METHOD FOR DETECTING BIOMOLECULE BY USING LOCALIZED SURFACE PLASMON RESONANCE

The present application the biomolecule-metal is formed a peptide decomposition of detecting nano-structre bond by treating the polyamide layer with biomolecule object; and said metal nano-structre topical of surface plasmon resonance for measuring the change in detecting of the biomolecules including step provides method. Topical surface plasmon resonance on by the signal change, metal nano-structre a metal nano-structre environment outside of the body, i.e. metal nano-structre around surface light absorption characteristics and that other medium being the fiber these characteristics using may detect biomolecules as a result of such streamlined. Specifically. off at the first and the second.

LSPR via telemetry signal, for detecting the targeted biomolecules as a result of such first metal nano-structre prepare silk fibroin fibre body. The method for producing metal nano-structre is known well to where a new file does not exist, a publicly known method without limited to.. Produced metal nano-structre of the object mark to be detected to biomolecule-decomposition form to a peptide.

Of the object mark to be detected biomolecule-decomposition a peptide detected by using a before introduced biomolecule object nano-structre metal of LSPR of targeted biomolecules as a result of such via telemetry signal may detect (in the embodiment 1). For example, specifically target protease from decomposition of a metallic binding peptides which after measuring the signal LSPR nano-structre, a metal target protease nano-structre the processing to said target protease is decomposed into specific and having a peptide that is reacting a metal salt of, LSPR measure the signal change before and after reaction. Said target protease decomposes and the binding peptides which by, the gap when the from nano-structre metal, metal nano-structre LSPR signal is the periphery of the of pixels is shifted by blue is electrically connected, through the surroundings of a target protease (also 1) can be. As well as, the degrees of LSPR signal change to the concentration of the mRNA target protease dependent so that target protease expression dose. and activity and (in the embodiment 1).

The present application-biomolecule at the peptide has a from decomposition biomolecule to be detected according to can be is advantageously present in the various, publicly known peptide to the web without restriction available. For example, the peptide has a specifically a proteolytic enzyme can be a peptide decomposition. For example, 9/MMP-2, MMP-7, MMP-13, MT1-MMP (membrane type 1 matrix serine protease; membrane-type 1 matrix metalloproteinase) or the like including enzyme matrix metalloproteinases (matrix metalloproteinases; MMP), thrombin, FXIIIa (factor Xiiia), caspase (caspase), the rain and dew height recovering sacrifice plasminogen activator (urokinase plasminogen activator, uPA), into FPGA (Fijian), cathepsin (cathepsins), HIV protease, proteasome or DPP-IV (dipeptidyl peptidase), such as by (proteasome) is decomposed into specific. may be to.

In one embodiment, the peptide has a seq ID no 1 to 13 selected from the group consisting of the amino acid sequence of one or more can be. For example, amino acid sequence of the peptide has a seq ID no 1 to 13 is formed either can be.

The present application in, biomolecules of limited to though it is not, can be biological enzymes and. In one embodiment, biological enzymes and proteolytic enzymes, enzyme metabolic-related per, glutamine can be metabolic-related enzyme. For example, biological enzymes and a substrate, a matrix (matrix metalloproteinases; MMP), thrombin, FXIIIa (factor Xiiia), caspase (caspase), the rain and dew height recovering sacrifice plasminogen activator (urokinase plasminogen activator, uPA), into FPGA (Fijian), cathepsin (cathepsins), HIV protease, the proteinase such as proteasome or DPP-IV (dipeptidyl peptidase) (proteasome); [...] (hexokinase), pos [...] (phosphofructokinase), M2 (pyruvatekinase M2) [...] , pyruvate dehydrogenase (pyruvate dehydrogenase) or lactate dehydrogenase (lactate dehydrogenase) per such as metabolic-related enzyme; or writing base hit it pushes but Oh sacrifice such as glutamine (glutaminase) can be metabolic-related enzyme.

Double even, enzyme matrix metalloproteinases (matrix metalloproteinase; MMPs) according to the enzymatic activity the extracellular matrix to dissolve deforming the substrates, non-invasive behavior of cancer cells adjusts the a is responsible for an important role. In particular, membrane type MMPs large substrate (membrane-type MMP) serine protease family among the tumour cells will directly but invasive tissue connected plays an integral role and is an enzyme.

