JP4249549B2 - Sample measuring apparatus and sample measuring method - Google Patents

Description

【００５９】
結果を表１に示す。表１は、実施例１，２の電流積分値、グルコース濃度０ｍｇ／ｄｌの電流積分値の平均値との差、および標準偏差、上記数１により求めたＣＶ値を示す。ＣＶ値は、３％以下であり、精度が良好であることがわかった。なお、表２は、グルコース濃度０ｍｇ／ｄｌの蒸留水での電流積分値を標準１−１，１−２，２−１，２−２として求めた表である。
【００６０】
【表１】

【００６１】
【表２】

【００６２】
【実施例３】
グルコース濃度をそれぞれ、１ｍｇ／ｄｌ、１９９ｍｇ／ｄｌ、４４５ｍｇ／ｄｌ、６６０ｍｇ／ｄｌ、９０１ｍｇ／ｄｌとしたサンプルを準備し、これらを５μＬを注入したサンプルを５個作製した。溶解時間８秒とし、この時間経過後直ちに、電圧走査を行った。走査方式５０ｍＶ／ｓで、電圧を０Ｖから−０．２Ｖにした後、同一の走査方式により−０．２Ｖから＋０．２Ｖに変化させた。これらのサンプルの検出電流値から、電圧との電流積分値を求めた。電流積分値を表３に記載する。
【００６３】
【比較例１】
グルコース濃度をそれぞれ、１ｍｇ／ｄｌ、１９９ｍｇ／ｄｌ、４４５ｍｇ／ｄｌ、６６０ｍｇ／ｄｌ、９０１ｍｇ／ｄｌとしたサンプルを準備し、これらを５μＬを注入したサンプルを５個作製した。溶解時間８秒とし、この時間経過後直ちに、電圧走査を行った。走査方式５０ｍＶ／ｓとし、電圧を−０．５Ｖから＋０．２Ｖに変化させた。これらのサンプルの検出電流値から、電圧との電流積分値を求めた。電流積分値を表３に記載する。なお、比較例１は、特開平２００１−３１１７１２号公報に開示される方法で測定したものである。
【００６４】
【表３】

【００６５】
図７に、実施例３及び比較例１とを、Ｘ軸をグルコース濃度、Ｙ軸を電流積分値とした座標のグラフで示した。比較例１が、グルコース濃度６００ｍｇ／ｄｌから直線性を失い、曲線を描くのに対し、本願発明の方法および装置による実施例３は、グルコース濃度１０００ｍｇ／ｄｌまで、直線性を保持することができることがグラフより解る。
【００６６】
【発明の効果】
従来の定電圧法、電圧走査法に比較して、高感度で検液中の被検成分量が測定でき、濃度の増加に対しても、精度良く直線性を保持することができる。
【図面の簡単な説明】
【図１】 本発明を実施するための装置のブロック図である。
【図２】 本発明を実施するための装置の回路図である。
【図３】 電圧電流曲線から、ピーク電流値を求める方法を示す。
【図４】 電圧電流曲線から、電流積分値を求める方法を示す。
【図５】 図５（ａ）は、ピーク電圧とピーク電流の相関性を示すグラフであり、図５（ｂ）は、ピーク電流と電流積分値との相関性を示すグラフである。
【図６】 走査速度の平方根とピーク電流との関係を示すグラフである。
【図７】 実施例３と比較例１のグルコース濃度に対する電流積分値を示すグラフである。
【図８】 一般に用いられている酵素センサの一例である。
【符号の説明】
１０；本発明の検体測定装置
１２；酵素センサ部
１４；検体測定装置の接続部
１６；電圧走査部
１８；電流検出部
２０；マイクロコンピュータ
２２ａ、２２ｂ；Ｄ／Ａ変換器
２２ｃ；Ａ／Ｄ変換器
２４ａ，２４ｂ；バッファ
２６ｃ；演算増幅回路
２８ｂ；電流電圧変換回路
１００；基本的な酵素センサ
１０２；電気絶縁性基板
１０４；測定極
１０６；対極
１０８；反応層
１１０；電気絶縁スペーサ
１１２；外面カバー
１１４；検体吸入口[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a sample measuring apparatus and method incorporating an enzyme sensor, and more particularly to a sample measuring apparatus and measuring method incorporating an enzyme sensor useful for measuring biological components in the medical field.
[0002]
[Background Art and Problems to be Solved by the Invention]
The enzyme sensor is a chemical sensor that mainly uses biological substances such as glucose, alcohol, lactic acid, uric acid, urea, sucrose as sensor materials. In many cases, a biological material having an excellent molecular recognition function is used, so that it can exhibit outstanding selectivity. An enzyme sensor is an electrochemical sensor that detects the consumption of oxygen or the production of hydrogen peroxide by an enzyme reaction. The method of transferring electrons between an enzyme and an electrode through an electron mediator is used. is there. This makes it possible to easily qualitatively or quantify the characteristics of glucose, alcohol, etc., or the amount of substance in the specimen. Examples of the specimen include blood, saliva, urine and the like, and specific components in these biological samples are detected.
