Patent ID: 12203863

BEST MODE

The present invention will now be described more fully with reference to the accompanying drawings and contents disclosed in the drawings. However, the present invention should not be construed as limited to the exemplary embodiments described herein.

The terms used in the present specification are used to explain a specific exemplary embodiment and not to limit the present inventive concept. Thus, the expression of singularity in the present specification includes the expression of plurality unless clearly specified otherwise in context. It will be further understood that the terms “comprise” and/or “comprising”, when used in this specification, specify the presence of stated components and/or steps, but do not preclude the presence or addition of one or more other components and/or steps thereof.

It should not be understood that arbitrary aspects or designs disclosed in “embodiments”, “examples”, “aspects”, etc. used in the specification are more satisfactory or advantageous than other aspects or designs.

In addition, the expression “or” means “inclusive or” rather than “exclusive or”. That is, unless otherwise mentioned or clearly inferred from context, the expression “x uses a or b” means any one of natural inclusive permutations.

In addition, as used in the description of the disclosure and the appended claims, the singular form “a” or “an” is intended to include the plural forms as well, unless context clearly indicates otherwise.

Although terms used in the specification are selected from terms generally used in related technical fields, other terms may be used according to technical development and/or due to change, practices, priorities of technicians, etc. Therefore, it should not be understood that terms used below limit the technical spirit of the present invention, and it should be understood that the terms are exemplified to describe embodiments of the present invention.

Also, some of the terms used herein may be arbitrarily chosen by the present applicant. In this case, these terms are defined in detail below. Accordingly, the specific terms used herein should be understood based on the unique meanings thereof and the whole context of the present invention.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present invention, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

In addition, in the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention unclear. The terms used in the specification are defined in consideration of functions used in the present invention, and can be changed according to the intent or conventionally used methods of clients, operators, and users. Accordingly, definitions of the terms should be understood on the basis of the entire description of the present specification.

A carboxylic acid-functionalized 3-dimensional SERS substrate according to the present invention is fabricated by functionalizing a 3-dimensional nanostructure, formed by laminating a metal nanowire array on a substrate several times, into the form of carboxylic acid. As the functionalized carboxylic acid binds to a target analyte, the presence or absence of the target analyte may be determined by checking a peak corresponding to the target analyte during SERS analysis.

FIG.1illustrates a perspective view of a carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

Referring toFIG.1, a carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention includes a substrate110, a 3-dimensional nanostructure120and a functionalized carboxylic acid derived from the 3-dimensional nanostructure120.

The substrate110is a working surface on which the 3-dimensional nanostructure120according to an embodiment of the present invention is to be formed.

The substrate110may be formed of, without being limited to, any of a polymer film, glass and a ceramic material.

In accordance with an embodiment, the substrate110may be coated with any one of gold (Au), silver (Ag), copper (Cu), nickel (Ni), platinum (Pt), chromium (Cr), cobalt (Co), and palladium (Pd).

The 3-dimensional nanostructure120according to an embodiment of the present invention may be formed on the substrate110.

The 3-dimensional nanostructure120includes a multistacked metal nanowire array121formed by alternately and repeatedly transfer printing a single-layer metal nanowire array121, laminated on the polymer mold220on which a pattern of a master mold210is duplicated, onto the substrate110to be perpendicular to each other.

In accordance with an embodiment, the polymer mold220may be formed of PMMA.

In accordance with an embodiment, metal (221) nanowires of the metal nanowire array121may have a diameter of 25 nm to 50 nm.

In accordance with an embodiment, the metal nanowire array121may be formed of the metal (221) nanowires having the same or different diameters.

In accordance with an embodiment, the 3-dimensional nanostructure120may be formed by laminating the metal nanowire array1214 to 10 times.

In accordance with an embodiment, the 3-dimensional nanostructure120may be formed of, without being limited to, any one of gold (Au), silver (Ag), copper (Cu), nickel (Ni), platinum (Pt), chromium (Cr), cobalt (Co), palladium (Pd) and an alloy of gold and silver.

The target analyte130is immobilized on the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention, and a laser irradiated from an objective lens10may detect a target analyte130using light scattered by the target analyte130, i.e., a Raman analysis signal through SERS measurement.

Hereinafter, a process of forming the 3-dimensional nanostructure120according to an embodiment of the present invention is described with reference toFIG.2.

FIG.2is a schematic diagram illustrating a process of forming a 3-dimensional nanostructure on a substrate according to an embodiment of the present invention.

Referring toFIG.2, a polymer material is deposited on the master mold210to form the polymer mold220on which an engraved concave and convex pattern is formed.

