KR20050103170A - Electrochemical electrode and instrumentation for low glucose concentration - Google Patents

Abstract

 Electrode preparation and measurement method for detecting low concentration glucose using electrochemical methods is an important technique that determines the performance and reliability of the device to quantitatively measure glucose, which is an important energy source of the living body. Typically, glucose concentration quantification is done electrochemically through selective enzymatic reactions. Glucose forms hydrogen peroxide and gluconic acid as reaction products through selective reaction with glucose oxidase, and indirectly determines the concentration of glucose by measuring the oxidation current generated when the hydrogen peroxide is electrochemically oxidized. In the present invention, a low concentration of glucose of less than 1 ppm was quantitatively determined using an efficient electrode production method consisting of platinum and carbon sensitive to low concentrations of hydrogen peroxide and a measuring device using a flow type electrochemical microcell using the electrode.

Description

 Electrochemical Electrode and Instrumentation for Low Glucose Concentration

The present invention provides an electrode manufacturing and measuring method required for the electrochemical method which is one of the methods for quantitatively analyzing glucose (Glucsoe) which is the main component of the human body.

In general, the quantification of glucose molecules can be qualitatively and quantitatively analyzed using liquid chromatography (LC) or ion chromatography (IC) using a column. These assays are qualitative and quantitative in the way that glucose transport differences occur through the interaction of glucose molecules with the column-filled material. The disadvantage of these chromatographic methods is that when other ions are present in the sample containing glucose, substances of other ions are also similar to glucose in the position similar to glucose, so that only the glucose molecules cannot be selectively separated due to interference. There are many disadvantages in over-quantification. In particular, the glucose present in the biological material is often present with many inorganic ions, so the interference effect is very large, and a special pretreatment for removing inorganic ions is required.These salts are difficult to selectively remove, so that they are 100 ppm by chromatography. There are many difficulties in achieving both qualitative and quantitative below concentrations.

Complementing the disadvantage of the chromatographic method is the determination of the concentration of glucose molecules only by measuring the oxidation current by an electrochemical method using an enzyme.

Glucose + Glucose oxidase + O ₂ ---> Gluconic acid + H ₂ O ₂ (1)

Gluconic acid + H ₂ O ₂ ---> 2H ⁺ + O ₂ + 2e ^- E = + 400-700 mV

Glucose molecules react with Glucose Oxidase, converting them into Gluconic acid and Hydrogen peroxide, which produce oxidation currents under oxidation voltages of 400 to 700 mV. The magnitude of the oxidation current is measured by the potentiostat to determine the concentration of glucose indirectly. The electrodes required for these electrochemistry are largely composed of three electrodes, a working electrode (WE), a counter electrode (CE) and a reference electrode (RE). The oxidation potential used in reaction (1) is based on the use of silver / silver chloride (Ag / AgCl) as the reference electrode. In general, the working electrode and the counter electrode use a platinum or carbon electrode with excellent stability. This electrochemical method is superior to the chromatographic method because it is not affected by other inorganic salts of the sample containing glucose, but it is difficult to analyze glucose at low concentrations of about 100 ppm. Therefore, the electrochemical electrode treatment method and measuring device to measure low concentration glucose quickly and quantitatively without external influence can be used as a source technology such as biosensor for glucose measurement. can do.

In the analysis of samples containing glucose molecules, the composition of the electrode is very important in the method of electrochemically analyzing glucose by converting it into hydrogen peroxide through selective enzymatic reaction. Generally, platinum and carbon are used as the working electrode and the counter electrode, but each has characteristics. Platinum is relatively good at detecting the oxidation current under oxidation potential of hydrogen peroxide converted by enzymes in glucose. In the case of the carbon electrode, the noise is small but the detection ability is insufficient, so it is not suitable for quantitative analysis of low concentration of glucose (Korea Patent Publication 10-2003-0047970). In the present invention, by using a 50% wt carbon / 50% wt platinum electrode to compensate for the shortcomings of the platinum electrode and the carbon electrode, a working electrode and a counter electrode were manufactured by a printing method to produce a three-phase electrochemical microcell. It is possible to quantitatively measure low concentration glucose below 1ppm mixed with high concentration inorganic salt by making it as detector of flow analysis device.

