KR101711600B1 - Peptide Sensors for Selective Detection of Explosives - Google Patents

Abstract

The present invention relates to a peptide sensor for selectively detecting explosives, a method for producing the same, and a detection apparatus using the same. The peptide sensor of the present invention is characterized in that the peptides according to the present invention immobilize the TNT and DNT derivatives on the surface thereof and then form a selective peptide sequence Was derived by the method of phage display. Since these peptides exhibit high selectivity for TNT or DNT for existing substances, they can be applied to devices such as Surface Plasmon Resonance (SPR), Cantilever and QCM to detect peptides contained in gas phase or liquid phase Can be used as a sensor.

Description

Peptide Sensors for Selective Detection of Explosives

The present invention relates to a peptide for a sensor for detecting explosives including TNT and DNT, and a sensor for detecting an explosive, characterized by using such a TNT or DNT-specific peptide.

The design and research of new sensors for explosives have been actively conducted so far. A typical compound used as an explosive is trinitrotoluene (TNT). Meanwhile, dinitrotoluene (DNT) is also present in some explosives through the process of synthesizing or decomposing TNT. Therefore, TNT and DNT are representative compounds present in explosives, and explosive sensors mainly detect these two substances as their main function. However, since TNT and DNT exist as solids and have a very low vapor pressure, the development of a receptor having a high sensitivity selectivity and a stable structure in the gas phase to detect TNT and DNT that is vaporized in the air and evaporated from explosives has been developed. It is the core technology of DNT sensor development. Recently, as a receptor capable of selectively binding to DNT and TNT with high sensitivity, it has shown the possibility that biopolymers such as peptides can be used as explosives sensors [ Langmuir 2008, 24 , 4938-4943].

Meanwhile, since the previously developed TNT and DNT-specific peptides were discovered from the phage display library for peptides that selectively bind to solid or crystalline TNT and DNT, it is estimated that there is a limitation in selectively binding to TNT or DNT in a monomolecular state. In particular, it will be somewhat less efficient in detecting TNT and DNT in the state of being vaporized and present in air.

Therefore, the technical problem to be achieved by the present invention is to obtain a peptide capable of selectively binding with trinitrotoluene (TNT) and dinitrotoluene (DNT) contained in explosives with high sensitivity, and to provide an explosive sensor using the same. In particular, it is to provide a peptide for detecting explosives useful for detecting TNT and DNT in a vaporized state as in the actual explosive detection situation, and a sensor including the same.

Another technical object of the present invention is to provide a peptide for selectively detecting contaminated TNT or DNT from environmental samples such as rivers, groundwater, soil, and the like, and a sensor including the same.

Another technical problem of the present invention is to provide a device for selective detection of TNT or DNT using such a peptide sensor.

In order to achieve the above technical problem, the present invention provides a TNT-specific peptide having any one of the amino acid sequences of SEQ ID NOs: 1 to 4 and a DNT-specific peptide having any one of the amino acid sequences of SEQ ID NOs: 9 to 11 . These peptides can be used as a peptide sensor to detect TNT or DNT.

Sequence number Amino acid sequence One H ₂ N-His-Pro-Leu-Lys-Gln-Tyr-Trp-Trp-Arg-Pro-Ser-Ile-COOH 2 H ₂ N-Val-Ser-Arg-His-Gln-Ser-Trp-His-Pro-His-Asp-Leu-COOH 3 H ₂ N-His-Arg-Asn-His-Leu-Met-Asp-Leu-Ser-Gly-Leu-Tyr-COOH 4 H ₂ N-Trp-Ser-Pro-Gly-Gln-Gln-Arg-Leu-His-Asn-Ser-Thr-COOH

Sequence number Amino acid sequence 9 H ₂ N-Lys-Met-His-Thr-Ala-Ser-Leu-Ser-Gln-Pro-Leu-Met-COOH 10 H ₂ N-Gln-Ser-Phe-Ala-Ser-Leu-Thr-Asn-Pro-Arg-Val-Leu-COOH 11 H ₂ N-Ala-Ser-Thr-Ala-Ser-Leu-His-Gln-Pro-Arg-COOH

