less than 1 nm is formed between the target substance and the metal nanoparticles, which can amplify and enhance the excellent SERS signal.

[0074] The step of forming a secondary capture structure as part of the centrifugal separation process can be repeated more than three times. More specifically, the centrifugal separation process can be repeated 3 to 7 times, 3 to 6 times, 3 to 5 times or 4 times. As a specific example, as the number of repetitions of the centrifugal separation increases, the signal in the SERS-based target substance detection process described later can also increase. However, since the signal amplification is saturated after repeating the process about four times, considering the ease and economy of detection reproducibility, the centrifugal separation process is preferably repeated four times.

[0075] At the same time, after the step of forming the secondary capture structure, the step of attaching a Raman dye on the surface of the metal nanoparticles may be included. Raman dye is a substance for detection by surface-enhanced Raman scattering (SERS) spectroscopy, which means a Raman-active organic compound, and any substance widely used in the technical field may be used without restriction.

[0076] For example, it can be selected from the group consisting of MGITC (malachite green isothiocyanate), RBITC (rhodamine B isothiocyanate), rhodamine 6G, adenine, 4-amino-pyrazole (3,4-d) pyrimidine, 2-fluoroadenine, N6-benzoyl adenine, kinetin, dimethyl-allyl-amino-adenine, zeatin, bromo-adenine, 8-aza-adenine, 8-azaguanine, 4-mercaptopyridine, 6-mercaptopurine, 4-amino-6-mercaptopyrazole (3,4-d) pyrimidine, 8-mercaptoadenine, 9-amino-acridine and a mixture thereof, but is not limited thereto.

[0077] Figure 3 FIG. 1 is a diagram showing a schematic diagram of a substrate for detecting a target substance based on surface enhanced Raman scattering (SERS) according to an embodiment of the present invention.

[0078] refer to Figure 3 , a target substance detection substrate 100 based on surface enhanced Raman scattering (SERS) according to the present invention comprises:

[0079] A substrate 110 to which a bioreceptor 120 is attached;

[0080] Target substance 130 captured by bioreceptor 120;

[0081] Metal nanoparticles 140 capping the target substance 130; and

[0082] The Raman dye (not shown) adheres to the metal nanoparticles 140 .

[0083] The target substance is sandwiched between the bioreceptor and the metal nanoparticles to form a capture structure.

[0084] Again, “the substrate 110 to which the bioreceptor 120 is adhered” may mean the substrate 110 to which the bioreceptor 120 is fixed by surface treatment of the substrate 110 .

[0085] The substrate 110 to which the bioreceptor 120 is attached may be a substrate 110 whose surface is modified by a surface modifying substance, for example, the substrate 110 to which the bioreceptor 120 is attached may be a substrate modified by an amino group (—NH 2 ).

[0086] Furthermore, the “bioreceptor” has specificity for the target substance, is fixed on the substrate 110 so as to capture the target substance 130, and may include a substance having binding affinity for the target substance 130. For example, the bioreceptor 120 may be an “antibody”, which means a protein composed of amino acids and sugar chains that can bind to the target substance 130.

[0087] The bioreceptor 120 is not limited to this type. As a specific example, it can be at least one selected from the group consisting of protein antibodies, aptamers, enzymes, nucleic acids, DNA, RNA, cells, cell mimics (biometic), proteins, organic compounds and polymers. A suitable bioreceptor can be selected according to the type of target substance. For example, when the target substance is cyt C, the bioreceptor can be a protein antibody.

[0088] Meanwhile, the substrate 110 may be at least one non-metallic material selected from the group consisting of silicon (Si), gallium arsenide (GaAs), glass, quartz, and polymer. It is not limited thereto, but as a specific example, the substrate may be glass.

[0089] On the one hand, the portion of the surface of the substrate 110 that is not attached with the bioreceptor 120 may further include a blocking molecule 121. The blocking molecule 121 may be at least one selected from the group consisting of BSA (Bovine Serum Albumin), casein, and skim milk. As a specific example, the blocking molecule 121 may be BSA (Bovine Serum Albumin).

[0090] Furthermore, the metal nanoparticles 140 may be at least one selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), tin (Sn), indium (In), gallium (Ga), thallium (Tl), lead (Pb), bismuth (Bi) and aluminum (Al), but are not limited thereto. As a specific example, the metal nanoparticles 140 may be gold (Au) nanoparticles.

[0091] On the one hand, the diameter of the metal nanoparticles 140 can be within the range of 10 to 100 nm on average, and the diameter of the metal nanoparticles 140 can be 20 to 95 nm, 40 to 90 nm, 60 to 95 nm, or 80 nm on average. For example, when the diameter of the metal nanoparticles is less than 10 nm, the process of precipitating the nanoparticles by centrifugal separation may not be performed, and when the diameter of the metal nanoparticles is greater than 100 nm, it may be difficult to consider the range of nanoparticles that can be synthesized in general.

