KR20200082428A - Metal Nanoparticle-Decorated Metallic Calcogenide-Bioreceptor and their Detection Method of Target Material using the same - Google Patents

Abstract

The present invention relates to a metal chalcogenide-bioreceptor treated with metal nanoparticles and a method for detecting a target material using the same. The present invention not only can detect the target material with high sensitivity and selectively, but also allows easy quantification of the target material, and thus can be effectively used for detection of the target material, particularly, in-situ detection of norovirus.

Description

Metal Nanoparticle-Decorated Metallic Calcogenide-Bioreceptor and their Detection Method of Target Material using the same}

The present invention relates to a metal chalcogenide-biooriceptor treated with metal nanoparticles and a target substance detection method using the same.

Food poisoning refers to an infectious or toxin-type disease caused or deemed to have occurred due to microorganisms or toxic substances harmful to the human body related to food intake, and is generally classified into food poisoning by microorganisms and food poisoning by chemicals according to the cause. Food poisoning by microorganisms is divided into bacterial and viral, and bacterial food poisoning is subdivided into toxin type and infection type. In particular, the main harmful viruses known to cause viral food poisoning include norovirus, rotavirus, and astro virus. . When eating food contaminated with these viruses, symptoms such as vomiting, abdominal pain, diarrhea, and fever appear.

Among them, Norovirus is the most common pathogen causing human enteritis in the world, and is known to cause more than 90% of non-bacterial enteritis in the United States and Europe. Norovirus was first discovered in a group of food poisoning patients at Norwalk Elementary School, Ohio, USA in 1968. In the United States, norovirus is caused by more than 70% of water-related enteritis patients and more than 50% of food-related enteritis patients. Is known. In Japan, the food poisoning that occurred from 2001 to 2007 was considered as the cause, and it was reported that the highest number of patients were caused by Norovirus. The British Food Standards Agency (FSA) also reported that norovirus was detected in 76% of oysters tested in oyster cultivation areas (39 locations, 800 cases). In addition, a recent epidemiologic investigation of diseases related to aquatic foods conducted by the US Centers for Disease Control and Prevention (CDC) showed that the ratio of causative agents to 188 food poisonings occurred in 1973-2006 was bacterial (76.1%), virus (21.3%), and parasite (2.6%).

According to the Korea Food and Drug Administration's announcement, 54% of distribution oysters are contaminated with microbiological hazards such as viruses and parasites due to various changes in fisheries environment such as climate change. Due to this increasing trend, food poisoning accidents caused by norovirus are the most known. Early food detection is important because norovirus may contain 10-100 particles when the shellfish or fish is consumed among foods causing norovirus food poisoning.

According to a conventional PCR-based (RT-PCR, multiplexed PCR) method capable of quickly and accurately detecting a trace amount of a foodborne disease-derived virus derived from aquatic fish and shellfish, simultaneous detection of multiple pathogens is possible, but present in a sample There is a disadvantage that false positive results can be derived due to the difficulty of quantification and quantification of the polymerase by the pollutant. In addition, since expensive fluorescent materials (Cy3, Cy5, Q-dot, etc.) have to be used as a marker, a lot of cost is consumed and accessibility is limited because they cannot be applied quickly in the field. In addition, the existing immunoassay technique using an antibody has the advantage of being easy to use, but the selectivity is not high for a specific microorganism, a lot of time and cost for antibody production and detection equipment installation is consumed, and the detection limit is low. There are disadvantages. In addition, since there is a possibility of a cross reaction, it is not suitable for the development of a new type of detection method that is easy to access in a high-speed field with high sensitivity. On the other hand, research is being conducted to detect microbial genes for food using DNA chips or DNA microarrays. However, the array chip, measurement system, and analysis program are separately configured, and thus, there is a disadvantage of low field access and time-consuming analysis. . Therefore, there is a need to develop a new type of target substance detection system by fusion with other methods.

Accordingly, the present inventor completed the present invention by conducting research to develop a material and method capable of detecting the target material on-site with selective and high sensitivity.