In order to discover a peptide that selectively binds to TNT or DNT that exists in a monomolecular state in the gas phase, the present inventors use TNT or DNT that is connected to a solid surface with a linker of a certain length and exists in the form of a single molecule. It was estimated that the present peptide could be excavated from the phage display library. Thus, the present inventors synthesized a derivative of TNT or DNT, immobilized it on a solid surface with a linker, and then identified a peptide sequence selective for TNT or DNT in a monomolecular state from a phage display library, and synthesized a peptide of the identified sequence. It was confirmed that it selectively binds to TNT and DNT. In addition, the present invention was completed by confirming that the peptide discovered by selectively measuring TNT or DNT present on the gas using a chip to which such a peptide is immobilized can be used as an explosive sensor.

In addition, the present invention provides a peptide for detecting TNT or DNT to which a linker for binding a solid support is further linked to the carboxy terminal of the TNT or DNT-specific peptide, and preferably such a linker is Gly-Cys. More preferably, the TNT or DNT-specific peptide of the present invention to which a linker is linked has an amino acid sequence of SEQ ID NO: 5-8 (TNT specific) or SEQ ID NO: 12-14 (DNT specific).

Sequence number Amino acid sequence 5 H ₂ N-His-Pro-Leu-Lys-Gln-Tyr-Trp-Trp-Arg-Pro-Ser-Ile-Gly-Cys-COOH 6 H ₂ N-Val-Ser-Arg-His-Gln-Ser-Trp-His-Pro-His-Asp-Leu-Gly-Cys-COOH 7 H ₂ N-His-Arg-Asn-His-Leu-Met-Asp-Leu-Ser-Gly-Leu-Tyr-Gly-Cys-COOH 8 H ₂ N-Trp-Ser-Pro-Gly-Gln-Gln-Arg-Leu-His-Asn-Ser-Thr-Gly-Cys-COOH

Sequence number Amino acid sequence 12 H ₂ N-Lys-Met-His-Thr-Ala-Ser-Leu-Ser-Gln-Pro-Leu-Met-Gly-Cys-COOH 13 H ₂ N-Gln-Ser-Phe-Ala-Ser-Leu-Thr-Asn-Pro-Arg-Val-Leu-Gly-Cys-COOH 14 H ₂ N-Ala-Ser-Thr-Ala-Ser-Leu-His-Gln-Pro-Arg-Gly-Cys-COOH

The present invention also provides an explosive sensor comprising a TNT or DNT-specific peptide according to the present invention, and preferably, in such a sensor, the peptide of the present invention has a carboxy end bound to a solid support (surface immobilization). do.

In various aspects, such as ease of surface immobilization and system design, the hydroxyl group (-OH) of the carboxy terminal (COOH) of the peptide according to the present invention is -NH ₂ , -NHR ₁ (R ₁ is an alkyl group having 1-18 carbon atoms), -OR ₁ (R ₁ is a C 1-18 alkyl group), or an amino acid may be substituted. The peptide having the carboxy terminal substituted may be prepared by a method well known in the field to which the present invention belongs, such as a solid-phase synthesis method and a liquid-phase synthesis method.

In addition, the shape of the solid support that can be used in the explosive sensor according to the present invention can be any shape such as beads, films, nanotubes, and porous particles, and the constituents of the solid support are gold, silica, glass, glass. Fiber, TiO ₂ , platinum, silver, chromium, nickel, platinum, aluminum, iron, copper, titanium, gold oxide, chromium oxide, silver oxide, or zirconium, etc., and polystyrene, nitrocellulose, polyvinyl chloride, polyethylene-terephthalate Polymer resins such as, etc. are also possible. As mentioned above, the carboxy end of the peptide according to the present invention, the end substituted with an amino group, or an end substituted with an alkyl ester group, etc. of the peptide according to the present invention may be immobilized on such a solid support (however, the present invention This is not limited.), an immobilization method well known in the field to which the present invention belongs may be used.

The present invention also provides an explosive detection method and an explosive detector, characterized in that using the TNT or DNT peptide according to the present invention or a sensor containing such a peptide. The detection sample may be in a liquid phase (aqueous solution environment), and preferably in a gas phase.