In accordance with an embodiment, a thin polymer film is coated on the master mold210on which the pattern has been formed, and then the thin polymer film may be manufactured in the form of an adhesive film-attached thin polymer film using an adhesive film by the polymer mold220.

Specifically, an adhesive film may be uniformly attached to one surface of the thin polymer film formed on the master mold210, and then the adhesive film-attached thin polymer film may be separated from the master mold210to manufacture the polymer mold220.

The thin polymer film is applied to the master mold210using at least one process of at least one process of spin coating, deep coating and spray coating, thereby having a concave and convex pattern.

In accordance with an embodiment, a polymer applied to the thin polymer film may have a solubility parameter of 20 MPa1/2to 40 MPa1/2and a glass transition temperature higher than room temperature.

Accordingly, the polymer may stably maintain a solid state at room temperature.

Next, a metal (221) material is selectively deposited on the engraved pattern of the polymer mold220to form the single-layer metal nanowire array121.

The polymer mold220on which the single-layer metal nanowire array121has been formed is transfer printed onto the substrate110.

Specifically, after positioning the polymer mold220such that the single-layer metal nanowire array121is in contact with the substrate110, the polymer mold220is only removed so that the single-layer metal nanowire array121is formed on the substrate110.

Specifically, an organic solvent vapor may be injected between an adhesive film and the polymer mold220to weaken adhesive force between the adhesive film and the polymer mold220.

Accordingly, the polymer mold220may be separated so that the metal nanowire array121remains on the substrate110.

In accordance with an embodiment, the polymer mold220and adhesive film on which the metal nanowire array121has been formed may be brought in contact with the polymer pad such that the metal nanowire array121is in contact with the polymer pad (not shown).

Next, the polymer mold220and the adhesive film may be separated from the polymer pad so that the metal nanowire array121remains on the polymer pad.

Next, the polymer pad on which the metal nanowire array121remains is brought in contact with the substrate110such that the metal nanowire array121is in contact with the substrate110, and then the polymer pad is separated from the substrate110to transfer print the metal nanowire array121.

The 3-dimensional nanostructure120may be formed to include the multistacked metal nanowire arrays121by repeating the aforementioned process such that the metal nanowire arrays121are to be perpendicular to each other.

FIG.3illustrates a sectional view of a master mold according to an embodiment of the present invention.

Referring toFIG.3, the master mold210may have concave and convex patterns having different heights.

The pattern of the master mold210may be formed using a patterning process including at least one of photolithography, block co-polymer self-assembly-based lithography and E-beam lithography, and a reactive ion etching (RIE) process.

The master mold210may be manufactured by directed self-assembly (DSA) of a block co-polymer (BCP).

In accordance with an embodiment, the master mold210may be coated with a PDMS brush polymer with low surface energy or a hydrophobic self-assembled monolayer (SAM) such as hexa methylene di silazane (HMDS) so that a surface of the master mold210has a low surface energy of 30 mJ/m2or less.

In accordance with an embodiment, the master mold210may be manufacture to have a concave and convex pattern with a line width of 15 nm and a line width of 8 nm by self-assembling a PS-PDMS block co-polymer on a silicon trench substrate110with a width of 1 um to 1 cm and a depth of 1 nm to 1 cm to form a linear surface pattern, and then by performing RIE process in an oxygen environment.

In accordance with an embodiment, the master mold210may be, without being limited to, a silicon wafer.

A specific fabrication method of the master mold210is described below with reference to the following examples.

FIG.4aillustrates a scanning electron microscope (SEM) image of a single-layered 3-dimensional nanostructure according to an embodiment of the present invention taken from a plane,FIG.4billustrates an SEM image, taken from a plane, of a 3-dimensional nanostructure formed by laminating a metal nanowire array according to an embodiment of the present invention twice, andFIG.4cillustrates an SEM image, taken from a plane, of a 3-dimensional nanostructure formed by laminating a metal nanowire array according to an embodiment of the present invention four times.

Referring toFIGS.4ato4c, the 3-dimensional nanostructure120according to an embodiment of the present invention is formed by alternately and repeatedly laminating the metal nanowire array121to be perpendicular to each other, thereby having a fabric-like shape when viewed from a plane.

Here, the metal (221) nanowires shown as relatively dark shades are metal (221) nanowires having a relatively large diameter.

A wave vector of a laser irradiated from the objective lens10is perpendicular to a surface of the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention.

By the laser, the free electron cloud of the metal (221) nanowires of the 3-dimensional nanostructure120horizontally vibrates.

In addition, a strong localized electromagnetic field is generated on a side of the metal (221) nanowires by the laser.

