JP3275504B2 - Flow type measuring device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a flow type measuring device for quickly and accurately measuring a specific component in an unknown sample.

[0002]

2. Description of the Related Art A sample is injected into a continuous stream and reacts with a specific component in the stream, and the resulting change in absorbance, change in fluorescence intensity, amount of chemiluminescence, or concentration of an electrode active substance is measured. The flow-type measuring device for detecting and quantifying a substance can perform quick measurement and can perform quantification with a small sample amount. At present, it is widely used in fields such as clinical analysis and food analysis.

As a reaction method, many methods utilizing reagents used in conventional colorimetric analysis and the like have been put to practical use. However, an immobilized enzyme is used to further improve the excellent substrate selectivity and reaction of the enzyme. Attention has been paid to flow type measurement devices that utilize selectivity and high speed.

[0004] Among them, an apparatus using an immobilized enzyme in a reaction part and an electrochemical detector in a detection part can perform high-precision analysis with a simple structure, and many research examples have been reported. For immobilized enzymes, besides using a single enzyme,
It has become possible to quantify a wider range of measurement targets by combining two or more types of enzymes. Similarly, by providing two or more electrochemical detection units, simultaneous quantification of two or more components is also possible.

For example, two-component measurement of glucose and sucrose using an electrode on which glucose oxidase is immobilized and an electrode on which glucose oxidase, invertase, and mutarotase are immobilized (Japanese Patent Publication No. 5-5581)
No. 6) and two electrodes having glucose oxidase immobilized thereon were arranged in series, and a column filled with a carrier immobilized with maltose phosphorylase, which performs a maltose-to-glucose conversion reaction, was provided in front of the rear glucose electrode. Measurement of two components of glucose and maltose
No. 8279) has already been disclosed.

In these measurements, a glucose electrode is arranged on the first electrode side, glucose present in the sample is measured in advance, and then sucrose or maltose is converted into glucose by an enzymatic reaction. The total amount of generated glucose is measured on the second electrode side. A calibration curve is created in advance, the glucose concentration is calculated from the output value of the first electrode, the contribution of glucose to the second electrode is corrected by calculation, and sucrose or maltose can be quantified from the corrected output value of the second electrode. This method has an advantage that two components can be measured by one sample injection.

[0007] Since the conversion of sucrose or maltose into glucose is performed by an enzyme reaction, when the enzyme concentration is sufficient and the reaction is performed at high speed, highly accurate measurement is possible. However, depending on the type, activity amount, and nature of the enzyme, the reaction rate is slow, and the peak position is shifted between the two types of standards, which may cause an error when analyzing the mixture. Also in the above example, when the amount of the enzyme is small, a delay occurs between the peak time of glucose and the peak time of sucrose or maltose, and when the mixed solution is injected, the calculated concentration of sucrose or maltose becomes lower. Was.

A first object of the present invention is to solve this problem and to provide an apparatus capable of performing accurate measurement when an output is obtained from an electrode based on a plurality of substances.

Further, for example, when the detecting section is a hydrogen peroxide electrode, there is a problem that a response value is shown not only for hydrogen peroxide but also for a measurement interfering substance such as ascorbic acid. In order to accurately measure hydrogen peroxide, a permselective membrane is provided to reduce the influence of interfering substances. In this case, the peak positions of interfering substances such as hydrogen peroxide and ascorbic acid to be measured may be shifted due to the difference in the diffusion rate in the film. There is no problem when the concentration of the interfering substance such as ascorbic acid is low, but when the concentration of the interfering substance increases, the peak position tends to be delayed due to the influence of the interfering substance peak.
For example, when a calibration curve is created using glucose at a hydrogen peroxide electrode having a glucose oxidase-immobilized membrane and a glucose solution containing a high concentration of ascorbic acid is analyzed as an unknown sample, an accurate peak is obtained due to the output of ascorbic acid. No accurate quantification was possible. The thickness of the film may be increased in order to remove the influence of the obstacle as much as possible, but in this case, there is a disadvantage that the sensitivity is reduced.

