JP2010286423A - Potential difference measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosample analyzer and a means capable of measuring at an optional point of time, relative to solution in which a chemical reaction is generated. <P>SOLUTION: Measurement is performed synchronously with reaction start (namely, adding/mixing of reagent solution to/with a dispensed specimen sample) by using a potential difference measuring method as a measuring method, and a concentration of a measuring object in the specimen sample is determined from the acquired potential difference. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a potentiometric device for measuring an analysis target component in a biological sample, particularly blood or urine, and a measurement method using the same.

  For the qualitative and quantitative analysis of biological samples such as blood and urine in the medical field, a colorimetric method is mainly used to measure the change in absorbance of the reaction solution obtained by reacting the sample sample with the reagent. It is done with an automatic analyzer. Various types of biochemical automatic analyzers have been developed from flow type to discrete type. At present, the discrete type in which all operations such as dispensing of a sample sample and a reagent solution into a reaction container, stirring of the sample sample and the reagent solution, and measurement of change in absorbance of the reaction solution are automated is the mainstream (for example, Patent Document 1). , 2). Since these instruments use the colorimetric method as a measurement principle, they require a large and expensive optical system. As a reagent used in the colorimetric method, an enzyme or a chemical substance that specifically reacts with an object to be measured in a specimen sample is used alone or in combination. For example, in the measurement of blood glucose level, phosphoric acid is added to glucose in the presence of ATP using hexokinase with the measurement target substance glucose as a substrate, and the resulting glucose-6-phosphate is converted to glucose-- in the presence of NADP. The target glucose concentration is obtained by increasing the absorbability of NADPH generated during the dehydrogenation reaction with 6-phosphate dehydrogenase.

  The end point method is usually used as a method for obtaining the concentration of the measurement object by the coloration method using the absorptiometry. The end point method is a method for obtaining the concentration of the measurement object from the measured value before the start of the reaction and the measured value after the end of the reaction. The measured value before the start of the reaction is a value obtained by measuring a mixed solution of the reagent sample (for example, a buffer solution) excluding the reagent component that reacts with the measurement target in the sample sample and the measured value after the reaction is completed. , And a value measured after completion of the reaction by adding a reagent containing a component that causes a reaction with the measurement object to the mixed solution.

  On the other hand, as a measurement method that does not require an optical system, there is a method of electrochemically measuring the concentration of a specimen sample. This method has the advantage that the measurement principle is an electrochemical measurement method, a large and complicated device is unnecessary, and the device system is small. Electrochemical measurement methods include a current measurement method for measuring the current value flowing through the working electrode in accordance with the oxidation-reduction reaction, and a potential difference measurement method for measuring the interface potential of the measurement electrode that changes with the chemical reaction.

  As reagents used in electrochemical measurement, enzymes and chemical substances that react specifically with a measurement object are used alone or in combination, as are reagents used in colorimetric methods. For example, in the measurement of blood glucose level using the current method, gluconolactone and hydrogen peroxide water are generated by oxidizing glucose in the presence of oxygen or mediator with glucose oxidase using glucose as a substrate as a measurement target substance. . By measuring the generated hydrogen peroxide solution as a current flowing through the electrode, the concentration of glucose as a measurement target substance is measured (Non-Patent Document 1). In this method, a reaction reagent containing an enzyme is held on the measurement electrode in advance, and the specimen sample introduced from the sample inlet passes through the flow path and reaches the measurement electrode surface to start a predetermined enzyme reaction. It is configured.

In the potentiometric method, the oxidation reaction by glucose oxidase is measured as a change in the redox potential of potassium ferricyanide and potassium ferrocyanide, which are mediators of the redox reaction, and the concentration of glucose as a measurement target substance is measured (Patent Document 4, Patent Document 4). 5). Even in the potentiometric method, a reaction reagent containing an enzyme is usually held on the measurement electrode in advance, and the introduced specimen sample is achieved on the surface of the measurement electrode, so that a predetermined enzyme reaction is started ( Patent Document 4). When a measurement container is used, a potential difference after a certain time after injecting the specimen sample into the measurement container in which a reaction solution containing a reagent such as an enzyme is previously held, or a potential difference before or just after the specimen sample is injected. The concentration of the measurement object was obtained using the amount of change between the voltage difference and the potential difference after a certain time (Patent Document 5).

Patent No. 2525063 WO2002 / 05962 JP 2005-345173 A Patent No. 3387926 JP 2008-128803 PR

Pure & Appl. Chem., Vol.68, (1996) pp.1837-1841

  However, with conventional biochemical automatic analyzers, it is not practical to perform measurements at arbitrary points in time. For example, in an apparatus using the absorbance method, a single absorbance measurement unit and a plurality of measurement containers are included in the apparatus, and thus the absorbance measurement of a plurality of containers is performed in time division. For this reason, it was not possible to measure at any point in time and to measure multiple measuring containers at the same time. Furthermore, in an apparatus using the absorbance method, the absorbance is usually measured after mixing and stirring the sample and the reagent. Since mixing / stirring and absorbance measurement were usually performed at different locations, absorbance could not be measured during mixing / stirring. In addition, even if mixing / stirring and absorbance measurement are performed at the same time, a stirrer such as a stirrer or bubbles generated by the stirrer may block the optical path, which may hinder stable measurement. .

