JP2011149878A - Potential difference measuring device and potential difference measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reference electrode, a potential measuring system and a potential difference measuring method capable of measuring a blood component with sufficient accuracy even when micronization of a measuring solution is performed. <P>SOLUTION: An ion selection electrode, according to circumstances, an ion selection electrode to positive ion or an ion selection electrode to a small quantity of ion included in a sample, is used as a reference electrode, and a potential difference between the reference electrode and a measuring electrode in contact with the measuring solution is measured. An ion concentration in the measuring solution is specified in order to acquire desired accuracy. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a measuring apparatus and a measuring method capable of performing electrical measurement and measuring a biological material with high accuracy.

  Blood tests are widely used for the purpose of understanding health conditions and early detection of diseases. In a blood test, a large biochemical automatic analyzer or a small point-of-care testing (POCT) device may be used depending on required accuracy, speed, cost, and the like. Large automatic biochemical analyzers are installed in general hospitals and testing centers, have high sample processing capacity per unit time, relatively high measurement accuracy, and relatively low running costs. Suitable for inspection. However, it takes some time to obtain results after setting the sample in the instrument for pipeline processing, and it also takes time to transport and wait for the sample, and to collect and send the test results. When considered, it usually takes several days to get results from the collection of the specimen. On the other hand, the device for POCT currently has a measurement accuracy that is not as good as that of a large-scale automatic biochemical analyzer, but it has the speed to obtain a test result immediately after the sample is collected. It is suitable for emergency tests such as outpatients, tests for outpatients, tests in ambulances, tests at clinics, self-tests at home such as self-blood glucose measurement.

  Regardless of which device is used for the inspection, there are various advantages if measurement is possible with a smaller amount of liquid. The test can be made less invasive by reducing the amount of specimen, the test cost can be reduced by reducing the amount of reagent, and the environmental burden can be reduced by reducing the amount of waste liquid. The amount of liquid to be measured that can be made very small depends greatly on the measurement method. The measurement methods currently used in inspection equipment are broadly divided into absorptiometry (including color reaction), current measurement, and potentiometry. The absorptiometry is a method for obtaining the concentration of a measurement object in a specimen from a change in absorbance caused by the reaction between a reagent and a specimen (specimen). Since the magnitude of the obtained signal depends on the optical path length and the light irradiation area, the sensitivity generally decreases due to the small amount of the measurement liquid. The current measurement method is an electrochemical measurement method in which a reaction product of a reagent and a sample is reacted with an electrode, measured as a current value, and a concentration of the measurement object is obtained. Since the current value depends on the area of the electrode, the sensitivity decreases due to the small amount of the measured solution in a simple scale-down. For example, a glucose sensor that measures a blood glucose level is not so high in sensitivity that it can be measured with a few drops of blood (for example, Patent Document 1), but for POCT having general measurement items. Testing equipment requires more blood volume to maintain measurement sensitivity. I-Stat (Non-patent Document 1) developed as an inspection apparatus for POCT required a blood volume of about 65 μl.

  Unlike these measurement methods, the potentiometric method is an electrochemical measurement method in which the signal does not depend on the electrode area. The potentiometric method is composed of a measurement electrode (working electrode) made of gold or platinum and a reference electrode, and an enzyme and a redox substance are present in the measurement solution (Patent Document 2). Further, the measurement electrode and the reference electrode are connected to a device for measuring a voltage such as a voltmeter. When the measurement target substance is added to the measurement solution, the measurement target substance is oxidized by the enzyme reaction, and at the same time, the oxidized redox substance is reduced. The surface potential of the measurement electrode generated at that time follows the following Nernst equation.

  The above equation does not include the electrode area, and the surface potential does not depend on the electrode area. Therefore, the electrode area can be reduced without reducing the signal intensity, and the required amount of specimen can be reduced.

  The difference between the current measurement method and the potential difference measurement method is not only the dependence of the signal on the electrode area, but also the difference in whether the signal depends on the reaction rate or the reaction amount of the enzyme reaction. In other words, the current measurement method obtains a signal proportional to the reaction rate of the enzyme reaction, whereas the potential difference measurement method obtains a signal that depends on the amount of reducing substance (or oxidation substance) generated by the enzyme reaction. Therefore, the current measurement method can use only the rate method for measuring the reaction rate of the enzyme reaction, while the potentiometric method can use the endpoint method for measuring the total amount of reaction products in addition to the rate method. As a characteristic of the enzyme, the reaction rate is proportional to the substrate concentration in the region where the substrate concentration is low, but the reaction rate is not proportional to the substrate concentration when the substrate concentration becomes high, and becomes constant when the substrate concentration becomes higher. The concentration at this boundary is called the Michaelis constant. If the concentration of the substance to be measured in the blood is equal to or higher than the Michaelis constant, the rate method must be used to dilute the sample, and the operation required for the measurement is complicated. It becomes. On the other hand, the end point method measures the total amount of reaction products, so there is no such limitation, and measurement without dilution is possible. One such enzyme is cholesterol dehydrogenase, and the potentiometric method is in principle a method that can measure cholesterol without dilution.

