JP2010117183A - Potential difference measuring device - Google Patents

Abstract

A potential difference measuring apparatus for measuring a component to be analyzed in a biological sample, particularly blood or urine, accurately measures the potential difference so that the concentration of the measurement target in the sample can be accurately quantified.
A potentiometric method is used as a measurement method, depending on the order of introduction of the sample sample and the reaction reagent solution into the reaction container, immediately after the introduction of the sample sample or after a certain time after the introduction, or start of stirring of the reaction solution. Immediately after measurement or in synchronization with a certain time after the start of stirring, the concentration of the measurement object in the sample is determined from the obtained potential difference.
[Selection] Figure 1

Description

  The present invention relates to a potentiometer for measuring an analysis target component in a biological sample, particularly blood or urine.

  For the qualitative and quantitative analysis of biological samples such as blood and urine in the medical field, the 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 carried out with an automatic chemical analyzer. Various types of biochemical automatic analyzers have been developed from flow type to discrete type. At present, the discrete type that automates all operations such as dispensing of a sample sample and a reagent solution into a reaction container, stirring the sample sample and the reagent solution, and measuring the change in absorbance of the reaction solution is the mainstream (for example, Japanese Patent No. 2525063). No., WO2002 / 059624). Since these apparatuses 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 using glucose as a measurement target substance 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 absorbance 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 an object to be measured by a coloration method using an 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 a reagent (for example, a buffer) excluding the reagent component that reacts with the measurement target in the sample and the sample, and the measured value after the end of the reaction is This is 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 an advantage that the measurement principle is an electrochemical measurement method, a large and complicated apparatus is unnecessary, and the apparatus system is small. Electrochemical measurement methods include a current measurement method for measuring a current value flowing through a working electrode in accordance with an oxidation-reduction reaction, and a potential difference measurement method for measuring an interface potential of the measurement electrode that changes with a chemical reaction. As reagents used in electrochemical measurements, enzymes and chemical substances that react specifically with the measurement object are used alone or in combination, as in the reagents used in the colorimetric method.

  For example, in the measurement of blood glucose level using a current method, gluconolactone and hydrogen peroxide 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 by the current flowing through the electrode, the concentration of glucose as a measurement target substance is measured (Pure & Appl. Chem., Vol. 68, (1996) pp. 1837-1841). 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 surface of the measurement electrode to start a predetermined enzyme reaction. It has become. In the potentiometric method, the oxidation reaction by glucose oxidase is measured as a change in the oxidation-reduction 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 No. 3387926). JP, 2008-128803, A). Even in the potentiometric method, a reaction reagent containing an enzyme is usually held in advance on the measurement electrode, and the introduced specimen sample is achieved on the measurement electrode surface, so that a predetermined enzyme reaction is started ( (Patent No. 3387926). When a measurement container is used, the potential difference after a certain time after injecting the specimen sample into the measurement container in which a reaction solution containing an enzyme or other reagent is previously held, or the 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 (Japanese Patent Laid-Open No. 2008-128803).

Patent No. 2525063 WO2002 / 05962 JP 2005-345173 A Patent No. 3387926 JP 2008-128803 JP Pure & Appl. Chem., Vol.68, (1996) pp.1837-1841