By using the electrochemical measurement method, it is easy to provide a measurement unit for each measurement container. This is because the absorbance measurement device requires an optical system, and the absorbance measurement device becomes larger, whereas the electrochemical measurement method is an electrical measurement method that does not require an optical system. Because you can. However, in the current measurement method, which is one of the electrochemical measurement methods, the signal intensity depends on the surface area of the electrode. Therefore, when multiple measurement units are installed, it is caused by slight differences in the electrode area between the measurement units. It is necessary to perform so-called calibration to correct the difference in signal strength. Furthermore, if the effective surface area of the electrode changes due to contamination on the electrode surface due to the measurement, it is necessary to recalibrate, which is troublesome. The present invention provides a biological sample analyzer and means capable of measuring a solution in which a chemical reaction occurs at an arbitrary time.

  In order to solve the above problems, the potential difference measurement method was used for measurement while mixing and stirring. An electrode used for measurement was placed in a measurement container, a solution was dispensed into the measurement container, and stirring and measurement were started in synchronization with the dispensing of the solution. For example, a specimen sample such as serum, plasma or whole blood is dispensed in advance in a measurement container, and a first reaction solution is further dispensed in the measurement container, and is synchronized with the dispensing of the first reaction solution. Stirring and potential measurement were started. In the case of using the second reaction solution, the second reaction solution was further dispensed into the measurement container, and the potential was measured in synchronization with the dispensing of the second reaction solution. As another procedure, the first reaction solution is dispensed in advance into the measurement container, the electrode is brought into contact with the first reaction solution, and the specimen sample is further dispensed into the measurement container. In synchronism with dispensing, the stirring and potential measurement were started. In these measurement procedures, it is desirable to continue stirring from the start of stirring until the end of measurement, but the measurement can be performed even if stirring is stopped halfway, and the above-described problems can be solved.

  The time required for mixing and the fluctuation of the measured value are obtained from the measured values, and compared with the database to determine whether the stirring has been performed normally. A deconvolution operation is performed on the measured values obtained as necessary, and a measured value, that is, a transfer function, obtained when the chemical reaction is assumed to be instantaneous, is obtained. From the obtained transfer function, obtain the time required for mixing and the fluctuation of the measured value, and determine whether the agitation was performed normally. The time required for mixing is, for example, the time from dispensing the solution to 90% of the final measured value. For the magnitude of fluctuation, for example, the maximum value and the minimum value are obtained from the measured values after dispensing of the solution, and the difference between them is obtained.

That is, the present invention includes the following.
(1) A first dispensing step of dispensing a sample solution containing a measurement object into a measurement container, and a first reaction solution containing a first reagent that reacts with the measurement object is dispensed into the measurement container. A second dispensing step, a stirring step for stirring the first reaction solution dispensed in the measurement container, and a plurality of measurements for measuring an interface potential of the measurement electrode disposed in the measurement container A potential difference characterized in that at least one of the measurement steps is started in synchronization with the second dispensing, and the stirring step is started in synchronization with the second dispensing. Measuring method.
(2) The method according to (1), wherein the stirring step is a step of stirring a mixed solution of the sample solution and the first reaction solution dispensed in the measurement container.
(3) The method according to (2), wherein at least one of the measurement steps does not involve the stirring step.
(4) Third dispensing for dispensing a second reaction solution containing a second reagent that reacts with the measurement object into the measurement container after the second dispensing step while continuing the stirring step. The method according to (1), wherein at least one of the measurement steps is started in synchronization with the third dispensing.
(5) a third dispensing step of dispensing a second reaction solution containing a second reagent that reacts with the measurement object into the measurement container after the second dispensing step; and in the measurement container A second agitation step of agitating the second reaction solution dispensed into the at least one of the measurement steps, the at least one of the measurement steps being started in synchronization with the third dispensing, At least one of them is not accompanied by the stirring step or the second stirring step, and the second stirring step is started in synchronization with the third dispensing. .
(6) The first dispensing step is included after the second dispensing step, and at least one of the measurement steps is started in synchronization with the first dispensing ( The method according to 1).
(7) having a second stirring step of stirring the sample solution dispensed in the measurement container, wherein at least one of the measurement steps is not accompanied by the stirring step or the second stirring step; The method according to (6), wherein the second stirring step is started in synchronization with the first dispensing step.
(8) The method according to (6), wherein the stirring step is started not in the second dispensing but in synchronization with the first dispensing.
(9) The method according to (6), wherein at least one of the measurement steps does not involve the stirring step.
(10) The method according to any one of (1) to (9), wherein the reaction generated by the first reaction solution or the second reaction solution is an enzyme reaction or an oxidation-reduction reaction.
(11) In the time-series data of the potential difference obtained by the measurement step, the method includes a step of comparing data for a predetermined time at the rising edge of the data with standard data and outputting a comparison result. (10) The method according to any one of the above.
(12) In the time-series data of the potential difference obtained by the measurement step, the difference between the maximum value and the minimum value of the data is compared with standard data, and a comparison result is output ( The method according to any one of 1) to (10).
(13) In the time series data of the potential difference obtained by the measurement step, a transfer function is derived from the time series data, an inverse function of the transfer function is derived, and input by calculation of the inverse function and the time series data The method according to any one of (1) to (10), further including a step of obtaining a function.
(14) The method according to (13), further comprising a step of comparing data for a predetermined time at the rising edge of the input function with standard data in the input function and outputting a comparison result.
(15) The method according to (13), further comprising a step of comparing a difference between the maximum value and the minimum value of the input function with standard data in the input function and outputting a comparison result.

In the potential difference measurement, the influence on the measurement by the stirring is small, so that the measurement can be performed while stirring by using the potential difference measuring method. By stirring and measuring in synchronization with the dispensing of the solution, the reaction process of the solution can be measured immediately after mixing reagents and specimens that could not be performed so far, and accurate measurement can be performed.