[0003]
A basic enzyme sensor 100 is shown in FIG. For example, an electrode system is disposed on the surface of an electrically insulating substrate 102 made of an insulating material such as plastic. In FIG. 8, the electrode system has two electrodes, one is the measurement electrode 104, and the other is the counter electrode 106 with respect to the measurement electrode, which are arranged in close proximity. Both end portions of the electrode systems 104 and 106 are electrode terminal portions connected to the measurement main body portion and end portions to which the reaction layer 108 is closely fixed. The reaction layer 108 includes at least an enzyme and an electron acceptor. An enzyme and an electron acceptor are supported on the immobilization film to form a substrate detection unit. The measurement electrode 104 and the counter electrode 106 are electrically connected to an external connection terminal via a lead portion. 110 is an electrically insulating spacer, and 112 is an outer cover. A sample fluid such as a body fluid is taken in from the sample inlet 114 and reacts with an enzyme immobilized on the reaction layer 108 and a substrate in the body fluid to cause an oxidation-reduction reaction. Function. A redox reaction also occurs. Since the amount of the substrate contained in the sample is correlated with the amount of the electron acceptor after the reaction, an electron transfer reaction occurs, and the current flows in proportion to the substrate concentration. This can be detected to know the substrate concentration.
[0004]
Utilizing such an enzyme reaction, an electrode system and an enzyme reaction layer are provided, and a biohaving function of electrochemically detecting a change in the concentration of a substance with an electrode system in the reaction of an enzyme, an electron acceptor and a sample solution. A sensor is disclosed (for example, Patent Document 1). In Patent Document 1, a disposable biosensor having a reaction layer including an electrode system is formed by forming an enzyme reaction layer on a surface of an electrode system, which is a mixture of a hydrophilic polymer solution and an electron acceptor. Thus, the substrate concentration can be measured very easily.
[0005]
Conventionally, an enzyme electrode method has been proposed as a method for detecting a reaction amount in a reaction layer of an enzyme sensor. In this enzyme electrode method, the oxidation-reduction current is detected by the potential step method (chronoamperometry, constant voltage method) in which a constant voltage is applied between the measurement electrode and the counter electrode, and the concentration of a specific component is measured. (For example, Patent Document 2).
[0006]
Also disclosed is a method of identifying a specific component amount by making the ground voltage constant, gradually increasing the voltage, scanning the voltage, detecting the current, and identifying the specific component amount (for example, Patent Document 3).
[0007]
[Patent Document 1]
Japanese Patent No. 2517153
[Patent Document 2]
Japanese Patent Laid-Open No. 3-85435
[Patent Document 3]
JP 2001-311712 A
[0008]
[Problems to be solved by the invention]
However, in the potential step method, the current that can be extracted is weak, and it is necessary to increase the amplification factor of the amplifier that converts the detected current into a voltage. Accordingly, there is a possibility that the noise current is also amplified, and as a result, there is a possibility that the measurement accuracy which is important for the sensor is adversely affected.
[0009]
Further, the voltage scanning method according to Patent Document 3 has a problem that the linearity is inferior in a region where the substrate concentration is wide.
[0010]
Here, the present inventors propose a method using a cyclic voltammetry method, a so-called linear voltage scanning method. Conventionally, in the cyclic voltammetry method, it is possible to extract a larger detection current than in the potential step method, but it has not been possible to obtain linearity from a low concentration region to a high concentration region.
[0011]
However, the present inventors aim to provide a sample measuring apparatus and a measuring method that solve the above problems, are less susceptible to noise current in measurement accuracy, and have a detection capability corresponding to a change in the concentration of the substrate. As a result of intensive studies, it has been found that in the cyclic voltammetry method, linearity can be obtained with high accuracy from a low concentration region to a high concentration region by scanning a voltage within a specific range. The present invention has been completed based on these findings.
[0012]
[Means for Solving the Problems]
InspectionAs an aspect of the body measuring device,
Forming an electrode portion including at least a measurement electrode and a counter electrode on an insulating substrate, and an enzyme sensor unit electrically bridging the measurement electrode and the counter electrode in a reaction layer including at least an enzyme and an electron acceptor;
A voltage scanning unit that generates a potential difference by applying a voltage that varies with time between the measurement unit and the counter electrode; and
A current detection unit for detecting a current flowing through the electrode unit;
A microcomputer for controlling the voltage scanning unit to generate a predetermined voltage, and determining a substrate component amount of the specimen based on an output of the current detection unit;
The voltage scanning unit increases the voltage applied to the measurement electrode of the enzyme sensor unit at a constant rate from 0 V to a preset voltage (E1) through a voltage capable of measuring the peak current of the electron acceptor with reference to the counter electrode. Alternatively, the voltage may be decreased or increased from the voltage E1 at a speed that is the same absolute value as the speed but reversed in sign, and scanned to a preset voltage (E2) through a voltage that can measure the peak current. .
