JPH04232452A - Measuring apparatus of two components - Google Patents

Abstract

PURPOSE:To make measurement of two components correct by calculating the contribution of a first measuring substance to a second enzyme electrode in the concentration of the first measuring substance obtained from a calibration curve, obtaining the concentration of a second measuring substance, and correcting the concentration of the second measuring substance by means of a function obtained by a standard solution. CONSTITUTION:When glucose as a first measuring substance is fed, hydrogen peroxide is generated by a first enzyme electrode 4, which is detected by a detector 7. When maltose as a second measuring substance passes through an immobilized enzyme coloumn 5, it is converted to glucose and hydrogen peroxide is generated by a second enzyme electrode 6 and detected. A first and a second calibration curves of the concentration of glucose are formed from the detecting values of the first and second enzyme electrodes 4, 6 with use of a glucose standard solution. The concentration of the first measuring substance is measured by the detecting value of the electrode 4 with using the calibration curves. The contribution of the first measuring substance to the electrode 6 is calculated. Then, the concentration of the second measuring substance is obtained from the value obtained by subtracting the contribution from the detecting value of the electrode 6. Moreover, a function of the concentration of the second measuring substance is obtained by using a standard solution of the second measuring substance, so that the concentration of the second measuring substance is corrected.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring device using an enzyme electrode for simultaneously measuring two components in a liquid.

0002

BACKGROUND OF THE INVENTION In recent years, immobilized enzymes have been widely applied in fields such as clinical testing, fermentation production, and analytical chemistry. In particular, immobilized enzyme electrodes are widely used because of the high selectivity of enzyme reactions, rapid measurement, and simplicity. Particularly in recent years, measurement devices that use enzyme electrodes to simultaneously measure two types of substances have been developed.
2-5172), etc., have been introduced. Furthermore, a method for measuring two components in the case where an enzyme reaction that produces or consumes an electrode active substance is common is disclosed (Japanese Patent Laid-Open No. 64-69944).

According to this method, for example, when measuring sucrose, sucrose is converted into fructose and α-
After hydrolysis to D-glucose, α
- converting D-glucose to β-D-glucose;
Further, hydrogen peroxide, which is an electrode active substance produced by oxidizing β-D-glucose with glucose oxidase, is electrochemically detected. When simultaneously measuring two substances, glucose (first measurement substance) and sucrose (second measurement substance), with a flow-type measuring device using an enzyme electrode, an enzyme for glucose measurement with immobilized glucose oxidase is used. An enzyme electrode for detecting both sucrose and glucose on which glucose oxidase, mutarotase, and invertase are immobilized is installed as a second electrode. Then, the concentration of the first measurement substance is directly measured by the first electrode,
For the concentration of the second analyte, use the concentration of the first analyte detected by the first enzyme electrode, calculate the contribution of the first analyte from the value calibrated in advance for the second electrode, and subtract this value from the detected value. It can be found by

[0004] In these methods, the first measuring substance responds to both the first electrode and the second electrode, and the second measuring substance itself does not directly respond to both electrodes, but is converted into the first measuring substance. The two-component measurement was based on the premise that only the second electrode responds after the signal is applied.

[0005]

[Problem to be Solved by the Invention] However, when the second analyte itself responds very slightly to the first enzyme electrode and the second enzyme electrode, this method for creating a calibration curve cannot be used when measuring a pure second analyte. However, since the first enzyme electrode responds slightly, a small amount of the first analyte that is not actually present is detected, and the detection value that contributes to this is subtracted, resulting in the second analyte being measured at a lower value, resulting in an inaccurate measurement value. I couldn't get it. Further, the detected value of the second measuring substance itself is small compared to the detected value of the first measuring substance, and it is difficult to create a calibration curve from the detected values of the second measuring substance. Furthermore, accurate measurement values cannot be obtained if the first measurement substance is mixed in the second measurement substance standard solution. The present invention aims to more accurately measure these two components.

