KR102189992B1 - 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, and not only can detect the target material with high sensitivity and selectively, but also enables easy quantification of the target material. Therefore, it can be usefully used for detection of a target substance, especially for on-site 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-bioceptor treated with metal nanoparticles and a method for detecting a target material using the same.

Food poisoning refers to an infectious or toxin-type disease that is caused or is believed to be caused by or caused by harmful microorganisms or toxic substances related to food intake, and is generally classified into food poisoning by microorganisms and food poisoning by chemical substances according to the cause. Food poisoning caused by microorganisms is divided into bacterial and viral, and bacterial food poisoning is subdivided into toxin type and infectious type. In particular, the main harmful viruses known to cause viral food poisoning include norovirus, rotavirus, and astro virus. . When eating foods 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 1968 in a group of food poisoning patients that occurred in Norwalk Elementary School in Ohio.In the United States, more than 70% of people with water-related enteritis and more than 50% of people with food-related enteritis are caused by norovirus. Is known. In the case of Japan, when looking at food poisoning from 2001 to 2007 by causative substance, it was reported that the number of patients caused by norovirus was the largest. The British Food Standards Agency (FSA) also reported that 76% of oysters tested in the UK oyster cultivation areas (39, 800 cases) had norovirus. In addition, in the recent in-depth epidemiologic investigation of seafood-related diseases conducted by the US Centers for Disease Control and Prevention (CDC), the ratio of causative substances to 188 cases of food poisoning that occurred between 1973-2006 was bacteria (76.1%), viruses (21.3%), and parasites. (2.6%).

In Korea, according to the announcement by the Korea Food and Drug Administration, 54% of distribution oysters are contaminated with microbiological risk factors such as viruses and parasites due to various changes in the fishery environment such as climate change. With the increasing trend, it is known that food poisoning accidents caused by norovirus are the most common. When eating shellfish or fish among the food poisoning causes of norovirus, even if only 10-100 particles of norovirus are present, infection symptoms may appear, so early detection is important.

According to the conventional PCR-based (RT-PCR, multiplexed PCR) method that can quickly and accurately detect a food poisoning-causing virus derived from a very small amount of fish and shellfish, it is possible to simultaneously detect multiple pathogens by using multiple analysis devices, but it is present in the sample. There is a disadvantage in that a false positive result may be derived due to the difficulty of quantification and inhibition of the polymerase by the pollutant. In addition, since expensive fluorescent materials (Cy3, Cy5, Q-dot, etc.) must be used as a labeling factor, a lot of cost is consumed, and accessibility is limited because it cannot be applied quickly in the field. In addition, although the conventional immunoassay technique using antibodies has the advantage of being able to use conveniently, it does not have high selectivity for specific microorganisms, consumes a lot of time and cost for antibody production and detection equipment, and has a low detection limit. There is a drawback. In addition, since there is a possibility of cross-reaction, it is not suitable for the development of a new type of detection method with easy access to the site with high sensitivity and high speed. On the other hand, research is being conducted to detect microbial genes that are harmful to food by using DNA chips or DNA microarrays, but there are disadvantages that the field access is low and the analysis takes a lot of time because the array chip, measurement system, and analysis program are separately configured. . Therefore, it is necessary to develop a new type of target substance detection system by fusion with other methods.

Accordingly, the present inventor completed the present invention by carrying out a study to develop a material and method capable of selectively and highly sensitively detecting a target substance on-site.

Korean 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, 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, wherein 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 comprising.

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 substance 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 image sample; Measuring an electrochemical signal of the complex in contact with the sample; And determining that the sample contains the target material when the size of the electrochemical signal increases.

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

The present invention relates to a nano-biocomposite for detecting a target substance based on a peptide acting as a bioreceptor and a metal chalcogenide-based surface treated with metal nanoparticles, wherein the complex of the present invention reacted with the target substance is treated on an electrode. In this case, since the electric signal is displayed even at a low concentration, the presence of the target material can be accurately and conveniently confirmed by using the complex of the present invention.

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

When the metal chalcogenide is treated with metal nanoparticles, the conductivity of the metal chalcogenide is improved, and by immobilizing the peptide on the metal chalcogenide treated with the metal nanoparticles, the target material is detected with high sensitivity due to excellent conductivity. Since this possible complex can be formed, target substances that bind to peptides can be analyzed by electrochemical methods.