The block diagram which shows an example of an electrical potential difference measuring apparatus. The block diagram which shows an example of an electrical potential difference measuring apparatus. The figure which shows an example of the measurement operation | movement using an electric potential difference measuring apparatus. The flowchart which shows an example of measurement operation. The flowchart which shows an example of measurement operation. The flowchart which shows an example of measurement operation. The flowchart which shows an example of measurement operation. The flowchart which shows an example of measurement operation. The flowchart which shows an example of measurement operation. The flowchart which shows an example of measurement operation. The flowchart which shows an example of measurement operation. The figure which shows an example of abnormality detection. The figure which shows an example of abnormality detection. The figure which shows an example of an inverse calculation. The figure which shows the structural example of the measurement electrode used for an electrical potential difference measuring apparatus. The block diagram which shows the other Example of an electrical potential difference measuring apparatus. The figure which shows an example of the structure of the analysis element used for the electric potential difference measuring apparatus using a FET sensor. The figure which shows an example of a measurement result. The figure which shows an example of a measurement result. The figure which shows an example of a measurement result. The figure which shows an example of a measurement result.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 is a block diagram showing an example of a potential difference measuring apparatus according to the present invention. The measuring apparatus of the present embodiment includes a potential difference measuring electrode 101, a reference electrode 102, a stirring device 103, a measuring container 104, a control device 105, a data processing device 106, a data display device 107, a specimen sample dispensing device 108, a reagent solution fraction. It comprises an injection device 109 and a cleaning device 110. As the potential difference measuring electrode 101, an electrode made of a noble metal such as gold or carbon can be used. In addition, in order to reduce the influence of contaminants when measuring biological components, a redox substance may be immobilized on the electrode surface. Ferrocene, pyridine, pyrimidine or the like may be used as the redox substance. The reference electrode 102 provides a reference potential in order to stably measure a potential change based on an equilibrium reaction or a chemical reaction occurring on the surface of the potential difference measuring electrode 101 in contact with the solution in the measurement container 104. Normally, the reference electrode 102 is a silver / silver chloride electrode or a calomel electrode using saturated potassium chloride as the internal solution, but the composition of the sample solution to be measured is constant. If the required measurement accuracy can be ensured, a silver / silver chloride electrode containing no internal solution can be used as a pseudo electrode. The stirring device 103 stirs the solution dispensed in the measurement container. The stirring device 103 is a so-called magnetic stirrer that rotates a magnet placed outside the measuring container and rotates a magnet placed in the solution even if the spatula attached to the tip of the rotating shaft is stirred in the solution. Also good. The solution may be stirred by rotating a cylindrical measurement container. An integrated electrode in which the potential difference measuring electrode 101 and the reference electrode 102 are integrated may be used, and the solution may be stirred by rotating the integrated electrode. The measurement container 104 is a container having a corner such as a square cell, and it is difficult to stir and mix the sample sample, the reagent solution, and the solution in the measurement container 104. Therefore, a container having a curve, for example, a cylindrical container is used. desirable. The control device 105 measures the potential between the potential difference measuring electrode and the reference electrode, and controls the movement and operation of the potential difference measuring electrode, the reference electrode, the stirring device, the specimen sample dispensing device, the reagent solution dispensing device, and the cleaning device. Do. It is desirable to use a voltmeter, OP amplifier, and FET (Field Effect Transistor) having an input impedance of 1 GΩ or more for the potential measuring device 111 that measures the potential between the potential difference measuring electrode 101 and the reference electrode 102. The potential difference measuring device 111 may be integrated with the potential difference measuring electrode 101. The data processing device 106 includes a calculation device 112, a temporary storage device 113, and a non-volatile storage device 114. The signal processing device 106 calculates a signal obtained by the potential difference measurement device 111 to determine the concentration of the measurement object in the sample sample, and performs abnormality determination. Or fitting or performing a deconvolution operation. The data display device 107 is connected to the data processing device 106 and displays various data. The sample sample dispensing device 108 is used to collect a sample sample separately prepared and dispense it into the measurement container 104. The reagent solution dispensing device 109 is used to dispense a separately prepared reagent solution and dispense it into the measurement container 104. The cleaning device 110 is used to discharge the measurement solution in the measurement container 104 or to clean the measurement container 104 using a separately prepared cleaning liquid. A syringe pump or a peristaltic pump (tube pump) can be used as a mechanism for dispensing and dispensing the liquid with the specimen sample dispensing device 108, the reagent solution dispensing device 109, and the cleaning device 110.

  FIG. 2 is a block diagram showing another example of the potential difference measuring apparatus according to the present invention. Although the basic configuration and their roles are the same as in Fig. 1, it has multiple sets of potentiometric electrodes, reference electrodes, agitators, and measurement vessels to measure different specimen samples and different substances to be measured simultaneously. can do. The number of potential difference measuring devices may be the same as the number corresponding to each set, and if the required accuracy can be ensured, each potential difference may be measured with a single potential difference measuring device using a multiplexer or the like. Unlike measurement methods that require optical systems such as absorbance measurement, it is easy to reduce the size of the electrodes in electrical measurement, so that the device becomes huge and the throughput per unit area and volume of the device is greatly reduced. Does not occur. Further, since the potential difference measurement theoretically does not depend on the electrode area for the signal intensity, the electrode can be reduced in size, and the increase in size of the apparatus due to the provision of a plurality of electrodes can be suppressed. The difference in the output signal intensity between the multiple electrodes is corrected by measuring the standard solution. So-called calibration is performed. In potentiometric measurement, even if the effective surface area changes due to contamination of the electrode surface, the output signal strength does not change if a voltmeter with a larger input impedance than the resistance value of the electrode surface is used, and calibration is performed again. There is no need to do.