[0006]

[Means for Solving the Problems] The present invention provides, from the upstream side,
A first enzyme electrode and a second enzyme electrode, both of which have an enzyme that produces a substance electrochemically detectable from the first measurement substance, are disposed, and further between the first enzyme electrode and the second enzyme electrode, or between the second enzyme electrode and the second enzyme electrode. A flow type measuring device having an enzyme electrode immobilized on an enzyme electrode to generate a first measuring substance from a second measuring substance, and (a) a standard solution of the first measuring substance and a first standard solution of the second measuring substance. A calibration curve is created from the detection values of the enzyme electrode and the second enzyme electrode, respectively. (a) Using the calibration curve, calculate the concentration of the first measuring substance based on the detection value of the sample at the first enzyme electrode. Calculate the contribution to the second enzyme electrode in the concentration of the first measuring substance, and calculate the concentration of the second measuring substance from the value obtained by subtracting the obtained contribution from the detected value of the second enzyme electrode of the sample, and (c) further The function of the set concentration of the standard solution of the second measuring substance and the concentration of the second measuring substance obtained by performing the measurement in (a) using this standard solution is determined, and the measurement sample obtained by performing (d) and (b) This is a two-component measuring device equipped with means for correcting the concentration of the second measuring substance in the sample using the function to obtain an accurate value.

[0007] Furthermore, the present invention provides that (a) to (b) are
A first calibration curve is created from the detection value of the first enzyme electrode regarding the standard solution of the first measurement substance, a second calibration curve is created from the detection value of the second enzyme electrode regarding the standard solution of the first measurement substance, and a second calibration curve is created from the detection value of the second enzyme electrode regarding the standard solution of the first measurement substance. A third calibration curve is created from the detection value of the second enzyme electrode regarding the standard solution of the two measurement substances, and (a) the concentration of the first measurement substance is calculated from the detection value of the sample at the first enzyme electrode and the first calibration curve. Then, the contribution to the second enzyme electrode at this first measurement substance concentration is calculated from the second calibration curve, and the obtained contribution is subtracted from the detection value of the second enzyme electrode of the sample and the third calibration curve. The above-mentioned two-component measuring device is disclosed, in which the concentration of the second measurement substance is determined by .

[0008] The present invention provides a first enzyme electrode and a second enzyme electrode on which an enzyme whose main substrate is a first measurement substance is immobilized;
An enzyme that generates the first measurement substance from the second measurement substance is immobilized on the upstream side of the enzyme electrode or on the second enzyme electrode,
A flow type two-component measuring device is disclosed that has a calculation storage means, and the calculation storage means performs the following measurements. [Calculation of calibration curve and correction formula] (a) Obtain the detection values of the first enzyme electrode and the second enzyme electrode using the first analyte standard solution,
Calculate a calibration curve and a second calibration curve for the first analyte at the second enzyme electrode; (b) Calculate the second calibration curve for the first analyte using the second analyte standard solution;
Calculate the detected value of the enzyme electrode, calculate a third calibration curve for the second measuring substance at the second enzyme electrode, and (c) calculate the detected value of the second measuring substance standard solution at the first enzyme electrode and the first calibration curve. , calculate the concentration of the first analyte detected from the second analyte standard solution, calculate the contribution to the second enzyme electrode from the first analyte concentration using the second calibration curve, Subtracting the contribution from the detection value of the second enzyme electrode of the standard solution, calculating the second measurement substance concentration X' from this subtraction value and a third calibration curve,
Furthermore, a correction formula for X' that is a relational formula between the set concentration X of the standard solution of the second measurement substance and X' is calculated. [Sample measurement] (d) Obtain the detection values of the first enzyme electrode and the second enzyme electrode when measuring the sample, and calculate the first measurement substance concentration from the detection value of the first enzyme electrode and the first calibration curve. , calculate the contribution of the first measuring substance at the second enzyme electrode from this value and the second calibration curve, and calculate the second measurement from the value obtained by subtracting the contribution from the detection value of the second enzyme electrode and the third calibration curve. The substance concentration is calculated, and the second measurement substance concentration is further corrected using the correction formula to calculate an accurate second measurement substance concentration.

[0009]

[Operation] In the present invention, the first substance to be measured is glucose, and the second substance to be measured is glucose.
Regarding the two components in which the measuring substance is maltose, we will mainly discuss a two-component measuring device using a platinum electrode with glucose oxidase immobilized on the electrode surface and a column with maltose phosphorylase immobilized on a silicic acid carrier. As a means of generating the first analyte from the second analyte without using the second analyte, the second analyte is
There are also methods such as using a second electrode as an electrode, or a second electrode on which two or more types of enzymes are immobilized at the same time. That is, the enzyme to be immobilized on the column in the above example can also be immobilized directly with the enzyme of the second enzyme electrode in a multilayered form or in a mixed manner.

