JP3991173B2 - Distinguishing liquid sample from control liquid - Google Patents

Description

表１からは、コントロール液１の最小値の方が血液の最大値よりも大きいため、例えば、比較値として１１０を選べば、比較値１１０よりも大きいα₄値を示すものをコントロール液１、小さいものを血液として両者を弁別することが出来る。同様にして、コントロール液１とコントロール液２も弁別可能である。しかし、コントロール液２と血液では、コントロール液２の最大値が血液の最小値よりも大きいために、弁別が不能である。
【００２０】
（演算２）
次は、演算１と同様にして得られる電流とその時間微分の比α_t（ｔ秒後電流から得られるαの意）を複数求め、それらの差を測定サンプル弁別のための指標値とする場合について説明する。０．５秒、２秒について、α_0.5，α₂を求める。式で表せば、それぞれ、α_0.5＝Ｉ_0.5／（Ｉ_0.5−Ｉ_0.6）、α₂＝Ｉ₂／（Ｉ₂−Ｉ_2.1）と表せる。これらを求めておいて、次に、これらの差を取る。本実施例では、α₂からα_0.5を減じた差を取った。ｔ₂秒のα_t2とｔ₁秒のα_t1の差をΔα（ｔ₁，ｔ₂）のように表すとして、本実施例での演算を式で表すと、Δα（０．５，２）＝α₂−α_0.5＝｛Ｉ₂／（Ｉ₂−Ｉ_2.1）｝−｛Ｉ_0.5／（Ｉ_0.5−Ｉ_0.6）｝となる。表２に各測定サンプルにおけるΔα（０．５，２）の値を示す。尚、基としたデータは、演算１に用いたものと同一で、コントロール液２のデータは、測定数３０の結果であり、血液のデータは、測定数９０の結果である。
【表２】

表２からは、コントロール液２の最大値の方が血液の最小値よりも小さいため、例えば、比較値として３０を選べば、比較値３０よりも小さいΔα（０．５，２）値を示すものをコントロール液２、大きいものを血液として両者を弁別することが出来る。
【００２１】
以上の結果から、コントロール液１，コントロール液２および血液の３つの測定サンプルを弁別することが可能となる。すなわち、本実施例に於いては、Δα（０．５，２）の値が３０未満であればコントロール液２であると判定し、３０以上であれば血液あるいはコントロール１であると判定し、更にα₄の値が１１０未満であれば血液で１１０以上であればコントロール液１であると判定することが出来る。これにより、コントロール液１，コントロール液２および血液の３つの測定サンプルを弁別出来たことになる。
【００２２】
これまでに述べてきた様な方法によって、コントロール液の弁別が可能となるので、弁別結果を表示することの出来るバイオセンサシステムを構築するのは容易である。また、コントロール液を用いたシステムチェックの結果表示は、本法によって測定された測定サンプルがコントロール液であることが弁別できるので、予めシステムにコントロール液の正常範囲電流値を記憶させておけば、濃度を調べるための電流測定の結果とその正常範囲電流値を比較した結果を表示することも可能になる。
【００２３】
【発明の効果】
本発明の方法を用いることによって、使用者に負担を掛けずコントロール液を測定した際には機器が自動的に血液サンプルなどの通常サンプルではなく、コントロール液であることを弁別してこれを表示し、使用者側の誤認識を防ぐことの出来るバイオセンサシステムを構築することが出来る。併せて、煩雑な作業を伴うこと無しにコントロール液によるシステムチェックの結果を知ることの出来るバイオセンサシステムを構築することが出来る。
【００２４】
【図面の簡単な説明】
【図１】時間と印加電位の関係を表すものである。
【図２】グルコース濃度９７ｍｇ／ｄｌであるものを測定した際の減衰電流を示す[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring an analyte concentration contained in a liquid sample. In particular, the present invention relates to a method for quantifying the concentration of, for example, glucose and cholesterol contained in a body fluid such as blood by amperometry.
[0002]
[Prior art]
Conventionally, a biosensor has been known as a method for easily and quickly quantifying the concentration of an analyte in a liquid sample, particularly a biological sample such as blood.
