KR101865428B1 - Method for measuring biomolecule using localized surface plasmon resonance - Google Patents

Abstract

The present application relates to a method for detecting biomolecules using local surface plasmon resonance. According to the present application, the existence, expression and activity of biomolecules can be measured using the local surface plasmon resonance phenomenon of the metal nanostructure. In addition, it is possible to diagnose diseases related to the biomolecules through the measurement of such biomolecules.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomolecule detection method using local surface plasmon resonance

The present application relates to a biomolecule detection method using local surface plasmon resonance.

Surface plasmon refers to the quantized vibration of free electrons propagating along the surface of a conductor, such as a metal film. The surface plasmon is excited by an incident light incident on the metal thin film at an angle equal to or greater than a critical angle of the dielectric medium through a dielectric medium such as a prism to cause resonance, which is referred to as surface plasmon resonance (SPR) .

When using monochromatic incident light, the incident angle (resonance angle) of resonance and the wavelength (resonance wavelength) at which resonance occurs are very sensitive to changes in the refractive index of a material close to the metal thin film. The SPR sensor has been used to measure the quantitative and qualitative analysis of a sample from the change of the refractive index of a substance close to a metal thin film, that is, the thickness of a thin film sample.

On the other hand, metal nanostructures of several nanometers to several hundreds of nanometers, such as nanoparticles, nanorods, and nanoholes, made of metals other than metal thin films, are formed by light of a specific frequency (wavelength) Collective oscillation of the electrons in the conduction band of the structure is induced, resulting in electrical dipole characteristics. As a result, the light in the corresponding frequency region is scattered and absorbed strongly, which is called localized surface plasmon resonance (LSPR).

Unlike SPR, scattering and absorption by LSPR can be measured by simple transmission spectroscopy without prism or diffraction grating. The absorbance characteristics of the metal nanostructure with respect to external incident light, that is, the light intensity, the line width of the absorption spectrum, and the wavelength of the light absorption, have a very strong dependence on the type of metal and the size and shape of the metal nanostructure. In addition, similar to SPR, their light absorbing properties are sensitive to the complex dielectric constant of the medium surrounding the surface of the metal nanostructure, that is, the external environment of the metal nanostructure. Biosensors using such properties are being developed.

Korea Patent Publication No. 2010-0026477 Korea Patent Publication No. 2011-0038215 U.S. Published Patent Application 2005/0181989

The object of the present invention is to provide a method for detecting biomolecules using local surface plasmon resonance.

The present invention provides a method for detecting a biomolecule comprising the steps of: treating a metal nanostructure on which a peptide specifically degraded by a biomolecule is formed, with a biomolecule to be detected; And

And measuring a local surface plasmon resonance change of the metal nanostructure.

The present application also relates to a metal nanostructure having a peptide specifically degraded by a biomolecule;

A light source unit for introducing light into the metal nanostructure; And

A light receiving portion for measuring a local surface plasmon resonance change of the metal nanostructure;

And a biomolecule detection sensor.

According to the present application, the existence, expression and activity of biomolecules can be measured using the local surface plasmon resonance phenomenon of the metal nanostructure. In addition, the measurement of the biomolecule can be used to diagnose a disease associated with the biomolecule.

FIG. 1 is a schematic view showing a process of measuring biomolecules through measurement of LSPR signal change of a metal nanostructure as an example of the present application.
2A shows an absorbance spectrum of PGNRs dispersed in water, 2b shows an obscuration microscope image (scale bar: 2 m) of PGNRs, and 2c shows a TEM image (scale bar: 50 mm) of PGNR adsorbed on a glass substrate.
3 shows a schematic diagram of an LSPR system as an example of the present application. In the drawings, reference numeral 1 denotes a light source, 2 and 7 denote optical fibers, 3 and 6 denote lenses and adapters, 4 denotes a diaphragm, 5 denotes a filter holder for a sample, and 8 denotes a light absorber.
4a shows the results of the absorbance measurement of PGNR according to the dielectric medium, and 4b shows the sensitivity analysis results of the LSPR substrate according to the change of the refractive index of the dielectric medium.
FIG. 5A shows changes in the maximum wavelength depending on MT1-MMP concentration and MT1-MMT inhibitor treatment, and 5b shows the protein degradation rate constant according to MT1-MMP concentration.
6A shows the maximum wavelength change due to proteolytic degradation of MT1-MMP in HT1080 and MCF7 cell lines, and 6b shows the protein degradation rate constant in HT1080 and MCF7 cell lines.

