KR101860259B1

KR101860259B1 - Nanotube array with fluorescent dyes inside and capping agents comprising peptide substrates specific to peptidases, Method of preparing the same, and Fluorometric Method for Quantatification of Enzyme concentration using the same

Info

Publication number: KR101860259B1
Application number: KR1020170042900A
Authority: KR
Inventors: 손상준; 이우승; 전미연
Original assignee: 가천대학교 산학협력단
Priority date: 2017-04-03
Filing date: 2017-04-03
Publication date: 2018-06-27

Abstract

The present invention relates to a nanotube array with a fluorescent material included therein and a peptide cap having substrate specificity to a protease on an outer surface, a manufacturing method thereof, and a quantifying method of a protease by fluorometry using the same. The present invention provides a nanotube array in which a peptide cap degradable by an enzyme is formed on a nanotube. According to the present invention, the nanotube array has a densely introduced hydrophobic cap material (peptide cap) with substrate specificity to an enzyme to properly store a fluorescent material therein. Also, the hydrophobic cap material is degraded when coming in contact with the enzyme to discharge the fluorescent material out of the nanotube. If a fluorescence intensity value of a collected dye is measured after coming in contact with the enzyme (incubation) for at least two hours, the trypsin concentration can be detected in a range of 1 ng. Quantification of a protease can be realized without a process of undergoing centrifugation of a reaction solution.

Description

A method of preparing a nanotube array comprising a nanotube array having a fluorescence substance on its inner surface and a peptide cap having a substrate specificity to a protein hydrolyzing enzyme on its outer surface, a method for producing the nanotube array, and a method for quantitatively determining a protein hydrolyzing enzyme using fluorescence analysis using the nanotube array with fluorescent dyes inside and capping agents comprising peptide substrates specific to peptidases, Method of preparing same, and Fluorometric Method for Quantification of Enzyme concentration using the same}

The present invention relates to a nanotube array carrying a fluorescent substance therein and having a peptide cap having a substrate specificity to a protein hydrolyzing enzyme on the outer surface, a method for producing the same, and a method for quantitatively determining a protein hydrolyzing enzyme using fluorescence analysis using the same And more particularly to a nanotube array in which an enzyme is detected by using a cap made of an amino acid sequence having an internal fluorescent material and formed at an entrance of a nanotube and having substrate specificity to a specific hydrolytic enzyme, And a method for quantitatively determining a protein hydrolyzing enzyme by fluorescence analysis using the same.

Protease is an enzyme that regulates protein activity or fate through hydrolysis of irreversible peptide bonds. Protease is a living organism that constitutes a protein that mediates a large number of life phenomena. It is a living organism such as protein catabolism, blood coagulation, cell growth and migration, protein activation, cell regulation and signal development, It plays a key role in pathological processes such as physiological phenomena and inflammation, cancer development and metastasis, and viral infection. Therefore, it is possible to develop an inhibitor that can diagnose diseases early or measure the activity of protease related enzymes by measuring the activity of the protease.

Recently, it has been newly found that protease plays a pivotal role in various human diseases such as cancer, dementia and AIDS. As an example, MMP (matrix metalloprotease) has been recognized as a factor that degrades the extracellular matrix in cells and the body in the past. However, in recent studies, it has been shown that they inhibit integrin signaling and pericellular matrix And it has been shown that it plays an important role in cancer growth such as new blood vessel formation, invasion of cancer cells, and metastasis.

In addition to the above-mentioned MMPs, it has been found that various proteases play a pivotal role in the pathogenesis of many diseases. Therefore, protease and its substrate proteins have attracted great interest as a main target of the development of new drugs. In particular, as new substrate proteins are discovered, the physiological functions of various proteolytic enzymes will be newly illuminated in the future, and new target protein proteases are expected to be discovered.

In the development of new drugs targeting such protease, it is very important to analyze the activity of the protease. Representative protease activity analysis methods currently used include peaks shift by spectroscopy using high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA), or a phospholipid conjugated polypeptide substrate. And the like. However, these methods have a disadvantage in that they are not economical and time efficient to use in screening many drugs as in the development of a new drug because a multi-step measurement protocol is required.

