KR100832522B1 - Detecting method for carbamate based and organic phosphate based agricultural chemicals - Google Patents

Abstract

Method for detecting carbamate and organophosphorus pesticide components according to the present invention comprises the steps of i) activating the surface of the metal thin film chip, ii) diluting the antibody with a pH buffer solution of pH 3.5 to 5.0 to prepare an antibody dilution solution (iii) immobilizing the antibody on the surface of the metal thin film by flowing the antibody diluent to the surface; and iv) immobilizing the antibody by providing an amine compound on the surface to stabilize the antibody; Providing a sample to be detected on the metal thin film chip to which the antibody is immobilized; A sample analysis step of analyzing the sample to be detected; And dissociating the sample to be detected from the metal thin film chip.

Description

Detecting method for carbamate based and organic phosphate based agricultural chemicals

1 is a conceptual diagram conceptually illustrating a detection method according to an embodiment of the present invention.

Figure 2 is a graph showing the change in RU value with pH and time.

3 is a three-dimensional image showing a state that the dextran is applied to the metal thin film chip according to an embodiment of the present invention.

FIG. 4 is a one-dimensional image showing the applied dextran of FIG. 3.

FIG. 5 is a three-dimensional image showing GST antibody immobilized on the dextran-coated metal thin film chip of FIG. 3.

FIG. 6 is a one-dimensional image showing a state in which the GST antibody of FIG. 5 is immobilized. FIG.

The present invention relates to a method for detecting carbamate-based and organophosphorus pesticides, and more particularly, to a method for detecting carbamate-based and organophosphorus-based pesticides, which has excellent detection sensitivity and can shorten the detection time.

Despite the inherent benefits of pesticides, pesticides remain in agricultural products and ecosystems, threatening environmental pollution and human health. In particular, residual pesticides present in agricultural products are of great concern both at home and abroad, and the safety of agricultural products is secured in advance by restricting the amount of pesticides used in agricultural products by restricting the amount of agricultural chemicals used in each country.

Conventional pesticide analysis relies mainly on gas chromatography (GC), high performance liquid chromatography (HPLC), and gas chromatography / mass spectroscopy (GC / MS). The sample is ground, extracted with a suitable organic solvent and purified. While these methods have the advantage of high sensitivity and accuracy, they require a great deal of expense, time, effort, expensive equipment and skilled skills.

Inspection for securing the safety of agricultural products requires rapid inspection technology due to the nature of distribution. Therefore, there is an urgent need to acquire a small, lightweight and economical equipment or biosensor development technology that can accurately and quickly measure residual pesticide components and is simple to operate.

Biosensors are "systems that use biological elements to obtain information from a measurement object, or convert objects into useful signals that can be recognized, such as color, fluorescence, electrical signals, etc., by using biological or mimic biological elements." Due to the ease of pretreatment and real-time measurement in the field, there is a growing interest in development in developed countries, especially in the health, medical and defense sectors.

 Based on these needs, studies on residual pesticides using biosensors in Korea include disc type detection kits with acetylcholinesterase adsorbed, optical biosensors for simultaneous measurement of two components using absorbance, and spectrophotometer. EBDC sterilant residual amount detection study and the like have been carried out using. Overseas, pesticide detection method using piezoelectric effect, surface plasmon resonance method and photothermal biosensor method were conducted.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for detecting carbamate-based and organophosphorus-based pesticides that can provide excellent detection sensitivity and can significantly improve detection efficiency by shortening the detection time.

Carbamate-based and organophosphorus pesticide detection method according to one aspect of the present invention is an antibody immobilization step, providing a sample to be detected on the metal thin film chip to which the antibody is immobilized, sample analysis to analyze the sample to be detected And dissociating the sample to be detected from the metal thin film chip. The antibody immobilization step is i) activating the surface of the metal thin film chip, ii) diluting the antibody with a pH buffer solution of pH 3.5 to 5.0 to prepare an antibody dilution, iii) flowing the antibody dilution to the surface And immobilizing the antibody on the metal thin film surface, and iv) stabilizing the antibody by providing an amine compound on the surface.

The metal thin film may be made of metal such as gold or silver. In addition, it is preferable to use the surface of the metal thin film chip is coated with dextran (dextran).