Republic of Korea Patent Publication No. 10-2017-0056006

One object of the present invention includes a metal nanoparticle, a metal chalcogenide and a peptide, the metal nanoparticle is bound to the metal chalcogenide, and the peptide is a complex for detecting a target substance bound to the metal nanoparticle Is to provide

Another object of the present invention includes a metal nanoparticle, a metal chalcogenide and a peptide, the metal nanoparticle is bound to the metal chalcogenide, and the peptide is a complex for detecting a target substance bound to the metal nanoparticle It is to provide a composition for detecting a target substance containing.

Another object of the present invention includes a metal nanoparticle, a metal chalcogenide and a peptide, the metal nanoparticle is bound to the metal chalcogenide, and the peptide is a complex for detecting a target substance bound to the metal nanoparticle It is to provide a kit for detecting a target material comprising a.

Another object of the present invention includes a metal nanoparticle, a metal chalcogenide and a peptide, the metal nanoparticle is bound to the metal chalcogenide, and the peptide is a complex for detecting a target substance bound to the metal nanoparticle Contacting the unknown sample; Measuring the electrochemical signal of the complex in contact with the sample; And when the size of the electrochemical signal is increased, to provide a target material detection method comprising the step of determining that the target material is included in the sample.

One aspect of the present invention includes a metal nanoparticle, a metal chalcogenide and a peptide, the metal nanoparticle is bound to the metal chalcogenide, and the peptide is a complex for detecting a target substance bound to the metal nanoparticle to provide.

The present invention relates to a nano-bio complex for detecting a target substance based on a metal chalcogenide treated with a metal nanoparticle and a peptide acting as a bioreceptor, and the electrode of the present invention reacting with the target substance In this case, since the electric signal is displayed even at a low concentration, the presence of the target substance can be accurately and simply confirmed using the complex of the present invention.

The term "metal chalcogenide" used in the present invention refers to a compound of a binary system or higher composed of an element and a metal element other than oxygen among elements of Group 6 of the periodic table, and refers to a material mainly classified as a semi-metal and a semiconductor. Since metal chalcogenide exhibits high mechanical properties, electrochemical properties, and biocompatibility, it can be usefully used for detection of target materials, particularly for detection of target materials in biological samples.

When the metal chalcogenide is treated with the metal chalcogenide, the conductivity of the metal chalcogenide is improved, and by immobilizing the peptide on the metal chalcogenide treated with the metal nanoparticle, the conductivity is excellent and the target material is detected with high sensitivity. The formation of this possible complex is possible, and the target material binding to the peptide can be analyzed by an electrochemical method.

The detection of the target material is achieved through, for example, reacting the complex of the present invention with a sample for a certain period of time, washing it, applying and drying the completed sample on the electrode, and then analyzing the Nafion-treated electrode through electrochemical impedance analysis. Can.

According to an embodiment of the present invention, the surface of the metal chalcogenide may be modified with a carboxyl group (-COOH) group.

According to one embodiment of the invention, the modification may be by reaction of the metal chalcogenide and 3-mercapto propionic acid (3-mercapto propionic acid).

The surface of the metal chalcogenide can be modified into a carboxyl group by the reaction of the metal chalcogenide with 3-mercaptopropionic acid, and the surface of the metal chalcogenide is modified with a carboxyl group, thereby minimizing the impedance value. It is possible to synthesize metal chalcogenide to which metal nanoparticles of sensitivity are bound.

According to an embodiment of the present invention, the metal nanoparticles may be one or more nanoparticles selected from the group consisting of gold nanoparticles, silver nanoparticles, and fluorescent nanoparticles.

The metal nanoparticles treated on the metal chalcogenide whose surface is modified with a carboxyl group improve the conductivity of the metal chalcogenide, and gold nanoparticles, silver nanoparticles and/or fluorescent nanoparticles may be used, but metal The nanoparticles are preferably gold(III) chloride trihydrate.

