KR20040017605A - Method of determining concentration of glucose using alternative current impedance - Google Patents

Abstract

PURPOSE: A method for measuring the density of glucose is provided to easily measure the density of glucose within a short period of time by using A/C impedance without utilizing ferment. CONSTITUTION: Alternate current voltage and direct current voltage are simultaneously applied to a bio sensor including an electrode and a platinum layer coated on the electrode. An alternate current waveform is obtained when the alternate current and direct current are simultaneously applied to the bio sensor. The alternate current waveform is analyzed so as to measure impedance. The impedance is calculated through a non-linear least square method. The alternate current voltage is about 5 to 10mV. The frequency of the alternate current voltage is about 10mHz to 100kHz.

Description

Method of measuring glucose concentration using the alternating impedance method {METHOD OF DETERMINING CONCENTRATION OF GLUCOSE USING ALTERNATIVE CURRENT IMPEDANCE}

[Industrial use]

The present invention relates to a method for measuring glucose concentration using an alternating current impedance method, and more particularly, to a glucose concentration using an alternating current impedance method that can accurately measure glucose concentration using a platinum-plated electrode and an alternating current impedance. It relates to a measuring method.

[Prior art]

A biosensor refers to a sensor in which a biomaterial is organically coupled with an electronic measuring device to convert chemical information in an arbitrary sample into an easily processed electrical signal. The biosensors are widely developed and widely used in the medical field because of the advantage that the quantitative information of the desired chemical species can be obtained very selectively in real time without the need for complicated chemical and biological treatment. In particular, there are many studies on biosensors for measuring glucose in diseases in which blood glucose should be frequently monitored, such as diabetes.

The biosensor for glucose measurement measures the concentration of glucose, an analyte, by means of a sensitized membrane (enzyme layer) immobilized with an enzyme that reacts with glucose, a biological substance, to an electrode.

As described above, a biosensor for measuring enzymatic glucose using an enzyme has been extensively studied and developed in various forms. However, enzymes are unstable because they are affected by temperature and pH and are affected by activity depending on ambient conditions.

Therefore, there is a need for a biosensor for measuring non-enzymatic glucose that does not use enzymes, but studies on it have not been actively conducted. In addition, most of the non-enzymatic glucose sensors studied are interfered with in vivo by the major interferers such as ascorbic acid, urea acid and aminoacetophenone. An example of a study on a biosensor for measuring non-enzymatic glucose is a sensor using glucose direct oxidation on a platinum surface (Anal. Chem. 2001, 73, 1599-1604). Since the method of directly oxidizing glucose in the platinum electrode is much slower than the oxidation rate of the interfering substance, it is very difficult to implement a non-enzymatic amperometric sensor using platinum as an electrode.

On the other hand, the AC impedance method is expected to reduce the effects of interference materials, unlike DC voltammetry method, and the study of measuring the concentration of the target substance in the living body by applying this method to the biosensor is urgently required.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a glucose concentration measuring method using a more convenient alternating current impedance method without using an enzyme.

Another object of the present invention is to provide a glucose concentration measuring method using the alternating current impedance method which can measure the concentration of the target substance in a very short time.

1 is a graph of glucose concentration using charge transfer resistance (RCT) and its inverse.

In order to achieve the above object, the present invention applies a direct current voltage and an alternating voltage to a biosensor comprising an electrode and platinum plated on the electrode, and analyzes the alternating current waveform obtained in the application process to measure the impedance, It provides a method for measuring glucose concentration using the alternating current impedance method comprising the step of calculating the impedance by a nonlinear least squares method.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for measuring the concentration of a substance in a living body using a non-enzymatic biosensor that does not use an enzyme. The present invention uses platinum and uses an alternating current impedance method.

To, as shown in equation (1), glucose oxidation current (i) is a potential (E) and a charge transfer resistance: and the values and relationships (charge transfer resistance R _CT), glucose oxidation current is proportional to the glucose concentration. Therefore, it can be seen that the inverse of the R _CT is proportional to the glucose concentration.

[Equation 1]

The present invention uses this principle to measure the glucose concentration according to the alternating current impedance method, wherein a platinum plated electrode is used as the electrode.

In the method for measuring the concentration of a substance in a living body of the present invention, first, a biosensor is immersed in a phosphate buffer solution containing glucose, and then a direct current voltage and an alternating voltage are simultaneously applied to the biosensor. The phosphate buffer solution uses a solution of about 0.1 M concentration and pH 7.2.

The DC voltage is preferably 0.3 to 0.4 V relative to the reference electrode, and the AC voltage is preferably 5 to 10 mV in amplitude. When the DC voltage is 0.3 to 0.4V relative to the reference electrode, the oxidation of glucose is relatively superior to other processes such as hydrogen adsorption and desorption, the formation and disappearance of platinum oxide, and the reduction of oxygen. Therefore, impedance measurement related to oxidation of glucose is measured. It is desirable because it can be performed successfully.

The biosensor is a non-enzymatic biosensor composed of a metal electrode and platinum plated on the metal electrode. The biosensor used in the present invention is preferable because the overvoltage required for electrochemically oxidizing glucose on the metal electrode is relatively low compared with other metal electrodes, and thus, platinum is plated, for example, deposited to facilitate the direct oxidation of glucose relatively easily.