  In the measurement using the principle of electrochemistry, an electrode that is a reference for potential in a solution, generally called a reference electrode, is used. The reference electrode is required for both the oxidation-reduction current method and the potentiometric method. However, the redox current method measures the current, whereas the potentiometric method measures the potential, so it is particularly expensive for the reference electrode. Accuracy is required. One of the commonly used reference electrodes is the internal liquid silver-silver chloride reference electrode. In a container such as glass, potassium chloride or sodium chloride solution with a certain concentration (saturated or 3M is often used) and silver-silver chloride. In general, an electrode is inserted and electrically connected to a solution to be measured through a liquid glass or porous liquid junction. Under specific conditions, a silver-silver chloride electrode alone was used as a pseudo reference electrode (Patent Document 2), and a chlorine ion selection electrode was used as a reference electrode (Patent Document 3).

JP-A-2-245650 Japanese National Patent Publication No. 9-500727 JP 2006-90785 A

Clin. Chem. 39/2 (1993) 283-287

  Using the potentiometric method with the above advantages, research and development of an enzyme sensor capable of measuring with high accuracy in a small amount of solution has resulted in insufficient accuracy when using a conventional reference electrode. It became clear that there was a case.

  When an internal liquid silver-silver chloride reference electrode is used, leakage of the internal liquid is the main cause, and the potential of the measurement electrode fluctuates. In order to prevent this, it is conceivable that the reference electrode, in particular the liquid junction, is miniaturized on the same scale as the measurement solution as well as the measurement solution. However, if the liquid junction is simply miniaturized, the liquid junction is liable to be clogged, resulting in a decrease in measurement accuracy. Moreover, since leakage of the internal liquid also occurs at the junction between the liquid junction and the reference electrode container, it cannot be prevented by simple miniaturization of the liquid junction.

  By using a pseudo-reference electrode such as a silver-silver chloride electrode that does not have internal liquid, it is possible to prevent fluctuations in measurement potential due to leakage of internal liquid. However, in that case, the accuracy of the reference electrode potential is lowered, and a new problem occurs that the measurement accuracy is lowered. Furthermore, since the reference electrode and the measurement solution are in direct contact, the accuracy of the reference electrode potential decreases due to the influence of interfering substances contained in the sample. Typical interfering substances are bromine and thiocyanic acid.

  Even if a chlorine ion selection electrode is simply used as the reference electrode as in Patent Document 3, it is difficult to solve the problem caused by the pseudo reference electrode. This is because, in Patent Document 3, since the chlorine ion selective electrode is in contact with the internal solution, it is unclear how a constant potential is generated when contacting the measurement solution containing the sample. It is also unclear how much accuracy can be obtained by removing the influence of interfering substances because no consideration is given to interfering substances.

  The present invention provides a reference electrode, a potential measurement system, and a potential measurement method that can measure blood components with sufficient accuracy even when the amount of the measurement solution is reduced.

  In the present invention, an ion selective electrode is used as the reference electrode. At that time, in consideration of the selection coefficient of the ion selective electrode, an ion selective electrode for positive ions was used as a reference electrode. In order to obtain the desired accuracy, the ion concentration in the measurement solution was defined. At that time, an ion selective electrode for ions with a small amount contained in the sample was used as a reference electrode. In one example, a sensor for organic substances such as glucose and cholesterol was used as a measurement electrode, and a sensor for inorganic ions was used as a reference electrode. In another example, a sensor for the redox substance was used as a measurement electrode, and a sensor for inorganic ions was used as a reference electrode.

  Considering the selection coefficient of the ion selection electrode used for the reference electrode, the response to the interfering substance can be suppressed, and sufficient measurement accuracy can be obtained. At that time, since most of the interfering substances are anions, the response to the interfering substances can be suppressed by using the ion selective electrode used for the reference electrode as an ion selective electrode for cations. By regulating the ion concentration in the measurement solution, fluctuations in the measurement potential with respect to interfering substances in the sample and fluctuations in the ion concentration can be suppressed. By using an ion selective electrode for ions with a small amount contained in the sample as the reference electrode, the ion concentration in the reagent can be dominant, and fluctuations in the measurement potential can be suppressed in response to fluctuations in the ion concentration in the sample. . By using a sensor for organic substances such as glucose and cholesterol as the measurement electrode and a sensor for inorganic ions as the reference electrode, the concentration of the inorganic substance in the measurement solution can be arbitrarily set to some extent while maintaining the accuracy of the measurement electrode potential. The reference electrode potential is stabilized and sufficient measurement accuracy can be obtained. By using the sensor for the redox substance as the measurement electrode and the sensor for the inorganic ions as the reference electrode, the inorganic substance concentration in the measurement solution can be arbitrarily set to some extent, while maintaining the accuracy of the measurement electrode potential. This will stabilize the measurement and provide sufficient measurement accuracy.