  FIG. 3 is a diagram showing an example of a measurement operation using the potential difference measuring apparatus according to the present invention. The measurement procedure is as follows. First, as shown in FIG. 3A, the specimen sample 303 is dispensed into the measurement container 301 using the specimen specimen dispensing device 302. Next, as shown in FIG. 3B, the potential difference measuring electrode 304, the reference electrode 305, and the stirring element 306 are installed in the measurement container 301. In this state, the dispensed specimen sample 303 is not in contact with the reference electrode 305 and the stirring element 306. Next, the reagent solution is dispensed into the measurement container 301 using the reagent solution dispensing device 307. In synchronization with the dispensing of the reagent solution, stirring and mixing by the stirring element 306 and potential measurement of the potential difference measuring electrode 304 are started (FIG. 3C). Furthermore, another reagent solution is dispensed into the measurement container 301 using the reagent solution dispensing device 307. At this time, the stirring by the stirring element 306 is continuously performed, and the potential measurement of the potential difference measuring electrode 304 is also continuously performed. After the measurement is completed, the potential difference measuring electrode 304, the reference electrode 305, and the stirring element 306 are moved out of the measuring container 301, the solution in the measuring container 301 is sucked out using the cleaning device 308, and the cleaning liquid is discharged / sucked from the cleaning device 308. By doing so, the measurement container 301 is washed.

  FIG. 4 is a flowchart showing an example of a measurement operation using the potential difference measuring apparatus of FIG. 1 or 2 according to the present invention. The vertical axis represents time, and time passes from top to bottom. First, a specimen sample is dispensed into the measurement container 104 by a dispensing means such as the specimen sample dispensing device 108 (401). Next, the reagent solution is dispensed by a dispensing means such as the reagent solution dispensing device 109 (402). In synchronism with the reagent dispensing, the sample sample and the reagent solution are agitated by the agitating means such as the agitating element 103 so that the sample sample and the reagent solution are well mixed (403). The potential difference is measured using the potential measuring device 111 (404). In order to keep the effect of stirring on the potential constant, stirring should be continued at a constant rate during measurement. However, if the effect is less than the required measurement accuracy, the sample sample and reagent solution should be mixed thoroughly. After that, stirring may be stopped. In that case, the flowchart is as shown in FIG. After that, measurement is continued (405), a certain period of time has elapsed from the start of measurement, the measured data is analyzed and the concentration of the analyte in the sample solution is determined within a certain error range, or there is an abnormality in the dispensing or reaction of the liquid If the condition for terminating the measurement is detected, the stirring is stopped and the measurement is terminated. The solution in the measurement container 104 is discharged by a liquid discharging means such as the cleaning device 110 (406), washed with a cleaning liquid (407), and prepared for the next measurement. When the measurement container 104 is made disposable, it is possible to omit the discharge of the liquid and the cleaning with the cleaning liquid. When there are a plurality of sets of potential difference measuring electrodes, reference electrodes, stirring devices, and measuring containers as shown in FIG. 2, the flow shown in FIG. 4 may be performed synchronously or asynchronously. . When the measurement is performed asynchronously, if the measurement in the group for some reason is completed earlier than the measurement in the other group, the measurement can be performed without waiting for the measurement in the other group to end.

  FIG. 6 is a flowchart showing another example of the measurement operation using the potential difference measuring apparatus of FIG. 1 or 2 according to the present invention. The point which measures by adding two types of reagents in order differs from the flowchart of FIG. First, a specimen sample is dispensed into the measurement container 104 by a dispensing means such as the specimen sample dispensing device 108 (601). Next, the reagent 1 solution is dispensed by a dispensing means such as the reagent solution dispensing device 109 (602). In synchronism with the reagent dispensing, the sample sample and the reagent 1 solution are agitated by the agitating means such as the agitating element 103 (603) and synchronized between the potential difference measuring electrode 101 and the reference electrode 102. Is measured by the potential measuring device 111 (604). Thereafter, the measurement is continued (605), and when a certain time has elapsed from the dispensing of the reagent 1 solution, or when it is determined that the reaction between the sample sample and the reagent 1 solution is completed by analyzing the measured data, The reagent 2 solution is dispensed into the measurement container by a dispensing means such as the injection device 109 (606). In synchronism with the dispensing of the reagent 2 solution, the potential difference between the potential difference measuring electrode 101 and the reference electrode 102 is measured by the potential measuring device 111 (607). Stir during the dispensing of 2 reagents. When a certain time has elapsed since the dispensing of the reagent 2 solution, or when the measured data is analyzed and the concentration of the measurement target in the sample solution is determined within a certain error range, the stirring and potential measurement are stopped and the measurement is terminated. Even if measurement is in progress, measurement is terminated if an abnormality is detected in the dispensing or reaction of the liquid. The solution in the measurement container 104 is discharged by a liquid discharging means such as the cleaning device 110 (609), washed with a cleaning liquid (610), and prepared for the next measurement. When the measurement container 104 is made disposable, it is possible to omit the discharge of the liquid and the cleaning with the cleaning liquid. In order to keep the effect of stirring on the potential constant, stirring should be continued at a constant rate during measurement. However, if the effect is less than the required measurement accuracy, the sample sample and reagent solution should be mixed thoroughly. After that, stirring may be stopped (FIG. 7). In that case, stirring is started so that the solution in the measuring vessel 104 and the reagent 2 solution are well mixed by stirring means such as the stirring element 103 in synchronization with the dispensing of the reagent 2 solution, and for potential difference measurement in synchronization with the same. A potential difference between the electrode 101 and the reference electrode 102 is measured by the potential measuring device 111.