The present invention provides a method for detecting a biomolecule comprising the steps of: treating a metal nanostructure on which a peptide specifically degraded by a biomolecule is formed, with a biomolecule to be detected; And measuring a change in local surface plasmon resonance of the metal nanostructure. The present invention also provides a method for detecting a biomolecule. Due to the change of the local surface plasmon resonance signal, the metal nanostructure exhibits different absorption characteristics depending on the external environment of the metal nanostructure, that is, the surrounding surface of the metal nanostructure, and biomolecules can be detected using these characteristics. Specifically, it is as follows.

Through LSPR signal measurement, metal nanostructures are first fabricated to detect target biomolecules. Methods for producing metal nanostructures are well known in the art and can be manufactured without limitation by known methods. Thereby forming a peptide that is specifically decomposed by the biomolecule to be detected in the produced metal nanostructure.

The target biomolecule can be detected by measuring the LSPR signal of the metal nanostructure before and after the introduction of the biomolecule to be detected using the peptide specifically degraded by the biomolecule to be detected (Example 1). For example, after measuring the LSPR signal of a metal nanostructure bound to a peptide that is specifically degraded to a target protease, the target proteinase is treated to a metal nanostructure, and the target proteinase is specifically After the degraded peptide is reacted, the change in LSPR signal before and after the reaction is measured. When the peptide is cleaved by the target protease and removed from the metal nanostructure, the LSPR signal around the metal nanostructure is changed to reveal blue shift, whereby the presence or absence of the target protease can be determined One). In addition, since the degree of the LSPR signal change depends on the concentration of the target protease, the expression level and activity of the target proteinase can be measured (Example 1).

In the present application, peptides that are specifically degraded by biomolecules may exist in various ways depending on the biomolecules to be detected, and known peptides can be used without limitation. For example, the peptide may be a peptide that is specifically degraded to a protease. For example, matrix metalloproteinases (MMPs), including MMP-2/9, MMP-7, MMP-13 and MT1-MMP (membrane type 1 matrix metalloproteinase) ; MMP), thrombin, FXIIIa (factor Xiiia), caspase, urokinase plasminogen activator (uPA), Fijian, cathepsins, HIV protease, DPP-IV or may be specifically cleaved by dipeptidyl peptidase or proteasome.

In one embodiment, the peptide may be one or more selected from the group consisting of the amino acid sequences of SEQ ID NOS: 1-13. For example, the peptide may be represented by any one of the amino acid sequences of SEQ ID NOS: 1 to 13.

In the present application, biomolecules may be biogenic enzymes, including, but not limited to, In one embodiment, the biological enzyme may be a protease, a sugar metabolism-related enzyme, or an enzyme related to glutamine metabolism. For example, the biosynthetic enzyme may be selected from the group consisting of matrix metalloproteinases (MMP), thrombin, FXIIIa (factor Xiiia), caspase, urokinase plasminogen activator (uPA) Fijian, cathepsins, HIV protease, dipeptidyl peptidase (DPP-IV) or proteasome; Related enzymes such as hexokinase, phosphofructokinase, pyruvate kinase M2, pyruvate dehydrogenase or lactate dehydrogenase, and the like. ; Or a glutaminase related enzyme such as glutaminase.

Among them, matrix metalloproteinases (MMPs) are substrates for degrading and transforming extracellular matrix by enzymatic activity, and play an important role in controlling the behavior of non-invasive cancer cells. In particular, among large families of MMPs, membrane-type MMPs are enzymes that play a direct and essential role in the invasion of tumor cells into connective tissue.

The peptide and the target biologic enzyme are known to be associated with certain diseases. Thus, by measuring the change of local surface plasmon resonance signal, presence or absence, expression level or activity of biological enzyme can be detected to provide useful information for diagnosis of the related disease. For example, by detecting the presence or expression level or activity of MT1-MMP, information for diagnosis of cancer can be provided. Peptide and target proteinase-related diseases are shown in Table 1 below.

[Table 1]

The present application also relates to a metal nanostructure having a peptide specifically degraded by a biomolecule; A light source unit for introducing light into the metal nanostructure; And a light receiving unit for measuring a local surface plasmon resonance change of the metal nanostructure.

In one embodiment, the metal nanostructure may be formed on a substrate. The metal nanostructure can be modified into an organic functional molecule in order to effectively bind the metal nanostructure to the substrate. For example, the metal nanostructure can be modified with an organic surface stabilizer or a surfactant (Production Example 1). Then, an LSPR substrate can be prepared by introducing an amine group to a substrate to bind the modified metal nanostructure to the substrate, and then bonding a probe, which reacts with the target biomolecule, to the substrate having the metal nanostructure bonded thereto (Production Example 2) .