Since the metal nanowire arrays121of the 3-dimensional nanostructure120are respectively laminated at an angle of 90 degrees, the polarization dependence of excitation laser is negligible.

When a surface of the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention is observed by a scattering-type near-field optical microscope (s-NSOM), the carboxylic acid-functionalized 3-dimensional SERS substrate100shows surface plasmon polariton (SPP).

The scattering-type near-field optical microscope (s-NSOM), surface plasmon polariton visualizes how SPP behaves depending on important parameters.

Here, the surface plasmon polariton increases as the period of the metal (221) nanowires decreases and the number of laminations of the metal nanowire array121increases.

Accordingly, the 3-dimensional nanostructure120formed by cross-laminating the metal nanowire array121to be perpendicular to each other may variously polarize light to produce the maximum plasmon effect and may increase a Raman analysis signal generated from the 3-dimensional nanostructure120.

In general, it is preferable to remove a residue of the polymer mold220existing on the surface by washing with acetone after transfer printing. However, it is almost impossible to completely remove the residue of the polymer mold220.

As the single-layer metal nanowire array121laminated on the polymer mold220is transfer printed onto the substrate110, a portion of the polymer mold220may remain on the surface of the metal nanowire array121.

Specifically, after the single-layer metal nanowire array121laminated on the polymer mold220is brought in contact with the substrate110, a residue of the polymer mold220may remain on the surface of the metal nanowire array121when the polymer mold220is separated from the metal nanowire array121in contact with the substrate110.

Accordingly, a residue of the polymer mold220may remain on the surface of the 3-dimensional nanostructure120formed by alternately and repeatedly laminating the metal nanowire array121, on which a residue of the polymer mold220remains, on the substrate110to be perpendicular to each other.

In accordance with an embodiment, a PMMA thin film, as the polymer mold220, absorbs energy from light and is easily cured by cross-linking or graphitization.

Accordingly, once the PMMA thin film is cured, it is easy to peel the PMMA thin film from the substrate110.

The metal nanowire array121formed on the polymer mold220may transfer thermal energy to the polymer mold220and may be cross-linked and graphitized.

FIG.5illustrates a Raman spectrum of a 3-dimensional nanostructure according to an embodiment of the present invention.

Here, the Raman spectrum of the 3-dimensional nanostructure120according to an embodiment of the present invention ofFIG.5illustrates D and G bands of graphite.

In the image inserted inFIG.5, a left circle denotes a location at which a Raman signal is measured.

Referring toFIG.5, Raman analysis signals (Raman signal in normal pattern) of general surfaces of the 3-dimensional nanostructure120have a constant intensity.

However, in can be confirmed that, when a residue of the PMMA thin film as the polymer mold220remains on the surface of the 3-dimensional nanostructure120, the Raman analysis signal (Raman signal in crumpled pattern) intensity is very large compared to those of general surfaces of the 3-dimensional nanostructure120.

Specifically, a strong peak is observed between about 1400 cm−1and about 1700 cm−1, which is due to the PMMA thin film residue present on the surface of the 3-dimensional nanostructure120.

The residue of the polymer mold220present on the 3-dimensional nanostructure120may be functionalized into a carboxylic acid through a functionalization process, thereby immobilizing the target analyte130.

Specifically, the residue of the thin polymer film present on the 3-dimensional nanostructure120may be functionalized into carboxylic acid by a process of reaction ion etching (RIE) the thin polymer film.

Hereinafter, functionalization of the 3-dimensional nanostructure120is described in detail with reference toFIG.6.

FIG.6is a schematic diagram illustrating a process of functionalizing a surface of a 3-dimensional nanostructure according to an embodiment of the present invention into carboxylic acid.

Referring toFIG.6, a residue of the polymer mold220present on the 3-dimensional nanostructure120has a —COOCH3functional group.

The redox reaction of the residue of the polymer mold220cured on the surface of the 3-dimensional nanostructure120occurs by an RIE process using an oxygen gas131so that CH3in —COOCH3of the residue of the polymer mold220is substituted with H and thus functionalized into carboxylic acid (—COOH).

The carboxylic acid-functionalized 3-dimensional nanostructure120according to an embodiment of the present invention may be coupled with an amine to immobilize the target analyte130.

FIG.7is a schematic diagram illustrating an amine coupling process of a carboxylic acid-functionalized 3-dimensional nanostructure according to an embodiment of the present invention.

Referring toFIG.7, a carboxylic acid-functionalized the 3-dimensional nanostructure120is modified to have an amine group through an amine coupling reaction according to an example, thereby immobilizing the target analyte130.

The amine coupling reaction may be performed by reacting a coupling agent including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) with the functionalized carboxylic acid.