[0010] Furthermore, a method such as injecting an enzyme solution into the channel in front of the second enzyme electrode may be considered, but of course this method is not a desirable method because it makes the expensive enzyme disposable. In addition to glucose and maltose, two-component measurements of glucose and sucrose using glucose oxidase, mutarotase, and invertase, two-component measurements of glucose and malto-oligosaccharides using glucose oxidase and glucoamylase, glucose oxidase and β-
Carbohydrate measurements such as glucose, cellobiose, and cellooligosaccharide two-component measurements using glucosidase, two-component measurements of urea and amino acids using an ammonia electrode and amino acid oxidase, and two-component measurements of amino acids and peptides using amino acid oxidase and peptidase. The present invention can be similarly applied to cases where an enzyme immobilized on an electrode for detecting a first substance to be measured also responds to a second substance to be measured.

FIG. 1 is a system diagram showing an example of the two-component measuring device of the present invention. Figure 1 shows a flow type system in which the buffer is pumped from the buffer tank (1) to the liquid pump (2).
The substance to be measured is injected from the sampler (3) and transferred from the first enzyme electrode (4) to the immobilized enzyme column (5).
), the second enzyme electrode (6), and the drainage tank (13).
sent to.

[0012] Each electrode is placed in a constant temperature bath (14) at 37°C, and a voltage is applied by a detector (7) based on instructions from a single board computer (8), and a current output value based on each measured substance is determined. The information is then sent to the single board computer (8) using a means for digitally converting minute currents, such as current amplification and A/D conversion. The single board computer (8) is also connected to the liquid pump (2) and sampler (3), where storage, judgment, settings, and calculations such as calibration and concentration determination are performed. Output the results etc.

In the following explanation, the first enzyme electrode and the second enzyme electrode are glucose detection electrodes in which glucose oxidase is immobilized on a platinum substrate, and the immobilized enzyme column (5) is an electrode in which maltose phosphorylase is immobilized on a silicic acid carrier. Perform this on an immobilized column. When glucose, which is the first substance to be measured, is injected, hydrogen peroxide is produced by the first enzyme electrode (4) on which glucose oxidase for glucose detection is immobilized, and the detector is detected by the current generated when hydrogen peroxide is electrolyzed. (7) is detected. Maltose, which is the second substance to be measured, is converted to glucose when passing through the column (5) on which maltose phosphorylase is immobilized.
A second enzyme electrode (6) immobilized with the same enzyme as the first enzyme electrode
) produces hydrogen peroxide, which is detected. Therefore, by determining the glucose concentration before and after passing through this immobilized enzyme column, it is possible to measure the maltose concentration and glucose concentration in the sample. In other words, the detection values of the first enzyme electrode and the second enzyme electrode using the glucose standard solution (YG1, YG2
), a first calibration curve and a second calibration curve are created for the glucose concentration (XG).

[0014]YG1=AG1×XG+BG1
...(1) YG2=AG2 × XG +BG2
(2) Next, a third calibration curve is created for the maltose concentration (XM) from the detection value (YM2) of the second enzyme electrode using the maltose standard solution. YM2=AM2×XM+BM2…(3)
The glucose and maltose concentrations can be measured from these three calibration curves, the detection value (Y1) of the first enzyme electrode using glucose, and the detection value (Y2) of the sample containing glucose and maltose at the second enzyme electrode.

[0015]Y1=YG1...(4)Y2=YG
2+YM2...(5) In the measurement of an unknown sample, the glucose concentration (XG) is first determined from the detection value of the first enzyme electrode. (4), (1)
Therefore, Y1 = YG1 = AG1 × XG + BG1.

[0016] The glucose contribution (YG2) at the second enzyme electrode is subtracted from the first calibration curve and the second calibration curve. (5),
From (2), YM2=Y2-YG2=Y2-(AG2×XG+BG2).

Then, the maltose concentration (XM) is determined from the remaining detected value (YM2). From (3), YM2=AM
2 × XM + BM2. However, this enzyme electrode for glucose detection slightly responds to not only glucose but also maltose and shows a detected value.

[0018]YM1=AM1×XM+BM1
(6) Therefore, the detected value of the first enzyme electrode is the sum of the detected value of glucose and the detected value of maltose. Y'1 = YG1 + YM1 ... (7) However, from (1), Y'1 = AG1 × X'G + BG1 ... (
8), and in samples containing maltose, the first
The detection value with the enzyme electrode will be slightly higher. (5), (2
), YM2=Y2 −YG2 Y′M2 =Y2 −(AG2 × X′G +
BG2)...(9), and the value subtracted from the detection value of the second enzyme electrode also becomes higher. From (3), Y'M2=AM2 × X'M +BM2...(
10), and the maltose concentration is measured at a lower value than the actual value.