According to an embodiment of the present invention, the metal chalcogenide is any one selected from the group consisting of molybdenum, tungsten, zinc, cadmium, titanium, vanadium, chromium and manganese; And at least one chalcogenide selected from the group consisting of sulfur, selenium and telenium.

In the complex of the present invention, a metal chalcogenide is used as a material for improving the conductivity of the bioreceptor peptide, and in particular, it is preferable that tungsten disulfide (WS ₂ ) is used as the metal chalcogenide.

According to an embodiment of the present invention, the target material may be selected from the group consisting of norovirus, microviral diarrhea virus, tuberculosis antigen, melamine and histamine.

According to one embodiment of the invention, the peptide may be to specifically bind to the target material.

When using a peptide that specifically binds a target substance to the complex of the present invention, for example, norovirus, microviral diarrhea virus, tuberculosis antigen, melamine or histamine, a peptide that specifically binds to the peptide is specific Norovirus, microviral diarrhea virus, tuberculosis antigen (e.g., CFP10 or Ag85B), melamine or histamine that binds with can be detected with high sensitivity and selectivity.

According to one embodiment of the invention, the peptide may be made of the amino acid sequence of SEQ ID NO: 1.

In the present invention, optimization conditions for the preparation of a metal chalcogenide-biooriceptor treated with metal nanoparticles as a complex for detecting a target substance using a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as a viroreceptor were established. Therefore, the complex according to one embodiment of the present invention can be particularly useful for the sensitivity and specific detection of norovirus.

Another aspect of the present invention includes a metal nanoparticle, a metal chalcogenide and a peptide, the metal nanoparticle is bound to the metal chalcogenide, and the peptide is a complex for detecting a target substance bound to the metal nanoparticle It provides a composition for detecting a target material containing.

Another aspect of the present invention includes a metal nanoparticle, a metal chalcogenide and a peptide, the metal nanoparticle is bound to the metal chalcogenide, and the peptide is a complex for detecting a target substance bound to the metal nanoparticle It provides a kit for detecting a target material comprising a.

Using the composition or kit for detecting a target substance of the present invention, it is possible to conveniently and accurately check whether a target substance is included in a sample. For example, by treating the food sample with the composition or kit of the present invention, it is possible to confirm in advance whether the food is infected with norovirus, and thus can be usefully used to prevent food poisoning in advance.

The target substance detection composition and kit may include tools and/or reagents generally used for target substance detection and/or analysis, in addition to the aforementioned target substance detection complex.

Such tools or reagents may include suitable carriers, labeling substances capable of generating a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, a substrate capable of measuring the activity of the enzyme and a reaction stopper may be further included. Suitable carriers include soluble carriers, such as physiologically acceptable buffers known in the art (eg PBS, etc.); Insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, etc.; Polymers such as magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and/or combinations thereof may be included.

In addition, the composition and kit for detecting a target substance may further include a carbon printed circuit (for example, Carbon-SPE) for analysis of the electrochemical signal in addition to the complex for detecting the target substance described above, and analyze the electrochemical signal. It may further include a sample and a device for.

Another aspect of the present invention includes a metal nanoparticle, a metal chalcogenide and a peptide, the metal nanoparticle is bound to the metal chalcogenide, and the peptide is a complex for detecting a target substance bound to the metal nanoparticle Contacting the unknown sample; Measuring the electrochemical signal of the complex in contact with the sample; And when the size of the electrochemical signal increases, determining that the target material is included in the sample.

The target material detection method of the present invention is a target material detection method based on electrochemical technology, and it is possible to quickly and easily detect a target material using a bioreceptor specific to the target material, and it is easier to detect than other optical equipment, and is portable. It has a high advantage that it can be applied in the field, and can be usefully used for rapid on-site analysis of target substances such as food poisoning bacteria.

According to one embodiment of the present invention, the method for measuring the electrochemical signal may be selected from the group consisting of an impedance method, a cyclic voltammetry method, and a time zone current method.