A second object of the present invention is to solve this problem, to reduce the influence of an obstacle, and to provide an apparatus for measuring with high sensitivity.

[0011] Although these techniques are described in the prior art using an immobilized enzyme and an electrode as an example, naturally, when a solution reaction is carried out in a flow-type measuring apparatus, peaks occur between a standard solution and an unknown sample containing interfering substances. Similarly, there are cases where accurate measurement cannot be performed due to the displacement.

[0012]

As described above, the conventional flow-type measuring apparatus has insufficient measurement accuracy. The present invention provides a more accurate flow-type measuring device.

[0013]

The gist of the present invention is illustrated by the following embodiments, but is not limited thereto.

The present invention provides a mechanism for continuously feeding a liquid (carrier), a sample injection mechanism for injecting a sample into a continuous carrier flow, and a reaction for causing a specific component in the injected sample to react in the flow. The present invention relates to a flow-type measuring device including a unit and a detecting unit for detecting a change resulting from the reaction of a specific component in a sample, and the flow-type measuring device has the following mechanism. A mechanism for storing the output waveform of the standard solution at the time of injecting the standard solution containing the specific component, a mechanism for detecting a peak position from the stored waveform and storing the peak position, and a mechanism for storing the peak position for a predetermined time. A mechanism for adding or subtracting a position and storing the result, a mechanism for storing an output value at the added or subtracted position, and a mechanism for creating a calibration curve using the output value and a specific component concentration in a standard solution And a flow-type measuring apparatus having a mechanism for calculating the concentration of a specific component in an unknown sample from the calibration curve and the output value of the unknown sample at the position added or subtracted in the above. This function enables higher accuracy.

Further, in the flow-type measuring apparatus having the mechanism of the present invention, the reaction section is an immobilized enzyme reactor filled with an immobilized enzyme carrier, and the detection section includes an electrode which responds to a substance increased or decreased by the immobilized enzyme reactor. It can be applied to the measuring device used.

Further, the flow-type measuring device having the mechanism of the present invention is applied to a measuring device using a reaction part having an immobilized enzyme membrane and a detection part using an electrode responding to a substance increased or decreased by the immobilized enzyme membrane. Can be.

In the present invention, in the measurement of an unknown sample, quantification is not always performed using the output value at the top of the peak, but is performed using the output value at a position predetermined by the substance to be measured. Thereby, the above-mentioned object of the present invention can be achieved.

[0018]

[Function] The flow-type measuring device is composed of a pump that sends liquid, an injection mechanism that automatically or manually injects a fixed amount of sample into the flow, and a specific component in the sample that reacts in the flow and changes. And a detection unit for detecting a change obtained in the reaction unit.

In the detecting section, a change in absorbance, a change in fluorescence intensity,
The amount of chemiluminescence or the concentration of the electrode active substance is detected by a spectrophotometer or an electrode. Regarding the method of calculating the detection value, a specific component in the sample is used as it is or a conversion reaction is performed in the reaction section, and a calibration curve is created from the relationship between the output of the detection section and the concentration of the standard. Conventionally, as this output value, the maximum value when the horizontal axis represents the time or the flow rate and the vertical axis represents the output value is used. That is, a calibration curve was created by obtaining the peak value from the output waveform. Similarly, in the case of an unknown sample, the peak value was detected, and the value was substituted into the calibration curve to calculate the concentration. An area method using the total area of the waveform may also be used.However, since it is necessary to continue the measurement until the waveform is completely returned to the baseline after the sample is injected, a method using the peak value is used. This is a disadvantageous method because the measurement time is longer than that of the method.

A problem in performing quantification using the peak value is the efficiency of the conversion part. In particular, in the case of a measurement method using an enzyme, the reaction rate may be slow depending on the type, activity amount, and properties of the enzyme, and the peak position may deviate between a standard containing a specific component and an unknown sample, making accurate measurement impossible. is there. In addition, the problem becomes more apparent when the detection unit detects a specific component and performs a conversion by an enzymatic reaction in the preceding stage to generate a component detectable by the detection unit.