[0019] Therefore, the first calibration curve, the second calibration curve, and the third calibration curve are
After creating the calibration curve, measure the maltose standard solution or
Determine the measured concentration from the detected values when creating the calibration curve. Determine the correction formula from the measured concentration of maltose and the concentration of the standard solution. XM = A × X'M + B (11) If the maltose concentration is corrected using this correction formula, an accurate maltose concentration can be determined.

The creation of a calibration curve and actual measurement will be explained in detail below based on a flowchart. [Step of determining a calibration curve and correction formula] Figure 3 shows the steps of determining the detection values of the first enzyme electrode and the second enzyme electrode using the first measurement substance standard solution, and calculating the detection values of the first measurement substance at the first enzyme electrode. 3 shows steps for calculating a first calibration curve and a second calibration curve for the first measurement substance in the second enzyme electrode.

FIG. 4 shows the first measurement using the second measurement substance standard solution.
FIG. 5 shows a step of determining the detected values of the enzyme electrode and the second enzyme electrode and calculating a third calibration curve for the second measuring substance at the second enzyme electrode. Although it is not an essential step, FIG. The step of determining detection values of the first enzyme electrode and the second enzyme electrode using a standard solution is shown. Comparing the case where the step of FIG. 5 is performed and the case where it is not performed, there is an advantage that when the step shown in FIG. 5 is performed, the accuracy is improved by acquiring the detected value of the second measurement substance standard solution twice.

FIG. 6 shows the second
The first analyte concentration of the second analyte standard solution is calculated from the detection value of the analyte standard solution at the first enzyme electrode and the first calibration curve, and the contribution from the first analyte concentration to the second enzyme electrode is calculated. The contribution is subtracted from the detection value of the second enzyme electrode of the second analyte standard solution, and the second analyte concentration X'M is calculated from the subtracted value and the third calibration curve. Calculate,
Furthermore, it shows a step of calculating the correction formula (11) from the set concentration XM of the standard solution of the second measuring substance.

The second analyte standard solution is detected by the first enzyme electrode because the first analyte is contained as an impurity in the second analyte standard solution, or because the second analyte itself is present in a small amount. It is thought that this is because it becomes a substrate for the enzyme of the first enzyme electrode. [Actual sample measurement] Figure 7 shows the detection values of the first enzyme electrode and the second enzyme electrode when measuring the sample, and the detection values of the first enzyme electrode and the first calibration curve are used to calculate the first measurement substance. Determine the concentration, calculate the contribution of the first measuring substance at the second enzyme electrode from this value and the second calibration curve, and calculate the contribution from the detection value of the second enzyme electrode by subtracting the contribution from the third calibration curve. Step of determining the second measurement substance concentration. Furthermore, a step of calculating an accurate second measurement substance concentration using correction formula (11) is shown.

[0024]

[Examples] The present invention will be explained in more detail with reference to Examples below, but of course the present invention is not limited thereto.

Example 1 Using the measuring device shown in FIG. 1, using distilled water as a blank sample, 10, 20, and 30 mM glucose standard solutions and 10,
After creating a calibration curve using 20, 30, and 40 mM maltose standard solutions, the maltose standard solutions were measured again and a correction formula was determined. The results are shown below, and the calibration curve is shown in FIG.

From the measured values in Table 1, the first, second and third calibration curves and the maltose calibration curve of the first enzyme electrode were determined. From (1), the first calibration curve YG1 = 8.83XG +0.66...(a) Correlation coefficient
r=1.000 From (2), the second calibration curve YG2=12.58XG -1.73...(b) r=1.
000 From (3), the third calibration curve YM2 = 3.49XM +0.15...(c) r = 1.0
00 From (6), the first enzyme electrode maltose calibration curve YM1=0.
17XM -0.49...(d) r=0.992

[0027]

[Table 1]

Next, the case of re-measuring the second measurement substance standard solution in FIG. 5 will be explained. The maltose standard solution was measured again and the results shown in Table 2 were obtained. Maltose measurement value X'M
and the standard solution concentration XM, the correction formula (11) was determined. Correction formula XM =1.064 × X'M -0.52r=
1.000

[0029]

[Table 2]

[0030] Using this correction formula, it is possible to accurately determine the maltose concentration of an actual sample.