The detection of the DNT or TNT is performed by measuring a change in mass according to the formation of a complex between the sensor on which the peptide is immobilized and DNT or TNT, a change in surface plasmon resonance, or a degree of warpage of the cantilever or a change in the frequency of the cantilever. May be, but is not limited thereto (for example, Burg, TP, etc. Nature 2007 , 446 , 1066; Grate, JW, etc. Anal . Chem . 1996 , 68 , 913; Patel, SV et al . Sens. Actuators , B 2003 , 96 , 541; Hagleitner, C. et al . Nature 2001 , 414 , 293; Freund, MS et al . Proc. Natl . Acad . Sci . USA 1995 , 92 , 2652; And Shekhawat, G. et al. Science 2006 , 311 , 1592).

The invention may also (not 2,4,6-trinitro phenoxy), the 3-in monomolecular state immobilized on a support surface, such as C _{2 - 5} is the 1-alkyl-amino group, preferably 3- (2, 4, It provides a method for detecting a TNT-specific peptide, characterized in that it uses a 6-trinitrophenoxy)propane-1-amino group.

The invention also in a unimolecular state immobilized on a support surface, such as 4- (2,4-dinitrophenyl) C _{2 - 6} 1-amino group to an alkyl, preferably 4- (2,4-dinitrophenyl It provides a method for detecting a DNT-specific peptide characterized by using a phenyl) butane-1-amino group.

The present invention provides a TNT or DNT-specific peptide having the following characteristics, an explosive detection sensor using the same, and an explosive detection method. First, the TNT or DNT-specific peptide developed in the present invention has higher selectivity for TNT and DNT than the explosive-specific polymer used in the conventional explosive sensor. In addition, it was confirmed that the selectivity for TNT and DNT was higher than that of the peptides detected using TNT and DNT crystals. Second, there is an advantage of developing a peptide that selectively binds to exposed TNT and DNT in the form of a single molecule, unlike a crystal state in which molecules are aggregated, by preparing a derivative of TNT or DNT, which is the main component of an explosive, and immobilized on the surface. This can be usefully used in the development of an explosive sensor with high selectivity by producing a peptide having a characteristic that effectively binds to TNT and DNT existing in the gas phase in the form of a single molecule, which is the same as the environment in which the explosive sensor is actually used.

1 is a view showing the synthesis of TNT/DNT derivatives, immobilization of the gold surface of these derivatives, and separation of phages selective to the TNT/DNT derivatives.
2 is a conceptual diagram of a coating in which the selected peptides according to the present invention are immobilized on the surface of a gold chip ((a) OEG + peptide, (b) peptide)).
3 is a conceptual diagram of a cantilever sensing system.
4 is a result of a comparison experiment of selectivity for DNT and NT in the gas phase of the produced peptide using the Cantilever sensing system.

Hereinafter, examples, etc. will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

<Example 1> Preparation of TNT derivative

3-(2,4,6-trinitrophenoxy)propane-1-ammonium chloride, a TNT derivative for surface immobilization, was synthesized from phenol using the synthetic route of Scheme 1 below. The synthesis process was to synthesize picric acid, 2-chloro-1,3,5-trinitrobenzene (1) from phenol, and 2-chloro-1,3,5-trinitrobenzene (1) and tert-butyl 3- Synthesis of 3-(2,4,6-trinitrophenoxy)propane-1-ammonium chloride is completed through the reaction of hydroxypropylcarbamate (2).

[Scheme 1]

1) Synthesis of picric acid: Phenol (10.0 g, 106.26 mmol) was dissolved in 98% H ₂ SO ₄ (100 mL). An ice bath was installed, and 98% HNO ₃ (60.26 g, 956.34 mmol) was slowly added dropwise. Then, the mixture was stirred at 90° C. for 5 hours, an ice bath was installed, and water (200 mL) was added to terminate the reaction. Extracted with ethyl acetate (250 mL) and water (100 mL × 5), ethyl acetate _{was dried over MgSO 4} , and ethyl acetate was distilled under reduced pressure to obtain a product. (21.18 g, 87%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 9.21 (s, 2H)