  However, in the potential difference measurement method using the measurement container described above, no consideration is given to the volume of the reaction reagent solution containing the reagent such as an enzyme and the sample sample when mounted in an actual apparatus, and the sample sample is not injected before or In the method of obtaining the concentration of the measurement object using the amount of change between the potential difference immediately after the injection and the potential difference after a certain time, there is a problem that the potential difference cannot be measured or the measured values vary. For example, when a sample sample with a small volume (usually several μL) is introduced first, and then a reaction reagent solution with a large volume (usually several tens to several hundred μL) is introduced, Since the measuring electrode is not immersed in the solution (i.e. it does not form a circuit), it is impossible to initiate a potentiometric measurement. In this case, by introducing the reaction reagent solution, the measurement electrode is immersed in the solution, and the potential difference measurement becomes possible for the first time. On the other hand, when a reaction reagent solution with a large volume is introduced first, potential difference measurement is possible, but since a sample sample with a small volume is injected into a reaction reagent solution with a large volume, mixing of the sample sample and the reaction reagent solution is possible. However, there was a problem that the reaction did not proceed well. In this case, since the reaction is not completed within a predetermined time, there is a problem that the measurement value varies. In addition, in the potentiometric method, noble metals such as gold and platinum are usually used as measurement electrodes. However, these electrodes are not very stable in a solution in an unmodified state, so that the potential difference drifts and measurement errors are likely to occur. There was a problem. As a countermeasure, the method of modifying the electrode surface with an electrochemically active substance is effective to some extent, but in the case of high-precision analysis, a slight drift in potential difference may be a problem. Therefore, there has been a demand for an apparatus configuration and a measurement procedure that are suitable for accurately measuring a potential difference.

  An object of the present invention is to perform a potentiometric measurement with high accuracy in a potentiometric method using two or more solutions including a specimen sample and a reaction reagent solution so that the concentration of the measurement object in the specimen sample can be accurately quantified. That is.

  In order to achieve the above object, in the present invention, in a potentiometric measurement method using two or more solutions including a sample sample and a reaction reagent solution, the sample is subjected to a potential difference immediately after the start of the reaction or after a certain time after the start of the reaction. Depending on the order in which the sample and reaction reagent solution are introduced into the reaction vessel, measurement is performed immediately after introduction of the specimen sample or after a certain time after introduction, or immediately after the start of stirring of the reaction solution or after a certain time after the start of stirring. The measurement procedure for determining the concentration of the measurement object in the sample was adopted from the obtained potential difference immediately after the start of the reaction and the amount of change in the potential difference after a certain time after the reaction was almost completed.

  For example, when a sample sample with a small volume (usually several μL) is introduced first, and then a reaction reagent solution with a large volume (usually several tens μL to several hundred μL) is introduced, the sample is added with the reaction reagent solution. Since the sample is quickly mixed in the reaction reagent solution and the reaction proceeds, the measurement of the interface potential of the measurement electrode is started immediately after dispensing the reaction reagent solution into the measurement solution or after a certain time after dispensing. At that time, means for dispensing the reaction reagent solution into the measurement container and a control device for controlling a signal for starting measurement of the interface potential of the measurement electrode in synchronization with the immersion of the measurement electrode and the reference electrode in the measurement solution. It has a device configuration. Furthermore, it is desirable to add a stirring means when the mixing efficiency of the specimen sample and the reaction reagent solution is poor.

  In addition, when a reaction reagent solution with a large volume is introduced first, and then a specimen sample with a smaller volume is injected, the reaction between the sample sample and the reaction reagent solution is insufficient and the reaction does not proceed well. It is necessary to leave it for a certain time after pouring or to provide a stirring mechanism. The measurement of the interfacial potential of the measurement electrode starts after a certain time after dispensing of the specimen sample, or immediately after the reaction solution starts stirring or after a certain time after starting stirring. In the case of leaving the specimen sample for a certain period of time after dispensing the specimen sample, the apparatus has a control device that controls a signal for starting measurement of the interface potential of the measurement electrode after a certain period of time after dispensing the specimen sample. In addition, in the case of having a stirring means, the apparatus has a control device for controlling a signal for starting measurement of the interface potential of the measurement electrode immediately after starting stirring of the reaction solution or after a certain time after starting stirring.

  The inner shape of the measurement container used in the present invention is preferably a cylindrical shape that can efficiently stir and mix the specimen sample and the reaction reagent solution. As a material of the electrode, a noble metal such as gold or silver can be used. Further, since the potential of the untreated electrode surface is unstable, it is desirable to fix a molecule having a redox substance such as ferrocene, pyridine, or pyrimidine to the electrode surface. The molecule having the redox substance may be immobilized on the electrode surface using a redox material derivative having an insulating molecule (for example, alkanethiol) that forms a self-assembled film on the electrode surface as a linker. Furthermore, an insulated gate field effect transistor formed on the same substrate as the electrode is used as a potential difference measuring device. At that time, it is desirable to perform measurement by superimposing an AC voltage of 1 KHz or more on the reference electrode.

