KR101340522B1 - Electrochemical biosensor for analyzing organophosphorus compounds and analytic method of organophosphorus compounds using it - Google Patents

Abstract

The present invention provides a lower substrate including a fixed electrode patterned with a gold thin film, a sample inlet, and an upper substrate including a channel providing a moving passage through which a sample can contact the fixed electrode, and a magnet to fix magnetic particles. The present invention relates to an electrochemical biosensor for detecting an organophosphate compound including a magnet part, and a method for quantitatively analyzing an organophosphate compound in a sample to be analyzed using the same.
Biosensor of the present invention can detect the organic phosphoric acid compound of 0.05 ppm level with only one device without an external reaction, it is expected that it can be widely applied in various fields such as agriculture, food, military.

Description

Electrochemical biosensor for analyzing organophosphorus compounds and analytic method of organophosphorus compounds using it}

The present invention relates to an electrochemical biosensor for detecting an organic phosphate compound capable of inducing an electrochemical signal by inducing multiple enzymatic reactions, and a method for quantitatively analyzing an organophosphate compound in a sample to be analyzed using the same.

Organophosphorus compounds are very harmful to the human body, so various attempts have been made to analyze them in various fields. In general, a material analysis technique using HPLC and GC is widely used for the detection of organophosphate compounds. Although the analytical method has the advantage of analyzing the sample very precisely and accurately, there are many difficulties in general use in daily life because it requires expensive equipment, trained manpower, and long analysis time. On the other hand, analytical methods using biosensors are less accurate than analytical methods such as HPLC or GC, but are easy to carry and can be easily used in everyday life because they can detect analytes quickly and easily.

Detection of Organophosphorus Toxic Compounds Mainly acetylcholine esterase (AChE) has been used in biosensor systems. In the early stages of study, AChE monoenzyme system based on acetylthiocholine (ATCh) was used. The enzyme reaction product, thiocholine, can be measured by optical and electrochemical methods. As the optical measuring method, a method using an "Ellman reaction" capable of detecting sulfhydryl (-SH) residues is widely used. The activity of the enzyme can be measured by quantifying thiocholine decomposed by AChE using a reaction that causes thiocholine with sulfhydryl residues to develop color with 2,2′-dinitro-5,5′-dithiobenzoic acid (DTNB). Can be. In the electrochemical measurement method, thiocholine is an electrochemically active substance, which can be directly measured through voltammetry. However, the direct oxidation of thiocholine requires a high potential, which can cause side reactions of other substances in the sample, which can negatively affect the quasi-chemical signal. Recently, in order to solve such side reactions, a method of applying a dual enzyme system using choline oxidase (ChOx) together with AChE has been studied. Acetylcholine (ACh) is used as a substrate to react with AChE to produce choline (Ch), and then choline is finally converted into betaine and hydrogen peroxide (H ₂ O ₂ ) by continuous enzymatic reaction before current. Decompose First generation biosensing using the generated hydrogen peroxide is possible, and the concentration of organic phosphoric acid can be determined by quantifying the generated hydrogen peroxide, which is the activity of AChE inhibited by organic phosphoric acid. However, in this case, too, a high oxidation potential must be applied for the oxidation of hydrogen peroxide, which may limit the type of electrode and induce side reactions. The existing AChE inhibition-based organophosphate compound reaction system consists of three steps: reaction of organic phosphoric acid and AChE, reaction of AChE and ACh, and electrochemical measurement of reactants. There is a problem that is difficult to apply for diagnostic purposes.

Therefore, a simpler and more accurate method of electrochemical measurement of organophosphate compounds is required.

Therefore, the present inventors have conducted research and efforts to develop a simple and accurate method for quantitative analysis of organophosphate toxic compounds. As a result, the present inventors induced an enzymatic reaction of acetylcholine esterase and choline oxidase to detect an electrochemical signal, and thereby the organic phosphorus in the sample. The present invention has been completed by the discovery that quantitative analysis of acidic compounds can be made.