  According to the present invention, by using an ion selective electrode as a reference electrode, leakage of internal liquid, which is a problem with conventional internal liquid type reference electrodes, can be suppressed, and sufficient measurement accuracy can be obtained even if the amount of the measurement solution is reduced. It becomes like this. In the conventional internal liquid type reference electrode, the potential between the internal liquid and the measurement solution is kept constant by movement of ions between the internal liquid and the measurement solution through the liquid junction. For this reason, if the leakage of the internal liquid is eliminated, the ions cannot move, so that the leakage of the internal liquid could not be eliminated in principle. On the other hand, in the ion selective electrode, since the potential between the solution and the ion selective membrane is determined by the equilibrium of ions at the interface between the solution and the ion selective membrane, it is not necessary in principle that ions pass through the inside of the ion selective membrane. Therefore, leakage of internal liquid can be suppressed while maintaining accuracy.

The figure which shows an example of the electrical potential difference measuring apparatus by this invention. The figure which shows an example of the electrical potential difference measuring apparatus by this invention. The figure which shows an example of the blood component measuring device by this invention. The side surface schematic diagram which shows an example of the blood component measuring device by this invention. The plane schematic diagram which shows an example of the blood component measuring device by this invention. The figure which shows an example of the electrical potential difference measuring apparatus by this invention. The figure which shows an example of the circuit of an electrical potential difference measuring apparatus. The figure which shows the comparison result of stability of the conventional internal liquid type silver-silver chloride reference electrode and the ion selective electrode of this invention. The figure which shows the solution amount dependence of the stability of the conventional internal liquid silver-silver-chloride reference electrode and the ion selective electrode of this invention. The figure which shows the structural example of an ion selective electrode.

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

  FIG. 1 is a diagram showing an example of a potential difference measuring apparatus according to the present invention. This apparatus has an ion selection electrode 101, a measurement electrode 102, a voltmeter 103, a container 104, and a measurement solution 105, and the terminals of the ion selection electrode 101 and the measurement electrode 102 are connected to the voltmeter 103, and between the two terminals. The potential difference can be measured. The ion sensitive part of the ion selective electrode 101 and the electrode part of the measurement electrode 102 are in contact with the measurement solution 105 in the container 104. As the ion selection electrode 101, a sodium selection electrode, a potassium selection electrode, a lithium selection electrode, a magnesium selection electrode, a calcium selection electrode, a rubidium selection electrode, a cesium selection electrode, a strontium selection electrode, a barium selection electrode, a chlorine selection electrode, or the like is used. It may or may not have an internal liquid. For example, Lithium, potassium, sodium, magnesium, calcium or ammonium described in Pure Appl. Chem., 72, 1851-2082 Umezawa et. Al., Pure Appl. Chem., 74, 923-994 Umezawa et. An ion selective electrode using an ion selective membrane is used.

  For example, as shown in FIG. 9, the ion selective electrode has a configuration comprising a container 901, an ion selective membrane 902, an internal solution 903, a silver / silver chloride electrode 904, and a terminal 905. The internal solution 903 contains chlorine ions for stabilizing the potential of the silver-silver chloride electrode 904 and ions to be measured by the ion-selective membrane 902 for stabilizing the potential of the ion-selective membrane 902.

  As the measurement electrode 102, an electrode using a noble metal such as gold, silver, or platinum, a carbon electrode, or an electrode obtained by modifying the electrode with an enzyme is used. The composition of the measurement solution 105 can be considered to be substantially the same at the part in contact with the ion selective electrode 101 and the part in contact with the measurement electrode 102.

  FIG. 2 is a diagram showing an example of a potential difference measuring apparatus according to the present invention. This apparatus includes a substrate 201, an ion selection electrode 202, a measurement electrode 203, a voltmeter 204, and a holding unit for the measurement solution 205. The ion selection electrode 202 and the measurement electrode 203 are on the same plane of the substrate 201, and the wiring from each electrode is connected to a voltmeter 204 so that the potential difference between the two electrodes can be measured. The ion sensitive part of the ion selective electrode 202 and the electrode part of the measurement electrode 203 are in contact with the measurement solution 205 disposed on the substrate 201. As the ion selection electrode 202, a sodium selection electrode, a potassium selection electrode, a lithium selection electrode, a magnesium selection electrode, a calcium selection electrode, a rubidium selection electrode, a cesium selection electrode, a strontium selection electrode, a barium selection electrode, a chlorine selection electrode, or the like is used. Even if the structure of the ion selective electrode 202 is such that the gelled solution corresponding to the internal liquid is sandwiched between the metal electrode and the ion selective membrane, the ion selective membrane is attached to a metal electrode called a so-called coat wire. It may be pasted. For example, the ion selective electrode described in Pure Appl. Chem., 72, 1851-2082 Umezawa et. Al., Pure Appl. Chem., 74, 923-994 Umezawa et. As the measurement electrode 102, an electrode using a noble metal such as gold, silver, or platinum, a carbon electrode, or an electrode obtained by modifying the electrode with an enzyme is used. The composition of the measurement solution 205 can be considered to be substantially the same between the portion in contact with the ion selective electrode 202 and the portion in contact with the measurement electrode 203.