  FIG. 8 is a flowchart showing another example of the measuring operation using the potential difference measuring apparatus of FIG. 1 or 2 according to the present invention. The order of dispensing the specimen sample and the reagent solution is different from the flowchart of FIG. First, a reagent solution is dispensed by dispensing means such as the reagent solution dispensing device 109 (801). Next, following the reagent dispensing, stirring by a stirring means such as a stirring element is started (802), and similarly, measurement of a potential difference between the potential difference measuring electrode 101 and the reference electrode 102 is started (803). . Thereafter, the measurement was continued (804), and a fixed time passed from the dispensing of the reagent 1 solution, or the measured data was analyzed and it was determined that the potential difference between the potential difference measuring electrode 101 and the reference electrode 102 was stable. In this case, the specimen sample is dispensed into the measurement container 104 by a dispensing means such as the reagent solution dispensing device 109 (805). In synchronism with the dispensing of the sample, the potential difference between the potential difference measuring electrode 101 and the reference electrode 102 is measured by the potential measuring device 111 (806). Stir during reagent dispensing. A certain amount of time has elapsed since the sample sample was dispensed, the measured data was analyzed and the concentration of the analyte in the sample solution was determined within a certain error range, or an abnormality was detected in the liquid dispensing or reaction, If the conditions for terminating the measurement are met, stop stirring and terminate the measurement. The solution in the measurement container is discharged by a liquid discharging means such as a cleaning device 110 (808), washed with a cleaning liquid (809), and prepared for the next measurement. When the measurement container is made disposable, it is possible to dispense with liquid discharge and cleaning with a cleaning solution. In the method in which reagent dispensing is performed after sample dispensing, the sample sample and the reagent solution are easily mixed by dispensing a large amount of reagent solution to a small amount of sample sample. On the other hand, in the present invention, since stirring and measurement can be performed at the same time, the time spent for stirring can be increased as compared with the prior art, and even if a small amount of specimen sample is dispensed, sufficient mixing is achieved. You can The advantage of the flow of FIG. 8 over the flow of FIG. 4 is that the surface potential of the measurement electrode is stable at the time of sample dispensing (805) even if it takes a certain amount of time to stabilize the surface potential of the measurement electrode. The reaction of the substance can be traced immediately after mixing the specimen sample and the reagent solution. As another stirring flow, when stirring is started in synchronization with reagent dispensing and sample dispensing as shown in FIG. 9 and stirring is stopped after sufficient stirring, sample dispensing is performed as shown in FIG. When stirring is started in synchronization and stirring is performed until the end of measurement, stirring is started in synchronization with sample dispensing as shown in FIG. 11, and stirring may be stopped after sufficient stirring. 8 and 9, the advantage of stirring after dispensing the reagent is that stirring is not performed between the surface state of the potential difference measuring electrode 101 and the reference electrode 102 and the state of the solution other than the electrode surface in the measurement container 104. It is to be able to make uniform in a shorter time than the case.

  The flowcharts of FIGS. 4 to 11 can be applied not only when the potential difference measuring device of FIGS. 1 and 2 is used, but also when a potential difference measuring device capable of performing the same operation is used.

  FIG. 12 is a diagram illustrating an example of abnormality detection according to the present invention. Based on the flowchart shown in FIG. 12, it was verified whether the signal change caused by the mixing of the sample sample and the reagent solution or the dispensing of the reagent 2 solution occurred in a reasonable time, and the mixing and stirring were performed correctly. Determine. First, low-pass filter processing is performed on the data array of measured values as necessary for the purpose of noise reduction. Next, the time at which a certain percentage of the final value obtained by measurement, for example 90%, is obtained, and the difference between the dispensing time and the time at which the maximum value is first obtained is taken as the rise time. Obtain and calculate the mixing standard time from the mixing standard time database based on the volume ratio and viscosity of the specimen sample and reagent solution, and compare them with the rise time obtained from the measured values. If the rise time obtained from the measured values is reasonable, it is judged that mixing and stirring of the sample sample and the reagent solution were performed normally. If the rise time was outside the appropriate range, mixing and stirring of the sample sample and the reagent solution were not performed. Judge that it was not performed normally, and re-measure the corresponding specimen. As an example of a method for determining whether the rise time obtained from the measured value is appropriate, there is a method of multiplying a coefficient and setting it as a threshold value such as 1.2 times the value of the mixed standard time database.

  FIG. 13 is a diagram illustrating another example of abnormality detection. The flow chart shown in the figure verifies whether fluctuations caused by abnormal stirring have occurred in the change in the signal generated by mixing the sample sample and reagent solution or by dispensing the reagent 2 solution. Determine if it was done correctly. First, low-pass filter processing is performed on the data array of measured values for the purpose of noise reduction. Next, the maximum value and the minimum value are detected from the measured values after the rise time detected by the flowchart of FIG. 12, and recorded together with the time as the maximum value / minimum value data array. The allowable difference / slope is acquired from the fluctuation database based on the quantity ratio / viscosity of the specimen sample and reagent solution, and it is determined whether the maximum value / minimum value data array is within the allowable value one by one. If it is determined that the value is within the allowable value until the end of the array, it is determined that mixing and stirring are correctly performed, and the analysis is terminated. If even one of the data exceeding the allowable value is found, it is judged that mixing / stirring was not performed correctly, and the corresponding specimen sample is measured again.