[0003]
A biosensor is one type of device for performing electrochemical measurements. Electrochemical measurement, as the name implies, measures chemical reaction as an electrical signal, such as current, voltage, and charge. The weak point of electrochemical measurement is its low specificity. That is, even if the substance generated by the chemical reaction is a substance that can be electrochemically measured, if another substance that can be electrochemically measured exists in the measurement sample, an error occurs immediately. In order to compensate for the weakness of the low specificity of the electrochemical measurement, the biosensor uses a biofunctional substance that can selectively and specifically react with the analyte, such as an enzyme. The biosensor has at least the following two parts. One is a part that selectively and specifically reacts with a target product as a selection function part. For example, enzymes and antibodies correspond to this. The other is a part that converts the chemical reaction of the selection function part into an electrical signal as a signal conversion part. Mainly corresponds to an electrode. In measurement of an analysis object using a biosensor, various electrical quantities such as current, voltage, and charge amount can be directly measured. Among them, there are many biosensors that measure current.
[0004]
A biosensor system is a combination of a device that applies a potential to a biosensor or measures a current that is a signal of the biosensor and a biosensor as a test cell. For example, in the case of a biosensor system for quantifying the concentration of lactic acid in a blood sample, a biosensor that uses an enzyme such as lactate oxidase (LOD) or lactate dehydrogenase (LDH) that reacts specifically with lactic acid as a selection function unit. As a test cell, a timer function that measures the time after the reaction between the blood sample and the enzyme starts, a function that applies a predetermined potential to the biosensor after a predetermined time has elapsed, and is determined from the start of the potential application. A function for measuring current after a lapse of time, a correlation between current and lactic acid concentration, for example, a function for storing a calibration curve, a function for determining the stored correlation, for example, a lactic acid concentration from the calibration curve and the measured current And constructing it by using it in combination with a device having a function to display the determined result on a display etc. Can. Such a biosensor system is not limited to the determination of the lactic acid concentration in a blood sample, and various types of biosensor systems are known.
[0005]
European Patent Application Publication No. 0230472 discloses a biosensor system that measures the glucose concentration of a blood sample using amperometry. In this system, a biosensor including a measurement electrode, a reference electrode, and a counter electrode is used as a test cell. The electrode is covered with a reagent layer containing glucose oxidase, potassium ferricyanide and other components. When a blood sample is placed in contact with the reagent layer, glucose in the sample reacts with potassium ferricyanide through the action of glucose oxidase to form potassium ferrocyanide. Thereafter, when a voltage is applied to the electrode, a reverse reaction occurs, and a current proportional to the concentration of potassium ferrocyanide generated in the first reaction flows. It is assumed that the measured value of this current corresponds to the concentration of glucose in the sample.
[0006]
In electrochemical measurement, a method of applying a potential in a rectangular wave with respect to time and measuring a current with respect to the potential is generally known as a potential step method. The term “rectangular wave potential application with respect to time” as used herein means that a constant potential that is substantially instantaneous is applied, and then the constant potential is continuously applied. In such a potential step method, a current that decays with the passage of time since the potential was applied, or a decaying current is observed. Since this decay current depends on time, it can be expressed as a function of time. On the other hand, this decay current also depends on the analyte concentration. Therefore, this decay current can also be expressed as a function of the analyte concentration. The decay current can be expressed as a function of time and analyte concentration. Therefore, as other factors affecting the decay current, for example, when the sample properties, sample temperature, electrode area, applied potential, etc. are kept constant, the decay current is I, the time is t, and the analyte. When the density is C, it can be expressed as I = (t, C). I (t, C) indicates that I is a function of t and C, and if the time t can be kept constant, in other words, the decay current measured at the same timing is the analyte. It can be expressed as a function of concentration only. Expressed by the formula, I (t = constant, C) = I (C). This principle is used in conventional biosensor systems. The current measurement time is determined in advance, and the current and the analyte concentration are correlated by using a calibration curve I (C) indicating the correlation between the current at that time and the analyte concentration. At this time, the current measurement is performed only once.