In accordance with an embodiment, the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention may be supported in the coupling agent solution so that an amine coupling reaction occurs and modification into an amine group occurs.

An amine-coupled 3-dimensional SERS substrate110binds the target analyte130to immobilize the target analyte130.

FIG.8ais an optical microscope image illustrating a state in which an amine-coupled 3-dimensional nanostructure according to an embodiment of the present invention is dip-coated on a target analyte and combined with the target analyte, andFIG.8bis an optical microscope image illustrating a state in which the amine-coupled 3-dimensional nanostructure according to an embodiment of the present invention is drop-coated on a target analyte and combined with the target analyte.

Referring toFIGS.8aand8b, in the case of dip coating, a target analyte is immobilized by an amine coupling reaction in a petri dish. In this case, it can be confirmed that the amine-coupled 3-dimensional nanostructure is cleanly coated without a white precipitate on a surface thereof.

However, in the case of drop-coated, a target analyte is dropped using a pipette without an amine coupling reaction. In this case, it can be confirmed that the target analyte is thickly aggregated on the carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

In accordance with an embodiment, the target analyte130may refer to a biological protein.

Specifically, the target analyte130may include, without being limited to, β-amyloid or tau protein used to diagnose Alzheimer's disease.

According to an embodiment, when the target analyte130is β-amyloid or tau protein, the presence or absence of β-amyloid or tau protein may be determined through binding of the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention to the target analyte130.

Accordingly, the presence or absence of β-amyloid or tau protein can be confirmed according to a Raman analysis signal generated through SERS using the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention, thereby being capable of diagnosing Alzheimer's.

Specifically, a Raman analysis signal generated from the 3-dimensional nanostructure120may include fibrillation information on the target analyte130.

In accordance with an embodiment, the amine coupling reaction may be performed at 4° C. to 10° C. for 4 hours to 8 hours.

When a target analyte according to an embodiment of the present invention is β-amyloid, the target analyte may become easily fibrous. To prevent fibrosis, it is preferred to perform an amine coupling reaction at a low temperature such as 4° C. to 10° C.

When the target analyte130is subjected to SERS measurement using the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention, the concentration of the target analyte130may be 10−12M or more.

In other words, when the target analyte130is subjected to SERS measurement using the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention, a detection limit concentration of the target analyte130may be 10−12M.

The β-amyloid protein known as the cause of Alzheimer's disease has an α-helix structure when it is normal.

However, due to some cause, the α-helix structure of the β-amyloid protein is denatured into a β-sheet form.

As the β-amyloid of the α-helix structure and β-amyloid of the β-sheet structure are agglomerated with each other, β-amyloid monomers are aggregated into oligomers, protofibrils or fibrils to cause dementia symptoms.

Accordingly, a process that monomers of β-amyloid or tau protein change into oligomers may be traced according to a Raman analysis signal generated through SERS using the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention.

FIG.9ais a TEM image illustrating monomers of a target analyte according to an embodiment of the present invention, andFIG.9billustrates a Raman spectrum of monomers of a target analyte according to an embodiment of the present invention.

Referring toFIGS.9aand9b, it can be confirmed that when the target analyte130bonded to the carboxylic acid-functionalized 3-dimensional SERS substrate100is a monomer, a Raman analysis signal peak in the α-helix structure is higher than that in the β-sheet.

FIG.10ais a TEM image illustrating oligomer type 1 of a target analyte according to an embodiment of the present invention, andFIG.10billustrates a Raman spectrum of oligomer type 1 of the target analyte according to an embodiment of the present invention.

Referring toFIGS.10aand10b, it can be confirmed that when a target analyte130bonded to a carboxylic acid-functionalized 3-dimensional SERS substrate100is partially agglomerated and is oligomer type 1, a Raman analysis signal peak in the β-sheet structure is slightly higher than a Raman analysis signal peak in the α-helix structure.

In addition, it can be confirmed that when the target analyte130is oligomer type 1, a Raman analysis signal peak in the β-sheet structure is higher than a Raman analysis signal peak when the target analyte130is a monomer.

FIG.11ais a TEM image illustrating oligomer type 2 of a target analyte according to an embodiment of the present invention, andFIG.11billustrates a Raman spectrum of oligomer type 2 of the target analyte according to an embodiment of the present invention.

Referring toFIGS.11aand11b, it can be confirmed that when a target analyte130bonded to a carboxylic acid-functionalized 3-dimensional SERS substrate100is more agglomerated than oligomer type 1 and thus becomes oligomer type 2, a Raman analysis signal peak in the β-sheet structure is remarkably higher than a Raman analysis signal peak in the α-helix structure.