Hereinafter, a solution to the first object of the present invention will be described. For example, a two-component measurement system of glucose and sucrose will be described as an example. In this measurement system, two electrodes are arranged in series or in parallel, a first electrode is a glucose electrode, and a second electrode is a sucrose electrode. And glucose oxidase is immobilized on the glucose electrode,
Glucose oxidase, invertase, and mutarotase are immobilized on the sucrose electrode.

First, the glucose in the sent sample is sent to the first electrode and is consumed by the action of glucose oxidase to generate hydrogen peroxide. The first electrode detects, for example, the amount of decrease in oxygen or the amount of increase in hydrogen peroxide. Note that the first electrode does not respond to sucrose in the sample.

Glucose sent to the second electrode undergoes the same reaction as glucose oxidase immobilized on the second electrode. On the other hand, sucrose is hydrolyzed by the invertase at the second electrode to generate α-D-glucose, and a conversion reaction is performed by mutarotase to β-D-glucose, which is a substrate of glucose oxidase,
It is detected at the second electrode.

Therefore, it is possible to prepare a calibration curve using glucose and sucrose standard solutions, and to measure an unknown sample in which both are mixed. The glucose concentration is determined by the first electrode. Since the second electrode reacts to glucose and sucrose, the glucose concentration determined at the first electrode and the second
The glucose contribution is corrected from the calibration curve for glucose at the electrode. Then, the corrected second electrode output value is applied to a sucrose calibration curve, and sucrose is quantified. As described above, since sucrose to glucose has an extra two-step reaction, if the invertase activity or the mutarotase activity is low, the peak position of the detected value at the second electrode is delayed for sucrose as compared to glucose. For this reason, when the mixed solution is injected, the output value on the second electrode side is not accurately detected, and the measured value of sucrose becomes lower.

Therefore, the present inventors have examined in detail the waveforms after injecting the sample, and found that a proportional relationship with the sample concentration is established at each point with respect to the output value at a certain time after the sample injection. found. At the same time, it was found that the shape of the waveform itself did not change significantly even when the peak position shifted. That is, a calibration curve can be obtained from an output value at an arbitrary point even if the output value is not the highest output value.

This phenomenon is considered to occur for the following reasons. First, the sample injected into the continuous flow initially forms a band-like band, but gradually mixes with the preceding and following medium (carrier), for example, a buffer solution. This mixing is governed by the diffusion phenomenon based on the concentration difference and the pulsation of the liquid sending section forming a flow. On the other hand, if the enzyme substrate in the sample has almost the same molecular weight, the degree of diffusion is almost equal, and it is considered that there is no great difference in distribution in the flow. However, when the enzyme reaction occurs on the electrode surface and the enzyme activity is low and the reaction rate is low, the product reaches the maximum concentration slowly after the maximum concentration point of the substrate has passed. Therefore, it seems that there is a delay when the electrode output value is viewed.
However, the lags occur at a uniform rate at each point of the substrate concentration, so that the waveforms themselves are similar.

In other words, the half widths of glucose and sucrose are almost the same, but only the time for detecting the maximum point of the output value differs.

In this case, it is desirable to obtain a detection value at the highest point of the sucrose waveform in terms of sensitivity. Therefore, it is preferable to also detect glucose at the position of the highest point of sucrose even when the conversion rate from sucrose to glucose is low.

However, in this case, if two standard solutions are injected in advance and a point at which the output value for each substance is maximized can be detected at, for example, the highest point of the sucrose waveform, but practically, Will perform unnecessary measurements. Therefore, it is desirable to determine the peak position from the standard solution to be measured first for preparing a calibration curve. That is, when measuring the standard solution in the order of glucose and sucrose, it is convenient to detect the highest point of the glucose output at the time of measuring the glucose and add a constant to the value to determine the point to be detected.

Therefore, a mechanism for storing the output waveform of the standard solution when the glucose standard solution is injected, a mechanism for detecting the peak position from the stored waveform, and storing the peak position, That is, a mechanism that presets a time corresponding to the expected shift in sucrose, adds the peak position to the peak position, and separately stores the output value, a mechanism that stores the output value at the position after the addition from the stored waveform, It has a mechanism to create a calibration curve using the specific component concentration in the standard solution, and has a mechanism to calculate the specific component concentration in the unknown sample from this calibration curve and the output value of the unknown sample at the waveform position calculated above. Can be realized.