In the activation step, N-hydroxysuccinimide, N-ethyl-N '(dimethylaminopropyl) carbodimide [N-ethyl-N' (dimethylaminopropyl) carbodimide] on the surface of the metal thin film chip. Shed.

As antibodies for detection of carbamate-based and organophosphorus-based pesticide components according to the present invention, glutathione s-transferase (GST), acetylcholinesterase (AChE) and the like are preferable.

Ethanol amine etc. can be used as said amine type compound.

In the sample analysis step, the surface plasmon resonance (SPR) is generated on the surface of the metal thin film and the incident light of monochromatic polarization is incident to analyze the SPR angle change.

In the dissociation step, PBS (Phosphate Buffered Saline) buffer white solution of sodium chloride (NaCl) and sodium hydroxide (NaOH) is poured on the surface of the metal thin film to selectively dissociate only antigen.

All of the above steps can be performed within a time of less than 15 minutes.

According to the detection method, carbamate-based and organophosphorus-based pesticide components remaining in vegetables and the like can be efficiently detected.

Hereinafter, with reference to the accompanying drawings to describe the present invention in detail.

Carbamate-based and organophosphorus-based pesticide detection method according to the present invention employs both enzyme immunoassay and surface plasmon resonance (SPR).

The detection method includes an antibody immobilization step, a detection target sample providing step, a detection step, and a dissociation step of the detection target sample.

The sample to be detected is a carbamate-based or organophosphorus-based pesticide component, and examples thereof include carbofuran which is a carbamate-based pesticide component. The sample to be detected functions as an antigen under enzyme immunoassay.

1 is a conceptual diagram conceptually illustrating a detection method according to an embodiment of the present invention.

Referring to FIG. 1, in order to detect the pesticide component, first, a metal thin film chip 100 having a metal thin film 20 formed on a parent substrate 10 is prepared. The metal thin film is formed by coating or depositing a metal material such as gold or silver (A).

Dextran is applied to the surface of the metal thin film 20 (B). As the metal thin film chip, for example, a CM5 chip (trade name, manufactured by Sweden Biocore Co., Ltd.) may be used. The CM5 chip generates surface plasmon resonance (SPR) by light having a wavelength of about 760 nm.

An activation material is provided on the dextran-coated metal thin film chip 100 so that the antibody can be bound well (C). Examples of the activating substance include N-hydroxysuccinimide (EDC) and N-ethyl-N '(dimethylaminopropyl) carbodimide [N-ethyl-N' (dimethylaminopropyl) carbodimide; NHS] can be used. By flowing the EDC and the NHS on the metal thin film chip 100, the metal thin film chip 100 may be activated.

An antibody is bound onto the activated metal thin film chip 100 (D). As the antibody for detecting the carbamate-based and organophosphorus-based pesticide components according to the present invention, glutathione s-transferase (GST), acetylcholinesterase (AChE) and the like can be used. More specifically, the GST is relatively advantageous for the detection of carbamate pesticide components, and the AChE is relatively advantageous for the detection of organophosphorus pesticide components.

The antibody is not directly provided on the surface of the metal thin film chip 100. The antibody flows on the surface of the metal thin film chip 100 in a dilution state diluted in a pH buffer solution.

The pH of the pH buffer solution is optimized through a concentration test or the like before the antibody is immobilized on the metal thin film chip 100 in order to fix the stable antibody.

The pH range for stable fixation of the GST and AChE is 3.5 to 5.0. If the pH is less than 3.5 or more than 5.0, not only stable fixation of the antibody is difficult but also there is a problem that the antigen is not completely dissociated in the dissociation step. The pH range is preferably 4.3 to 4.7.

As the pH buffer, sodium acetate can be used.

The detection target sample is provided to the antibody of the metal thin film chip 100 (E). The carbamate and organophosphorus pesticide components in the sample to be detected are reacted with the antibody as an antigen. The detection of the pesticide component can be quantified using surface plasmon resonance and computer processing.

Surface plasmon resonance is a collective vibration of electrons occurring on the surface of a metal thin film, and the surface plasmon wave is a surface electromagnetic wave propagating along the interface between a metal and an adjacent dielectric material. Excitation of surface plasmon is caused by discontinuity of the vertical component of the electric field at the interface, and the surface charge is induced when the electric field is applied to the interface of two media with different dielectric functions from outside. The vibration of the charge appears as a surface plasma wave. This surface plasma wave, unlike electromagnetic waves in free space, is a wave that vibrates parallel to the plane of incidence and has a p-polarization component. Therefore, surface plasmons can be excited only by polarized electromagnetic waves.