[0007]
A biosensor system that performs current measurement a plurality of times is disclosed in Japanese Patent Publication No. 2651278. In the present invention, information obtained by performing current measurement a plurality of times is used for detecting an abnormal sample supply state. Information other than the analyte concentration is being obtained from the relationship between the currents at different times, not the current magnitude itself. In other words, rather than the current I, it is an attempt to obtain information from a function of time I (t). This system takes two consecutive current measurements and compares the ratio of the measured currents to the ratio of the reciprocal of the square root of the time at which the measurement was taken to determine whether the system is working properly. I have an indicator. This is because the theoretical formula of I (t, C) in the potential step method under the conditions that the sample amount is semi-infinite, the analyte concentration distribution is uniform immediately before the potential is applied to the electrode, and the sample is not stirred is the time ( Based on being proportional to the inverse of the square root of t). For example, if the blood sample does not cover the entire surface of the detection electrode, or if the reaction zone is hydrated before or during the test, a leak will occur and the blood sample will not only cover the electrodes in the reaction zone but also outside the reaction zone. If there is an error in the reading, the ratio of the current obtained from the two consecutive measurements is compared with the ratio of the reciprocal of the square root of the time at which the measurement was performed. It is said that it is possible to determine that an error has occurred by using the index.
[0008]
As another conventional technique for checking whether or not the biosensor system is operating normally, a method using a control solution is known. This technique has a purpose slightly different from that of the invention disclosed in Japanese Patent Publication No. 2651278, and is mainly intended to check whether or not a device in the biosensor system is operating normally. A system check method using a control solution will be described below by taking a biosensor system for quantifying the glucose concentration of a blood sample as an example.
[0009]
The control solution is prepared to a known glucose concentration, and the response of a normal system, for example, a glucose concentration display is examined in advance. Further, some control liquids have a viscosity increased by adding a water-soluble polymer, and some have a dye added in order to approximate the properties of a blood sample. The system user measures the control solution by the same means as when measuring a blood sample periodically or as necessary. Then, it is determined whether the system response is normal. The determination of whether or not the system response is normal depends on comparing the actual system response value with the normal range displayed on the container of the control liquid or the package insert. For example, when a control solution having a normal range of 70 to 120 mg / dL is measured, if the response value of the system is 80 mg / dL, 110 mg / dL, etc., it is determined that the system is operating normally, and the response value of the system Is 50 mg / dL, 140 mg / dL, etc., it is determined that the system is abnormal. In this way, it is determined whether or not the biosensor system is operating normally.
[0010]
[Problems to be solved by the invention]
In the biosensor system check method using the control liquid as described above, whether the control liquid is measured for the system check or the measurement of a normal sample such as a blood sample, the response of the system Is the same with only the indication of the analyte concentration, so if the user does not know what the sample was used, it was the result of measuring the control solution, or a normal sample such as a blood sample was measured There was a problem that it was not possible to determine whether it was a result. For this reason, for example, a result of measuring a normal sample such as a blood sample is misrecognized as a result of measuring a control solution, or it is determined to be abnormal although the system is normal It is the result of measuring a normal sample such as a blood sample even though it is determined to be normal even though the system is abnormal, or the result of measuring the control solution There was a risk that a doctor would make a wrong diagnosis. Especially in the latter case, the risk of wrong diagnosis based on wrong blood test data was serious and serious.
[0011]
Further, when determining whether or not the system is normal, it is necessary to compare the measurement results of the control liquid with the normal range displayed on the control liquid container or the package insert, and the work is complicated. It was particularly troublesome when the system check had to be performed frequently, when the system check was performed using a control solution with multiple concentrations, or when the system check had to be performed for a large number of biosensor systems. .
[0012]
The object of the present invention is not to put a burden on the user, and when the control solution is measured, the system automatically discriminates that the control solution is not a normal sample such as a blood sample, and displays this. Provided is a method for constructing a biosensor system that can prevent misrecognition on the user side. In addition, the present invention provides a method for constructing a biosensor system that can know the result of a system check using a control solution without complicated operations.