That is, at the stage of injecting the glucose standard solution, instead of performing the usual quantitative work at the peak position,
For example, quantification at a point optimized for sucrose can be performed. This processing flow is shown in FIG.

Here, a two-component system of glucose and sucrose is described. In a similar measurement system, a glucose electrode on which glucose oxidase is immobilized, a column filled with a carrier on which maltose phosphorylase is immobilized, and another column. A glucose and maltose two-component measurement system in which a glucose electrode is arranged, and an L-lactic acid measurement electrode in which the electrode and a column packed with a carrier on which L-lactic acid oxidase is immobilized are arranged; and an L-lactic acid dehydrogenase. And a column packed with an immobilized dehydrogenase such as glutamate dehydrogenase, another set of electrodes for measuring L-lactic acid, and pyruvine necessary for producing L-lactic acid by L-lactic acid dehydrogenase A measurement system for glutamic acid and the like is performed in a system in which an acid and nicotinamide adenine dinucleotide oxidized form are present.

In particular, in a measurement system using the above-described dehydrogenase reaction, the reaction hardly progresses due to the equilibrium constant of the enzyme, and a peak shift easily occurs. Therefore, it is desirable to obtain a detection value by this measuring method.

For example, when the conversion rate from the first component (lactic acid) to the second component (glutamic acid) is 24% and the peak of the second component is 7 seconds later than the peak of the first component, the first component and the second component FIG. 4 shows the change of the measurement time and the detection value for the standard solution and the mixed solution of FIG. The total concentrations of the standard solution and the mixed solution of the first component and the second component were the same. When the output values of the respective standard solutions are superimposed, the output value of the mixed solution is obtained, and it can be seen that as the ratio of the second component increases, the position of the peak shifts behind the peak of the first component.

In FIG. 4, A is a first component standard solution, B
Is a sample in which the first component and the second component are mixed at a ratio of 2: 1, C is a sample in which the first component and the second component are mixed at a ratio of 1: 2, and D is a second component standard solution. It is.

The measurement of two components of L-lactic acid and glutamic acid using the dehydrogenase system will be described below. L-lactic acid can be detected by a reaction with L-lactic acid oxidase (hereinafter abbreviated as LOD).

LOD reaction: L-lactic acid + O ₂ → pyruvic acid + H ₂ O ₂ and glutamic acid are two kinds of enzymes, glutamate dehydrogenase (hereinafter abbreviated as GLDH) and L-lactate dehydrogenase (hereinafter abbreviated as L-LDH). After being converted to lactic acid, it is likewise detected by LOD.

GLDH reaction: glutamic acid + NAD ⁺ → α-ketoglutaric acid + ammonia + NADH + H ⁺

L-LDH reaction: pyruvate + NADH + H ⁺ → L-lactic acid + NAD ⁺

An error may occur when the mixture is analyzed, but accurate measurement is possible by using the measuring apparatus of the present invention.

Although there is no problem when the highest point of each component is determined in advance as described above, in order to perform analysis efficiently, the first component (in the above example, glucose or lactic acid corresponds to the first component). Empirically 30 for the detection time of the highest point
It is desirable to add (or subtract, for example, for the second purpose described below) one half to one half of the time. In many cases, the first component is a reaction system having a sufficient speed, and the highest point is detected at a time expected from the cross-sectional area and length of the flow path and the flow rate. If the addition time is too short, the contribution of the first component increases, and the effect of improving accuracy shown in the present invention is lost.
On the other hand, if the time is too long, the analysis time increases, the efficiency decreases, and the output value of the second component itself may be reduced, which is disadvantageous in terms of accuracy.