In addition, it can be confirmed that when the target analyte130is oligomer type 2, a Raman analysis signal peak in the β-sheet structure is higher than a Raman analysis signal peak when the target analyte130is a monomer or oligomer type 1.

FIG.12is a TEM image illustrating a protofibril shape of a target analyte according to an embodiment of the present invention.

Referring toFIG.12, it can be confirmed that the target analyte is further agglomerated beyond the state of oligomer type 2 to form a protofibril.

FIG.13is a TEM image illustrating a fibril shape of a target analyte according to an embodiment of the present invention.

Referring toFIG.13, the target analyte130is further agglomerated beyond a protofibril state to form a fibril shape.

Accordingly, fibrillation information on the target analyte130may be obtained according to a Raman analysis signal generated through SERS using the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention.

Specifically, it may be confirmed from a Raman analysis signal generated through SERS using the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention how much the oligomer, protofibril or fibril of the target analyte130has progressed.

For a theoretical understanding of the Raman measurement results, it is desirable to view SERS analysis from the point of view of electromagnetic enhancement (EM) which is known to be a more important factor than chemical enhancement.

Incident electromagnetic radiation induces vibration dipoles in nanowires, and the dipoles intensify an incident laser by creating a strong local electric field on a surface.

At the same time, a scattered beam caused by vibrational transition of the target analyte130is enhanced by the same mechanism as an incident beam.

SERS enhancement considering the two contributions may be expressed by Equation 1 below:

Electromagnetically⁢enhanced⁢SERS≅MLoc⁡(wL)⁢MLoc⁡(wR)[Equation⁢1]

In Equation 1, MLoc(wL)and MLoc(wR)are enhancement factors (EF) of local electric field strength at an excitation laser frequency and Raman frequency.

However, the target analyte130is not directly located in an enhanced local electromagnetic field region such as a laser-irradiated point.

Instead, the target analyte130is at most 50 nm away from the carboxylic acid-functionalized 3-dimensional SERS substrate100.

It was found by prior studies that SERS intensity has a distance dependence of 1/r10(r is a distance from a surface of the substrate110to the target analyte130).

Nevertheless, the carboxylic acid-functionalized 3-dimensional SERS substrate100enables excellent SERS improvement because the metal nanowire array121constituting the three-dimensional nanostructure120is spread over a wide area.

Accordingly, most of the incident laser and scattered beam induce vibration dipoles and can contribute to enhancing the Raman analysis signal.

This can be proved by calculating reinforcement factors.

Among calculation methods of various enhancement factors such as a single-molecule enhancement factor (SMEF), a SERS substrate enhancement factor (SSEF), and an analytical enhancement factor (AEF), AEF is the most appropriate index for the carboxylic acid-functionalized 3-dimensional SERS substrate100.

AEF may be expressed by Equation 2 below:
AEF=ISERS/I×C/CSERS[Equation 2]

In Equation 2, ISERSand CSERSrepresent the strength and concentration of the molecules of the target analyte130on the carboxylic acid-functionalized 3-dimensional SERS substrate100, and IRMand CRMrepresent the strength and number of the molecules of the target analyte130on the substrate110.

When ISERS=921, CSERS=10−9M, IRM=166, and CRM=10−4M, AEF is estimated to be 5.5×105.

This AEF value is the highest AEF value of the carboxylic acid-functionalized 3-dimensional SERS substrate100when a material constituting the substrate110is gold.

In addition, according to a prior report by Mubeen et. al., it can be seen that Raman analysis signal intensity is increased when a bottom surface of the substrate110is a thin gold film compared to when it is made of silicon.

In addition, according to prior reports, it can be confirmed that a gold substrate110can effectively reflect an excitation beam and a scattered beam, thereby contributing to the enhancement of a Raman signal.

Accordingly, it is desirable to use a gold substrate110as the substrate110of the carboxylic acid-functionalized 3-dimensional SERS substrate100according to an embodiment of the present invention.

Hereinafter, the present invention is described in more detail with reference to examples of demonstrating the characteristics and effects of the carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention. The following examples are only presented to experimentally prove the effects of the present invention, and the present invention is not limited by the following examples.

EXAMPLE

Master Mold Fabrication

A concave and convex pattern of a master mold was formed by direct self-assembly (DSA) of a block co-polymer.

A silicon wafer was patterned using KrF photolithography to prepare a guiding pattern having a depth of 40 nm, a width of 1 μm and a period of 1.25 μm.

Poly(styrene-b-dimethylsiloxane (PS-b-PDMS, molecular weight=48 kgmol−1, fps=66.3%, polydispersity index (PDI)=1.19) purchased from Polymer Source Inc. was used as a block co-polymer.

