JP4625946B2 - pH measuring device and pH measuring method - Google Patents

Description

  The present invention relates to a pH measurement device that simultaneously measures a pH change derived from an acid or alkali passing through a gas permeable membrane and a pH change derived from all acids or alkalis by using a plurality of pH electrodes. . The present invention also relates to a pH measurement method using the same. The pH measurement device of the present invention is suitable for quantifying the amount of acid or alkali secreted from a biological sample or the like to be measured, such as medicine, physiology, cell biology, as well as fermentation, brewing, culture, etc. It is suitably used in the bioindustry field.

  Some acids and alkalis, when dissolved in water, have almost zero vapor pressure, such as lactic acid, phosphoric acid, fatty acid, sodium hydroxide, sodium bicarbonate, etc. There are some which have a vapor pressure such as carbon dioxide gas, hydrogen sulfide, ammonia and the like (hereinafter, this may be called “gaseous”). In general, gaseous acids and alkalis have a low molecular weight and are insufficiently dissociated into ions. On the other hand, liquid acids and alkalis have a large molecular weight, or have a large dissociation into ions like hydrogen chloride even if the molecular weight is small. In a living body, a gaseous acid or alkali such as carbon dioxide, hydrogen sulfide, or ammonia and a liquid acid or alkali such as lactic acid, fatty acid, hydrochloric acid, or sodium bicarbonate are often mixed. In addition, some have a low vapor pressure, such as acetic acid and formic acid, though they are low values.

  So far, a so-called Severinghaus type sensor in which a gas permeable membrane and a pH electrode are combined has been used for measuring a gaseous acid or alkali. In this case, a chamber covered with a gas permeable membrane is filled with an internal solution whose pH changes due to dissolution of the gas to be measured, and a pH electrode and a reference electrode are accommodated in the chamber. In this type of gas sensor, the gas concentration is generally displayed as the partial pressure of the gas phase in equilibrium with the analyte. A gas sensor that measures the partial pressure of a gaseous acid or alkali in a specimen by intermittently circulating and stopping the internal solution by applying the principle of the Severinghaus gas sensor is disclosed (Patent Document 1). .

  A gas sensor described in Patent Document 1 includes a gas exchange section having a gas exchange chamber that is provided with an inlet and an outlet for circulating a carrier solution containing ions at least conjugated with a measurement target gas and is isolated from the outside by a gas permeable membrane. And a forward path portion connected to the inlet of the gas exchange chamber and leading the carrier solution to the gas exchange chamber; a return path portion connected to the outlet of the gas exchange chamber and leading the carrier solution from the gas exchange chamber to the outside; It is a gas sensor comprising a reference pH electrode installed inside the forward path section and a measurement pH electrode installed either inside the gas exchange chamber or inside the return path section. This makes it easy to automate the zero point calibration and compensate for the baseline drift and temperature drift of the pH electrode.

  On the other hand, when the sample to be measured contains both liquid and gaseous acids and alkalis, a method and apparatus for measuring the rate of total acid secretion without separating the two are well known. An example of such a case is the measurement of extracellular acidification. Measurement of extracellular acidification is important as an indicator of cellular activity. Cells metabolize glucose, glutamic acid, pyruvic acid, lipids, etc., and synthesize ATP (adenosine triphosphate), which is an energy medium necessary for cell activity. As a result, lactic acid, carbon dioxide, etc. The acidic substance is discharged out of the cell. This process is called energy metabolism. In the most important glucose metabolism, metabolism from glucose to lactic acid proceeds by a glycolytic reaction in the cytoplasm outside the mitochondria, and finally metabolized to carbon dioxide and water by a respiratory reaction in the mitochondria. In both processes, 38 molecules of ATP are synthesized from one glucose molecule. Since both lactic acid and carbon dioxide, which are metabolites, are acidic substances, it is possible to know the energy metabolism of cells by measuring the rate of extracellular acidification.

  There have been many reports on the measurement of extracellular acidification so far, most of which are using “Microphysiometer” manufactured by Molecular Devices. This uses a kind of pH electrode called LAPS (Light Addressable Potentiometric Sensor) as a detector to measure the pH change of the culture solution in the vicinity of the cultured cells (Patent Document 2, Non-Patent Document 1). In one example of this method, a pH electrode is disposed facing the substrate to which the cells are adhered, and a pH change immediately after stopping the flow of the solution by flowing or stopping a medium having a weak pH buffering capacity in the gap. From this rate, the rate of extracellular acidification is determined. The cultured cells secrete carbon dioxide, which is a gaseous acid, lactic acid, which is a liquid acid, and bicarbonate ions, which are liquid alkalis. Since only one pH electrode is used as a detector in this extracellular acidification measurement system, only the sum of gaseous and liquid acids and alkalis can be measured.

