KR20160006636A - Sensor System for Detecting Organophosphorous Residues By Inducing Coagulation of Gold Nanoparticles - Google Patents

Abstract

The present invention relates to a sensor system for detecting organophosphorus pesticide residues using gold nanoparticles, imidazole or green fluorescent protein (GFP) and, more specifically, to an organophosphorus pesticides detection method which induces gold nanoparticles by reacting organophosphorus pesticides with imidazole or GFP, and accordingly uses a change in an absorption spectrum to detect organophosphorus pesticides. According to the present invention, a pesticide residue detecting system has a fast detection speed and an excellent optical change of a sample according to a presence of organophosphorus pesticides, and a range of a detection limit concentration is broad, so the invention can be useful for a pesticide residues biosensor for site analysis.

Description

Technical Field [0001] The present invention relates to a sensor system for detecting pesticide residues in organic poultry by inducing coagulation of gold nanoparticles,

The present invention relates to a residual organophosphorus pesticide detection sensor system through induction of aggregation of gold nanoparticles, and more particularly, to a method of detecting gold (Ag) phosphorus pesticide by a reaction between an organic phosphorus pesticide and a green fluorescent protein (GFP) And a method for detecting an organophosphorus pesticide using the change of the absorption spectrum.

Organophosphorus pesticides are the most widely used pesticides, and they are commercially available for the purpose of insecticides for pest control. Compared with the organic chlorine system, it has less persistence and less risk of residuals and stronger insecticide. However, there are many agents which are toxic to human and livestock, and it is weak in persistence and requires a relatively large amount of use. Examples of organophosphorus pesticides include diazinon, iprobenfos, edifenphos, dichlorovos, parathion, malathion or EPN ( o- ethyl- o - ( p- nitrophenyl) -phenyl phosphothioate) have been widely used.

Residual pesticides are low-molecular toxic chemicals, which are concentrated and toxic in the body in small amounts, adversely affecting human health and ecosystems. Therefore, continuous analysis and environmental monitoring to monitor the presence of pesticides are indispensable and introduction of a system for this is required.

However, existing pesticide analysis requires high-priced equipments and professional manpower. Since it is mostly time-consuming and expensive analytical method, it is simple and convenient in the field, and bio- Sensor system development is required.

Conventional organophosphorus pesticide detection systems using AChE (acetylcholinesterase) or OPH (organophosphate hydrolase) require preparation of expensive enzymes, complicated experimental procedures, and a large number of sample solutions, and require reaction times of enzymes and substrates There are disadvantages.

For example, a disinfectant detection biosensor using liposomes (Vicky Vamvakaki et al ., Biosens . Bioelectron ., 22 (12): 2848-2853 , 2007) reported that AChE hydrolyzes nanosized liposome complexes And the fluorescence intensity is decreased as the degree of hydrolysis is decreased when the enzyme activity is inhibited by the pesticide, so that the principle of measuring the change of fluorescence is used. However, the process of forming a liposome complex is complicated and many kinds of reagents such as buffer solution, liquid nitrogen, 5,5'-dithiobis-nitro-benzoic acid (DTNB) There are disadvantages.

In addition, an organophosphate hydrolase (OPH) and an organophosphate compound detection optical biosensor using gold nanoparticles (AL Simonian et al ., Anal. Chim. Acta., 534 (1): 69-77, 2005) will be. Hydroxy-9H- (1, 3-dichloro-9,9-dimethylacetidin-2-one (7-hydroxy-9H- (1,3-dichloro-9,9-dimethylacridin-2-one, DDAO phosphate), the DDAO phosphate binds to OPH, and the gold nanoparticles bound to OPH exhibit fluorescence. , The presence of organophosphorus pesticides causes OPH to be separated from DDAO phosphate while being bound to pesticides, and at the same time, the distance from the gold nanoparticles becomes farther away to decrease the intensity of fluorescence, It has the disadvantage that it needs reagent and time to bind OPH to gold nanoparticles and shows low detection level.

On the other hand, gold nanoparticles are the most widely used nanomaterials in the field of biosensing, and have many unique properties that are not seen in common organic materials, and many sensor systems have been developed using them. Gold nanoparticles have an extinction coefficient of about 10 ³ -10 ⁵ as compared with general organic dyes. They have a unique UV / vis spectrum. They have excellent strength and sensitivity. It occurs rapidly in blue, and makes it possible to measure a small color change, which can not be confirmed by the eye, through the measurement of the spectrum.

Accordingly, the present inventors have made intensive efforts to solve the above problems. As a result, they have found that, instead of using an enzyme such as AChE or OPH, the organophosphorus pesticide is treated with imidazole, an organic compound, or GFP (Green Fluorescent Protein) The agglomeration size of gold nanoparticles is increased. Therefore, it is possible to detect pesticides up to the unit of ppb because the change in pesticide concentration is small even at a small concentration of pesticide.