Another method for analyzing the activity of proteolytic enzymes is fluorescence quenching and measurement methods based on the characteristics of fluorescent dyes such as nanomaterials. For example, Cupp-Enyard et al. Proposed a protease kit applicable to serine protease, metalloproteinase, aspartic protease, cysteine protease, and the like, targeting the FITC label (labeled) calcein substrate Respectively. The protease kit compares the control sample, which is a non-enzyme system, with the trypsin-containing sample, and measures their trypsin activity to measure the efficacy and sensitivity. However, the method using the proposed protease kit can be performed before the fluorescent substance is exposed to the enzyme, and even if the enzyme specificity is shown, the concentration (content) of the fluorescent substance attached to the kit is small and the fluorescent substance degraded by the enzyme There was a limit to measuring the enzyme concentration from the optical phenomenon.

An object of the present invention is to provide an array structure capable of storing therein a sufficient amount of dye necessary for reliable enzyme concentration measurement and releasing a dye upon enzyme reaction.

An object of the present invention is to provide an enzyme concentration measuring method and a sensor capable of measuring a more reliable and accurate enzyme concentration.

One aspect of the present invention is

Performing a plasma treatment on the anodized aluminum (AAO) template in which a plurality of pores are formed to form a bottleneck (a region narrower than the inlet) under the pore entrance;

Coating the inside surface of the pores to form a nanotube;

Modifying the bottleneck with an amine group;

And binding a specific amino acid sequence to the amine group to form an amino acid sequence stopper on the bottleneck.

In another aspect,

An array of a plurality of nanotubes, wherein the nanotubes have a bottleneck that is recessed inward at an inlet portion and has a diameter smaller than a diameter of the tube main body, the nanotubes including an amino acid sequence plug formed in the bottleneck portion And the amino acid sequence plug is attached to an amine group-containing compound attached to the bottleneck.

In yet another aspect,

Immersing the nanotubes in a fluorescent dye solution to load the fluorescent dye into the nanotubes; Contacting a solution containing an unknown enzyme to a cap portion of the nanotube for a predetermined time; And collecting the discharged fluorescent dye or nanotube to measure the fluorescence intensity of the dye.

The present invention provides a nanotube array in which a peptide cap capable of being degraded by an enzyme is formed on a nanotube. The nanotube array of the present invention is capable of storing a fluorescent material in the inside of a nanotube array having a specific substance of an enzyme substrate (a peptide stopper) densely inserted therein, and in contact with an enzyme, the hydrophobic stopper material is decomposed, Can be released outside the nanotube.

The conventional kit structure requires at least a few hundred microliters of reagent and requires complicated steps of preparation of two or more complicated solutions such as enzyme substrate solution, buffer solution, and small mineral solution and solution preparation, reaction, separation and fluorescence measurement There has been a difficulty in detecting the final fluorescence signal. On the other hand, the nanotube array of the present invention consists of two steps of measuring fluorescence by nano drop immediately after the sample is dropped for 1 hour or 4 hours after dropping a sample amount of 1 to 5 을 onto the surface of the nanotube array. It is possible to guarantee a comparatively small sample size, a simple measurement procedure, and a fast measurement time.

In addition, the nanotube array of the present invention can reduce the contamination or decomposition of amino acid sequences because the amino acid sequence caps are fixed to the inner bottleneck of the tube (relatively little external exposure), compared to a structure in which peptides are fixed on the surface of a conventional flat kit .

1 shows a nanotube array having an amino acid sequence stopper according to the present invention.
Fig. 2 shows the manufacturing method of Fig.
FIG. 3 shows the measurement of the fluorescence intensity of a dye using FIG.
4 is a fluorescence image of the template measured in Example 2 and Comparative Examples 1 to 3. Fig.
FIG. 5 shows the fluorescence intensities according to the respective concentrations and time (0, 1, 2, 4, 8 hours).
6 shows the fluorescence intensities of the dyes according to the contact time by trypsin concentration.
FIG. 7 shows the relationship between the fluorescence intensity and the concentration according to each time, with reference to the graph of FIG.
8 shows the fluorescence intensity of the dye according to the plug structure.

The present invention relates to a method for producing a nanotube array comprising an amino acid sequence stopper. FIG. 1 shows a nanotube array having an amino acid sequence stopper according to the present invention, and FIG. 2 shows a manufacturing method of FIG.

Referring to FIG. 2, the nanotube manufacturing method of the present invention includes a bottleneck forming step, a nanotube forming step, a reforming step, and a cap forming step.