  FIG. 14 is a diagram illustrating an example of the inverse operation. If the reaction rate of the chemical reaction used for the measurement is sufficiently faster than the mixing time, the abnormality detection as shown in FIGS. 12 and 13 can be performed using the measured raw data. However, if the reaction rate of the chemical reaction is about the same or less than the mixing time, the rise time delay and fluctuations due to mixing / stirring failure will be lost, making it difficult to detect abnormalities from raw data. End up. Therefore, the raw data is considered to be a function obtained from the convolution operation of the solution mixing function and the reaction function, and the inverse operation for obtaining the mixing function from the raw data is performed. The method will be described with reference to FIG. First, each parameter of the ideal reaction function obtained by convolution of the ideal mixture function and reaction function is obtained by fitting to the raw data. For fitting, for example, fitting such as least squares can be used. A transfer function G (s) is a reaction function obtained from the determined parameters. The inverse transfer function G-1 (s) is obtained from the transfer function G (s). As an example of how to obtain, there is a method using an inverse matrix. A mixed function is obtained by performing a convolution operation on the obtained inverse transfer function G-1 (s) on the raw data. For the obtained mixing function, the abnormality determination as shown in FIGS.

  FIG. 15 is a diagram showing a structural example of a measurement electrode used in the potential difference measuring apparatus of the present invention. In this embodiment, gold is used as an electrode material, and a ferrocene derivative is fixed to the gold electrode surface as an oxidizing substance. The ferrocene derivative 1502 was immobilized on the gold electrode 1501 by bonding of gold and thiol through an alkanethiol 1503 having a thiol group at the terminal. The ferrocene derivative was immobilized on the gold electrode surface by the following procedure. First, the gold electrode used for immobilization was washed with 1N nitric acid, pure water, and ethanol in this order, and the gold electrode surface was purged with nitrogen. Next, it was immersed in a ferrocene derivative solution (11-ferrocenyl-1-undecanethiol, concentration: 0.5 mM, solvent: ethanol) for 1 hour. After immobilization, the cells were washed with ethanol and pure water and stored in an aqueous solution or buffer solution containing a supporting salt until use.

  FIG. 16 is a block diagram showing another embodiment of the potential difference measuring apparatus. The measurement apparatus of the present embodiment includes a potential difference measurement electrode 1601, a reference electrode 1602, a measurement container 1603, a control device 1604, a data processing device 1605, a data display device 1606, and a sample sample dispenser for dispensing a sample sample into the measurement container 1603. An injector 1607, a reagent solution dispenser 1608 that dispenses the reagent solution into the measurement container 1603, and a stirring element 1609 that stirs the dispensed specimen sample and the reagent solution. The control device 1604 measures the potential between the potential difference measuring electrode 1601 and the reference electrode 1602 using the potential difference measuring device 1610, the potential difference measuring electrode 1601, the reference electrode 1602, the specimen sample dispenser 1607, and the reagent solution dispensing. The movement and operation control of the vessel 1608 and the stirring element 1609 are performed. The data processing device 1605 includes a calculation device 1611, a temporary storage device 1612, and a nonvolatile storage device 1613. The signal processing device 1605 calculates a signal obtained by the potential difference measurement device 1610 to determine the concentration of the measurement object in the specimen sample and performs abnormality determination. Or fitting or performing a deconvolution operation. The potential difference measuring electrode 1601 is placed in contact with the bottom of the measurement container 1603. The potential difference measuring electrode 1601 uses gold as a material, and a ferrocene derivative 1614 is fixed to the gold surface via an alkanethiol. In this embodiment, a ferrocene derivative is used as the redox substance, but pyridine, pyrimidine, or the like may be used. The reference electrode 1602 provides a reference potential in order to stably measure a potential change based on an equilibrium reaction or a chemical reaction occurring on the surface of the potential difference measurement electrode 1601 in contact with the solution in the measurement container 1603. Usually, the reference electrode is a silver / silver chloride electrode or calomel electrode using saturated potassium chloride as the internal solution. However, if the composition of the sample solution to be measured is constant, There is no problem even if only a silver / silver chloride electrode is used as an electrode. The measurement container 1603 is preferably a container having a curve, for example, a cylindrical container, because it is difficult to stir and mix the sample sample and reagent solution dispensed in a container having a corner such as a square cell. The sample sample dispenser 1607 and the reagent solution dispenser 1608 can use a syringe pump or a peristaltic pump (tube pump). Here, a stirring element 1609 having a rotating tip spatula was used.

