KR20110110497A - Method of manufacturing sensor for detection of target material(s), sensor manufactured by the method and method of detecting target material(s) using the sensor - Google Patents

Abstract

The present invention relates to a method for manufacturing a sensor for selectively detecting a specific hazardous chemical species or non-selectively simultaneously detecting harmful chemicals belonging to a specific hazardous chemical group. According to the manufacturing method, it is cheaper to provide a sensor that can be easily performed on site for selective detection of a specific hazardous chemical species or non-selective detection of all hazardous chemicals belonging to a specific hazardous chemical group. It can be built quickly and quickly, and the sensors manufactured in this way can be effectively used to quickly and accurately detect on-site the presence of specific hazardous chemical species or hazardous chemical groups to be detected.

Description

Manufacturing method of sensors for detecting hazardous chemicals, sensors manufactured by such methods and methods for detecting hazardous chemicals using such sensors {SENSOR OF MANUFACTURING SENSOR FOR DETECTION OF TARGET MATERIAL (S), SENSOR MANUFACTURED BY THE METHOD AND METHOD OF DETECTING TARGET MATERIAL (S) USING THE SENSOR}

The present invention relates to the detection of hazardous chemicals, and more particularly, to a method for manufacturing a sensor for species or group specific detection of misused hazardous chemicals, Sensors and methods for using these sensors to quickly and accurately detect specific hazardous chemical species or groups of hazardous chemicals on site.

In recent years, misuse of harmful chemicals in foods represented by concentrated aquatic products and processed products is a serious threat to the health and safety of humankind, and thus the reliability of food is already very low. In order to ensure the credibility of these foods and to restore the public's right to health, regulations on more aggressive and effective misuse of hazardous chemicals should be made, which can be realized through rapid and accurate on-site low molecular sensor system.

Representative hazardous chemicals that need to be regulated for abuse include antibiotics, pesticides, preservatives, environmental pollutants, and environmentally hazardous substances.

For example, in the case of antibiotics, the use of antibiotics in the domestic livestock industry is the highest against livestock production, compared with some developed countries such as Denmark, Japan, and the United States. Is a serious threat to your health.

In order to reduce the use of antibiotics, there is an urgent need for regulation of reckless use not only in the manufacture of feed but also in farms. In addition, establishing a rapid and accurate antibiotic detection system has become a very important task contributing to the public's health and safety.

Conventional techniques for regulating the misuse of antibiotics are difficult to apply in general techniques for various hazardous chemicals, or for selective detection of individual hazardous chemicals and for non-hazardous chemicals in specific hazardous chemical groups. It has technical limitations that selective detection is not easy or that on-site verification is not easy through rapid visual confirmation.

Accordingly, an object of the present invention is to provide a method for manufacturing a sensor that can specifically detect species or group of low-molecular harmful chemicals that are misused, such as antibiotics.

Another object of the present invention is to provide a sensor manufactured using the method.

Another object of the present invention relates to a method for specifically detecting a species or a group of low-molecular harmful chemicals being misused using a sensor manufactured using the method.

In accordance with the above object, the present invention provides a method of manufacturing a sensor for selectively detecting a specific hazardous chemical species or non-selective simultaneous detection of harmful chemicals belonging to a specific hazardous chemical group, 1) labeling a biotin to a target material to be detected, wherein the target material is one or more harmful chemicals having a specific molecular region or two or more harmful chemicals having a common molecular region, and the specific molecular region or common Labeling biotin on the target material to expose the molecular site; 2) selecting a receptor protein from an antibody display library that recognizes the exposed specific or common molecular region and binds to the biotin-labeled target material and at the same time has a higher binding capacity to the target material than the biotin-labeled target material ; 3) synthesizing a target substance analog labeled with a fluorescent substance having a lower binding force than that of the target substance to the receptor protein; And 4) immobilizing the receptor protein on a solid support and binding the fluorescent substance-labeled target substance analog to the receptor protein.

In accordance with the above object, the present invention also provides a method of manufacturing a sensor for selectively detecting individual antibiotic species belonging to a quinolone antibiotic or non-selective simultaneous detection of antibiotics belonging to a quinolone antibiotic group. 1) labeling a biotin to a target substance to be detected, wherein the target substance is at least one selected from the group consisting of quinolone antibiotics having a specific molecular region and a common molecular region, and the specific molecular region or common molecule Labeling the biotin to the target material to expose the site; 2) selecting a receptor protein from an antibody display library that recognizes the exposed specific or common molecular region and binds to the biotin-labeled target material and at the same time has a higher binding capacity to the target material than the biotin-labeled target material ; 3) synthesizing a target substance analog labeled with a fluorescent substance having a lower binding force than that of the target substance to the receptor protein; And 4) immobilizing the receptor protein on a solid support and binding the fluorescent substance-labeled target substance analog to the receptor protein.

According to the above another object, the present invention is a solid support; Receptor proteins immobilized on said solid support; And a target substance analog labeled with a fluorescent substance that binds to the receptor protein and selectively detected by a specific hazardous chemical species prepared according to the above-described method or belonging to a specific hazardous chemical group. Sensors for non-selective simultaneous detection of chemicals, e.g. selective detection of individual antibiotic species belonging to the quinolone antibiotics or non-selective simultaneous detection of antibiotics belonging to the quinolone antibiotic group Provide a sensor for

In accordance with another object, the present invention comprises the steps of 1) collecting a sample; And 2) subjecting the sample to a sensor as described above to identify a change in color or fluorescence, selectively detecting a specific hazardous chemical species or in a specific hazardous chemical group. Detection methods for non-selective simultaneous detection of harmful chemicals belonging to, for example, selective detection of individual antibiotic species belonging to the quinolone antibiotics or non-selective antibiotics belonging to the quinolone antibiotic group A detection method for detecting at the same time is provided.

The present invention also provides a compound represented by the following Chemical Formula 1:

Where

A _1, A _2, A ₃ and A ₄ each independently is hydrogen and the biotin-binding, biotin, or a linker, wherein the linker is C _{1 -20} alkyl chain and in the alkyl chain is a heteroatom, amide, reverse amide, ester (ester), carbamate and urea, and may include a group selected from the group except that A ₁ , A ₂ , A ₃ and A ₄ are simultaneously hydrogen;

X ₁ and X ₂ are each independently carbon or nitrogen;

Y is hydrogen or fluorine but when X ₂ is nitrogen, Y is absent;

R ₁ is an unsubstituted or a straight-chain alkyl chain of a C _{1 -8} alkyl substituted with a straight chain or a halogen C _{1 -5,} unsubstituted or substituted with a straight chain alkyl or halogen substituted C _{1 -5} C _{3 -} cyclic alkyl chains of _8, unsubstituted or C _{1 -5} straight-chain, and the aryl or heteroaryl of _-8 C ₆ alkyl chain substituted by a halogen or a, in the alkyl chain may include a hetero atom, X ₁ Can be linked to form a heterocycle;

R ₂ is hydrogen, amine, C _{1 - 8} alkyl, or C _{1 - 8} alkoxy;

R ₃ is hydrogen, straight chain alkyl of C _{1 -8,} cyclic alkyl chain in C _{3 -8,} or a heterocycle,

And wherein the heterocyclic ring is unsubstituted or substituted, N, O, S, SO and SO _2, 1 ring system or an aromatic or non-aromatic ring system of the compound of 2 _{5 -12} C containing at least one selected from the group consisting of the heterocyclic ring substituents are halogen, hydroxy, amino, nitro, cyano, C _{1 -} is selected from the _six die group consisting of alkyl _amino-6 alkyl, C _{1 - 6} alkoxy, C _{1 - 6} monoalkylamino and C _1.

The present invention also provides an antibody that can specifically bind to a molecular region other than the molecular region to which the biotin of the compound of Formula 1 is bound.

The present invention also provides a compound represented by the following formula (2):

Where

B ₁ , B ₂ , B ₃ and B ₄ are each independently hydrogen, Cy 3, Cy 5, FAM, TET, TxR, ROX, SYBR Green, ALEXA, FITC, BODIPY, TAMRA and Texas Red or and a fluorescent material in combination with the linker, the linker may be a C _{1 -20} alkyl chain in the alkyl chain comprises a functional group selected from heteroatoms, amide, reverse amide, ester (ester), a carbamate and a urea group consisting of Except that B ₁ , B ₂ , B ₃ and B ₄ are hydrogen at the same time;

X ₁ and X ₂ are each independently carbon or nitrogen;

Y is hydrogen or fluorine but when X ₂ is nitrogen, Y is absent;

R ₁ is an unsubstituted or a straight-chain alkyl chain of a C _{1 -8} alkyl substituted with a straight chain or a halogen C _{1 -5,} unsubstituted or substituted with a straight chain alkyl or halogen substituted C _{1 -5} C _{3 -} cyclic alkyl chains of _8, unsubstituted or C _{1 -5} alkyl, a straight chain or an aryl or heteroaryl group in the C _{6 -8} substituted by halogen in the alkyl chain may contain a hetero atom, and X ₁ Can be joined to form a heterocycle;

R ₂ is hydrogen, amine, C _{1 - 8} alkyl, or C _{1 - 8} alkoxy;

R ₃ is hydrogen, straight chain alkyl of C _{1 -8,} cyclic alkyl chain in C _{3 -8,} or a heterocycle,

And wherein the heterocyclic ring is unsubstituted or substituted, N, O, S, SO and SO _2, 1 ring system or an aromatic or non-aromatic ring system of the compound of 2 _{5 -12} C containing at least one selected from the group consisting of the heterocyclic ring substituents are halogen, hydroxy, amino, nitro, cyano, C _{1 -} is selected from the _six die group consisting of alkyl _amino-6 alkyl, C _{1 - 6} alkoxy, C _{1 - 6} monoalkylamino and C _1.