After contacting PDMS-OH brush (5 kgmol−1, Polymer Source Inc.) with the patterned silicon wafer, annealing was performed at 150° C. for 90 minutes in a vacuum chamber, and then an unreacted block co-polymer was removed by washing with toluene.

Next, PS-b-PDMS dissolved in toluene (0.8 wt %) was coated on the patterned silicon wafer.

BCP was solvent-annealed at room temperature for 12 hours using toluene vapor, and reactive ion etching (RIE) using CF4plasma (source power of 50 W, etching for 21 seconds) and O2plasma (source power of 60 W, etching for 30 seconds) was performed.

Operation pressure and gas flow rate were maintained at 15 mTorr and 30 sccm, respectively.

A master mold pattern has a trench width of 1 μm, a mesa sidewall width of 0.25 μm, and BCP stripes of a trench.

A master mold was manufactured to include 28 BCP stripes with a diameter of 15 nm and an interval of 20 nm.

Carboxylic Acid-Functionalized 3-Dimensional SERS Substrate Fabrication through Transfer Printing

A 3-dimensional nanostructure was formed using a simple transfer printing process.

A PMMA solution composed of 4 wt % PMMA (MW 100,000) dissolved in a mixture of acetone and toluene was spin-coated on a master mold to be replicated.

The replicated PMMA was peeled off using a polyimide (PI) adhesive film.

Gold (Au) nanowires were formed by angle deposition of gold via electron beam evaporation on a PMMA replica.

Next, the gold nanowires were exposed to a vapor mixture of acetone and heptane for 20 seconds.

Because the adhesive force between the PMMA and the PI films was weakened due to the previous solvent annealing step, a single layer of gold nanowires was transfer printed to a PDMS pad, which is a polymer pad, to form a 3-dimensional nanostructure.

The substrate was a p-type Si wafer (Si substrate) or thin gold film-deposited p-type Si wafer (Au substrate).

Next, the 3-dimensional nanostructure was functionalized into carboxylic acid by an RIE process using an oxygen gas.

Accordingly, a carboxylic acid-functionalized 3-dimensional SERS substrate was fabricated.

Amine Coupling

Amine coupling was performed using EDC/NHS.

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents were purchased from Sigma-Aldrich Inc.

EDC 20 mM and NHS 20 mM were dissolved in a (2-(N-morpholino)ethanesulfonic acid (MES) buffer solution and a phosphate-buffered saline (PBS) buffer solution.

After concentrating both the buffer solutions to 1 M, the carboxylic acid-functionalized 3-dimensional SERS substrate was immersed in the buffer solution, cooled, and coupled with amine for 4 hours.

Materials and Characteristics

Both β-amyloid and tau protein were purchased from Sigma-Aldrich Inc.

An aptamer used to prove the superiority of the carboxylic acid-functionalized 3-dimensional SERS substrate was purchased from GenoTech Corp. (Daejeon, Korea).

Dispersive Raman spectroscopy (ARAMIS, Horiba) with excitation lasers at 514 nm and 785 nm was used for SERS measurement.

Raman analysis signals (SERS signals) measured by the dispersive Raman spectroscopy were collected for 5 seconds for the measurement of examples.

An enhanced local electromagnetic field of the carboxylic acid-functionalized 3-dimensional SERS substrate was measured by a scattering scanning near-field optical microscope (s-SNOM, Anasys, Instruments, CA, US) with a quantum cascade laser (QCL), excitation wavelengths were 1200 to 1800 cm−1, and top-view images were obtained using a field emission scanning electron microscope (FE-SEM, Hitachi, S-4800).

Characteristic Evaluation

Confirmation of Amine Coupling and Target Analyte Immobilization

To confirm whether amine coupling was successfully performed and a target analyte was immobilized on a carboxylic acid-functionalized 3-dimensional SERS substrate, an oligonucleotide having an amine at the 5′ end thereof and TAMRA at 3′ end thereof was used.

TAMRA, a type of Raman dye, was used to eliminate the possibility of errors that may occur due to the small Raman cross-section area of a protein.

FIG.14illustrates RIE process time-dependent SERS spectra of a carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

Referring toFIG.14, a surface of the carboxylic acid-functionalized 3-dimensional SERS substrate was etched with O2plasma to have various RIE times (0 sec, 30 sec, 60 sec, 90 sec).

At each RIE time condition, immobilization was carried out under conditions of 90 W source power, 45 W bias power and 15 mTorr gas pressure of 30 sccm flow.

After the RIE process, an amine coupling reaction through EDC/NHS was performed.

A TAMRA peak was not observed in a carboxylic acid-functionalized 3-dimensional SERS substrate which was not subjected to RIE (i.e., RIE time: 0 sec).