  FIG. 17 is a diagram showing an example of the structure of an analysis element used in a potential difference measuring device using an FET sensor. FIGS. 17A and 17B show a cross-sectional structure and a planar structure, respectively. An insulated gate field effect transistor 1701 has a source 1702, a drain 1703, a gate insulator 1704 formed on the surface of a silicon substrate, and a gold electrode 1705. A gold electrode 1705 and a gate 1706 of an insulated gate field effect transistor are connected by a conductive wiring 1707. Preferably, the insulated gate field effect transistor is a metal-oxide semiconductor field effect transistor (FET) using silicon oxide as an insulating film, but there is no problem even if a thin film transistor (TFT) is used. By adopting this structure, the redox substance can be easily immobilized on the gold electrode 1705 via the alkanethiool. The insulated gate field effect transistor used here is a depletion type FET having an insulating layer using SiO2 (thickness: 17.5 nm), and a gold electrode is fabricated in a size of 400 μm × 400 μm. Since normal measurement uses an aqueous solution, the device must operate in solution. When measuring in a solution, it is necessary to operate in an electrode potential range of −0.5 to 0.5 V, which hardly causes an electrochemical reaction. Therefore, in this embodiment, the fabrication condition of the depletion type n-channel FET, that is, the ion implantation condition for adjusting the threshold voltage (Vt) is adjusted, and the threshold voltage of the FET is set around −0.5V. In place of the gold electrode, an electrode made of other noble metal such as silver may be used. When an FET sensor is used as a measurement electrode, it is necessary to apply a voltage to the reference electrode. However, in order to reduce the influence of external fluctuations on the gold electrode surface, it is preferable to apply an AC component. At that time, the surface potential of the gold electrode can be stabilized by superimposing an AC voltage of 1 KHz or more on the DC component. Further, since the FET sensor responds to light, it is preferable to use an opaque material for the measurement container or to shield the measurement container itself.

  18, 19, and 20 are diagrams showing examples of measurement results obtained using the measurement method according to the present invention. Using the apparatus shown in FIG. 16, the glucose concentration in horse serum was measured according to the flow shown in FIG. First, 30 μL of the specimen sample was injected into the measurement container. Next, 300 μL of the reagent solution was injected into the measurement container. The composition of the reagent solution was as shown in (Table 1).

In synchronization with the dispensing of the reagent solution, stirring with a stirring bar and measurement of potential difference with a voltmeter were started. The potential difference measurement was performed every 300 seconds for 1 second. As a result, the time change of the potential difference shown in FIG. Next, using a data processor, the ferrocyan concentration in the measurement solution was determined from the potential difference. Specifically, the Nernst equation

Solved. As a result, the time change of the ferrocyan concentration shown in FIG. 18 (b) was obtained. Ferrocyan,

(Where NAD: nicotinamide adenine dinucleotide (oxidized form), NADH: nicotinamide adenine dinucleotide (reduced form))
Thus, when the reaction efficiency is 100%, 2 mol of ferrocyan is produced from 1 mol of glucose. In this reaction system, the reaction efficiency was determined separately and found to be 90%. Based on the glucose molecular weight of 180 and the dilution factor of 11 times, the produced ferrocyan concentration [mM] was converted to the glucose concentration in the sample [mg / dL] and multiplied by the reaction efficiency to obtain FIG. 18 (c). Through the above process, the glucose concentration in the sample was measured to be 112 mg / dL. Subsequently, an input function was obtained according to the flow of FIG. First, the transfer function is defined as follows.

The reaction curve of FIG. 18 (c) was fitted using G (t),

Got. The result is shown in FIG. An inverse function G-1 (t) of G (t) was obtained numerically, and the reaction curve of FIG. 18 (c) was calculated to obtain FIG. 19 (b). This is a reaction curve when it is assumed that the enzyme reaction that has proceeded in a moment like G (t) is actually finished, that is, the state of mixing of the solution other than the enzyme reaction. Here, the input function Call it. In Fig. 19 (b), when the rise time was determined according to the flow of Fig. 12, the maximum value was 121mg / dL, and it was 112mg / dL, which exceeded 90% of the value at 4 seconds after the start of the reaction. Therefore, the rise time was 4 seconds. When the same experiment was conducted relatively gently, another experimental result shown in FIG. 20 was obtained. It can be seen from FIG. 20 (a) that the rise of the reaction curve is slightly delayed. This was analyzed according to the flow of FIG. 14 to obtain an input function shown in FIG. Similarly, the rise time was found to be 17 seconds, and the rise delay due to gentle stirring was quantitatively obtained. In this example, data was acquired at 1-second intervals, so analysis was possible in 1-second units. However, since the reaction solution and the electrode are always in contact, the measurement interval can be shortened. The degree of agitation can be analyzed with higher time resolution than the case. Further, when FIG. 19B is compared with FIG. 20B, a relatively large overshoot is observed in FIG. 20B. This is considered to be fluctuations that occur in the process until the solution is uniformly mixed, and analysis is performed according to the flow of FIG. FIG. 19 (b) and FIG. 20 (b) differ in the degree of fluctuation due to the state of stirring, but if this occurs under the same conditions, there is some abnormality in mixing / stirring. Suspected to have occurred. An abnormality can be detected by detecting this in accordance with the flow of FIG.

  FIG. 21 is a diagram showing an example of measurement results obtained using the measurement method according to the present invention. Using the apparatus shown in FIG. 16, the glucose concentration in horse serum was measured according to the flow shown in FIG. First, 30 μL of the specimen sample was injected into the measurement container. Next, 270 μL of the reagent 1 solution was injected into the measurement container. After 300 seconds, 60 μL of the reagent 2 solution was injected into the measurement container. Each composition was as shown in (Table 2).