In the method for detecting a target substance of the present invention, a sample to be checked to see if a target substance is included is brought into contact with the target substance detecting complex according to one embodiment of the present invention, so that the target substance reacts with the complex, and treatment with the electrode Therefore, it can be performed by analysis of the electrochemical signal, and when the size of the electrochemical signal increases, it can be determined that the target material is included in the sample.

According to an embodiment of the present invention, the target material may be norovirus.

When using a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the complex for detecting a target substance used in the method for detecting a target substance of the present invention, the complex specifically binds to norovirus, and thus, rotavirus, dengue virus serotype 1, Norovirus can be specifically detected even in samples in which various substances such as 2, 3, and 4 are mixed.

According to the metal chalcogenide-biooriceptor treated with metal nanoparticles and a method for detecting the target material using the same, the target material is not only capable of being sensitively and selectively detected, but also can be easily quantified, so that the target material It can be useful for the detection of, in particular, on-site detection of norovirus.

1 shows a method of synthesizing (a) a metal chalcogenide-biolyceptor treated with metal nanoparticles, and (b) a method for electrochemical detection of norovirus using a metal chalcogenide-biolyceptor treated with metal nanoparticles. This is a schematic diagram of Korea.
Figure 2 after the metal chalcogenide synthesis according to temperature, (a) X-ray diffraction analysis (X-Ray Diffraction) and (b to e) field emission scanning electron microscope (Field Emission Scanning Electron Microscope) to analyze the structure and shape One result ((b) 260°C, (c) 270°C, (d) 280°C, (e) 290°C).
3 is a graph showing the results of X-ray diffraction analysis of the metal chalcogenide sample obtained according to the reaction time, with the reaction temperature fixed at 280°C.
FIG. 4 is a field emission scanning electron microscope image of a metal chalcogenide sample obtained according to a reaction time while the reaction temperature is fixed at 280° C. ((a) 2 hours, (b) 4 hours, (c) 8 hours. , (d) 12 hours, (e) 16 hours, (f) 20 hours).
5 is a graph showing the gas adsorption method (Brunauer-Emmett-Teller) of the metal chalcogenide obtained according to the reaction temperature ((a) 270°C, (b) 280°C, (c) 290°C).
6 is a graph of the gas adsorption method (Brunauer-Emmett-Teller) of the metal chalcogenide obtained according to the reaction time ((a) 12 hours, (b) 16 hours, (c) 20 hours).
7 is a graph showing (a) metal chalcogenide impedance graphs coated with metal nanoparticles and (b) impedance values according to 3-Mercaptopropionic acid (MPA) concentration.
FIG. 8 is a graph of (a) metal chalcogenide impedance coated with metal nanoparticles and (b to g) scanning electron microscope images according to the concentration of gold(III) chloride trihydrate ((b) 0mg, (c) ) 0.6mg, (d) 1.2mg, (e) 2.4mg, (f) 5.0mg, (g) 10.0mg).
9 is a graph showing the results of X-ray diffraction analysis of metal chalcogenide and metal chalcogenide coated with metal nanoparticles.
FIG. 10 is (a) impedance value according to the concentration of a bioreceptor used in manufacturing a metal chalcogenide-biooriceptor treated with metal nanoparticles, (b) when manufacturing a metal chalcogenide-biooriceptor treated with metal nanoparticles The impedance value according to the binding reaction time between the metal chalcogenide treated with the metal nanoparticles and the bioreceptor, and (c) the impedance value according to the binding reaction time between the metal chalcogenide treated with the metal nanoparticles-bioreceptor and norovirus. .
11 is a graph showing (a) impedance spectrum according to norovirus concentration and (b) norovirus detection limit using the same. In addition, (c) is a graph showing the impedance spectrum according to the norovirus concentration extracted from the actual oyster and (d) the limit of detection of the norovirus using the same.
12 is an impedance graph obtained after reacting a metal chalcogenide-biooriceptor treated with metal nanoparticles and dengue virus serotypes 1, 2, 3, 4 and rotavirus, respectively.

