Nanocube assembly SERS substrate for label-free assay of biomolecule and manufacturing method therefor

Abstract

A surface-washed nanocube assembly SERS substrate according to one embodiment of the present invention comprises: a base substrate; and a plurality of nanocube aggregates arranged on the substrate at intervals of several nm, wherein the surface of the nanocube is washed to remove noise from the Raman spectrum.

Description

Nanocube assembly SERS substrate for label-free biomolecule analysis and its manufacturing method

[0001] The present invention relates to a substrate for SERS and a method for manufacturing the same.

[0002]

[0003] Plasmonic nanostructures exhibiting localized surface plasmon resonance (LSPR) have unique properties and are applied in various fields such as sensing, imaging, diagnosis, treatment, and catalysis. Among the technologies for creating these nanostructures, the bottom-up method, which assembles nanoparticles, allows access to structures on a molecular level, down to several nanometers, which are inaccessible with the top-down method, and thus has high utility, and related research has been actively conducted in the past.

[0004] As an example, application number KR10-2018-0076446A discloses a technology for gold nanoparticles characterized by having multiple branches.

[0005] The plasmonic nanoparticle self-assembly method, one of the representative bottom-up methods, is a method of designing nanoparticles, which are unit chains, to spontaneously assemble into a specific pattern, and structures with various patterns and materials have been developed.

[0006] Of particular note in the structure of these nanoparticle assemblies is the nanoscale space created between the particles. This space, called a plasmonic nanogap, is known to generate a strongly enhanced electromagnetic field due to plasmon resonance, leading to various phenomena, including enhanced optical signal enhancement and enhanced catalytic performance, within the gap. This electromagnetic field enhancement phenomenon is generally more advantageous at smaller sizes (up to a nanometer), and is highly sensitive to size and shape, requiring precise structural control.

[0007] However, most self-assembly technologies require additional complex surface modification processes such as exchange, change, and substitution of surface protection agents (capping agents), and these processes have limitations such as additional processing steps, loss of units during surface modification, and limitations in the minimum size of nanogaps due to limitations in the size of surface protection agents.

[0008] Furthermore, fabricating an ideal SERS substrate requires high-density hotspot formation, a laser-tuned resonance wavelength, and mass production capabilities. However, existing technologies have failed to simultaneously satisfy these requirements.

[0009]

[0010] Under the above-described background, the present applicants studied a method for simply forming a regular and controllable nanopattern using only nanoparticles and a depletant.

[0011] The purpose of the present invention is to fabricate two-dimensional or three-dimensional nanocube self-assemblies that are not limited by the size and sharpness of the unit particles. Furthermore, the present invention proposes a next-generation SERS substrate and its fabrication method, which are superior in terms of ease of fabrication, signal amplification, and signal reproducibility, and have structural, optical, and spectroscopic advantages. The present invention also aims to utilize the substrate for label-free biomolecule detection, disease diagnosis, and various other applications.

[0012]

[0013] A surface-cleaned nanocube assembly SERS substrate according to one embodiment of the present invention comprises: a base substrate; and a plurality of nanocube aggregates arranged at intervals of several nm on the substrate; wherein the nanocube surfaces are cleaned to remove noise from a Raman spectrum.

[0014] The above surface cleaning may be performed using sodium borohydride (NaBH4) according to the reductive desorption principle.

[0015] The above SERS substrate has a Raman spectrum of 950 cm -1 1100 cm inland -1 The peak intensity in may be less than twice the largest peak intensity among the peaks outside the above region.

[0016] The above nanocube aggregate may comprise an array of single-layer or multi-layer nanocubes.

[0017] The above nanocube aggregate may include a self-assembly formed by interaction with a coagulant and a surfactant.

[0018] The above nano cube aggregate may have a two-dimensional single-layer structure or a three-dimensional multi-layer structure depending on the surface roughness of the substrate.

[0019] The base substrate may include one of a silicon wafer, a metal-deposited surface, mica, glass, and a quartz slide.

[0020] The above SERS substrate may have plasmon resonance in the range of 750 nm to 900 nm.

[0021]

[0022] An optical member comprising a surface-washed nanocube assembly SERS substrate proposed in one aspect of the present invention comprises a substrate of one of the above embodiments, and the optical member is one of a color filter, a fluorescent light-emitting member, and a molecular signal amplifier.

[0023]

[0024] In another aspect of the present invention, a nanoprocessing member comprising a surface-washed nanocube assembly SERS substrate comprises a substrate of one of the above embodiments.