  FIG. 3 is a diagram showing an example of a blood component measuring apparatus according to the present invention. The apparatus includes a measurement unit 301 and a control unit 302. The measurement unit 301 includes an ion selection electrode 311, a measurement electrode 312, a voltmeter 313, a measurement solution container 314, a measurement solution 315, a sample dispenser 316, and a sample dispenser. The container 317 includes a sample solution 318, a reagent dispenser 319, a reagent container 320, and a reagent solution 321. The ion selection electrode 311 and the measurement electrode 312 are the same as those shown in FIG. As the sample solution 318, whole blood, serum, plasma, urine, or body fluid of human or other organisms is used. The reagent solution 321 is adapted to the sample solution 318 and used in accordance with measurement items such as glucose and cholesterol. Before measurement, the measurement solution container 314 is empty, and the sample solution 318 and the reagent solution 321 are respectively dispensed into the measurement solution container 314 by the sample dispenser 316 and the reagent dispenser 319, and mixed well. After the measurement solution 315 is obtained, the ion selective electrode 311 and the measurement electrode 312 are brought into contact with the measurement solution 315. A potential between the ion selection electrode 311 and the measurement electrode 312 is measured with a voltmeter 313 to obtain a measured value.

  4A and 4B are diagrams showing an example of a blood component measuring apparatus according to the present invention, FIG. 4A is a schematic side view, and FIG. 4B is a schematic plan view. This apparatus includes a substrate 401, an ion selection electrode 402, a measurement electrode 403, a voltmeter 404, and a filter paper 405 as a measurement solution holding unit. The ion selection electrode 402 and the measurement electrode 403 are on the same plane of the substrate 401, and the wiring from each electrode is connected to the voltmeter 404 so that the potential difference between the two electrodes can be measured. As the ion selection electrode 402 and the measurement electrode 403, those shown in FIG. The filter paper 405 contains a reagent and may be dry or wet. When a specified amount of the sample solution is added to the filter paper 405, the sample solution dissolves or mixes the reagent in the filter paper 405 and becomes a reaction and measurement solution. The sample solution is whole blood, serum, plasma, urine, or body fluid from humans or other organisms. As the reagent in the filter paper 405, a reagent according to measurement items such as glucose and cholesterol is used. The measurement solution comes into contact with the ion selection electrode 402 and the measurement electrode 403, and the potential between the ion selection electrode 402 and the measurement electrode 403 is measured with a voltmeter 404 to obtain a measured value.

  FIG. 5 is a diagram showing an example of a potential difference measuring apparatus according to the present invention. This apparatus includes a substrate 501, an ion selection electrode 502, a measurement electrode 503, a field effect transistor (FET) 504, a source electrode 505, a drain electrode 506, conductive wiring 507 and 508, a terminal 509, and a measurement solution holding. The measurement solution 510 held in the part is included. The ion selection electrode 502 and the measurement electrode 503 are on the same plane of the substrate 501. The surfaces of the conductive wirings 507 and 508 are insulated so as not to directly touch the measurement solution 510. The potential of the measurement electrode 503 can be measured as a voltage-current characteristic between the source electrode 505 and the drain electrode 506 by the FET 504, whereby the potential difference between the ion selection electrode 502 and the measurement electrode 503 can be measured. The ion sensitive part of the ion selective electrode 502 and the electrode part of the measurement electrode 503 are in contact with the measurement solution 510 held on the substrate 501.

  As the ion selection electrode 502, a sodium selection electrode, a potassium selection electrode, a lithium selection electrode, a magnesium selection electrode, a calcium selection electrode, a rubidium selection electrode, a cesium selection electrode, a strontium selection electrode, a barium selection electrode, a chlorine selection electrode, or the like is used. Even if the structure of the ion selective electrode 502 is such that the gelled solution corresponding to the internal liquid is sandwiched between the metal electrode and the ion selective membrane, the ion selective membrane is attached to a metal electrode called a so-called coat wire. It may be pasted. For example, an ion selective electrode described in Pure Appl. Chem., 72, 1851-2082 Umezawa et. Al., Pure Appl. Chem., 74, 923-994 Umezawa et. As the measurement electrode 503, an electrode using a noble metal such as gold, silver, or platinum, a carbon electrode, or an electrode obtained by modifying the electrode with an enzyme is used. The composition of the measurement solution 510 can be considered to be substantially the same between the part in contact with the ion selective electrode 502 and the part in contact with the measurement electrode 503.

