JP2012026812A - Potential difference measuring instrument - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly accurate analysis method with a minute amount of a sample in a potential difference measurement method.SOLUTION: Anions in an electrolyte solution are prevented from infiltrating into an ion exchange resin and an ion exchange film of them by electrostatic repulsion to the ion exchange group having an opposite charge. Further, cations are difficult to infiltrate into the ion exchange resin and the ion exchange film by electrostatic repulsion to the ion exchange group having an opposite charge if an ion exchange resin and an ion exchange film having an anion exchange group are arranged side by side therewith. It is considerable that the arrangement of the ion exchange resin and the ion exchange film having the cation exchange group or the anion exchange group in this way can demonstrate an effect for preventing ion transmission. It is considered that an effect on a measurement potential given by ions leaked out of a reference electrode by soaking into a measurement solution can be reduced by providing a region obtained by laminating the ion exchange resin or the ion exchange film between the reference solution and the measurement solution.

Description

  The present invention relates to a potential difference measuring apparatus applicable to analysis of biological samples such as blood and urine, and more particularly to a potential difference measuring apparatus suitable for measuring a very small amount of sample.

  One method for diagnosing a human disease using blood, urine, or the like as a sample is a wet chemistry analysis method. This is a method using a so-called solution reagent, which has a long history, and detection reagents have been developed for many items, and there are various measuring instruments ranging from simple small machines to large fully automatic machines.

  In the wet chemistry analysis method, the reagents are divided into several groups in consideration of stability, and can be mixed at the time of dissolution and preparation, or the reagent addition procedure can be divided into several steps. Furthermore, since an appropriate amount of reagent can be dissolved and prepared according to the number of measurement specimens, the reagent cost per measurement can be reduced. It is complicated and cumbersome to automate the handling of a large number of solutions such as reagents, but the development of clinical laboratory equipment has a history and high social demand, so it already has a large, medium and small processing capacity. Efficient biochemical automatic analyzers using wet chemistry analysis methods have also been developed and put to practical use in fields that require water.

  As a measurement principle, a biochemical automatic analyzer mainly uses a colorimetric method in which a biological sample is quantitatively analyzed by reacting a specimen with a reagent and measuring a change in absorbance of the reaction solution. In the colorimetric method, a component in a biological sample can be quantified by applying light having a specific wavelength to a spectroscopic cell containing a measurement solution and measuring the absorbance of the specific component.

  In addition to the colorimetric method, there is a method of measuring the current associated with the oxidation-reduction reaction of the oxidation-reduction reagent by reacting with the sample and a reaction reagent containing the oxidation-reduction reagent, and a method of measuring the interfacial potential of the electrode that changes with the chemical reaction There is an electrochemical measurement method. Since these methods do not require an optical system, there is an advantage that the measurement system becomes small. These techniques are described in Patent Document 1, Non-Patent Document 1, and the like.

Japanese Patent No. 3387926

Donald T. Sawyer, Andrzej Sobkowiak and Julian L. Roberts, Jr. “Basics of Electrochemical Measurements” Maruzen Co., Ltd. 2003

  When aiming to reduce the amount of sample or the amount of liquid to be measured, the colorimetric method can reduce the size of the spectroscopic cell. However, it is necessary to consider a decrease in the sensitivity of the absorbance measurement due to the miniaturization of the spectroscopic cell. In other words, the optical path length of a cell that is shortened for miniaturization of a spectroscopic cell has a lower limit of length in order to avoid a decrease in sensitivity. At present, the minimum measurement liquid volume is limited to about 50 μL. In addition, in the current measurement method, the absolute amount of the substance to be measured contained with the decrease in the amount of the measurement liquid decreases and the current value decreases, so the sensitivity decreases. On the other hand, in the potential difference measurement method, the electrode voltage does not depend on the amount of the measurement liquid in principle, so it is considered that there is a high possibility that the amount will be reduced.

  An object of the present invention is to provide an analysis method with high accuracy with a small amount of sample in a potentiometric method.