[0025]

[0026] According to another embodiment of the present invention, a method for manufacturing a surface-washed nanocube assembly SERS substrate comprises the steps of: applying a first solution containing a surfactant and a coagulant and a second solution containing metal nanocube units onto a substrate to form a nanocube self-assembly; and washing away residues of at least one of the surfactant and the coagulant on the surface of the nanocube self-assembly using a reduction and desorption method.

[0027] The above washing step may include washing the substrate on which the nanocube self-assembly is formed with sodium borohydride (NaBH4).

[0028] The above washing step may include a step of washing the substrate on which the nanocube self-assembly is formed with oxygen plasma.

[0029]

[0030] A method for label-free analysis of biomolecules and human-derived substances using a surface-washed nanocube assembly SERS substrate proposed in one aspect of the present invention comprises the steps of: preparing a SERS substrate including a nanocube assembly; washing the surface of the SERS substrate; and dropping a solution or plasma containing biomolecules onto the surface of the washed SERS substrate and then acquiring a Raman spectrum.

[0031]

[0032] The above descriptions all represent various implementation examples of the present invention, and it is also included in the technical idea of ​​the present invention that one or more implementation examples are combined and implemented as one embodiment.

[0033]

[0034] The present invention proposes a SERS substrate and demonstrates its potential for optical and spectroscopic applications. In particular, the embodiments of the present invention demonstrate the utility of the proposed SERS substrate as a next-generation SERS substrate by utilizing it as a molecular Raman signal amplifier, demonstrating its applicability as a label-free detection method for biomolecules and as a next-generation diagnostic technology.

[0035] However, the effects of the present invention are not limited to the effects described above, and include all effects naturally implemented due to the various configurations proposed in the present invention.

[0036]

[0037] Figure 1 is a schematic diagram suggesting a schematic form of a SERS substrate (NAM Substrate) proposed in one embodiment of the present invention and the possibility of diagnosing various diseases using Raman spectra.

[0038] FIG. 2 is a front-view SEM image (a), a side-view SEM image (b) of a SERS substrate including single-layer nanocubes according to one embodiment of the present invention, and a GISAXS (grazing-incidence small-angle scattering) spectrum graph measured to confirm the periodicity of the SERS substrate.

[0039] FIG. 3 is an image showing calculated and experimental values ​​(a) of the UV-Vis spectrum of a SERS substrate according to one embodiment of the present invention and an electric field distribution (b) at the plasmon peak (801 nm) of the SERS substrate.

[0040] FIG. 4 is a graph showing the Raman spectrum of a rhodamine 6G molecule measured after correction with an internal standard material of a SERS substrate according to one embodiment of the present invention and the enhancement factor on the SERS substrate.

[0041] Figure 5 is an image showing the Raman spectrum of various biomolecules on a SERS substrate simply treated with O2 plasma (left) and the Raman spectrum of BDAC (benzyldimethyammonium chloride) molecules used as a coagulant on a SERS substrate washed only with purified water (DIW) (right), which corresponds to a comparative example of the present invention.

[0042] FIG. 6 is an image showing Raman spectra of 12 biomolecules through a SERS substrate whose surface was washed with NaBH4 according to an embodiment of the present invention, in contrast to FIG. 5.

[0043] Figure 7 is a table showing the Raman peak values ​​of biomolecules measured in the Raman spectrum of Figure 6.

[0044] FIG. 8 is an image comparing the Raman spectra of a SERS substrate before (gray dotted line) and after (red solid line) washing with NaBH4 according to one embodiment of the present invention.

[0045] Fig. 9 is 1000 cm from Fig. 8 -1 This is a box plot image comparing the intensity changes of nearby Raman spectrum signals.

[0046]

[0047] The embodiments of the present invention are provided for the purpose of illustrating the technical concept of the present invention. The scope of the rights of the present invention is not limited to the embodiments presented below or the specific descriptions of these embodiments.

[0048] All technical and scientific terms used in this invention, unless otherwise defined, have the meanings commonly understood by those skilled in the art to which this invention pertains. All terms used in this invention have been selected for the purpose of more clearly explaining the invention and are not intended to limit the scope of the rights provided for in this invention.

[0049] Expressions such as “comprising,” “having,” and the like used in the present invention should be understood as open-ended terms that imply the possibility of including other embodiments, unless otherwise stated in the phrase or sentence in which the expression is included.

[0050] The singular expressions described in the present invention may include plural meanings unless otherwise stated, and this also applies to the singular expressions described in the claims.