Particular diseases, said peptide and a targeting biological enzymes and relates to a constitution: know. Therefore, topical by measuring the surface plasmon resonance signal change, with the presence or absence of an biological enzymes and, expression amount or activity of its associated by sensing the useful information for diagnosing a disease may provide a. For example, with the presence or absence of an MT1-MMP, expression amount or activity for the diagnosis of a cancer by sensing the information may provide a. Peptide and a targeting protease related disorders enabled to operate close to the loop table 1 shown in equal.

[Table 1]

The present application the in addition, biomolecule-decomposition a peptide is formed metal nano-structre body; said metal nano-structre light source unit which emits an incident light on the body; and said metal nano-structre topical of for measuring the change in surface plasmon resonance using a dry-etching process provides including biomolecule detection sensor.

In one embodiment, metal nano-structre on a substrate can be formed. Metal nano-structre nano-structre on a substrate; a metal binds efficiently to functional organic a nano-structre. can modify acid molecules. For example, metal nano-structre organic a surface stabilizer or surfactant can modify. (manufacturing example 1). Furthermore, substrate then introduced to a amine group bonded to the modified metal nano-structre a substrate after, metal by a probe reaction target biomolecule nano-structre is joined is coupled to a substrate the LSPR substrate can be produced (manufacturing example 2).

In one embodiment, glass, plastic, metal, silicon, quartz, alumina, oxide crystal to ultra high degree can be, or a mixture of these. For example, plastic PMMA, PC, such as COC, a rail, and, a nickel metal, aluminum, iron, may be copper, the oxide crystal to ultra high degree SiO₂, TiO₂, Ta₂ O₅, Al₂ O₂, the logical source including the device, not limited to blood.

In one embodiment, a gold metal nano-structre, the, copper, aluminum, platinum, silicon, germanium or can be mixtures thereof.

The present application in, metal nano-structre is not limited to the shape of the body. For example, cylindrical, square column, focusing, hexagon column, hexagonal prism, sloped, opening, hemi, portion of, formed between a deck, half oval and knobbly, portion of formed between a deck, truncated quadrangular pyramid whose, square pair horn , truncated quadrangular pyramid whose to, triangular pyramid, triangular pair horn , triangular pyramid to, conical, frustum or ring can be.

Metal nano-structre of form, size or kinds of has wavelength region plasmon band according to the upper electrode in addition can be changed.

In one embodiment, metal nano-structre a gold nanorods, opening skeletal muscle relaxant, skeletal muscle relaxant faced cube, skeletal muscle relaxant may it will be a tripodal pair horn. Metal nano-structre is 100 nm to 150 nm, 140 nm to 110 nm, or 125 nm to 135 nm range of aperture ratio of a liquid crystal display 60 nm to 70 nm, of 45 nm to 35 nm or 50 nm to 60 nm having a diameter ranging between when the nanorods, plasmon band wavelength region upper electrode unit. can be enhanced.

In one embodiment, gold nanorods the aspect ratio of the major axis: 10:1 to 1:1 length of shortened, 1:1 to 8:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, or can be 1:1 to 3:1. Groove of gold nanoparticles 3:1 aspect ratio is maximum absorbance is prevented on 1000 nm to 650 nm is formed at a, in water low absorbance detection efficiency of the biomolecules a supply part has first and second.

In one embodiment, a peptide decomposition specifically biomolecule seq ID no 1 to 13 selected from the group consisting of the amino acid sequence of one or more can be.

The present application in, metal nano-structre a organic functional molecule is combined with the receiving container, may be proposed. A metal functional molecule organic said nano-structre can be for stabilising organic surface stabilizer or surfactant can be.

For example, surface stabilizers may be amphiphilic compound, specifically, polyvinyl alcohol (PVA), polyethylene glycol hexa of tetradecyltrimethylammonium bromide, dodecyl sodium monolithic brush grief glycol and polyethylene sulfite selected from the group consisting of compounds of the radioactive part into contact with one or more but, not limited to blood.