The detection of the target material is, for example, performed by reacting the complex of the present invention with the sample for a certain period of time, washing, coating and drying the reaction-completed sample on the electrode, and then analyzing the electrochemical impedance of the electrode treated with Nafion. I can.

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

The surface of the metal chalcogenide can be modified into a carboxyl group by the reaction of the metal chalcogenide and 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 with sensitive metal nanoparticles.

According to one 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.

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

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

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

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

According to an embodiment of the present invention, the peptide may specifically bind to the target material.

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

According to an embodiment of the present invention, the peptide may be composed of the amino acid sequence of SEQ ID NO: 1.

In the present invention, optimization conditions for preparing a metal chalcogenide-bioceptor treated with metal nanoparticles, 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 an embodiment of the present invention may be particularly useful for high sensitivity and specific detection of norovirus.

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

Another aspect of the present invention includes a metal nanoparticle, a metal chalcogenide, and a peptide, wherein 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 substance 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 contained in a sample. For example, by treating a food sample with the composition or kit of the present invention, it is possible to confirm in advance whether or not the food is infected with a norovirus, so it can be usefully used to prevent food poisoning in advance.

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

Such tools or reagents may include a suitable carrier, a labeling substance capable of generating a detectable signal, a solubilizing agent, a detergent, a buffering agent, a stabilizer, and the like. When the labeling material is an enzyme, it may further include a substrate capable of measuring the activity of the enzyme and a reaction terminator. 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, and the like; Polymers such as magnetic particles plated with metal on latex, other paper, glass, metal, agarose, and/or combinations thereof may be included.

In addition, the target material detection composition and kit may further include a carbon printed circuit (eg, Carbon-SPE) for analysis of an electrochemical signal in addition to the above-described target material detection complex, and the analysis of 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, wherein 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 image sample; Measuring an electrochemical signal of the complex in contact with the sample; And determining that the sample contains the target material when the size of the electrochemical signal increases.

The target substance detection method of the present invention is a target substance detection method based on electrochemical technology, which enables rapid and simple detection of a target substance using a bioreceptor specific to the target substance, and is easier to detect and more portable than other optical equipment. It has the advantage of being high so that it can be applied to the field, so it can be usefully used for rapid field analysis of target substances such as food poisoning bacteria.

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

In the method for detecting a target substance of the present invention, a sample to be checked whether a target substance is contained is brought into contact with a target substance detection complex according to an embodiment of the present invention, so that the target substance reacts with the complex, and the electrode is treated Thus, it may be performed by analyzing the electrochemical signal, and when the size of the electrochemical signal increases, it may be determined that the target material is contained in the sample.

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

When a peptide consisting of the amino acid sequence of SEQ ID NO: 1 is used in the target substance detection complex used in the method for detecting a target substance of the present invention, the complex binds specifically 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-bioceptor treated with metal nanoparticles and the detection method of the target material using the same, not only can the target material be highly sensitive and selectively detected, but also the target material can be easily quantified. It can be usefully used for detection of, in particular, field detection of norovirus.

1 shows a method for synthesizing (a) a metal chalcogenide-bioceptor treated with metal nanoparticles and (b) a method for electrochemical detection of norovirus using a metal chalcogenide-bioceptor treated with metal nanoparticles It is a schematic diagram for
Figure 2 is after the synthesis of metal chalcogenide 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 a metal chalcogenide sample obtained according to the reaction time while the reaction temperature is fixed at 280°C.
4 is a field emission scanning electron microscope image of a metal chalcogenide sample obtained according to the 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 results of a gas adsorption method (Brunauer-Emmett-Teller) of metal chalcogenide obtained according to the reaction temperature ((a) 270°C, (b) 280°C, (c) 290°C).
6 is a graph of a gas adsorption method (Brunauer-Emmett-Teller) of metal chalcogenide obtained according to the reaction time ((a) 12 hours, (b) 16 hours, (c) 20 hours).
7 is a graph of (a) metal chalcogenide impedance coated with metal nanoparticles and (b) impedance values according to the concentration of 3-Mercaptopropionic acid (MPA).
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 shows (a) impedance values according to the concentration of a bioreceptor used in manufacturing a metal chalcogenide-bioreceptor treated with metal nanoparticles, (b) a metal chalcogenide-bioreceptor treated with metal nanoparticles. It is the impedance value according to the binding reaction time between the metal chalcogenide treated with metal nanoparticles and the bioreceptor, (c) the impedance value according to the binding reaction time between the metal chalcogenide-bioreceptor and norovirus treated with metal nanoparticles. .
11 is a graph showing (a) an impedance spectrum according to a norovirus concentration and (b) a norovirus detection limit using the same. In addition, (c) the impedance spectrum according to the concentration of norovirus extracted from the actual oyster and (d) a graph showing the detection limit of norovirus using the same.
12 is an impedance graph obtained after reacting metal chalcogenide-bioceptors treated with metal nanoparticles with dengue virus serotypes 1, 2, 3, 4, and rotavirus, respectively.