In addition, due to the nature of gold nanoparticles, which exhibit minute optical changes according to the distance between particles, we have tried to find out a small amount of organophosphorus pesticides more quickly and easily. As a result, we have found that a mixture of organophosphorus pesticides and gold nanoparticles When the imidazole solution is added to the solution, the absorption spectrum of the gold nanoparticles changes sharply as the gold nanoparticles aggregate by the added imidazole, so that the presence of the organic phosphorus pesticide can be visually recognized easily Thereby completing the present invention.

It is an object of the present invention to provide a method for detecting organophosphorus pesticides which can detect a small concentration of organophosphorus pesticide more quickly and easily and has a wide range of detection limit concentration and can perform on-site analysis.

Another object of the present invention is to provide a method for quantifying the concentration of an agricultural chemical based on the absorbance measured using the method.

In order to achieve the above object, the present invention provides a method for producing a pesticidal composition, comprising the steps of: (a) adding at least one solution selected from the group consisting of imidazole solution, histidine solution, pyrazole solution, histamine solution and GFP solution to a mixed solution of a pesticide contaminated sample and gold nanoparticles To induce aggregation of the gold nanoparticles; And (b) measuring the absorbance of the gold nanoparticles by agglutination at 600 to 700 nm.

(A) adding at least one solution selected from the group consisting of an imidazole solution, a histidine solution, a pyrazole solution, a histamine solution, and a GFP solution to a mixed solution of a pesticide contaminated sample and gold nanoparticles to form gold nanoparticles To induce flocculation of < / RTI > (b) measuring the absorbance of the gold nanoparticles by agglomeration at 600 to 700 nm; And (c) quantifying the concentration of the pesticide based on the measured absorbance.

The present invention also relates to a gold nanoparticle solution; And at least one solution selected from the group consisting of imidazole solution, histidine solution, pyrazole solution, histamine solution and GFP solution.

The residual pesticide detection system according to the present invention is advantageous as a residual pesticide biosensor for on-site analysis because the optical change of the reagent is remarkably excellent according to the presence of the organophosphorus pesticide, the detection speed is high, and the range of the detection limit concentration is wide.

Fig. 1 shows the change of the absorption spectrum when the pesticide and imidazole are sequentially added to the gold nanoparticle solution.
FIG. 2 is a graph showing changes in absorption spectrum by specific components when gold nanoparticles and / or imidazole are added to an organophosphorus pesticide. FIG.
FIG. 3 shows the results obtained by adding gold nanoparticles and imidazole after varying the concentration of each of diazinon, edifenphos, iprovenphos, malathion, tebuconazole, acetamiprid, penitrothion, Of the absorbance spectrum.
FIG. 4 is a standard curve graph showing the absorbance measured at 670 nm when gold nanoparticles and imidazole were added after varying the concentration of each of diazinon, ediphene, and ivovenose.
FIG. 5 is a graph showing the absorbance measured at 670 nm when gold nanoparticles and imidazole were added to the non-pesticide compound and three organophosphorus pesticides, respectively.
6 is a graph showing changes in zeta potential of gold nanoparticles when gold nanoparticles and / or imidazole are added to an organophosphorus pesticide.
7 is a graph showing changes in particle size due to agglomeration of gold nanoparticles when gold nanoparticles and / or imidazole are added to an organophosphorus pesticide.
8 is a graph showing absorbance change at 670 nm measured by pH change of organophosphorus pesticide, gold nanoparticle, and imidazole mixture.
9 shows the change of the absorption spectrum when the pesticide and EGFP are sequentially added to the gold nanoparticle solution.
10 is a graph showing the effect of the organophosphorus pesticide on the activity of EGFP.
11 is a graph showing changes in absorption spectrum by which elements when gold nanoparticles and / or EGFP are added to an organophosphorus pesticide.
Fig. 12 is a graph showing the absorption spectra of gold nanoparticles and EGFP after different concentrations of diazinon, ediphene, and ivovenose, respectively.
13 is a standard curve graph showing the absorbance measured at 670 nm when gold nanoparticles and EGFP were added after varying the concentration of each of diazinon, ediphene, and ivovenose.
14 is a graph showing the absorbance measured at 670 nm when gold nanoparticles and EGFP were added to the non-pesticide compound and three organophosphorus pesticides, respectively.
FIG. 15 is a graph showing absorbance changes at 670 nm of organic phosphorus pesticides, gold nanoparticles, and EGFP mixtures with different concentrations over time.
16 is a graph showing changes in particle size due to agglomeration of gold nanoparticles when gold nanoparticles and / or EGFP are added to an organophosphorus pesticide.
FIG. 17 is a graph showing the change in absorbance of the organophosphorus pesticide, gold nanoparticles, and EGFP mixture according to pH change at 670 nm.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, gold nanoparticle solution, imidazole solution or GFP solution was added to an organic phosphorus pesticide, and an experiment was performed to measure the change of the absorption spectrum with an optical measuring instrument. As a result, it was confirmed that the organophosphorus pesticide, imidazole or GFP induced aggregation of gold nanoparticles, and a new spectrum showing the strongest peak at 600 to 700 nm was generated. As a result, It was confirmed that pesticides can be detected easily and quickly.