  According to the present invention, in a potentiometric measurement method using two or more solutions including a specimen sample and a reaction reagent solution, immediately after or after introduction of the specimen sample, depending on the order of introduction of the specimen sample and the reaction reagent solution into the reaction container. By starting the measurement after a certain time after, or immediately after starting the stirring of the reaction solution or after a certain time after starting the stirring, the measurement can be performed immediately after starting the reaction or after a certain time after starting the reaction, Since the concentration of the analyte in the sample can be determined from the obtained potential difference immediately after the start of reaction and the amount of change in potential difference after a certain period of time when the reaction is almost complete, a highly accurate potential difference measurement method with little effect of drift Thus, the concentration of the measurement object in the sample can be accurately quantified. At that time, a stirring element is generally used to mix the dispensed specimen sample and the reagent solution. However, in the potential difference measuring method of the present invention, since the stirring element does not affect the potential measurement, the reaction from the start of the reaction. Data can be acquired and potential difference can be measured with high accuracy. In the case of absorbance measurement, the stirring element is on the optical path in the reaction vessel, and during the stirring and mixing, the absorbance cannot be measured due to reflection / scattering of the measurement light. Further, by immobilizing a molecule having a redox substance such as ferrocene, pyridine, or pyrimidine on the electrode surface, the potential of the redox reaction in the reaction solution can be measured with high accuracy. Furthermore, by using an insulated gate field effect transistor formed on the same substrate as the electrode as the potential difference measuring device, leakage current can be reduced and the interface potential on the electrode surface can be measured stably. At that time, by superimposing an AC voltage of 1 KHz or more on the reference electrode, the interface potential on the gold electrode surface can be stabilized, and the measurement accuracy is improved.

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 measurement apparatus according to the present embodiment includes a potentiometric electrode 101, a reference electrode 102, a measurement container 103, a control device 104, a data processing device 105, a specimen sample dispenser 106 that dispenses a specimen sample into the measurement container 103, and a reagent. A reagent solution dispenser 107 that dispenses the solution into the measurement container 103 and a stirring element 108 that stirs the dispensed specimen sample and the reagent solution are provided. The control device 104 controls the potential measurement of the potential difference measurement electrode 101 and the movement and start of operation of the potential difference measurement electrode 101, the reference electrode 102, the specimen sample dispenser 106, the reagent solution dispenser 107, and the stirring element 108. I do. The data processing device 105 performs data processing on the signal acquired by the potential difference measuring electrode 101 and calculates the concentration of the measurement object in the sample.

  As the potential difference measuring electrode 101, an electrode made of a noble metal such as gold or carbon can be used. Further, in order to reduce the influence of foreign substances at the time of measuring biological components, a redox substance may be immobilized on the electrode surface. As the redox substance, ferrocene, pyridine, pyrimidine, or the like may be used. 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 103. 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 103 is preferably a container having a curved surface, for example, a cylindrical container, because it is difficult to stir and mix the sample sample and the reagent solution in a container having a corner such as a square cell. As the specimen sample dispenser 106 and the reagent solution dispenser 107, a syringe pump or a peristaltic pump (tube pump) can be used. As the stirring element 108, a device in which a spatula at the tip rotates can be used, but a stirrer that rotates by a magnetic force may be used.

  FIG. 2 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. 2A, the specimen sample 203 is dispensed into the measurement container 201 using the specimen sample dispenser 202. Next, as shown in FIG. 2B, the potential difference measuring electrode 204, the reference electrode 205, and the stirring element 206 are installed in the measurement container 201. In this state, the dispensed specimen sample 203 is not in contact with the reference electrode 205 and the stirring element 206. Next, the reagent solution is dispensed into the measurement container 201 using the reagent solution dispenser 209. In synchronization with the dispensing of the reagent solution, the stirring and mixing by the stirring element 206 and the potential measurement of the potential difference measuring electrode 204 are started (FIG. 2 (c)).