Hereinafter, the present invention will be described in more detail through one or more examples. 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 optimization conditions for synthesis of metal chalcogenide

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

In order to prepare a metal chalcogenide-bioceptor treated with metal nanoparticles for electrochemical detection of a target material, a metal chalcogenide was synthesized according to the reaction temperature to confirm the optimum 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. It was reacted for 16 hours at different temperatures (270°C, 280°C, 290°C) using a hydrothermal synthesis method. After the reaction was over, in order to remove impurities, the supernatant was removed using a centrifuge at 4,500×g for 10 minutes (repeated 3 times). Then, the obtained WS ₂ NF (tungsten disulfide nanoflower) powder was dried at 60° C. for 10 hours.

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

As a result of the X-ray diffraction analysis 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 scanning microscopy 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 the reaction temperature of 290° C., the decomposition of the nano flower particles due to high temperature was confirmed (FIGS. 2(b) to 2(e)).

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

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

In order to prepare a metal chalcogenide-bioceptor treated with metal nanoparticles for electrochemical detection of a target material, the optimum 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. Using a hydrothermal synthesis method, the reaction was performed at 280° C. for different times (2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours). After the reaction was over, in order to remove impurities, the supernatant was removed using a centrifuge at 4,500×g for 10 minutes (repeated 3 times). Then, the obtained WS ₂ NF (tungsten disulfide nanoflower) powder was dried at 60° C. for 10 hours.

As a result of X-ray diffraction analysis, when the reaction time was 2 hours, it was confirmed that 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 scanning microscopy analysis, when the reaction time was 2, 4 or 8 hours, a rod shape was formed, whereas when the reaction time was 12, 16 or 20 hours, it was confirmed that the nano flower particle shape was formed (Fig. 4 ).

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

<1-3> Confirmation of optimization conditions for the synthesis of metal chalcogenide according to surface area analysis

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

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

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, it was confirmed that the surface area appeared the widest as 14.8 m ² /g when the reaction temperature was 280°C (Fig. 5). . On the other hand, as a result of analyzing the surface area of the metal chalcogenide according to the reaction time by fixing the reaction temperature at 280°C, it was confirmed that the surface area was the widest when the reaction time was 16 hours.

Accordingly, it was confirmed that 280° C. and 16 hours, when the surface area of the metal chalcogenide was synthesized with the largest amount, was the optimum reaction condition for the synthesis of metal chalcogenide.

Example 2. Confirmation of optimization conditions for synthesis of metal chalcogenide treated with metal nanoparticles

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

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

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

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

In addition, as a result of X diffraction analysis, a 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 of using 3-mercaptopropionic acid 60mM and chloroauric acid 2.4 mg (0.1 mg/ml), which have the lowest impedance value, are the optimum conditions for the synthesis of metal chalcogenide treated with metal nanoparticles.

Example 3. Confirmation of optimization conditions for synthesis of metal chalcogenide-bioceptors treated with metal nanoparticles and confirmation of reaction time optimization conditions with norovirus

In order to confirm the optimization conditions for the synthesis of metal chalcogenide-bioceptors treated with metal nanoparticles, and to confirm the optimization conditions for the reaction time of metal chalcogenide-bioceptors treated with norovirus and metal nanoparticles, On the surface of the metal chalcogenide treated with particles, peptides of various concentrations were treated with various reaction times.

