KR20190123974A - A biosensor using localized surface plasmon resonance and a method for analyzing biomolecules using the same - Google Patents

Abstract

The present invention relates to a localized surface plasmon resonance-based biosensor and a method for analyzing a biological material using the same, comprising: a substrate; a three-dimensional metal nanostructure arranged on the substrate and shielding an upper surface; and a receptor of an analysis biological material immobilized on a side surface of the metal nanostructure. Therefore, the present invention is capable of remarkably improving a sensitivity when compared to a conventional method.

Description

Biosensor using local surface plasmon resonance and analysis method of biomolecule using same {A BIOSENSOR USING LOCALIZED SURFACE PLASMON RESONANCE AND A METHOD FOR ANALYZING BIOMOLECULES USING THE SAME}

The present invention relates to a biosensor using local surface plasmon resonance and a method for analyzing biomolecules using the same.

Surface plasmons refer to quantized vibrations of free electrons that propagate along a conductor surface, such as the surface of a metal thin film. Such surface plasmons are excited by incident light incident on a metal thin film through a dielectric medium such as a prism at an angle greater than or equal to the critical angle of the dielectric medium, and cause resonance. Surface Plasmon Resonance (SPR) It is called). The surface plasmon generated at this time has a property of propagating several micrometers along the metal thin film, and is also called surface plasmon polariton (SPP).

When using monochromatic incident light, the angle of incidence (resonance angle) of the incident light with resonance and the wavelength at which resonance occurs (resonance wavelength) are very sensitive to the change in refractive index of the material close to the metal thin film. Surface plasmon resonance (SPR) sensors have been used to quantitatively and qualitatively analyze a sample from a change in refractive index of a material, that is, a sample close to a metal thin film using this property.

Meanwhile, metal nanostructures having a size of several nm to several hundred nm, such as nanoparticles (nanoparticles or nanodots), nanorods, or nanoholes, which are made of metal, and not metal thin films, may have a specific frequency ( Wavelength) causes collective oscillation of electrons in the conduction band of the nanostructure, resulting in electrical dipole characteristics. As a result, the light in the frequency region is strongly scattered and absorbed, which is called Localized Surface Plasmon Resonance (LSPR). Scattering and absorption by LSPR, unlike SPP, can measure extinction by simple transmission spectroscopic method without prism or diffraction grating. Line width, absorption center wavelength and the like show a very strong dependence on the type of metal and the size and shape of the metal nanostructure. In addition, similar to SPP, their absorbance properties are sensitive to the external environment of the metal nanostructure, i.e. the complex permittivity of the medium around the surface of the metal nanostructure, which is used to detect biomolecules or chemical components. Research on chemical sensors is being actively conducted.

However, unlike SPP-based bio / chemical sensors, LSPR-based bio / chemical sensors have a very low sensitivity, which is only about 1/10 to 1/100 of SPP-based sensors. The reason is that the range of the electromagnetic field generated by LSPR is only about 1/10 of the SPP. That is, in the case of SPP, the depth of plasmon electromagnetic field penetration from the surface of the metal thin film to the medium to be measured reaches about 200 to 300 nm depending on the wavelength, whereas in the case of LSPR, the depth of electromagnetic field penetration from the surface of the metal nanostructure to the medium is increased. It is only about 20-30 nm. Even more seriously, unlike in the case of the SPP where the electromagnetic field is formed evenly on the surface of the metal thin film, the LSPR has a characteristic that the generated electromagnetic field is concentrated only in a specific area such as the side of the metal nanostructure. If the biological material to be analyzed is uniformly adsorbed on the entire surface of the metal nanostructure with the same probability, there is a problem that causes a significant loss in terms of sensitivity.

Therefore, in order to solve the above problems, there is still a need for research to further improve the sensitivity of the biosensor, in particular, using local surface plasmon resonance.

United States Patent Application Publication No. 2012/0188551

An object of the present invention is to provide a biosensor using a local surface plasmon resonance, the sensitivity of which is significantly improved compared to the conventional method, and a method for analyzing a biomaterial using the same.