In one embodiment, the substrate can be glass, plastic, metal, silicon, quartz, alumina, oxide crystals, or mixtures thereof. For example, plastic, and the number of days, such as PMMA, PC, COC, metal may be nickel, aluminum, iron, copper, oxide crystals are _{SiO 2, TiO 2, Ta 2} O 5, Al 2 O , but can be _2, But is not limited thereto.

In one embodiment, the metal nanostructure may be gold, silver, copper, aluminum, platinum, silicon, germanium, or mixtures thereof.

In the present application, the shape of the metal nanostructure is not limited. For example, it is possible to use a column, a square column, a triangular column, a hexagonal column, a hexagonal column, an octagonal column, a sphere, a hemisphere, a part of a sphere, an ellipsoid, a half ellipsoid, a part of an ellipsoid, a quadrangular pyramid, Triangular pyramids, cones, truncated cones or rings.

Depending on the type, size, or type of the metal nanostructure, the plasmon band wavelength region is changed, and thus the measurement sensitivity may be changed.

In one embodiment, the metal nanostructure can be a gold nanorod, a gold nanowire, a gold nanowell, or a gold nanotagonal triangular pyramid. When the metal nanostructure is a nanorod having a height in the range of 100 nm to 150 nm, 110 nm to 140 nm, or 125 nm to 135 nm and a diameter in the range of 60 nm to 70 nm, 50 nm to 60 nm, or 35 nm to 45 nm, the plasmon band wavelength region becomes longer The measurement sensitivity can be increased.

In one embodiment, the aspect ratio of the gold nanorods is in the range of 1: 1 to 10: 1, 1: 1 to 8: 1, 1: 1 to 6: 1, 1: 1 to 5: : 1 to 4: 1, or 1: 1 to 3: 1. When the aspect ratio of the gold nanorods is 3: 1 or more, the maximum absorbance is formed on the range of 650 nm to 1000 nm. Therefore, the absorbance against water is low and the biomolecule detection efficiency can be enhanced.

In one embodiment, the peptide specifically degraded to the biomolecule may be at least one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 1 to 13.

In the present application, the metal nanostructure may be bonded to an organic functional molecule. The organic functional molecule may be an organic surface stabilizer or a surfactant capable of stabilizing the metal nanostructure.

For example, the surface stabilizer may be an amphipathic compound, and more specifically, it may be composed of polyvinyl alcohol (PVA), polyethylene glycol hexadecyl trimethyl ammonium bromide, sodium dodecyl sulfite, and polyethylene glycol sorbitan monolith type compounds But is not limited thereto.

Surfactants include cationic surfactants including alkyl trimethylammonium halides; Saturated or unsaturated fatty acids such as oleic acid, lauric acid, or dodecylic acid; Trialkylphosphines such as trioctylphosphine oxide (TOPO), trioctylphosphine (TOP) or tributylphosphine; Alkylamines such as trialkylphosphine oxide, oleic amine, trioctylamine or octylamine; alkylamines such as trialkylphosphine oxide, oleic amine, trioctylamine or octylamine; A neutral surfactant comprising an alkyl thiol; Alternatively, anionic surfactants can be used including, but not limited to, sodium alkyl sulfate or sodium alkyl phosphate.

Particularly, in consideration of the stabilization of the nanoparticles and the uniform size distribution, it is preferable to use polyethylene glycol.

Hereinafter, the present application will be described in detail by way of examples. The following examples are illustrative of the present application and the scope of the present application is not limited to the following examples.

[ Manufacturing example  One] Polyethylene glycol  Coated Gold nonarods (GNRs) Manufacturing