The metal electrode may be an inert metal such as carbon, gold, or negative metal, or a metal having resistance to acid such as stainless steel. Ag / AgCl is used as the reference electrode.

At this time, the frequency of the AC voltage is preferably set to 10mHz to 100kHz.

The impedance is measured by analyzing the phase difference compared to the DC voltage applied to the obtained AC current waveform.

The measured impedance value is applied to the nonlinear least-squares method according to the equivalent circuit of the following Scheme 1 to measure the concentration of the substance in vivo. A system composed of a solution, a working electrode and an auxiliary electrode inserted therein can be thought of as an electric circuit, and this electric circuit can be considered as the following Scheme 1. In practice, the values of the configuration variables in Equation 1 can be determined so that the measured impedance changes in the same way as the AC frequency changes. The method used is the well-known nonlinear least-squares method. At this time, one of the configuration variables, Rct, is a charge transfer resistance at the electrode, which decreases as electrons enter and exit due to an electrochemical reaction at the electrode. Therefore, by measuring the value of this resistance, the rate of redox reaction at the electrode can be measured. In the DC voltage region where glucose oxidation is the main reaction, this resistance value decreases as the oxidation of glucose becomes active.

[Circuit Formula 1]

In the formula 1, R _CT means a charge transfer resistance,

R _S means the resistance value of the solution,

C _DL is the capacitance of the electrical double layer,

R _OX and CPE _OX are the circuit variables added to compensate for the remaining electrical components.

Hereinafter, preferred embodiments of the present invention will be described. However, the following examples are only one preferred embodiment of the present invention and the present invention is not limited to the following examples.

(Example 1)

After immersing the biosensor prepared by electroplating platinum on the gold metal electrode in 0.1 M phosphate buffer solution (pH 7.2), the biosensor was + 0.4V DC voltage compared to the reference electrode (Ag / AgCl) and alternating current having an amplitude of 10 mV. Applied with voltage. At this time, the frequency of the AC voltage was changed between 10mHz-100 kHz.

The phase difference was analyzed by comparing the obtained DC current waveform with the applied DC voltage and the impedance was measured accordingly.

The measured impedance values were calculated by the nonlinear least-squares method according to the equivalent circuit of the following Scheme 1.

[Circuit Formula 1]

At this time, the value of R _CT was measured while increasing the glucose concentration of the buffer solution to 0, 10, 20 mM, and the results are shown in FIG. 1. If also the reciprocal value of the R _CT according to the results shown in the first measurement, and thus it can be seen that R _CT is proportional to the glucose concentration can be seen that it is possible to quantify the glucose.

In order to determine the effect of the interfering substance in the method according to Example 1, the sensitivity to glucose concentration for the presence of 0.1 mM ascorbic acid when the R _S value in the 0.1 M phosphate buffer solution was determined is shown in Table 1 below. It was. In Table 1 below, the numbers outside the parentheses represent relative Rct values, which are measured with 1 as the absence of glucose in 0.1M phosphate release solution, and the numbers in parentheses represent the inverse values of Rct. As shown in Table 1, it can be seen that as the glucose concentration increases, the effect of ascorbic acid decreases.

solution Glucose concentration [mM] 0 10 20 0.1M phosphate buffer solution 1.000 (1.00) 0.294 (3.74) 0.194 (5.39) 0.1mM Ascorbic Acid / 0.1M Phosphoric Acid Buffer Solution 0.694 (1.44) 0.233 (4.29) 0.190 (5.27)

Table 2 shows the results of response to glucose measured by direct current method. Comparing Tables 1 and 2, it can be seen that the response by the AC impedance method is less affected by ascorbic acid than the response by the DC current method.

Response to glucose concentration by direct current method (nA) solution Glucose concentration [mM] 0 5 10 20 0.1M phosphate buffer solution 0 420 698 804 0.1mM Ascorbic Acid / 0.1M Phosphoric Acid Buffer Solution 250 362 420 554

In Table 2 above, the current in the 0.1 M phosphate buffer solution was calculated by setting it as 0.

As described above, the present invention can measure the concentration of glucose by using an alternating current impedance method using a platinum-plated electrode without using an enzyme, and the interference of the interfering substances commonly encountered in non-enzymatic glucose sensors is alternating. Using the impedance method can be significantly suppressed. In addition, observing the response to the alternating current is likely to improve the shortcomings of conventional amperometric sensors that wait tens of minutes because the DC current is stabilized.

Claims (4)

Applying a direct current voltage and an alternating voltage to a biosensor comprising an electrode and platinum plated on the electrode; Analyzing the alternating current waveform obtained in the applying process to measure impedance; The impedance is calculated by nonlinear least squares method Method of measuring glucose concentration using an alternating current impedance method comprising the step. The measuring method of claim 1, wherein the AC voltage is 5 to 10 mV. The method of claim 1, wherein the frequency of the AC voltage is 10 mHz to 100 kHz. The method of claim 1, wherein the DC voltage is 0.3 to 0.4 V relative to a reference electrode.