  As described above, even in the prior art, it was possible to measure only the secretion rate of gaseous acid or alkali. It was also possible to measure the total secretion rate of gaseous acid or alkali and liquid acid or alkali. However, it was impossible to separate and simultaneously measure the secretion rate of a gaseous acid or alkali and a liquid acid or alkali.

JP 2001-289811 A Japanese Patent No. 2993982 N. J. W. Parce, “Detection of Cell-Affecting Agents with a Silicon Biosensor”, Science, October 1989, Vol. 246, p. 243-247

  The present invention has been made in order to solve the above problems, and a pH change derived from a gaseous acid or alkali and a pH change derived from a liquid acid or alkali can be obtained by a single composite sensor. The purpose is to separate and measure simultaneously.

The above-mentioned problem is a pH measurement device comprising a plurality of pH electrodes and means for supplying and stopping a baseline solution;
A pH electrode (A) and a pH electrode (B) are disposed in the flow path of the baseline solution,
The comparative electrode is disposed at a position where the pH electrode (A) and the pH electrode (B) can be in liquid junction,
A gas permeable membrane is disposed between the pH electrode (A) and the measurement target,
A gas permeable membrane is not arranged between the pH electrode (B) and the measurement target,
While the supply of the baseline solution is stopped, the pH electrode (A) changes the pH of the baseline solution derived from the acid or alkali passing through the gas permeable membrane, and the pH electrode (B) This can be solved by providing a pH measuring device having means for simultaneously measuring pH changes of the baseline solution derived from acid or alkali.

  In the pH measurement device, it is a preferred embodiment that the pH electrode (A) is covered with a tube made of a gas permeable membrane, and the baseline solution flows inside the tube. A preferred embodiment is provided with a measurement chamber in which the pH electrode (A) and the pH electrode (B) are incorporated, and the measurement chamber has an inlet and an outlet for the baseline solution, and a measurement window in contact with the measurement object. In particular, the measurement chamber has two measurement windows, one of the measurement windows is covered with a gas permeable membrane, the pH electrode (A) is arranged inside thereof, and the other measurement window is covered with a gas permeable membrane. It is preferable that the pH electrode (B) is disposed on the inside without breaking. It is also a preferred embodiment that both the pH electrode (A) and the pH electrode (B) are formed on a single semiconductor substrate.

  In the pH measurement device, it is preferable that the path length from the pH electrode (A) to the pH electrode (B) is at least five times the distance from the gas permeable membrane to the pH electrode (A). It is also preferable that a reference electrode is disposed upstream of the pH electrode (A) and the pH electrode (B) in the baseline solution flow path. It is also preferable that the comparison electrode is composed of a combination of a reference pH electrode and a pseudo comparison electrode. It is also preferred that the pH of the baseline solution is 10 to 500 μM / pH.

  In addition, the above problem is that the pH electrode (A) and the pH electrode (B) are arranged in the flow path of the baseline solution, and the comparison electrode is arranged at a position where the pH electrode (A) and the pH electrode (B) can be in liquid junction Then, after the baseline solution having the same composition was passed around the pH electrode (A) and the pH electrode (B) and replaced, the flow of the baseline solution was stopped, and the pH electrode (A) generated gas from the object to be measured. A pH measurement method characterized by simultaneously measuring pH changes derived from an acid or an alkali passing through a permeable membrane, and pH changes derived from all acids or alkalis generated from a measurement object at the pH electrode (B), respectively. It is also solved by providing. A secretion quantification method for quantifying acid or alkali secreted from living tissue by such a pH measurement method is also a preferred embodiment of the present invention, and at this time, the amount of carbon dioxide gas and the amount of lactic acid are respectively quantified. Is particularly preferred.

  According to the pH measurement apparatus of the present invention, the pH change derived from a gaseous acid or alkali and the pH change derived from a liquid acid or alkali are separated and measured simultaneously by one composite sensor. Can do. As a result, for example, it is also possible to quantify the amount of lactic acid and carbon dioxide corresponding to the glycolytic activity and respiratory activity of cells secreted from the living body, respectively. Obtainable.