[0013]
Also, InspectionAs another aspect of the body measuring device,
An apparatus for measuring the basic mass of a specimen by a programmed computer and displaying the basic mass value,
Forming an electrode portion including at least a measurement electrode and a counter electrode on an insulating substrate, and an enzyme sensor unit electrically bridging the measurement electrode and the counter electrode in a reaction layer including at least an enzyme and an electron acceptor;
A voltage scanning unit that generates a potential difference by applying a voltage that varies with time between the measurement unit and the counter electrode; and
A current detection unit for detecting a current flowing through the electrode unit;
A sample measuring device including a microcomputer that controls the voltage scanning unit to generate a predetermined voltage and discriminates a substrate component amount of the sample based on an output of the current detection unit;
The microcomputer is
(1) Means for generating a potential difference between both electrodes of the voltage scanning unit and the current detection unit in a predetermined constant voltage range (E1 to E2), and the voltage of the counter electrode in the enzyme sensor unit is used as a reference The measuring electrode is scanned from the voltage 0 V to the voltage (E1) at a constant voltage scanning speed (v1), and then continuously, the voltage scanning speed (v1) and the absolute value are the same from the voltage (E1). A voltage signal that continuously scans up to a voltage (E2) at a voltage scanning speed (v2 = −v1) in which
(2) means for analog conversion of the voltage signal;
(3) means for digitally converting the current value detected by the current detector;
(4) means for calculating a peak current value or a current area value detected from a voltage-current curve of the digitally converted current value and the scanned electrode voltage;
(5) means for comparing the peak current value or current area value with a reference calibration curve prepared in advance, and determining the component amount of the substrate concentration of the specimen; and
(6) Means for displaying the substrate concentration of the specimen
It may have a control means consisting of
[0014]
Also, InspectionAs an aspect of the body measurement method,
A method for measuring a basic mass of a specimen using the apparatus and displaying the measured basic mass value,
Supplying a sample for measurement onto the reaction layer of the enzyme sensor unit;
Scanning the voltage between the electrodes;
Detecting the current flowing through the enzyme sensor unit;
The computer can perform the following control steps.
(1) A step of generating a potential difference between both electrodes of the voltage scanning unit and the current detection unit within a preset constant voltage range (E1 to E2), wherein the voltage of the counter electrode in the enzyme sensor unit is used as a reference The measuring electrode is scanned from the voltage 0 V to the voltage (E1) at a constant voltage scanning speed (v1), and then continuously, the voltage scanning speed (v1) and the absolute value are the same from the voltage (E1). A voltage signal for continuously scanning up to the voltage (E2) at a voltage scanning speed (v2 = −v1) obtained by reversing
(2) converting the voltage signal into analog;
(3) a step of digitally converting the current value detected by the current detection unit;
(4) calculating a peak current value or a current area value detected from a voltage-current curve of the digitally converted current value and the scanned electrode voltage;
(5) a step of determining a component amount of a substrate concentration of the specimen by comparing the peak current value or the current area value with a reference calibration curve prepared in advance; and
(6) A step of displaying the substrate concentration of the specimen.
[0015]
Here, the step (1) of the control step using the computer reaches E1 from the voltage 0V via the voltage at which the peak current of the electron acceptor can be observed in the voltage scan of the measuring electrode, and further the voltage It may be a step of scanning from E1 to E2 via a voltage at which the peak current of the electron acceptor can be observed.
[0016]
In the enzyme sensor unit, the enzyme may be glucose oxidase and the electron acceptor may be potassium ferricyanide.
[0017]
Alternatively, in the voltage scanning of the microcomputer, the voltage E1 may be −0.2V, the voltage E2 may be 0.2V, and the voltage scanning speed may be 10 mV / s to 200 mV / s.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an enzyme sensor having an insulating substrate, a reaction layer in which an electrode system is adhered and fixed, and a disposable chip that can be replaced for each specimen, and a measuring apparatus incorporating a chip composed of an enzyme sensor. Compared to the above, there are provided a sample measuring apparatus and a sample measuring method for detecting the substrate concentration of the sample with high accuracy.
[0019]
Here, the sample measuring apparatus used in the present invention will be described. An example of an embodiment of the sample measuring apparatus according to the present invention is shown according to FIG. Reference numeral 10 denotes a sample measurement apparatus according to the present invention, which is an outline of an enzyme sensor unit 12 having a bipolar electrode system of a measurement electrode and a counter electrode for the measurement electrode, and a sample measurement device in which this enzyme sensor unit is incorporated in the sample measurement device body. FIG. The enzyme sensor unit 12 is, for example, a chip-shaped enzyme sensor, and is connected to the connection unit 14 of the sample measurement device according to the present invention. In the enzyme sensor 12, a voltage is applied to the counter electrode of the enzyme sensor unit, a potential difference is generated between the electrodes, and the voltage is scanned, and a current generated from the measurement electrode of the enzyme sensor unit is detected. A current detection unit 18 is connected, and the voltage scanning unit 16 is supplied with a measurement electrode voltage to be applied between the electrodes. The current detector 18 detects a current. A microcomputer (hereinafter referred to as “microcomputer”) 20 is connected to the voltage scanning unit 16 and the current detection unit 18.