Accordingly, in one aspect, the present invention provides a method for producing a pesticidal composition, comprising: (a) adding at least one solution selected from the group consisting of an imidazole solution, a histidine solution, a pyrazole solution, a histamine solution and a GFP solution to a mixed solution of a pesticide contaminated sample and gold nanoparticles Thereby inducing aggregation of gold nanoparticles; And (b) measuring the absorbance of the gold nanoparticles by agglutination at 600 to 700 nm.

The pesticide contaminated sample of the present invention means a sample obtained from a crop or the like suspected of contamination by residual pesticide.

In one embodiment of the present invention, gold nanoparticles can be synthesized by the citric acid reduction method of gold (III) tetrachloride. The gold nanoparticles can be prepared in liquid phase by a method of reducing the metal salt under a high temperature condition without using a template and a method of synthesizing at room temperature using a template.

A method of not using a template is to dissolve a metal salt in a solvent, and then a strong reducing agent such as citrate is added under strong stirring at a boiling point or higher to obtain metal nanoparticles. Another method is to use surface stabilizers or templates to overcome thermodynamically unstable conditions in the formation of gold nanoparticles. In some cases, a soft template such as a hard template and a surfactant such as mesoporous alumina, silica or carbon nanotube (CNT) is used. Methods for preparing nanoparticles using a template include seed-mediated crystal growth methods using a seed and a growth solution, and a polyol process.

Gold nanoparticles are the most widely used nanomaterials in the field of biosensing. Gold nanoparticles have a unique UV / vis spectrum with an extinction coefficient as high as 10 ³ -10 ⁵ as compared with general organic dyes. Under visible light, gold nanoparticles have a unique color and cause specific aggregation of gold nanoparticles to aggregate themselves, resulting in changes in the permittivity of gold nanoparticle surfaces, As the local surface plasmon (LSP) absorption conditions change, the color of the gold nanoparticle solution itself changes under visible light. In other words, as the distance of the nanoparticles becomes closer, the absorption wavelength of the light is red-shifted to become blue gradually as the reaction proceeds from the original red color.

In the present invention, a colorimetric sensor capable of measuring a target sample without any special analysis equipment using the color change of the solution itself when there is a specific reaction between organic phosphorus pesticide, imidazole and gold nanoparticles .

Gold nanoparticles are characterized by their strongest peak at 519 nm when measured by UV / vis spectroscopy. However, the gold nanoparticles aggregated as a result of the reaction of the imidazole to the pesticide, and the spectrum was changed to have the strongest peak at a wavelength of 600 to 700 nm, preferably 670 nm, using a UV / vis spectrophotometer. Respectively.

The GFP used in the present invention is a green fluorescent protein, and preferably EGFP (Enhanced green fluorescent protein) may be used as in the embodiment of the present invention. EGFP is a type of GFP fluorescent protein, which means an enhanced green fluorescent protein in which one or two amino acids are replaced by another in GFP. It has the characteristic of showing fluorescence stronger than general GFP.

In one embodiment of the present invention, EGFP expression was induced and purified from Escherichia coli transformed with recombinant DNA of EGFP gene to obtain EGFP. Fluorescent proteins that react with organophosphorus pesticides and gold nanoparticles are not necessarily limited to GFP or EGFP but may be red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP) , Enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), enhanced red fluorescent protein (ERFP), or enhanced blue fluorescent protein .

(A) adding at least one solution selected from the group consisting of an imidazole solution, a histidine solution, a pyrazole solution, a histamine solution, and a GFP solution to a mixed solution of a pesticide contaminated sample and gold nanoparticles, Inducing aggregation of nanoparticles; (b) measuring the absorbance of the gold nanoparticles by agglomeration at 600 to 700 nm; And (c) quantifying the concentration of the pesticide based on the measured absorbance.

In one embodiment of the present invention, when the gold nanoparticle solution, the imidazole solution or the EFGP solution were mixed with the organophosphorus pesticide, there was a large difference in the absorbance depending on the concentration of the pesticide contained therein. At a wavelength of 670 nm The difference is most obvious. Therefore, as shown in Figs. 4 and 13, the standard curve according to the concentration of the pesticide was shown using the absorbance at the wavelength of 670 nm, and the 3 sigma method ) Was calculated to be 27.9 ppb or 17 ppb, respectively, and plateau was observed at a concentration near 3 ppm, so that the detectable range for the organophosphorus pesticide was found to be 0.01 to 3 ppm .

In another embodiment of the present invention, when the results of the quantitative determination of the organic phosphorus pesticide using the present invention and the results of quantitative determination of the organic phosphorus pesticide using HPLC (High-performance liquid chromatography) are compared with the gold nanoparticles and imidazole or GFP It was confirmed that the method of quantifying pesticides of the present invention is as accurate as HPLC and can replace HPLC quantitation as a faster and more efficient method than HPLC.