According to the manufacturing method of the sensor for detecting hazardous chemicals of the present invention, the selective detection of specific hazardous chemical species or non-selective detection of all hazardous chemicals belonging to a specific hazardous chemical group is performed. It is possible to build a cheaper and faster sensor that can be easily implemented in the sensor, and the sensor manufactured in this way is effectively used to quickly and accurately detect on-site the presence of a specific hazardous chemical species or a group of hazardous chemicals to be detected. Can be.

1 is a flow chart schematically showing a manufacturing method of a sensor for detecting a hazardous chemical according to an embodiment of the present invention.
FIG. 2 is a schematic diagram illustrating a process of labeling biotin to expose specific molecular sites of enlofloxacin for specific detection of enlofloxacin belonging to a (fluoro) quinolone antibiotic.
FIG. 3 is a schematic diagram illustrating the process of labeling biotin to specific molecular sites of nofloxacin such that the common molecular sites of (fluoro) quinolone antibiotics are exposed for simultaneous detection of all antibiotics belonging to (fluoro) quinolone antibiotics. It is a schematic diagram.
4 is a schematic diagram illustrating a screening process of scFv that recognizes and binds a common or specific molecular region of a biotin-labeled target material.
FIG. 5 shows a process for preparing an enlofloxacin derivative labeled Cy3 at a common molecular site for specific detection of enlofloxacin belonging to a (fluoro) quinolone antibiotic and a compound formed by such a process (CMY-001- It is a schematic diagram which shows Cy3).
FIG. 6 shows a process for preparing Cy3-labeled nofloxacin derivatives on specific molecular sites of nofloxacin for simultaneous detection of all antibiotics belonging to (fluoro) quinolone antibiotics and the compounds formed by such a process (CMY-002- It is a schematic diagram which shows Cy3).
7 is a schematic diagram showing a sensor according to an embodiment of the present invention and a target material detection process using the sensor.
8 shows the results of the primary selection process of antibody clones that bind CMY-001-CY3 or CMY-002-CY3 from an antibody display library.
Figure 9 shows the selectivity of clones (A2, C3) selected by competitively binding the first selected antibody clones (enclofloscin (CMY-003) and nofloxacin analogues (CMY-004)) , F6, F9, G10) shows the absorbance results.
10 shows SDS-PAGE results for clones with good selectivity (A2, F9, G10).
Figure 11 shows the fluorescence intensity on the surface of the substrate according to the concentration of enlofloxacin after treatment with the enrofloxacin in the sensor manufactured in Example 1.
12 shows the fluorescence intensity at the substrate surface according to the concentration of nofloxacin after treatment with nofloxacin in the sensor fabricated in Example 1. FIG.
Figure 13 shows the fluorescence intensity at the surface of the substrate according to the concentration of these antibiotics after treatment with enlofloxacin and nofloxacin to the sensor prepared in Example 1.
FIG. 14 shows the fluorescence intensity at the surface of the substrate according to the concentration of these antibiotics after treatment with enlofloxacin and nofloxacin in the sensor fabricated in Example 2. FIG.

A method for manufacturing a sensor for detecting a hazardous chemical substance of the present invention will be described in detail with reference to the accompanying drawings.

1 is a flow chart schematically showing a method of manufacturing a sensor for detecting a hazardous chemical substance according to an embodiment of the present invention.

Referring to FIG. 1 , first, biotin is labeled on a target material to be detected (S100).

The target substance is one or more hazardous chemicals having a specific molecular site or two or more hazardous chemicals having a common molecular site. In the case of specific species-specific detection, biotin is labeled on the target material to expose the specific molecular site, and in the case of specific group-specific detection, the common molecular site is exposed. Label biotin on the target material.

In the present invention, "specific molecular site" refers to a characteristic moiety unique to each of one or more hazardous chemicals, and "common molecular site" refers to all chemicals belonging to a specific hazardous chemical group. It means the molecular part which exists in common.

In order to simultaneously and non-selectively detect chemicals belonging to a specific chemical group, from the harmful chemical group consisting of two or more harmful chemicals having the common molecular region (e.g., fluoroquinolone antibiotics) It may consist of selecting one hazardous chemical (eg nofloxacin) and then labeling the biotin with the previously selected hazardous chemical so that the common molecular sites are exposed.

Specifically, the target material may be a drug including antibiotics, pesticides, preservatives, environmental hormones and the like. In particular, (fluoro) quinolone antibiotics, which are most frequently found in foods such as chicken, can be used as detection targets. Specific examples of (fluoro) quinolone antibiotics include enrofloxacin, ciprofloxacin, pyromidic acid, flumequine, ocfloxacin, and nofloxacin. ), Nalidixic acid (Nalidixic acid), pipemidic acid (Pipemidic acid) and the like and their structure is as follows.

When the target material is a quinolone antibiotic, the biotin-bound target material may be a compound of Formula 1 below:

[Formula 1]

Where

A _1, A _2, A ₃ and A ₄ each independently is hydrogen and the biotin-binding, biotin, or a linker, wherein the linker is C _{1 -20} alkyl chain and in the alkyl chain is a heteroatom, amide, reverse amide, ester (ester), carbamate and urea, and may include a group selected from the group except that A ₁ , A ₂ , A ₃ and A ₄ are simultaneously hydrogen;

X ₁ and X ₂ are each independently carbon or nitrogen;

Y is hydrogen or fluorine but when X ₂ is nitrogen, Y is absent;

R ₁ is an unsubstituted or a straight-chain alkyl chain of a C _{1 -8} alkyl substituted with a straight chain or a halogen C _{1 -5,} unsubstituted or substituted with a straight chain alkyl of C _1-5, or a halogen-substituted C _{3 -} cyclic alkyl chains of _8, unsubstituted or C _{1 -5} straight-chain, and the aryl or heteroaryl of _-8 C ₆ alkyl chain substituted by a halogen or a, in the alkyl chain may include a hetero atom, X ₁ Can be linked to form a heterocycle;

R ₂ is hydrogen, amine, C _{1 - 8} alkyl, or C _{1 - 8} alkoxy;

R ₃ is hydrogen, straight chain alkyl of C _{1 -8,} cyclic alkyl chain in C _{3 -8,} or a heterocycle,

And wherein the heterocyclic ring is unsubstituted or substituted, N, O, S, SO and SO _2, 1 ring system or an aromatic or non-aromatic ring system of the compound of 2 _{5 -12} C containing at least one selected from the group consisting of the heterocyclic ring substituents are halogen, hydroxy, amino, nitro, cyano, C _{1 -} is selected from the _six die group consisting of alkyl _amino-6 alkyl, C _{1 - 6} alkoxy, C _{1 - 6} monoalkylamino and C _1.

As used herein, "heteroatom" means an atom other than carbon or hydrogen, such as oxygen, nitrogen, sulfur, etc., "heteroaryl" means aryl containing said heteroatom, and "amide"- C (= 0) NH, "reverse amide" means -NHC (= 0) and "halogen" means fluorine, chlorine, bromine or iodine.

When selectively detecting a specific hazardous chemical species, biotin can be bound to a terminal functional group of a molecular site (eg, non-specific molecular site) other than the specific molecular site of the target material, and the specific hazardous chemical material When non-selective simultaneous detection of hazardous chemicals belonging to the group (group), one hazardous chemical substance belonging to the harmful chemicals group is selected, and then the molecular sites other than the common molecular sites (eg, specific molecular sites) Biotin can be bound to a terminal functional group of.

When the compound of Formula 1 selectively detects individual antibiotic species belonging to, for example, quinolone antibiotics, A ₄ of the compound is biotin and antibiotics belonging to the quinolone antibiotic group are non- When selectively detected simultaneously, A ₃ of the compound may be biotin.

More specifically, when the fluoroquinolone antibiotic represented by the following formula (3) is described as an example, the part shown in red on the right is a common molecular region and the part shown in black on the left is an antibiotic belonging to a fluoroquinolone antibiotic. Each corresponds to a distinctive molecular site.

Wherein R ₁ is ethyl or cyclopropyl, X is a carbon atom unsubstituted or substituted with CH (CH ₃ ) CH ₂ CH ₂ , CH (CH ₃ ) CH ₂ O, etc., and R ₂ is hydrogen, amine , such as a methyl, methoxy, R ₃ is substituted such as an unsubstituted or substituted by C _{1 -3} alkyl piperazine, unsubstituted or substituted with an amine (NH ₂₎ pyrrolidine.)

In the case of labeling the biotin to expose the common molecular region, the biotin may be bound to a molecular site other than the common molecular region, for example, a terminal functional group of the specific molecular region. For example, in the structure of Chemical Formula ₃ , biotin may be bonded to the left portion of black, preferably R ₃ . On the other hand, when biotin is labeled to expose specific molecular sites, biotin can be bound to terminal functional groups other than specific molecular sites, for example, non-specific molecular sites. For example, in the structure of Chemical Formula 3, biotin may be bonded to the right side, preferably, the carboxyl terminal, indicated in red.