[0020]
The enzyme sensor unit 12 is mainly composed of an electrode system and a reaction layer. The electrode system includes at least two electrodes, a measurement electrode and a counter electrode, and is provided on an insulating substrate.
[0021]
The reaction layer is tightly fixed on the electrode system and contains at least the enzyme and the electron acceptor. Examples of the carrier include microcrystalline cellulose. The enzyme reacts selectively with the substrate in the sample, and the electron acceptor reacts in correlation with the electron transfer associated with the reaction between the substrate and the enzyme. The enzyme is selected depending on the substrate in the specimen to be measured, for example, glucose component, alcohol component, lactic acid component, uric acid component, etc. For example, if glucose is to be detected, glucose oxidase, which is an oxidoreductase, is selected. Can be mentioned. The electron acceptor is an inorganic or organic compound that activates the reaction of the enzyme. For example, alkali metal ferricyanate, ferrocene or an alkyl-substituted product thereof, p-benzoquinone, methylene blue, β-naphthoquinone 4- Examples include potassium sulfonate, phenazine methosulfate, and 2,6-dichlorophenolindophenol. From the viewpoint of solubility in a solvent such as water, ferricyanide alkali metal salts, particularly potassium salts and ferrocenes are preferable. For example, when measuring glucose in blood, potassium ferricyanide is reduced to potassium ferrocyanide when glucose reacts with glucose oxidase and is oxidized. The amount of potassium ferrocyanide produced is approximately proportional to the glucose concentration.
[0022]
The microcomputer of the sample measurement apparatus of the present invention controls voltage control, detection of current measured by the enzyme sensor unit, correction of current value, calculation of the amount of test component from the current value, and the like. The microcomputer 20 determines a voltage to be applied to the enzyme sensor unit 12, plays a role of calculating and displaying the current applied to the enzyme sensor unit 12 and further detected by the current detection unit. The microcomputer 20 incorporates both conversion functions of D / A (digital / analog) conversion and A / D (analog / digital) conversion. The D / A (digital / analog) conversion function plays a role of D / A converting a current for applying a predetermined voltage to the enzyme sensor to the voltage scanning unit. During the measurement, a series of voltages are scanned and applied from the voltage scanning unit 16 to the enzyme sensor. The A / D conversion function plays a role of A / D converting the current generated by the reaction between the detected substrate component and the enzyme. The current value detected by the current detection unit within a certain period of time is calculated and processed in relation to the applied voltage by the calculation function in the microcomputer, converted into the basic mass in the sample, and displayed.
[0023]
Specifically, the voltage scanning is performed by scanning the measurement electrode at a constant voltage scanning speed (v1) from a voltage 0 V to a preset voltage (E1) with reference to the counter electrode voltage in the enzyme sensor unit, and then continuously. Then, scanning from the voltage (E1) to the preset voltage (E2) at the voltage scanning speed (v2 = −v1) whose sign is the same as that of the voltage scanning speed (v1) and whose sign is the same, Can be supplied.
[0024]
During the measurement, a predetermined voltage is always controlled by the microcomputer so that the voltage changes at a constant speed within a certain range, and the voltage signal from the microcomputer is converted into an analog signal and applied from the voltage scanning unit 16 to the enzyme sensor unit 12. Yes.
[0025]
Next, the operation procedure of the sample measurement apparatus of the present invention will be described. An enzyme sensor unit for infiltrating body fluid to be detected is prepared. The enzyme sensor unit connects, for example, a chip-shaped enzyme sensor to the connection unit 14 of the detection device according to the present invention. When the enzyme sensor unit 12 is immersed in the sample liquid and the sample liquid is guided to the reaction layer, the reaction between the test component and the enzyme is started, and a current i proportional to the amount of the test component is generated. This current i is detected by the current detector 18. This value is digitally converted by the A / D conversion function of the microcomputer 20. A peak current or a current integral value is calculated from a current value obtained by digital conversion in time series. This value is compared with a set table in the microcomputer, the basic mass in the specimen is determined, and displayed as a measured value.
[0026]
The measurement mechanism and the measurement procedure will be specifically described with reference to FIG. In FIG. 2, the electric circuits corresponding to the functional units 14, 16, 18, and 20 in FIG. 1 are surrounded by dotted lines, and their mutual relationships are displayed.
[0027]
When the enzyme sensor unit 12 is connected to the connection unit 14 of the enzyme sensor unit, a predetermined voltage P1 is output from the D / A converter 22a through the buffer 24a under the control of the microcomputer 20.