Use the equation A = εLc when calculating the specific concentration of pesticide using absorbance. Where A is the absorbance and is defined as A = -log (I / I ₀ ). I ₀ is the intensity of light before passing through the specimen, and I is the intensity of the light after passing through the specimen. L is the distance that light passes through the cuvette containing the sample, ε is the molar absorbance of the material, and c is the molar concentration of the sample. When L and ε are known, the solute concentration can be obtained by measuring the absorbance (A) of the solution.

In the present invention, it is possible to additionally include a pretreatment step of diluting the contaminated sample of agricultural chemicals with methanol, ethanol, or an aqueous solution thereof.

In one embodiment of the present invention, the organophosphorus pesticide was pretreated with 10% methanol before it was reacted with gold nanoparticles and imidazole solution or EFGP solution. This is to extract organic phosphorus pesticides from polluted samples of pesticide contamination. Instead of methanol, ethanol, which is a solvent having similar properties, may be used, or an aqueous solution of methanol or ethanol may be used.

In the present invention, the diameter of the gold nanoparticles may be 10 to 50 nm. When gold nanoparticles are 10 ~ 50nm in size, they show red color. When gold particles are aggregated due to organophosphorus pesticides and imidazole, embolization phenomenon can be best observed.

In the present invention, the organophosphorus pesticide may be diazinon, edifenphos or iprobenfos. Organophosphorous compounds are compounds in which an alkyl or aryl group is bonded to a phosphorus atom (P) and include diclovos, parathion, malathion, tebuconazole, acetamiprid, EPN , Fenitrothion, or fenthion, can be applied to the detection method and the quantification method of the present invention.

In the present invention, the concentration of the gold nanoparticle solution may be 8 to 12 nM, the concentration of the imidazole solution may be 0.1 to 0.4 mM, the detection concentration of the organophosphorus pesticide may be 0.01 to 3 ppm, The optimum pH of pesticide detection is 7.4 ~ 8.9, more preferably 7.4 ~ 8.4.

In the embodiment of the present invention, when the concentration of the gold nanoparticle solution was adjusted to 10 nM and the concentration of the imidazole solution was set to 0.3 mM, the reaction with the organophosphorus pesticide occurred most effectively when the volume ratio was 1: 1. In addition, when the pH of the mixed solution was 8.4, the difference in absorbance depending on the presence or absence of the organic phosphorus pesticide was large and it was found to be the most suitable pH for the detection of the organic phosphorus pesticide.

In another aspect, the present invention provides a gold nanoparticle solution; And at least one solution selected from the group consisting of an imidazole solution, a histidine solution, a pyrazole solution, a histamine solution and a GFP solution.

In the present invention, the concentration of the gold nanoparticle solution is 8 to 12 nM, and the concentration of the at least one solution selected from the group consisting of imidazole solution, histidine solution, pyrazole solution, histamine solution and GFP solution is 0.1 to 0.4 mM Lt; / RTI >

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Examples using gold nanoparticles and imidazole

Preparation of samples and equipment

Trisodium citrate dehydrate used in the preparation of gold nanoparticles was purchased from BIO BASIC (CANADA INC.) And gold (III) chloride hydrate (HAuCl ₄ ) was purchased from Sigma- Gt; Aldrich < / RTI > (USA). Gold nanoparticles were synthesized by the citric acid reduction method of gold (III) tetrachloride, and gold nanoparticles with a diameter of 13 nm were used in the following experiments.

Organophosphorus pesticides such as diazinon, edifenphos, iprobenfos and phosphate buffered saline (tablet) were purchased from Sigma-Aldrich (USA).

Methanol was purchased from Merck chemicals, imidazole from BIO BASIC (CANADA INC.), And Milli-Q grade water (18.2 MΩ cm, Millipore) was used in all experiments.

Absorbance was measured with a Synergy H1 Hybrid Reader purchased from Biotek (Korea).

Example  1: Measurement of the absorption spectrum change by addition of gold nanoparticles and / or imidazole to organophosphorus pesticides

Diazinon, an organic phosphorus pesticide, was dissolved in 10 mL of 10% methanol to adjust the concentration to 1000 ppm. This was diluted with 10% methanol to prepare a sample diluted to a concentration of 1 ppm.

When gold nanoparticles and imidazole were added to organophosphorus pesticides, abrupt changes in absorption spectrum (peak shift) occurred, and an experiment was conducted to determine which factors caused these changes.

1-1. When diazinon and gold nanoparticle solution were reacted

First, a diluted solution of diazinon, an organic phosphorus pesticide, at a concentration of 1 ppm and a 10 nM gold nanoparticle solution were mixed at a volume ratio of 2: 1 and reacted at room temperature.

As a result, as shown in FIG. 2, the intrinsic absorption spectrum of the gold nanoparticles shows a peak at about 520 nm. Even when the organic phosphorus pesticide, diazinon, was reacted with the gold nanoparticles, the peak position of the gold nanoparticles Appeared at the same 520 nm. Therefore, it was confirmed that diazinon alone does not directly affect the absorption spectrum of gold nanoparticles.