Next, the second object of the present invention will be described. Taking the case where the detection unit is a hydrogen peroxide electrode as an example, the influence of an obstruction can be reduced by using the measurement device of the present invention. The hydrogen peroxide electrode has a problem that it exhibits a response value not only to hydrogen peroxide but also to interfering substances such as ascorbic acid. For this reason, it has been practiced to provide a permselective membrane to reduce the influence of obstructions. In this case, the peak positions of the hydrogen peroxide to be measured and the interfering substance such as ascorbic acid often shift due to the difference in the diffusion rate in the film. By utilizing this characteristic, the influence of the concentration of interfering substances such as ascorbic acid can be reduced. Naturally, the thickness of the permselective membrane may be increased in order to remove the influence of the obstacle as much as possible, but there is a disadvantage that the sensitivity is lowered at the same time. On the other hand, by setting the detection position before the peak position of hydrogen peroxide, the effect of ascorbic acid can be reduced and the sensitivity does not decrease. Generally, the diffusion rate of the interfering substance is lower than that of hydrogen peroxide to be measured, so that an output is obtained with a delay.

These are described using an example of an immobilized enzyme and an electrode. However, when a solution reaction is performed in a flow-type measuring device, the peak positions of the standard solution and the unknown sample containing an interfering substance are shifted. Sometimes, accurate measurement is possible by using the device of the present invention. The peak position is fixed not only when there is a difference in the reaction time in the reaction section as described above, but also when there is an obstacle that affects the standard and the peak position of the interferer is different from the standard. Doing so is effective in improving the measurement accuracy.

Various chemical reactions and enzyme reactions can be used for the reaction in the reaction section of the measuring device in the present invention.
There is no particular limitation. However, it is particularly effective to fix the peak detection position in a measuring device having a reaction section using an immobilized enzyme.

The method of immobilizing the enzyme used in the reaction section is not particularly limited, and an adsorption method, a chemical bonding method, an inclusive method, or the like can be used. Among them, a chemical bonding method capable of producing a strong immobilized body is preferable.

As a carrier used for immobilization, diatomaceous earth, silica gel, glass beads, alumina, ceramic, carbon, activated carbon, molecular sieve, silicon rubber, cellulose, agarose, amino acid polymer and the like can be used.

The chemical bonding method is a method in which an amino group is introduced into the surface of a carrier with an aminosilanating reagent, formylation is performed using a polyfunctional aldehyde such as glutaraldehyde, and then immobilized by contacting with an enzyme. Can be exemplified.
The method of filling the column with the carrier immobilized can increase the amount of enzyme that can be immobilized as compared with the case of immobilizing it on the electrode surface, and can easily immobilize a sufficient amount of enzyme for the reaction. As the material of the column, an acrylic resin, a fluororesin, a vinyl chloride resin, a metal such as glass or stainless steel, or a combination thereof can be used.

In addition to the form of a reactor immobilized on a carrier and packed in a column, a method of immobilization on a membrane on the electrode surface is also effective.

Regarding the electrodes, for measuring oxygen,
Various known oxygen electrodes such as carbani type and Clark type, and carbon, platinum,
A known hydrogen peroxide electrode using nickel, palladium or the like and using silver or the like on the cathode side can be used. Generally, platinum is often used as the anode because of its low overvoltage to hydrogen peroxide and high sensitivity. An electrode having a permselective membrane such as a polysiloxane membrane, an acrylic resin membrane, a protein membrane, or an acetylcellulose membrane on the electrode surface is desirable from the viewpoint of more complete removal of obstacles.

The electrode system is composed of a working electrode and a counter electrode.
A hydrogen peroxide electrode or an oxygen electrode can be used. From the viewpoints of stability and accuracy, a three-electrode structure including a working electrode, a reference electrode, and a counter electrode is preferable.

Further, the detecting section of the measuring apparatus according to the present invention is not limited to an electrode, and a spectrophotometer, a fluorometer, a chemiluminescence detector and the like can be used. When measuring multiple components simultaneously using a flow-type measurement device, two or more components can be measured in series or in parallel as in the above-described example.
The present invention can be implemented by providing at least two detection units. Second
In the detection unit, the first component and the second component are obtained by changing to the first component. Therefore, in order to obtain the detection value,
The measurement is performed for each of the component detection unit and the second component detection unit, and two components can be measured by using two detection values obtained for one sample.