In the potentiometric method, the following Nernst equation E = E0 + RT / nF × ln (Cox / Cred) is used in a potentiometric method using two or more solutions including a specimen sample and a reaction reagent solution.
E: potential of electrode surface, E0: standard potential of redox substance, R: gas constant, T: temperature, n: number of electrons in chemical reaction, F: Faraday constant, Cox: oxidation type concentration of redox substance, Cred: It is known that a reduced concentration of a redox substance is established. From the above formula, the redox substance changes from the oxidized form to the reduced form with the chemical reaction between the target object and the reagent, so the potential change of the system is obtained from the ratio of Cox / Cred and correlated with the concentration of the desired reactant. be able to. In the Nernst equation, since the potential difference is a function of temperature and concentration, the amount of liquid to be measured is irrelevant in principle.

  In the potentiometric measurement method, the interfacial potential in a measurement electrode is measured by combining a reference electrode with a measurement electrode in a measurement solution. When a very small amount of internal liquid leaks from the junction (junction) at the tip of the reference electrode in the measurement liquid, an electrical connection with the measurement liquid is made and the potential difference with the measurement electrode is measured. When aiming to reduce the amount of the sample or the amount of the measuring solution, if the amount of the measuring solution itself is small, even if the salt solution used in the internal solution leaks in a very small amount, the effect of the leaked salt on the potential appears remarkably. The amount of leakage differs depending on the structure and material of the reference electrode, and is considered to be particularly related to the material of the joint.

  At the same time that the reference electrode is in contact with the measurement solution, ions contained in the internal solution of the reference electrode (generally using a saturated aqueous potassium chloride solution) diffuse into the measurement solution from the joint of the reference electrode. The potential fluctuates as the diffused ions reach the surface of the measurement electrode, but if the amount of the solution to be measured is excessive compared to the leaked ions, the effect of a very small amount of leaked ions is detected as the measured potential fluctuation. Hateful. When the amount of the measurement liquid is reduced to about 10 μL, for example, the amount of leaked ions from the reference electrode is very small, and even if the measurement time is 1 minute, it is considered that the influence on the potential cannot be ignored. In order to make it less susceptible to such leakage ions, it is only necessary to limit the diffusion of leakage ions from the reference electrode.

  Since ions have a positive or negative charge, if a similar positive or negative charge exists in the surrounding area, an electrostatic Coulomb force is generated between the charge of the ion and the surrounding charge. In the case of a cation, if there is a positive charge of the same sign around it, a repulsive force is generated, and if it is a negative charge, an attractive force is generated. Similarly, in the case of anions, a repulsive force is generated with a negative charge of the same sign, and an attractive force is generated with a positive charge. When the electrolyte solution is brought into contact with an ion exchange resin or ion exchange membrane having a cation exchange group, the cation in the electrolyte solution is exchanged with the cation of the ion exchange group by electrostatic interaction with the ion exchange group of a different sign charge. Ion exchange occurs. On the other hand, anions in the electrolyte solution are prevented from penetrating into these ion exchange resins and ion exchange membranes by electrostatic repulsion with ion exchange groups having opposite charges. Furthermore, when an ion exchange resin or ion exchange membrane having an anion exchange group is arranged next to it, the cation penetrates into these ion exchange resin or ion exchange membrane by electrostatic repulsion with an ion exchange group having an opposite charge. It becomes difficult to do. Thus, it is thought that the effect which prevents permeation | transmission of ion can be exhibited by arranging the ion exchange resin and ion exchange membrane which have a cation exchange group or an anion exchange group.

  By providing a region where the above-mentioned ion exchange resin or ion exchange membrane is laminated between the reference electrode solution and the measurement solution, it is possible to reduce the influence of ions leaking from the reference electrode to the measurement solution and affecting the measurement potential. It is thought that. There is also a concern that by providing a buffer region constituted by an ion exchanger, an ion exchange occurs and a liquid potential that cannot be ignored is generated. Therefore, in actual sample measurement, after measuring with only the reagent that does not contain the sample, measure the solution in which the sample reacted with the reagent and calculate the difference (change) between them. It is possible to obtain the result.