The bottleneck forming step is a step of plasma-treating an AAO template formed with a plurality of pores to form a bottleneck (a region narrower than the inlet) under the foreskin inlet.

The anodic aluminum oxide (AAO) template with multiple pores can be prepared by known methods. For example, an aluminum foil is put into an oxalic acid solution, an anodic oxidation reaction is performed by applying a voltage, and irregularly grown alumina is removed. Subsequently, the aluminum foil is again placed in a solution of oxalic acid, and an anodic oxidation reaction is further performed to obtain an AAO template having many pores.

The bottleneck may be formed by treating the AAO template with argon plasma. For example, when the AAO template is processed in a chamber with a power of 150 W to 300 W or less, alumina at the pore inlet portion is abutted against the high energy argon plasma particles and peels off the surface, but is rearranged next to the entrance A bottleneck portion with a narrow diameter is formed.

Referring to FIG. 1, the bottleneck 2 is a region that is narrower in diameter than the inlet just under the pore inlet. The present invention can narrow the entrance of the nanotube through the formation of the bottleneck. When the entrance of the nanotube is wide, the effect of blocking the outflow of the fluorescent dye aqueous solution solvent of the amino acid sequence stopper (peptide stopper) composed of the hydrophobic amino acid is deteriorated. Therefore, in the present invention, the entrance of the nanotube is reduced, The blocking function can be enhanced.

The forming of the nanotubes may be performed by coating various materials on the inside of the pores. For example, the nanotube may be manufactured by coating one material selected from the group consisting of silica, carbon, titanium oxide, tungsten oxide, and silicon on the inside of the pore.

More specifically, the silica nanotubes can be prepared by coating the pores of the AAO template with silica by a sol-gel method. The silica sol solution can be prepared by polymerizing a silica precursor with stirring in alcohol and / or water. As the silica precursor, chlorosilane or tetraalkoxysilane may be exemplified. In this case, it is preferable that the alkoxy group is a linear or branched alkoxy group of C1-C5. Any silica adsorbed on the AAO template and capable of forming silicon dioxide during the drying and oxidation process can be used as the silica precursor.

The plurality of nanotubes formed in the AAO template may have a diameter of 5 nm to 100 nm and a length of 100 nm to 30 microns, but the present invention is not limited thereto.

The method of the present invention includes modifying the bottleneck surface with an amine group (which may also be referred to as partial reforming).

The reforming step is a step of selectively introducing an amine group into the vicinity of the bottleneck, rather than reforming the entire interior of the nanotube with an amine group.

On the other hand, when the AAO template is immersed in an amine group-containing polymer solution (APTS solution), amine groups are introduced throughout the entire nanotubes, making it difficult to introduce the peptide only into the bottleneck.

Accordingly, the present invention relates to a method and apparatus for wetting APTS on a surface of a microbead having a size larger than that of a nanotube inlet, preferably a bottleneck, and then contacting the bead with the AAO template, Group can be introduced.

More specifically, the step of modifying with an amine group

Mixing an aqueous solution of a compound having an amine group with a microbead, separating the microbead on the surface of the compound from the solution, separating the nanotube-formed anodized aluminum (AAO) template and the microbead Into the microtube, and a hammering step of vibrating the microtube.

The amine group-containing compound may be at least one selected from the group consisting of aminopropyltriethoxysilane (APTES), aminopropyltrimethoxysilane, ethylenediamine trimethoxysilane 3- (2-Aminoethylamino) propyltrimethoxysilane, diethylenetriamine methoxysilane (3-trimethoxysilylpropyl) diethylenetriamine.

The microbeads are preferably alumina, silica, titanium oxide, tungsten oxide, or the like. The size of the microbead may be 0.2 to 100 microns, but it may be spherical or elliptical having a diameter larger than that of the nanotube inlet.

The microtube is generally a material that can withstand the vibration applied thereto, and is not limited thereto. In general, plastic products which are easy to manufacture and have high strength can be used. There is no particular limitation on the vibration speed of the microtubes, but it is preferable to vibrate at a speed of 60 to 2000 rpm for 10 minutes to 72 hours.

There is no particular limitation on the temperature in the above-mentioned hammering step, but it is easy to carry out by about 5 to 80 ° C. The method of the present invention can appropriately adjust the time for hammering according to the hardness, size, and speed of the microbead.