  FIG. 6 is a diagram illustrating an example of a circuit when the potential difference measuring apparatus illustrated in FIG. 5 is used. The substrate 601 has a terminal 602 connected to the ion selection electrode, a terminal 603 connected to the source of the FET, and a terminal 604 connected to the drain of the FET. The terminals 602 and 603 are connected to the ground, and a constant voltage is applied between the terminals 603 and 604. The current value of the terminal 604 at that time is measured. The potential difference between the measurement electrode and the ion selection electrode can be obtained from the voltage-current characteristics of the FET measured separately.

  Measurement accuracy can be improved by defining the ion concentration in the measurement solution at the time of measurement using the apparatus described in FIGS. 1 to 6 as follows. The potential E of the ion selective electrode is obtained from the following Nicholsky-Eisenman equation.

  The measurement solution is a mixture of a reagent and a sample. Even if the ion concentration in the reagent is constant, if the ion concentration in the sample varies, the ion concentration in the measurement solution varies. As a result, the potential of the ion selective electrode fluctuates, resulting in a decrease in measurement accuracy. If the ion concentration in the sample is completely unknown, it is difficult to remove this fluctuation. However, focusing on the fact that the sample is whole blood, serum, plasma, or urine, the ion concentration range necessary to maintain life is limited, so the ion concentration in the sample can be predicted to some extent. That is, the potential variation width of the ion selective electrode is estimated using the above equation, and the reagent composition and the reagent-sample mixing ratio may be devised so that the variation width is less than the required accuracy.

The ion concentration in the measurement solution derived from the reagent is _cr and _{cr, j} , the minimum value of the ion concentration in the sample is c _{s, min} and c _{s, j, min} , and the maximum value of the ion concentration in the sample is c _{s. , max} and c _{s, j, max} , and the sample dilution rate is d (for example, the dilution rate when mixing 9 μl of reagent and 1 μl sample is 10, dilution when mixing dry reagent and 10 μl sample) If the activity coefficient is 1 for simplicity, the minimum potential (E _min ) and maximum value (E _max ) that can be taken by the ion selective electrode are obtained from the Nicholsky-Eisenmann equation.

である。すなわち，イオン選択電極の電位変動幅(Ｅは，

It is. That is, the potential variation width of the ion selective electrode (E is

から求まる。(Ｅが必要な精度以下に収まるように，試薬由来の測定溶液中のイオン濃度ｃ_r及びｃ_r,jを設定する。また，上式から分かるように，試薬由来の測定溶液中のイオン濃度を高くすれば試料由来のイオン濃度変動が相対的に小さくなり，(Ｅも小さくなる。そのため，試薬由来の測定溶液中のイオン濃度をなるべく大きくしたいが，溶解度や酵素に至適なイオン濃度範囲があるため，これらの濃度を超えないようにする。

Obtained from (The ion concentrations _cr and _{cr, j} in the reagent-derived measurement solution are set so that E falls below the required accuracy. Also, as can be seen from the above equation, the ion concentration in the reagent-derived measurement solution is set. The ion concentration variation from the sample becomes relatively small if the value is increased (E also becomes small. Therefore, I want to increase the ion concentration in the reagent-derived measurement solution as much as possible, but the ion concentration range optimal for solubility and enzyme Therefore, do not exceed these concentrations.

  An example of the fluctuation range of the ion concentration in the sample is shown below.

  From the viewpoint of stabilizing the potential of the ion selective electrode, it is desirable to use an ion selective electrode for ions having a low ion concentration in the sample. (1) Ions with a low ion concentration in the sample have a small fluctuation range, and the ion fluctuation rate can be reduced by including a certain amount of ions in the reagent (the potential of the ion selective electrode is the logarithm of the ion concentration). (2) Because there is an upper limit for ions that can be included in the reagent.

  Pure Appl. Chem., 72, 1851-2082 Umezawa et. Al., Pure Appl. Chem., 74, 923-994 Umezawa et. Al. The selectivity is better than that of the electrode. For example, there are few ionic species that significantly lower the selectivity, such as the response of the chlorine ion electrode to bromine and thiocyan. Therefore, it is desirable that the ion selective electrode used as the reference electrode is a positive ion selective electrode.

  As an example, the conditions under which serum glucose is measured are described below.