  As described above, the present invention is characterized in that a buffer region composed of an ion exchanger is provided between the reference electrode and the measurement solution in order to prevent potential fluctuation due to leakage of the internal liquid of the reference electrode.

  According to the present invention, in a potentiometric measurement method using two or more solutions including a sample sample and a reaction reagent solution, a highly accurate potentiometric measurement method with little potential fluctuation is realized even in a small amount of measurement solution. It becomes possible to accurately quantify the concentration of the measurement object.

It is a block diagram which shows an example of a measurement cell (Example 1). (Example 2) which is a figure which shows arrangement | positioning of an ion exchange membrane. It is a figure which shows arrangement | positioning of an ion exchange resin (Example 3). (Example 4) which is a block diagram which shows the example of a multichannel structure of a measurement cell. (Example 5) which is a block diagram which shows a measuring apparatus.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples.

FIG. 1 is a block diagram showing an example of a measurement cell according to the present invention. It consists of a measurement electrode 1, a measurement liquid 2, an ion permeable membrane 3, an ion exchanger 4, a reference electrode liquid layer 5, a reference electrode 6, and a reference electrode lead wire 7. As the measurement electrode 1, an electrode made of a noble metal such as gold or carbon can be used. Further, a redox substance may be immobilized on the electrode surface in order to reduce the influence of impurities in the measurement liquid. Ferrocene, pyridine, pyrimidine, etc. can be used as the redox material. The reference electrode 6 gives a reference potential to stably measure a potential change on the measurement electrode 1 in contact with the measurement liquid 2.

Potential stability in ion exchange membrane At the same time that the reference electrode is in contact with the measurement solution, ions contained in the internal solution of the reference electrode (generally using a saturated potassium chloride aqueous solution) diffuse into the measurement solution from the junction of the reference electrode. The potential fluctuates as the diffused ions reach the surface of the electrode for measurement. However, if the amount of the solution to be measured is excessive compared to the leaked ions, the influence of a very small amount of leaked ions is detected as the measured potential fluctuation. It is hard to be done. When the amount of the measurement liquid is reduced, it is considered that the influence on the potential cannot be ignored even if the amount of leaked ions from the reference electrode is small. In order to make it less susceptible to such leakage ions, it is considered that the diffusion of leakage ions from the reference electrode may be limited.

  The ions in the internal solution leaking from the reference electrode diffuse into the measurement solution according to the concentration gradient, but in the present invention, a buffer region composed of an ion exchanger is provided between the measurement solution and the reference electrode in order to suppress the diffusion rate. . As the ion exchanger 4, an anion exchanger and a cation exchanger are used, and the respective exchangers are stacked and used. Ions leaking from the reference electrode ooze into the reference electrode liquid layer and then reach the ion exchanger through the ion permeable membrane 3. A part of the anion is ion-exchanged with an anion exchange group bonded to the anion exchanger, and the rest of the anion is impeded from permeation of the anion because the cation exchange group is bonded to the adjacent cation exchanger. On the other hand, permeation of the cation is hindered because the anion exchange group is bonded to the anion exchanger. Some cations are ion exchanged with a cation exchange group attached to an adjacent cation exchanger. Since the ions leaking from the reference electrode are blocked by the ion exchanger, the flow from the ion permeable membrane 8 to the measurement liquid 2 can be suppressed.

  It is called ion exchange that either a cation or an anion forming an ionic bond exchanges with another ion of the same sign. Solids having the ability to exchange ions and having positive charges and trapping anions are called anion exchangers, and those having negative charges and trapping cations are called cation exchangers. The ion exchanger has a functional group capable of forming an ionic bond, and examples of the cation exchange group include a sulfonic acid group, a carboxyl group, and an anion exchange group such as an amino group and an ammonium group.

  There are two types of anion exchange groups: strong base type and weak base type. The strong base type ion exchange group is an ammonium group, and is strongly dissociated in water and has a strong basic property. Similarly, there are two types of cation exchanger exchange groups: strong acid type and weak acid type. The strong acid type ion exchange group is a sulfonic acid group, and is strongly dissociated in water and has a strongly acidic property.