[0013]
[Means for Solving the Problems]
The present invention is a method used in a biosensor system for quantifying an analyte concentration of a liquid sample by measuring an electric current, which calculates a ratio of the measured current and its time derivative, and uses this as an index for the liquid sample This is a method for discriminating between the control liquid and the control liquid . The time derivative of the measured current may be a strict sense derivative obtained from analog-continuous measurement data, or may be a derivative value substantially from digitally discontinuous measurement data. It may be calculated. For example, the current can be measured at a relatively short time interval, and the difference between the two can be used as a time derivative. In other words, in the definition equation of time differentiation, lim (Δt → 0) = {I (t + Δt) −I (t)} / Δt, Δt must be infinitesimal, so that strict time differentiation is obtained from discontinuous measurement. Although it is impossible to obtain, it is possible to obtain an approximate time derivative from the current measured at a relatively short time interval Δt. If Δt cannot be shortened for some reason, for example, the time derivative can be obtained approximately by measuring three or more points and taking a moving average of the differences. The ratio between the time derivative thus obtained and the current is taken as an index for discriminating between the liquid sample and the control liquid . The ratio of time derivative and current may be differentiated / current or current / differentiated as desired, and constants may be included in these four arithmetic operations. There is no essential difference between them, only the way of selecting the comparison values described later is different. The time derivative may be a strict derivative (dI / dt) or an approximate derivative (ΔI / Δt), and the current derivative is also used when calculating an index for discriminating between the liquid sample and the control liquid. And the ratio of the time derivative is {I / (dI / dt)} or {I / (ΔI / Δt)}, {(dI / dt) / I} or {(ΔI / Δt) / I} However, these ratios may be (aα ± b), where α, a, and b are constants. This is because there is no essential difference in selecting any of the above as an index for discriminating between the liquid sample and the control liquid, and only the method of obtaining the comparison value described later is different.
[0014]
As a method for obtaining another index for discriminating between the liquid sample and the control liquid, the ratio of the current and its time derivative can be calculated a plurality of times and the difference between them can be taken. Assuming that the ratio α of the current and its time derivative is calculated n times (n times), if the k-th and the first are α _k and α ₁ and the difference between them is Δα, Δα (k, l) = α It can also be expressed as _k −α ₁ . However, the parentheses of Δα (k, l) represent the difference between the kth and lth α. In this case, the sign may be changed to obtain an index for discriminating between the liquid sample and the control liquid, and even if Δα (k, l) = α _k −α ₁ , Δα (k, l) = Α ₁ −α _k may be used. Also, constants may be included in terms of four arithmetic operations, c and d may be certain constants, and {c (Δα) ± d} may be used as an index for discriminating between the liquid sample and the control liquid . This is because there is no essential difference in selecting any of the above as an index for discriminating between the liquid sample and the control liquid, and only the method of obtaining the comparison value described later is different.
[0015]
The liquid sample and the control liquid can be discriminated by comparing the index obtained by the method described so far with the comparison value. Since the comparison value is an experimentally obtained value, in order to discriminate the liquid sample from the control liquid using this method, the index value of the liquid sample or the control liquid is examined in advance. I have to leave. For example, when discriminating between a control liquid and a liquid sample using an electric current and its time derivative ratio α as an index, a threshold value such that an α value of less than α ₀ is observed in the control liquid and α ₀ or more is observed in the sample. As a result, α ₀ may be selected as a comparison value. If the comparison value determined in this way is stored in the system in advance, the liquid sample and the control liquid can be discriminated by comparing the index value observed during actual measurement with the comparison value. If discrimination is possible, it is easy to display the result on the system.
[0016]
If it this method is a control solution with is discriminated can be displayed together with the determination result of the system check. The determination result may be displayed as a binary choice such as ◯, ×, OK or NG, or may be specifically displayed such as control High · 270 mg / dl · OK.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
【Example】
Hereinafter, the present invention will be described in more detail with reference to specific examples. As an example of a biosensor system, a glucose sensor system that quantifies the glucose concentration of a blood sample will be described. The glucose sensor which is a test cell of the glucose sensor system has the following configuration. On an insulating substrate made of PET (polyethylene terephthalate), an electrode system (carbon) including a lead (silver), a working electrode, and a counter electrode, and an electrical insulating layer are formed by screen printing. The electrically insulating layer has a constant area of the exposed portion of the working electrode and the counter electrode, and partially covers the lead. A carboxymethyl cellulose (CMC) layer, which is a hydrophilic polymer, is formed on the electrode portion thus formed. Furthermore, an enzyme and mediator layer made of glucose oxidase (GOD) as an enzyme and potassium ferricyanide as an electron carrier (mediator) is formed on the CMC layer. (Hereinafter, the CMC layer and the enzyme and mediator layer are collectively referred to as a reaction layer.) Further, an insert composed of a cover and a spacer is formed, and when the sample touches the insert, the reaction layer and the electrode system are fixed by capillarity. About 3 μL of sample is supplied as a volume. On the other hand, a general-purpose potentiostat, BAS100B / W (manufactured by BAS) was used as a measuring instrument. In the case of a normal biosensor system, a dedicated device is used in combination with one test cell. In this embodiment, a general-purpose device is used. However, it is easy to reproduce the present embodiment even with a simple device using a known technique.