[0052]

EXAMPLES The contents of the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Example for the first object of the present invention) Measurement was performed using the apparatus shown in FIG. 1) Production of LOD-immobilized column 150 mg of fired diatomaceous earth refractory brick (30 to 60 mesh) was thoroughly dried, immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, and then thoroughly diluted with toluene. Wash and dry. The aminosilane-treated carrier is immersed in 5% glutaraldehyde for 1 hour to formylate. Thereafter, the well is washed well with distilled water and finally replaced with a 100 mM sodium phosphate buffer having a pH of 7.0, and this buffer is removed as much as possible. This formylated refractory brick has a pH of 7.0,
200 μl of a solution obtained by dissolving LOD (manufactured by Sigma) at a concentration of 50 units / ml in 100 mM sodium phosphate buffer
1 and allowed to stand at 0-4 ° C. for 1 day to immobilize. This enzyme-immobilized carrier was packed in an acrylic column having an inner diameter of 3.5 mm and a length of 30 mm to obtain an LOD-immobilized column. L
The OD-immobilized column is indicated by (4) and (7) in FIG.

2) Production of GLDH, L-LDH fixed column The refractory brick used in 1) is formylated in the same manner as 1). PH 7.0, 10
200 μl of a solution prepared by dissolving GLDH (Boehringer) at 1500 units / ml and L-LDH (Boehringer) at 100 units / ml in 0 mM sodium phosphate buffer was brought into contact with the solution and left at 0 to 4 ° C. for 1 day. Immobilize. This enzyme-immobilized carrier is 3.5 mm in inner diameter and 30 m in length.
m and packed in an acrylic column to make a GLDH, L-LDH immobilized column (6).

3) Manufacture of hydrogen peroxide electrode The side of a platinum wire having a diameter of 2 mm was covered with a heat-shrinkable Teflon,
Finish one end of the line with a file and 1500 emery paper. Using the platinum wire as a working electrode, a 1 cm square platinum plate as a counter electrode, and a saturated calomel electrode as a reference electrode, an electrolytic treatment is performed for 10 minutes at +2.0 V in 0.1 M sulfuric acid. Thereafter, the platinum wire is thoroughly washed with water, dried at 40 ° C. for 10 minutes, and dried.
After immersing for 1 hour in an anhydrous toluene solution of 0% γ-aminopropyltriethoxysilane, the substrate is washed. 20 mg of bovine serum albumin (Fraction V, manufactured by Sigma) is dissolved in 1 ml of distilled water, and glutaraldehyde is added thereto in an amount of 0.2 mg.
%. 5 μl of this mixed solution was quickly placed on the previously prepared platinum wire, and dried and cured at 40 ° C. for 15 minutes to obtain a hydrogen peroxide selective permeable membrane, which was used as hydrogen peroxide electrodes (5) and (8).

An Ag / AgCl reference electrode was used as a reference electrode, and a conductive pipe was used as a counter electrode.

4) Measuring device The flow type measuring device shown in FIG. 2 was used. The composition of the buffer was 100 mM sodium phosphate, 50 mM potassium chloride,
It contains 5 mM sodium azide, 1 mM NAD, and 1 mM pyruvate and has a pH of 7.0. The flow rate of the pump (2) was 1.0 ml / min. The microcomputer (11) incorporated in the measurement device performs constant storage, waveform storage, peak detection, constant adjustment, waveform reanalysis, output value detection, and calibration curve creation, and quantitative calculation when the sample is actually injected. Has functions. This result is obtained by using the personal computer (1
It is transferred to 2) and recorded.

5) Measurement of Samples For GLDH and L-LDH, when this activity was fixed and used under these conditions, it was recognized that the peak time at the second electrode was about 6 seconds later for glutamic acid than for L-lactic acid. Was done. 4) Distilled water, 1, 2,
Inject 3 mM L-lactic acid, 1, 2, 3 mM L-glutamic acid, add 6 seconds to the peak detection time at the time of the first measurement of L-lactic acid at the second electrode (8), and then set the detection position to that time. It was fixed and the measurement was performed. The current values obtained at the first electrode (5) and the second electrode (8) were as shown in Table 1, and the following calibration curves were obtained.