1 is a flow type electrochemical measuring device capable of quantitative analysis of glucose at a low concentration of less than 1 ppm. The apparatus consists of five parts: an eluent (a), a sample injector (b), a microfluidic pump (C), an electrochemical microcell (d) used as a detector, and an electrostatic potential (e). Eluents are water-soluble liquids that contain an inorganic salt that can act as an electrolyte in which an electrochemical reaction can occur and does not generate a signal by participating in an electrochemical reaction. The sample injector contains a 0.26 ml sampling loop. Glucose samples in inorganic salts were pretreated to react with glucose oxidase and converted to hydrogen peroxide, and then introduced into the sampling loop of the sample injector. Microfluidic pumps serve to quantitatively transfer eluents and samples to a three-phase electrochemical microcell consisting of a work, reference and counter electrode. The transferred pretreated sample generates an oxidation current through the oxidation reaction of hydrogen peroxide in the electrochemical microcell, and the signal magnitude allows determination of the concentration of low concentration glucose molecules.

<Example>

Eluent (a) used in the present invention is a 0.1 M-phosphate and 77 mM sodium chloride (NaCl) solution commonly used as an electrolyte. 0.5, 1.0 and 2.0 ppm standard solutions were prepared using D-glucose (Aldrich. Co.). At this time, the solvent used for preparing the standard solution was the same electrolyte containing 0.1M-phosphate and 77mM sodium chloride as the eluent. Before injecting the standard solution into the sample injector (b), the 0.4 ml-conjugated sample was converted to 0.1 ml-glucose oxidase (Sigma, 50 unit / ml, USA) and pretreatment via selective enzymatic reaction. The pretreated sample is transferred to an electrochemical microcell via a micro flow pump (flow rate 0.3 ml / min). At this time, the current generated by the oxidation of hydrogen peroxide at the oxidation potential in the electrochemical microcell is detected through an electrostatic crisis (Elbio Co. Korea). The electrochemical microcells used were composed of working electrode, reference electrode and counter electrode. At this time, the working electrode and the counter electrode were mixed with platinum and carbon ink (Ercon Co. USA) in a ratio of 50% wt: 50% wt and printed with a thickness of 0.1 mm by a printing method. At this time, the electrode was dried for 20 seconds using hot air at 120 degrees Celsius to complete the electrode. The area of each electrode was 2.2 mm x 1.6 mm. The reference electrode was printed in the same size as the reference electrode by printing using Ag / AgCl ink (Ercon Co. USA). The oxidation voltage used was 600 mV based on the Ag / AgCl electrode and the volume of the total electrochemical microcell was 0.015 ml. The calibration curve of the standard solution containing the inorganic salt detected using this flow type electrochemical measuring apparatus is shown in FIG. The signal measured at the electrostatic potential is shown as a box inside the figure. Determination of glucose concentration was made by calculating the area through integration through time x oxidation current, it can be seen that glucose concentration of less than 0.5 ppm can be measured.

It was shown that the glucose concentration of less than 1 ppm can be determined by the electrochemical method through the manufacturing method of the flow type electrochemical measuring apparatus and the working electrode of the present invention. In particular, glucose concentrations in many biological samples containing inorganic salts can also be measured.

1 is a flow type electrochemical measuring device

a is eluent

b is the injector

c is a peristaltic pump

d is an electrochemical micro cell

e is the potentiostat

CE: counter electrode, RE: reference electrode, WE: working electrode.

2 is a calibration curve of signals and glucose using a flow type electrochemical measuring apparatus.

Claims (2)

Low concentration glucose measurement device of less than 1ppm minimizing the interference of inorganic ions using the flow type electrochemical microcell detector having the configuration of FIG. The electrode according to claim 1, wherein the working electrode and the counter electrode of the flow type electrochemical microcell are formed of a printing method using a platinum ink and a carbon ink in a weight ratio of 50:50 mixed ink.