Accordingly, the present invention provides a magnet for fixing a magnetic substrate and an upper substrate including a lower substrate including a fixed electrode patterned with a gold thin film, a sample inlet, and a channel providing a moving passage through which a sample can contact the fixed electrode. An object of the present invention is to provide an electrochemical biosensor for detecting an organophosphate compound including a magnet part including a magnet part.

In addition, the present invention is introduced into the biosensor choline oxidase (ChOx), acetylcholine esterase (AChE) magnetic particles, the substrate sample and the sample to be analyzed in turn, and analyzed the electrochemical signal according to the organophosphate compound in the sample to be analyzed It is another object to provide a method for quantitative analysis of analyses.

The present invention

A lower substrate including a fixed electrode patterned with a gold thin film;

An upper substrate including a sample inlet and a channel providing a moving passage through which the sample may contact the fixed electrode; And

An electrochemical biosensor for detecting an organic phosphate compound is characterized by including a magnet part including a magnet to immobilize magnetic particles.

Further, according to the present invention,

1) inserting choline oxidase into the sample inlet of the biosensor and fixing it to a gold electrode;

2) washing the acetylcholine esterase magnetic particles into the sample inlet and then washing them;

3) inserting a substrate sample containing acetylcholine and an electron transporter into the sample inlet, detecting and washing an electrochemical signal;

4) adding a sample to be analyzed in the sample inlet and reacting;

5) inserting a substrate sample containing acetylcholine and an electron carrier into the sample inlet and detecting an electrochemical signal; And

6) comparing the electrochemical signals of steps 3) and 5)

It is another feature of the quantitative analysis method of an organic phosphate compound in a sample comprising a.

Biosensor of the present invention can detect the organic phosphoric acid compound of 0.05 ppm level with only one device without an external reaction, it is expected that it can be widely applied in various fields such as agriculture, food, military.

1 is a diagram illustrating a form in which choline oxidase is immobilized on a fixed electrode of a biosensor.
Figure 2 is a schematic of the acetylcholine esterase magnetic particles.
3 is a diagram illustrating a state in which acetylcholine esterase magnetic particles are immobilized on a fixed electrode by a magnet.
4 is a plan view schematically illustrating a lower substrate of the biosensor of the present invention.
5 is a plan view schematically illustrating the upper substrate of the biosensor of the present invention.
6 is a plan view schematically illustrating a biosensor in which the lower substrate and the upper substrate are combined.
7 is a graph comparing the concentration of diazinon measured in Example 1 with the actual concentration.
8 is a plan view schematically illustrating the position of the magnet changed in Example 2. FIG.
9 is a graph comparing a voltammogram according to the position of a magnet.
10 is a graph comparing the current at 400mV according to the position of the magnet.

Hereinafter, the present invention will be described in detail.

The present invention provides a lower substrate including a fixed electrode patterned with a gold thin film, a sample inlet, and an upper substrate including a channel providing a moving passage through which a sample can contact the fixed electrode, and a magnet to fix magnetic particles. The present invention relates to an electrochemical biosensor for detecting an organophosphate compound including a magnet part, and a method for quantitatively analyzing an organophosphate compound in a sample to be analyzed using the same.

First, the biosensor comprises: a lower substrate including a fixed electrode patterned with a gold thin film; An upper substrate including a sample inlet and a channel providing a moving passage through which the sample may contact the fixed electrode; And a magnet including a magnet to fix the magnetic particles.

In the fixed electrode patterned with the gold thin film, acetylcholine esterase and choline oxidase are immobilized to induce an enzyme-linked reaction. Therefore, immobilization of each enzyme is essential. Therefore, it is preferable to form a self assembly monolayer (SAM) having a specific reactor for the immobilization of the enzyme. The self-assembled monolayer film may be formed by treating one or more compounds selected from cystamine, cysteamine, and 4-aminothiophenol.

In addition, the lower substrate includes an electrode to which an electrochemical measuring device can be connected to detect an electrochemical signal.

On the other hand, the upper substrate made of a transparent polymer or glass-based material includes a sample inlet and a channel for providing a moving passage for contacting the sample to the fixed electrode.

In particular, the channel is a fine channel of the channel may be made of a control unit for controlling the flow of the sample from the sample inlet, a signal detector for detecting a signal from the fixed electrode, the inlet of the cleaning solution, the waste solution reservoir and the air outlet, etc. have.