The term hammering refers to vibrating the microtube to strike the closure material at a rapid rate with the microbeads therein.

In the cap forming step, a specific amino acid sequence is bound to the amine group to form an amino acid sequence stopper on the bottleneck.

In the cap forming step, amino acid may be attached to the bottleneck part by using amino group as a starting point and by known solid phase peptide synthesis (SPPS) method.

The amino acid sequence stopper is a polypeptide consisting of an amino acid sequence that is degraded to a specific protease, and the polypeptide functions as a cap for the nanotube.

The amino acid sequence stopper may be prepared so as to have a sequence capable of being degraded by a specific protease to be measured.

Examples of the protease include trypsin, cardeptine, MMP (matrix metalloproteinase), caspase, secretase, urokinase, HIV-1 protease (HIV-1 protease), HSV-1 protease, TEV protease, prostate specific antigen (PSA), anthrax lethal factorendopeptidase, or thrombin ) And the like.

For example, the amino acid sequence stopper may include an amino acid sequence of Phe-Trp-Phe which is decomposable to trypsin. In addition, the amino acid sequence stopper may include an amino acid sequence of Gly-Phe-Leu-Gly degradable to cardiffin B.

In the cap forming step, the amino acid sequence plug may be formed of a hydrophobic amino acid. The hydrophobic plug can effectively block the release of the hydrophilic solution stored inside the nanotube.

The cap forming step may form three or more amino acid sequences by a solid phase peptide synthesis (SPPS) method. When the number of amino acid sequences is two or less, some of the hydrophilic solution stored in the nanotubes may be released even before the reaction with the protease.

The cap forming step may include attaching a hydrophobic compound and a hydrolysis promoting linker to the amino acid sequence.

The hydrophobic compound may be a hydrophobic compound selected from the group consisting of bile acid, fatty acid and cholesterol. Examples of the fatty acids include oleic acid, linolenic acid, linolenic acid, stearic acid, arachidonic acid, palmitic acid, and lauric acid.

The hydrolysis promoting linker may be an amide bondable compound including an alkyl compound having 4 to 8 carbon atoms. For example, the hydrolysis linker may be an aminohexanoic acid having 6 carbon atoms.

Proteolytic enzymes (eg, trypsin) are too large to access amino acids. Therefore, the present invention can facilitate the access and connection of the amino acid sequence with the enzyme using a hydrolysis linker having an alkyl chain.

The method of the present invention provides nanotubes formed with amino acid sequence caps (peptide caps) and nanotube arrays in which they are arranged. The method of the present invention can provide an amino acid sequence stopper that stores a fluorescent substance inside the nanotube and prevents the fluorescent substance from being leaked to the outside.

The method of the present invention provides a new method for improving the blocking efficiency of a fluorescent material of a cap by making a bottleneck near a nanotube inlet and forming an amino acid sequence in the bottleneck. The method of the present invention provides a method of forming amino acid sequence caps by selectively binding an amine group only to a bottleneck to implement the method.

In another aspect, the present invention provides an array in which a plurality of nanotubes are arranged. The nanotube array can be referred to the manufacturing method described above.

Referring to FIG. 1, the nanotube array of the present invention includes a plurality of nanotubes 10 formed on an AAO template, and an amino acid sequencer 20 formed on the nanotube bottleneck A.

The bottleneck A is a region where the inner surface of the inlet portion of the nanotube protrudes inward to narrow the inlet. That is, the bottleneck A has a diameter smaller than the diameter of the main body of the nanotube 10.

The amino acid sequence stopper (20) is formed by binding to an amine group-containing compound attached to the bottleneck portion (A).

The amino acid sequence stopper may be represented by the following formula (1).

[Chemical Formula 1]

R-A-B

Wherein R is a hydrolysis promoting linker, A is an amino acid sequence in which three or more standard amino acids are peptide-linked, and the three or more amino acid sequences may be the same or different standard amino acids, and B is a hydrophobic compound.

The A is a polypeptide consisting of an amino acid sequence that is degraded to a specific protease, and the polypeptide functions as a cap for the nanotube.

For example, the A may comprise an amino acid sequence of -Phe-Trp-Phe- which is capable of degrading to trypsin. In addition, the A may include an amino acid sequence of -Gly-Phe-Leu-Gly- which is capable of degrading to carduxin B.

The A may be one in which at least three hydrophobic amino acids are peptide-bonded.