Of course, an accurate value can be obtained with a value other than the added value of 6 seconds, but the apparatus of this embodiment used this value, which is the peak position at the time of measuring glutamic acid.

[0060]

[Table 1]

Calibration curve 1 First electrode L-lactic acid calibration curve
Y = 26.60X + 0.60 Calibration curve 2 Second electrode L-lactic acid calibration curve Y = 1
6.09X + 0.22 Calibration curve 3 Second electrode L-glutamic acid calibration curve Y =
4.02X + 0.23

Here, Y is the current value (nA), and X is the concentration of L-lactic acid or L-glutamic acid (mM) in the sample.
In addition, the respective concentrations of the mixture of L-lactic acid and glutamic acid were determined from the measured current values. Table 2 shows the current values, peak positions, and measured concentrations of the mixed solution obtained at the first electrode (5) and the second electrode (8). As can be seen from Table 2, accurate measurements have been made.

[0063]

[Table 2]

Comparative Example 1 Using the same measuring apparatus as in Example 1 and using the same standard solution, without fixing the peak position of the second electrode (without adding a constant), the output value at the peak maximum position It measured using. The current values obtained at the first electrode and the second electrode were as shown in Table 3, and the following calibration curves were obtained. For example, as shown in FIG. 4, the peak positions of the second electrode are L-lactic acid and glutamic acid, and the peak positions of the respective standard solutions are different.
There was a delay of about 6 seconds compared to lactic acid.

[0065]

[Table 3]

Calibration curve 1 First electrode L-lactic acid calibration curve
Y = 27.48X + 0.58 Calibration curve 2 Second electrode L-lactic acid calibration curve Y = 1
8.32X + 0.29 Calibration curve 3 Second electrode L-glutamic acid calibration curve Y =
4.25X + 0.34

Here, Y is the current value (nA), and X is the L-lactic acid concentration (mM) in the sample. In addition, the respective concentrations of the mixture of L-lactic acid and glutamic acid were determined from the measured current values. Table 4 shows current values and measured concentrations of the mixed solution obtained at the first electrode and the second electrode. Obviously, the error of the measured value of glutamic acid is larger than that of Example 1. In the case of the mixed liquid measurement, as shown in FIG. 4, there was a difference in the peak position depending on the mixing ratio, and a shift in the detection position was recognized.

[0068]

[Table 4]

Embodiment 2 (Embodiment for the second object of the present invention) This will be described with reference to FIG. 1) Production of hydrogen peroxide electrode The same electrode used in Example 1 was replaced with a hydrogen peroxide electrode (2
4).

2) Measuring device The flow type measuring device shown in FIG. 3 was used. Buffer tank (2
1) The composition of the buffer solution sent by the pump (22) is 100 mM sodium phosphate, 50 mM potassium chloride,
PH 7.0 with 1 mM sodium azide. The flow rate of the pump (22) was 1.0 ml / min. A sample is injected from a sampler (23) and guided to a hydrogen peroxide electrode (24) in a thermostat (25) to detect hydrogen peroxide.

3) Measurement of sample In the measuring device of 2), 1, 2, 5 mM hydrogen peroxide and 1
0, 20, and 50 mM ascorbic acid was injected, and an output value was detected by adding -2 seconds to the first peak time of hydrogen peroxide. The prepared calibration curve is shown below.

From the results, it was found that the calibration curve 1 vs. hydrogen peroxide Y = 50.1X +
0.10 Calibration curve 2 vs. ascorbic acid Y = 0.2X +
0.05 where Y is the current value (nA) and X is the hydrogen peroxide or ascorbic acid concentration (mM) in the sample. When the slope ratio of the calibration curve was calculated, ascorbic acid was 0.1% of hydrogen peroxide.
It can be seen that the gradient is 40%, and the effect of the obstacle is extremely small.

Comparative Example 2 1) Production of Hydrogen Peroxide Electrode The same electrode as used in Example 2 was used (FIG. 3). 2) Measuring device Using the same measuring device as in Example 2 and having no mechanism of the present invention, measurement was performed under the same conditions.

