KR20000000791A - Sensor for measuring l-glutamic acid - Google Patents

Abstract

PURPOSE: A sensor for measuring L-glutamic acid is provided to quickly and exactly measure the consistency of L-glutamic acid secreted from a synapse part inside a brain tissue. CONSTITUTION: The sensor for measuring L-glutamic acid using the carbon fiber handling the surface by an enzymatic film as an electrode material comprises steps of:pre-processing a carbon fiber electrode in electric chemical; forming an enzyme and a shared union film of a nonconductive high polymer polymeric film in the surface of the carbon fiber electrode pre-processed in electric chemical.

Description

Glutamate Measuring Sensor

The present invention relates to a sensor for measuring glutamate, and more particularly, to a sensor capable of measuring the concentration of glutamate secreted at synaptic sites in brain tissue.

Recently, efforts are being actively made to approach brain science, an unexplored field in the 21st century, and in this case, as a basic analysis tool, a sensor technology that can directly measure neurotransmitters in vivo The importance of sensors that can be inserted directly into brain tissue is very active.

In particular, the glutamate sensor is very important in the field of neuroscience, such as neurological disorders related to brain damage, and the demand for a glutamate sensor that can be measured in vivo is increasing. Glutamate (L-glutamic acid), which is known as one of the essential amino acids, is one of the representative neurotransmitters in the brain and is known to play a major role in the stimulation process at synapses. In particular, the homeostasis (plasticity) at the synaptic region and the growth and degeneration of neurons (neuron) unit neurons are deeply involved.

Recently, glutamate binds with N-methyl-D-aspartic acid (NMDA) receptors to increase the calcium ion (Ca ²⁺ ) concentration in the body, eventually producing NO (nitric oxide), a NO syntase. Research has been reported to be related to the activation of.

NO is found as a free radical ubiquitous in the mammal's body and is a molecule synthesized from arginine. The NO is a molecule that participates in various biological activities such as neurotransmission, vasodilation, inhibition of cell disruption and platelet aggregation by intracellular toxins of macrophages, and is a substance that can cause neuropathy. .

Since glutamate is deeply involved in neurological disorders related to brain damage, many efforts have been made to measure the concentration of glutamate in the brain, particularly in the field of neuroscience.

A representative effort in this regard is a method of quantifying glutamate as an analyzer such as high performance liquid chromatograpy (HPLC) in vitro using microdialysis. However, it is difficult to track the concentration change of glutamate in real time in the case of samples collected using the microdialissis method. In general, neurotransmitters such as glutamate have inadequate concentration changes and difficult measurement.

Meanwhile, glutamate is a substance that cannot be oxidized / reduced by itself compared with other neurotransmitters such as catecholamines, that is, dopamine, norepinephrine, and epinephrine.

Therefore, in general, the following enzyme reaction can be used to measure the concentration change of glutamate.

The enzymes available for measuring changes in glutamate concentrations include glutamate oxidase, glutamate dehydrogense, glutamate decarboxylase, and glutamine syntase, which are used as catalysts. Glutamate is quantified using the biochemical reaction of.

As a method of quantifying glutamate,

One of them is to measure the amount of glutamate by measuring the concentration of hydrogen peroxide (H ₂ O ₂ ) or oxygen (O ₂ ) produced by the reaction according to Scheme 1 below, wherein glutamate oxidase is used.

L-glutamate + O ₂ + H ₂ O ---------> α- ketoglutarate + NH ₃ + H ₂ O ₂

Glutamate oxidase

In addition, in the α-ketoglutarate produced by the glutamate oxidase reaction as described above, glutamate is regenerated by using a reaction according to Scheme 2 using a glutamate dehydrogenase as a catalyst. As a result, studies have been reported to increase the measurement sensitivity of glutamate quantification (Talanta; Article 38) (49): 49-55, 1991).

L-glutamate + NAD (P) ⁺ + H ₂ O <----------------> α-ketoglutarate + NH ₄ ⁺ + NAD (P) H glutamate dehydrogenase

On the other hand, there is a method of measuring the amount of carbon dioxide generated through the reaction according to the glutamate decarboxylase reaction scheme 3 using an optical fiber probe, and quantifying glutamate through the measured amount.

L-glutamate ----------------> γ-aminobutyrate + CO ₂ glutamate decarboxylase

As another method, a technique for quantifying glutamate by fixing glutamine syntase on a pH electrode or an ion-selective field effect transistor (ISFET) has been reported.