[0028]
When the gap between the measurement electrode and the counter electrode is immersed in the test solution, a current i flows. This current i is detected. When the current i is detected, voltage application between the measurement electrode and the counter electrode is stopped under the control of the microcomputer 20. Subsequently, voltage application between the measurement electrode and the counter electrode is stopped until the enzyme is dissolved in the test solution for a certain time.
[0029]
In the present invention, although depending on the reaction conditions of the enzyme or electron acceptor supported on the enzyme sensor and the substrate, the certain time is usually 2 seconds to 10 seconds, corresponding to the reaction time of the substrate and the enzyme. After a predetermined time has elapsed, under the control of the microcomputer 20, the D / A converter 22a passes through the buffer 24a and the predetermined voltage P1 is output. The predetermined voltage P2 is output from the other D / A converter 22b through the buffer 24b.
[0030]
Since the voltage of the counter electrode (3d) of the enzyme sensor unit is P1, the voltage of the measurement electrode (3c) is P2, and the-and + terminals of the operational amplifier circuit 26c are virtual short circuits, the enzyme sensor unit terminals (external terminals 3c, 3d) is given a voltage (P2-P1).
[0031]
When the enzyme sensor unit is in a conducting state, a current i flows through the substrate detection unit of the enzyme sensor unit. As a result, the output terminal of the current-voltage conversion circuit 28b formed by the resistor Rx, the capacitor C1, and the operational amplifier 26c is connected to the output terminal.
Px = P2 + i · Rx
Will be observed.
Since P2 is added here, the subtraction circuit 30 outputs a new voltage P3 excluding the voltage P2.
This P3 is
(Px + P4) −P2 = [(P2 + i · Rx) + P4] −P2 = takes a value calculated by i · Rx + P4. P4 in this arithmetic expression is a ground voltage, and is set to a positive voltage in the case of a single power supply driving circuit.
[0032]
The voltage P3 corresponding to the current i is A / D converted by the A / D conversion circuit 22c and further digitized by the microcomputer 20.
[0033]
Here, a feature of the present invention is that, in the above-described apparatus, the voltage scanning means inverts the voltage from minus to plus or from plus to minus according to the cyclic voltammetry method. The voltage P2-P1 is shown in FIG. The detected current is plotted against the voltage.
[0034]
The present invention is characterized by a voltage scanning procedure. That is, the voltage (P2-P1) is 0 V at the start of voltage application, and the measured value from 0 V to the peak current of the electron acceptor is measured with respect to the counter electrode at a constant scanning speed (unit: mV / s). The voltage is scanned at a constant speed up to a preset voltage (−E1) via a voltage that can be observed, and then continuously with the counter electrode as a reference, at the same voltage scanning speed with the same sign and the opposite sign. The voltage scanning is set to a preset voltage (E2 = E1) through a voltage at which the peak current can be observed.
[0035]
For example, the substrate is glucose, the enzyme is glucose oxidase, the electron acceptor is ferricyan [Fe (CN)₆]^3-Will be described.
[0036]
(P2-P1) is set to 0 V at the start of voltage application. When negative scanning is performed at a constant voltage scanning speed, Felicyan [Fe (CN)₆]^3-A reduction takes place that receives electrons from the measuring electrode.
[0037]
The voltage is scanned to -E1 through a voltage at which the peak current can be observed. As a result, in the vicinity of the measurement pole, ferrocyan [Fe (CN)₆]^4-Will increase.
At this time, in the vicinity of the counter electrode, ferrocyan [Fe (CN)₆]^4-Oxidation passes electrons to the counter electrode, and as a result, ferricyan [Fe (CN)₆]³-Increases. Ferrocyan [Fe (CN)₆]^4-Is only the amount produced by reaction with glucose oxidase, and ferricyan [Fe (CN)₆]^3-The amount to be finite is all ferrocyan near the counter electrode [Fe (CN)₆]^4-But Felician [Fe (CN)₆]^3-It is oxidized to.
[0038]
Ferrocyan increase at measuring electrode [Fe (CN)₆]^4-Is a decrease in the ferrocyan [Fe (CN)₆]^4-As a result, in the vicinity of the measurement pole, ferrocyanine produced by enzymatic reaction of glucose oxidase and glucose [Fe (CN)₆]^4-Twice as much ferrocyan [Fe (CN)₆]^4-Exists.
[0039]
From P2−P1 = −E1V, when scanning positively at the same voltage scanning speed as described above, from a certain voltage, ferrocyan [Fe (CN)₆]^4-Oxidation passing electrons to the measuring electrode occurs, and the voltage is scanned up to E2V through a voltage at which a peak current can be observed. As a result, in the vicinity of the measurement pole, ferricyan [Fe (CN)₆]^3-Will increase.