Hereinafter, the present invention will be described in more detail through one or more embodiments. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 Confirmation of Synthesis Optimization Conditions for Metal Chalcogenide

<1-1> Metal chalcogenide synthesis optimization conditions according to the reaction temperature

In order to prepare a metal chalcogenide-biolyceptor treated with metal nanoparticles for the electrochemical detection of the target material, the metal chalcogenide was synthesized according to the reaction temperature to determine the optimal reaction temperature.

Specifically, 0.4 g of tungsten hexachloride and 0.37 g of thioacetamide were added to 10 ml of tertiary distilled water and stirred for 1 hour. The reaction was performed for 16 hours at different temperatures (270°C, 280°C, 290°C) using a hydrothermal synthesis method. After the reaction was completed, to remove impurities, the supernatant was removed using a centrifuge at 4,500 x g for 10 minutes (repeat 3 times). Then, the obtained WS ₂ tungsten disulfide nanoflower (NF) powder was dried at 60° C. for 10 hours.

For X-ray diffraction (XRD) analysis, a New D8-Advance device of Bruker AXS was used, and WS ₂ NF was placed on Cu-Kα and analyzed to 5 to 70°C. Meanwhile, the structure and shape of WS ₂ NF were analyzed using a K-alpha scanning microscope from ThermoFisher Scientific. On the other hand, the structure and shape of WS ₂ NF was analyzed using a SIGMA field emission scanning microscope from Carl Zeiss.

As a result of X-ray diffraction analysis, it was confirmed that a WO ₃ (ICSD No. 80634) structure was formed at a reaction temperature of 260° C., and a hexagonal (ICSD No. 08-0237) structure was formed at a reaction temperature of 270° C. or higher. It was confirmed (Fig. 2(a)). In addition, as a result of the scanning microscope analysis, it was confirmed that nano flower particles were not formed at 260° C., but nano flower particles were formed at 270° C. or higher. On the other hand, at a reaction temperature of 290° C., decomposition of nano flower particles due to high temperature was confirmed (FIGS. 2( b) to 2( e )).

Therefore, it was confirmed that the metal chalcogenide structure and nano flower particle shape of 280°C, which were most stably synthesized, are the optimum reaction temperature for metal chalcogenide synthesis.

<1-2> Confirmation of optimization conditions for metal chalcogenide synthesis according to reaction time

In order to prepare a metal chalcogenide-biolyceptor treated with metal nanoparticles for electrochemical detection of a target material, an optimal reaction time was confirmed by synthesizing metal chalcogenide according to the reaction time.

Specifically, 0.4 g of tungsten hexachloride and 0.37 g of thioacetamide were added to 10 ml of tertiary distilled water and stirred for 1 hour. The reaction was performed at 280°C for different times (2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours) using a hydrothermal synthesis method. After the reaction was completed, to remove impurities, the supernatant was removed using a centrifuge at 4,500 x g for 10 minutes (repeat 3 times). Then, the obtained WS ₂ tungsten disulfide nanoflower (NF) powder was dried at 60° C. for 10 hours.

As a result of X-ray diffraction analysis, it was confirmed that when the reaction time was 2 hours, a WO ₃ (monoclinic) structure was formed. When the reaction time was 4 or 8 hours, a WO ₃ (hexagonal) structure was formed, and when the reaction time was 12 hours or more, it was confirmed that a WS ₂ (hexagonal) structure was formed (FIG. 3 ). In addition, as a result of the scanning microscopic analysis, when the reaction time was 2, 4 or 8 hours, a rod shape was formed, while when the reaction time was 12, 16 or 20 hours, it was confirmed that a nano flower particle shape was formed (FIG. 4 ). ).

Therefore, it was confirmed that the 16 hours that the metal chalcogenide structure and nano flower particle shape were most stably synthesized were the optimal reaction times for metal chalcogenide synthesis.

<1-3> Synthesis optimization conditions of metal chalcogenide according to surface area analysis

In order to prepare a metal chalcogenide-biolyceptor treated with metal nanoparticles for electrochemical detection of a target material, the surface area of the metal chalcogenide prepared at an optimum reaction temperature or an optimum reaction time is analyzed to optimize The reaction temperature and reaction time were confirmed.