2) Synthesis of 2-chloro-1,3,5-trinitrobenzene: picric acid (20.0 g, 87.3 mmol) was added to the reactor and dissolved in MC (250 mL). An ice water bath was installed, and _{POCl 3} (12 mL, 130.95 mmol) and pyridine (8.55 mL, 104.76 mmol) were slowly added dropwise. When the dropwise addition was completed, the mixture was stirred at 40° C. for 2 hours. Then, an ice water bath was installed, and the reaction was terminated with water (50 mL). Then, extraction was performed with ethyl acetate (100 mL) and water (50 mL × 4), and ethyl acetate was _{dried over MgSO 4} , and then ethyl acetate was distilled under reduced pressure to obtain a crude product. The crude product is recrystallized using MC and hexane to obtain a pure product. (17.94 g, 83%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.86 (s, 2H)

3) Synthesis of tert-butyl 3-hydroxypropylcarbamate: 3-aminopropan-1-ol (10.0 g, 133.13 mmol) is dissolved in MC (200 mL). Set up an ice bath, and slowly add di-tert-butyl dicarbonate (43.58 g, 199.69 mmol) dropwise. After the dropwise addition is complete, the mixture is stirred at room temperature for 2 hours. After inversion, extract with ethyl acetate (200 mL) and water (50 mL × 3), _{dry ethyl acetate with MgSO 4} , and distill ethyl acetate under reduced pressure to obtain a product. (20.98 g, 90%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 3.61 (t, 2H), 3.29 (q, 2H), 1.64 (m, 2H), 1.40 (s, 9H)

*4) Synthesis of tert-butyl 3-(2,4,6-trinitrophenoxy)propylcarbamate: 2-chloro-1,3,5-trinitrobenzene (5.0 g, 20.19 mmol) in THF (75 mL) Dissolved in, KOH (5.33 g, 80.76 mmol) was added and stirred. Then, tert-butyl 3-hydroxypropylcarbamate (4.25 g, 24.23 mmol) was added, followed by heating to reflux for 4 hours. When the reaction was completed, TFH was distilled under reduced pressure, and extracted with ethyl acetate (100 mL) and water (50 mL x 4). Ethyl acetate was _{dried over MgSO 4} and then ethyl acetate was distilled under reduced pressure to obtain a crude product. The crude product was purified through column chromatography (silica gel 60, 70-230 mesh ASTM) to obtain a pure product. (2.88 g, 37%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.86 (s, 2H), 4.32 (t, 2H), 3.29 (q, 2H), 2.04 (m, 2H), 1.40 (s, 9H)

5) Synthesis of 3-(2,4,6-trinitrophenoxy)propane-1-ammonium chloride: tert-butyl 3-(2,4,6-trinitrophenoxy)propylcarbamate (2.0 g, 5.18 mmol ) Was dissolved in MC (30 mL), and an ice water bath was installed. Dry HCl gas was blown into this solution for 1 hour. When a solid was formed in the solution, the reaction was terminated. Then, the solid was filtered to obtain a product. (1.25 g, 75%) ¹ H NMR (300 MHz, CD ₃ OD) δ 9.08 (s, 2H), 4.44 (t, 2H), 3.19 (t, 2H), 2.23 (m, 2H)

By analyzing the NMR spectrum of the synthesized TNT derivative (3-(2,4,6-trinitrophenoxy)propane-1-ammonium chloride), it was confirmed that the TNT derivative having the desired structure was synthesized.

<Example 2> Preparation of DNT derivative

4-(2,4-dinitrophenyl)butan-1-amine, a DNT derivative for surface immobilization, was synthesized from phenol using the synthetic route of Scheme 3 below. In the synthesis process, but-3-enylbenzene is synthesized from benzyl bromide, 4-phenylbutan-1-ol is synthesized from but-3-enylbenzene, and 1-chloro- 4-phenylbutane was synthesized, 1-(4-chlorobutyl)-2,4-dinitrobenzene was synthesized from 1-chloro-4-phenylbutane, and 1-(4-chlorobutyl)-2,4- 4-(2,4-dinitrophenyl)butan-1-amine was synthesized from dinitrobenzene.