[0051]

[0052] Raman scattering refers to the inelastic scattering of light generated when incident light interacts with the vibrations of a molecule. This causes the vibrational modes of the molecule to be observed as sharp peaks in the Raman spectrum. Each molecule has a unique Raman spectrum, which is called its "molecular fingerprint." This characteristic has attracted significant attention in the field of molecular sensing. However, the very low probability of Raman scattering occurrence makes detection of the Raman signal itself difficult, limiting its practical application.

[0053] Surface-Enhanced Raman Scattering (SERS) is a phenomenon that amplifies Raman signals using plasmonic nanostructures. When plasmonic metals such as gold or silver are fabricated with a size smaller than the wavelength of light, localized surface plasmon resonance (LSPR) occurs. The resonance wavelength varies depending on the shape and size of the nanostructure, and a strong electric field is formed on the metal surface at the resonance wavelength. This electric field can amplify the Raman signal, and by controlling the shape and size of the nanostructure, the Raman signal can be amplified by up to 10 10Amplification of more than two orders of magnitude is also possible. The most commonly used structure for Raman signal amplification, or SERS, is a nanogap, and the region where the Raman signal is strongly amplified is called a hot spot.

[0054] SERS substrates are a variety of nanostructures designed to amplify the weak Raman signals of molecules. These substrates have been manufactured using bottom-up, top-down, or a combination of both methods. Commercialized examples include SERSpace (Nanoscope Systems), a film of silver nanospheres, a 3D crosspoint metal nanowire substrate (PiCO Foundry), a nanoforest-structured SERStrate (Silmeco), and an inverted pyramid-structured Klarite (UK).

[0055]

[0056] The present inventors have fabricated such a SERS substrate in a novel manner and named it a NAM (nanocube-assembled multi- or monolayer) substrate. The substrate (NAM substrate) proposed in the present invention is a SERS substrate that utilizes the nanogaps formed between nanocubes as hot spots by self-assembling synthesized nanocubes through depletion force.

[0057] When large solid building blocks and depletants such as small polymers or micelles coexist within a system, if the distance between the building blocks becomes closer than the size of the depletant, an excluded area where the depletant cannot exist can be formed. This causes a difference in the concentration of the depletant within the system, and this difference in concentration can create an osmotic pressure between the excluded area and the surroundings, which can generate a depletion force between the building blocks. The depletion force-mediated assembly proposed by the present inventors is a technology that utilizes this cohesive force to induce self-assembly of the building blocks, and the present inventors used this to form self-assembled nanocube aggregates on a base substrate.

[0058] The SERS substrate manufactured in this way has a signal amplification factor (enhancement factor), which is a key performance of the SERS substrate, of 10 as a result of self-evaluation. 6 Above, the signal intensity deviation was confirmed to be at least 6.5%. In addition, it was experimentally demonstrated that the above SERS substrate can measure Raman-active molecules, biomolecules, plasma, etc. through various surface modification methods.

[0059]

[0060] A surface-cleaned nanocube assembly SERS substrate according to one embodiment of the present invention comprises: a base substrate; and a plurality of nanocube aggregates arranged on the substrate at intervals of several nm; wherein the surface of the nanocubes is cleaned to remove noise from a Raman spectrum.

[0061] The above nanocubes may be arranged in a square lattice structure with spacings of 10 nm or less.

[0062]

[0063] The above surface cleaning may be performed using sodium borohydride (NaBH4).

[0064] For label-free detection of biomolecules, adsorbing molecules onto the surface of a nanostructure can be crucial. As shown in the examples described below, the Raman spectrum of the SERS substrate of the above example, manufactured without any special surface cleaning, strongly detected signals from the BDAC molecule used as a coagulant. It is presumed that the BDAC molecules were electrostatically adsorbed around the halide ions during the fabrication of the SERS substrate of the example.

[0065] The binding energy of gold and halide ions is approximately 30-35 kcal / mol, which is greater than or similar to the binding energies of most biomolecules. Therefore, the residual halide ions can interfere with the adsorption of biomolecules in plasma. For this reason, the halide ions and BDAC molecules adsorbed on the surface of the SERS substrate must be removed for accurate detection.

[0066] However, these ions or molecules also play a role in reducing the surface energy of nanoparticles, helping them maintain their structure. Therefore, a method may be needed to simultaneously remove and replace these ions and facilitate the adsorption of biomolecules.

[0067] The above NaBH4 has a high binding energy (118 kcal / mol) comparable to that of molecules with thiol functional groups, which have high binding energies with gold. Therefore, when NaBH4 is added to a solution containing gold nanoparticles with thiol functional groups, the hydride ions can act as ligands while simultaneously desorbing the adsorbed molecules through ligand exchange.