  FIG. 3 is a view showing an example of another stirring means according to the present invention, where (a) shows a side view and (b) shows a bird's-eye view. A stirring spatula 303 is attached to the stirring element 302 installed on the stirring element base 301, and the solution in the reaction vessel 304 is moved by rotating or vertically moving the stirring element base 301 as shown by an arrow in the figure. Is stirred and mixed. At that time, since the potential difference measuring electrode 305 and the reference electrode 306 are inserted into the central hole of the stirring element base 301 and do not contact the stirring element 302, there is no problem in potential measurement.

  FIG. 4 is a view showing an example of another stirring means according to the present invention, wherein (a) is a side view and (b) is a bird's eye view. A stirring spatula 402 is attached to the tip of the stirring element 401. By rotating the stirring element 401, the solution in the measurement container 403 is stirred and mixed. At that time, the potential difference measuring electrode 404 and the reference electrode 405 are arranged so as not to touch the stirring element 401, and there is no problem in potential measurement.

  FIG. 5 is a diagram showing an example of the structure 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 a redox substance. The ferrocene derivative 502 was immobilized on the gold electrode 501 by bonding gold and thiol via an alkanethiol 503 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 the immobilization, it was washed with ethanol and pure water and stored in a buffer solution until used.

  FIG. 6 is a block diagram showing another embodiment of the potential difference measuring apparatus according to the present invention. The measurement apparatus according to the present embodiment includes a potential difference measurement electrode 601, a reference electrode 602, a measurement container 603, a control apparatus 604, a data processing apparatus 605, a specimen sample dispenser 606 that dispenses a specimen sample into the measurement container 603, and a reagent. A reagent solution dispenser 607 for dispensing the solution into the measurement container 603 and a stirring element 608 for stirring the dispensed specimen sample and the reagent solution are provided. The control device 604 measures the potential of the potential difference measuring electrode 601 and controls the operation start of the potential difference measuring electrode 601, the reference electrode 602, the specimen sample dispenser 606, the reagent solution dispenser 607, and the stirring element 608. . The data processing device 605 performs data processing on the signal acquired by the potential difference measuring electrode 601 and calculates the concentration of the measurement object in the specimen sample.

  The potential difference measurement electrode 601 is disposed in contact with the bottom of the measurement container 603. The potential difference measuring electrode 601 uses gold as a material, and a ferrocene derivative 609 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 602 provides a reference potential in order to stably measure a potential change based on an equilibrium reaction or a chemical reaction that occurs on the surface of the potential difference measuring electrode 601 in contact with the solution in the measurement container 603. 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 measuring container 603 is preferably a container having a curved surface, 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 specimen sample dispenser 606 and the reagent solution dispenser 607 can use a syringe pump or a peristaltic pump (tube pump). The stirring element 608 used was one whose tip spatula rotates.

  FIG. 7 is a block diagram showing an example of the potential difference measuring apparatus of the present invention using an FET sensor. The measurement apparatus of this embodiment includes a potential difference measurement FET sensor 701, a reference electrode 702, a measurement container 703, a control apparatus 704, a data processing apparatus 705, a specimen sample dispenser 706 for dispensing the specimen sample into the measurement container 703, A reagent solution dispenser 707 for dispensing the reagent solution into the measurement container 703, a stirring element 708 for stirring the dispensed specimen sample and the reagent solution, and a power source 709 for applying a voltage to the reference electrode are provided. The control device 704 measures the current change between the source 710 and the drain 711 of the potential difference measuring FET sensor 701, the potential difference measuring FET sensor 701, the reference electrode 702, the specimen sample dispenser 706, and the reagent solution dispenser. 707, the operation start of the stirring element 708 and the power source 709 are controlled. The data processing device 705 performs data processing on the signal acquired by the potential difference measuring FET sensor 701 to calculate the concentration of the measurement object in the specimen sample. The potential difference measuring FET sensor 701 is installed in contact with the bottom of the measurement container 703. The sensing part of the FET sensor 701 uses a gold electrode 712, and a ferrocene derivative 713 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. When the FET sensor 701 is used as a measurement electrode, it is necessary to apply a voltage between the FET sensor 701 and the reference electrode 702 using the power source 709. In order to reduce the influence of external fluctuations on the gold electrode surface. In addition, it is preferable to apply an alternating current 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.