Dryer and control method thereof alkyl surfactant cationic surfactant including a methyl ammonium halide (alkyl trimethylammonium halide) ; (oleic acid) oleic, [...] (lauric acid), or use of Saccharomyces cerevisiae the thread it buys (dodecylic acid), saturated or unsaturated fatty acid having such as; tree [...] oxide (trioctylphosphine oxide: TOPO), tree [...] (trioctylphosphine: TOP) or butyl phosphine (tributylphosphine) such as trialkyl phosphine; tree alkyl gun spin oxide , [...] oleate (oleic amine), tree [...][...] or (trioctylamine) (alkyl amine) alkylamines such as (octylamine); alkyl thiol (alkyl thiol) for including neutral surfactant; or, sodium alkyl sulfate (sodium alkyl sulfate) or sodium alkyl phosphate (sodium alkyl phosphate) including a the anionic surfactants, limited to blood not.

In particular, nanoparticles of stabilizing, and, when considered in the uniform size distribution, polyethylene glycol is preferably a.

Hereinafter, the present application for. as further described to embodiment. Embodiment relate to the present application is exemplified application the present is a range of a aspect limited not embodiment.

[Manufacturing e.g. 1] rod nanosturecture coated polyethylene glycol are linked (gold nonarods; GNRs) for manufacturing

Seeded growth method using monodispersed skeletal muscle relaxant (seed-mediated growth method) have been synthesised rod. In order to produce a (gold-seed solution) [...] skeletal muscle relaxant, gold mine chloride solution (10 mm) 250 micro l methylhexadecyl a dryer and control method thereof ammonium bromide (CTAB) solution behind placed in (93 mm) 7.5 ml, with sodium boron hydride solution (10 mm) 600 micro l with a strong stirring the into the additional. [...] nanosturecture generated behind obtain a lead line having a 2 minutes, 4 reacted time at room temperature. For (growth solution) liquid crystals are grown then shown as follows. In a minor axis and a 9.5 ml CTAB solution is capable of strongly stirring, reagent is coulometric titrated solution (10 mm) 80 micro l, gold mine chloride solution (10 mm) 50 micro l, ascorbic acid solution (100 mm) 55 micro l, skeletal muscle relaxant l micro 12 sequentially dropped [...] putting the finishing, reacted seconds 30. The solution thus obtained is then resulting product the 24 time behind from the cell compartment, 15,000 rpm in 30 minutes the repetitively 3 process centrifugal separator, then excess of CTAB solution is removed, the DW (deionized water) 5 ml redispersable in. GNRs the surface of the substrate to effectively bond the, skeletal muscle relaxant (PEG) polyethylene glycol rod is coated with polyethylene glycol (PEG) coated with nanosturecture rod (polyethylene glycol coated GNRs (PEG); PGNRs) have been prepared. Polyethylene coated groups for synthesis of rod nanosturecture, at the both end parts of other functional groups of the ground terminal of as a stabilizing (cm-PEG-SH) polyethylene glycol. PEG GNRs mote this year of is which is conjugated to a water-surface of applicants, and modified with PEG is GNRs. Cm-PEG-SH 50 mg of then into the 5 ml solution rod skeletal muscle relaxant, 48 visitor is checked through a stirring time at room temperature. 30 minutes to the solution the 15,000 rpm in of the plural shapes are centrifugal separator which are not bound to cm-PEG-SH then molecules, DW 5 ml to coated with, polyethylene glycol redispersion of the back rod nanosturecture obtained.

DW PGNRs dispersed in a it was determined that of the absorption edge of the spectrum (Shimadzu, UV-1800). According each longitudinal axis and a transverse axis PGNRs PGNRs of electrons around oscillation, absorbance peak occurs. Also as shown in the 2a, 520 nm and 780 nm PGNRs of the absorption edge of the picks out.

[Manufacturing e.g. 2] LSPR for manufacturing

Opening is (12 mm φ) the bloom or solution (3:1 H₂ SO₄/30% H₂ O₂) the washed by means of using. After rinsing, includes a transparent cover corresponding DW key, a repetitively and re-washing the behind, the drying. Cover glass surface to coat the an amine functional group, 100 micro l APTMS (aminopropyltrimethoxysilane) solution an aqueous solution is included 24 reacted time. After reaction, excess of includes a transparent cover corresponding DW and oil mixture in a vacuum separator to, washing the behind, was very dry. After, a cover glass coated with amine groups [...] glycol coated rod nanosturecture and age reacted time 24 the dipped into a strong acid to aqueous solution, then then being washed with DW, was very dry.