The labeling process of biotin may be performed using chemical synthesis methods well known in the art such as amide or ester bonding, and may be directly connected to the target material, but mainly to the target material through a linker (Bai He, Subash Velaparthi, Gilles Pieffet, Chris Pennington, Aruna Mahesh, Denise L. Holzle, Michael Brunsteiner, Richard van Breemen, Sylvie Y. Blond, and Pavel A. Petukhov, J. Med. Chem . 2009, 52 , 7003-7013, Tadashi Honda, Tomasz Janosik, Yukiko Honda, Jie Han, Karen T. Liby, Charlotte R. Williams, Robin D. Couch, Amy C. Anderson, Michael B. Sporn, and Gordon W. Gribble J. Med . Chem . 2004, 47, 4923- 4932).

For example, the process of labeling biotin at the molecular site of enlofloxacin, which is a common molecular site of (fluoro) quinolone antibiotics, for the specific detection of enrofloxacin belonging to (fluoro) quinolone antibiotics. And the compound (CMY-001, Formula 1a) formed by such a process is shown in FIG. 2 , and is a type of (fluoro) quinolone antibiotic for the simultaneous detection of all antibiotic species belonging to the (fluoro) quinolone antibiotic. The process of labeling biotin on specific molecular sites of floxacin and the compound formed by such a process (CMY-002, Formula 1b) are shown in FIG. 3 .

[Formula 1a]

[Chemical Formula 1b]

Next, a receptor protein is recognized from the antibody display library to bind the biotin-labeled target material by recognizing the exposed specific or common molecular site, and at the same time, a receptor protein having a higher binding capacity to the target material than the biotin-labeled target material. Performs a process of selecting (S200).

The receptor protein may be an antibody selected from the group consisting of scFv, Fab and whole antibody, in particular scFv may be used. Such antibodies are advantageous in that they exhibit high selectivity for the group of molecules that can be bound. In addition, these antibodies are relatively inexpensive because they can be produced in large quantities using current genetic engineering techniques.

The antibody display library is a technique for displaying an antibody (scFv, etc.) library on a surface of a phage, cell or spore and searching for clones having high binding ability to antigens at high speed. It is used to select excellent receptor proteins.

If a common molecular moiety or molecule-specific portions of the biotin-labeled target substance with exhibited the selection process of the scFv that binds to this schematic as Figure 4.

Screening of receptor proteins (scFv, etc.) finds antibody clones that bind to biotin-labeled targets from antibody display libraries and competes with actual harmful chemicals (such as antibiotics) to screen for suitable clones. Competence assays can finally select harmful chemical species specific antibodies or harmful chemical group specific antibodies with good selectivity / binding ability (see Examples 1 and 2).

For example, the antibody may have an amino acid sequence encoded by a nucleic acid sequence represented by SEQ ID NO: 1 or 2. The antibody is also interpreted to include similar sequences that serve the same function, for example, a nucleic acid sequence having at least 90%, more preferably at least 95% identity, of the nucleic acid sequence of SEQ ID NO: 1 or 2 It is interpreted as including the encoded amino acid sequence.

Next, a target substance analog labeled with a fluorescent substance having a binding strength lower than that of the target substance to the receptor protein is synthesized (S300).

Target substance analogs bind to the aforementioned receptor proteins (scFv, etc.) but correspond to molecules with lower binding constants than the target substance to be detected.

The target substance analog may be a derivative of the target substance. Such derivatives may be bulky to induce structural changes at the binding site of the target material by adding bulky or ionic groups (which can reduce binding energy), by adding steric or charge interference, or Can be an artificial derivative such as a molecule modified by bonding a large group.

Fluorescent materials play an important role in visual inspection of foods containing harmful chemicals such as antibiotics. Such fluorescent materials include Cy3, Cy5, FAM, TET, TxR, ROX, SYBR Green, Fluorescent dyes such as ALEXA, FITC, BODIPY, TAMRA, Texas Red, and rhodamine may be used, but are not limited thereto.

For example, the target substance analog labeled with a fluorescent substance may be a compound represented by the following Chemical Formula 2:

[Formula 2]

Where

B ₁ , B ₂ , B ₃ and B ₄ are each independently hydrogen, Cy 3, Cy 5, FAM, TET, TxR, ROX, SYBR Green, ALEXA, FITC, BODIPY, TAMRA and Texas Red or and a fluorescent material in combination with the linker, the linker may be a C _{1 -20} alkyl chain in the alkyl chain comprises a functional group selected from heteroatoms, amide, reverse amide, ester (ester), a carbamate and a urea group consisting of Except that B ₁ , B ₂ , B ₃ and B ₄ are hydrogen at the same time;

X ₁ and X ₂ are each independently carbon or nitrogen;

Y is hydrogen or fluorine but when X ₂ is nitrogen, Y is absent;

R ₁ is an unsubstituted or a straight-chain alkyl chain of a C _{1 -8} alkyl substituted with a straight chain or a halogen C _{1 -5,} unsubstituted or substituted with a straight chain alkyl or halogen substituted C _{1 -5} C _{3 -} cyclic alkyl chains of _8, unsubstituted or C _{1 -5} alkyl, a straight chain or an aryl or heteroaryl group in the C _{6 -8} substituted by halogen in the alkyl chain may contain a hetero atom, and X ₁ Can be joined to form a heterocycle;

R ₂ is hydrogen, amine, C _{1 - 8} alkyl, or C _{1 - 8} alkoxy;

R ₃ is hydrogen, straight chain alkyl of C _{1 -8,} cyclic alkyl chain in C _{3 -8,} or a heterocycle,

And wherein the heterocyclic ring is unsubstituted or substituted, N, O, S, SO and SO _2, 1 ring system or an aromatic or non-aromatic ring system of the compound of 2 _{5 -12} C containing at least one selected from the group consisting of the heterocyclic ring substituents are halogen, hydroxy, amino, nitro, cyano, C _{1 -} is selected from the _six die group consisting of alkyl _amino-6 alkyl, C _{1 - 6} alkoxy, C _{1 - 6} monoalkylamino and C _1.

Specifically, for the specific detection of enlofloxacin belonging to the (fluoro) quinolone antibiotics, a process for preparing an enlofloxacin derivative labeled Cy3 at a common molecular site and a compound formed by such a process (CMY-001- Cy3, Formula 2a) is shown in Figure 5 , the process for the preparation of the enlofloxacin derivatives Cy3-labeled to the specific molecular region of nofloxacin for the simultaneous detection of all antibiotic species belonging to (fluoro) quinolone antibiotics and Compound (CMY-002-Cy3, Formula 2b) formed by such a process is shown in FIG. 6 .

(2a)

[Formula 2b]

Finally, the process of immobilizing the receptor protein on a solid support and binding the target substance analog labeled with the fluorescent substance to the receptor protein (S400).

The solid support may be, but is not limited to, resins, beads, or nanoparticles.

The receptor protein can bind to the solid support via random covalent bonds. For example, a functional group such as a carboxyl group of a solid support and a lysine residue (amine group) of a receptor protein may bind through a covalent bond (amide bond).

The target substance analog is bound through an ambient temperature reaction with a receptor protein bound to a solid support. In this state the periphery of the solid support remains uncolored. However, in the presence of a target substance (eg, antibiotic) to be detected, the receptor protein binds to the target substance having a higher binding force, thereby releasing a fluorescent substance-labeled analog compound. In this case, color changes that can be seen with the naked eye appear due to the released fluorescent substance, so that the presence of hazardous chemicals can be confirmed quickly and accurately in the field.

Synthesis of the target substance analog (S300) and immobilization of the receptor protein on the solid support (part of S400) may be performed in a reversed order.

7 schematically illustrates a sensor according to an embodiment of the present invention and a target material detection process using the sensor.

When the solid support is nanoparticles, the target material may be detected through the color change of the solid support itself. For example, the nanoparticles are originally green, but the target substance analog labeled with the fluorescent material bound thereto shows only the color of the fluorescent material according to the Forster resonance energy transfer (FRET) phenomenon. However, in the presence of the target material, the receptor protein binds to the more binding target material, and the target material analog labeled with the fluorescent material is released so that the distance between the two chromophores is increased. Going back to green. This release of the fluorescent material is visible color change can be seen with the naked eye can quickly and accurately confirm the presence of hazardous chemicals in the field. In particular, scFv used as a receptor protein is a small protein, such as 25 kDa, is a useful protein that can maintain a suitable distance in the FRET phenomenon.

The present invention also provides a sensor produced by such a method and a species or group specific detection method for hazardous chemicals using such a sensor.

The sensor according to an embodiment of the present invention is for selectively detecting individual antibiotic species belonging to a quinolone antibiotic or non-selectively simultaneously detecting antibiotics belonging to a quinolone antibiotic group. A solid support prepared by; Receptor proteins immobilized on said solid support; And a target substance analog labeled with a fluorescent substance that binds to the receptor protein.

The detection method of the present invention includes the steps of 1) taking a sample and 2) processing the sample to the sensor described above to determine the presence or absence of color change or fluorescence intensity change.