  The reference electrode 702 provides a reference potential in order to stably measure a potential change based on an equilibrium reaction or a chemical reaction that occurs on the surface of the sensing portion of the FET sensor 701 that is in contact with the solution in the measurement container 703. 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 703 is preferably a container having a curved surface, 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 706 and the reagent solution dispenser 707 can use a syringe pump or a peristaltic pump (tube pump). As the stirring element 708, a device in which a spatula at the tip rotates was used.

  FIG. 8 is a diagram showing an example of the structure of an analytical element used in the potential difference measuring apparatus of the present invention using an FET sensor. FIGS. 8A and 8B show a cross-sectional structure and a planar structure, respectively. In the insulated gate field effect transistor 801, a source 802, a drain 803, and a gate insulator 804 are formed on a surface of a silicon substrate, and a gold electrode 805 is provided. A gold electrode 805 and an insulated gate field effect transistor gate 806 are connected by a conductive wiring 807. 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 805 via the alkanethiool.

The insulated gate field effect transistor used in this example is a depletion type FET having an insulating layer using SiO ₂ (thickness: 17.5 nm), and a gold electrode is fabricated to a size of 400 μm × 400 μm. is there. 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 that 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 to around −0.5V. In place of the gold electrode, an electrode made of another noble metal such as silver may be used.

  FIG. 9 is a block diagram showing another embodiment of the potential difference measuring apparatus according to the present invention. In this embodiment, the mixing of the specimen sample and the reagent solution and the potential difference measurement are performed at different locations. First, as shown in FIG. 9A, a predetermined amount of the measurement solution in the measurement container 902 on the measurement container table 901 is dispensed by the dispenser 903. The dispenser 903 moves on the measurement chip 906 of the detection unit 905 at the stage 904, drops the measurement solution, and starts the potential difference measurement (FIG. 9B).

  The procedure of stirring and mixing the specimen sample and the reagent solution at that time will be described with reference to FIG. First, as shown in FIG. 10A, the specimen sample 1003 is dispensed into the measurement container 1001 using the specimen sample dispenser 1002. Next, as shown in FIG. 10 (b), the stirring element 1004 is installed in the measurement container 1001. Next, the reagent solution is dispensed into the measurement container 1001 using the reagent solution dispenser 1005. After stirring and mixing with the stirring element 1004 for a certain time (for example, 1 to 2 seconds) in synchronization with the dispensing of the reagent solution, a part of the reaction solution is immediately separated and added to the measuring chip to measure the potential. Start.

  The structure of the measuring chip used in the present invention will be described with reference to FIG. FIGS. 11A and 11B respectively show a cross-sectional structure and a planar structure. The measurement chip has a gold electrode 1102, a reference electrode 1103, terminal pads 1104 and 1105, a conductive wiring 1106 for connecting the gold electrode 1102 and the terminal pad 1104, a reference electrode 1103 and a terminal pad 1105 on a silicon substrate 1101. Conductive wiring 1107 to be connected is formed. Portions other than the gold electrode 1102, the reference electrode 1103, and the terminal pads 1104 and 1105 are covered with high heat resistant polyimide. A ferrocene derivative was fixed to the gold electrode 1102 via an alkanethiol to obtain a measurement electrode. In this example, 11-ferrocenyl-1-undecaniol was used as the ferrocene derivative, but pyridine, pyrimidine and the like may be used as other electrochemically active substances. The reference electrode 1103 is a pseudo reference electrode by forming silver silver chloride on the surface of a gold electrode. After the reference electrode 1103 is manufactured, the measurement cell member 1108 is attached to form the measurement cell 1109. In this embodiment, the measurement cell member is a polydimethylsiloxane sheet having a circular through hole, but a flexible material such as silicon rubber may be used. Further, a square through hole may be used instead of the circular through hole. In this example, the gold electrode was formed with a size of 1 mm × 1 mm and a thickness of 100 nm. However, in the potentiometric method, the measurement sensitivity does not depend on the surface area of the electrode, so the area of the gold electrode depends on the volume of the measurement solution. There is no problem even if it is changed. Moreover, if the thickness of the gold electrode is 50 nm or more, no pinhole or the like can be formed.