In order to achieve the above object, an aspect of the present invention provides a substrate, a metal nanostructure of a three-dimensional structure arranged on the substrate, the upper surface is shielded and the receptor of the analyte biological material immobilized on the side of the metal nanostructure To provide, it provides a topical surface plasmon resonance (LSPR) based biosensor.

In addition, another aspect of the present invention comprises the step of forming a three-dimensional metal nanostructure of the top surface shielded on the substrate, and immobilizing the receptor of the biological material to be analyzed on the side of the metal nanostructure In addition, the present invention provides a method of manufacturing an LSPR-based biosensor.

In addition, another aspect of the present invention is a living body using LSPR comprising the step of processing the biological material to be analyzed in the above-described LSPR-based biosensor and local surface plasmon resonance (LSPR) in the LSPR-based biosensor Provide a method for analyzing the substance.

The local surface plasmon resonance-based biosensor of the present invention intentionally analyzes only the side of the metal nanostructure in which the surface is exposed and the electromagnetic field is concentrated by shielding the upper surface of the three-dimensional metal nanostructure arranged on the substrate. By immobilizing the receptor of the biological material of interest, even a small amount of biological material can be detected or analyzed with a very high sensitivity.

1 is a schematic diagram illustrating an LSPR based biosensor according to an embodiment of the present invention.
2A is a schematic diagram of a metal nanostructure according to an embodiment of the present invention, and (B) occurs in the metal nanostructure of (A) using a Finite-difference time-domain (FDTD) program. This is a graph showing the distribution and intensity of electromagnetic fields.
3 is a schematic view of a metal nanostructure according to Comparative Example 1 of the present invention.
4 is a schematic view of a metal nanostructure according to Comparative Example 2 of the present invention.
5 is a view showing the results of measuring the LSPR signal for each AFP concentration in Examples and Comparative Examples 1 and 2 of the present invention.

Hereinafter, the present invention will be described in detail.

1. Topical Surface Plasmon  resonance( LSPR A) based biosensor

One aspect of the invention provides a topical surface plasmon resonance based biosensor.

The LSPR-based biosensor includes a substrate, a metal nanostructure, and a receptor of the biomaterial to be analyzed.

The substrate may be glass, plastic, metal, silicon, quartz, alumina, oxide crystals, or a mixture thereof.

The plastic may be polyethylene phthalate (PET), polymethylmethacrylate (PMMA), polycarbonate (PC), or cyclic olefin copolymer (COC).

The metal may be nickel, aluminum, iron, or copper, and the oxide crystal may be silicon dioxide (SiO ₂ ), titanium dioxide (TiO ₂ ), tantalum pentoxide (Ta ₂ O ₅ ), or dialuminum dioxide (Al _2). O ₂ ) and the like.

It is preferable that the said substrate is transparent, and it is preferable that the thickness is 50 micrometers-5 mm. If the thickness of the substrate is less than 50㎛ there is a problem that the strength is difficult to handle, if it exceeds 5mm may be a problem in mounting on the spectroscope and other devices for measurement.

At least one metal nanostructure is formed on at least a portion of the substrate. When there are a plurality of metal nanostructures, the metal nanostructures may be aligned or arranged in a predetermined pattern on the substrate.

The metal nanostructure may be in a standardized shape or an atypical shape. Particularly, when the metal nanostructure is a shaped shape, the metal nanostructure is a cylinder, or a polygonal column such as a square column, a triangular column, a pentagonal column, a hexagonal column, an octagonal column, or a truncated cone, or a triangular pyramid, a square pyramid, It may be a three-dimensional structure having various shapes such as a polygonal pyramid such as a pentagonal pyramid, a hexagonal pyramid, or an octagonal pyramid.

The metal nanostructure may be made of a metallic material made of gold, silver, copper, aluminum, platinum, or a mixture thereof, and more preferably, may be made of a material such as gold, silver, copper, aluminum, and the like.

The height (thickness) of the metal nanostructure may be 1 nm to 500 nm, and preferably 10 nm to 200 nm. If the height of the metal nanostructure is out of range, there may be a problem in observing the LSPR phenomenon.