In the channel, each sample is injected through the sample inlet, and during washing, the washing solution is introduced into the inlet of the washing solution.

The material forming the channel may be PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), polycarbonate, polystyrene, quartz (Quartz), glass, and the like.

The upper substrate and the lower substrate may be combined through a conventional method, but preferably, the upper substrate and the lower substrate are combined with the upper substrate through a plasma treatment on the lower substrate.

In addition, the biosensor of the present invention includes a magnet capable of fixing the magnetic particles as an essential configuration. If the magnet is generally a permanent magnet commonly used, its use is not limited, and it is preferable to use a magnet having a thickness of 5 mm or less and a diameter of 1 cm or less. The magnet may be positioned at the bottom of the lower substrate, but the position thereof may be adjusted in consideration of the electrochemical signal level. However, since the signal is detected more as the position of the magnetic particles is closer to the electrode, the magnet may be disposed below the fixed electrode. It is a good idea to position them.

The present invention provides a method for quantitatively analyzing an organophosphate compound in a sample using the biosensor.

First, choline oxidase (ChOx) is added to the sample inlet of the biosensor and fixed to the gold electrode. In this case, when the self-assembled monolayer film formed on the gold electrode is formed, the immobilization of the enzyme may be more advantageously achieved by a reactor formed on the surface. In particular, when the binding support polymer or binder is mixed with choline oxidase, Covalent bonds are induced, leading to better immobilization. As the binding support polymer, one or more polymers selected from poly-L-lysine (PLL), poly-L-ornithine, polyethyleneimine, and chitosan may be used. The binder may be at least one compound selected from glutaraldehyde, disuccinimidyl tartarate (DST), and bis [sulfosuccinimidyl] subberate (Bis [sulfosuccinimidyl] suberate (BS ³ )). Can be used.

The choline oxidase is fixed to the gold electrode is shown in Figure 1 shown.

Next, an acetylcholine esterase (AChE) magnetic particle is introduced into the sample inlet, and the particle is immobilized at a specific position by a magnet. The acetylcholine esterase magnetic particles may be prepared by mixing magnetic particles and acetylcholine esterase. At this time, the material of the magnetic particles is preferably selected from iron oxide (Iron oxide), nickel oxide (Nickel oxide) and iron-nickel oxide (Iron-nickel oxide), the particles having a diameter in the range of 1 ~ 10 ㎛ It is good to use In addition, the magnetic particles include an amine group, a carboxylate group, an aldehyde group, a maleimide group, an N-hydroxysuccinimide group (NHS), and the like on the surface to form an effective covalent bond with an acetylcholine esterase. It is preferable to use it by surface treatment so that it may be exposed.

The immobilization process of the acetylcholine esterase is shown in FIGS. 2 and 3.

The acetylcholine esterase magnetic particles are added and washed by adding a washing solution. In the present invention, the washing solution may be a phosphate buffer solution (Phosphate buffer solution) between pH 6-9.

 Thereafter, a substrate sample containing acetylcholine and an electron carrier is introduced into the sample inlet, and an electrochemical signal is detected.

The acetylcholine is converted to choline by an enzymatic reaction on the surface of the acetylcholine esterase magnetic particles introduced in advance, and the choline is oxidized at a gold electrode to which choline oxidase is fixed to display an electrical signal. The acetylcholine is added at a concentration of 0.1 to 2.0 mM, more preferably at a concentration of 0.5 to 1.0 mM, which is advantageous for proper signal detection.

Cyclic voltammetry is used to detect electrochemical signals. Cyclic voltammetry is a method widely used as an electrochemical signal detection method, and it is advantageous to manufacture a sensor and a system at a low cost because the system is simpler than other methods such as spectroscopic methods. In order to detect an electrochemical signal from the electrode through cyclic voltammetry, the electrode is used as a working electrode, and a three-electrode system including a silver / silver chloride reference electrode and a platinum wire electrode as an auxiliary electrode is used for signal detection. . In carrying out cyclic voltammetry, many of the water-soluble materials having electroactivity are useful as detection materials for measurement. To this end, the electron transporter is used as an electrochemically active species, and as the electron transporter, a ferrocene derivative, a ferricyanide compound, or the like may be used. Preferably, the ferrocene methanol, potassium ferricyanide (K ₃ [Fe (CN)) ] ₆ ) or sodium ferricyanide (Na ₃ [Fe (CN)] ₆ ) is preferably used and prepared to have a concentration of several millimoles or less in the electrolyte solution.