As the hydrolysis promoting linker, R may be an amide bondable compound including an alkyl compound having 4 to 8 carbon atoms. For example, the hydrolysis linker may be an aminohexanoic acid having 6 carbon atoms.

The B may be a hydrophobic compound selected from the group consisting of bile acid, fatty acid and cholesterol. Examples of the fatty acids include oleic acid, linolenic acid, linolenic acid, stearic acid, arachidonic acid, palmitic acid, and lauric acid.

The nanotube array of the present invention is a structure in which a plurality of nanotubes are vertically gathered. The nanotube array can store fluorescent dyes in each of a plurality of nanotubes and can be used for screening new drugs such as high-speed mass screening because it can be decomposed by a specific enzyme to provide fluorescence generation or intensity.

In another aspect, the present invention relates to a method for detecting an enzyme.

The enzyme detection method of the present invention includes a step of loading a fluorescent dye, a step of opening a hydrophobic stopper by a protein hydrolyzing action on the enzyme, and a step of measuring the fluorescence intensity of the released fluorescent dye using nano drop.

The fluorescent dye loading step is a step of immersing the nanotube array in a fluorescent dye solution. For example, the fluorescent dyes may be selected from the group consisting of biotin, fluorescein 5 (6) -isothiocyanate, rhodamine, Cy5, Cy3, EDANS (5- (2'- Amino-1-naphthalene sulfuric acid), tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), and the like.

The fluorescent dye may be dissolved in dimethylsulfoxide (DMSO), alcohols including methanol and ethanol, acetonitrile, dimethylformamide (DMF) and the like.

When the nanotube array is immersed in the fluorescent dye solution, the fluorescent dye solution is very slowly filled into the nanotubes despite the presence of the amino acid sequence plug. The loading step may be performed for about 1 to 4 hours.

The enzyme contacting step is a step of separating the nanotube array from the fluorescent dye solution, and then contacting a solution containing the enzyme with the stopper portion of the nanotube for a predetermined period of time.

The enzyme contacting step may be carried out in various ways.

For example, a plurality of holes are made in a thin film (Parafilm) with a micropunch, and the holes are formed so as to correspond to the entrance (cap portion) of the nanotubes. When the thin film is covered with a nanotube array and the enzyme is dropped into the hole, the enzyme can be brought into contact with the stopper portion. The enzyme contacting step can be carried out for several hours.

When the amino acid sequence plug is specifically reacted with the enzyme by the enzyme contacting step, the fluorescent dye solution loaded inside the nanotube is released from the nanotube. More specifically, if the nanotube has a hydrophobic stopper, the water molecules in the solution can not pass through the stopper, so that the dye inside is left intact, but when the hydrophobic stopper is removed by the enzyme, .

On the contrary, when the enzyme does not specifically react with the amino acid sequencer, there is no fluorescent dye emission.

When a hydrophobic plug composed of a substrate having a selectivity for a protein hydrolyzing enzyme is decomposed by an enzyme, it is possible to transfer a solvent water molecule, which is restrained by a hydrophobic plug, so that a fluorescent dye existing inside the nano array is released to the enzyme solution Resulting in an increase in the fluorescence signal. To measure this fluorescence signal, 1 μL of the enzyme solution present on the surface was taken and measured by Nano Drop.

The method can determine whether the enzyme has substrate specificity in the amino acid sequence plug according to the fluorescence intensity of the collected fluorescent dye.

The nanotube array can be used as an enzyme detection sensor.

The present invention can detect the trypsin concentration to a range of 1 ng by measuring the fluorescence intensity value of the collected dyes after contacting (incubating) the enzyme for 2 hours or more.

The present invention can quantify protein hydrolyzing enzyme without the process of centrifuging the reaction solution.

Hereinafter, the present invention will be described in detail with reference to the embodiments and drawings. It should be understood, however, that the appended claims are illustrative of the specific embodiments of the invention and are not intended to limit the scope of the invention.

Example One

AAO Template manufacturing

An aluminum film having a thickness of 0.25 mm was subjected to a one-step anodic oxidation reaction in a 0.3 M oxalic acid solution at 10 ° C and 40 V for 7 hours. Subsequently, the irregularly grown alumina in the first stage oxidation reaction was removed by etching. The AAO template was prepared by performing the two - step oxidation reaction under the same conditions as in step 1. The prepared alumina template was immersed in a 5% phosphoric acid solution to enlarge the hole size.

argon plasma process

The prepared AAO template was treated with argon plasma at 250 W and 20 cc for 7 minutes to prepare a SNT template of a gourd-shaped SNT and a bottlenecked structure as a thin layer (GSNT template).