The development of the sensor for measuring glutamate using the glutamate quantitative method is a clinical diagnostic sensor for discriminating liver damage and hepatitis or hepatic function, or glutamate is used as a representative food additive to enhance the flavor of food. It has been mainly focused on food control sensors.

In the case of glutamate sensors developed for clinical use or food use, glutamate oxidase, glutamate dehydrogenase, glutamatetal, etc. are described in the sensitive areas of these transducers by using a platinum electrode, a dissolved oxygen electrode, or an ISFET as a transducer. It was developed in the form of a membrane attached with immobilized carbonate and glutamine synthetase.

The sensor developed for clinical or food use is not suitable for brain research because its shape electrode size is too large to be inserted directly into brain tissue.

Therefore, efforts have been made to detect glutamate concentrations in vitro by incorporating the sensor and microdialis- tion, but the concentrations of glutamate that are measured in vitro can not distinguish the rapid concentration change of glutamate actually released in the body. There is a disadvantage. Moreover, the concentration of the buffer solution through dialysis tubing affects the actual concentration of glutamate, which prevents accurate concentration measurement.

Therefore, in the case of the sensor for brain research, the use of micro electrode material is required, but the problem of using a small amount of enzyme, the exclusion of electrode interference material, the biocompatibility of electrode material And problems with the period of use thereof may occur.

An example of a sensor that can be inserted directly into brain tissue is at the University of Kansas, USA, where glutamate oxidase and ascorbate oxidase are immobilized on a platinum / iridium (Pt / Ir) wire with a diameter of 250 μm. (Brain Research 659: 117-125, 1994) However, the sensor is bothersome to use two enzymatic reactions and biocompatibility of Pt / Ir electrodes used as transducers. The degree of biocompatibility has not been verified and is pointed out as a disadvantage.

Carbon fibers have been studied as electrode materials for measuring neurotransmitters in biological samples. Carbon fiber electrodes have excellent electrode characteristics, easy bioinsertion, and biocompatibility. good. Therefore, the surface of the carbon fiber electrode is treated with nafion (nafion), but it is used for the measurement of neurotransmitters of catecholamines such as dopamine, but in the case of glutamate, research is being conducted in several universities.

The measurement of glutamate using the carbon fiber electrode has the following problems.

First, in the case of measuring the glutamate concentration, as described above, an enzyme such as glutamate oxidase must be immobilized on the surface of the carbon fiber electrode.

Second, the signal interference effect by other electrode active materials present in the biological sample should be excluded. Because carbon fiber electrodes also react with substances such as ascorbic acid and dopamine, which are present in many biological samples such as brain tissue, these reactions do not allow accurate measurement of glutamate levels.

Third, when measuring the concentration of glutamate using a glutamate oxidase reaction, it is required to increase the response characteristics for the hydrogen peroxide (H ₂ O ₂ ) generated in the above-described reaction. The response characteristic to hydrogen peroxide is the most important element for improving the sensitivity of glutamate sensor, but the carbon fiber electrode responds well to catecholamines such as ascorbic acid or dopamine, but the response to hydrogen peroxide is relatively good. It is necessary to improve the response characteristics.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a sensor for measuring glutamate that can quickly and accurately measure the concentration of glutamate secreted from synaptic sites in brain tissue.

1 is a schematic diagram of a sensor for measuring glutamate according to the present invention.

Explanation of symbols for main parts of the drawings

1.Connector

3 ... glass pipette

5.Copper ship

7.insulating epoxy

9.Carbon Fiber

11 ... Sense site consisting of enzyme / polymer polymer membrane

In order to achieve the above object, the glutamate measuring sensor of the present invention is an electrochemical pretreatment, and also uses carbon fiber whose surface is treated with an enzyme membrane covalently bonded to an enzyme and a nonconductive polymer polymerization membrane. .

In this case, the carbon fiber may be electrochemically modified using the Prussian blue.

The enzyme may be glutamate oxidase.

The non-conductive polymer polymer membrane may be a polymer membrane composed of a polymer membrane composed of m-phenylenediamine and resorcinol.

In addition, the manufacturing method of the sensor for glutamate measurement is as follows.

That is, (a) electrochemically pretreating the carbon fiber electrode;

(b) forming a covalent bond film of an enzyme and a non-conductive polymer polymer film on the surface of the electrochemically pretreated carbon fiber electrode;

It is made of a manufacturing method.