The cyclic voltammetry is performed to measure the change in the waveform of the voltage current obtained from the detected electrochemical signal or the change in the maximum current value to quantify the signal.

When the electrochemical signal by the above process is detected, the sample to be analyzed is added to the sample inlet including the organophosphate compound to be actually analyzed after being washed again. At this time, it is preferable to induce the reaction to proceed for 3 to 10 minutes after the addition of the sample to be analyzed.

In addition, a substrate sample containing acetylcholine and an electron transporter is added thereto, and an electrochemical signal is detected. When the degree of inhibition according to the input of the sample to be analyzed is compared with the detected electrochemical signal before the input of the sample to be analyzed, the concentration of the organophosphate compound may be quantified.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.

Preparation Example: Preparation of Biosensor

The bio-sensing chip includes a lower substrate 100 including a fixed electrode 110 patterned with a gold thin film and a pad 120 for connecting an analyzer of an electrochemical signal, and an upper substrate 200 including a microchannel of PDMS material. (Figs. 4 and 5).

The upper substrate includes a sample injection port 210 into which a sample is injected, a control unit 220 controlling a flow of a sample, a signal detection unit 230 at which a signal is detected, a cleaning solution injection port 240 to inject a cleaning solution, and a waste It consists of a reservoir 250 in which the solution is stored and an outlet 260 through which air can be discharged.

Meanwhile, the magnet 400 is positioned under the fixed electrode 110 of the lower substrate.

6 is a schematic view of the biosensor of the present invention combining the lower substrate and the upper substrate.

Example 1 Quantitative Analysis of Organophosphate Compounds Using a Biosensor

Three units of choline oxidase were placed in a 1 mg / 150 μl poly-L-lysine (PLL) solution, and 10 μl of 0.2 wt% glutaraldehyde solution was further added to obtain a choline oxidase mixture.

Next, the mixed solution was added to the sample inlet of the biosensor prepared in Preparation Example and the reaction was performed for 2 hours.

Meanwhile, 1 mg of acetylcholine esterase of 0.1 mg / ml was added to 30 mg of an aldehyde-modified magnetic particle (iron oxide, 5 μm in diameter) solution, and then reacted for 1 hour to obtain acetylcholine esterase magnetic particles. In order to increase the stability of the imine covalent bond formed at this time, 10 μl of a 5 mol sodium cyanoborohydride (NaCNBH ₃ ) solution was added as a reducing agent.

50 μl of the acetylcholine esterase magnetic particles were placed in a sample inlet, and then fixed in a desired position using a magnet. Then, 50 mM phosphate buffer solution (pH 7.2) was added to the washing solution inlet, and the washing process was performed.

Subsequently, a substrate sample mixed with 0.8 mM of acetylcholine and 0.1 mM of ferrocene methanol was injected into the sample inlet, and the electrochemical signal was measured. The electrochemical signal was measured using an electrochemical analyzer model 630B of CH instruments connected to a laptop computer. Used.

After 50 mM phosphate buffer solution (pH 7.2) was added to the washing solution inlet, the washing process was carried out, and reaction was performed for 5 minutes while adding diazinon (DZN) at a concentration of 0 to 0.8 ppm as an organic phosphate compound sample. Proceeded.

Subsequently, a substrate sample mixed with 0.8 mM of acetylcholine and 0.1 mM of ferrocene methanol was injected into the sample inlet, and the electrochemical signal was measured.

By comparing the detected electrochemical signal, the degree of inhibition by diazinon was analyzed and its concentration was confirmed. The results are shown in FIG. 7.

As shown in FIG. 7, it was confirmed that the concentration of diazinon can be analyzed almost accurately by the analysis method of the present invention.