Specifically, Brunauer-Emmett-Teller (BET) surface area analysis was performed using the Nova 1200e model of Quantachrome, and Example <1-1> except that the reaction time was fixed at 16 hours or the reaction temperature was fixed at 280°C. Alternatively, WS ₂ NF was obtained in the same manner as in <1-2>, and after drying at 60° C. for 12 hours, the surface area was measured.

The reaction time was fixed to 16 hours, and as a result of analyzing the surface area of the metal chalcogenide according to the reaction temperature, when the reaction temperature was 280°C, it was confirmed that the surface area was most wide at 14.8 m ² /g (FIG. 5). . On the other hand, by fixing the reaction temperature at 280°C, and analyzing the surface area of the metal chalcogenide according to the reaction time, when the reaction time is 16 hours, it was confirmed that the surface area is the largest.

Therefore, it was confirmed that 280° C. and 16 hours synthesized with the largest surface area of the metal chalcogenide were optimal reaction conditions for the synthesis of the metal chalcogenide.

Example 2. Synthesis optimization conditions of metal chalcogenide treated with metal nanoparticles

To confirm the optimization conditions for the synthesis of metal chalcogenide treated with metal nanoparticles, the metal chalcogenide surface was modified with a carboxyl group (-COOH), and metal chalcogenide treated with metal nanoparticles was synthesized.

Specifically, n-butyllithium/hexane solution (1.25M, 2ml) was added to a metal chalcogenide of 20mg and stirred for 1 hour, and then 3-mercaptopropionic acid was added at different concentrations (25, 50, 60). , 80, 100, 150mM) and stirred for 1 hour. To remove unbound 3-mercaptopropionic acid, it was dialyzed in a 1000Da dialysis bag for 3 days. After dialysis, 1 ml of 0.3 M sodium borohydride solution and gold(III) chloride trihydrate were added to metal chalcogenide solution substituted with carboxyl groups in different amounts (0.6, 1.2, 2.4, 5.0, 10.0 mg). After adding and reacting for 2 hours, in order to remove impurities, after centrifugation at 4,500×g for 10 minutes, the supernatant was discarded and tertiary distilled water was added to the metal chalcogenide treated with metal nanoparticles, followed by dispersion. Used for analysis.

Impedance analysis was performed using a CHI 750E instrument from CH Instruments. 10 μl and 0.1% Nafion were placed on the working electrode of the carbon printed circuit and analyzed. Before the impedance analysis, the OCP (Open Circuit Potential) was measured, and the value was input to the impedance.

After synthesizing the metal chalcogenide treated with metal nanoparticles according to the concentration of 3-mercaptopropionic acid, the impedance was analyzed, and when using 60 mM of 3-mercaptopropionic acid, the impedance was found to be the lowest (Fig. 7). ). As a result of analyzing the metal chalcogenide treated with metal nanoparticles according to the concentration of 3-mercaptopropionic acid at a concentration of 60 mM and the concentration of gold chloride, it was confirmed that when gold chloride of 2.4 mg or more was used, the impedance value did not decrease any more. (Fig. 8(a)). In addition, as a result of the scanning electron microscope analysis, it was confirmed that the size of the metal nanoparticles increased as the concentration of gold chloride increased (FIG. 8(b) to FIG. 8(g)).

In addition, as a result of X diffraction analysis, the metal nanoparticle peak (JCPDS No. 04-0784) was confirmed in the metal chalcogenide treated with metal nanoparticles, confirming that the metal chalcogenide treated with metal nanoparticles was synthesized. Became (Fig. 9).

Therefore, it was confirmed that the conditions for using 3-mercaptopropionic acid 60mM and 2.4 mg (0.1 mg/mL) of gold chloride with the lowest impedance value are optimal conditions for the synthesis of metal chalcogenide treated with metal nanoparticles.