Ion selective electrode : sodium selective electrode (selection coefficient (log): K ⁺ , −2.4; Li ⁺ , −3.0; NH ₄ ⁺ , −4.2; Ca ²⁺ , −3.8; Mg ^{2) +} , -4.0)
Measurement electrode : 11-ferrocenyl-1-undecanethiol modified gold electrode
Voltmeter : Input impedance 10GΩ or more
Reagent 1 :
Potassium ferricyanide (9.9 mM)
Potassium ferrocyanide (0.1 mM)
Sodium chloride (137 mM)
Potassium chloride (3 mM)
Magnesium chloride (10 mM)
Disodium hydrogen phosphate (10 mM)
Potassium dihydrogen phosphate (1.8 mM)
Adenosine triphosphate (4 mM)
Hexokinase (10U / ml)
Glucose 6-phosphate dehydrogenase (40 U / ml)
Diaphorase (3.75 U / ml)
Nicotinamide adenine dinucleotide (0.5 mg / ml)
Reagent 2 :
Potassium ferricyanide (9.9 mM)
Potassium ferrocyanide (0.1 mM)
Potassium chloride (140 mM)
Magnesium chloride (10 mM)
Dipotassium hydrogen phosphate (10 mM)
Potassium dihydrogen phosphate (1.8 mM)
Adenosine triphosphate (4 mM)
Hexokinase (10U / ml)
Glucose 6-phosphate dehydrogenase (40 U / ml)
Diaphorase (3.75 U / ml)
Nicotinamide adenine dinucleotide (0.5 mg / ml)
Sample : human serum Regardless of whether reagent 1 or reagent 2 is used, potassium ferrocyanide is generated according to the glucose concentration in human serum according to the following reaction formula.

Glucose + adenosine triphosphate → glucose 6 phosphate + adenosine diphosphate (catalyzed by hexokinase)
Glucose 6-phosphate + nicotinamide adenine dinucleotide (oxidized form) → Glucose 1,5-lactone 6-phosphate + nicotinamide adenine dinucleotide (reduced form) (catalyzed by glucose 6-phosphate dehydrogenase)
2 potassium ferricyanide + nicotinamide adenine dinucleotide (reduced form) → 2 ferrocyanide potassium + nicotinamide adenine dinucleotide (oxidized form) (catalyzed by diaphorase)

  The quantity ratio between potassium ferricyanide and potassium ferrocyanide can be measured as the potential difference between the measuring electrode and the ion selective electrode according to the Nernst equation. The electrolyte was adjusted so that sodium ions were dominant in reagent 1 and potassium ions were dominant in reagent 2. Enzymatic reactions are almost unaffected by this difference in electrolyte, especially in endpoint measurements that can be used for potentiometric measurements.

  The fluctuation range of each ion in human serum was estimated as shown in the above table. When reagent 1 and the sample are mixed at 9: 1, according to the equation, the potential fluctuation range of the ion selective electrode is 0.23 mV. Here, for the sake of simplicity, the activity coefficients were all calculated as 1. On the other hand, when the reagent 2 and the sample are mixed at 9: 1, the potential fluctuation range of the ion selective electrode is 2.45 mV according to the Nicholsky-Eisenman equation. According to the Nernst equation, a potential variation of 0.23 mV corresponds to a concentration variation of 0.9%, and a potential variation of 2.45 mV corresponds to a concentration variation of 10%. Therefore, before the ion concentration in the reagent is optimized ( It can be seen that the measurement accuracy changes 10 times or more between the reagent 2) and the optimized (reagent 1). Moreover, since the accuracy of about 1 to 3%, that is, the accuracy of about 0.25 to 0.75 mV is required in the measurement of the normal blood component, it can be understood that the accuracy before the optimization cannot withstand practical use.

  Such reagent optimization can be used because ions are not the object of measurement. If the ions to be measured are included in the reagent in the measurement of ions, the measurement accuracy is lowered due to the principle of the ion selective electrode. On the other hand, when measuring organic substances such as glucose and cholesterol, even if ions are contained in the reagent for the purpose of stabilizing the potential of the ion selective electrode used as the reference electrode, the measurement is not directly affected.

  An example of the optimized reagent composition when measuring cholesterol and neutral fat in the same measurement system is described below.

Ion selective electrode : potassium selective electrode (selection coefficient (log): Na ⁺ , −4.0; Li ⁺ , −4.0; NH ₄ ⁺ , −1.5; Ca ²⁺ , −4.2; Mg ^{2 +} , -6.0)
Measurement electrode : 11-ferrocenyl-1-undecanethiol modified gold electrode
Voltmeter : Input impedance 10GΩ or more
Cholesterol measurement reagent :
Potassium ferricyanide (9.9 mM)
Potassium ferrocyanide (0.1 mM)
Potassium chloride (100 mM)
Tris-HCl (0.2M)
Cholesterol dehydrogenase (40 U / ml)
Diaphorase (3.75 U / ml)
Nicotinamide adenine dinucleotide (0.5 mg / ml)
Neutral fat measurement reagent :
Potassium ferricyanide (9.9 mM)
Potassium ferrocyanide (0.1 mM)
Potassium chloride (140 mM)
Magnesium chloride (10 mM)
Disodium hydrogen phosphate (10 mM)
Potassium dihydrogen phosphate (1.8 mM)
Adenosine triphosphate (4 mM)
Lipase (10U / ml)
Glycerol kinase (10U / ml)
Glycerol 3-phosphate oxidase (40 U / ml)