At this time, the manufacturing method may further include the step of electrochemically modifying the electrochemically pretreated carbon fiber electrode using the Prussian blue, the enzyme may be glutamate oxidase, the vision The conductive polymer polymer film may be a polymer film of m-phenylenediamine and resorcinol.

Hereinafter, the operation of the configuration of the present invention will be described in detail.

Glutamate in the biological sample produces alpha-ketoglutarate by glutamate oxidase reaction and simultaneously converts oxygen dissolved in the sample into hydrogen peroxide (H ₂ O ₂ ).

At this time, the quantification of glutamate is possible by measuring a current that changes when hydrogen peroxide produced by glutamate oxidase is oxidized on an electrode to which a constant voltage is applied.

Therefore, in order to measure the appropriate glutamate, glutamate oxidase should be immobilized on the surface of the carbon fiber, and interference by an electrode active material other than glutamate, such as ascorbic acid or dopamine, present in biological samples such as brain tissue should be eliminated. In addition, as described above, since the quantification of glutamate is performed through the measurement of the current change when the hydrogen peroxide produced by the reaction is oxidized or reduced, it is necessary to increase the response characteristic of the electrode material to hydrogen peroxide.

The present invention adds a certain amount of glutamate oxidase together when m-phenylenediamine, a non-conducting polymer, and resorcinol are electrochemically polymerized on a carbon fiber surface, and the enzyme molecule is m-phenylene. The above problem is solved by providing a microelectrode covalently bonded to a polymer film of diamine and resorcinol.

The polymer polymerization membrane of m-phenylenediamine and resorcinol serves as a binder for fixing the enzyme in the carbon fiber electrode.

In addition, the polymer polymerization membrane can selectively permeate only a material having a small molecular weight due to the characteristics of the polymer membrane. According to the experiment, permeation of catecholamines such as ascorbic acid or dopamine with a relatively high molecular weight decreased. Therefore, the polymer membrane of m-phenylenediamine and resorcinol responds well to catecholamines such as ascorbic acid or dopamine, while the electrochemically pretreated carbon fiber electrode. The response to hydrogen peroxide may provide a function to remove the electrode interference material to solve the conventional disadvantages that were relatively poor.

In addition, in the present invention, in order to improve the response characteristics of the electrode material to the hydrogen peroxide itself, carbon fiber, an electrode material, was modified as prussian blue. This equation also helps to determine the proper polarizing voltage.

The use of the polymer membrane of m-phenylenediamine and resorcinol of the present invention further provides an electrode surface by a protein or an electrode generating material present in a sample during a long time measurement of a biological sample in a conventional sensor for neurotransmitter measurement. This problem can be solved.

Hereinafter, the manufacturing method of the sensor for glutamate measurement through an embodiment of the present invention. The following examples are provided to illustrate the present invention in more detail, but the scope of the present invention is not limited thereto.

(Example)

Fabrication of Carbon Fiber Electrodes

A carbon fiber having a diameter of 8-30 μm was cut into a size of about 5 cm and used as a basic electrode of the present invention. The glass tube (outer diameter: 1.5mm, inner diameter: 1mm) used as the electrode support was adjusted slightly larger than the outer diameter of the carbon fiber after microfabrication using a micropipette puller.

One end of the carbon fiber was connected to a conductive copper wire using a conductive silver paste to form a connector that can be connected to a measuring device, and then sintered at 100 ° C. for 10 minutes. Next, carbon fibers were inserted into glass microtubes formed in advance, and the inside of the glass tube was treated with an appropriate amount of epoxy to be sintered and insulated. The carbon fiber exposed to the outside of the glass microtubule was cut under a microscope so that the tip length was about 100-500 μm to complete the carbon fiber electrode (see FIG. 1).

The finished carbon fiber electrode was electrochemically oxidized to pretreat the surface because impurities may be present on the surface. In other words, the surface of the electrode is alternately washed with acetone and distilled water, and then a reference electrode (Ag / AgCl), a counter electrode made of platinum wire, and a carbon fiber electrode are formed as working electrodes. Using a three-electrode system, 70 Hz power was continuously applied at + 3V for 20 seconds, + 2.4V for 15 seconds, and finally at + 1.8V for 10 seconds in a pH 7.4 phosphate buffered saline (0.1M phosphate buffered saline). Was applied electrochemically. The pretreated carbon fiber electrode was able to obtain electrochemically stable and reproducible electrode characteristics.

Carbon fiber electrode formula using prussian blue

In order to improve the response characteristics to hydrogen peroxide, carbon fibers were modified as Prussian blue as follows.