On the other hand, the strength of weak acid type or weak base type is expressed by proton dissociation constant Ka, and can be examined by chemical handbook. The smaller the Ka value, the weaker the degree is considered. The weak acid type carboxyl group has a different Ka depending on the compound, for example, benzoic acid is 6.6 × 10 ⁻⁵ (25 ° C.). It becomes 1.8 × 10 ⁻¹¹ (25 ° C.).

  The ion exchangers that can be used in the present invention can be broadly classified into ion exchange membranes and ion exchange resins. Since anion exchangers and cation exchangers need to be laminated alternately, they are bonded together in ion exchange membranes. However, in ion exchange resins, anion exchange resins and cation exchange resins are put in separate containers so that they do not mix. The contact is made through the membrane (FIGS. 2 and 3).

  The anion exchanger has two types of exchange groups: strong base type and weak base type. Strong base type is divided into trimethylamine type (compound 1) and dimethylethanol type (compound 2) of ammonium salt. The weak base type represents an amine in (Compound 3).

  The degree of basicity is weak base type <dimethylethanol type <trimethyl type, and ion exchange ability is different.

  The cation exchange groups of the cation exchanger include a strong acid type of sulfonic acid (compound 4) and a weak acid type of carboxyl (compound 5).

  The ion exchange membrane or ion exchange resin having the ion exchange group mentioned above can be used in combination.

  In order to prevent substances other than ions in the measurement solution from entering the ion exchange membrane or ion exchange resin and adsorbing to the ion exchange groups, the ion exchanger and the reference electrode solution or measurement solution are separated by an ion permeable membrane. . For the ion permeable membrane, a material such as that used for the junction of the reference electrode can be used. Generally, porous ceramic, porous Vycor glass, ceramic frit, quartz, cellulose pulp, glass frit, and the like.

  Table 1 shows the results of examining the variation in measurement potential using the measurement cell and ion exchange membrane shown in FIGS. 1 and 2 in combination of strong acid type and strong base type and weak acid type and weak base type ion exchange membranes. Astom Neoceptor CMX was used as the strong acid ion exchange membrane, and Neoceptor AMX Astom was used as the strong base ion exchange membrane. Asahi Glass Co., Ltd. HSF was used as the weak acid type ion exchange membrane, and Asahi Glass Co., Ltd. AAV was used as the weak base type ion exchange membrane. The measurement conditions used in this experiment are as follows.

Reference electrode: BAS RE-1C
Working electrode: BAS ring disk electrode Au
Ion permeable membrane: Porous Vycor glass Reference electrode solution: Saturated aqueous solution of potassium chloride Measurement solution: Concentration ratio of 10 mM potassium ferricyanide and 10 mM potassium ferrocyanide is 100: 1
Measurement liquid volume: 10 μL
Measurement value: After adding the measurement solution on the measurement electrode, the potential change in mV / min was shown in 1 minute immediately after the reference electrode was immersed in the measurement solution.

  It was shown that potential fluctuations can be suppressed by using an ion exchange membrane.

Potential stability in ion exchange resin We investigated fluctuations in measurement potential using the measurement cell and ion exchange membrane shown in Fig. 1 and Fig. 3 in combination of strong acid type and strong base type and weak acid type and weak base type ion exchange resin. The results are shown in Table 2. Mitsubishi Chemical Corporation Diaion SK1B was used as the strong acid type ion exchange resin, and Mitsubishi Chemical Corporation Diaion SA10A was used as the strong base type ion exchange resin. Mitsubishi Chemical Corporation WK10 was used as the weak acid type ion exchange resin, and Mitsubishi Chemical Corporation WA10 was used as the weak base type ion exchange resin. The measurement conditions used in this experiment are as follows.