[0040]
In the present invention, the reason for scanning in the opposite direction to -E1V is that the peak current is in the vicinity of 0V. Also, by scanning in the opposite direction, ferrocyan [Fe (CN)₆]^4-As a result, the current value that can be detected increases, and even if it is affected by noise, the numerical value for each sample can be obtained with good accuracy. Note that the same effect can be obtained by scanning the voltage once it is swung to the minus and then reversed to the plus, or first swung to the plus and then reversed to the minus.
[0041]
By correlating the voltage with the detected current value, the substrate component amount to be detected is determined by the voltage-current curve. The following two methods are mentioned as this determination method.
[0042]
In one method, as shown in FIG. 3, when the voltage of P2-P1 is scanned from 0V to -E1, the peak changes linearly until it rises after passing through -E1.
[0043]
The intersection point between the tangent line from −E1 until the peak rises and the straight line passing through the voltage at which the maximum current (imax) is observed and parallel to the i-axis is i.₀And The peak current is
ip = imax−i₀
Calculate with A high correlation is obtained between the peak current ip and the test component concentration.
Since the enzyme activity is generally temperature dependent, the peak current value ip at the actual measurement temperature is changed to the peak current value ip at the reference temperature.₀To correct. For temperature correction, a temperature correction table created in advance by the microcomputer 100 is used. After that, the microcomputer 100 uses the peak current ip₀Is compared with a reference calibration curve prepared in advance, and the component amount of the detected substrate is calculated.
[0044]
In another method, as shown in FIG. 4, when the voltage (P2-P1) is scanned from 0V to -E1 to the turn-back E1, the tangent line in the interval until the peak rises after passing through -E1. The area surrounded by the voltage-current curve obtained by voltage scanning from -E1 to E1 is defined as a current area value S. A high correlation is obtained between the current area value and the amount of the substrate component in the specimen.
[0045]
Thereafter, the microcomputer 20 calculates the test component amount by comparing the current integration value S0 with a reference calibration curve created in advance.
[0046]
The amount of the test component in the test solution is measured by the mechanism as described above. The amount of components determined by either corresponds linearly to the substrate concentration. FIG. 5A is a diagram showing the correlation between the peak voltage and the peak current, and FIG. 5B is a diagram showing the correlation between the peak current and the current integrated value. Thus, it can be seen that the peak current and the peak voltage are correlated, and further, the peak current and the current integrated value are also correlated.
[0047]
The voltage scanning speed is not particularly limited, but is preferably 50 to 200 mV / s in order to shorten the measurement time. If it is too fast, the output current will be larger than necessary. As the output current increases, the peak voltage shifts in the direction of increasing. When the peak voltage reaches 0.15 V or more, the linearity between the glucose concentration and the current integrated value is lost. In this range, the peak current and the scanning speed are correlated. FIG. 6 is a graph showing the relationship between the peak current and the scanning speed, in which the peak current value is plotted against the square root of the scanning speed when the scanning speed is changed at 50, 100, and 200 mV / s. . In the present invention, it is necessary for the voltage scanning speed to be always constant in the process of voltage scanning in a certain range in order to obtain an accurate peak current or current integral value.
[0048]
It is a feature of the present invention that the voltage scanned in the present invention is changed from the voltage 0 V to minus or plus and scanned to the reverse voltage after the peak voltage exceeds the observed peak voltage. When potassium ferricyanide is selected as the electron acceptor, the scanned voltage is preferably −0.3 V to +0.3 V, particularly preferably −0.2 V to +0.2 V. This operation method is set in the microcomputer.
[0049]
The obtained peak current value or current integrated value is digitized by an A / D conversion function included in the microcomputer. Here, since the enzyme activity generally has temperature dependence, the peak current value or current integral value at the actual measurement temperature is corrected to the peak current value or current integral value at the reference temperature. For the temperature correction, a temperature correction table created in advance by the microcomputer 20 is used.
[0050]
The corrected digitized peak current value or current integral value is compared with a reference measurement value prepared in advance in the microcomputer, and the amount of the component of the substrate in the sample is determined. The determined component amount is displayed by the display means.
[0051]
Although the present invention has been described above, the present invention is not limited to these embodiments, and various improvements, changes, and modifications can be made based on the knowledge of those skilled in the art without departing from the spirit of the present invention. It can implement in the aspect which added.
[0052]
【Example】
Hereinafter, embodiments of the method and apparatus of the present invention will be described in detail with reference to the following examples.
[0053]
The sample measuring device shown in FIG. 2 was used as the enzyme sensor and the sample measuring device.
Redox enzyme: glucose oxidase (active 165 units (u) / mg, manufactured by Toyobo Co., Ltd.), electron acceptor: pure potassium ferricyanide (manufactured by Nacalai Tesque, Inc.), microcrystalline cellulose solution: Zeolus cream (Asahi Kasei Corporation) What was dissolved in an aqueous solution of FP-03 (crystalline cellulose 10 wt%) was dripped and dried onto a PET film and used as a reaction layer of an enzyme sensor.