1-2. When an imidazole solution is reacted with a gold nanoparticle solution

Except for the organophosphorus pesticide, only the 0.3 mM imidazole solution and 10 nM gold nanoparticle solution were mixed at a volume ratio of 1: 1, and the absorbance was measured. As a result, no specific peak migration was found as shown in FIG. 2, Can not affect the absorption spectra of gold nanoparticles alone.

1-3. Diazinone ( diazinon ), Imidazole solution, and gold nanoparticle solution were mixed and reacted

When a solution in which a solution of diazinon, an organic phosphorus pesticide, diluted to a concentration of 1 ppm, a 0.3 mM imidazole solution, and a 10 nM gold nanoparticle solution were mixed at a volume ratio of 2: 1: 1 and reacted at room temperature and the absorption spectrum was measured , A new peak was generated at 670 nm as shown in FIG. 2, from which imidazole induces agglomeration of gold nanoparticles in response to pesticides and influences the absorption spectrum of gold nanoparticles to detect organophosphorus pesticides .

For edifenphos, iprobenfos, malathion, tebuconazole, acetamiprid, fenitrothion, and fenthion as well, The same experiment was performed and the same type of results were obtained.

Example 2: Measurement of absorbance by concentration of each organophosphorus pesticide

Diazinon, edifenphos, iprobenfos, malathion, tebuconazole, acetamiprid, fenitrothion, and pention, fenthion Each of the organophosphorus pesticides was prepared in the same manner as in Example 1, and pesticide samples having various concentrations ranging from 0.01 to 10.0 ppm were prepared. Subsequently, organic phosphorus pesticide samples at respective concentrations, 10 nM gold nanoparticle solution and 0.3 mM imidazole solution were mixed at a volume ratio of 2: 1: 1, and the change of the absorption spectrum was measured in the range of 400 to 700 nm with an optical measuring instrument (Fig. 3), thereby obtaining a standard curve (Fig. 4).

As a result, as shown in FIG. 3, the peak position in the absorption spectrum was 670 nm, which was the same regardless of the concentration of the pesticide. However, the absorbance showed a large difference depending on the concentration of the pesticide, The most obvious. Therefore, as shown in FIG. 4, the standard curve according to the concentration of the pesticide was shown using the absorbance at the wavelength of 670 nm, and the 3 sigma method (a method of determining the limit by three times the standard deviation at the top and bottom of the average value) The minimum detection limit used was calculated to be 27.9 ppm, and plateau was observed at a concentration near 3 ppm, so that the detectable range for the organophosphorus pesticide was found to be 0.01 to 3 ppm.

Comparative Example: Detection of Organophosphorus Pesticide Detection Specificity Using Non-Pesticide Compound Control

In order to confirm that the pesticide detection sensor system containing gold nanoparticles and imidazole shows a specific reaction only to organic phosphorous pesticides, benzene, which is a non-agrochemical compound composed of each functional group contained in the formula of organic phosphorous pesticide, A solution prepared by diluting phenol, toluene, xylene, dichlorobenzene, and phosphoric acid in 10% methanol to a concentration of 1 ppm, and a solution of 10 nM gold nanoparticles, 0.3 mM imidazole solution was mixed at a volume ratio of 2: 1: 1.

As a result of analyzing the absorbance at 670 nm, as shown in FIG. 5, the absorbance of benzene, phenol, toluene, xylene, dichlorobenzene, phosphoric acid, etc. which are non-agricultural chemical compounds was remarkably low as 0.33 or less, (Diazinon) was found to be higher than 0.41, and it was confirmed that the organophosphorus pesticide had a specific reaction.

Example  3: Zeta potential changes of organophosphorus pesticides, gold nanoparticles, and imidazole mixtures

When the organophosphorus pesticide and the imidazole solution were sequentially added to the gold nanoparticle solution (concentration and mixing ratio were the same as in Example 1-3), the zeta potential of the gold nanoparticles according to each addition was measured by a zeta potential particle analyzer Respectively. The zeta potential of the gold nanoparticles was -35 mV and there was little change to -36 mV when the organophosphorus pesticide was added. However, when imidazole was added to each of the above samples, both were greatly increased to -12 mV, It was confirmed again after Example 1-3 that the aggregation of the gold nanoparticles was greatly influenced (Fig. 6).

Example 4: Change in particle size of gold nanoparticles

When the organophosphorus pesticide and imidazole solution were sequentially added to the gold nanoparticle solution (concentration and mixing ratio were the same as in Example 1-3), the size of the gold nanoparticles according to each addition was measured with a zeta potential particle analyzer Respectively. However, when imidazole was added to each of the above samples, it was greatly increased to 202 nm and 235 nm, respectively, and it was found that imidazole was increased to gold nanoparticles It was confirmed again after Example 1-3 that the agglomeration of the particles was greatly influenced (Fig. 7).