A characteristic peak of TAMRA appeared after a surface of the carboxylic acid-functionalized 3-dimensional SERS substrate was etched with O2plasma.

This demonstrates that the amine coupling reaction occurred between the oligonucleotide and the carboxylic acid-functionalized 3-dimensional SERS substrate surface.

The Raman analysis signal rapidly decreased with increasing RIE time, which is probably due to removal of the PMMA residue.

Detection Limit Concentration of Target Analyte

FIG.15illustrates target analyte concentration-dependent SERS spectra of a carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

Referring toFIG.15, a Raman analysis signal of the carboxylic acid-functionalized 3-dimensional SERS substrate was measured while varying the concentration (10−6M, 10−7M, 10−8M, 10−9M, 10−10M) of a target analyte.

As a result of Raman measurement, it was confirmed that a Raman analysis signal peak is higher with increasing concentration of the target analyte.

In addition, it can be confirmed that, when the concentration of the target analyte is 10−10M, a Raman analysis signal peak is hardly visible.

Accordingly, it can be confirmed that a detection limit concentration for a target analyte of the carboxylic acid-functionalized 3-dimensional SERS substrate is 10−10M.

SERS Analysis Dependent Upon Number of Laminated Metal Nanowire Arrays

FIG.16illustrates metal nanowire array lamination number-dependent SERS spectra of a carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

Here, “silicon wafer” spectra represent Raman spectrum results measured while increasing the number of metal nanowire arrays laminated on a silicon wafer.

In addition, “gold substrate” spectra represent Raman spectrum results measured while increasing the number of metal nanowire arrays laminated on a gold substrate.

Referring toFIG.16, in both the silicon wafer and the gold substrate, the optimal lamination number of the metal nanowire arrays is 4.

However, when laminated more than 4 times, a different tendency is shown depending upon a substrate type.

FIG.17illustrates substrate type-dependent SERS intensity of a carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

Here, a SERS analysis signal intensity for each substrate type was measured at 1650 cm−1.

In the case of the silicon wafer, the saturation of Raman analysis signal intensity started immediately after the fourth lamination, but in the case of the gold substrate, Raman analysis signal intensity decreased once significantly and then was saturated.

However, when the number of metal nanowire array layers exceeds four, a thin gold film on the bottom of the gold substrate effectively reflects a laser beam, thereby reducing the ability to improve the Raman analysis signal.

Accordingly, the metal nanowire array is laminated preferably 4 to 10 times, most preferably 4 times, on the substrate.

SERS Analysis of Carboxylic Acid-Functionalized 3-Dimensional SERS Substrate on which β-Amyloid or Tau Protein is Immobilized

Label-free SERS measurement of β-amyloid and tau protein as biomarkers of Alzheimer's was performed using the unique properties of a carboxylic acid-functionalized 3-dimensional SERS substrate.

It is very difficult to obtain Raman spectra directly from proteins due to small Raman cross sections as well as complex chemical components.

However, several Raman spectrum characteristics were confirmed in both β-amyloid and tau protein by using the carboxylic acid-functionalized 3-dimensional SERS substrate.

Similar to the case of an aptamer, β-amyloid was immobilized on a surface of the carboxylic acid-functionalized 3-dimensional SERS substrate by an amine bond.

Since β-amyloid generally aggregates rapidly at room temperature, amine coupling was performed at 4° C. to avoid aggregation of β-amyloid.

A Raman spectrum of β-amyloid was measured in a spectral range of 800 cm−1to 1600 cm−1.

FIG.18illustrates reaction time-dependent SERS spectra of a carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

Here, the reaction time means a time for which the carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention reacts with a target analyte.

Referring toFIG.18, it can be confirmed that a reaction time between β-amyloid, as a target analyte, and the carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention does not change significantly after 4 hours.

In addition, it can be confirmed that various peaks appear in Raman spectra of β-amyloid.

It can be confirmed that in Raman spectra ofFIG.18, the Raman analysis signal peaks in 1270 cm−1band are most prominent.

A broad band of 1200 cm−1to 1340 cm−1designated as amide III includes information on a secondary structure of protein.

An amide III band mainly reflects Cα—C stretching and C═O in-plane bending as well as N—H in-plane bending and C—N stretching.

Together with the amide I band and the amide II band, an amide III band provides information on a change in a protein structure.

However, the amide I band is poorly detected in SERS, and it is difficult to observe the amide II band under near-infrared (˜785 nm) laser excitation due to the small Raman cross-section area of a protein.

The strong peak at 1270 cm−1shows that β-amyloid has an α-helix structure.