Example 2 Analysis of Electrochemical Signals According to the Position of Magnets

In the biosensor manufactured in the above preparation example, the magnet was changed to the position of 1 to 2 of FIG. 8 (the preparation example is present at the position 3), and then 0.8 mM of acetylcholine and 0.1 mM of ferrocene methanol were mixed. The substrate sample was injected into the sample inlet to measure the electrochemical signal, and the results are shown in FIGS. 9 and 10.

As shown in FIGS. 9 and 10, the closer the magnet is located to the electrode, the more strongly the electrochemical signal is measured. As shown in the manufacturing example, the magnet is positioned below the gold electrode to improve the accuracy of the analysis. It is considered to be more preferable.

100: lower substrate 110: fixed electrode
111 self-assembled monolayer 112 choline oxidase
120: pad for connecting the measuring device 200: upper substrate
210: sample injection port 220: control unit
230: signal detection unit 240: washing solution inlet
250: waste solution reservoir 260: air outlet
300: magnetic particles 310: acetylcholine esterase
400: magnet

Claims (9)

In an organophosphate compound detection system comprising an electrochemical biosensor and a sample, the electrochemical biosensor
A lower substrate including a fixed electrode patterned with a gold thin film;
An upper substrate including a sample inlet and a channel providing a moving passage through which the sample may contact the fixed electrode; And
It comprises a magnet portion including a magnet to fix the magnetic particles, a self-assembled monolayer film is formed on the surface of the gold thin film, the sample comprises choline oxidase, acetylcholine esterase magnetic particles, the acetylcholine esterase magnetic particles Organic phosphate compound detection system that is at least one selected from iron oxide (Iron oxide), nickel oxide (Nickel oxide) and iron-nickel oxide (Iron-nickel oxide).
delete The organophosphate system of claim 1, wherein the self-assembled monolayer film is formed of at least one compound selected from cystamine, cysteamine, and 4-aminothiophenol. Compound detection system.
The organophosphate compound detection system of claim 1, wherein the magnet part is positioned under the fixed electrode.
In the quantitative analysis method of an organophosphate compound using the organophosphate compound detection system of any one of Claims 1, 3, and 4,
1) inserting choline oxidase (ChOx) into the sample inlet of the electrochemical biosensor to fix the gold electrode;
2) washing the acetylcholine esterase magnetic particles into the sample inlet and then washing them;
3) inserting a substrate sample containing acetylcholine and an electron transporter into the sample inlet, detecting and washing an electrochemical signal;
4) adding a sample to be analyzed in the sample inlet and reacting;
5) inserting a substrate sample containing acetylcholine and an electron carrier into the sample inlet and detecting an electrochemical signal; And
6) comparing the electrochemical signals of steps 3) and 5)
Method for quantitative analysis of organophosphate compounds in a sample comprising a.
The method of claim 5, wherein the electron transporter is ferroceneemethanol, Potassium ferricyanide (K ₃ [Fe (CN)] ₆ ) and Sodium ferricyanide (Na ₃ [Fe (CN)] ₆ Method for quantitative analysis of organophosphate compounds in a sample, characterized in that the selected.
The method of claim 5, wherein the acetylcholine esterase magnetic particles are prepared by mixing acetylcholine esterase with magnetic particles selected from iron oxide, nickel oxide and iron-nickel oxide. Method for quantitative analysis of organophosphate compounds in a sample, characterized in that.
The method of claim 7, wherein the magnetic particles are surface-treated to expose a functional group selected from amine group, carboxylate group, aldehyde group, maleimide group, N-hydroxysuccinimide (NHS) group A method for quantitative analysis of organophosphate compounds in a sample, which is prepared by mixing with acetylcholine esterase.
The method of claim 5, wherein when the choline oxidase (ChOx) of step 1) is added, poly-L-lysine (PLL), poly-L-ornithine, polyethyleneimine and chitosan ( Chitosan) Further selected from among polymers, or a binder selected from glutaraldehyde, disuccinimidyl tartarate (DST), and bis [sulfosuccinimidyl] subberate (Bis [sulfosuccinimidyl] suberate, BS ³ ) Method for quantitative analysis of organophosphate compounds in a sample, characterized in that the mixture is added.

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