If the two media are coated with a very thin metal, and the incident light is polarized and monochromatic light, the light reflected at a specific incident angle will cause an SPR phenomenon in which the light density is significantly reduced. The incident angle is called SPR angle. The SPR angle depends on the refractive index of the solution in which the electromagnetic wave is penetrated. The refractive index changes as the buffer changes, but also depends on the mass change of the sensor chip surface. Therefore, the combination of immobilized material (ligand. Antibody) and analyte (analyte) on the surface of the sensor chip results in a change of mass and thus the SPR angle is different from the first. Data that continuously records the change of the SPR angle is called a sensorgram. In the actual sensogram, the SPR angle is converted into a RU (resonance unit) and displayed. At this time, 1,000 RU is 1ng / mm 2, SPR angle change is 0.1 °.

As described above, the pesticide component to be detected according to the present invention can be detected by analyzing the SPR incident angle. The detection system can be changed in various ways depending on the antigen, the type of antibody and the intention of the operator.

When the detection step is completed, the antigen reacted with the antibody undergoes a dissociation step to dissociate from the antibody (F). In the dissociation step, only the antigen is selectively dissociated so that the metal thin film chip 100 in which the antigen is dissociated may be repeatedly reused in a subsequent detection operation.

In order to dissociate, a dissociation solution must be flowed onto the metal thin film chip 100. The dissociation solution is a dilution solution in which sodium chloride (NaCl) and sodium hydroxide (NaOH) are diluted in PBS (Phosphate Buffered Saline) buffer.

The molar ratio and concentration of the sodium chloride and sodium hydroxide can be appropriately adjusted according to the dissociation target.

All of the detection work is completed and the metal thin film chip 100 is released from the antigen can be recycled (G).

Detection of pesticide components up to at least 0.001 ppm in accordance with the detection method is possible. In addition, all of the above steps can be completed within 15 minutes, no matter how long.

Hereinafter, the present invention will be described in more detail with reference to specific embodiments. However, the technical spirit of the present invention is not limited by the following examples.

Example 1

Immobilization Condition Experiment

Concentration tests were performed to select the optimal pH buffer to ensure that the GST to be immobilized was easily accessible to the nagative charged. To this end, 1,000 μl of 10 mM sodium acetate was prepared in the range of pH 7.0 to 3.5. Measurement experiments were conducted to find the optimal pH value by diluting with 50 g / ml GST for each pH. After concentration test, 50 mM NaOH was flowed for 1 minute to prepare antibody immobilization.

Immobilization Condition Experiment Results

In order to find a pH value in which GST can easily approach the nagative charged, the sodium acetate buffer and the GST mixture were experimented with different pH from pH 7.0 to pH 3.5 as shown in FIG. 2. Experimental results showed high peak RU (resonance unit) at pH 4.5, 4.0, 3.5. PH 4.5 clearly showed a distinction between RU values when reacting and dissociating. On the other hand, pH 4.0 and pH 3.5 react well, but do not dissociate completely in the dissociation section, so that the RU value caused by GST residue on the chip surface is continuously measured, indicating that it has excessive negative charge. These results indicate that pH 4.5 sodium acetate buffer provides the best GST activity with moderate negative charge on the chip surface.

Example 2

Immobilization

50 μl of EDC and NHS are mixed on the surface of the metal thin film chip and flowed at a flow rate of 5 μl / min to activate the carboxyl group of about 30 to 40% so that the amine group of the protein can be covalently bonded. It was. The ligand, GST, was diluted and flowed into the pH buffer employed in the concentration test. The concentration of GST used in the experiment was 50 g / ml and 200 μl was prepared. Finally, 50 μl of 1 M ethanolamine was flowed to stabilize the immobilized GST.

Example 3

Metal thin film  Surface observation of the chip

The dextran was applied and the GST was fixed on the metal thin film chip manufactured according to the embodiment of the present invention.