  Hereinafter, the present invention will be described more specifically with reference to the drawings. FIG. 1 is a schematic view of a composite sensor portion of an example of the pH measurement device of the present invention having a measurement chamber and a measurement window. FIG. 2 is a schematic diagram of a composite sensor portion of an example of the pH measuring device according to the present invention, in which the reference electrode is composed of a combination of a reference pH electrode and a pseudo-reference electrode. FIG. 3 is a schematic view of a composite sensor portion of an example of the pH measurement device of the present invention that does not have a measurement chamber. FIG. 4 is a schematic view of a composite sensor portion of an example of the pH measurement device of the present invention, in which the entire measurement chamber is formed of an elongated annular body. 1 to 4 show only the composite sensor portion of the pH measuring device of the present invention. The pH measurement apparatus of the present invention has supply and stop means for baseline solution, electrical wiring connected to each electrode, data processing means, etc. in addition to the composite sensor portion, but illustration is omitted here. ing.

  First, the basic configuration of the pH measuring device of the present invention will be described with reference to FIG. FIG. 1 shows a composite sensor portion of an example of the pH measuring device of the present invention. The sensor body 1 includes a front chamber 2 and a measurement chamber 3 and has an inlet 4 and an outlet 5 for a baseline solution. A pH electrode (A) 6 and a pH electrode (B) 7 are accommodated inside the measurement chamber 3, and the pH electrode (A) 6 is surrounded by a tube 8 made of a gas permeable membrane. Furthermore, the comparison electrode 9 is installed at a position where the liquid electrode can be connected to the pH electrode (A) 6 and the pH electrode (B) 7 (in this case, inside the front chamber 2). A measurement window 10 is provided in the vicinity of both the pH electrode (A) 6 and the pH electrode (B) 7 of the sensor body 1, and a sample 11 to be measured is in close contact therewith.

  A part of the baseline solution entering from the inlet 4 flows through the tube 8 while replacing the periphery of the pH electrode (A) 6, and is discharged from the outlet 5. Further, the remaining portion of the baseline solution that has entered from the inlet 4 flows outside the tube 8 while replacing the periphery of the pH electrode (B) 7, and is discharged from the outlet 5. While the supply of the baseline solution is stopped, the acid or alkali generated from the sample 11 and passing through the gas permeable membrane reaches the pH electrode (A) 6 by diffusion. At this time, all the acid or alkali generated from the sample 11 reaches the pH electrode (B) 7 by diffusion.

Taking the case where a liquid acid LH and a gaseous acid GH are secreted from the sample 11 as an example, acidification _AL and _AG of the baseline solution by each acid is obtained by the composite sensor described in FIG. The method is described. A sample solution 11 is brought into close contact with the measurement window 10 of the composite sensor shown in FIG. When the inside of the sensor body 1 is sufficiently replaced with a fresh baseline solution, the potential difference between the pH electrode (B) 7 and the pH electrode (A) 6 with respect to the reference electrode 9 is read, and V _T0 (mV) and V _G0 are respectively read. (MV). Next, the feeding of the baseline solution is stopped, and both outputs after a predetermined time t are set to V _Tt (mV) and V _Gt (mV).

For simplicity, assuming that both electrodes have the same pH sensitivity S (mV / pH), the pH change ΔpH _T of the solution outside the tube 8 made of a gas permeable membrane is expressed by the following formula (1).
ΔpH _T = (V _Tt −V _T0 ) / S (1)
The pH change ΔpH _G of the solution inside the tube 8 is expressed by the following formula (2).
ΔpH _G = (V _Gt −V _G0 ) / S (2)

Since the tube 8 allows the gaseous acid GH to permeate but does not allow the liquid acid LH to permeate, ΔpH _G indicates the acidification of the baseline solution by the gaseous acid GH alone. On the other hand, since all the acids including both GH and LH can be dissolved in the baseline solution outside the tube 8, ΔpH _T represents acidification by all acids. That is, the following formulas (3) and (4) are established.
ΔpH _G = A _G (3)
ΔpH _T = _{AG + L} (4)

Here, A _{G + L} is acidification by both LH and GH. Assuming that A _{G + L} is approximately equal to the sum of A _G and A _L , that is, if additivity holds for acidification, equation (4) can be rewritten into equation (5) below.
_{ΔpH T = A G + A L} (5)