The diameter of the metal nanostructure or the radius of the circumscribed circle may be 5 nm to 500 nm, preferably 50 nm to 250 nm. When the metal nanostructure is a truncated cone, a triangular pyramid, a square pyramid, a pentagonal pyramid, a hexagonal pyramid, or an octagonal truncated cone, the radius of the bottom diameter or the circumscribed circle is 5 nm to 500 nm, and the diameter of the top surface or the circumscribed circle is 5 nm to 400 nm. In this case, the top surface diameter or radius of the circumscribed circle may be smaller than the bottom diameter or radius of the circumscribed circle. If the diameter of the metal nanostructure or the length of the radius of the circumscribed circle is out of the range, there is a problem in observing the LSPR phenomenon.

The wavelength region of the surface plasmon band (Surface Plasmon Absorption Band) varies according to the shape, material, size, etc. of the metal nanostructure, and thus measurement sensitivity may also vary. In general, the longer the wavelength, the higher the measurement sensitivity, but in reality it must be adjusted to the specifications of the measurement system.

The metal nanostructure may include nano imprint lithography (NIL), electron beam lithography (ebl), focused ion beam (fob), soft lithography (sl), or block copolymers. It may be arranged at regular intervals on the substrate by a method such as self-assembly. When a plurality of metal nanostructures are formed on the substrate, the spacing in which the metal nanostructures are arranged is not particularly limited, but the diameter or circumscribed circle of the metal nanostructures is such that they do not affect the LSPR properties of other adjacent metal nanostructures. It is preferably 0.7 to 1.5 times the radius of (or the diameter of the lower end or the radius of the circumscribed circle).

An upper surface of the metal nanostructure may be shielded. In this case, the shielding material for shielding the upper surface of the metal nanostructure as described above, may be a metal oxide or non-metal oxide that inhibits the adsorption of the biological material, such as the biological material to be analyzed and the receptor selectively coupled thereto. The shielding material may be an oxide such as silicon oxide (SiO ₂ ), aluminum oxide (Al ₂ O ₃ ), and most preferably silicon oxide. In this case, the upper surface of the metal nanostructure may be deposited on the upper surface of the metal nanostructure of the three-dimensional structure using the vertical deposition of the electron beam evaporator (electron beam evaporator).

Referring to (b) of FIG. 2, in the case of the metal nanostructure according to the embodiment of the present invention, the electromagnetic field of the local surface plasmon generated by the incident light is distributed more intensively on the side than the upper surface of the metal nanostructure. You can see that the strength is also great. After all, when detecting or analyzing a biological material using LSPR, the binding reaction of the biological material can be concentrated in terms of the metal nanostructure as described above, thereby further amplifying the signal change of the LSPR. . Therefore, by shielding the upper surface of the metal nanostructure as described above, the metal of the upper surface is blocked and the metal is exposed only on the side of the metal nanostructure, thereby combining the reaction of the receptor of the analyte to be described below with the analyte to be analyzed. This can happen intensively in terms of metal nanostructures.

The upper surface shielding material may be applied in a thickness of 1 nm to 30 nm. If the thickness of the material that shields the upper surface of the metal nanostructure is less than 1 nm, the effect of shielding the upper surface is not sufficiently exhibited. If the thickness is greater than 30 nm, the excessive local effective refractive index increase of the metal nanostructure and the measurement resonance wavelength resulting therefrom are It is possible to move excessively long wavelengths out of the observation range.

The receptor of the biological material to be analyzed is a receptor that can specifically bind to the material to be analyzed, and is immobilized on the side of the metal nanostructure.

The receptor of the biological material to be analyzed may be any receptor that can specifically bind to the biological material to be analyzed, and may be, for example, in the form of DNA, aptamer, peptide, protein (antibody, etc.).

The receptor of the biological material to be analyzed may be directly immobilized on the surface of the side surface of the metal nanostructure, or after immobilizing a linker serving as an intermediate crosslinking, the receptor of the biological material to be analyzed is immobilized on the linker. It may be immobilized through the steps.

The linker may be 11-mercaptoundecanoic acid (MUA), 11-mercaptoundecanol (MUOH), 3-mercaptopropionic acid (MPA), thiol oligo ethylene glycol (OEG), thiol polyethylene glycol (PEG), or the like.