[Scheme 2]

1) Synthesis of but-3-enylbenzene: benzyl bromide (5.0 g, 29.23 mmol) and allylmagnesium bromide 1.0 M diethyl ether solution (43.85 mL, 43.84 mmol) were added to the reactor under nitrogen, and 50°C. Reacted at for 2 hours. After that, the reaction was terminated with water (10 mL), followed by extraction with water (20 mL × 3). Diethyl ether was _{dried over MgSO 4} , and diethyl ether was distilled under reduced pressure to obtain a product. (3.48 g, 90%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.25 (m, 5H), 5.84 (m, 1H), 5.01 (m, 2H), 2.69 (m, 2H), 2.38 (m, 2H)

2) Synthesis of 4-phenylbutan-1-ol: But-3-enylbenzene (3.0 g, 22.69 mmol) was added to the reactor under nitrogen, and then an ice bath was installed. Then, borane-THF complex 1.0 M solution (35 mL, 35 mmol) was slowly added dropwise, followed by stirring for 1 hour. After 1 hour, NaOH (1.2 g) and 30% H ₂ O ₂ (3.75 mL) were slowly added and stirred for 1 hour. Thereafter, extraction was performed with ethyl acetate (20 mL) and water (15 mL × 3), and ethyl acetate was _{dried over MgSO 4} , and then ethyl acetate was distilled under reduced pressure to obtain a product. (2.89 g, 85%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.20 (m, 5H), 3.62 (t, 2H), 2.62 (t, 2H), 1.62 (m, 4H).

3) Synthesis of 1-chloro-4-phenylbutane: 4-phenylbutan-1-ol (2.8 g, 17.97 mmol) was added to a reactor and dissolved in 20 ml of methylene chloride (MC). An ice water bath was installed, and _{POCl 3} (2.47 mL, 26.96 mmol) and pyridine (1.76 mL, 21.56 mmol) were slowly added dropwise. When the dropwise addition was completed, the mixture was stirred at 40° C. for 2 hours. After that, an ice water bath was installed, and the reaction was terminated with water (10 mL). After extraction with diethyl ether (30 mL) and water (25 mL × 4), diethyl ether was _{dried over MgSO 4} , and diethyl ether was distilled under reduced pressure to obtain a product. (2.87 g, 95%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.22 (m, 5H), 3.58 (t, 2H), 2.63 (t, 2H), 1.81 (m, 4H).

4) Synthesis of 1-(4-chlorobutyl)-2,4-dinitrobenzene: 1-chloro-4-phenylbutane (2.5 g, 14.82 mmol) was dissolved in 98% H ₂ SO ₄ (20 mL). An ice water bath was installed, and 70% HNO ₃ (5.66 mL, 88.92 mmol) was slowly added dropwise. Then, the mixture was stirred at 80° C. for 3 hours, and water (10 mL) was added to an ice bath to terminate the reaction. Extracted with ethyl acetate (50 mL) and water (50 mL × 4), ethyl acetate was _{dried over MgSO 4} , and then ethyl acetate was distilled under reduced pressure to obtain a crude product. The crude product was purified through column chromatography (silica gel 60, 70-230 mesh ASTM) to obtain a pure product. (2.49 g, 65%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.72 (d, 1H), 8.38 (dd, 1H), 7.61 (d, 1H), 3.60 (t, 2H), 3.02 (t, 2H), 1.95 (m, 4H).

5) Synthesis of 4-(2,4-dinitrophenyl)butan-1-amine: 1-(4-chlorobutyl)-2,4-dinitrobenzene (1.0 g, 3.86 mmol) and 2.0 M methanol solution of ammonia (15 mL) was introduced into a reactor dedicated to a microwave reactor. It was reacted at 120° C. for 20 minutes using a microwave reactor. After the reaction was completed, distillation under reduced pressure to remove methanol, and extracted with ethyl acetate (20 mL) and water (10 mL × 3). And the water layer was made neutral, and extracted using MC (15 mL × 4). MC was _{dried using MgSO 4} , and then MC was distilled under reduced pressure to obtain a product. (0.32 g, 35%) ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.78 (d, 1H), 8.38 (dd, 1H), 7.61 (d, 1H), 3.01 (t, 2H), 2.73 (t, 2H), 1.62 (m, 4H)

By analyzing the NMR spectrum of the synthesized DNT derivative (4-(2,4-dinitrophenyl)butan-1-amine), it was confirmed that the DNT derivative having the desired structure was synthesized.