Since the pH electrode (A) 6 and the pH electrode (B) 7 are replaced by a common baseline solution, it is assumed that the gaseous acid has reached a sufficient equilibrium state inside and outside the gas permeable membrane at time t. Assuming that _AG in equation (3) is equal to _{AG in} equation (5). At this time, acidification by a liquid acid is represented by the following formula (6) from the formulas (3) and (5).
A _L = ΔpH _T −ΔpH _G (6)

As described above, can be obtained by respectively separating A _G and A _L from equation (3) and (6). When the surroundings of the pH electrode (A) 6 and the pH electrode (B) 7 are replaced with different baseline solutions, the degree of acidification differs even if the same concentration of gaseous acid is dissolved. Therefore, the acidification by the liquid acid cannot be separated by the above formula (6). From this, the importance of the device of the present invention for replacing the periphery of the pH electrode (A) 6 and the pH electrode (B) 7 with a common baseline solution can be understood.

  Next, each component in the pH measuring device of the present invention will be described. First, as pH electrode (A) 6 and pH electrode (B) 7, glass electrode, metal oxide / metal electrode, ISFET (Ion Sensitive Field Effect Transistor), LAPS (Light Addressable Potentiometric Sensor) Various types known so far are used. Among them, semiconductor sensors such as ISFET and LAPS are suitable as a pH electrode used in the present invention because they can be miniaturized and have a high response speed. In view of the fact that light irradiation is not required and the apparatus can be simplified, ISFET is particularly preferably used. At this time, it is preferable that the pH electrode (A) 6 and the pH electrode (B) 7 have substantially the same pH sensitivity, temperature characteristics, and drift characteristics. As a result, it is not necessary to individually set calibration parameters (pH sensitivity, temperature correction coefficient, drift correction coefficient, etc.) for each of the pH electrode (A) 6 and the pH electrode (B) 7. It can be simplified.

  As the comparison electrode 9, a conventional saturated electrode, a liquid junction type comparison electrode having a silver / silver chloride electrode or the like as an internal electrode, or the like is used. At this time, it is possible to use a conventional liquid junction type reference electrode in which a container having a liquid junction part is filled with an internal solution containing a constant concentration of chlorine ions, but the baseline solution always has a constant concentration of chlorine ions. Can be used, a bare silver / silver chloride electrode disposed directly in the flow path of the baseline solution can be used, thereby simplifying the apparatus. The location of the comparison electrode 9 may be anywhere as long as it can be in liquid junction with the pH electrode (A) 6 and the pH electrode (B) 7. However, it is preferable that the comparison electrode 9 is disposed at a position upstream of the pH electrode (A) 6 and the pH electrode (B) 7 in the flow path of the baseline solution. This is because the periphery of the reference electrode 9 is always filled with a fresh baseline, and contamination of the liquid junction by the sample is avoided. In the example of FIG. 1, the inside of the front chamber 2 that is always filled with a fresh baseline is suitable as a place for installing the reference electrode 9.

  Furthermore, it is also preferable that the reference electrode is composed of a combination of a reference pH electrode and a pseudo comparison electrode. FIG. 2 shows a composite sensor portion of an example of the pH measuring device in this case. In the example of FIG. 2, a reference pH electrode 12 and a pseudo comparison electrode 13 are arranged inside the anterior chamber 2 instead of the comparison electrode 9 in FIG. 1. In this case, the potential difference (Va) between the pH electrode (A) 6 and the pseudo comparison electrode 13, the potential difference (Vb) between the pH electrode (B) 7 and the pseudo comparison electrode 13, the reference pH electrode 12 and the pseudo comparison electrode 13 The potential difference (Vr) is measured. At this time, Va-Vr corresponds to the potential difference between the pH electrode (A) 6 and the comparison electrode 9 in the example of FIG. 1, and Vb-Vr is the pH electrode (B) 7 in the example of FIG. Therefore, the acidification of the baseline solution can be calculated in the manner described above. Thus, by reading the differential outputs of the reference pH electrode 12, the pH electrode (A) 6 and the pH electrode (B) 7, noise other than the acidification of the baseline solution, such as induction noise, temperature noise, and the like. It is possible to perform measurement with higher accuracy by canceling out flow noise and the like. Such a method of differential pH measurement is disclosed in detail in Japanese Patent Laid-Open No. 2001-289811 (Patent Document 1).