3) Measurement of Sample A calibration curve was prepared by performing exactly the same measurement as in Example 2 except that the maximum value of the output waveform was automatically analyzed as a peak value. The prepared calibration curve is shown below. Calibration curve 1 vs. hydrogen peroxide Y = 50.1X +
0.10 Calibration curve 2 vs. ascorbic acid Y = 1.1X +
0.00 where Y is the current value (nA) and X is the hydrogen peroxide or ascorbic acid concentration (mM) in the sample. When the slope ratio of the calibration curve was calculated, ascorbic acid was found to be 2.
A gradient of 20% results. Naturally, since there is a permselective membrane, the influence of the obstruction is small, but the relative ratio is larger than that of the second embodiment. Therefore, in Example 2, the influence of the interfering substance can be reduced without deteriorating the sensitivity of the target component even with a high concentration of interfering substance. As a result, extremely accurate measurement of hydrogen peroxide is possible.

[Brief description of the drawings]

FIG. 1 shows a processing flow of the apparatus of the present invention.

FIG. 2 shows a flow type L-type used in Example 1 and Comparative Example 1.
It is a figure of a glutamic acid and L-lactic acid measuring device.

FIG. 3 is a diagram of a flow-type hydrogen peroxide measuring device used in Example 2 and Comparative Example 2.

FIG. 4 shows an example in which the peak time of the first component (lactic acid) and the second component (glutamic acid) have a difference of 7 seconds, and the conversion ratio from the second component to the first component is 24%. It is a figure showing the peak when the total concentration of each of the standard solution and the mixed solution of the first component and the second component is the same.

[Explanation of symbols]

 Reference Signs List 1 buffer solution tank 2 pump 3 sampler 4 LOD immobilized column 5 hydrogen peroxide electrode (first electrode) 6 GLDH, L-LDH immobilized column 7 LOD immobilized column 8 hydrogen peroxide electrode (second electrode) 9 constant temperature bath DESCRIPTION OF SYMBOLS 10 Detector 11 Single board computer 12 Personal computer 13 RS232C code 14 Sampler control signal 15 Liquid feed pump control signal 16 Waste liquid 21 Buffer tank 22 Pump 23 Sampler 24 Hydrogen peroxide electrode 25 Constant temperature bath 26 Detector 27 Single board computer 28 RS232C Code 29 Personal computer 30 Sampler control signal 31 Liquid feed pump control signal 32 Waste liquid

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-7-98295 (JP, A) JP-A-7-92137 (JP, A) JP-A-7-39395 (JP, A) JP-A-6-98 169798 (JP, A) JP-A-6-18478 (JP, A) JP-A-4-232452 (JP, A) JP-A-64-69944 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/26 371 G01N 27/416

Claims (3)

(57) [Claims] 1. A mechanism for sending a carrier, a sample injecting mechanism for injecting a sample into a carrier, a reaction unit for reacting a specific component in the injected sample, and detecting a change resulting from a reaction of the specific component in the sample. A flow-type measuring device having a detecting unit for detecting a standard solution containing a specific component, a mechanism for detecting a peak position from the stored waveform, and storing the peak position, for a predetermined time. Add to or subtract from the peak position,
A mechanism for storing the result, a mechanism for storing the output value at the position where the addition or subtraction is performed, and a mechanism for creating a calibration curve using the output value and the concentration of the specific component in the standard solution. And a flow-type measuring device having a mechanism for calculating the concentration of a specific component in an unknown sample from the output value of the unknown sample at the waveform position added or subtracted as described above. 2. The flow-type measuring apparatus according to claim 1, wherein the reaction unit is an immobilized enzyme reactor filled with an immobilized enzyme carrier, and the detection unit is an electrode that responds to a substance that increases or decreases by the immobilized enzyme reactor. 3. The flow-type measurement device according to claim 1, wherein the reaction section is an immobilized enzyme membrane, and the detection section is an electrode that responds to a substance increased or decreased by the immobilized enzyme membrane.