[0018]
As a measurement sample, control liquid 1, control liquid 2, and blood were used. The control liquid 1 is an aqueous solution to which benzoic acid is added as a preservative and Red No. 2 and glucose are added as pigments. Two glucose concentrations of 97 mg / dl and 325 mg / dl were used. The property of the control liquid 1 was close to water and relatively low in viscosity. The control liquid 2 is an aqueous solution of polyvinyl pyrrolidone (PVP), which is a water-soluble polymer, and glucose. Two types of glucose concentrations, 83 mg / dl and 256 mg / dl, were used. The property of the control liquid 2 was relatively high in viscosity. Six types of blood having different blood cell component concentrations and glucose concentrations were used. Blood cell component concentration (indicated by hematocrit value) and glucose concentration are (30%, 102 mg / dl), (44%, 101 mg / dl), (58%, 103 mg / dl), (28%, 268 mg / dl). (45%, 263 mg / dl), (62%, 266 mg / dl). Moreover, heparin sodium was added as an anticoagulant. These measurement samples were supplied to the reaction layer and electrode system of the test cell, and allowed to stand without applying potential for 25 seconds. During this time, glucose in the measurement sample reacts with potassium ferricyanide in the reaction layer through the action of glucose oxidase to produce potassium ferrocyanide. Then, a constant potential of 500 mV was applied between the working electrode and the counter electrode in a rectangular wave with respect to time for 5 seconds after 25 seconds, and the current was measured at intervals of 0.1 seconds. The current observed at this time is due to the reverse reaction from potassium ferrocyanide to potassium ferricyanide. Since this current is proportional to the concentration of potassium ferrocyanide produced in the initial reaction, the measured value of this current corresponds to the glucose concentration of the measurement sample. In addition, this current is an attenuation current that decays with the passage of time from the application of the potential. FIG. 1 shows the relationship between time and applied potential. FIG. 2 shows the decay current when measuring the control solution 1 having a glucose concentration of 97 mg / dl. The time unit on the horizontal axis is seconds, and the moment when the potential is applied is 0 seconds.
[0019]
(Calculation 1)
An operation value for measurement sample discrimination is obtained by the method described below. In the present embodiment, a current after 4 seconds from the voltage application is used, but the present invention is not limited to this, and a current suitable for each measurement sample valve can be used. First, the difference obtained by subtracting the current after 4.1 seconds from the value after 4 seconds of the measured decay current was taken as the time derivative of the current after 4 seconds. In this case, Δt = 0.1 seconds is used instead of infinitesimal Δt in the definition equation of time differentiation, lim (Δt → 0) = {I (t + Δt) −I (t)} / Δt, Corresponds to the replacement. Thus obtained time derivative of the denominator, the current after 4 seconds a value of the ratio of the molecule, did this in the discrimination index alpha _4. The subscript ₄ of α ₄ indicates an index obtained from the current after 4 seconds. Table 1 shows the value of the index α ₄ in each measurement sample. The data of the control solution 1 and the control solution 2 are the results of the number of measurements 30, respectively, and the blood data are the results of the number of measurements 90. Table 1 shows the α ₄ value for each measurement sample.
[Table 1]

From Table 1, since the minimum value of the control solution 1 is larger than the maximum value of blood, for example, if 110 is selected as the comparison value, the control solution 1, which shows an α ₄ value greater than the comparison value 110, Both can be discriminated by using small blood as blood. Similarly, the control liquid 1 and the control liquid 2 can be discriminated. However, the control liquid 2 and blood cannot be discriminated because the maximum value of the control liquid 2 is larger than the minimum value of blood.
