This invention relates to a method for electrochemically measuring a concentration of an analyte in a sample. More specifically, this invention relates to a method for electrochemical measurement to obtain a concentration of analyte in a sample by correcting errors caused by sample physical properties and sensor sensitivity.
Recently in the fields of biochemistry, clinical medicine or the like, electrochemical measurement has been used to measure a concentration of an analyte in a sample in a rapid and simple manner. An electrochemical measurement is a method to measure an analyte in a sample by using an electric signal such as a current that is obtained from a chemical reaction or an enzymatic reaction.
For example, the concentration of glucose in a blood is measured in the following process. Glucose as an analyte in a blood sample reacts with glucoseoxidase (GOD) to generate a GODxc2x7H2 complex, from which electrons are liberated by an electron carrier such as potassium ferricyanide. The free electrons are captured in an electrode to calculate a current value, and thus, a glucose concentration is obtained by calculation from a calibration curve, a calibration formula or the like that is produced previously.
In an electrochemical measurement, a disposable device called a biosensor is commonly used. A biosensor has a reaction layer and an electrode system.
A reaction layer comprises a reaction reagent, an enzyme or a matrix to react specifically with an analyte in a sample, and provides a reaction site.
An electrode system comprises a working electrode and a counter electrode, and by applying voltage to perform an oxidation-reduction reaction, the electrode system captures electrons that are generated by the chemical reaction occurring in the reaction layer, where the electrons are electric signals of a current from an electron carrier to the electrode.
A biosensor is used in a combination with a measurement apparatus having various functions such as providing a certain voltage in a predetermined period, measuring electric signals transferred from the biosensor, and converting the electric signals into a concentration of the analyte. Such a system is called a biosensor system.
Among various methods to apply voltage to an electrode system, a method to apply voltage like a rectangular wave with respect to time is called a potential step method. In a typical biosensor system, sample feeding to the biosensor is detected and subsequently, an open circuit or a voltage with substantially no current flow is provided to promote a chemical reaction. After a predetermined period of time, a fixed voltage is applied to deliver electrons between the electron carrier and the electrode, i.e., to perform an oxidation-reduction reaction. A state where the biosensor is provided with a voltage to perform a desired oxidation-reduction reaction is called an excitation state. In general, a current value at an arbitrarily-predetermined point of time during the excitation state is measured, and the current value is converted into a concentration of the analyte, using a calibration curve and a calibration formula that are produced previously.
Methods in which plural excitations and plural current measurements are performed are disclosed, for example, in JP-2651278, JP-A-8-304340, and JP-A-10-10130.
JP-2651278 provides a method to determine whether a current flowing in a reaction site changes in accordance with a relationship with a certain Cottrell current.
JP-A-8-304340 suggests reduction of measurement errors caused by a reduction type intermediate product that is generated during a storage of the sensor.
JP-A-10-10130 provides a method for discriminating a whole blood sample and an aqueous solution as a control in a biosensor system.
Measurement results obtainable by using the biosensor can include errors due to various factors. One of the factors causing such errors is a sample physical property.
For example, when the sample is whole blood and the analyte is glucose in the blood, the hematocrit value (Hct) as a volume ratio of erythrocyte to the whole blood is found to cause errors in the measurement result, and differences between individuals are great. The reason appears to be a rising viscosity of the sample.
Concentrations of neutral fat and protein in the blood also affect the measurement result. In a measurement of an analyte in a blood, sample physical properties, such as blood cells, lipid and protein, will cause measurement errors.
Generally in a conventional method to avoid any influences by such errors, the composition of a chemical reaction layer or of an electrode system in a biosensor is improved. For example, JP-A-62-64940 discloses a biosensor in which an enzyme to detoxify a measurement-interfering substance is immobilized on an enzyme-immobilizing membrane. JP-A-61-3048 discloses a biosensor comprising not only a biocatalytic electrode but an electrode to detect the quantity of measurement-interfering substances. JP-A-60-211350 discloses a biosensor comprising two electrode systems including an electrode system containing an enzyme and a pigment and also an electrode system provided with a porous material containing a pigment only. However, these biosensors with complicated structures require complicated manufacturing processes, and the manufacturing cost is also raised.