  FIG. 12 is a block diagram showing another embodiment of the potential difference measuring apparatus according to the present invention. In this embodiment, the mixing of the specimen sample and the reagent solution and the potential difference measurement are performed at different locations. First, a predetermined amount of the measurement solution in the measurement container 1202 on the measurement container table 1201 is dispensed by the dispenser 1203. The dispenser 1203 moves onto the measurement solution introduction unit 1205 at the stage 1204 and injects the measurement solution into the measurement solution introduction unit 1205. The measurement solution introduced into the measurement solution introduction unit 1205 is introduced and held in the flow cell unit 1207 through the introduction tube 1206. In synchronism with the introduction / holding of the measurement solution to the flow cell unit 1207, the potential difference measurement is started by the potential measuring device 1208. At the time of the potential difference measurement, the flow of the solution is stopped, and the measurement solution is held in the flow cell so that there is no influence such as disturbance of the potential due to the flow. In this example, the potential difference when the flow was stopped before introducing the measurement solution was blank. The acquired signal is subjected to data processing by the data processing device 1209 to calculate the concentration of the measurement object in the sample. A series of operations such as introduction / holding of the measurement solution to the flow cell unit 1207 and liquid supply of the measurement solution after the potential difference measurement to the waste liquid tank 1210 are controlled by the liquid supply unit 1211. The liquid feeding unit 1211 can use a syringe pump or a peristaltic pump (tube pump). The procedure of stirring and mixing the specimen sample and the reagent solution used in the present invention was performed according to the procedure shown in FIG.

  The structure of the flow cell used in the present invention will be described with reference to FIG. FIG. 13 shows a cross-sectional structure of the flow cell. The flow cell has a structure in which a solution introduction pipe 1302, a solution waste liquid pipe 1303, and a reference electrode 1304 are connected to a flow cell member 1301. In this embodiment, a sheet made of polydimethylsiloxane is used as the flow cell member 1301, but a flexible material such as silicon rubber may be used. A measurement electrode 1305 is attached to the inner surface of the flow cell member 1301 and is in contact with the flow channel 1306 of the flow cell member 1301. A signal from the measurement electrode 1305 can be extracted to the outside through a wiring 1307. The reference electrode 1304 is attached so as to be in contact with the flow path 1306 in the flow cell member 1301 through the fine flow path 1308 formed in the flow cell member 1301. The reference electrode 1304 can be electrically connected to the outside through a wiring 1309. In this example, a gold electrode on which 11-ferrocenyl-1-undecaniol was immobilized was used as the measurement electrode 1305. Instead of 11-ferrocenyl-1-undecaniol, a ferrocene derivative, pyridine, pyrimidine, or the like having a different number of carbon chains of other alkanethiols may be used. As the reference electrode 1304, a silver / silver chloride electrode using saturated potassium chloride as an internal solution is used. However, there is no problem even if only a calomel electrode or a silver / silver chloride electrode is used.