Monodisperse gold nanorods were synthesized by seed-mediated growth method. To prepare a gold-seed solution, 250 μL of chloroauric acid solution (10 mM) was added to 7.5 mL of hexadecyltrimethylammonium bromide (CTAB) solution (93 mM) and sodium borohydride solution 10 mM) was further added under strong stirring. The resulting gold nanosidation solution was stirred for 2 minutes and reacted at room temperature for 4 hours. Then, the growth solution was prepared as follows. 50 μl of chloroauric acid solution (10 mM), 55 μl of ascorbic acid solution (100 mM), and 12 μl of gold nanosilicate were sequentially added in the same manner as in Example 1, except that 9.5 ml of the CTAB solution was vigorously stirred After dropping, it was reacted for 30 seconds. The resulting solution was stored for 24 hours and then centrifuged for 30 minutes at 15,000 rpm for 3 times to remove excess CTAB solution and redispersed in 5 mL of deionized water (DW). To effectively bind GNRs to glass substrates, polyethylene glycol (PEG) coated polyethylene glycols (PEG) coated GNRs (PGNRs) were prepared by coating polyethylene glycol (PEG) on gold nanorods. Polyethylene glycol (CM-PEG-SH) having different terminal functional groups was used as a stabilizer in order to synthesize a polyethylene-coated gold nanorod. The thiol group of PEG was conjugated to the surface of GNRs and GNRs was modified with PEG. 50 mg of CM-PEG-SH was added to 5 mL of gold nano-rods solution, and the mixture was stirred at room temperature for 48 hours. The solution was centrifuged at 15,000 rpm for 30 minutes to remove unbound CM-PEG-SH molecules and redispersed in 5 mL of DW to obtain a gold nanorod coated with polyethylene glycol.

The absorbance spectrum of PGNRs dispersed in DW was measured (Shimadzu, UV-1800). Electrons in the vicinity of PGNRs are vibrated according to the longitudinal and transverse axes of PGNRs, resulting in an absorbance peak. As shown in Fig. 2A, the absorbance peaks of PGNRs appeared at 780 nm and 520 nm.

[ Manufacturing example  2] LSPR  Fabrication of Substrate

The cover glass (12 mm φ) was washed with a pyran solution (3: 1 H ₂ SO ₄ /30% H ₂ O ₂ ). After washing, the cover glass was repeatedly washed three times with DW and then dried. To coat the surface of the cover glass with an amine functional group, an aqueous solution containing 100 μl of APTMS (aminopropyltrimethoxysilane) solution was reacted for 24 hours. After the reaction, the cover glass was washed with excess DW and ethanol, and then dried. Thereafter, the cover glass coated with the amine functional group was immersed in an aqueous solution of gold nanorods coated with poly (ethylene glycol), reacted for 24 hours, washed with DW, and then dried.

A high numerical aperture dark field condenser (U-DCW, Olympus), which combines PGNRs on a glass substrate with an amino group incorporated and transfers the adsorbed PGNRs to a sample surface with a very narrow beam of white light from a tungsten halogen lamp ) Were observed with dark field microscopy (BX51, Olympus). The absorbance spectrum of PGNRs shows a peak at 520 nm in the visible region, so PGNRs on the glass substrate strongly absorb light at a wavelength of 520 nm. Because PGNRs strongly absorb light at 520 nm, they scatter light with the same wavelength. As shown in FIG. 2B, PGNRs immobilized on an aminated glass substrate were observed with green color dots on dark field microscope images by optical properties, particularly absorbance. The size and shape of the PGNRs fixed on the glass substrate were observed with a scanning electron microscope (JSM-7001F, JEOL Ltd) (Fig. 2C). As a result, the major axis length of PGNRs was 35.2 ± 1.5 nm and the minor axis length was 10.8 ± 0.9 nm ( n = 100). The aspect ratio (major axis length / minor axis length) of PGNR was about 3.5. These results suggest that PGNRs are uniformly immobilized on a glass substrate, and that the prepared sample substrate can be used as a substrate for measuring proteolytic activity of MT1-MMP in tumor cells.

The sample substrate was then reacted with a solution containing the MT1-MMP specific cleavage peptide (MSCP) consisting of the amino acid sequence of SEQ ID NO: 1. The MSCP solution was prepared as follows. 1 mg of MSCP was dissolved in 10 mL of PBS buffer. Subsequently, 1 mL of dopamine solution (0.9 mg / mL) was added to the solution and vigorously stirred, and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (1.1 mg) -Hydroxysulfosuccinimide (1.2 mg) was added thereto at a later time, and the mixture was reacted for 4 hours with stirring. As a result, an LSPR substrate having a gold nanorod and a peptide specifically degraded to MT1-MMP was bonded (FIG. 1).

[ Example  1] of the gold nanorod LSPR  Measurement of biomolecules by signal analysis

Enzyme and cell lysate preparation

On the mother liquor, the MT1-MMP enzyme solution dissolved at a concentration of 0.2 mg / mL was diluted with PBS buffer solution to a concentration of 100 nM, 10 nM, and 1 nM, respectively, at room temperature. A radioimmunoprecipitation assay (RIPA) buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF (2 x ¹⁰ < ⁶ > cells / mL) was prepared.