Sensor sensitivity can be another factor causing measurement errors. In many cases, sensor sensitivity varies from one manufacturing lot of biosensors to another. Manufactures make sensor sensitivity correction tips for the respective lots, and ship the tips with their biosensors.
When a lot for a biosensor is changed, a user should correct sensitivity by using a correction tip corresponding to the changed lot before he performs an ordinary measurement (JP-A-4-357452).
However, such an operation will impose extra work on the user. Moreover, since the range corrected with such a correction tip is determined based on the sensitivity during manufacture of the sensor, changes of the sensor sensitivity over time, which occur after shipping, will not be corrected.
Since an electrochemical measurement includes a chemical reaction, it is affected also by an environmental temperature and a sample temperature. Namely, an environmental temperature and sample temperature also can be factors of measurement errors. JP-2748196 provides a method of correcting nonlinear temperature dependency of a chemical sensor.
Therefore, the purpose of the present invention is to provide an electrochemical measurement method that can decrease work for users and manufacturers without requiring any complicated structure or process of manufacturing a biosensor and a measurement apparatus. Such a method can provide highly accurate results by correcting errors of concentration of an analyte in a sample.
To achieve the purpose, the present invention provides an electrochemical measurement to measure a concentration of an analyte in a sample by using a biosensor having an electrode system and a chemical reaction layer. The measurement comprises calculation to obtain as parameters a current value obtained as a result of application of a fixed voltage to the sensor after feeding the sample and the ratio of the current value, and calculation of the concentration of the analyte by using a statistical technique. The parameters are set adequately for each error factor affecting the measurement results, and error factors can be corrected directly by selecting parameters that will be affected greatly by the sample physical properties, or by selecting current values that will be affected greatly by the sensor sensitivity.
For the above-mentioned parameters, for example, the following parameters P1 and P2 are preferably used, and the parameters are obtained by applying a predetermined voltage twice to a biosensor after feeding a sample in order to promote an electrochemical reaction.
P1: a ratio (If/Ib), where (If) is the value of maximum current or a current occurring after the maximum in a first excitation, and (Ib) is the value of current (Ib) read at any point in a second excitation
P2: a current value (Ib) read at any point in the second excitation
With a statistical technique using these two parameters, errors caused by sample physical properties and sensor sensitivity can be corrected with high accuracy. Therefore, the present invention provides measurement with high reliability without requiring any complicated structures or methods of manufacturing sensors, or imposing extra work on users.
In addition to the above-mentioned parameters P1 and P2, the following parameters P3 and P4 also can be used.
P3: a value (I/xcex94I(xcex3)) obtained by normalizing a derivative value or a differential value of a current at any point in a second excitation, with a current value at the same point.
Here, xe2x80x9cnormalizationxe2x80x9d means adaptation of a ratio of either the derivative value or the differential value to a current value at the same point of time in order to make the derivative value or the differential value a parameter not dependent on the concentration of the analyte. When a ratio of the differential value to a current value is adapted, the current value can be read at any point between two points having a finite difference.
P4: a ratio (Ib(xcex1)/Ib(xcex2)) of an initial current value (Ib(xcex1)) to a terminal current value (Ib(xcex2)) in a second excitation
The inventors consider that these parameters are indices as shown below. However, as it still remains in the realm of speculation, one parameter will not always correspond to one index.
P1: mainly, a current value affected greatly by sample physical properties
P2: mainly, a current value to indicate a concentration of an analyte in a sample
P3: mainly, a current value indicating diffusion of a compound and a mixture of a sample and a reactant inside a sensor at a chemical reaction part
P4: mainly, a current value affected greatly by the electrode sensitivity of a sensor
In the present invention, the current value Ib at any point in the second excitation, which is used for calculating the parameters P1 and P2, is preferably the terminal current value (Ib(xcex2)) in the second excitation.
When a value of at least either the parameter P3 or P4 is out of the expectation range, the value is preferably substituted by a boundary value of a closest expectation range. This correction will be regarded as an xe2x80x9coff-value correctionxe2x80x9d. An expectation range in the present invention is a parameter range that is expected from the concentration of an analyte.