The sample may be food such as agricultural products, aquatic products, livestock products, food and beverages, and can be quickly detected by the naked eye on the field using a sample obtained by simple washing of the food surface.

When the hazardous chemicals are detected using the sensor manufactured by the above-described method, it is possible to promptly detect the chemicals in the field regardless of the type of the hazardous chemicals so that it can be usefully used for safety management of food as a whole.

Hereinafter, examples of (fluoro) quinolone antibiotics as harmful chemicals and examples of scFv as receptor proteins are described. However, these are merely to help the understanding of the present invention and the scope of the present invention. Is not limited to the following examples.

Example  One: ( Fluoro A kind of quinolone antibiotic ( species )sign Enrofloxacin  Fabrication of Sensors for Specific Detection

<Step 1> ( Fluoro Corresponding to the common molecular region of quinolone antibiotics Enrofloxacin  At the molecular site Biotin Labeled  Compound (Formula 1a, CMY -001)

As shown in FIG . 2 , a compound of Formula 1a (5,14-dioxo-18-((3a S , 4 S , 6a R ) -2-oxohexahydro- 1H -thieno [3,4- d] imidazol-4-yl) -7,10-dioxa-4,13-diazaoctadecyl 1-cyclopropyl-7- (4-ethylpiperazin-1-yl) -6-fluoro-4 -Oxo-1,4-dihydroquinoline-3-carboxylate) was prepared by the following method.

<1-1> 3- ( tert - Butoxycarbonylamino Profile 1- Cyclopropyl -7- (4- Ethyl piperazine -1-yl) -6- Fluoro -4-oxo-1,4- Dihydroquinoline -3- Carboxylate  Produce

Enlofloxacin (3.0 g, 8.34 mmol), tert-butyl 3-hydroxypropylcarbamate (1.9 g, 10.80 mmol), 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride ( 2.4 g, 12.52 mmol) and 4-dimethylaminopyridine (102 mg, 0.83 mmol) were diluted in pyridine (70 mL) and stirred at 100 ° C. for 6 hours. When the reaction was completed, distillation under reduced pressure to remove pyridine, chloroform was added and washed with water and saturated brine. The separated organic layer was dried over anhydrous magnesium sulfate, and then filtered under reduced pressure and distilled under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: chloroform: methanol = 7: 7: 1) to obtain 2.51 g (yield: 59%) of the title compound.

¹ H NMR (500 MHz, CDCl 3) δ 8.57 (s, 1H), 8.04 (d, 1H, J = 13 Hz), 7.29 (d, 1H, J = 7 Hz), 6.68 (bs, 1H), 4.38 (t , 2H, J = 6 Hz), 3.45 (m, 1H), 3.39 (m, 2H), 3.34 (m, 4H), 2.75 (m, 4H), 2.57 (m, 2H), 1.97 (m, 2H) , 1.47 (s, 9H), 1.33 (m, 2H), 1.16 (m, 5H).

<1-2> 3-aminopropyl 1- Cyclopropyl -7- (4- Ethyl piperazine -1-yl) -6- Fluoro -4-oxo-1,4- Dihydroquinoline -3- Carboxylate  Produce

The compound (2.51 g, 4.90 mmol) prepared in <Step 1-1> was diluted with dichloromethane (16 mL), trifluoroacetic acid (8 mL) was added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solvent was removed by distillation under reduced pressure and basified with 2N aqueous sodium hydroxide solution (pH = 8). The obtained residue was extracted three times with chloroform, and the separated organic layer was dried over anhydrous magnesium sulfate, and then filtered under reduced pressure and distilled under reduced pressure. The obtained residue was dried to give 0.79 g (yield: 39%) of the title compound.

¹ H NMR (500 MHz, CDCl ₃ ) δ 8.82 (s, 1H), 8.01 (d, 1H, J = 13.5 Hz), 7.34 (d, 1H, J = 7 Hz), 3.62 (m, 4H), 3.47 (m, 1H), 3.34 (m, 4H), 2.68 (m, 4H), 2.52 (m, 2H), 1.76 (m, 2H), 1.33 (m, 2H), 1.15 (m, 5H);

MS (ESI ⁺ ): m / z = 417.13 [M + H] ⁺ .

<1-3> 1- (9 H - Fluorene -9-day) -3,12- Dioxo -2,7,10- Trioxa -4,13- Diahexahexadecane -16-day 1- Cyclopropyl -7- (4- Ethyl piperazine -1-yl) -6- Fluoro 4-oxo-1,4- Dihydroqui Nolin-3- Carboxylate  Produce

Compound (0.23 g, 0.55 mmol) prepared in <Step 1-2>, 2- [2- (fluorenylmethyloxycarbonyl-amino) ethoxy] ethoxyacetic acid (0.23 g, 0.55 mmol), 1 -Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (0.16 g, 0.83 mmol) and pyridine (0.14 mL, 1.66 mmol) were diluted in dichloromethane (4 mL) and kept at room temperature for 1 hour. Stirred. When the reaction was completed, water was added and extracted three times with chloroform. The separated organic layer was washed with water and saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulfate, filtered under reduced pressure and distilled under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: chloroform: methanol = 7: 7: 1) to obtain 0.24 g (yield: 56%) of the title compound.

¹ H NMR (500 MHz, CDCl ₃ ) δ 8.78 (s, 1H), 8.01 (d, 1H, J = 13.5 Hz), 7.73 (d, 2H, J = 7.5 Hz), 7.59 (d, 2H, J = 7.5 Hz), 7.37 (t, 2H, J = 7.5 Hz), 7.29 (m, 3H), 5.61 (bt, 1H), 4.35 (d, 2H, J = 7.5 Hz), 4.28 (t, 2H, J = 6 Hz), 4.21 (s, 2H), 4.19 (m, 1H), 3.75 (m, 2H), 3.68 (m, 2H), 3.60 (t, 2H, J = 5 Hz), 3.55 (m, 2H), 3.42 (m, 2H), 3.33 (m, 4H), 2.72 (m, 4H), 2.55 (m, 2H), 1.99 (m, 2H), 1.28 (m, 3H), 1.15 (m, 5H);

MS (ESI ⁺ ): m / z = 785.09 [M + H] ⁺ .

<1-4> 3- (2- (2- (2- Aminoethoxy ) Ethoxy ) Acetamido Profile 1- Cyclopropyl -7- (4- Ethyl piperazine -1-yl) -6- Fluoro -4-oxo-1,4- Dihydroquinoline -3- Carboxylate  Produce

The compound (0.23 g, 0.29 mmol) prepared in <Step 1-3> was diluted with dichloromethane (4 mL), triethylamine (1 mL) was added, and the mixture was stirred at room temperature for 1 hour. When the reaction was completed, the resultant was concentrated by distillation under reduced pressure, and the obtained residue was purified by column chromatography (chloroform: methanol = 9: 1, containing 1% triethylamine) to obtain 0.077 g (yield: 47%) of the title compound.

¹ H NMR (500 MHz, CDCl ₃ ) δ 8.80 (s, 1H), 8.00 (m, 1H), 7.33 (d, 1H, J = 7 Hz), 4.27 (m, 2H), 4.20 (s, 2H), 3.76-3.45 (m, 10H), 3.34 (m, 4H), 2.70 (m, 4H), 2.53 (m, 2H), 1.77 (m, 2H), 1.33 (m, 3H), 1.17 (m, 5H) .

<1-5> compound CMY -001 (5,14- Dioxo -18-((3 a S ,4 S , 6 a R )-2- Oxohexahydro -One H -Cheap Ieno [3,4-d] Zol-4-yl) -7,10- Dioxa -4,13- Diamond octadecyl  One- Cyclopropyl -7- (4- Ethyl piperazine -1-yl) -6- Fluoro -4-oxo-1,4- Dihydroquinoline 3-carboxylate)

The compound (77 mg, 0.14 mmol) and (+)-biotin N -hydroxysuccinimide ester (47 mg, 0.14 mmol) prepared in <Step 1-4> were N, N -dimethylformamide (2 ml). Diisopropylethylamine (48 μl, 0.27 mmol) was added thereto, followed by stirring at room temperature for 1 hour. When the reaction was completed, water was added and extracted with chloroform. The separated organic layer was dried over anhydrous magnesium sulfate, filtered under reduced pressure, and distilled under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: chloroform: methanol = 7: 7: 3) to obtain 67 mg (yield: 62%) of the title compound.

¹ H NMR (500 MHz, CDCl ₃ ) δ 10.14 (bt, 1H, J = 5.5 Hz, 8.80 (s, 1H), 7.99 (d, 1H, J = 13.0 Hz), 7.34 (d, 1H, J = 7.0 Hz), 7.00 (bt, 1H, J = 5.5 Hz), 6.58 (s, 1H), 5.87 (s, 1H), 4.49 (m, 1H), 4.29 (m, 3H), 4.19 (s, 2H), 3.72 (m, 2H), 3.67 (m, 2H), 3.60-3.52 (m, 4H), 3.50 (m, 1H), 3.43 (m, 2H), 3.36 (m, 4H), 3.12 (m, 1H) , 2.88 (m, 1H), 2.71 (m, 5H), 2.56 (m, 2H), 2.22 (t, 2H, J = 7.5 Hz), 2.01 (m, 4H), 1.75-1.59 (m, 4H), 1.42 (m, 2H), 1.33 (m, 2H), 1.16 (t, 3H, J = 7.0 Hz);

¹³ C NMR (125 MHz, CDCl ₃ ) δ 175.68, 173.74, 170.84, 165.47, 164.41, 152.68, 147.00, 145.11, 138.72, 121.93, 112.73, 111.24, 105.06, 71.07, 70.26, 70.21, 68.63, 63.15, 62.02 , 55.78, 52.52, 52.38, 49.90, 40.75, 39.37, 36.30, 36.07, 35.02, 28.99, 28.42, 28.26, 25.80, 11.86, 9.05;

MS (ESI ⁺ ), m / z 788.49 [M + H] ⁺ .