[0092] At this time, a metal layer may be formed on the target substance by the metal nanoparticles 140 , and the thickness of the metal layer may be 10 to 100 nm on average.

[0093] In most of the prior art, the target substance 13 and the metal nanoparticles 140 are kept tens of nanometers apart by antibodies. However, in the present invention, the metal nanoparticles 140 are directly coated on the target substance 130, so that a distance of less than 1 nm is formed between the target substance 130 and the metal nanoparticles 140, which can amplify and enhance the excellent SERS signal.

[0094] The present invention provides a method for detecting a target substance using a target substance detection substrate based on surface enhanced Raman scattering (SERS).

[0095] The target substance may be detected by SERS spectroscopy. In this case, the target substance may be quantitatively analyzed by measuring the intensity of Raman scattering caused by the target substance binding to the substrate.

[0096] For reference, SERS is obtained by amplifying the inherent SERS signal transmitted by the vibration state of the molecule when light enters the substance on the surface of the metal nanoparticle. The specificity of each substance is often used in the qualitative determination of body substances by SERS signals. According to the present invention, the average 500 to 2000 cm can be measured by surface enhanced Raman scattering (SERS). -1The target substance on the target substance detection substrate based on surface enhanced Raman scattering (SERS) is detected by measuring the intensity of Raman signals in the wavelength range of 785 nm. More specifically, the SERS signal can be measured by irradiating the substrate with an average laser of 785 nm.

[0097] The target substance can be a protein, nucleic acid, DNA, RNA, enzyme, organic molecule, virus, extracellular vesicles, microvesicles, exosomes, cells, fat, metabolite or pathogen. More specifically, the target substance can be a protein, and as a specific example, it can be a protein.

[0098] That is, the present invention can make metal nanoparticles adhere to target substances through centrifugal separation, thereby enhancing SERS signals and improving detection sensitivity, and can provide a SERS signal improvement method for target substance detection, which includes: (a) a step of reacting and adhering a substrate of a bioreceptor that specifically binds to the target substance and the target substance to form a primary capture structure (bioreceptor-target substance); and (b) a step of centrifuging the substrate forming the primary capture structure in the presence of a solution containing metal nanoparticles to form a secondary capture structure having a sandwich structure of bioreceptor-target substance-nanoparticles.

[0099] In the present invention, SERS signal improvement includes signal amplification, and the amplified SERS signal can help improve the detection sensitivity of the target substance.

[0100] In addition, the present invention provides a method for detecting a target substance using the SERS signal improvement method.

[0101] Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

[0102] However, the following embodiments and experimental examples are merely for illustration of the present invention, and the contents of the present invention are not limited to the following embodiments and experimental examples.

[0103] [Example] Fabrication of protein sensor

[0104] Step 1: Adhesion of target protein

[0105] The method introduced in "One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics." (Scientific reports, 2014, 4, 4407) was applied to the surface treatment method of a glass substrate protein chip for immobilizing a target protein for antigen-antibody reaction on the substrate.

[0106] refer to Figure 4 , illustrating the adhesion process of the target protein on the substrate.

[0107] First, a 5 cm × 5 cm cover glass was washed with water and ethanol and dried. Then, in order to hydroxylate the substrate, it was immersed in a piranha solution (solution (H2SO4:H2O2=7:3 (v / v)) for 30 minutes, washed with water, and then immersed in an ethanol solution of a mixture of 1% 3-aminopropyltrimethoxysilane (3-Aminopropyltrimethoxysilane, APTMS) and 8 μg / ml antibody diluent for 30 minutes, thereby functionalizing the surface organosilane, that is, the substrate amino group and adhering the bioreceptor.

[0108] Then, the substrate was immersed in a 1% BSA (bovine serum albumin) solution for 30 minutes to block the APTES in the area where the antibody was not attached. Then, the substrate was immersed in a solution containing cyt C as an antigen to induce immune binding, thereby producing a substrate to which the target protein was attached.

[0109] Step 2: Adhesion of nanoparticles

[0110] Figure 5 1 is a diagram showing the nanoparticle adhesion process based on centrifugal separation in an embodiment of the present invention (the left side is before centrifugal separation, and the right side is after centrifugal separation).

[0111] refer to Figure 5 , a support made of a material such as PDMS is located on the bottom surface of the tube and is flatly placed on the substrate bound to the target protein antigen prepared in step 1 to make the bottom surface of the centrifugal separation tube flat.

[0112] Then, the tube was filled with 400 μl of a colloidal solution of gold nanoparticles (BBI gold) having an average diameter of 80 nm, and then centrifuged. At this time, the centrifugal separation was performed at 1000×g for 3 minutes.