It is preferable in the present invention that correction formulas including a plurality of statistical techniques corresponding to an environmental temperature or a sample temperature are prepared and an optimum correction formula is selected therefrom in order to perform an adequate correction corresponding to either the environmental temperature or the sample temperature. The reason is that the current value obtained by applying voltage to the sensor to make an excitation state varies considerably by the environmental temperature or the sample temperature, and the parameters also vary.
Also, when the environmental temperature or the sample temperature is within the boundary region for a temperature used in the correction, it is preferable to select both the correction methods adjacent to the boundary in order to calculate the respective correction ranges, so that a concentration of the analyte in the sample is calculated based on a value obtainable by adding either an average value or a weighted average value to a current value to be converted into a concentration. When the environmental temperature or the sample temperature corresponds to the boundary region, measurement accuracy will be improved further by selecting both the correction methods adjacent to the boundary in order to obtain an average value or a weighted average value of the obtained correction value rather than selecting one of the methods.
In the present invention, for example, a discriminant function or a Mahalanobis"" distance can be used as a statistical technique.
The following explanation concerns a case to use a discriminant function as a statistical technique.
A discriminant function means a linear expression Z=f(x1, x2, . . . ) defined by plural parameters (x1, x2, . . . ) in order to discriminate to which of two previously-produced groups (e.g., G1 and G2) the measurement data belong.
Z value code is used to discriminate to which of the groups the data belong. For example, a function to discriminate the level of Hct of a blood sample is defined by using a low Hct group G1 and a high Hct group G2 as populations.
It is preferable to obtain a measurement result by preparing plural discriminant functions in accordance with a concentration of an analyte in a sample, selecting an adequate discriminant function from the discriminant functions using Ib of the parameter P2 as an index, calculating a discriminant score Z value from the selected discriminant function, and by correcting errors with the Z value.
When plural discriminant functions are selected by the indices, it is preferable that Z value is calculated from each of the discriminant functions, and a correction range corresponding to the Z value is calculated, so that the measurement result is obtained from either an average value or a weighted average value of the correction range. As mentioned above, the measurement accuracy will be improved by using an average value or a weighted average value of the correction range rather than using a correction range of any one of the functions.
In a correction using the discriminant function, the correction can be performed to determine to which population the data belong, by using the Z value code. However, it is preferable that the correction range is calculated based on the discriminant score Z value. For example, the discriminant score Z value can be divided into a range to be corrected and a range not to be corrected (correction range is 0).
The range to be corrected can be divided further into a range to be corrected by a correction range proportional to the discriminant score Z value, and a range to be corrected by a fixed correction range without reference to the discriminant score Z value. This can achieve a correction with higher accuracy when compared to a technique performing correction by a grouping depending on the code of the discriminant score. This correction technique will be regarded as xe2x80x9cnonlinear correction methodxe2x80x9d in this description.
In the measurement of the present invention, similarly, a measurement with high accuracy can be achieved by a selection of Mahalanobis"" space and a Mahalanobis"" distance D in place of the selection of the above-mentioned discriminant function and calculation of the discriminant score Z value as a statistical technique.
In the present invention, preferably, errors caused by sensor sensitivity are corrected after errors caused by sample physical properties are corrected. Since Ib as the parameter P2 is considered to reflect the concentration of the analyte in the sample, it is preferable that errors caused by the sample physical properties and sensor sensitivity with respect to the Ib are corrected, and subsequently, the concentration of the analyte in the sample is calculated based on a value obtained by multiplying this correction value by a temperature correction coefficient.
Furthermore, a measurement apparatus of the present invention comprises a means to measure either an environmental temperature or a sample temperature, a means to detect sample feeding, a means to apply a certain voltage at a predetermined point of time, a means to measure a current value generated by an electrochemical reaction, and a means to convert the measured value of the current into a concentration of an analyte in a sample, wherein the temperature measuring means, the means for detecting sample feeding, the applying means, the means for measuring current value and the conversion means are controlled to perform the present invention. The control is performed typically with a microcomputer having the programmed procedures.