  FIG. 14 shows the results of measuring glucose using the potentiometer of the present invention. Here, the measurement was performed with the apparatus configuration shown in FIG. FIG. 15 shows the flow of the measurement operation performed in this example. In this example, after dispensing the specimen sample into the measurement container and the reagent solution into the measurement container, the potential difference measurement electrode, the reference electrode, and the stirring element were inserted into the measurement container at the same time, and after a certain time, stirring The potential difference measurement was started in synchronism with the operation start of the element. In this example, the potential difference measurement electrode, the reference electrode, and the stirring element are simultaneously set to 1 second after the insertion into the measurement container, and the measurement time is 3 minutes. In consideration of the reaction time of the enzyme to be used, the characteristics of the reagent, and the like, the fixed time and measurement time after the potential difference measurement electrode, the reference electrode, and the stirring element are simultaneously inserted into the measurement container may be changed. As shown in FIG. 14, it can be seen that a constant potential difference is exhibited about 150 seconds after the start of the reaction. A calibration curve prepared in advance under the measurement conditions of this example is shown in FIG. A calibration curve was prepared using two samples with known glucose concentrations (concentration, 70 mg / dl and 150 mg / dl). A dotted line in FIG. 16 indicates a calibration curve, and a black circle indicates a measured value, and 115 mg / dL was obtained as a glucose concentration.

The measurement conditions used in this experiment are as follows.
Reagent volume: 30 μL
Reagent composition: 100 mM phosphate buffer, pH 7.0
37.5 mM potassium ferricyanide
1.25 mg / mL glucose dehydrogenase
2% Triton X-100
Sample volume: 7.5 μL
Sample: Control serum (autonorm)

  Although glucose measurement was demonstrated in this invention, the enzyme illustrated in Table 1 can be used according to a measuring object.

The block diagram which shows an example of the electrical potential difference measuring apparatus by this invention. The figure which shows an example of the measurement operation | movement using the electric potential difference measuring apparatus by this invention. It is a figure which shows an example of the other stirring means by this invention, (a) is a side view, (b) Bird's-eye view. It is a figure which shows an example of the other stirring means by this invention, (a) is a side view, (b) Bird's-eye view. The figure which shows the structural example of the measurement electrode used for the electric potential difference measuring apparatus of this invention. The block diagram which shows the other Example of the electric potential difference measuring apparatus by this invention. The block diagram which shows an example of the electric potential difference measuring apparatus of this invention using FET sensor. It is a figure which shows an example of the structure of the analysis element used for the potentiometer of this invention using a FET sensor, (a) is a cross-sectional structure, (b) is a planar structure. It is a block diagram which shows the other Example of the potentiometric device by this invention, (a) Dispensing a measurement solution, (b) Addition of a measurement solution to a measurement chip. The figure which shows the procedure of the stirring mixing of the sample sample and reagent solution by this invention. It is a figure which shows an example of the structure of the measurement chip | tip used for the potentiometric device of this invention, (a) is a cross-sectional structure, (b) is a planar structure. The block diagram which shows the other Example of the electric potential difference measuring apparatus by this invention. The figure which shows an example of the structure of the flow cell used for the electric potential difference measuring apparatus of this invention. The figure which shows the result of having measured glucose using the potentiometric device which is another Example of this invention. The figure which shows the glucose concentration measurement operation | movement flow using the potentiometer which is the other Example of this invention. The figure which shows the calibration curve with respect to the acquired glucose concentration using the potentiometric device which is another Example of this invention.

Explanation of symbols

  101, 204, 305, 404, 601 ... Potential difference measuring electrodes, 102, 205, 306, 405, 602, 702, 1103, 1304 ... Reference electrodes, 103, 201, 304, 403, 603, 703, 902, 1001, 1202 ... Measurement container, 104,604,704 ... Control device, 105,605,705,1209 ... Data processing device, 203,1003 ... Sample sample, 106,202,606,706,1002 ... Specimen sample dispenser, 107, 209, 607, 707, 1005 ... Reagent dispenser, 108, 206, 302, 401, 608, 708, 1004 ... Stirring element, 203 ... Sample sample, 205 ... Reagent solution, 301 ... Stirring element stage, 303 402, stirring spatula, 501, 712, 805, 1102 ... gold electrode, 502, 609, 71 ... Ferrocene derivative, 503 ... Alkanethiol, 701 ... FET sensor for potential difference measurement, 709 ... Power source, 710, 802 ... Source, 711, 803 ... Drain, 801 ... Insulated gate field effect transistor, 804 ... Gate insulator, 806 ... Insulation Gate of gate field effect transistor, 807, 1106, 1107 ... conductive wiring, 901, 1201 ... measurement container base, 903, 1203 ... dispenser, 904, 1204 ... stage, 905 ... detector, 906 ... measurement chip, 1101 DESCRIPTION OF SYMBOLS ... Silicon substrate, 1104, 1105 ... Terminal pad, 1108 ... Measurement cell member, 1109 ... Measurement cell, 1205 ... Measurement solution introduction part, 1206 ... Introduction pipe, 1207 ... Flow cell part, 1208 ... Potential measurement device, 1210 ... Waste liquid Tank, 1211 ... Liquid feeding part, 1301 ... Flow Members, 1302 ... solution introducing pipe, 1303 ... solution waste pipes, 1305 ... measurement electrodes, 1306 ... passage, 1307,1309 ... wiring, 1308 ... microchannel