SNT - AA0 template Produce

The plasma treated AAO template was immersed in a solution of 95% ethanol, ammonium hydroxide, and TEOS (Tetraethyl orthosilicate, 98%, Sigma Aldrich) in a volume ratio of 100: 32: 2. A silica-coated AAO template (SNT-AA0 template) was obtained by the sol-gel method.

SNT - AA0 template Opening Partial reforming near the inlet ( APTES Partial reforming)

(Volume ratio = 100: 20: 1) of toluene, (3-aminopropyl) triethoxysilane (APTES, 98%, Sigma Aldrich) and DIEA (N1- (3- trimethoxysilylpropyl) diethylenetriamine) The AAO template and the microphone bead were inserted into the tube and then subjected to a hammering treatment for about 30 minutes. After the reforming reaction, the template was washed with methanol and cured at 120 < 0 > C.

Fixation of amino acid sequence

The SPPS method for fixing the amino acid sequence bound to the amine group formed at the entrance end of GSNT is as follows. The GSNT template was immersed in a 10% TEA / MC (v / v) (Triethylamine (TEA) solution) and MC and IPA were used to wash the template alternately. The washed template was immersed in the reaction solution (Boc-Amino acids / Boc-Ahx-OH / Oleic acid, HOBt, mixture of DCC and DIEA, 200 μM each) and shaken for 1 hour. After the reaction, the template was washed with DMF, IPA, and MC. The washed template was reacted with 50% TFA / MC solution and boc protecting groups were separated. The template was washed alternately with MC and IPA and the template was immersed in the TEA solution for 10 minutes. The previous SPPA procedure was repeated until the desired amino acid sequence was fixed. The octadecyl group was then fixed with oleic acid to form a highly reactive hydrophobic covalent plug. The obtained template is the nanotube array of the present invention.

Dye loading

The nanotube array was immersed in a fluorescent dye solution (50 mM RB in DMSO (dimethylsulfoxide)) for about 2 hours. DI.

Enzyme contact

The holes in the parafilm were made into micro-punches, which were placed on dye-loaded nanotube arrays. The enzyme solution (Enzyme: trypsin, buffer solution: 50 mM Tris-HCl buffer solution) was dropped onto the parafilm (10 μL). It was incubated for 4 hours under dark conditions.

RB (FITC) Fluorescence intensity Measure

The enzyme solution was collected and fluorescence was measured with a fluorescence meter.

Example 2

In order to observe whether or not the bottleneck was formed, the APTES partial modification with the argon plasma treatment, the silica coating and the microbead was carried out in Example 1, and the template was immersed in the fluorescent dye FITE and collected and washed (Example 1 No sequencing and no enzyme contact was made). Fluorescence was observed with an optical microscope by irradiating 485nm wavelength.

Comparative Example 1

The nanotubes were formed in the AAO template without performing the argon plasma treatment and the APTES partial modification in Example 2, and the template was immersed in the fluorescent dye FITE and collected and washed.

Comparative Example 2

APTES modification was performed on the entire interior of the nanotubes by immersing the AAO template in the APTES solution without performing the APTES partial modification in Example 2. The template was immersed in the fluorescent dye FITE and collected and washed.

Comparative Example 3

The AAO template was obtained in Example 2 without performing the argon plasma treatment (APTES partial modification with microbeads was performed). After immersing the template in the fluorescent dye FITE, it was collected and washed.

4 is a fluorescence image of the template measured in Example 2 and Comparative Examples 1 to 3. Fig. 4a (dark field image) and 4b (fluorescence image) were Comparative Example 1, and almost no fluorescence could be observed with a fluorescence optical microscope since they did not have an amine group portion to react with the isothiocyanate group of FITC. 4C and 4D are fluorescence images of Comparative Example 2. Fig. FIGS. 4C and 4D show that the FITC molecules react with the amine groups of the APTES modified to the inside of the nanotubes and attach all over the inside of the SNT, so that the fluorescence is observed by the entire length (size) of the nanotubes. 4E and 4F are fluorescence images of Comparative Example 3. Fig. In the case of FIG. 2F, the magnitude of fluorescence is much smaller than that of 4c and 4d because APTES is partially modified near the entrance. Figs. 4G and 4H are fluorescence images of Example 2. Fig. 4h shows a smaller and uniform fluorescence intensity as compared to FIG. 4f of Comparative Example 3. FIG. This is because FITC is attached to the entire inlet side in the comparative example 3, whereas FITC is formed only in the bottleneck in the second embodiment.