In glass substrate have been introduced amino group through the combination of the PGNRs, adsorbed PGNRs a tungsten halogen lamp of white light of transferring the nanotubes onto the surface sample beam very narrow aperture super capacitor dark (high numerical aperture dark field condenser; U-DCW, Olympus) using microscope dark (dark field microscopy; BX51, Olympus) has been observed on in. PGNRs of the absorption edge of the spectrum, the peak of a 520 nm in visible part represents the 520 nm on a glass substrate the PGNRs. is capable of strongly absorbing light in wavelength. 520 nm is PGNRs since is capable of strongly absorbing light in, scatter the light with a wavelength such as.. Also as shown in the 2b, a board are secured to glass amination PGNRs an optical properties, in particular on Image microscope dark in extinction by revealed that dots green color. Furthermore, glass board are secured to a form and size of PGNRs scanning electron microscope has been observed on in (JSM-7001F, JEOL Ltd) (also 2c). As a result, the length of the major axis of PGNRs the length of the and the uniaxial 35.2±1.5 nm 10.8±0.9 nm was (n = 100). PGNR aspect ratio of (major axis length/as long as a minor) was about 3.5. The results indicate that PGNRs to be removably secured to a glass to form a uniform, MT1-MMP in tumor cells produced sample substrate is substrate for measuring proteolytic activity of can be used..

The corresponding sample and substrate is after amino acid sequence of seq ID no:1 peptide specific cleavable MT1-MMP made of (MT1-MMP specific cleavage peptide; MSCP) having reacted to a solution. MSCP solution of works as follows. A MSCP 1 mg was dissolved in 10 ml PBS buffer solution. Next, dopamine solution (0.9 mg/mL) then additional to the solution to a 1 ml, strongly which agitates the, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (1.1 mg) and alcoholic beverage gun -N [...] -hydroxy (1.2 mg) merging and then input additional to, reacted the stirring time 4. Thereby skeletal muscle relaxant specifically MT1-MMP rod and combined with a peptide decomposition LSPR been produced with at substrate (also 1).

[In the embodiment 1] groove of gold nanoparticles LSPR measuring of the biomolecules through analysis signal

Preparing solution and cell enzyme

On the mother liquor at a concentration of 0.2 mg/mL MT1-MMP enzyme solution dissolved in the demonstrated in solution for to make, respectively PBS buffer solution 100 nm, 10 nm, 1 nm and the dilution each at room temperature at a concentration of. 50 mm tris (Tris)-HCl (pH 7.4), 150 mm NaCl, 1% (Triton) X-100 tone tree, 1 mm EDTA, 1 mm sodium [...] , RIPA (radioimmunoprecipitation assay) comprised of 1 mm PMSF using buffer solution of whole cells solution (2X10⁶ cell/mL) manufacturing processes and the cost of production.

Detection of the LSPR signal

Two chip sample have been configured glass cover, is coated with a field PGNR cd1a. glass cover state, the other field asymmetry correction apparatus and method the first voice portion out of an opening is. A vacuum opening is two the n bit parallel data inputted sealed using a [...] , the corresponding sample and chip tray reversing unit vertically raises in QTH (quartz tungsten halogen) lamp and portable light absorption LSPR system is set up to come in placed between the parts. Also using experiments to 3 is shown that was LSPR signal detecting system. The abstract, the QTH in a position separated from a light source via optical fibers to a collection lens (focusing lens) after reaching the, is transmitting the sample. Light passing through the sample via optical fibers lens and collection is again, and again by being in light absorption , LSPR by measuring the absorbance through groups. signal.

Measuring sensitivity

Invasive cancer cells to economize in MT1-MMP in proteolytic activity of 2 e.g. for LSPR is prepared upon a, we have demonstrated that by using such AlN crystal substrate. Refractive index different from (refractive index; RI) with a variety of dielectric medium (air: 1.000, water: 1.333, ethanol: 1.362, 1-propanol: 1.387, dimethyl formamide: 1.428, and chloroform: 1.490) using sensitivity of substrate coated with PGNRs LSPR index recognize the for measuring the spectrum (extinction) absorbance (also 4a). Dielectric medium amount of time increases with an increasing RI of (dielectric media), absorbance spectrum has come about, transition were to red-to peak wavelength of. Furthermore, peripheral dielectric of the medium RI change according to LSPR substrate sensitivity of the bill. LSPR substrate sensitivity was with 169.8 nm/RI unit (RIU) (also 4b). This result may, PGNRs RI around the through measuring changes in an, LSPR sensitivity substrate is produced media is manufactured into low concentration of biomolecule an outside power source to the device. suitable for.