[0068] Furthermore, because NaBH4 is highly reactive with water, it can react with water and disappear from the gold surface after a certain period of time. Therefore, in the present invention, NaBH4 can be used as a surface cleaning agent, making it a highly effective means for removing halide ions, BDAC molecules, and other contaminants.

[0069]

[0070] The above SERS substrate has a Raman spectrum of 950 cm -1 1100 cm inland -1 The peak intensity in may be less than twice the largest peak intensity among the peaks outside the above region.

[0071] As will be explained again in the examples to be described later, if the surface of the nanocubes is effectively washed with a component such as NaBH4, the intensity of the Raman spectrum due to the coagulant or surfactant that was essentially used in the manufacturing step of the nanocubes may decrease overall. In another embodiment, the peak intensity may be 1.5 times or less, and in another embodiment, the 950 cm -1 1100 cm inland -1 The peak intensity in may be less than the largest peak among the peaks outside the above region.

[0072] The residual traces of the above-mentioned BDAC molecules on the nanocube surface are observed at 950 cm in the Raman spectrum. -1 1100 cm inland -1 It appears as a very high peak value in .

[0073]

[0074] The above nanocube aggregate may comprise an array of single-layer or multi-layer nanocubes.

[0075] The self-assembled nanocube aggregate proposed in the present invention is named NAM (Nanocube-Assembled Multi- or Monolayer) because the nanocubes, which are the units, have a single-layer or multi-layer structure. The nanocube aggregate can be controlled to have a multi-layer structure or a single-layer structure depending on the surface roughness of the substrate surface on which it is formed.

[0076]

[0077] As described above, the nanocube aggregate may include a self-assembled aggregate formed through interaction with a coagulant and a surfactant. The formation of such a self-assembled nanocube aggregate may be a unique method of the SERS substrate proposed in the embodiments of the present invention.

[0078]

[0079] The above nano cube aggregate may have a two-dimensional single-layer structure or a three-dimensional multi-layer structure depending on the surface roughness of the substrate.

[0080] In an embodiment of the present invention, the cohesion between the metal nanocube unit and the self-assembly forming substrate can be controlled, which can be accomplished by controlling the surface roughness of the base substrate on which the self-assembly is formed.

[0081] In one embodiment of the present invention, the structure of the nanocube self-assembly that is ultimately formed can be controlled differently in two or three dimensions by varying the surface roughness of the base substrate. The term "surface roughness" in the present invention is defined as the centerline average roughness (Ra) of irregular irregularities that occur on the surface of a material with a short period and a relatively small amplitude, and is understood as the opposite meaning of the flatness or evenness of the material surface.

[0082] In the present invention, the surface roughness (Ra) can be understood as a value obtained when measuring an area of ​​1 um x 1 um in air at room temperature using a scanning probe microscope, unless otherwise stated.

[0083]

[0084] The base substrate may include one of a silicon wafer, a metal-deposited surface, mica, glass, and a quartz slide.

[0085] Specifically, when a substrate having a relatively small surface roughness is used as the base substrate, the defect-induced cohesion between the metal nanoparticle unit and the surface of the self-assembly forming substrate is strengthened, so that a single-layer two-dimensional nanocube self-assembly can be formed.

[0086] More specifically, the substrate having a small surface roughness may specifically mean a substrate having a surface roughness of less than 0.12 nm, and more specifically, may mean a substrate having a surface roughness in a range consisting of one upper limit and / or lower limit selected from the group consisting of 0 nm, 0.01 nm, 0.02 nm, 0.03 nm, 0.04 nm, 0.05 nm, 0.06 nm, 0.07 nm, 0.08 nm, 0.09 nm, 0.1 nm, and 0.11 nm. When a substrate having a small surface roughness in such a range is used as a substrate for forming a self-assembly, the cohesive force between the substrate and the self-assembly unit is improved, so that a single-layer two-dimensional nanocube self-assembly can be formed.

[0087] More specifically, the substrate having a small surface roughness may be one selected from the group consisting of a silicon wafer, a metal-deposited substrate, and mica, but is not limited thereto as long as it is a material having a small surface roughness and capable of performing the same function.

[0088]

[0089] In addition, specifically, when a substrate having a large surface roughness is used as the base substrate, the defect-induced cohesion between the metal nanoparticle unit and the surface of the substrate for forming the self-assembly is weakened, so that a multilayer supercrystalline three-dimensional nanocube self-assembly can be formed.