Compound CMY-001 thus prepared was immobilized on paramagnetic beads to which streptavidin was bound.

<Step 2> Biotin Labeled Enrofloxacin  compound( CMY Combined with -001) scFv  Selection

See Shim, H.'s lab, Yang, HY, Kang, KJ, Chung, JE, and Shim, H. Mol . According to the method described in Cells , 2009, 27 , 225, a phage display scFv library was added to beads immobilized with compound CMY-001, incubated at room temperature for 1 hour, and washed to separate bound phage particles. Came out.

Single phage particle colonies were inoculated into SB-ampicillin (200 μl) contained in each well of a 96-well plate and then grown under stirring (400 rpm) at 37 ° C. IPTG (final concentration 1 mM) was added to each well and then incubated for 16 hours with stirring at 30 ° C. The following day, the supernatant was removed after centrifugation for 15 minutes at 3,000 rpm. Resuspend the bacterial pellet in each well in ice-cold 1x TES buffer (40 μl, 20% sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0), then add 0.2 x TES (60 μl) and ice Incubated for at least 30 minutes in the phase. After 15 min centrifugation at 3,000 rpm, the supernatant containing soluble scFv was carefully recovered. Avidin (10 μg / ml in PBS) was immobilized in each well of an ELISA plate (Corning Costar 3690, 25 μl / well) and then blocked with mPBS. Biotin treated antigen (CMY-001, 1 μg / ml, in PBS) was added to the ELISA plate and incubated for 15 minutes at room temperature. After unbound antigen was removed by washing, the supernatant (25 μl) containing the soluble scFv obtained above was added to each well of the ELISA plate and incubated for 1 hour at room temperature. The plate was washed three times with PBST and then anti-HA-HRP antibody (Santa Cruz Biotechnology, 1: 3000 in mPBS) was added to each well. Plates were incubated for 1 hour at room temperature, washed three times with PBST and then boundimetrically detected bound scFv clones (ELISA-positive scFv clones) using tetramethylbenzimidine (TMB) substrate. . These clones are described in Shim, H.'s lab, Yang, HY, Kang, KJ, Chung, JE, and Shim, H. Mol . Purified according to the method described in Cells , 2009, 27 , 225]. The results are shown in Fig.

Looking at the avidin plate (A) of Figure 8 , it can be seen that 10 antibody clones show a good affinity with biotin-labeled enlofloxacin (CMY-001).

The 10 antibody clones selected in this way were finally selected through a competition assay with nofloxacin, a similar antibiotic, nofloxacin, to finally select antibody clones having excellent selectivity / affinity.

Specifically, for competitive analysis, biotin treated antigen (CMY-001, 1 μg / ml, in PBS) was added to streptavidin-coated ELISA plates and incubated for 15 minutes at room temperature. After unbound antigen was removed by washing, each of the 10 antibody clones (50 μl, 20 μg / ml in PBS) was added and incubated for 1 hour at room temperature. After washing three times with PBST, serially diluted analytes CMY-003 and CMY-004 each (see FIG. 9 ) were added to each well and incubated for 1 hour at room temperature. Plates were washed three times with PBST, and then the extent of binding was detected colorimetrically using anti-HA-HRP antibody and TMB substrate.

As a result, as shown in FIG . 9 , it can be seen that three antibody clones (A2, C3, F6) among the ten antibody clones obtained above have excellent selectivity.

Of the three antibodies thus selected, clone A2 was finally identified via SDS-PAGE (see FIG. 10 ) and primer OMPSEQ (see Steinberger, P., Rader, C., and Barbas, CF, III. Analysis of Selected Antibodies.In Phage Display : A Laboratory Manual , CF Barbas, III, DR Burton, JK Scott, and GJ Silverman, eds. (Cold Spring Harbor, USA; Cold Spring Harbor Press), pp. 11.24]) was used to analyze the DNA sequence. As a result, it was confirmed that antibody clone A2 had an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 1.

<Step 3> Cy3 in Labeled Enrofloxacin  Analogs (Formula 2a, CMY -001- Cy3 ) Synthesis

As shown in FIG . 5 , the compound of formula 2a (CMY-001-Cy 3, 2-((1 E , 3 E ) -3- (1- (1- (1-cyclopropyl-7- (4-ethyl) Piperazin-1-yl) -6-fluoro-4-oxo-1,4-dihydroquinolin-3-yl) -1,7,16-trioxo-2,9,12-trioxa-6, 15-diazaic acid-20-yl) -3,3-dimethylindoline-2-ylidene) prop-1-enyl) -1,3,3-trimethyl- 3H -indoleium) It was prepared by the method.

First, 2-((1 E , 3 E ) -3- (1- (4-carboxybutyl) -3,3-dimethylindoline-2-ylidene) prop-1-enyl) -1,3 , 3-trimethyl- 3H -indolium (22mg, 0.05mmol) and 2- ( 1H- 7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyl uronium hexafluoro Phosphate methanealuminum (HATU, 23mg, 0.06mmol) was diluted with N, N -dimethylformamide (2ml), diisopropylethylamine (24µl, 0.14mmol) was added and stirred at room temperature for 1 hour. . The compound (25mg, 0.05mmol) prepared in <Step 1-4> was added thereto and stirred at room temperature for 2 hours. When the reaction was completed, the solvent was removed by distillation under reduced pressure, and the obtained residue was purified by preparative high performance liquid chromatography to obtain 6.9 mg (yield: 14%) of the title compound.

¹ H NMR (500 MHz, CDCl ₃ + CD ₃ OD) δ 8.82 (s, 1H), 8.40 (t, 1H, J = 13.5 Hz), 8.04 (d, 1H, J = 13 Hz), 7.42 (m, 3H ), 7.38 (m, 2H), 7.26 (m, 2H), 7.18 (m, 2H), 6.46 (m, 2H), 4.27 (t, 2H, J = 6.5 Hz), 4.19 (s, 2H), 4.09 (bt, 2H, J = 6.5 Hz), 3.75 (bs, 4H), 3.69 (m, 2H), 3.65 (s, 3H), 3.64 (m, 2H), 3.58 (t, 2H, J = 5.5 Hz) , 3.56-3.46 (m, 5H), 3.43 (m, 2H), 3.24 (q, 2H, J = 7 Hz), 3.24 (m, 2H), 2.37 (bt, 2H, J = 6.5 Hz), 1.98 ( m, 2H), 1.82 (m, 4H), 1.73 (s, 6H), 1.72 (s, 6H), 1.44 (t, 3H, J = 7 Hz), 1.37 (m, 2H), 1.15 (m, 2H) ;

¹³ C NMR (125 MHz, CD ₃ OD) δ 175.53, 174.79, 174.27, 171.18, 170.58, 165.82, 156.40, 154.58, 152.61, 150.94, 147.39, 143.38, 142.88, 142.17, 141.00, 140.91, 138.86, 128.86, 128. 125.61, 125.54, 122.34, 122.19, 111.88, 111.24, 111.10, 110.36, 106.72, 102.63, 102.45, 71.10, 69.95, 69.27, 68.00, 62.68, 56.28, 52.09, 51.22, 49.45, 43.74, 39.14, 36.00, 35.25, 35.06 30.23, 29.57, 28.60, 27.14, 26.96, 26.61, 22.88, 8.36, 7.37;

MS (MALDI), m / z 987.967 [M + H] ⁺ .

<Step 4> Agarose  Receptor protein ( scFv  Clone A2) and Cy3 in Labeled  Enlofloxacin analogs ( CMY -001- Cy3 ) Immobilization

Dry cyanogen bromide agarose resin (0.53 g) was washed 5 times with cold 1 mM hydrochloric acid solution, inflated for 1 hour in the same solution, then washed 10 times with water and twice with 0.1 M sodium bicarbonate / sodium chloride buffer. Thereafter, the reaction was performed with clone A2 (1.73 mg) in 0.1 M sodium bicarbonate / sodium chloride buffer (1.5 mL) at room temperature for 2 hours. After removing the reaction buffer and reacted with 1M ethanolamine aqueous solution at room temperature for 3 hours. The reaction solution was removed and then washed with 0.1 M sodium bicarbonate / sodium chloride buffer followed by 0.1 M acetate buffer. This washing process was repeated five times to obtain agarose resin covalently bound to clone A2.

Next, the compound CMY-001-Cy3 (100 uM PBS solution) obtained in <Step 3> was incubated for 2 hours at room temperature with agarose resin to which the clone A2 was bound, and then washed several times with PBS buffer Cy3 fluorescent material An agarose resin bonded to A2 was obtained, to which an enlofloxacin analog (CMY-001-Cy3) labeled with.