LSPR  Detection of signal

The sample chip consisted of two cover glasses, one with cover glass coated with PGNR and the other with an empty cover glass. The two cover glasses were sealed using a vacuum grease, and the sample chip was vertically erected and positioned between a quartz tungsten halogen (QTH) lamp on a LSPR system and a portable absorber. The LSPR signal detection system used in the experiment is shown in Fig. In summary, the light source from the QTH lamp reaches the focusing lens through the optical fiber and then passes through the sample. The light passing through the sample again reaches the light absorber through the collecting lens and the optical fiber, and the LSPR signal can be measured by measuring the absorbance through the light absorber.

Sensitivity measurement

In order to examine the proteolytic activity of MT1-MMP in invasive cancer cells, LSPR substrate prepared according to Preparation Example 2 was used. Coated with PGNRs using various dielectric media (air: 1.000, water: 1.333, ethanol: 1.362, 1-propanol: 1.387, dimethylformamide: 1.428, and chloroform: 1.490) having different refractive indexes The sensitivity index of the substrate was examined by measuring the LSPR extinction spectrum (FIG. 4A). As the RI of the dielectric media increased, a red-shift occurred at the peak wavelength of the absorbance spectrum. Then, the sensitivity of the LSPR substrate to the change in RI of the surrounding dielectric medium was calculated. The sensitivity of the LSPR substrate was calculated to be 169.8 nm / RI unit (RIU) (FIG. 4B). These results indicate that the LSPR substrate prepared by measuring the change in RI around PGNRs is highly sensitive and suitable for detecting low concentration of biomolecules.

MT1 - MMP Of proteolytic activity

Prior to measuring the proteolytic activity of MT1-MMP, MT1-MMP detection ability of the LSPR substrate prepared according to Preparation Example 2 was examined (FIG. 5A). In order to investigate the interaction between MT1-MMP and PGNRs with MSCP, the change of the maximum wavelength (λ _max ) was measured by varying MT1-MMP concentration and reaction time. Based on the change in the maximum wavelength (λ _max ) of the LSPR spectrum due to the proteolytic activity of MT1-MMP, it was confirmed that the efficiency of proteolysis was dominantly influenced by MT1-MMP concentration. As the concentration of MT1-MMP increased, the lambda _max change of the LSPR spectrum also increased, resulting in a higher plateau value. In order to obtain a quantitative kinetic constant of proteolysis for each condition , Situ (in situ) were calculated for proteolytic activity. A Langmuir kinetic model was applied to understand the kinetics of proteolytic activity determined by the change in lambda _max of the LSPR spectrum. The Langmuir kinetic model describes the rate equation for the dissociation of molecules on the sample surface. Enzyme activity and proteolytic activity are irreversible processes and that the cleaved peptides can not bind to the residual peptides of PGNRs. The Langmuir kinetic model provides the expression N (t) = N ₀ exp (- k _p t ) . Where N ₀ is the number of peptides bound to the surface of the original PGNRs. The number of cleaved peptides is thus obtained through ΔN _p (t) = N ₀ (1-exp (-k _p t)) . The protein degradation rate constant ( k _p ) under each condition was determined using a Langmuir kinetic model expressed as Δ N _p (t) = N ₀ (1 exp (- k _p t ) 5b). In order to verify the specific activity of MT1-MMP, the change in λ _max of LSPR spectra was examined using GM6001, an MT1-MMP inhibitor. Before treating MT1-MMP, the LSPR substrate was incubated with GM6001 to block MT1-MMP specific degradation. Even when the concentration of MT1-MMP was 100 nM, no degradation of the MSCP occurred, and thus no change in the? _Max of the LSPR spectrum was observed. Thus, the protein degradation rate constant is close to zero. This result indicates that sensitive measurement of the proteolytic activity of MT1-MMP is possible using the manufactured LSPR-based nano-biosensor.