[0020]
(Calculation 2)
Next, a plurality of ratios α _t (meaning α obtained from the current after t seconds) of the current obtained in the same manner as the calculation 1 and its time derivative are obtained, and the difference between them is used as an index value for measurement sample discrimination. The case will be described. Α _0.5 and α ₂ are obtained for 0.5 seconds and 2 seconds. Expressed by the formula, they can be expressed as α _0.5 = I _0.5 / (I _0.5 −I _0.6 ) and α ₂ = I ₂ / (I ₂ −I _2.1 ), respectively. Find these and then take these differences. In this example, the difference obtained by subtracting α _0.5 from α ₂ was taken. The difference between t ₂ seconds alpha _t2 and t ₁ seconds alpha _t1 as represented as [Delta] [alpha] (t _1, t _2), to represent the operation of the present embodiment by the formula, [Delta] [alpha] (0.5, 2) = Α ₂ −α _0.5 = {I ₂ / (I ₂ −I _2.1 )} − {I _0.5 / (I _0.5 −I _0.6 )}. Table 2 shows the value of Δα (0.5, 2) in each measurement sample. Note that the base data is the same as that used in the calculation 1, the data of the control solution 2 is the result of the measurement number 30, and the blood data is the result of the measurement number 90.
[Table 2]

From Table 2, since the maximum value of the control solution 2 is smaller than the minimum value of blood, for example, if 30 is selected as the comparison value, a Δα (0.5, 2) value smaller than the comparison value 30 is shown. Both can be discriminated by using the control liquid 2 as the substance and the blood as the larger one.
[0021]
From the above results, it is possible to discriminate between the three measurement samples of the control liquid 1, the control liquid 2 and the blood. That is, in this example, if the value of Δα (0.5, 2) is less than 30, it is determined that it is a control liquid 2, and if it is 30 or more, it is determined that it is blood or control 1. Furthermore, if the value of α ₄ is less than 110, it can be determined that it is the control solution 1 if it is 110 or more for blood. As a result, the three measurement samples of the control liquid 1, the control liquid 2 and the blood can be discriminated.
[0022]
Since the control liquid can be discriminated by the method as described above, it is easy to construct a biosensor system capable of displaying the discrimination result. In addition, since the result of the system check using the control liquid can be discriminated that the measurement sample measured by this method is the control liquid, if the normal range current value of the control liquid is stored in advance in the system, It is also possible to display the result of comparing the current measurement result for examining the concentration with the normal range current value.
[0023]
【The invention's effect】
By using the method of the present invention, when measuring the control liquid without imposing a burden on the user, the instrument automatically distinguishes and displays that it is not a normal sample such as a blood sample but a control liquid. It is possible to construct a biosensor system that can prevent misrecognition on the user side. In addition, it is possible to construct a biosensor system that can know the result of the system check using the control solution without complicated operations.
[0024]
[Brief description of the drawings]
FIG. 1 represents the relationship between time and applied potential.
FIG. 2 shows the decay current when measuring a glucose concentration of 97 mg / dl

Claims (8)

A method used in a biosensor system for quantifying an analyte concentration of a liquid sample by measuring a current, the ratio of the measured current and its time derivative being calculated, and the ratio of the measured current and its time derivative being calculated. the method of discrimination of a liquid sample and a control solution, characterized in that to distinguish between the liquid sample and the control liquid using. The method for discriminating between a liquid sample and a control liquid according to claim 1, wherein a ratio of the measured current and its time derivative is calculated a plurality of times. Wherein the plurality of times calculated any method discrimination of the liquid sample and control solution according to claim 2 you and calculates the difference between the two ratios. 2. The method for discriminating between a liquid sample and a control liquid according to claim 1, wherein the calculated ratio is compared with a predetermined comparison value stored in advance in the system, and the result is displayed. 3. The method for discriminating between a liquid sample and a control liquid according to claim 2, wherein the ratio calculated a plurality of times is compared with a predetermined comparison value stored in advance in the system and the result is displayed. Compared to any given comparison value which is previously system stores the difference between the two of the plurality of ratio the calculated, the liquid sample and control solution according to claim 3, characterized in that to display the results Discrimination method. The method of discrimination of the liquid sample and control solution according to any one of claims 1 to 6 wherein the liquid sample is blood. 8. The method for discriminating between a liquid sample and a control liquid according to claim 4, wherein the determination result of the system check by the control liquid is displayed.

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