[0090] More specifically, the substrate having a large surface roughness may refer to a substrate having a surface roughness of 0.12 nm or more and less than 1.2 nm, and more specifically, may refer to a substrate having a surface roughness in a range consisting of one upper limit and / or lower limit selected from the group consisting of 0.12 nm, 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1.0 nm, and 1.1 nm. When a substrate having a large surface roughness in the above range is used as a substrate for forming a self-assembly, a supercrystalline three-dimensional nanocube self-assembly may be formed. At this time, if the surface roughness is less than 0.12 nm, the cohesion between the self-assembly forming surfaces may be strengthened, so that a three-dimensional self-assembly may not be formed. On the other hand, if the surface roughness is excessively large, such as 1.2 nm or more, the cohesion may be extremely weakened, so that two-dimensional and three-dimensional self-assemblies themselves may not be formed.

[0091] More specifically, the substrate having a surface roughness of 0.12 nm or more and less than 1.2 nm may be one selected from the group consisting of glass and quartz slides, but is not limited thereto as long as it is a material having a large surface roughness and capable of performing the same function.

[0092] The above SERS substrate may have plasmon resonance in the range of 750 nm to 900 nm.

[0093]

[0094] An optical member comprising a surface-washed nanocube assembly SERS substrate proposed in one aspect of the present invention comprises a substrate of one of the above embodiments, and the optical member is one of a color filter, a fluorescent light-emitting member, and a molecular signal amplifier.

[0095] In another aspect of the present invention, a nanoprocessing member comprising a surface-washed nanocube assembly SERS substrate comprises a substrate of one of the above embodiments.

[0096]

[0097] According to another embodiment of the present invention, a method for manufacturing a surface-washed nanocube assembly SERS substrate comprises the steps of: applying a first solution containing a surfactant and a coagulant and a second solution containing metal nanocube units onto a substrate to form a nanocube self-assembly; and washing away residues of at least one of the surfactant and the coagulant on the surface of the nanocube self-assembly using a reduction method.

[0098] The manufacturing method of the present invention may be characterized by cleaning the surface of the nanocube self-assembly. However, such surface cleaning has been performed conventionally using O2 plasma or purified water (DIW), but this has been problematic because it cannot effectively remove noise from coagulants or surfactants that are inevitably used in the Raman spectrum of a SERS substrate manufactured by the self-assembly method.

[0099]

[0100] The above washing step may include washing the substrate on which the nanocube self-assembly is formed with sodium borohydride (NaBH4).

[0101] The above washing step may include a step of washing the substrate on which the nanocube self-assembly is formed with oxygen plasma. The step of washing with oxygen plasma may be performed together with the step of washing with sodium borohydride.

[0102]

[0103] A method for label-free analysis of biomolecules and human-derived substances using a surface-washed nanocube assembly SERS substrate proposed in one aspect of the present invention comprises the steps of: preparing a SERS substrate including a nanocube assembly; washing the surface of the SERS substrate; and dropping a solution or plasma containing biomolecules onto the surface of the washed SERS substrate and then acquiring a Raman spectrum.

[0104] When a washing step is included using the method described above, detection of various molecules can be achieved simply by dropping plasma or other biological substances on the surface of the SERS substrate proposed in the present invention and then analyzing the Raman spectrum over time.

[0105]

[0106] <Example>

[0107] Hereinafter, embodiments of the present invention will be described in detail using FIGS. 1 to 9.

[0108] Figure 1 is a schematic diagram suggesting a schematic form of a SERS substrate (NAM Substrate) proposed in one embodiment of the present invention and the possibility of diagnosing various diseases using Raman spectra.

[0109] As suggested in Fig. 1, in one embodiment of the present invention, a high-performance SERS substrate formed of single-layer nanocubes was fabricated using a defect-induced aggregation-based self-assembly technique via simple spotting. An example of one specific fabrication method is as follows.

[0110]

[0111] Example 1-1: Seed particle synthesis

[0112] A solution of Au nanospheres with a diameter of 1-2 nm was prepared by mixing 100 mM CTAB (Cetyltrimethylammonium bromide), 10 mM HAuCl4, and 10 mM NaBH4. In this process, CTAB is used as the first surfactant, NaBH4 as the reducing agent, and HAuCl4 as the metal ion.

[0113] A 10 nm diameter nanosphere solution was prepared by mixing 200 mM CTAC (Cetyltrimethylammonium chloride), 100 mM ascorbic acid, 1-2 nm diameter Au nanosphere solution, and 0.5 mM HAuCl4, and purified through centrifugation.

[0114] In this process, CTAC is used as a first surfactant, ascorbic acid as a reducing agent, HAuCl4 as a metal ion, and Au nanospheres with a diameter of 1-2 nm are used as seed particles.