Specifically, 100 µl of each peptide (0.1, 0.3, 0.5, 0.7, 1.0, 2.0 mg/ml) of different concentrations was added to a metal chalcogenide solution treated with 0.1 mg/ml of metal nanoparticles, and then 90 minutes Reacted for a while. In addition, in order to confirm the reaction time of the metal chalcogenide and the peptide treated with the metal nanoparticles, peptides at a concentration of 0.7 mg/ml were used for different reaction times (60, 90, 120, 180, 240, 300, 360 minutes). Reacted. 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 did not increase any more when the peptide concentration was 1 mg/ml. (Fig. 10(a)). In addition, as a result of fixing the peptide concentration at 1 mg/ml and checking the impedance graph according to the reaction time, it was confirmed that the impedance value did not increase at 240 minutes (FIG. 10(b)). Therefore, it was confirmed that the conditions of the peptide concentration of 1.0 mg and the reaction time of 240 minutes were the optimum conditions for the synthesis of metal chalcogenide-bioceptors treated with metal nanoparticles.

Meanwhile, in order to confirm the optimum reaction time of the metal chalcogenide-bioceptor treated with metal nanoparticles to the norovirus, norovirus was added to the metal chalcogenide-bioceptor solution treated with 0.1 mg/ml of metal nanoparticles. (10 ⁵ copies/ml) 100 µl was added and reacted with different reaction times (20, 30, 40, 50, 60, 90, 120 minutes), and it was confirmed that the impedance did not increase any more at 60 minutes. , It was confirmed that the optimal reaction time of the metal chalcogenide-bioceptor treated with norovirus and metal nanoparticles was 60 minutes.

Example 4. Confirmation of Electrochemical Detection of Metal Chalcogenide-Bioceptors Treated with Metal Nanoparticles According to Changes in Norovirus Concentration

To confirm whether norovirus can be detected in a concentration-dependent manner using a metal chalcogenide-bioceptor treated with metal nanoparticles prepared in Example 3, metal chalcogenide-bioceptor treated with metal nanoparticles Viruses were treated by concentration to analyze the tendency of impedance.

Specifically, norovirus (control group, 10 ¹ , 10 ² , 10 ³ , 10 ⁴ copies/ml) diluted with buffer in DMEM, respectively, in metal chalcogenide-bioceptors treated with 0.1 mg/ml of metal nanoparticles. Put 100 µl and react for 60 minutes. Thereafter, 200 µl of third distilled water was added to disperse the metal chalcogenide-bioceptor treated with the metal nanoparticles bound with the virus. A metal chalcogenide-bioceptor treated with 10 µl of metal nanoparticles and 4 µl of 0.1% Nafion were placed on the working electrode of the carbon printed circuit, and impedance was analyzed.

As a result, it was confirmed that the impedance value increased as the norovirus concentration increased (FIGS. 11(a) and 11(b)), and the detection limit was 2.37 copies/ml (R ² =0.9875). In addition, as a result of analyzing the impedance by separating norovirus from the oyster by ISO TS 1527-1 method and applying it to a metal chalcogenide-bioceptor treated with metal nanoparticles, the impedance value is dependent on the concentration of the norovirus. 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 a concentration dependent and high sensitivity using a metal chalcogenide-bioceptor treated with metal nanoparticles.

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

In order to confirm that the metal chalcogenide-bioreceptor treated with metal nanoparticles specifically binds to norovirus, norovirus, rotavirus that exhibits symptoms similar to norovirus, and dengue virus, which are other types of viruses, are used as metal nanoparticles. Impedance was analyzed by applying to a metal chalcogenide-bioceptor treated with particles.

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

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

Through these results, the high sensitivity and selectivity of the metal chalcogenide-bioceptor treated with the metal nanoparticles of the present invention against norovirus was confirmed.

So far, the present invention has been looked at around the examples. Those of ordinary skill in the art to which the present invention pertains will be able to 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 from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto 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)

Including metal nanoparticles, metal chalcogenides 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 nanoparticle,
The target material is a norovirus,
The peptide consists of the amino acid sequence of SEQ ID NO: 1,
Complex for detection of target substances.
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 performed by a reaction of the metal chalcogenide and 3-mercapto propionic acid.
The complex for detecting a target substance according to claim 1, wherein the metal nanoparticles are at least one nanoparticle selected from the group consisting of gold nanoparticles, silver nanoparticles, and fluorescent nanoparticles.
The method of claim 1, wherein the metal chalcogenide,
Any one 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
The target substance detection complex consisting of.
delete delete delete 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 an electrochemical signal of the complex in contact with the sample; And
When the size of the electrochemical signal increases, determining that the sample contains a target material
Target substance detection method comprising a.
The method of claim 11, wherein the method of measuring the electrochemical signal is any one method selected from the group consisting of an impedance method, a cyclic voltammetry method, and a time vs. current method.
The method of claim 11, wherein the target material is a norovirus.

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