Example  2: ( Fluoro Group of quinolone antibiotics group Fabrication of Sensors for Specific Detection

<Step 1> Nofloxacin  On specific molecules Biotin Labeled  Preparation of Compound (Formula 1b, CMY-002)

As shown in FIG . 3 , the compound of Formula 1b (CMY-002, 7- (4- (4,13-dioxo-17-((3a S , 4 R , 6a R ) -2-oxohexahydro-1 H -thieno [3,4-d] imidazole-4 -Yl) -6,9-dioxa-3,12-diazaheptadecyl) piperazin-1-yl) -1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3 -Carboxylic acid) was prepared by the following method.

<1-1> 7- (4- (1- (9 H - Fluorene -9-day) -3,12- Dioxo -2,7,10- Trioxa -4,13- Diapentadecane -15-yl) piperazin-1-yl) -1-ethyl-6- Fluoro -4-oxo-1,4- Dihydroquinoline Preparation of 3-carboxylic acid

Nofloxacin (0.24 g, 0.74 mmol) and 2- (tert-butoxycarbonylamino) ethyl 4-methylbenzenesulfonate (0.29 g, 0.90 mmol) were added to N, N -dimethylformamide (2 mL). Diluted and triethylamine (0.31 mL, 2.23 mmol) was added dropwise and stirred at 100 ° C. for 2 hours. Upon completion of the reaction, chloroform was added and washed with water and saturated brine. The separated organic layer was dried over anhydrous magnesium sulfate, filtered under reduced pressure, and distilled under reduced pressure. The obtained residue was dried and diluted with dichloromethane (2 mL), trifluoroacetic acid (1 mL) was added, and the resultant was stirred at room temperature for 2 hours. When the reaction was completed, the reaction solvent was removed by distillation under reduced pressure, and the obtained residue was dried, diluted with N, N -dimethylformamide (2 mL), and diisopropylethylamine (0.42 mL, 2.42 mmol) was added dropwise. To this was stirred 2- [2- (fluorenylmethyloxycarbonyl-amino) ethoxy] ethoxyacetic acid (233 mg, 0.61 mmol) and stirred in N, N -dimethylformamide (2 mL) in advance for 30 minutes and A mixed solution of 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (128 mg, 0.62 mmol) was added and stirred at room temperature for 1 hour. Upon completion of the reaction, chloroform was added and washed with water and saturated brine. The separated organic layer was dried over anhydrous magnesium sulfate, filtered under reduced pressure, and distilled under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: chloroform: methanol = 7: 7: 3, containing 1% acetic acid) to obtain 103 mg (yield: 19%) of the title compound.

¹ H NMR (500 MHz, CDCl ₃ + CD ₃ OD) δ 8.86 (s, 1H), 7.90 (m, 1H), 7.74 (d, 2H, J = 7.5 Hz), 7.59 (d, 2H, J = 7 Hz) , 7.38 (t, 2H, J = 7 Hz), 7.29 (t, 2H, J = 7.5 Hz), 6.66 (m, 1H), 4.40 (d, 2H, J = 6.5 Hz), 4.21 (m, 3H), 4.02 (s, 2H), 3.68 (m, 2H), 3.65 (m, 2H), 3.57 (m, 2H), 3.43 (m, 2H), 3.37 (m, 2H), 3.20 (m, 4H), 2.68 (m, 4H), 2.58 (m, 2H), 1.44 (m, 3H);

MS (ESI ⁺ ), m / z 730.39 [M + H] ⁺ .

<1-2> 7- (4- (4,13- Dioxo -17-((3 a S ,4 R , 6 a R )-2- Oxohexahydro -One H - Thieno [3,4-d] imidazole -4-yl) -6,9- Dioxa -3,12- Diazaheptadecyl Piperazin-1-yl) -1-ethyl-6- Fluoro -4-oxo-1,4- Dihydroquinoline -3- Carboxylic acid  Produce

The compound (0.10 g, 0.14 mmol) prepared in <Step 1-1> was diluted with dichloromethane (4 mL) and piperidine (1 mL) was added thereto, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solvent was removed by distillation under reduced pressure, and the obtained residue was dried. The resulting residue was dried over (+)-biotin N -hydroxysuccinimide ester (47 mg, 0.14 mmol) and N, N -dimethylformamide (3 ml). Diluted and triethylamine (78 μl, 0.56 mmol) was added, followed by stirring at 70 ° C. for 20 hours. When the reaction was completed, the solvent was removed by distillation under reduced pressure, and the obtained residue was purified by preparative high performance liquid chromatography to obtain 14.8 mg (yield: 14%) of the title compound.

¹ H NMR (500 MHz, CD ₃ OD) δ 8.92 (s, 1H), 8.08 (d, 1H, J = 13 Hz), 7.29 (d, 1H, J = 6.5 Hz), 4.58 (m, 2H), 4.50 (m, 2H), 4.31 (m, 2H), 4.09 (s, 2H), 3.76 (m, 3H), 3.68 (m, 4H), 3.58 (m, 2H), 3.46 (m, 4H), 3.40 ( m, 2H), 3.23 (m, 2H), 2.93 (m, 2H), 2.69 (d, 2H, J = 12.5 Hz), 2.24 (m, 2H), 1.66 (m, 2H), 1.56 (m, 2H ), 1.45 (m, 2H), 1.32 (t, 3H, J = 7.5 Hz);

MS (ESI ⁺ ), m / z 734.50 [M + H] ⁺ .

Compound CMY-002 thus prepared was immobilized on paramagnetic beads to which streptavidin was bound.

<Step 2> Biotin Labeled With nofloxacin (CMY-002)  Combined scFv  Selection

Using the phage display library in the same manner as in <Step 2> of Example 1, scFv clones having excellent affinity with the compound CMY-002 synthesized in <Step 1> were selected. The results are shown in Fig.

Looking at the avidin plate (B) of Figure 8 , it can be seen that nine antibody clones show excellent affinity with biotin-labeled nofloxacin (CMY-002).

In this first screened 9 antibody clones, in the same manner as in <Step 2> of Example 1 competition assay with enlofloxacin and nofloxacin, an antibiotic having a common molecular region thereof Finally, antibody clones with no difference in selectivity were finally selected. The result is shown in Fig.

Referring to FIG. 9 , it can be seen that two antibody clones (F9, G10) among the nine antibody clones obtained above exhibit specific responses to the (fluoro) quinolone antibiotic group.

Both selected antibody clones were finally identified via SDS-PAGE (see FIG. 10 ). For clone F9, primer OMPSEQ (Steinberger, P., Rader, C., and Barbas, CF, III. Analysis of Selected Antibodies.In Phage Display: A Laboratory Manual , CF Barbas, III, DR Burton, JK Scott, and GJ Silverman, eds. (Cold Spring Harbor, USA; Cold Spring Harbor Press), pp. 11.24]) was used to analyze the DNA sequence. As a result, antibody clone F9 was found to have an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 2.

<Step 3> Cy3 in Labeled Nofloxacin  Analogs (Formula 2b, CMY -002- Cy3 ) Synthesis

As shown in Figure 6 , the compound of formula 2b (CMY-002-Cy3, 2-((1 E , 3 E ) -3- (1- (1- (4- (3-carboxy-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-7- Yl) piperazin-1-yl) -4,13-dioxo-6,9-dioxa-3,12-diazaheptadecane-17-yl) -3,3-dimethylindoline-2-yl Den) prop-1-enyl) -1,3,3-trimethyl- 3H -indolium) was prepared by the following method.

First, 2-((1 E , 3 E ) -3- (1- (4-carboxybutyl) -3,3-dimethylindoline-2-ylidene) prop-1-enyl) -1,3 , 3-trimethyl-3 H -indolium (81mg, 0.18mmol) and 2- ( 1H- 7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyl uronium hexafluoro Phosphate methanealuminum (HATU, 82 mg, 0.22 mmol) was diluted with N, N -dimethylformamide (3 mL), diisopropylethylamine (90 μl, 0.50 mmol) was added, and the mixture was stirred at room temperature for 1 hour. .

Next, 7- (4- (2- (2- (2- (2-aminoethoxy) ethoxy) acetamido) ethyl) piperazin-1-yl) -1-ethyl-6-fluoro- 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (84 mg, 0.17 mmol) was added and stirred at room temperature for 16 hours. When the reaction was completed, the solvent was removed by distillation under reduced pressure, and the obtained residue was purified by preparative high performance liquid chromatography to obtain 19 mg (yield: 12%) of the title compound.

¹ H NMR (500 MHz, CD ₃ OD) δ 8.84 (s, 1H), 8.54 (t, 1H, J = 13 Hz), 7.96 (d, 1H, J = 14 Hz), 7.54 (d, 2H, J = 7 Hz ), 7.44 (m, 2H), 7.35 (d, 2H, J = 8 Hz), 7.31 (m, 2H), 7.23 (m, 1H), 6.43 (d, 2H, J = 13.5 Hz), 4.54 (bs, 2H), 4.16 (t, 2H, J = 7Hz), 4.10-3.33 (m, 25H), 2.32 (t, 2H, J = 7.5Hz), 1.84 (m, 2H), 1.78 (m 2H), 1.763 ( s, 6H), 1.759 (s, 6H), 1.53 (m, 3H);

¹³ C NMR (125 MHz, CD ₃ OD) δ 175.58, 174.77, 174.35, 173.12, 168.17, 152.66, 150.98, 148.53, 142.86, 142.16, 141.00, 140.91, 137.56, 128.81, 125.65, 125.55, 122.37, 122.20, 122.20, 122.20 111.85, 111.20, 111.12, 107.43, 106.41, 102.65, 102.42, 70.75, 70.12, 69.45, 56.67, 51.99, 49.78, 49.45, 46.94, 43.74, 39.10, 35.10, 33.64, 30.59, 27.14, 26.96, 26.72, 22.85, 22.61

MS (MALDI), m / z 933.149 [M + H] ⁺ .