This observation was observed with an atomic force microscope called AFM (Atomic Force Microscope). The AFM is used to measure the shape of extremely fine nanosize and to characterize the physical and chemical. It is also widely used to measure biological activity, including genes, proteins and living cells. In particular, non-contact AFM mechanically vibrates the microscope's cantilever near natural frequencies without damaging the sample, allowing high-resolution viewing of up to three-dimensional images. In this embodiment, the immobilization state of the antibody covalently bonded to the surface of the gold chip as the metal thin film chip was observed and analyzed using the AFM.

3 is a three-dimensional image showing the appearance of the dextran is applied on the metal thin film chip according to an embodiment of the present invention. FIG. 4 is a one-dimensional image showing the applied dextran of FIG. 3.

3 and 4, dextran is uniformly coated on the surface of the gold chip having a width × length of 5 μm, respectively. The dextran is uniformly applied to induce antibodies to be more efficiently bound to the dextran.

FIG. 5 is a three-dimensional image showing GST antibody immobilized on the dextran-coated metal thin film chip of FIG. 3. FIG. 6 is a one-dimensional image showing a state in which the GST antibody of FIG. 5 is immobilized. FIG.

The GST antibody was immobilized by an amine coupling immobilization method. Referring to FIGS. 5 and 6, it was found that the GST was relatively evenly immobilized on the surface of dextran. The average immobilization height of the GST antibody was 0.15 μm and the volume was 2.85 μm ³ .

The detection method of the carbamate-based and organophosphorus-based pesticide components according to the present invention is not only very excellent in detection sensitivity, but also can shorten the detection time. In the future, the method may be further developed to develop a simple and accurate biosensor.

More specifically, the detection method according to the present invention can accurately detect the pesticide component of less than 0.001ppm, and the detection time is also very efficient since it takes only about 10 to 15 minutes.

In addition, by dissociating the pesticide component as an antigen participating in the reaction completely with the antibody, it is possible to increase the accuracy of the subsequent detection reaction, and semi-permanent use of the biochip.

As described above, the present invention has been described by way of a limited embodiment, but the present invention is not limited to the above embodiments, and those skilled in the art to which the present invention pertains may make various modifications and variations from this description. Do. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be determined not only by the claims below but also by the equivalents of the claims.

Claims (10)

i) activating the surface of the metal thin film chip; ii) diluting the antibody with a pH buffer solution of pH 3.5 to 5.0 to prepare an antibody dilution solution; iii) immobilizing the antibody on the surface of the metal thin film chip by flowing the antibody diluent to the surface; And iv) immobilizing the antibody by providing an amine compound on the surface to stabilize the antibody; Providing a sample to be detected on the metal thin film chip to which the antibody is immobilized; A sample analysis step of analyzing the sample to be detected; And And a dissociation step of dissociating the sample to be detected from the metal thin film chip. The method for detecting carbamate-based and organophosphorus-based pesticides according to claim 1, wherein the preparing of the antibody diluent comprises using a pH buffer solution of pH 4.3 to 4.7. The method of claim 1, wherein the metal thin film chip is made of gold (Au). The method for detecting carbamate-based and organophosphorus-based pesticides according to claim 1, wherein dextran is applied to the surface of the metal thin film. The method of claim 1, wherein the activating step is N-hydroxysuccinimide and N-ethyl-N '(dimethylaminopropyl) carbodimid [N-ethyl-N on the surface of the metal thin film chip. A method for detecting carbamate-based and organophosphorus-based pesticides, characterized by flowing '(dimethylaminopropyl) carbodimide'. The method of claim 1, wherein the antibody is any one selected from the group consisting of glutathione s-transferase (GST) and acetylcholinesterase (AChE). The method for detecting carbamate-based and organophosphorus-based pesticides according to claim 1, wherein the amine-based compound is ethanol amine. The method of claim 1, wherein the sample analysis step is to generate a surface plasmon resonance (SPR) on the surface of the metal thin film and a carbamate-based and organophosphorus pesticide component characterized in that for analyzing the change in the SPR angle by incident light of monochromatic polarization Detection method. The carbamate according to claim 1, wherein the dissociation step comprises selectively dissociating only antigen by flowing a PBS (Phosphate Buffered Saline) buffer white solution of sodium chloride (NaCl) and sodium hydroxide (NaOH) onto the surface of the metal thin film. Method for detecting component and organic phosphorus pesticides The method of claim 1, wherein all the steps are performed within a time of 15 minutes or less.

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