FIG.19illustrates β-amyloid concentration-dependent SERS spectra of a carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

Referring toFIG.19, it can be confirmed that the α-helix structure of the β-amyloid structure is no longer dominant as the concentration of β-amyloid increases.

The α-helix structure, which is the original secondary structure of β-amyloid, tends to be misfolded, so that β-amyloid may be oligomerized.

Misfolded β-amyloid may often interact with other normal proteins to cause toxicity.

Since normal β-amyloid with α-helix structure mainly exists when the concentration of 3-amyloid is low, a strong band may be confirmed at 1270 cm−1as a result of SERS analysis.

However, since the number of misfolded β-amyloids increases as the concentration of β-amyloid increases, it can be confirmed that rapid band increase is observed at 1145 cm−1and 1390 cm−1as the concentration of β-amyloid with a deformed α-helix structure increases.

FIG.20illustrates β-amyloid concentration-dependent SERS intensity according to an embodiment of the present invention.

Here, a graph denoted by ▪ represents an intensity at 1270 cm−1, a graph denoted by ● represents an intensity at 1390 cm−1, and a graph denoted by ▴ represents an intensity at 1145 cm−1.

Referring toFIG.20, it can be confirmed that Raman analysis signal peaks become very strong in 1145 cm−1and 1390 cm−1bands as the concentration of β-amyloid increases.

A 1390 cm−1band called amide S band is due to Cα-H bending.

In the amide S band, it can be confirmed that the Raman analysis signal intensity is weak when a protein has a large Cα-H bending such as an α-helix.

However, it can be confirmed that Raman analysis signal intensity is strong in a β-sheet structure or a structure having small Cα-H bending such as a random coil.

In a band at 1145 cm−1due to Cα-C—N modification, it can be confirmed that a Raman analysis signal is intensified when the α-helix structure is deformed.

FIG.21illustrates tau protein concentration-dependent SERS spectra of a carboxylic acid-functionalized 3-dimensional SERS substrate according to an embodiment of the present invention.

In addition to β-amyloid, tau protein, which is another biomarker of Alzheimer's disease, on a surface of the carboxylic acid-functionalized 3-dimensional SERS substrate was observed by SERS.

Tau protein is abundant in the human brain and is involved in stabilizing microtubules.

Abnormal tau phosphorylation causes polypeptides to self-aggregate, resulting in fibrosis.

Tau protein consists of 441 amino acids and thus is the longest protein of six isomers.

Referring toFIG.21, it can be confirmed that, despite the very complex chemical structure of the tau protein, the Raman spectrum thereof shows a peak at the same wavenumber with increasing concentration.

In addition, it can be confirmed that a band, such as an amide band, including structural information does not appear clearly in the Raman spectrum of the tau protein.

The secondary structure of tau protein is naturally unfolded or essentially disordered, which means that the content of secondary structures (α-helices and β-sheets) in the polypeptide chain thereof is low.

Accordingly, it can be confirmed that the amide III band is almost invisible when the concentration of tau protein is high, i.e., at a tau protein concentration of 10−7M.

It can be confirmed that both the appearance of the amide S band at 1390 cm−1and the appearance of the Cα-C—N modified band at 1145 cm−1are due to increase of tau protein with disordered structures.

Compared to β-amyloid, the Raman band of aromatic amino acid is well distinguished in the case of tau protein.

In particular, the phenylalanine (Phe) band of 1000 cm−1is not sensitive to structural changes, so it can be used for quantitative analysis of proteins without labeling.

Other Raman bands due to aromatic amino acids or peptide bonds were assigned based on the existing literature.

As described above, the carboxylic acid-functionalized 3-dimensional SERS substrate to be used for label-free SERS measurement of proteins enables protein structure analysis of SERS by simple spectroscopic analysis.

It can be confirmed that, compared with conventional analysis methods, such as NMR, small X-ray scattering (SAXS), and low-temperature EM, which require delicate sample preparation and complex analytical procedures, SERS measurement using the carboxylic acid-functionalized 3-dimensional SERS substrate according to the present invention has significant advantages.

Although the present invention has been described through limited examples and figures, the present invention is not intended to be limited to the examples. Those skilled in the art will appreciate that various modifications, additions, and substitutions are possible, without departing from the scope and spirit of the invention. Therefore, the scope of the present invention should not be limited by the embodiments, but should be determined by the following claims and equivalents to the following claims.

DESCRIPTION OF SYMBOLS

10: objective lens100: carboxylic acid-functionalized 3-dimensional SERS substrate110: substrate120: 3-dimensional nanostructure121: metal nanowire array130: target analyte131: oxygen gas210: master mold220: polymer mold221: metal atom