[0115]

[0116] Synthesis of gold nanocube units

[0117] Gold nanocubes were synthesized by mixing appropriate concentrations and volumes of CTAC, sodium bromide (NaBr), 10 nm nanosphere solution, ascorbic acid, and HAuCl4. In the process, CTAC was used as a first surfactant, ascorbic acid as a reducing agent, HAuCl4 as a metal ion, NaBr as a surface protectant, and 10 nm diameter Au nanospheres as seed particles.

[0118] In the above process, nanocubes with controlled size and edge sharpness index were synthesized by controlling the amount of metal ions and the amount of surface protective agent. The nanocubes of the examples were synthesized using the technology of Nano Letters, 18, 6475 (2018) and 'Method for producing metal nanocubes with controlled edge sharpness index', but nanocubes manufactured by other methods can also function as units of the final product, two-dimensional / three-dimensional self-assembled bodies, and are not limited to a specific method.

[0119]

[0120] Fabrication of two-dimensional nanocube self-assemblies

[0121] A self-assembly solution was prepared by mixing CTAB, BDAC, and nanocube solutions. The self-assembly solution was placed on a base substrate and aged for about three hours at room temperature and high humidity to form a self-assembly. The base substrate used, a silicon wafer, was sonicated in the order of acetone, ethanol, and distilled water, and then dried with nitrogen. To confirm that the self-assembly formation was not limited by the structural factors of the unit, self-assembly was formed by controlling the size and sharpness. According to the above-described principle, an appropriate coagulant concentration was used according to the size and shape of the nanocube unit. Two-dimensional self-assembly was formed on a silicon wafer using nanocube units of various sizes and sharpnesses, and two-dimensional nanocube self-assemblies were obtained by washing away the floating nanocube units and the second surfactant through a high-concentration coagulant solution.

[0122] FIG. 2 is a front-view SEM image (a), a side-view SEM image (b) of a SERS substrate including single-layer nanocubes according to one embodiment of the present invention, and a GISAXS (grazing-incidence small-angle scattering) spectrum graph measured to confirm the periodicity of the SERS substrate.

[0123]

[0124] FIG. 3 is an image showing calculated and experimental values ​​(a) of the UV-Vis spectrum of a SERS substrate according to one embodiment of the present invention and an electric field distribution (b) at the plasmon peak (801 nm) of the SERS substrate.

[0125]

[0126] Fabrication of three-dimensional nanocube self-assemblies

[0127] The base substrate was made of glass, and ultrasonic treatment was performed in the same manner as the silicon wafer used for forming a two-dimensional self-assembly. At this time, CTAB, as a second surfactant, has the same effect as the first surfactant and, as explained in the principle above, is adsorbed onto the nanocube units and the self-assembly forming substrate, thereby changing the surface properties of the self-assembly forming substrate.

[0128] Additionally, BDAC acts as a depleting agent, causing the 50 nm nanocubes, which are the unit units, to aggregate and grow into self-assemblies. The self-assembly solution was placed on a surface-modified self-assembly forming substrate, and self-assemblies were formed after an appropriate aging time under room temperature and high humidity conditions.

[0129] When the substrate for forming self-assemblies is a silicon wafer, it was confirmed that a single-layer two-dimensional nanocube self-assembly was formed under appropriate CTAB and BDAC conditions. In contrast, when the substrate for forming self-assemblies is glass, only three-dimensional self-assemblies were confirmed to be formed.

[0130]

[0131] The advantages of the present invention obtained through this experimental process are as follows.

[0132]

[0133] Ease of production

[0134] NAM, a self-assembly technology based on defect-induced aggregation via simple spotting, is fabricated via defect-induced aggregation, and self-assembly is induced in solution. Since NAM is fabricated by simply spotting a self-assembly forming solution onto a substrate on which self-assembly will be formed, there is no need for specialized equipment requiring a high vacuum environment like top-down processing, and it can be fabricated at low cost under room temperature and pressure conditions.

[0135] In addition, the nanostructures produced through these examples were formed over a large area with a high integration rate and had nanogaps of 10 nm or less at regular intervals, and the integration area could be freely controlled depending on the spotting area.

[0136] Therefore, it was confirmed that, unlike existing technologies, this technology can uniformly and easily produce nanostructures with gaps of several nanometers over a large area at low cost and can freely control the contact area.