[0054]
The voltage scan was performed as follows. P2-P1 is 0 V at the start of voltage application, is negatively scanned at a voltage scanning speed of 50 mV / s, and a voltage at which peak current can be observed: P2-P1 = −0.2 V, 50 mV / s The voltage was scanned positively at a voltage scanning speed, and the voltage was scanned to 0.2 V through a voltage at which a peak current can be observed.
[0055]
[Example 1]
A 100 mg / dL glucose standard solution was prepared as a specimen. Three samples were prepared by using the sample as a glucose standard solution and injecting 5 μL of the glucose standard solution from the inlet of the enzyme sensor chip (Sample 1-1) (Sample 1-2) (Sample 1-3). The dissolution time was 4 seconds, and voltage scanning was performed immediately after this time. The voltage was changed from 0 V to -0.2 V at a scanning method of 100 mV / s, and then changed from -0.2 V to +0.2 V by the same scanning method. The peak voltage of (Sample 1-1) and the measured current at the peak voltage were −0.26 μA at −0.06 V and +34.8 μA at 0.075 V.
The peak voltage of (Sample 1-2) and the measured current at the peak voltage were −2.26 μA at −0.06 V and +33.92 μA at 0.075 V. The peak voltage of (Sample 1-3) and the measured current at the peak voltage were −22.2 μA at −0.06 V and +33.43 μA at 0.075 V.
[0056]
[Example 2]
The sample was the glucose standard solution prepared in Example 1, and two samples were prepared by injecting 5 μL of the glucose standard solution from the inlet of the enzyme sensor chip (Sample 2-1) (Sample 2-2). The dissolution time was 8 seconds, and voltage scanning was performed immediately after this time had elapsed. The voltage was changed from 0 V to -0.2 V at a scanning method of 50 mV / s, and then changed from -0.2 V to +0.2 V by the same scanning method.
The peak voltage of (Sample 2-1) and the measured current at the peak voltage were −16.84 μA at −0.08 V and +24.4 μA at 0.045 V. The peak voltage of (Sample 2-2) and the measured current at the peak voltage were -16.84 μA at −0.055 V and +25.38 μA at 0.07 V.
[0057]
A current integrated value was obtained from the measured current, and a CV value was obtained by the following equation.
[0058]
[Expression 1]

[0059]
The results are shown in Table 1. Table 1 shows the difference between the current integrated value of Examples 1 and 2 and the average value of the current integrated value of glucose concentration 0 mg / dl, the standard deviation, and the CV value obtained by the above formula 1. The CV value was 3% or less, and it was found that the accuracy was good. In addition, Table 2 is a table | surface which calculated | required the current integral value in distilled water with a glucose concentration of 0 mg / dl as standard 1-1, 1-2, 2-1, 2-2.
[0060]
[Table 1]

[0061]
[Table 2]

[0062]
[Example 3]
Samples having glucose concentrations of 1 mg / dl, 199 mg / dl, 445 mg / dl, 660 mg / dl, and 901 mg / dl were prepared, and five samples were prepared by injecting 5 μL of these samples. The dissolution time was 8 seconds, and voltage scanning was performed immediately after this time had elapsed. The voltage was changed from 0 V to -0.2 V at a scanning method of 50 mV / s, and then changed from -0.2 V to +0.2 V by the same scanning method. From the detected current values of these samples, the current integrated value with the voltage was obtained. The current integration values are listed in Table 3.
[0063]
[Comparative Example 1]
Samples having glucose concentrations of 1 mg / dl, 199 mg / dl, 445 mg / dl, 660 mg / dl, and 901 mg / dl were prepared, and five samples were prepared by injecting 5 μL of these samples. The dissolution time was 8 seconds, and voltage scanning was performed immediately after this time had elapsed. The scanning method was 50 mV / s, and the voltage was changed from −0.5 V to +0.2 V. From the detected current values of these samples, the current integrated value with the voltage was obtained. The current integration values are listed in Table 3. Comparative Example 1 was measured by the method disclosed in JP-A-2001-311712.
[0064]
[Table 3]

[0065]
In FIG. 7, Example 3 and Comparative Example 1 are shown in a graph of coordinates with the X axis as the glucose concentration and the Y axis as the current integration value. Comparative Example 1 loses linearity from a glucose concentration of 600 mg / dl and draws a curve, whereas Example 3 according to the method and apparatus of the present invention can maintain linearity up to a glucose concentration of 1000 mg / dl. Is understood from the graph.
[0066]
【The invention's effect】
Compared to the conventional constant voltage method and voltage scanning method, the amount of the test component in the test solution can be measured with high sensitivity, and the linearity can be maintained with high accuracy even when the concentration increases.
[Brief description of the drawings]
FIG. 1 is a block diagram of an apparatus for carrying out the present invention.
FIG. 2 is a circuit diagram of an apparatus for carrying out the present invention.
FIG. 3 shows a method for obtaining a peak current value from a voltage-current curve.
FIG. 4 shows a method for obtaining an integrated current value from a voltage-current curve.