In synchronization with the dispensing of the reagent 1 solution, stirring with a stirring bar and measurement of a potential difference with a voltmeter were started. The potential difference measurement was performed every 5 seconds for 1 second. As a result, the time change of the potential difference shown in FIG. Next, with respect to the potential difference measured 300 seconds after the reagent 2 solution was injected using the data processing apparatus, the Nernst equation was used in the same manner as in the case of obtaining FIGS. 18 (b) and 19 (b). The ferrocyan concentration in the measurement solution was determined from the potential difference. Based on the separately determined reaction efficiency of 90%, glucose molecular weight of 180, and dilution factor of 12 times, the produced ferrocyan concentration [mM] is converted into the glucose concentration in the sample [mg / dL], and FIG. 21 (b) is obtained. It was. FIG. 21 (b) displays the time of reagent 2 solution injection as 0 second. In the same manner as when obtaining FIG. 19 (c) and FIG. 20 (c) from FIG. 19 (b) and FIG. 20 (b), reaction curve fitting and reverse calculation were performed for FIG. A reaction curve (FIG. 21 (c)) was obtained when it was assumed that the reaction was completed in an instant. The rise time at this time was 1 second or less. As described above, even when two reagents are used, the time required for mixing the solutions can be obtained by performing the analysis using the time of the reagent 2 solution injection as the time of starting the reaction. Even when the specimen is injected later, the time required for mixing the solution can be obtained if the reaction is started by the injection of the specimen.

DESCRIPTION OF SYMBOLS 101 Potential difference measurement electrode 102 Reference electrode 103 Stirring device 104 Measuring container 105 Control device 106 Data processing device 107 Data display device 108 Specimen sample dispensing device 109 Reagent solution dispensing device 110 Cleaning device 111 Potential measuring device 112 Computing device 113 Temporary storage Device 114 Non-volatile memory device 301 Measuring container 302 Specimen sample dispensing device 303 Specimen sample 304 Potential difference measuring electrode 305 Reference electrode 306 Stirring element 307 Reagent solution dispensing device 308 Cleaning device 401 to 407 Operation in flowchart 601 to 610 Operation 801 to 809 Operation in the flow chart 1501 Gold electrode 1502 Ferrocene derivative 1503 Alkanethiol 1601 Potential difference measurement electrode 1602 Reference electrode 1603 Measurement container 1604 Control device 1605 Data processing Device 1606 Data display device 1607 Specimen sample dispenser 1608 Reagent solution dispenser 1609 Stirring element 1610 Potential difference measurement device 1611 Arithmetic device 1612 Temporary storage device 1613 Non-volatile storage device 1614 Ferrocene derivative 1701 Field effect transistor 1702 Source 1703 Drain 1704 Gate Insulator 1706 Gate 1707 Conductive wiring

Claims (15)

A first dispensing step of dispensing a sample solution containing a measurement object into a measurement container;
A second dispensing step of dispensing a first reaction solution containing a first reagent that reacts with a measurement object into the measurement container;
An agitation step of agitating the first reaction solution dispensed into the measurement container;
A plurality of measurement steps for measuring an interface potential of a measurement electrode disposed in the measurement container,
At least one of the measuring steps is started in synchronization with the second dispensing,
The potential difference measuring method, wherein the stirring step starts in synchronization with the second dispensing.   The potential difference measuring method according to claim 1, wherein the stirring step is a step of stirring a mixed solution of the sample solution and the first reaction solution dispensed in the measurement container.   The potential difference measurement method according to claim 2, wherein at least one of the measurement steps does not involve the stirring step. While continuing the stirring step, after the second dispensing step, there is a third dispensing step for dispensing a second reaction solution containing a second reagent that reacts with the measurement object into the measurement container. And
The potential difference measuring method according to claim 1, wherein at least one of the measuring steps is started in synchronization with the third dispensing. A third dispensing step of dispensing a second reaction solution containing a second reagent that reacts with the measurement object into the measurement container after the second dispensing step;
A second stirring step of stirring the second reaction solution dispensed into the measurement container,
At least one of the measuring steps is started in synchronization with the third dispensing,
At least one of the measurement steps is not accompanied by the stirring step or the second stirring step,
The potential difference measuring method according to claim 1, wherein the second stirring step starts in synchronization with the third dispensing. Having the first dispensing step after the second dispensing step;
The potential difference measuring method according to claim 1, wherein at least one of the measuring steps is started in synchronization with the first dispensing. A second stirring step of stirring the sample solution dispensed into the measurement container;
At least one of the measurement steps is not accompanied by the stirring step or the second stirring step,
The potential difference measuring method according to claim 6, wherein the second stirring step is started in synchronization with the first dispensing step.   The potential difference measuring method according to claim 6, wherein the stirring step is started not in the second dispensing but in synchronization with the first dispensing.   The potential difference measuring method according to claim 6, wherein at least one of the measuring steps does not involve the stirring step.   10. The potential difference measuring method according to claim 1, wherein the reaction generated by the first reaction solution or the second reaction solution is an enzyme reaction or an oxidation-reduction reaction.   The potential difference measuring method according to any one of claims 1 to 10, wherein in the time series data of the potential difference obtained by the measuring step, the data for a predetermined time at the rising edge of the data is compared with the standard data, and the comparison result is obtained. A potential difference measuring method comprising a step of outputting.   The potential difference measuring method according to any one of claims 1 to 10, wherein in the time series data of the potential difference obtained by the measuring step, the difference between the maximum value and the minimum value of the data is compared with standard data, A potential difference measuring method comprising a step of outputting a comparison result.   The potential difference measuring method according to any one of claims 1 to 10, wherein in the time series data of the potential difference obtained by the measuring step, a transfer function is derived from the time series data, and an inverse function of the transfer function is derived, A potential difference measuring method comprising a step of obtaining an input function by computing the inverse function and the time series data.   14. The potential difference measuring method according to claim 13, further comprising a step of comparing data of a predetermined time at the rising edge of the input function with standard data in the input function and outputting a comparison result. Method.   14. The potential difference measuring method according to claim 13, further comprising a step of comparing a difference between a maximum value and a minimum value of the input function with standard data and outputting a comparison result in the input function. Potential difference measurement method.

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