[0137]

[0138] High signal reproducibility

[0139] To induce highly crystalline self-assemblies free of structural defects, benzyldimethylhexadecylammonium chloride (BDAC) and hexadecyltrimethylammonium bromide (CTAB) molecules were introduced to form a charged bilayer on the surface of the charged depletant and building blocks. These molecules generate electrostatic repulsion between the building blocks and can induce toggling interactions during self-assembly. These toggling interactions guide the building blocks to energetically stable positions through repeated assembly and disassembly. The highly crystalline self-assemblies exhibited few structural defects, as confirmed by the patterns and peaks observed in the electron microscope reciprocal lattice image and the peaks observed in the GI-SAXS spectrum. In addition, signal amplification is significantly affected by structural defects at hot spots. The highly crystalline, defect-free NAM substrate exhibited a signal deviation of up to 6.5% at 611.5 cm-1 as confirmed by rhodamine 6G (R6G) molecules.

[0140]

[0141] FIG. 4 is a graph showing the Raman spectrum of a rhodamine 6G molecule measured after correction with an internal standard material of a SERS substrate according to one embodiment of the present invention and the enhancement factor on the SERS substrate.

[0142] To verify its utility as a SERS substrate, the Raman spectrum of rhodamine 6G (R6G) molecules was measured. To compensate for equipment errors, a silicon wafer, the bottom surface of the NAM substrate, was used as an internal standard. The peak of the internal standard, 520.5 cm -1The signal position was adjusted based on the internal standard and the signal intensity was normalized for correction. The spectrum corrected with the internal standard showed high reproducibility. All major peaks of R6G showed signal deviations of less than 10%, and among them, 612 cm -1 In the commercially available substrates, the signal deviation was not presented or was published as a literature value. Considering that the signal deviation quantified with Raman-active molecules such as R6G in the literature is about 14% (methylene blue) for Hammatsu's product, about 16% (methylene blue) for EnSpectr's product, and 11.3% (rhodamine 6G) for the 3D multilayer structure of the Korea Institute of Machinery and Materials, the NAM substrate can be said to be a SERS substrate with considerably high reproducibility. In addition, the amplification factor quantified through the six characteristic peaks of the R6G molecule is on average 3.1x10 6 It was confirmed by ship. 10 7 The amplification rate was judged to be a fairly high value, as it could enable single molecule measurement.

[0143]

[0144] Signal amplification using nano gaps of less than 10 nm

[0145] The crystal structure and high crystallinity of the SERS substrate can have a positive effect on signal amplification. The lattice structure of the nanocubes, the building blocks of the NAM, was confirmed to be a square lattice, and the entire region of the nanogaps (less than 10 nm) formed between these nanocubes was confirmed to be a hotspot capable of signal amplification.

[0146] This structure has a hotspot density that is approximately 171 times higher than that of nanosphere self-assemblies having the same particle size and lattice structure, and approximately 150 times higher than that of nanosphere self-assemblies arranged in a honeycomb structure. In other words, the SERS substrate proposed in the present invention can secure a higher analyte accommodation space than existing nanosphere-based SERS substrates.

[0147] The inventors conducted an experiment to maximize signal amplification by adjusting the size of the building block to match the resonance wavelength with the commercially available laser wavelength (785 nm), and as a result, the signal was increased by about 10 6 It was confirmed that the amplification was approximately 10 times.

[0148]

[0149] Confirmation of the NaBH4 washing effect

[0150] In addition, the inventors confirmed the possibility of label-free detection of biomolecules through the SERS substrate of the above example. (i) Water containing eight types of biomolecules and (ii) water not containing biomolecules were sampled side by side on the SERS substrate (comparative example) that had not been washed according to the above example, and the Raman signals were measured. Even when washed using O2 plasma, the Raman spectra of the sample containing eight types of biomolecules and the Raman spectra of the sample containing only water did not differ significantly, and only slight changes that were almost impossible to distinguish with the naked eye were observed.

[0151] Figure 5 is an image showing the Raman spectrum of various biomolecules on a SERS substrate simply treated with O2 plasma (left) and the Raman spectrum of BDAC (benzyldimethyammonium chloride) molecules used as a coagulant on a SERS substrate washed only with purified water (DIW) (right), which corresponds to a comparative example of the present invention.

[0152] 1025 cm in BDAC molecule -1Given that the strong ring breathing mode of benzene-based molecules was observed, it was assumed that most of the signals originated from BDAC molecules used as coagulants.

[0153]

[0154] To distinguish the sample from the comparative examples described above, the sample was prepared by performing a biomolecule label-free detection step after the NaBH4 washing step. Raman spectra were then measured, and results distinct from the comparative examples described above were obtained.