Invasiveness  Detection of proteolytic activity in tumor cells

To determine whether the LSPR-based nano-biosensor can be used for cancer diagnosis, we examined the proteolytic activity of MT1-MMP using whole cell lysate extracted from liver cancer cells. Using a whole cell lysate from two other tumor cell lines (HT1080 and MCF7 cells), the change in the λ _max of the LSPR spectrum due to proteolysis of MSCP by MT1-MMP was measured (FIG. 6A). The protein degradation rate constant ( k _p ) was determined in each tumor cell line using the Langmuir kinetic model (Fig. 6b). For MCF7 cells (MT1-MMP deficiency), k _p value of HT1080 cells (MT1-MMP over-expression), whereas the k _p value of 6.10 min ^-1 was 11.58 min ^-1. This means that the proteolytic activity of HT1080 cells is about two times higher than in MCF7 cells. That is, the manufactured LSPR-based nano-biosensor can detect MT1-MMP expressing cancer cells by detecting the presence or absence of MT1-MMP expression.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Method for measuring biomolecule using localized surface plasmon          resonance <130> P130381 <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> MT1-MMP specific peptide seqence <400> 1 Gly Pro Leu Pro Leu Arg Ser Trp Gly Leu Lys   1 5 10 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> MMP-2/9 specific peptide seqence <400> 2 Pro Leu Gly Leu Arg   1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> MMP-7 specific peptide seqence <400> 3 Val Pro Leu Ser Leu Thr Met   1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MMP-13 specific peptide seqence <400> 4 Pro Leu Gly Met Arg Gly   1 5 <210> 5 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Cathepsins B specific peptide seqence <400> 5 Lys Lys   One <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Cathepsins D specific peptide seqence <400> 6 Pro Ile Cys Phe Phe Arg Leu   1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> PSA specific peptide seqence <400> 7 His Ser Ser Leu Gln   1 5 <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Caspase-1 specific peptide seqence <400> 8 Trp Glu His Asp   One <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Caspase-3 specific peptide seqence <400> 9 Asp Glu Val Asp   One <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Thrombin specific peptide seqence: the P letter means pipecolinic          acid. <400> 10 Phe Pro Arg Ser   One <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Fijian specific peptide seqence <400> 11 Asn Gln Glu Gln Val Ser   1 5 <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> DPP-IV specific peptide seqence <400> 12 Gly Pro Gly Pro   One <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HIV protease specific peptide seqence <400> 13 Gly Val Ser Gln Asn Tyr Pro Ile Val Gly   1 5 10

Claims (11)

A substrate into which an amine group is introduced;
A metal nanostructure which is bonded to the substrate by the amine group and has a peptide specifically decomposed by biomolecules;
A light source unit for introducing light into the metal nanostructure; And
A light receiving portion for measuring a local surface plasmon resonance change of the metal nanostructure;
A biomolecule detection sensor is used,
Treating the metal nanostructure on which a peptide specifically degraded by the biomolecule is formed with biomolecules to be detected; And
Measuring a local surface plasmon resonance change of the metal nanostructure,
Wherein the aspect ratio of the metal nanostructure is in the range of 3: 1 to 10: 1 in the major axis and the minor axis.
The method according to claim 1,
Wherein the peptide is at least one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 1 to 13.
The method according to claim 1,
The biomolecule may be selected from the group consisting of matrix metalloproteinases (MMP), thrombin, FXIIIa (factor Xiiia), caspase, urokinase plasminogen activator (uPA), Fijian, The compounds of the present invention can be used in combination with other therapeutic agents such as cathepsins, HIV protease, dipeptidyl peptidase (DPP-IV), proteasome, hexokinase, phosphofructokinase, pyruvate kinase M2, A method for detecting biomolecules such as pyruvate dehydrogenase, lactate dehydrogenase, or glutaminase.
A substrate into which an amine group is introduced;
A metal nanostructure which is bonded to the substrate by the amine group and has a peptide specifically decomposed by biomolecules;
A light source unit for introducing light into the metal nanostructure; And
A light receiving portion for measuring a local surface plasmon resonance change of the metal nanostructure;
/ RTI &gt;
Wherein the aspect ratio of the metal nanostructure is in the range of 3: 1 to 10: 1 in the major axis and the minor axis.
delete 5. The method of claim 4,
Wherein the substrate is glass, plastic, metal, silicon, quartz, alumina, oxide crystals or mixtures thereof.
5. The method of claim 4,
Wherein the metal nanostructure is gold, silver, copper, aluminum, platinum, silicon, germanium or a mixture thereof.
5. The method of claim 4,
The shape of the metal nanostructure may be selected from the group consisting of a cylinder, a square pillar, a triangular pillar, an octagonal pillar, a hexagonal pillar, an octagonal pillar, an elliptical portion, a half elliptical portion, a portion of an elliptical portion, a quadrangular pyramid, a quadrangular pyramid, Truncated cone or ring biomolecule detection sensor.
5. The method of claim 4,
The metal nanostructure is a gold nanorod or gold nanotriangular horn biomolecule detection sensor.
delete 5. The method of claim 4,
Wherein the peptide is at least one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 1 to 13.

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