FIG. 5A is a graph showing the correlation between the peak voltage and the peak current, and FIG. 5B is a graph showing the correlation between the peak current and the current integrated value.
FIG. 6 is a graph showing the relationship between the square root of the scanning speed and the peak current.
7 is a graph showing current integration values with respect to glucose concentrations in Example 3 and Comparative Example 1. FIG.
FIG. 8 shows an example of a commonly used enzyme sensor.
[Explanation of symbols]
10; Sample measuring apparatus of the present invention
12: Enzyme sensor unit
14; Connection part of sample measuring device
16: Voltage scanning unit
18; Current detector
20; Microcomputer
22a, 22b; D / A converter
22c; A / D converter
24a, 24b; buffer
26c; operational amplifier circuit
28b; current-voltage conversion circuit
100; Basic enzyme sensor
102; Electrically insulating substrate
104; Measuring pole
106; counter electrode
108; reaction layer
110; electric insulating spacer
112; outer cover
114; sample inlet

Claims (5)

An apparatus for measuring the basic mass of a specimen by a programmed computer and displaying the basic mass value,
Forming an electrode portion including at least a measurement electrode and a counter electrode on an insulating substrate, and an enzyme sensor unit electrically bridging the measurement electrode and the counter electrode in a reaction layer including at least an enzyme and an electron acceptor;
A voltage scanning unit that generates a potential difference by applying a voltage that varies with time between the measurement electrode and the counter electrode; and
A current detection unit for detecting a current flowing through the electrode unit;
A sample measuring device including a microcomputer that controls the voltage scanning unit to generate a predetermined voltage and discriminates a substrate component amount of the sample based on an output of the current detection unit;
The microcomputer is
(1) Means for generating a potential difference between both electrodes of the voltage scanning unit and the current detection unit in a predetermined constant voltage range (E1 to E2), and the voltage of the counter electrode in the enzyme sensor unit is used as a reference The measuring electrode is scanned from the voltage 0 V to the voltage (E1) at a constant voltage scanning speed (v1), and then continuously, the voltage scanning speed (v1) and the absolute value are the same from the voltage (E1). A voltage signal that reverses the voltage from minus to plus or from plus to minus by continuously scanning up to the voltage (E2) at a voltage scanning speed (v2 = −v1) in which
(2) means for analog conversion of the voltage signal;
(3) means for digitally converting the current value detected by the current detector;
(4) means for calculating a peak current value or a current area value detected from a voltage-current curve of the digitally converted current value and the scanned electrode voltage;
(5) Control comprising: means for determining the component amount of the substrate concentration of the sample by comparing the peak current value or current area value with a reference calibration curve prepared in advance; and (6) means for displaying the substrate concentration of the sample. A sample measuring apparatus having means. A method for measuring a basic mass of a specimen using the apparatus according to claim 1 and displaying the measured basic mass value.
Supplying a sample for measurement onto the reaction layer of the enzyme sensor unit;
Scanning the voltage between the electrodes;
Detecting the current flowing through the enzyme sensor unit;
A method in which a computer performs the following control steps to measure a base mass of a specimen and display the base mass measurement value .
(1) A step of generating a potential difference between both electrodes of the voltage scanning unit and the current detection unit within a preset constant voltage range (E1 to E2), wherein the voltage of the counter electrode in the enzyme sensor unit is used as a reference The measuring electrode is scanned from the voltage 0 V to the voltage (E1) at a constant voltage scanning speed (v1), and then continuously, the voltage scanning speed (v1) and the absolute value are the same from the voltage (E1). A voltage signal that reverses the voltage from minus to plus or from plus to minus by continuously scanning up to the voltage (E2) at a voltage scanning speed (v2 = −v1) that is reversed .
(2) converting the voltage signal into analog;
(3) a step of digitally converting the current value detected by the current detection unit;
(4) calculating a peak current value or a current area value detected from a voltage-current curve of the digitally converted current value and the scanned electrode voltage;
(5) a step of determining the component amount of the substrate concentration of the sample by comparing the peak current value or the current area value with a reference calibration curve prepared in advance; and (6) a step of displaying the substrate concentration of the sample. In step (1) of the control step using the computer, in the voltage scan of the measuring electrode, E1 is reached from a voltage of 0 V through a voltage at which the peak current of the electron acceptor can be observed. The method for measuring a base mass of an analyte and displaying the measured base mass value according to claim 2 , which is a step of scanning to reach E2 via a voltage at which a peak current of a receptor can be observed. 4. The method for measuring a base mass of a specimen according to claim 2 or 3 , wherein the enzyme is glucose oxidase and the electron acceptor is potassium ferricyanide and displaying the base mass measurement value . In the voltage scanning of the microcomputer, the voltage E1 -0.2V, the voltage E2 is 0.2V, claims 2 to 4 the voltage scanning rate, characterized in that a 10mV / s~200mV / s A method for measuring the base mass of the specimen according to any one of the above and displaying the measured base mass .

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