[0113] Next, the prepared protein sensor was taken out from the tube, washed with distilled water, and then dried with nitrogen gas.

[0114] Figure 6 The left side is a scanning electron microscope (SEM) photograph of the substrate after centrifugation. Figure 6 The right side of the figure is a dark field microscope photo of the substrate after centrifugation. Figure 6 As shown, it can be confirmed that the gold nanoparticles are uniformly adhered to the substrate in step 1.

[0115] [Comparative Example]

[0116] The protein sensor was fabricated by immobilizing antibodies on gold nanoparticles and attaching cyt C as an antigen.

[0117] More specifically, a gold nanoparticle solution was provided to a glass substrate washed with a piranha solution (H2SO4:H2O2=7:3 (v / v)), dried, treated with 10mM MPA (mercaptopropionic acid) for 12 hours and washed. Next, a solution of 0.2M EDC and 0.2M NHS mixed in a ratio of 1:1 was treated for 1 hour, and a substrate to which antibodies could adhere was manufactured through a washing process. Furthermore, 0.1mg / ml of antibodies were treated for 1 hour and then washed to introduce antibodies onto the substrate.

[0118] Then, in order to block the adhesion of cyt C as an antigen, 1% (w / v) BSA was treated for 20 minutes to prepare a protein sensor.

[0119] <Experimental example>

[0120] Experimental Example 1. Signal amplification by repeated centrifugation process

[0121] When manufacturing a protein sensor, the centrifugal separation process is repeated to repeat the nanoparticle adhesion process on the substrate to which the target protein is adhered. In addition, the SEM image and visible color of the substrate according to the repeated process (number of cycles) are confirmed, and the SERS signal of the target protein is measured.

[0122] The result is Figure 7 Displayed in.

[0123] Figure 7 A is a schematic diagram showing the centrifugal separation process. Figure 7 B is the color change of the substrate according to the number of centrifugal separation repetitions (cycles) and SEM images, Figure 7 C is a graph showing the SERS signal of the target protein according to the repetition (cycle number) of centrifugation.

[0124] refer to Figure 7 A and Figure 7 In B, it can be confirmed through SEM images and visual colors that the number of attached nanoparticles gradually increases as the nanoparticle attachment process is repeated.

[0125] In addition, reference Figure 7 C shows that the SERS signal of the target protein increases with the increase in the number of centrifugal separation repetitions (cycles), and it can be confirmed that the signal increase decreases after about 3 repetitions.

[0126] That is, it can be seen that sufficient signal amplification can be performed by repeating the process about three times.

[0127] Experimental Example 2. Analysis of Nanoparticle Coating Uniformity

[0128] Gold nanoparticles were applied to the surface into which amino groups were introduced by APTES, and 4-aminothiophenol (4-ATP) was treated to coat the SERS detection surface.

[0129] Furthermore, the spectrum was analyzed. More specifically, the spectrum was analyzed by moving the detection point by 2 μm to a total of 120 μm, and the spectrum was analyzed at randomly set detection points. The results are as follows: Figure 8 Displayed in.

[0130] Figure 8 (a) is a graph showing the results of analyzing the spectrum when the detection point is moved by 2 μm to a total of 120 μm. Figure 8 (b) is a graph showing the results of analyzing spectra at randomly set detection points.

[0131] refer to Figure 8 , the results of analyzing the spectra with the detection point moved by 2 μm up to a total of 120 μm showed that the signal of 4-ATP derived from the substrate was very uniform.

[0132] At the same time, the results of obtaining signals from randomly set detection points also showed the same results, thereby confirming that the technology of the present invention is a technology for manufacturing a uniform substrate.

[0133] Experimental Example 3. Signal detection of target protein

[0134] The signal of C-reactive protein (target protein) was detected by Surface Enhanced Raman Scattering (SERS) using the protein sensors prepared in Examples and Comparative Examples.

[0135] Specifically, the surface enhanced Raman scattering spectrum (SERS spectra) was observed using a Raman microscope (Nicolet Raman spectrometer (Almeca XR)), and the antigen signal detection efficiency was calculated based on this.

[0136] And the result is Fig. 9 Displayed in.

[0137] Fig. 9 is a graph showing the results of detecting target protein signals using the protein sensors prepared in Examples and Comparative Examples ( Fig. 9 (a) is an embodiment, Fig. 9 (b) is a comparative example).

[0138] The antigen signal detection efficiency can be calculated by the following [Formula 1].

[0139] [Formula 1]

[0140] S total =W ab ×S ab +W cytc ×S cytc

[0141] Here, S total means that the combined signal,

[0142] W ab is the weighted value of the antibody signal, S ab It's an antibody signal.

[0143] W cytc is the weighted value of the antigen signal, S cytc It is an antigen signal.