Example  5: Changes in absorbance of organophosphorus pesticides, gold nanoparticles and imidazole mixtures according to pH

In order to examine whether the gold nanoparticles react with the organophosphorus pesticide and the imidazole, the change of the absorbance at 670 nm according to the pH change was analyzed (concentration and mixing ratio were the same as in Example 1-3) ).

As a result of analyzing the absorbance of the pH adjusted from 3.4 to 10.4, it was confirmed that the difference in the absorbance value between the organic phosphorous pesticide 0 ppm and 1 ppm 670 nm was increased as the pH was higher, as shown in FIG. The highest difference was observed at pH 8.4.

Example  6: Deionized water ( deionized  water and tap water, and the results of quantitative determination of organic phosphorus pesticides using HPLC

The gold nanoparticles were reacted with organophosphorus pesticides and imidazoles and the absorbance was measured to determine the concentrations of the pesticides in deionized water and tap water. The results were analyzed by high-performance liquid chromatography (HPLC) (The concentrations and mixing ratios of the gold nanoparticles and imidazole solutions are the same as in Example 1-3). The results are shown in Table 1 below.

The results of quantitative determination of pesticides applied to deionized water and tap water were compared with those of organic pesticide using HPLC.

Samples One 2 3 Diazinon conc. Added (ppm) 0.080 0.170 0.300 In deionized water
mean 占 SD (ppm) ^* 0.080 0.024 0.173 + - 0.056 0.295 + 0.042 Recovery (%) 100.4 101.6 98.3 In tap water
mean 占 SD (ppm) ^* 0.079 0.012 0.146 + 0.012 0.340 ± 0.001 Recovery (%) 98.5 86.3 113.5 HPLC
mean 占 SD (ppm) ^* 0.082 ± 0.002 0.149 + 0.004 0.263 + 0.064 Recovery (%) 96.2 87.6 87.7

* Mean value for 3 results; SD: standard deviation

As a result, as shown in Table 1, the concentration of the organophosphorus pesticide was diluted to 0.080 ppm, 0.170 ppm and 0.300 ppm, and quantitatively analyzed using the method of the present invention and HPLC, respectively. As a result, . The average recovery rate when using the quantitative method of the present invention was 100.1% for deionized water and 99.4% for tap water, and the error rate was smaller than 90.5% of the HPLC determination method. As a result, it was confirmed that the pesticide quantitation method of the present invention is as accurate as HPLC, and can replace HPLC quantitation as a faster and more efficient method than HPLC.

Examples using gold nanoparticles and GFP

Preparation of samples and equipment

Trisodium citrate dehydrate used in the preparation of gold nanoparticles was purchased from BIO BASIC (CANADA INC.) And gold (III) chloride hydrate (HAuCl ₄ ) was purchased from Sigma- Gt; Aldrich < / RTI > (USA). Gold nanoparticles were synthesized by the citric acid reduction method of tetrachloromethane (?) Acid, and gold nanoparticles having a diameter of 13 nm were used in the following experiments.

Organophosphorus pesticides diazinon, edifenphos, iprobenfos and phosphate buffered saline (tablet) were purchased from Sigma-Aldrich (USA).

Methanol was purchased from Merck chemicals and Milli-Q grade water (18.2 MΩ cm, Millipore) was used in all experiments.

EGFP was used to induce expression from E. coli transformed with recombinant DNA and purified.

Fluorescence and absorbance were measured with a Synergy H1 Hybrid Reader from Biotek (Korea).

Example 7: Determination of the effect of organophosphorus pesticides on the activity of EGFP

First, 10 μl of the pesticide was dissolved in 10 mL of 10% methanol to a concentration of 1000 ppm with respect to diazinon, an organic phosphorus pesticide. This was diluted with 10% methanol and samples were diluted to various concentrations of 10 ppb, 100 ppb, 1 ppm and 10 ppm, respectively.

To confirm whether the fluorescence intensity of EGFP was affected by the organophosphorus pesticide, EGFP was added to each of the pesticide samples having different concentrations prepared above, and after 3 minutes at room temperature, 10% methanol was used as a control, The fluorescence intensity of the EGFP protein was measured at excitation 480 nm and emission 510 nm wavelength (FIG. 10).

As a result, as shown in FIG. 10, it was confirmed that the intensity of fluorescence emitted by EGFP varies depending on the concentration of the pesticide. The higher the concentration of pesticide, the stronger the fluorescence intensity, and the stronger the intensity of fluorescence at the concentration of 10ppm.

The same experiment was performed on edifenphos and iprobenfos, and the same results were obtained.

Example  8: gold nanoparticles in organophosphorus pesticides and / or EGFP  Upon addition Absorbance  Measurement of spectral change

When gold nanoparticles and EGFP were added to organophosphorus pesticides, a sudden change in absorption spectrum (peak shift) occurred.