The biological material to be analyzed may be used without particular limitation, and may be various cancer disease markers, cardiovascular disease markers, hormones, toxins, various cytokines (cytokine), biomolecules such as environmentally harmful low molecular substances, and the like. Depending on the substance, a receptor specifically binding thereto may be appropriately selected and used.

The LSPR-based biosensor can detect or analyze a biomaterial by forming an electromagnetic field of LSPR on a surface of a metal nanostructure in which a receptor of the biomaterial to be analyzed is fixed and measuring a change in the LSPR signal. At this time, as in the present invention, when shielding the upper surface of the metal nanostructure arranged on the substrate to block the exposure of the metal on the upper surface, only the side of the metal nanostructure in which the electromagnetic field generated from the LSPR is concentrated Since the receptor of the biological material to be analyzed can be immobilized, in the process of detecting or analyzing the biological material using the LSPR, a change in the LSPR signal can be detected and analyzed even with a small amount of the biological material.

2. LSPR  Analytical Method of Biological Materials Using Biobased Sensors

Another aspect of the present invention provides a method for analyzing a biomaterial using an LSPR-based biosensor.

The method for analyzing a biomaterial using the LSPR-based biosensor may include processing the biomaterial to be analyzed in the LSPR-based biosensor and measuring LSPR in the LSPR-based biosensor.

The above-described LSPR-based biosensor may further include amplifying the LSPR signal after processing the biological material to be analyzed and before measuring the LSPR.

The amplifying the LSPR signal may include reacting a receptor to which an enzyme specifically binding to the biological material to be analyzed is bound and adding a precipitate forming material that reacts with the enzyme to form a precipitate, thereby forming the metal nanostructure. Forming a precipitate on the surface of may include. Since the LSPR-based biosensor detects a change in refractive index near the surface of the metal nanostructure, the LSPR signal can be amplified by maximizing the change in the refractive index of the surface of the metal nanostructure, and thus, deposits are deposited on the surface of the metal nanostructure. This makes the surface refractive index change large, so that the LSPR signal (change in resonance wavelength) can be amplified.

The receptor to which the enzyme is bound may include a ligand capable of binding the enzyme to a portion other than a portion that binds the biological material to be analyzed. The enzyme may specifically bind to a ligand of the receptor to which the enzyme is bound or a receptor to which the enzyme is bound, wherein the enzyme is previously linked with a linker molecule capable of specifically binding to a ligand of the receptor to which the enzyme is bound. May be combined. At this time, the linker molecule may be DNA, PNA, aptamer, low molecular weight compounds, amino acids, peptides, proteins and the like. The enzyme may be biotin or alkaline phosphatase, but is not limited thereto.

The precipitate forming material is a substrate that specifically reacts with the enzyme, and the product obtained by reacting the enzyme with the precipitate forming material may precipitate and adsorb onto the surface of the metal nanostructure to amplify the LSPR signal.

The precipitate forming material may be a single material or a mixture of two or more. In particular, the precipitate-forming material includes all of the benzene ring and bromine element in the finally formed precipitate by using a compound composed of a benzene ring having a high refractive index and bromine (Br), an element having a high atomic polarizability. It is possible to maximize the refractive index change of the surface of the metal nanostructure. Specifically, the precipitate-forming material may be 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP), nitro blue tetrazolium (NBT), or a mixture thereof, but reacts with the enzyme to form a precipitate. If it can, it is not limited to this.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments.

However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited thereto.

< Example >

<1> Gold (Au) nanostructure array fabrication

On the transparent glass substrate (5-inch glass wafer), an array of gold nanostructures having a top surface of a cone-shaped gold nanostructure 150 mm in diameter and 40 nm in height was shielded with silicon oxide having a thickness of 5 nm.