[0155] FIG. 6 is an image showing Raman spectra of 12 biomolecules through a SERS substrate whose surface was washed with NaBH4 according to an embodiment of the present invention, in contrast to FIG. 5.

[0156] Figure 7 is a table showing the Raman peak values ​​of biomolecules measured in the Raman spectrum of Figure 6.

[0157]

[0158] FIG. 8 is an image comparing the Raman spectra of a SERS substrate before (gray dotted line) and after (red solid line) washing with NaBH4 according to one embodiment of the present invention.

[0159] When washing with NaBH4, it was confirmed that the intensity of the Raman spectrum generated by the depletant decreased overall. The difference was 1000 cm -1 It can be distinguished by the change in peak intensity near 1000 cm -1 The nearby peak corresponds to the breathing mode of the benzyl group of the depletant BDAC molecule (blue shaded in the left graph). This peak is not observed in the Raman spectra of other molecules without a benzene ring.

[0160]

[0161] Fig. 9 is 1000 cm from Fig. 8 -1This is a box plot image comparing the intensity changes of nearby Raman spectrum signals.

[0162] Quantitatively, as shown in the box plot of Figure 9, the intensity before and after washing differed by an average of more than 12 times, and the t-test results confirmed that there was a statistically significant difference.

[0163]

[0164] The above description is merely an illustrative example of the technical idea of ​​the present invention, and those skilled in the art will appreciate that various modifications and variations can be made without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are intended to illustrate rather than limit the technical idea of ​​the present invention, and the scope of the technical idea of ​​the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Claims

1. Base substrate; and A plurality of nano-cube aggregates arranged at intervals of several nm on the substrate; The above nanocube surface is washed and noise is removed from the Raman spectrum. Surface-cleaned nanocube assembly SERS substrate.

2. In paragraph 1, The above surface cleaning was performed using sodium borohydride (NaBH4). Surface-cleaned nanocube assembly SERS substrate.

3. In paragraph 1, The above SERS substrate has a Raman spectrum of 950 cm -1 1100 cm inland -1 The peak intensity in is less than twice the largest peak intensity among the peaks outside the above region, Surface-cleaned nanocube assembly SERS substrate.

4. In paragraph 1, The above nanocube aggregate comprises an array of single-layer or multi-layer nanocubes, Surface-cleaned nanocube assembly SERS substrate.

5. In paragraph 1, The above nano cube aggregate comprises a self-assembly formed by interaction with a coagulant and a surfactant. Surface-cleaned nanocube assembly SERS substrate.

6. In paragraph 1, The above nano cube aggregate is determined to have a two-dimensional single-layer structure or a three-dimensional double-layer structure depending on the surface roughness of the substrate. Surface-cleaned nanocube assembly SERS substrate.

7. In paragraph 1, The base substrate comprises one of a silicon wafer, a metal-deposited surface, mica, glass and quartz slide. Surface-cleaned nanocube assembly SERS substrate.

8. In paragraph 1, The above SERS substrate has a plasmon resonance in the range of 750 nm to 900 nm. Surface-cleaned nanocube assembly SERS substrate.

9. In optical materials, Including the substrate of paragraph 1, The above optical member is one of a color filter, a fluorescent light-emitting member, and a molecular signal amplifier. An optical member comprising a SERS substrate of nanocube assembly having a washed surface.

10. In the case of materials for nano-processing, Using the substrate of the first clause as a patterning mask, A nanoprocessing component comprising a SERS substrate of nanocube assembly having a washed surface.

11. A method for manufacturing a nanocube assembly SERS substrate, A step of applying a first solution containing a surfactant and a coagulant and a second solution containing metal nanocube units onto a substrate and forming a nanocube self-assembly; and A step of washing away residues of one or more surfactants and coagulants on the surface of the nanocube self-assembly using a reduction and desorption method; Method for manufacturing a SERS substrate of nanocube assembly with a washed surface.

12. In paragraph 11, The above washing step is, Including washing the substrate on which the above nanocube self-assembly is formed with sodium borohydride (NaBH4). Method for manufacturing a SERS substrate of nanocube assembly with a washed surface.

13. In paragraph 11, The above washing step includes a step of washing the substrate on which the nanocube self-assembly is formed with oxygen plasma. Method for manufacturing a SERS substrate of nanocube assembly with a washed surface.

14. Step of preparing a SERS substrate including a nanocube assembly; a step of washing the surface of the SERS substrate; and A step of obtaining a Raman spectrum after dropping a solution or plasma containing biomolecules on the surface of the washed SERS substrate; A label-free analysis method for biomolecules and human-derived substances using a surface-washed nanocube assembly SERS substrate.

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