Example 3. Synthesis optimization conditions of metal chalcogenide-biooriceptor treated with metal nanoparticles and reaction time optimization conditions with norovirus

In order to confirm the synthesis optimization conditions of the metal chalcogenide-biooriceptor treated with the metal nanoparticles, and to determine the optimization conditions of the reaction time of the norovirus and the metal chalcogenide-biooriceptor treated with the metal nanoparticles, the metal nano Various concentrations of peptides were treated on the surface of the metal chalcogenide treated with particles at various reaction times.

Specifically, 100 μl of peptides of different concentrations (0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 mg/ml) were added to the metal chalcogenide solution treated with 0.1 mg/ml metal nanoparticles, followed by 90 minutes. During the reaction. In addition, in order to confirm the reaction time of the metal chalcogenide treated with the metal nanoparticles and the peptide, the peptides at the concentration of 0.7 mg/ml were used for different reaction times (60, 90, 120, 180, 240, 300, 360 minutes). To react. The amino acid sequence of the used peptide is shown in Table 1.

designation Amino acid sequence (5'→3') Sequence number receptor peptide QHKMHKPHKNTKEKEKEKEGGGGSGGGGSC SEQ ID NO: 1

As a result of analyzing the impedance tendency according to the peptide concentration for the metal chalcogenide solution treated with 0.1 mg/ml metal nanoparticles, it was confirmed that the impedance value does not increase any more when the peptide concentration is 1 mg/ml. (Fig. 10(a)). In addition, when the peptide concentration was fixed at 1 mg/ml and the impedance graph according to the reaction time was confirmed, it was confirmed that the impedance value did not increase at 240 minutes (FIG. 10(b)). Therefore, it was confirmed that the peptide concentration of 1.0 mg and the reaction time condition of 240 minutes are optimal conditions for the synthesis of the metal chalcogenide-biolyceptor treated with metal nanoparticles.

On the other hand, in order to confirm the optimal reaction time for the norovirus of the metal chalcogenide-biooriceptor treated with the metal nanoparticles, the norovirus in the metal chalcogenide-biooriceptor solution treated with the metal nanoparticles of 0.1 mg/ml (10 ⁵ copies/ml) After adding 100 µl and reacting with different reaction times (20, 30, 40, 50, 60, 90, 120 minutes), it was confirmed that the impedance no longer increases at 60 minutes. , It was confirmed that the optimal reaction time of norovirus and metal chalcogenide-biooriceptor treated with metal nanoparticles was 60 minutes.

Example 4. Confirmation of electrochemical detection according to norovirus concentration change of metal chalcogenide-biooriceptor treated with metal nanoparticles

Using the metal chalcogenide-biooriceptor treated with the metal nanoparticles prepared in Example 3, in order to confirm whether norovirus can be detected in a concentration-dependent manner, the metal chalcogenide-biooriceptor treated with the metal nanoparticle The virus was treated by concentration to analyze the tendency of the impedance.

Specifically, norovirus (control, 10 ¹ , 10 ² , 10 ³ , 10 ⁴ copies/ml) diluted with buffer in DMEM, respectively, in a metal chalcogenide-bioreceptor treated with 0.1 mg/ml metal nanoparticles 100 µl was added and reacted for 60 minutes. Then, 200 μl of tertiary distilled water was added to disperse the metal chalcogenide-biolyceptor treated with virus-bound metal nanoparticles. Impedance was analyzed after raising the metal chalcogenide-biolyceptor treated with 10 μl of metal nanoparticles and 4 μl of 0.1% Nafion on the working electrode of the carbon printed circuit.

As a result, as the norovirus concentration increased, the impedance value increased (Figs. 11(a) and 11(b)), and the detection limit was found to be 2.37 copies/ml (R ² =0.9875). In addition, after the norovirus was separated from the oyster by the ISO TS 1527-1 method, the impedance was analyzed by applying the metal nanoparticles to the treated metal chalcogenide-biolyceptor, and the impedance value was dependent on the norovirus concentration. It increased, and the detection limit was confirmed to be 6.21 copies/ml (R ² =0.9698) (FIGS. 11(c) and 11(d)).