<2> Linker Immobilization on Gold Nanostructure Array Surfaces

The prepared gold nanostructure array was supported by piranha solution (piranha solution, H ₂ SO ₄ : H ₂ O ₂ = 3: 1) at 120 ° C. for 2 minutes to remove contaminants adsorbed on the surface. After washing, the array of gold nanostructures was immersed in 10 mM 11-mercaptoundecanoic acid (MUA) dissolved in ethanol for 12 hours or longer to bind MUA to the surface of gold. Thereafter, 0.1 M of 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride, EDC) and 0.025 M of n-hydrosuccinic acid were dissolved in distilled water. Immersion (n-hydrosuccinimide, NHS) was dissolved in a solution for 15 minutes to activate the carboxyl group (COOH) of MUA.

<3> Specific Receptor Binding to Gold Nanostructures

The prepared linker (MUA) -conjugated gold nanostructure array was immersed in a 1X PBS (phosphate buffered saline) solution containing 0.1 mg / ml AFP (alphaferoprotein) antibody, and then reacted for 30 minutes. AFP antibodies were immobilized on the surface of the construct. In order to prevent nonspecific binding, 1X PBS (pH 7.4) containing 1% of bovine serum albumin (BSA) was reacted for 30 minutes.

<4> Binding of Specific Receptors to Biological Materials

AFP (alphaferoprotein) (in 1 mg / ml BSA) was applied to the gold nanostructure array to which the AFP antibody was immobilized and reacted for 30 minutes.

<5> measurement of LSPR

AFP antibody, which binds specifically to AFP and binds to biotin and alkaline phosphatase, was reacted for 30 minutes. AP Buffer containing 0.1 M BCIP (5-bromo-4-chloro-3-indolyl phosphate p-toluidine) and 1 mg / mL nitro blue tetrazolium (NBT), an enzyme that causes enzyme precipitation with alkaline phosphatase The solution (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl 2, pH 9.5) was reacted to produce a precipitate proportional to the AFP concentration on the surface of the gold nanostructures. Then, the results of measuring the LSPR signal at the concentration of AFP at 1 ng / ml, 10 pg / ml, and 1 fg / ml are shown in FIG. 5.

< Comparative example  1>

An array of conical gold nanostructures having a diameter of 150 nm and a height of 40 nm was prepared on a transparent glass substrate (5-inch glass wafer).

In the same manner as in Examples <2> to <5>, the results of measuring LSPR signals are shown in FIG. 5.

< Comparative example  2>

A cylindrical gold nanostructure array having a diameter of 150 nm and a height of 20 nm was prepared on a transparent glass substrate (5-inch glass wafer).

In the same manner as in Examples <2> to <5>, the results of measuring LSPR signals are shown in FIG. 5.

As shown in FIG. 5, the embodiment according to the present invention can confirm that the signal change (wavelength change) of LSPR is larger than that of Comparative Examples 1 and 2 at all the AFP concentrations tested, and thus, more sensitive and precise bio Sensing was possible.

100: substrate
200: metal nanostructure
300: top surface shielding material
400: Receptor of the biological material to be analyzed

Claims (6)

Board;
A three-dimensional metal nanostructure arranged on the substrate, the upper surface of which is shielded; And
A receptor of the biological material to be immobilized on the side of the metal nanostructure;
Topical surface plasmon resonance based biosensor that comprises.
The method according to claim 1,
Wherein said receptor is directly immobilized on the side of said metal nanostructure, or immobilized via a linker.
The method according to claim 1,
The three-dimensional structure is a topical surface plasmon which is any one selected from the group consisting of cylinder, triangular prism, square pillar, pentagonal pillar, hexagonal pillar, octagonal pillar, truncated cone, triangular pyramid, square pyramid, pentagonal pyramid, hexagonal pyramid and octagonal pyramid Resonance based biosensor.
The method according to claim 1,
Local surface plasmon resonance-based biosensor that shields the upper surface of the three-dimensional structure of the metal nanostructure with a metal oxide or non-metal oxide.
Processing the biomaterial to be analyzed in the topical surface plasmon resonance based biosensor of claim 1; And
Measuring local surface plasmon resonance in the local surface plasmon resonance based biosensor;
Method of analysis of a biological material using a topical surface plasmon resonance-based biosensor.
The method according to claim 5,
And amplifying a signal of local surface plasmon resonance after measuring the local surface plasmon resonance after treating the biological material to be analyzed.

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