Depending on concentration Fluorescence intensity Experiment

The enzyme concentration measurement experiment was performed as in Example 1. SPPS is a method of peptide sequences stopper Ahx-Phe-Trp-Phe- C 18 (Ahx-FWFC 18) made of a (Ahx (aminohexanoic acid), Phenylalanine (Phenylalanine, Phe), tryptophan (Tryptophan, Trp)) Im. Enzymes were tested as in Example 1 using trypsin and the concentrations were varied at 0, 7.4, 14.8, 74.2, 146.7, 741.2, 1467.0 and 3710.7 ng.

FIG. 5 shows the fluorescence intensities according to the respective concentrations and time (0, 1, 2, 4, 8 hours). Referring to FIG. 5, when trypsin treatment was not performed, the dye molecules were still retained in the nanotubes, and the fluorescence intensity was almost constant. That is, when the trypsin treatment is not carried out, it can be confirmed that there is no degradation or damage of the amino acid sequence stopper. When 7.4 ng of trypsin is added, the RB molecules trapped in the nanotube (CGSNT) are released because the sequence plug exposed by trypsin is degraded. However, since the amount of trypsin is not sufficient, the CGSNT can not decompose all the sequences attached to the wall of the plug, and the fluorescence intensity is not so high. On the other hand, when 14.8 ng or more of trypsin was added, fluorescence intensity increased sharply compared to 0 or 7.4 ng of trypsin. This is because amino acid sequence caps are almost completely degraded by trypsin, and the dye inside the nanotubes is discharged outside the tube.

Over time Fluorescence intensity Experiment

6 shows the fluorescence intensities of the dyes according to the contact time by trypsin concentration. Referring to FIG. 4, there was no change in fluorescence intensity in the absence of the enzyme, and it was confirmed that the fluorescence intensities were increased in proportion to the enzyme concentration in the case of the contact (incubated) for 1 hour or more.

FIG. 7 shows the relationship between the fluorescence intensity and the concentration according to each time, with reference to the graph of FIG. Referring to FIG. 7, when the enzyme is contacted (incubated) for 2 hours or more, the fluorescent dye according to the enzyme concentration shows almost linearity. The graph of FIG. 7 shows that the fluorescence intensity value of the collected dyes can be measured to detect the trypsin concentration to the range of 1 ng.

Depending on the plug structure Fluorescence intensity Experiment

The enzyme concentration measurement experiment was performed as in Example 1. SPPS is a method of peptide sequences stopper Ahx-Phe-Trp-Phe- C 18 (Ahx-FWFC 18) made of a (Ahx (aminohexanoic acid), Phenylalanine (Phenylalanine, Phe), tryptophan (Tryptophan, Trp)) Im. The enzyme was prepared in the same manner as in Example 1, except that trypsin was used and the trypsin concentration was dropped on the parafilm at 0, 0.37, 37, and 371 ng, respectively. Comparative Example 4 has a nucleotide sequence plug of Ahx-Phe-Trp-Phe-C ₁₈ (Ahx-FWFC ₁₈ ) as in Example 1, but does not have a bottleneck near the entrance of the nanotube due to no argon plasma treatment Structure template. In this case, the fluorescence intensity is very low as compared with Comparative Examples 4 to 8 and Example 3. This is because Comparative Example 4 has the same amino acid sequence plug as that of Example 3, but the dye molecules are escaping out of the nanotubes during cleaning because the plasma is not treated (no bottleneck) and the hole on the inlet side of the nanotube is too wide.

In Comparative Example 5, the plasma treatment was performed, but the amino acid sequence plug was Ahx-FC ₁₈ . In Comparative Example 6, plasma treatment was performed, but the amino acid sequence plug was Ahx-FWC ₁₈ . In Comparative Examples 5 and 6, even if the bottleneck is formed at the inlet of the nanotube, the number of amino acids serving as a cap is one to two, which does not provide a sufficient sealing function.