Therefore, it was confirmed that norovirus can be detected with concentration-dependent and high sensitivity using a metal chalcogenide-biooriceptor treated with metal nanoparticles.

Example 5. Confirmation of detection specificity of metal chalcogenide-biolyceptor treated with metal nanoparticles

To confirm that the metal chalcogenide-biooriceptor treated with the metal nanoparticles specifically binds to the norovirus, the norovirus, rotavirus showing norovirus-like symptoms, and dengue virus, another type of virus, are metal nanoparticles. Impedance was analyzed by applying to a particle treated metal chalcogenide-biooriceptor.

Specifically, rotavirus, dengue virus serotypes 1, 2, 3, 4 and norovirus were used by diluting (10 ⁴ copies/ml) in DMEM buffer. The prepared virus was diluted with a metal chalcogenide-biooriceptor treated with 0.1 mg/ml metal nanoparticles, and 100 µl of each virus was added and reacted for 60 minutes. To remove impurities, the supernatant was removed by centrifugation at 4500×g for 7 minutes. Thereafter, 10 μl of a metal chalcogenide-biolyceptor treated with metal nanoparticles reacted with a virus on a working electrode of a carbon printed circuit and 4 μl of 0.1% Nafion were raised and then analyzed for impedance.

As a result, the impedance value increased only in the norovirus sample, and it was confirmed that the impedance value did not increase in dengue virus and rotavirus (FIG. 12).

Through these results, it was confirmed the distress and selectivity of the metal chalcogenide-biolyceptor treated with the metal nanoparticles of the present invention for norovirus.

So far, the present invention has been focused on the embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Metal Nanoparticle-Decorated Metallic Calcogenide-Bioreceptor and their Detection Method of Target Material using the same <130> PN180469 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> receptor peptide <400> 1 Gln His Lys Met His Lys Pro His Lys Asn Thr Lys Glu Lys Glu Lys 1 5 10 15 Glu Lys Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys 20 25 30

Claims (13)

Metal nanoparticles, metal chalcogenide and peptides,
The metal nanoparticles are bound to the metal chalcogenide,
The peptide is a complex for detecting a target substance bound to the metal nanoparticles.
The complex for detecting a target substance according to claim 1, wherein the surface of the metal chalcogenide is modified with a carboxyl group (-COOH) group.
The complex for detecting a target substance according to claim 2, wherein the modification is by reaction of the metal chalcogenide with 3-mercapto propionic acid.
The complex for detecting a target substance according to claim 1, wherein the metal nanoparticles are one or more nanoparticles selected from the group consisting of gold nanoparticles, silver nanoparticles, and fluorescent nanoparticles.
According to claim 1, The metal chalcogenide,
Any metal selected from the group consisting of molybdenum, tungsten, zinc, cadmium, titanium, vanadium, chromium and manganese; And
At least one chalcogenide selected from the group consisting of sulfur, selenium and telenium
It is composed of a target substance detection complex.
The complex for detecting a target substance according to claim 1, wherein the target substance is any substance selected from the group consisting of norovirus, microviral diarrhea virus, tuberculosis antigen, melamine and histamine.
The complex for detecting a target substance according to claim 1, wherein the peptide specifically binds to the target substance.
According to claim 1, The peptide is a complex for detecting a target substance consisting of the amino acid sequence of SEQ ID NO: 1.
A composition for detecting a target substance comprising the complex of claim 1.
A kit for detecting a target substance comprising the complex of claim 1.
Contacting an unknown sample with the complex of claim 1;
Measuring the electrochemical signal of the complex in contact with the sample; And
When the size of the electrochemical signal increases, determining that the target material is included in the sample
Target material detection method comprising a.
12. The method of claim 11, wherein the method for measuring the electrochemical signal is any one selected from the group consisting of an impedance method, a cyclic voltammetry method, and a time zone current method.
The method of claim 1, wherein the target material is norovirus.

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