Example 2 In FIG. 5, a method of determining a correction formula using the detected value of the previously obtained maltose standard solution without re-measuring the second test substance standard solution will be described. After creating a calibration curve using the measured values in Table 1 of Example 1, the measured values were calculated using the detected values of the maltose standard solution, and a correction formula was determined. The results are shown below. Table 3 is calculated from the measured values in Table 1.

[0032]

[Table 3]

Correction formula XM = 1.077 × X'M -0.90r=
0.999 Table 4 shows the results of measurements of actual samples using the results of Examples 1 and 2 above.

Values measured on samples using the first to third calibration curves of Example 1 (before correction), values corrected using the correction formula obtained by the method of Example 1 (Example 1), and the method of Example 2 The values corrected using the correction formula obtained in (Example 2) are shown below. Also HPL
Measured values by C are shown.

[0035]

[Table 4]

In Examples 1 and 2, the same results as the HPLC measurement values were obtained. On the other hand, before correction without using a correction formula, the maltose value was a small value.

[0037]

[Effects of the Invention] By using the two-component measuring device of the present invention, when the second substance to be measured responds very slightly to the first enzyme electrode and the second enzyme electrode, the second substance to be measured responds to the first enzyme electrode and the second enzyme electrode very slightly. It became possible to obtain more accurate measurement values for the two measured substances.

[Brief explanation of the drawing]

FIG. 1 is a system diagram of a glucose/maltose two-component measuring device which is an example of the present invention.

[Fig. 2] Fig. 2 shows the detection values of the standard solutions of glucose and maltose in Example 1 at the first enzyme electrode and the second enzyme electrode, (a) is the first calibration curve, and (b) is the 2 calibration curve,
(c) is the third calibration curve, and (d) is the maltose first electrode calibration curve.

FIG. 3 shows steps for calculating a first calibration curve and a second calibration curve.

FIG. 4 shows the step of calculating a third calibration curve.

[Fig. 5] Fig. 5 shows that the first
The steps of obtaining detection values of the enzyme electrode and the second enzyme electrode are shown.

FIG. 6 shows steps for calculating a correction formula.

FIG. 7 shows the step of determining the second measurement substance concentration;
and a step of calculating an accurate second measurement substance concentration using a correction formula.

[Explanation of symbols]

1 Buffer tank 2 Liquid pump 3 Sampler 4 First enzyme electrode 5 Immobilized enzyme column 6 Second enzyme electrode 7 Detector 8 Single board computer 9
RS232C code 10 Personal computer 11 Sampler control signal 12 Liquid pump control signal 13 Drainage tank 14 Constant temperature tank

Claims (2)

[Claims] Claim 1: From the upstream side, a first enzyme electrode and a second enzyme electrode are disposed, both electrodes having an enzyme that generates an electrochemically detectable substance from a first measuring substance, and further comprising a first enzyme electrode and a second enzyme electrode. A flow type measuring device having an immobilized enzyme that generates a first analyte from a second analyte between a second enzyme electrode or on the second enzyme electrode, and (a) a standard solution of the first analyte, a first analyte, 2. Create a calibration curve from the detection values of the first enzyme electrode and the second enzyme electrode regarding the standard solution of the two measuring substances, and (a) use the calibration curve to perform the first measurement based on the detection value of the sample at the first enzyme electrode. Calculate the concentration of the substance, calculate the contribution to the second enzyme electrode in this first measurement substance concentration, and subtract the obtained contribution from the detection value of the second enzyme electrode of the sample. (c) Furthermore, find the function of the set concentration of the standard solution of the second analyte and the concentration of the second analyte obtained by performing the measurement in (b) using this standard solution, and (d) ( A two-component measuring device comprising means for correcting the concentration of the second substance to be measured in the measurement sample obtained by performing (b) using the function to obtain an accurate value. [Claim 2] (A) to (B) include: (a) creating a first calibration curve from the detection values of the first enzyme electrode regarding the standard solution of the first measurement substance; Create a second calibration curve from the detected values of the two enzyme electrodes,
In addition, a third calibration curve is created from the detection value of the second enzyme electrode regarding the standard solution of the second measurement substance, and (a) the concentration of the first measurement substance is calculated from the detection value of the sample at the first enzyme electrode and the first calibration curve. Calculate the contribution to the second enzyme electrode at this first measurement substance concentration from the second calibration curve, and subtract the obtained contribution from the detection value of the second enzyme electrode of the sample and the third 2. The two-component measuring device according to claim 1, wherein the concentration of the second measuring substance is determined by a calibration curve.