  In these reagents, potassium ions are the dominant electrolyte in order to improve accuracy when using a potassium selective electrode as a reference electrode. Each reaction equation is shown below.

cholesterol:
Cholesterol + nicotinamide adenine dinucleotide (oxidized form) → cholestenone + nicotinamide adenine dinucleotide (reduced form) (catalyzed by cholesterol dehydrogenase)
2 potassium ferricyanide + nicotinamide adenine dinucleotide (reduced form) → 2 ferrocyanide potassium + nicotinamide adenine dinucleotide (oxidized form) (catalyzed by diaphorase)

Neutral fat:
Triglyceride + 3H ₂ O → Glycerol + 3RCOOH (catalyzed by lipase)
Glycerol + adenosine triphosphate → glycerol triphosphate + adenosine diphosphate (catalyzed by glycerol kinase)
Glycerol triphosphate + potassium ferricyanide → dihydroxyacetone phosphate + potassium ferrocyanide (catalyzed by glycerol triphosphate oxidase)

  The potassium ferrocyanide produced as shown in each reaction formula is measured as a change in potential difference between the measurement electrode and the ion selective electrode.

FIG. 7 shows the results of comparing the stability of a conventional internal liquid silver chloride reference electrode and the ion selective electrode of the present invention using the measurement system of FIG. In the figure, the line indicated by silver-silver chloride is that of the internal liquid-type silver-silver chloride reference electrode, and that indicated by ISE is the result of using the ion selective electrode. The measurement was performed under the following conditions.
Reference electrode : internal liquid silver chloride reference electrode (internal liquid: saturated potassium chloride, liquid junction: porous glass) or sodium ion selective electrode
Measurement electrode : 11-ferrocenyl-1-undecanethiol modified gold electrode
Measurement solution : 10 μl
Sodium sulfate (0.1M)
Potassium ferricyanide (5 mM)
Potassium ferrocyanide (5 mM)

  During the measurement for 50 seconds, a potential drift of about 0.5 mV was observed when the internal liquid silver-silver chloride reference electrode was used, but a potential drift of about 0.1 mV was observed when the ion selective electrode was used. It was.

FIG. 8 shows the results of measuring the solution amount dependency of the stability (potential drift) of the conventional internal liquid silver-silver chloride reference electrode and the ion-selective electrode (ISE) of the present invention using the measurement system of FIG. Show. The measurement conditions are
Silver silver chloride : Internal liquid silver-silver chloride reference electrode (internal liquid: saturated potassium chloride, liquid junction: porous glass)
ISE : Sodium selective electrode
Measurement electrode : 11-ferrocenyl-1-undecanethiol modified gold electrode
Measurement solution :
Sodium sulfate (0.1M)
Potassium ferricyanide (5 mM)
Potassium ferrocyanide (5 mM)
It was. It can be seen that the potential drift amount increases in inverse proportion to the amount of solution in the internal liquid silver chloride reference electrode, whereas the potential drift amount does not depend on the amount of solution in the ion selective electrode.

  As shown in FIGS. 7 and 8, when the conventional internal liquid-type silver-silver chloride reference electrode was used, the amount of potential drift increased as the measured solution amount decreased. The cause may be leakage of internal liquid. By using the ion selective electrode as the reference electrode, no significant potential drift was observed with a measurement liquid volume of at least about 10 μl. In the conventional internal liquid type reference electrode, the potential between the internal liquid and the measurement solution is kept constant by movement of ions between the internal liquid and the measurement solution through the liquid junction. Therefore, if the leakage of the internal liquid is eliminated, the ions cannot move, so that the leakage of the internal liquid cannot be eliminated in principle. On the other hand, in the ion selective electrode, since the potential between the solution and the ion selective membrane is determined by the equilibrium of ions at the interface between the solution and the ion selective membrane, it is not necessary in principle that ions pass through the inside of the ion selective membrane. Therefore, it is considered that leakage of internal liquid could be suppressed while maintaining accuracy.