<Example 3> Selection and amino acid sequence analysis of phage having TNT-specific peptide

In order to select a TNT-specific phage, 3-(2,4,6-trinitrophenoxy)propane-1-ammonium chloride, a TNT derivative, was used as a linker, 2,5-dioxopyrrolidin-1-yl 23-mercapto. Gold using -3,6,9,12-tetraoxatricosan-1-oate (2,5-dioxopyrrolidin-1-yl 23-mercapto-3,6,9,12-tetraoxatricosan-1-oate) It was immobilized on a thin film. After washing the gold thin film (1.7cm×1.7cm) with ethanol, 2 mM 2,5-dioxopyrrolidin-1-yl 23-mercapto-3,6,9,12-tetraoxatrico dissolved in ethanol It was allowed to react with acid-1-oate at room temperature for 14 hours under the condition of no light. After washing the gold thin film with the linker attached with ethanol, 1 mM 3-(2,4,6-trinitrophenoxy)propane-1-ammonium chloride and 3 mM glycine were added to 50 mM NaHCO ₃ (pH9.1). After melting, it was reacted with the gold thin film for 14 hours. ^{A phage display library (NEB, 1.5×10 13} pfu, 10ul) was mixed with 90ul of TBS and reacted for 1 hour on the gold thin film on which the TNT derivative was immobilized, and 500ul of TBS was poured and washed 10 times. The phage adsorbed on the surface of the gold thin film on which the TNT derivative is immobilized was eluted with 0.2M glycine-HCl (pH2.2) solution, and then 5ml of E. coli suspension (ER2738, overnight culture) was diluted in LB medium solution at a ratio of 1/100. . Then, the phage eluate was added and incubated for 4.5 hours, followed by centrifugation and separation of the phage bound to the immobilized TNT derivative by separating the phage using PEG/NaCl. The separation process of the phage binding to the TNT derivative was repeated 4 times, and the finally separated phage was inoculated into ER2738 E. coli culture medium and then cultured on an agar plate. The phages were separated from individual phage plaques formed on the agar plate, inoculated into ER2738 E. coli culture, and cultured for 5 hours. The phages were separated and phage DNA was purified from the separated phages. The nucleotide sequence of the purified phage DNA was determined to determine the peptide sequence attached to the phage surface protein.

Through the experiment, the TNT-specific peptide sequences of SEQ ID NOs: 1 to 4 were selected.

<Example 4> Selection and amino acid sequence analysis of phage having DNT-specific peptide

In order to select DNT-specific phage, a DNT derivative 4-(2,4-dinitrophenyl)butan-1-amine) was added to a gold thin film with 2,5-dioxopyrrolidin-1-yl 23-mercapto-3. ,6,9,12-tetraoxatricosan-1-oate (2,5-dioxopyrrolidin-1-yl 23-mercapto-3,6,9,12-tetraoxatricosan-1-oate) was immobilized using. After washing the gold thin film (1.7cm×1.7cm) with ethanol, 2 mM 2,5-dioxopyrrolidin-1-yl 23-mercapto-3,6,9,12-tetraoxatricosan-1- After Oate was dissolved in ethanol, it was reacted with the gold thin film for 14 hours at room temperature under the condition of no light. After washing the gold thin film with the linker attached with ethanol, dissolve 1 mM of 4-(2,4-dinitrophenyl)butan-1-amine and 3 mM glycine in 50 mM NaHCO ₃ (pH9.1), and then the gold thin film And reacted for 14 hours. ^{A phage display library (NEB, 1.5×10 13} pfu, 10ul) was mixed with 90ul of TBS and reacted for 1 hour on a gold thin film immobilized with 4-(2,4-dinitrophenyl)butan-1-amine, and 500ul of TBS was added. Poured and washed 10 times. Phage adsorbed on the surface of the gold thin film was eluted with 0.2M glycine-HCl (pH2.2) solution, and then 5 ml of E. coli suspension (ER2738, overnight culture) was diluted in LB medium solution at a ratio of 1/100. Then, the phage eluate was added and cultured for 4.5 hours, followed by centrifugation and separation of phage bound to the immobilized DNT derivative by separating the phage using PEG/NaCl. The separation process of the phage binding to the DNT derivative was repeated four times, and the finally separated phage was inoculated into ER2738 E. coli culture medium, and then cultured on an agar plate. The phages were separated from individual phage plaques formed on the agar plate, inoculated into ER2738 E. coli culture, and cultured for 5 hours. The phages were separated and phage DNA was purified from the separated phages. The nucleotide sequence of the purified phage DNA was determined to determine the peptide sequence attached to the phage surface protein.