8-1. When diazinon and gold nanoparticle solution were reacted

First, a diluted solution of diazinon, an organic phosphorus pesticide, at a concentration of 1 ppm and a 10 nM gold nanoparticle solution were mixed at a volume ratio of 2: 1 and reacted at room temperature.

As a result, as shown in FIG. 11, the intrinsic absorption spectrum of gold nanoparticles shows a peak at about 520 nm. Even when the organic phosphorus pesticide, diazinon, and gold nanoparticles were reacted, the peak positions of gold nanoparticles were found to be the same at 520 nm . Therefore, it was confirmed that diazinon alone does not directly affect the absorption spectrum of gold nanoparticles.

8-2. When the EGFP solution is reacted with the gold nanoparticle solution

As a result of measuring the absorbance by mixing 5 μg / mL EGFP solution and 10 nM gold nanoparticle solution only at a volume ratio of 1: 1 except for the organic phosphorus pesticide, no specific peak shift was found as shown in FIG. 11, and EGFP Can not affect the absorption spectra of gold nanoparticles alone.

8-3. Diazinone ( diazinon ), EGFP  Solution, and gold nanoparticle solution were mixed and reacted

When 5 μg / mL EGFP solution and 10 nM gold nanoparticle solution were mixed at a volume ratio of 2: 1: 1 in a diluted solution of 1 ppm of diazinon, an organic phosphorus pesticide, and reacted at room temperature, the absorption spectrum was measured , A new peak was generated at 670 nm as shown in FIG. 11, from which EGFP induces aggregation of gold nanoparticles in response to pesticides and influences the absorption spectrum of gold nanoparticles to detect organophosphorus pesticides I could confirm.

The same experiment was performed on edifenphos and iprobenfos and the same results were obtained (Fig. 12).

Example 9: Measurement of absorbance by concentration of each organophosphorus pesticide

Pesticide samples having various concentrations ranging from 0.01 to 10.0 ppm were prepared for each of the organophosphorus pesticides of diazinon, edifenphos and iprobenfos in the same manner as in Example 7 . Then, as in Example 8, organic phosphorus pesticide samples at respective concentrations, 10 nM gold nanoparticle solution and 5 μg / mL EGFP solution were mixed at a volume ratio of 2: 1: 1, and an optical measuring instrument was used to measure the absorption spectrum The change was measured in the range of 400 to 700 nm (FIG. 12), and a standard curve was obtained (FIG. 13).

As a result, as shown in FIG. 12, the peak position in the absorption spectrum was 670 nm, which was the same regardless of the concentration of the pesticide. However, there was a large difference in the absorbance depending on the concentration of the pesticide, The most obvious. Therefore, as shown in FIG. 13, the standard curve according to the concentration of the pesticide was shown using the absorbance at the wavelength of 670 nm, and the 3 sigma method (a method of determining the limit by three times the standard deviation at the top and bottom of the average value) The minimum detection limit was calculated to be 17 ppb, and plateau was observed at a concentration near 3 ppm. Therefore, it was confirmed that the detection range for the organophosphorus pesticide was 0.01 to 3 ppm.

Comparative Example: Detection of Organophosphorus Pesticide Detection Specificity Using Non-Pesticide Compound Control

In order to confirm whether the pesticide detection and colorimetric sensor system including gold nanoparticles and EGFP show a specific reaction only to the organic phosphorus pesticide, the non-pesticidal compounds benzene, A solution prepared by diluting phenol, toluene, xylene, dichlorobenzene, and phosphoric acid in 10% methanol to a concentration of 1 ppm, and a solution of 10 nM gold nanoparticles, 5 / / mL of EGFP solution was mixed at a volume ratio of 2: 1: 1.

As a result of analyzing the absorbance at 670 nm, the absorbance of benzene, phenol, toluene, xylene, dichlorobenzene, phosphoric acid, etc., which are non-pesticide compounds, was remarkably low as 0.35 or less as shown in FIG. 14 and Diazinon ), Edifenphos, and Iprobenfos were found to be higher than 0.41, indicating that they were peculiar to organophosphorous pesticides.

Example  10: organophosphorus pesticides, gold nanoparticles, and EGFP  Change in absorbance of the mixture with time

Experiments were carried out to determine the most suitable reaction time by measuring the change of the absorbance value with time when the concentration of the organic phosphorus pesticide was different at 670 nm, where the change of absorbance was largest. Absorbance values were measured every minute after adding all of the organophosphorus pesticides (diazinon, 0, 0.01, 0.1, 1.0 ppm concentration), gold nanoparticles and EGFP.

As a result, as shown in FIG. 15, it was confirmed that the rate of change of absorbance was high at the beginning of the reaction, and the reaction rate was rapidly increased. Accordingly, when the reaction time was shortened, the difference of the absorbance value according to the concentration of the pesticide was remarkable Respectively. The reaction time can be measured within a very short time, so that it can be detected within a short time, and it can be confirmed that the reaction continues continuously for a long time.