Comparative Example 7 is a case in which no hydrophobic compound, fatty acid (oleic acid) is present as Ahx-FWF-GSNT, and Comparative Example 8 is in the absence of aminohexanoic acid as a hydrolysis promoting linker (FWFC ₁₈ -GSNTs). In the case of Comparative Examples 7 and 8, the rate of fluorescence intensity increase compared to the trypsin concentration was lower than that in Example 4. [ Example 4 has all three amino acids, a hydrophobic compound and a hydrolysis promoting linker, and can increase rapidly in proportion to the amount of trypsin even when the fluorescence intensity is 40 ng or less.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments.

Claims

Performing a plasma treatment on the anodized aluminum (AAO) template in which a plurality of pores are formed to form a bottleneck (a region narrower than the inlet) under the pore entrance;
Coating the inner surface of the pores to form a nanotube;
Modifying the bottleneck with an amine group;
And binding a specific amino acid sequence to the amine group to form an amino acid sequence stopper on the bottleneck.

2. The method of claim 1, wherein the plasma treatment step is performed with a high energy plasma of 150 to 300 W.

The method of claim 1, wherein the step of modifying with an amine group comprises:
Mixing a solution in which a compound having an amine group is dissolved into a microbead;
Separating the microbeads with the compound on the surface from the solution;
Inserting the micro-bead into an anodized aluminum (AAO) template having a nanotube formed therein; And
And a hammering step of vibrating the microtubes.

The nanotube array according to claim 3, wherein the microbead has a size larger than an entrance of the nanotube

4. The method of claim 3, wherein the compound having an amine group is selected from the group consisting of aminopropyltriethoxysilane (APTES), aminopropyltrimethoxysilane, ethylenediaminetetramethylsilane (3- (2-Aminoethylamino) propyltrimethoxysilane ) Or diethylenetriamine ((3-Trimethoxysilylpropyl) diethylenetriamine).

The method according to claim 1, wherein the amino acid sequence stopper is formed of a hydrophobic amino acid.

The method according to claim 1, wherein the step of forming the amino acid sequence stopper comprises forming three or more amino acid sequences by a solid phase peptide synthesis (SPPS) method, wherein a hydrophobic compound and an alkyl compound are attached to the amino acid sequence Method of manufacturing a tube array

An array in which a plurality of nanotubes are arranged,
Wherein the nanotube includes a bottleneck portion which is recessed inward at an inlet portion and has a diameter smaller than a diameter of the nanotube body, the nanotube includes an amino acid sequence plug formed in the bottleneck portion, Is bonded to an amine group-containing compound attached to the nanotube array.

9. The nanotube array according to claim 8, wherein the amino acid sequence stopper is represented by the following formula (1).
[Chemical Formula 1]
RAB
Wherein R is an alkyl compound having 4 or more carbon atoms, A is an amino acid sequence in which three or more standard amino acids are peptide-bonded, and the three or more amino acid sequences may be the same or different standard amino acids, and B is a hydrophobic compound.

10. The nanotube array of claim 9, wherein the A is peptide-linked with three or more hydrophobic amino acids.

10. The nanotube array according to claim 9, wherein the A has substrate specificity to a specific protease and is degraded only to the protease.

12. The nanotube array of claim 11, wherein A comprises -Phe-Trp-Phe- or -Gly-Phe-Leu-Gly-.

The nanotube array according to claim 9, wherein R is an amide bondable compound including an alkyl compound having 4 to 8 carbon atoms.

10. The nanotube array according to claim 9, wherein B is a hydrophobic compound selected from the group consisting of bile acids, fatty acids and cholesterol.

Immersing the nanotube array of any one of claims 8 to 14 in a fluorescent dye solution to load the fluorescent dye into the nanotube;
Contacting a solution containing an unknown enzyme to a cap portion of the nanotube for a predetermined time; And
Collecting the emitted fluorescent dye or nanotube and measuring the fluorescence signal signal of the dye,
Wherein the contacting step comprises an open step of hydrolyzing and opening the nanotube cap with the enzyme when the unknown enzyme has substrate selectivity to the nanotube plug.

16. The enzyme detection method according to claim 15, wherein the enzyme detection method determines whether or not the enzyme has substrate specificity in the amino acid sequence stopper according to intensity of the collected fluorescent dye or nanotube.