<Step 4> Agarose  Receptor protein ( scFv  Clone F9) and Cy3 in Labeled  Nofloxacin analog ( CMY -002- Cy3 ) Immobilization

The same procedure as in <Step 4> of Example 1 was performed except for using F9 instead of A2 as a receptor protein and CMY-002-Cy3, a nofloxacin analog labeled with Cy3 instead of CMY-001-Cy3. F9-bound agarose resin to which nofloxacin analog (CMY-002-Cy3) labeled with a Cy3 fluorescent material was bound was obtained.

Evaluation example  1: with sensor Fluoro Species of quinolone antibiotics species Specific detection

Specific (selective) detection of enlofloxacin, a type of (fluoro) quinolone antibiotic, was performed using the sensor prepared in Example 1.

Specifically, in a 96-well plate, 50 μl of a suspension containing CMGA-001-Cy3 and A2 bound agarose resin in PBS buffer was aliquoted into each of 20 wells. Prepare rofloxacin and nofloxacin at different concentrations (0, 0.001, 0.01, 0.1, 1, 10, 33, 100, 333, 1000 uM), add 50 µl each, shake several times. The fluorescence intensity was quantified by observing with a fluorescence microscope and an optical microscope.

As a result, as shown in FIG . 11 , when enrofloxacin was treated in the sensor manufactured in Example 1, fluorescence decreased on the surface of the resin substrate as the concentration of enrofloxacin was increased. On the other hand, referring to Figure 12 , when treated with nofloxacin to the sensor produced in Example 1, it was shown that the fluorescence intensity is kept constant on the surface of the resin substrate regardless of the antibiotic concentration. This result can be clearly seen through the graph of FIG. 13 . This result shows that the sensor fabricated in Example 1 effectively performs species-specific detection of (fluoro) quinolone-based antibiotics.

Evaluation example  2: with sensor Fluoro Group of quinolone antibiotics group Specific detection

Specific detection of the (fluoro) quinolone antibiotic group was performed using the sensor prepared in Example 2 in the same manner as in Evaluation Example 1.

As a result, as shown in FIG . 14 , when enrofloxacin and nofloxacin were treated in the sensor fabricated in Example 2, the fluorescence was increased on the surface of the resin substrate as the antibiotic concentration increased, regardless of the type of these antibiotics. It appeared to decrease. These results show that the sensor fabricated in Example 2 effectively performs specific detection for the (fluoro) quinolone antibiotic group.

<110> Seoul National University R & DB Foundation <120> METHOD OF MANUFACTURING SENSOR FOR DETECTION OF TARGET          MATERIAL (S), SENSOR MANUFACTURED BY THE METHOD AND METHOD OF          DETECTING TARGET MATERIAL (S) USING THE SENSOR <130> PC-09131 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 738 <212> DNA <213> Artificial Sequence <220> <223> Clone A2 <400> 1 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattatgata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcg atctcttctg gtggtggtag taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaagatctt 300 gattcttcta ggagtaatga tttcgactac tggggccagg gtacacgggt caccgtgagc 360 tcaggtggag gcggttcagg cggaggtgga tccggcggtg gcggatcgca gtctgtgctg 420 actcagccac cctcagcgtc tgggaccccc gggcagaggg tcaccatctc ttgtagtggc 480 tcttcatcta atattggcag taatgctgtc aactggtacc agcagctccc aggaacggcc 540 cccaaactcc tcatctatgc tgatagtaat cggccaagcg gggtccctga ccgattctct 600 ggctccaagt ctggcacctc agcctccctg gccatcagtg ggctccggtc cgaggatgag 660 gctgattatt actgtggttc ttgggattat agcctgagtg gttatgtctt cggcggaggt 720 accaagctga cggtccta 738 <210> 2 <211> 738 <212> DNA <213> Artificial Sequence <220> <223> Clone F9 <400> 2 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc gattattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctcttatg ataatggtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagatggt 300 acgacgtcgc tgtggattcc gttcgactac tggggccagg gtacactggt caccgtgagc 360 tcaggtggag gcggttcagg cggaggtgga tccggcggtg gcggatcgca gtctgtgctg 420 actcagccac cctcagcgtc tgggaccccc gggcagaggg tcaccatctc ttgtactggc 480 tcttcatcta atattggcaa taatactgtc aactggtacc agcagctccc aggaacggcc 540 cccaaactcc tcatctattc tgataataag cggccaagcg gggtccctga ccgattctct 600 ggctccaagt ctggcacctc agcctccctg gccatcagtg ggctccggtc cgaggatgag 660 gctgattatt actgtgctgc ttgggatgct agcctgagtg cttatgtctt cggcggaggc 720 accaagctga ccgtccta 738

Claims (23)

In the method of manufacturing a sensor for selectively detecting a specific hazardous chemical species or non-selective simultaneous detection of harmful chemicals belonging to a specific hazardous chemical group,
1) labeling a biotin to a target material to be detected, wherein the target material is one or more harmful chemicals having a specific molecular region or two or more harmful chemicals having a common molecular region, and the specific molecular region or common Labeling biotin on the target material to expose the molecular site;
2) selecting a receptor protein from an antibody display library that recognizes the exposed specific or common molecular region and binds to the biotin-labeled target material and at the same time has a higher binding capacity to the target material than the biotin-labeled target material ;
3) synthesizing a target substance analog labeled with a fluorescent substance having a lower binding force than that of the target substance to the receptor protein; And
4) immobilizing the receptor protein on a solid support and binding the fluorescent substance labeled target substance analog to the receptor protein
Manufacturing method comprising a. The method of claim 1,
In the step 1), when selectively detecting the harmful chemical species (species), the biotin is bonded to the terminal functional groups of the molecular site other than the specific molecular site, and harmful chemicals belonging to the harmful chemical group (group) When non-selective and simultaneous detection, the production method characterized in that to bind the biotin to the terminal functional groups of the molecular region other than the common molecular region. The method of claim 1,
When non-selective simultaneous detection of hazardous chemicals belonging to the hazardous chemical group, step 1)
1-1) selecting one harmful chemicals from two or more harmful chemicals having the common molecular region; And
1-2) labeling biotin to one of the harmful chemicals to expose common molecular sites
Manufacturing method comprising a. The method of claim 1,
The target material is a manufacturing method, characterized in that selected from the group consisting of drugs, pesticides, preservatives and environmental hormones. The method of claim 1,
The target material is Enrofloxacin (Enrofloxacin), Ciprofloxacin (Ciprofloxacin), pyromidic acid (Piromidic acid), Flumequine (Flumequine), Ofloxacin (Nfloxacin), nofloxacin (Norfloxacin), Nalidixic acid (Nalidixic) acid) and pipemidic acid (Pipemidic acid), characterized in that at least one quinolone antibiotic selected from the group consisting of. The method of claim 1,
The receptor protein is a manufacturing method, characterized in that the antibody selected from the group consisting of scFv, Fab and whole antibody (whole antibody). The method of claim 1,
The solid support is selected from the group consisting of resins, beads and nanoparticles. 1) taking a sample; And
2) processing the sample to a sensor manufactured by the method of any one of claims 1 to 7, characterized in that it comprises a step of identifying a color or fluorescence change, specific hazardous chemical species (species) A detection method for selective detection or non-selective simultaneous detection of hazardous chemicals belonging to a specific hazardous chemical group. The method of claim 8,
The sample is a detection method, characterized in that the food. A method of manufacturing a sensor for selectively detecting individual antibiotic species belonging to a quinolone antibiotic or non-selectively simultaneously detecting antibiotics belonging to a quinolone antibiotic group
1) labeling a biotin to a target substance to be detected, wherein the target substance is at least one selected from the group consisting of quinolone antibiotics having a specific molecular region and a common molecular region, and the specific molecular region or a common molecular region Labeling the biotin to the target material to expose the target material;
2) selecting a receptor protein from an antibody display library that recognizes the exposed specific or common molecular region and binds to the biotin-labeled target material and at the same time has a higher binding capacity to the target material than the biotin-labeled target material ;
3) synthesizing a target substance analog labeled with a fluorescent substance having a lower binding force than that of the target substance to the receptor protein; And
4) immobilizing the receptor protein on a solid support and binding the fluorescent substance labeled target substance analog to the receptor protein
Manufacturing method comprising a. The method of claim 10,
In the step 1), the biotin is labeled on the target material to be detected to obtain a compound of Formula 1 below:
[Formula 1]