[0144] The signal detected by the protein sensor will appear as a linear combination of the Raman signal of the antigen (cytochrome c) and the signal of the antibody. Therefore, the SERS signal of the antibody was first obtained when only the antibody was attached, and an experiment was performed to find the signal that was closest to the signal detected by linearly combining the Raman signal of the antigen (cytochrome c) and the signal of the antibody obtained separately.

[0145] The results confirm that the protein sensor prepared in the embodiment has a better antigen signal detection efficiency than the protein sensor prepared in the comparative example. More specifically, the comparative example only includes about 3.61% of antigen signals, while the signal detected by the embodiment of the present invention includes about 6 times higher antigen signals.

[0146] In summary, it can be judged that the protein sensor of the present invention has excellent signal detection efficiency compared with the comparative example because the single layer of gold nanoparticles is directly attached to the target protein, so the distance between the gold nanoparticles and the target antigen (target protein) is small.

[0147] Industrial Applicability

[0148] The present invention relates to a method for manufacturing a substrate for detecting a target substance based on surface enhanced Raman scattering (SERS), etc. By providing the present invention, the detection sensitivity of the target substance can be improved, and the present invention is expected to be widely used in molecular diagnostic detection methods, etc.

Claims

1. A method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering, comprising the following steps: Immersing the substrate with the bioreceptor attached into a sample solution containing the target substance, thereby forming a primary capture structure that combines the target substance and the bioreceptor; placing the substrate forming the primary capture structure inside a centrifuge, and placing a solution containing metal nanoparticles into the centrifuge; and By operating the centrifuge, a secondary capture structure in which metal nanoparticles are directly coated on the surface of the target substance is formed.

2. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 1, characterized in that: The substrate for adhering bioreceptors is obtained by the preparation method of the following steps: immersing the substrate in a solution comprising a surface modification substance and a bioreceptor, thereby adhering the bioreceptor to the substrate; and The blocking molecule is adhered to the portion of the surface of the substrate to which the biological receptor is not adhered.

3. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 2, characterized in that: The blocking molecule is at least one selected from the group consisting of bovine serum albumin, casein and skim milk.

4. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 1, characterized in that: The concentration range of the solution containing metal nanoparticles is an average of 1×10 9 Up to 1×10 12 Particles / ml.

5. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 1, characterized in that: The step of forming a secondary capture structure operates a centrifuge in the range of 1000 xg to 6000 xg and in the range of 2 to 5 minutes.

6. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 5, characterized in that: The step of forming the secondary capture structure is repeated three or more times.

7. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 1, characterized in that: In the step of forming the secondary capture structure, the target substance is sandwiched between the bioreceptor and the metal nanoparticles, thereby forming a capture structure.

8. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 1, characterized in that: After the step of forming the secondary capture structure, the method further includes the following step: adhering Raman dye on the surface of the metal nanoparticles.

9. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 1, characterized in that: The metal nanoparticles are at least one selected from the group consisting of gold, silver, platinum, tin, indium, gallium, thallium, lead, bismuth and aluminum.

10. The method for manufacturing a substrate for target substance detection based on surface enhanced Raman scattering according to claim 1, characterized in that: The diameter of the metal nanoparticles is in the range of 10 to 100 nm on average.

11. A substrate for target material detection based on surface enhanced Raman scattering, comprising: substrates for adhesion of biological receptors; Target substances captured by biological receptors; Metal nanoparticles capped with target substances; and Raman dyes attached to metal nanoparticles, The target substance is sandwiched between the bioreceptor and the metal nanoparticles to form a capture structure. The metal nanoparticles are directly coated on the surface of the target substance by centrifugal separation.

12. The target substance detection substrate based on surface enhanced Raman scattering according to claim 11, characterized in that: Blocking molecules are also included on the portion of the surface of the substrate to which the bioreceptors are not attached.

13. The substrate for target substance detection based on surface enhanced Raman scattering according to claim 12, characterized in that: The blocking molecule is at least one selected from the group consisting of bovine serum albumin, casein and skim milk.

14. The substrate for target substance detection based on surface enhanced Raman scattering according to claim 11, characterized in that: The metal nanoparticles are at least one selected from the group consisting of gold, silver, platinum, and aluminum.

15. A target substance detection method based on surface enhanced Raman scattering, characterized in that: A target substance is detected using the substrate for detecting a target substance based on surface enhanced Raman scattering according to claim 12.

16. The target substance detection method based on surface enhanced Raman scattering according to claim 15, characterized in that: The following steps are involved: The average 500 to 2000 cm -1 The Raman signal intensity in the wavelength range is measured to detect the target substance.

17. The target substance detection method based on surface enhanced Raman scattering according to claim 15, characterized in that: The target substance is a protein, a nucleic acid, a DNA, a RNA, an enzyme, an organic molecule, a virus, an extracellular vesicle, a microvesicle, an exosome, a cell, a fat, a metabolite or a pathogen.

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