Reference electrode: BAS RE-1C
Working electrode: BAS ring disk electrode Au
Ion permeable membrane: Porous ceramic Reference electrode solution: Saturated aqueous potassium chloride solution Measuring solution: Concentration of 10 mM potassium ferricyanide and 10 mM potassium ferrocyanide
The ratio is 100: 1
Measurement liquid volume: 10 μL
Measurement value: After adding the measuring solution on the measuring electrode, immediately after immersing the reference electrode in the measuring solution for 1 minute
The potential fluctuation in mV / min was shown.

  It was shown that potential fluctuations can be suppressed by using an ion exchange resin.

Multi-channel FIG. 4 is a block diagram showing an example of a measurement cell according to the present invention, in which a plurality of measurement cells shown in FIG. 1 are arranged on a substrate and correspond to multi-item or multi-analyte measurement. The measuring electrode 1, the measuring liquid 2, the ion permeable membranes 3 and 8, the ion exchanger 4, the reference electrode liquid layer 5, and the substrate 13 are included. As the measurement electrode 1, an electrode made of noble metal such as gold or carbon can be used. Further, a redox substance may be immobilized on the electrode surface in order to reduce the influence of impurities in the measurement liquid. Ferrocene, pyridine, pyrimidine, etc. can be used as the redox material. One reference electrode corresponds to each measurement cell.

FIG. 5 is a block diagram showing an example of a measuring apparatus according to the present invention. The measurement electrode 1, the measurement liquid 2, the reference electrode 6, the control device 14, and the data processing device 15 are included. The data processor 15 processes the signal acquired by the measurement electrode 1 and calculates the concentration of the measurement object in the sample. As the measurement electrode 1, an electrode made of a noble metal such as gold or carbon can be used. Further, a redox substance may be immobilized on the electrode surface in order to reduce the influence of impurities in the measurement liquid. Ferrocene, pyridine, pyrimidine, etc. can be used as the redox material. The reference electrode 6 gives a reference potential to stably measure a potential change on the measurement electrode 1 in contact with the measurement liquid 2.

DESCRIPTION OF SYMBOLS 1 Measuring electrode 2 Measuring liquid 3,12 Ion permeable membrane 4 Ion exchanger 5 Reference electrode solution layer 6 Reference electrode 7 Reference electrode lead wire 8 Anion exchange membrane 9 Cation exchange membrane 10 Anion exchanger 11 Cation exchanger 13 Substrate 14 Control device 15 Data processing device

Claims (10)

A measurement container containing the reaction solution;
A measurement electrode provided in contact with the reaction solution in the measurement container;
A reference electrode provided in contact with the reaction solution in the measurement vessel;
In a potential difference measuring device comprising a measuring means for measuring an interface potential of the measuring electrode,
An electric potential difference measuring apparatus comprising an ion exchanger provided in the reaction container so that the reference electrode and the reaction solution are not in direct contact with each other. The potential difference measuring apparatus according to claim 1,
A potentiometric device characterized in that the ion exchanger is a combination of acidic and basic ion exchangers. In the potential difference measuring device according to claim 2,
A potentiometric device characterized in that the ion exchanger is a combination of a strongly acidic and strongly basic ion exchanger. In the potential difference measuring device according to claim 2,
A potentiometric device characterized in that the ion exchanger is a combination of a weakly acidic and weakly basic ion exchanger. The potential difference measuring apparatus according to claim 1,
The potentiometer is characterized in that the ion exchanger is a combination of acidic and basic ion exchange membranes. In the potential difference measuring apparatus according to claim 5,
A potentiometric device characterized in that the ion exchange membrane is a combination of a strongly acidic and strongly basic ion exchange membrane. In the potential difference measuring apparatus according to claim 5,
A potentiometric device characterized in that the ion exchanger is a combination of weakly acidic and weakly basic ion exchange membranes. The potential difference measuring apparatus according to claim 1,
A potentiometric device characterized in that the ion exchanger is a combination of acidic and basic ion exchange resins. In the potential difference measuring device according to claim 8,
A potentiometric device characterized in that the ion exchange membrane is a combination of strongly acidic and strongly basic ion exchange resins. In the potential difference measuring device according to claim 8,
A potentiometric device characterized in that the ion exchanger is a combination of weakly acidic and weakly basic ion exchange resins.

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