Measuring proteolytic activity of MT1- MMP

MT1-MMP prior to measurement of the protein-degrading activity which is administrated orally, 2 e.g. manufacturing an electrochemical method for LSPR recognize ability detecting MT1-MMP substrate (also 5a). PGNRs with MT1-MMP and a MSCP to see the interaction between, MT1-MMP wavelength maximum and thus reaction time and concentration of (λ_max) it was determined that change of. MT1-MMP due to proteolytic activity of LSPR spectrum maximum wavelength (λ_max) on the basis of the variation of, proteolytic efficiency is MT1-MMP dominant concentration identifying influence can be. Increases concentration of MT1-MMP, LSPR spectrum λ_max change in addition improve, as a result, higher plateau (plateau) value is shown. Furthermore, quantitative of degradation proteins to conditions reaction rates (kinetic) in situto obtain constant (in situ)protein-degrading activity which is administrated orally the bill. LSPR spectrum λ_max change determined by proteolytic activity kinetics of for to understand, Langmuir positive photosensitive anionic the delay circuit delays an approximation model (Langmuir kinetic model) the lower substrate in a plane form. Langmuir kainate dissociated of its molecules on a sample surface are the delay circuit delays an approximation model reaction rates according to the described expressions. Enzyme activity and a proteolytic activity are irreversibly the peptide has a and a chopped process variable comparison voltage generator of fdram PGNRs unavailable for binding to peptide that hypothesis set up. The delay circuit delays an approximation model positive photosensitive anionic Langmuir N (t) =N₀exp(-k_pt) provides modified. Here N₀ has initially PGNRs number peptide bonded to the surface.. The number of the peptide cutting, thus, Δ N_p (t) =N₀ (1-exp (-k_pt)) are obtained through. ΔN_p (t) =N₀ (1-exp(-k_pt)) which appears as the Langmuir positive photosensitive anionic the delay circuit delays an approximation using a model, the decomposition rate constant conditions of proteins in (k_p) (also 5b) was obtained. MT1-MMP of specific activity for for verifying, using inhibitors GM6001 MT1-MMP LSPR spectrum λ_max had a visit from change. MT1-MMP prior to processing, specific MT1-MMP for shielding it from degradation to GM6001 substrate LSPR [...] section. Even when 100 nm concentration of MT1-MMP, does not take place in the reveal the degradation of MSCP, thus LSPR spectrum λ_max change in addition did not shown. Therefore, proteolytic rate constants for arranging the copies in the frequency space was close to 0. As a result the manufactured electrospun fiber based LSPR MT1-MMP using biosensor of proteolytic activity of sensitive. indicates an a detection unit.

Invasive detection of proteolytic activity in tumor cells

Produced LSPR based nano-biosensor can be used in method for rapidly and conveniently diagnosing cancer find to, hepatocellular carcinoma cell extracted from a cell lysate in using protein MT1-MMP, we have demonstrated that despite detection of proteolytic activity. The two other tumor cell lines, both of (HT1080 and MCF7 cells) by using cell lysate in proteolytic of MSCP MT1-MMP according to LSPR spectrum λ_max it was determined that change (also 6a). Langmuir positive photosensitive anionic the delay circuit delays an approximation using a model each tumor cell lines, both rate constants for proteolytic in (k_p) resolved to fund (also 6b). MCF7 cells in the case of (MT1-MMP deficient), k_p value 6.10 min^-1, while the higher (overexpressing MT1-MMP) of HT1080 cells k_p value 11.58 min^-1 was. This proteolytic activity of cells than in cells HT1080 MCF7 about two times more high.. I.e., LSPR produced MT1-MMP a biosensor nano based on whether and extent by detecting the expression of, MT1-MMP may detect cancer cells expression.