Using a three-electrode system consisting of a reference electrode (Ag / AgCl), a counter electrode made of platinum wire, and a carbon fiber electrode as an indicator electrode, Fe (NO ₃ ) ₃ dissolved in 1 mM K ₃ [Fe (CN) ₆ ] and 1 mM HCl Cyclic voltammetry was performed by applying a voltage of 0mV to + 2V scan rate of 50mV / s in a solution of the solution dissolved to 1mM, and then 0.1M KCl containing 3mM HCl. Under the electrolyte, a voltage was applied at a scan rate of 50 mV / s from 0 to +0.4 V to form a Purutian blue film.

The blue fiber-modified carbon fiber electrode produced hydrogen peroxide reduction at + 0.2V and hydrogen peroxide oxidation at + 0.9V.

Immobilization of Glutamate Oxidase Using Electrochemical Polymerization

The enzyme uses glutamate oxidase EC 1.4.3.11 extracted from streptomyces. After dissolving 200 mg of glutamate oxidase in 1 ml of a phosphate buffer solution, the enzyme is adsorbed onto the surface of a carbon fiber modified with a blue light, which is electrochemically used as a current source of 5 mA / cm ² using a carbon fiber electrode. Next, as a polymer solution for electrochemical polymerization, m-phenylenediamine and resorcinol were prepared in a phosphate buffer solution in which dissolved oxygen was removed in advance so as to be 3 mM each, and then mixed 1: 1. The carbon fiber electrode is immersed in a polymer solution to undergo an electrochemical polymerization to form a covalent bond layer between the polymer and the enzyme. The electrochemical polymerization is performed by applying +900 mV to the carbon fiber electrode for 10 minutes compared to the Ag / AgCl reference electrode.

As another method of enzyme immobilization, the electrochemical polymerization can be carried out under the same conditions under the condition that the enzyme solution and the polymer mixture solution are mixed at the same time without the enzyme adsorption reaction. In this case, however, it is very important to control the concentration of the enzyme.

In another method, the glutamate oxidase solution prepared above is adsorbed on the surface of a carbon fiber modified with Prussian blue with 0.1% Nafion solution, and then subjected to an electrochemical polymerization reaction in the same manner. A covalent bond layer can be formed.

After the electrochemical polymerization, the electrode is washed two or three times with distilled water and stored at + 4 ℃.

In the measurement of glutamate, in the case of using the reduction reaction of hydrogen peroxide, the sensor standard calibration curve of the glutamate standard solution at 37 ° C. can be obtained by using pH 7.4 phosphate buffered saline, which is +0.2 compared to the reference electrode. The concentration of glutamate can be determined by measuring the reduction reaction of hydrogen peroxide under a voltage of about V.

In addition, when the oxidation reaction of hydrogen peroxide is used, the concentration of glutamate can be obtained by measuring the oxidation reaction of hydrogen peroxide at a voltage of about + 0.9V with respect to the reference electrode.

As described above, according to the present invention, in the sensor for glutamate measurement using the electrochemical measurement method, the enzyme is properly fixed to the electrode material, the signal interference effect by other electrode active materials is excluded, and the response to the hydrogen peroxide of the electrode As a result, the sensitivity of the glutamate sensor can be increased.

Claims (8)

A sensor for glutamate measurement using electrochemical pretreatment and carbon fiber whose surface is treated with an enzyme membrane in which an enzyme and a nonconductive polymer polymerized membrane are covalently bonded. The sensor for glutamate measurement according to claim 1, wherein the carbon fiber is a carbon fiber electrode which is electrochemically modified by using Prussian blue. The sensor for measuring glutamate according to claim 1, wherein the enzyme is glutamate oxidase. The sensor for measuring glutamate according to claim 1, wherein the non-conductive polymer polymer film is a polymer film made of m-phenylenediamine and resorcinol. (a) electrochemically pretreating the carbon fiber electrode; (b) forming a covalent bond film of an enzyme and a non-conductive polymer polymer film on the surface of the electrochemically pretreated carbon fiber electrode; Method for producing a sensor for measuring glutamate consisting of. 6. The method of claim 5, further comprising electrochemically modifying the electrochemically pretreated carbon fiber electrode by using Prussian blue. The method of claim 5, wherein the enzyme is a glutamate oxidase. The method of claim 5, wherein the non-conductive polymer polymer film is a polymer film of m-phenylenediamine and resorcinol.