Example 11: Change in particle size of gold nanoparticles

When the organophosphorus pesticide and the EGFP solution were sequentially added to the gold nanoparticle solution (concentration and mixing ratio were the same as in Example 8-3), the size of gold nanoparticles according to each addition was measured by a nanoparticle analyzer. The size of the gold nanoparticles was 11.7 nm. When the organophosphorus pesticide was added, the size of the nanoparticles was not changed to 12 nm. However, when EGFP was added, the amount of gold nanoparticles increased to 520.8 nm. It was confirmed once again after Example 8-3 (Fig. 16).

Example  12: Organophosphorus pesticides, gold nanoparticles, and EGFP  Absorbance change according to pH of mixture

In order to determine whether the gold nanoparticles are reacted with the organic phosphorus pesticide and EGFP, the change in absorbance at 670 nm according to the pH change was analyzed (the concentration and mixing ratio were the same as in Example 8-3) .

As shown in FIG. 17, when the pH of the organic phosphorus pesticide was increased, the absorbance at 670 nm tended to increase with increasing pH, and the organic phosphorus pesticide Diazinon), the difference in the absorbance values was increased. The highest difference was observed at pH 8.4.

Example  13: Results of quantitative determination of organic phosphorus based pesticides using the present invention HPLC  Comparison of Pesticide Determination Results by Organophosphorus Analysis

The gold nanoparticles were reacted with the organophosphorus pesticide and EGFP, and the absorbance was measured to determine the concentration of the pesticide. The result was compared with the quantitative results obtained by HPLC (High-performance liquid chromatography) (Concentration and mixing ratio of the gold nanoparticles and the EGFP solution were the same as in Example 8-3).

The concentrations of the organic phosphorus pesticides were diluted to 0.085, 0.170, and 0.300 ppm, respectively, and quantitatively analyzed using the method of the present invention and HPLC. As a result, as shown in Table 2, similar results .

Samples One 2 3 Diazinon conc. Added (ppm) 0.085 0.170 0.340 the present method
mean 占 SD (ppm) ^* 0.124 + 0.015 0.156 + 0.010 0.248 + 0.012 Recovery (%) 145.9 91.8 82.7 HPLC
mean 占 SD (ppm) ^* 0.082 ± 0.002 0.149 ± 0.002 0.263 + 0.014 Recovery (%) 96.2 87.6 87.7 Reliability (%) 151.6 104.7 94.3

* Mean value for 3 results; SD: standard deviation

When using the quantitative method of the present invention, the average recovery was 106.8%, which is less than the error rate of 90.5% of the HPLC quantitation method. As a result, it was confirmed that the pesticide quantitation method of the present invention is as accurate as HPLC, and can replace HPLC quantitation as a faster and more efficient method than HPLC.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

A method for detecting an organophosphorus pesticide comprising the steps of:
(a) inducing agglomeration of gold nanoparticles by adding at least one solution selected from the group consisting of imidazole solution, histidine solution, pyrazol solution and histamine solution to a mixed solution of pesticide contaminated sample and gold nanoparticles; And
(b) measuring the absorbance of the gold nanoparticles by agglutination at 600 to 700 nm. A method for quantifying an organophosphorus pesticide comprising the steps of:
(a) inducing agglomeration of gold nanoparticles by adding at least one solution selected from the group consisting of imidazole solution, histidine solution, pyrazol solution and histamine solution to a mixed solution of pesticide contaminated sample and gold nanoparticles;
(b) measuring the absorbance of the gold nanoparticles by agglomeration at 600 to 700 nm; And
(c) quantifying the concentration of the pesticide based on the measured absorbance. 3. The method according to claim 1 or 2, further comprising a pre-treatment step of diluting the contaminated sample with methanol, ethanol or an aqueous solution thereof. The method according to claim 1 or 2, wherein the diameter of the gold nanoparticles is 10 to 50 nm. 3. The method according to claim 1 or 2, wherein the organophosphorus pesticide is selected from the group consisting of diazinon, edifenphos, iprobenfos, malathion, tebuconazole, parathion, acetamiprid, fenitrothion, or fenthion. < Desc / Clms Page number 13 > The method according to claim 1 or 2, wherein the concentration of the gold nanoparticle solution is 8 to 12 nM, and the concentration of the at least one solution selected from the group consisting of imidazole solution, histidine solution, pyrazole solution, 0.4 mM. ≪ / RTI > The method according to claim 1 or 2, wherein the detection concentration of the organophosphorus pesticide is 0.01 to 3 ppm. 3. The method according to claim 1 or 2, wherein the optimum pH of the organophosphorus pesticide detection is 7.4 to 8.4. Gold nanoparticle solution; And
At least one solution selected from the group consisting of an imidazole solution, a histidine solution, a pyrazole solution and a histamine solution. 10. The method of claim 9, wherein the concentration of the gold nanoparticle solution is 8 to 12 nM, and the concentration of the at least one solution selected from the group consisting of imidazole solution, histidine solution, pyrazole solution and histamine solution is 0.1 to 0.4 mM A kit for detecting organophosphorus pesticides.

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