101 ion selection electrode 102 measurement electrode 103 voltmeter 104 container 105 measurement solution 201 substrate 202 ion selection electrode 203 measurement electrode 204 voltmeter 205 measurement solution 301 measurement unit 302 control device 311 ion selection electrode 312 measurement electrode 313 voltmeter 314 container 315 measurement Solution 316 Dispensing device 317 Container 318 Reagent liquid 319 Dispensing device 320 Container 321 Reagent liquid 401 Substrate 402 Ion selection electrode 403 Measurement electrode 404 Voltmeter 405 Measurement solution 501 Substrate 502 Ion selection electrode 503 Measurement electrode 504 Field effect transistor 505 Source 506 Drain 507 Conductive wiring 508 Conductive wiring 509 Terminal 510 Measurement solution 601 Substrate 602 Terminal 603 connected to ion selection electrode Terminal 604 connected to FET source Contacted to drain of FET Have been terminal

Claims (21)

A container or a part into which a measurement solution containing a measurement object is introduced;
A measurement electrode in contact with the measurement solution;
A reference electrode in contact with the measurement solution;
A potentiometer for measuring a potential difference between the measurement electrode and the reference electrode;
The potential difference measuring apparatus, wherein the reference electrode is an ion selective electrode.   The potential difference measuring device according to claim 1, wherein the ion selective electrode is an ion selective electrode for positive ions.   The potentiometer according to claim 1, wherein the ion selective electrode is an ion selective electrode for lithium, potassium, sodium, magnesium, calcium, or ammonia.   The potentiometer according to claim 1, wherein the measurement solution is a mixture of a sample and a reagent. Of the ions in the measurement solution,
Among the ions to be measured by the ion selective electrode, the concentration c _r of ions derived from the reagent and the concentration c _{r, j} of ions derived from the reagent among the interfering ions j of the ion selective electrode are:
The assumed minimum concentration of ions in the sample is c _{s, min} for ions to be measured by the ion selective electrode, and c _{s, j, min} for interfering ions j of the ion selective electrode,
The assumed maximum concentration of ions in the sample is c _{r, max} for ions to be measured by the ion selective electrode, and c _{r, j, max} for interfering ions j of the ion selective electrode,
The dilution rate of the sample is d,
The gas constant is R, the absolute temperature of the measurement solution is T, the valence of ions to be measured by the ion selective electrode is z, the Faraday constant is F, the selection coefficient of the interfering ions j of the ion selective electrode is K _j , When the valence of interfering ions j of the ion selective electrode is z _j ,

The potential difference measuring device according to claim 4, wherein _cr and _{cr, j} do not increase solubility in water.   The potentiometer according to claim 4, wherein the reagent contains a chemical substance or an enzyme that reacts with an organic substance.   The potential difference measuring apparatus according to claim 4, wherein the sample is a biological material.   The potentiometer according to claim 4, wherein the sample is whole blood, serum, plasma, urine, or body fluid.   The potentiometer according to claim 1, wherein an organic substance is measured by the measurement electrode.   The potential difference measuring apparatus according to claim 1, wherein the redox substance is measured by the measurement electrode. Introduce the measurement solution containing the measurement object into the container or part,
Bringing a measurement electrode and a reference electrode into contact with the measurement solution;
In a potential difference measuring method for measuring a potential difference between the measurement electrode and the reference electrode using a potentiometer,
The potential difference measuring method, wherein the reference electrode is an ion selective electrode.   The potential difference measuring method according to claim 11, wherein the ion selective electrode is an ion selective electrode for positive ions.   12. The potential difference measuring method according to claim 11, wherein the ion selective electrode is an ion selective electrode for lithium, potassium, sodium, magnesium, calcium or ammonia.   The potential difference measuring method according to claim 11, wherein the measurement solution is prepared by mixing a sample and a reagent. Of the ions in the measurement solution,
Among the ions to be measured by the ion selective electrode, the concentration c _r of ions derived from the reagent and the concentration c _{r, j} of ions derived from the reagent among the interfering ions j of the ion selective electrode are:
The assumed minimum concentration of ions in the sample is c _{s, min} for ions to be measured by the ion selective electrode, and c _{s, j, min} for interfering ions j of the ion selective electrode,
The assumed maximum concentration of ions in the sample is c _{r, max} for ions to be measured by the ion selective electrode, and c _{r, j, max} for interfering ions j of the ion selective electrode,
The dilution rate of the sample is d,
The gas constant is R, the absolute temperature of the measurement solution is T, the valence of ions to be measured by the ion selective electrode is z, the Faraday constant is F, the selection coefficient of the interfering ions j of the ion selective electrode is K _j , When the valence of interfering ions j of the ion selective electrode is z _j ,

The potential difference measuring method according to claim 14, wherein _cr and _{cr, j} do not increase the solubility in water.   The potential difference measuring method according to claim 15, wherein the reagent includes a chemical substance or an enzyme that reacts with an organic substance.   The potential difference measuring method according to claim 15, wherein the sample is a biological material.   The potential difference measuring method according to claim 15, wherein the sample is whole blood, serum, plasma, urine, or body fluid.   The potential difference measuring method according to claim 11, wherein an organic substance is measured by the measurement electrode.   The potential difference measuring method according to claim 11, wherein the redox substance is measured by the measurement electrode.   The potential difference measuring method according to claim 11, wherein the measurement solution has substantially the same composition at a site in contact with the measurement electrode and a site in contact with the reference electrode.

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