Claims (19)

Means for dispensing a measurement solution containing a measurement object into a measurement container;
Means for dispensing a reaction solution containing an enzyme or a reagent that selectively reacts with the measurement object into the measurement container;
A measuring electrode in contact with the solution in the measuring vessel;
A reference electrode in contact with the solution in the measurement vessel;
Means for measuring the interface potential of the measurement electrode,
And a control device that controls a signal for starting measurement of the interface potential of the measurement electrode immediately after the start of the reaction with the enzyme or the reagent or after a certain time after the start of the reaction. Characteristic potential difference measuring device.   2. The potential difference measuring apparatus according to claim 1, wherein an inner shape of the measurement container is a cylindrical shape.   The potential difference measuring apparatus according to claim 1, wherein the measurement container is a flow cell.   The potential difference measuring apparatus according to claim 1, wherein the reaction container is formed on a measurement electrode.   The potential difference measuring apparatus according to claim 1, wherein the measurement electrode is disposed in contact with the measurement container.   2. The potentiometric apparatus according to claim 1, wherein an oxidation-reduction substance is immobilized on the measurement electrode.   7. The potentiometric apparatus according to claim 6, wherein the redox substance has a thiol group.   2. The potentiometric apparatus according to claim 1, wherein the measurement is started in synchronization with the start of dispensing of the measurement solution or the reaction solution into the measurement container.   2. The potentiometric apparatus according to claim 1, further comprising a stirring means for mixing the dispensed measurement solution and the reaction solution in the measurement container.   10. The potential difference measuring apparatus according to claim 9, wherein the stirring means is a stirring element that rotates or moves up and down.   10. The potential difference measuring apparatus according to claim 9, wherein the stirring element is disposed adjacent to the measurement electrode.   10. The potentiometric device according to claim 9, wherein the stirring element starts operating in synchronization with the dispensing of the measurement solution or the reaction solution.   2. The potential difference measuring apparatus according to claim 1, wherein the measurement electrode is a field effect transistor and an electrode connected to a gate of the field effect transistor by a wiring.   14. The potential difference measuring apparatus according to claim 13, wherein the electrode material is a noble metal.   14. The potential difference measuring apparatus according to claim 13, wherein the reference electrode is formed on the same substrate as the field effect transistor.   14. The potential difference measuring apparatus according to claim 13, wherein the measurement container is covered with a light shielding member.   14. The potential difference measuring apparatus according to claim 13, further comprising a power source that applies a voltage between the electrode and the reference electrode.   18. The potential difference measuring apparatus according to claim 17, wherein an AC voltage having a frequency of 1 kHz or more is applied by the power source. A step of dispensing a measurement solution containing a measurement object into a measurement container;
Dispensing a reaction solution containing an enzyme or reagent that selectively reacts with the measurement object into the measurement container;
Mixing the dispensed measurement solution and the reaction solution in the measurement container;
Measuring the interfacial potential of the measurement electrode disposed in the measurement container,
A potential difference measuring method, wherein the measurement of the interfacial potential of the measurement electrode is started in synchronism with the mixing of the dispensed measurement solution and the reaction solution in the measurement container.

Images