Through the experiment, the DNT-specific peptide sequences of SEQ ID NOs: 9 to 11 were selected.

<Example 5> TNT/DNT-specific Peptide Selectivity Analysis for TNT/DNT

After dissolving the prepared peptide (1 mg) in a buffer solution, it was reacted on the surface of a gold thin film used in a measuring device such as SPR, QCM (Quartz Crystal Microbalance) or Cantilever as shown in FIG. 2. After that, each selectivity was analyzed through the following process.

1) The cantilever coated with a gold thin film was cleaned in the following process. First, UV cleaning was performed for 20 minutes. Then, the chips were immersed in 2N NaOH solution for 30 minutes, then immersed in deionized water for 10 minutes, immersed in 1N HCl for 5 minutes, and finally washed with demineralized water.

2) The peptide was dissolved in PBS buffer (pH about 7.2) to make a 1mM solution, and the cantilever was soaked for about 12 hours, and then washed well with PBS buffer and demineralized water.

3) Before use in the experiment, the experiment was performed through the Cantilever sensing system as shown in FIG. 3 after immersing in PBS buffer and storing at 4°C.

In the experiment, after injecting a certain amount of PBS buffer into the peptide-coated Cantilever sensing system, the same concentration (0.55 mM) of DNT and NT (2-nitrotoluene) was dissolved in the PBS buffer, and a certain amount of each was injected. As a method of measuring the amount of deflection of the cantilever, the injected DNT and NT as shown in FIG. 3 undergo a selective reaction with the peptide through a vaporization process. The results using the peptide of SEQ ID NO: 12 are shown in FIG. 4.

<110> Kookmin University Industry Academy Cooperation Foundation <120> Peptide Sensors for Selective Detection of Explosives <130> P12-204 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of TNT <400> 1 His Pro Leu Lys Gln Tyr Trp Trp Arg Pro Ser Ile 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of TNT <400> 2 Val Ser Arg His Gln Ser Trp His Pro His Asp Leu 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of TNT <400> 3 His Arg Asn His Leu Met Asp Leu Ser Gly Leu Tyr 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of TNT <400> 4 Trp Ser Pro Gly Gln Gln Arg Leu His Asn Ser Thr 1 5 10 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of TNT <400> 5 His Pro Leu Lys Gln Tyr Trp Trp Arg Pro Ser Ile Gly Cys 1 5 10 <210> 6 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of TNT <400> 6 Val Ser Arg His Gln Ser Trp His Pro His Asp Leu Gly Cys 1 5 10 <210> 7 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of TNT <400> 7 His Arg Asn His Leu Met Asp Leu Ser Gly Leu Tyr Gly Cys 1 5 10 <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of TNT <400> 8 Trp Ser Pro Gly Gln Gln Arg Leu His Asn Ser Thr Gly Cys 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of DNT <400> 9 Lys Met His Thr Ala Ser Leu Ser Gln Pro Leu Met 1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of DNT <400> 10 Gln Ser Phe Ala Ser Leu Thr Asn Pro Arg Val Leu 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of DNT <400> 11 Ala Ser Thr Ala Ser Leu His Gln Pro Arg 1 5 10 <210> 12 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of DNT <400> 12 Lys Met His Thr Ala Ser Leu Ser Gln Pro Leu Met Gly Cys 1 5 10 <210> 13 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of DNT <400> 13 Gln Ser Phe Ala Ser Leu Thr Asn Pro Arg Val Leu Gly Cys 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide for Selective Detection of DNT <400> 14 Ala Ser Thr Ala Ser Leu His Gln Pro Arg Gly Cys 1 5 10

Claims (7)

delete delete delete delete delete delete Characterized in that a monomolecular 3- (2,4,6-trinitrophenoxy) propane-1-amino group immobilized on the surface of a support is used.

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