Where
A _1, A _2, A ₃ and A ₄ each independently is hydrogen and the biotin-binding, biotin, or a linker, wherein the linker is C _{1 -20} alkyl chain and in the alkyl chain is a heteroatom, amide, reverse amide, ester (ester), carbamate and urea, and may include a group selected from the group except that A ₁ , A ₂ , A ₃ and A ₄ are simultaneously hydrogen;
X ₁ and X ₂ are each independently carbon or nitrogen;
Y is hydrogen or fluorine but when X ₂ is nitrogen, Y is absent;
R ₁ is an unsubstituted or a straight-chain alkyl chain of a C _{1 -8} alkyl substituted with a straight chain or a halogen C _{1 -5,} unsubstituted or substituted with a straight chain alkyl or halogen substituted C _{1 -5} C _{3 -} cyclic alkyl chains of _8, unsubstituted or C _{1 -5} alkyl, a straight chain or an aryl or heteroaryl group in the C _{6 -8} substituted by halogen in the alkyl chain may contain a hetero atom, and X ₁ Can be joined to form a heterocycle;
R ₂ is hydrogen, amine, C _{1 - 8} alkyl, or C _{1 - 8} alkoxy;
R ₃ is hydrogen, straight chain alkyl of C _{1 -8,} cyclic alkyl chain in C _{3 -8,} or a heterocycle,
And wherein the heterocyclic ring is unsubstituted or substituted, N, O, S, SO and SO _2, 1 ring system or an aromatic or non-aromatic ring system of the compound of 2 _{5 -12} C containing at least one selected from the group consisting of the heterocyclic ring substituents are halogen, hydroxy, amino, nitro, cyano, C _{1 -} is selected from the _six die group consisting of alkyl _amino-6 alkyl, C _{1 - 6} alkoxy, C _{1 - 6} monoalkylamino and C _1. The method of claim 11,
When selectively detecting individual antibiotic species belonging to quinolone antibiotics, when A ₄ of the compound is biotin and non-selective simultaneous detection of antibiotics belonging to the quinolone antibiotic group, A ₃ is a manufacturing method characterized in that the biotin. The method of claim 10,
In step 2), the receptor protein is characterized in that the antibody is selected from the group consisting of scFv, Fab and whole antibody (whole antibody). The method of claim 10,
In step 3), the target substance analog labeled with a fluorescent substance is a compound represented by the following formula (2):
(2)

Where
B ₁ , B ₂ , B ₃ and B ₄ are each independently hydrogen, Cy 3, Cy 5, FAM, TET, TxR, ROX, SYBR Green, ALEXA, FITC, BODIPY, TAMRA and Texas Red or and a fluorescent material in combination with the linker, the linker may be a C _{1 -20} alkyl chain in the alkyl chain comprises a functional group selected from heteroatoms, amide, reverse amide, ester (ester), a carbamate and a urea group consisting of Except that B ₁ , B ₂ , B ₃ and B ₄ are hydrogen at the same time;
X ₁ and X ₂ are each independently carbon or nitrogen;
Y is hydrogen or fluorine but when X ₂ is nitrogen, Y is absent;
R ₁ is an unsubstituted or a straight-chain alkyl chain of a C _{1 -8} alkyl substituted with a straight chain or a halogen C _{1 -5,} unsubstituted or substituted with a straight chain alkyl or halogen substituted C _{1 -5} C _{3 -} cyclic alkyl chains of _8, unsubstituted or C _{1 -5} alkyl, a straight chain or an aryl or heteroaryl group in the C _{6 -8} substituted by halogen in the alkyl chain may contain a hetero atom, and X ₁ Can be joined to form a heterocycle;
R ₂ is hydrogen, amine, C _{1 - 8} alkyl, or C _{1 - 8} alkoxy;
R ₃ is hydrogen, straight chain alkyl of C _{1 -8,} cyclic alkyl chain in C _{3 -8,} or a heterocycle,
And wherein the heterocyclic ring is unsubstituted or substituted, N, O, S, SO and SO _2, 1 ring system or an aromatic or non-aromatic ring system of the compound of 2 _{5 -12} C containing at least one selected from the group consisting of the heterocyclic ring substituents are halogen, hydroxy, amino, nitro, cyano, C _{1 -} is selected from the _six die group consisting of alkyl _amino-6 alkyl, C _{1 - 6} alkoxy, C _{1 - 6} monoalkylamino and C _1. The method of claim 14,
When selectively detecting individual antibiotic species belonging to the quinolone antibiotic, the compound B ₄ is a fluorescent substance and when the non-selective simultaneous detection of antibiotics belonging to the quinolone antibiotic group, the compound B ₃ is a manufacturing method characterized in that the fluorescent material. Solid support;
Receptor proteins immobilized on said solid support; And
A target substance analog labeled with a fluorescent substance that binds to the receptor protein
16. A method for selectively detecting individual antibiotic species belonging to a quinolone antibiotic, prepared according to the method of any one of claims 10 to 15, or for non-selective antibiotics belonging to a quinolone antibiotic group. Sensor for simultaneous detection with 1) taking a sample; And
2) confirming the color or fluorescence change by treating the sample to the sensor according to claim 16
A detection method for selectively detecting individual antibiotic species belonging to a quinolone antibiotic, or non-selectively simultaneously detecting antibiotics belonging to a quinolone antibiotic group. Compound represented by the following formula (1):
[Formula 1]

Where
A _1, A _2, A ₃ and A ₄ each independently is hydrogen and the biotin-binding, biotin, or a linker, wherein the linker is C _{1 -20} alkyl chain and in the alkyl chain is a heteroatom, amide, reverse amide, ester (ester), carbamate and urea, and may include a group selected from the group except that A ₁ , A ₂ , A ₃ and A ₄ are simultaneously hydrogen;
X ₁ and X ₂ are each independently carbon or nitrogen;
Y is hydrogen or fluorine but when X ₂ is nitrogen, Y is absent;
R ₁ is an unsubstituted or a straight-chain alkyl chain of a C _{1 -8} alkyl substituted with a straight chain or a halogen C _{1 -5,} unsubstituted or substituted with a straight chain alkyl or halogen substituted C _{1 -5} C _{3 -} cyclic alkyl chains of _8, unsubstituted or C _{1 -5} alkyl, a straight chain or an aryl or heteroaryl group in the C _{6 -8} substituted by halogen in the alkyl chain may contain a hetero atom, and X ₁ Can be joined to form a heterocycle;
R ₂ is hydrogen, amine, C _{1 - 8} alkyl, or C _{1 - 8} alkoxy;
R ₃ is hydrogen, straight chain alkyl of C _{1 -8,} cyclic alkyl chain in C _{3 -8,} or a heterocycle,
And wherein the heterocyclic ring is unsubstituted or substituted, N, O, S, SO and SO _2, 1 ring system or an aromatic or non-aromatic ring system of the compound of 2 _{5 -12} C containing at least one selected from the group consisting of the heterocyclic ring substituents are halogen, hydroxy, amino, nitro, cyano, C _{1 -} is selected from the _six die group consisting of alkyl _amino-6 alkyl, C _{1 - 6} alkoxy, C _{1 - 6} monoalkylamino and C _1. The method of claim 18,
The compound is characterized in that represented by the formula 1a or 1b:
[Formula 1a]

[Chemical Formula 1b]

An antibody capable of specifically binding to a molecular region other than the molecular region to which the biotin of the compound according to claim 19 is bound. The method of claim 20,
The antibody has an amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 1 or 2. A compound represented by formula
(2)

Where
B ₁ , B ₂ , B ₃ and B ₄ are each independently hydrogen, Cy 3, Cy 5, FAM, TET, TxR, ROX, SYBR Green, ALEXA, FITC, BODIPY, TAMRA and Texas Red or and a fluorescent material in combination with the linker, the linker may be a C _{1 -20} alkyl chain in the alkyl chain comprises a functional group selected from heteroatoms, amide, reverse amide, ester (ester), a carbamate and a urea group consisting of Except that B ₁ , B ₂ , B ₃ and B ₄ are hydrogen at the same time;
X ₁ and X ₂ are each independently carbon or nitrogen;
Y is hydrogen or fluorine but when X ₂ is nitrogen, Y is absent;
R ₁ is an unsubstituted or a straight-chain alkyl chain of a C _{1 -8} alkyl substituted with a straight chain or a halogen C _{1 -5,} unsubstituted or substituted with a straight chain alkyl or halogen substituted C _{1 -5} C _{3 -} cyclic alkyl chains of _8, unsubstituted or C _{1 -5} alkyl, a straight chain or an aryl or heteroaryl group in the C _{6 -8} substituted by halogen in the alkyl chain may contain a hetero atom, and X ₁ Can be joined to form a heterocycle;
R ₂ is hydrogen, amine, C _{1 - 8} alkyl, or C _{1 - 8} alkoxy;
R ₃ is hydrogen, straight chain alkyl of C _{1 -8,} cyclic alkyl chain in C _{3 -8,} or a heterocycle,
And wherein the heterocyclic ring is unsubstituted or substituted, N, O, S, SO and SO _2, 1 ring system or an aromatic or non-aromatic ring system of the compound of 2 _{5 -12} C containing at least one selected from the group consisting of the heterocyclic ring substituents are halogen, hydroxy, amino, nitro, cyano, C _{1 -} is selected from the _six die group consisting of alkyl _amino-6 alkyl, C _{1 - 6} alkoxy, C _{1 - 6} monoalkylamino and C _1. The method of claim 22,
The compound is characterized in that represented by the formula 2a or 2b:
(2a)

(2b)

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