  In particular, when an ISFET is used as the pH electrode (A) 6 and the pH electrode (B) 7, it is preferable to use the ISFET that is the reference pH electrode 12 and the pseudo comparison electrode 13 instead of the comparison electrode 9. In this case, as the ISFET for the reference pH electrode 12, use one having substantially the same pH sensitivity, temperature characteristics, and drift characteristics as the ISFET used as the pH electrode (A) 6 and the pH electrode (B) 7. Is desirable. As the pseudo comparison electrode 13, a noble metal electrode such as platinum, silver, or gold, a carbon electrode, or the like can be used.

  As the gas permeable membrane, a homogeneous membrane excellent in gas permeability such as silicone rubber or a porous membrane made of a hydrophobic resin such as polytetrafluoroethylene or polypropylene is suitable. Although the tube 8 is illustrated as a gas permeable film in FIG. 1, the form is not specifically limited. A gas permeable membrane is disposed between the pH electrode (A) 6 and the measurement object, and no gas permeable film is disposed between the pH electrode (B) 7 and the measurement object. Among acids or alkalis generated from the measurement object, The arrangement may be such that only the gaseous acid or alkali selectively reaches the pH electrode (A) 6. For example, in FIG. 1, instead of arranging the tube 8 surrounding the pH electrode (A) 6, the pH electrode (A) 6 and the pH electrode (B) 7 may be partitioned by a flat membrane-shaped gas permeable membrane. . Further, a flow path for storing the pH electrode (A) 6 may be provided, and a flat film-shaped gas permeable film may be attached thereon.

  It is desirable that the path length from the pH electrode (A) 6 to the pH electrode (B) 7 is sufficiently larger than the distance from the gas permeable membrane to the pH electrode (A) 6. By doing so, the liquid acid or alkali is transferred to the pH electrode until the gaseous acid or alkali reaches the pH electrode (A) 6 through the gas permeable membrane within the stop time of the baseline solution. (B) Only 7 can be reached, and the influence thereof does not reach the pH electrode (A) 6. This is important for separating and quantifying a gaseous acid or alkali and a liquid acid or alkali. Specifically, the path length from the pH electrode (A) 6 to the pH electrode (B) 7 is preferably at least 5 times the distance from the gas permeable membrane to the pH electrode (A) 6, and more than 10 times. More preferably, it is more preferably 15 times or more. In the example of FIG. 1, the path length includes the distance from the pH electrode (B) 7 to the pH electrode (A) 6 via the upper end 14 of the tube 8 and the lower end of the tube 8 from the pH electrode (B) 7. This is the shorter distance of the distance to reach the pH electrode (A) 6 via 15. At this time, the distance from the gas permeable membrane to the pH electrode (A) 6 is about half of the inner diameter of the tube 8.

  As shown in the example of FIG. 1, it is preferable to include a measurement chamber 3 in which a pH electrode (A) 6 and a pH electrode (B) 7 are built. Since it is also possible to measure by suspending or suspending the sample to be measured in the measurement chamber 3, it is possible to deal with various samples such as biological tissue pieces and microorganisms. Moreover, it is also preferable that the measurement chamber 3 has the measurement window 10 which the sample which is a measuring object contacts. In this case, since the measurement can be performed simply by bringing the sample 11 into contact with the measurement window 10, it is also possible to measure a secretion from the body by bringing a part of the body into contact therewith. Since the amount of secretion from the living tissue is usually a very small amount, it is preferable that the pH fluctuates sufficiently with a small amount of acid or alkali. In order to increase the response speed and shorten the measurement time, The capacity of 3 is preferably smaller. Specifically, the volume is preferably 1000 μL or less, and more preferably 100 μL or less. On the other hand, in the case where a plurality of electrodes are formed on a single semiconductor substrate, the lower limit value of the capacity of the measurement chamber 3 can be set to about 0.1 μL. If so, it is usually 1 μL or more. Moreover, the one where the area of the measurement window 10 is wide is preferable because the measurement time can be shortened. As shown in the example of FIG. 1, the front chamber 2 is preferably provided upstream of the measurement chamber 3 in which the pH electrode (A) 6 and the pH electrode (B) 7 are incorporated, It is preferable to arrange the reference electrode 9.

  The embodiment shown in FIG. 3 does not have the measurement chamber 3 and is surrounded by a tube 8 made of a gas permeable membrane and a pH electrode (B) disposed outside the tube 8. ) 7 is surrounded by a cylindrical sample 11 and measured. This is suitable not only when using a sample that is a tubular body such as a cut blood vessel, but also when using a sample in which cells are adhered and cultured on the inner wall of the tubular body.

  In the embodiment shown in FIG. 4, the measurement chamber 3 is formed inside the sensor body 1 consisting of an elongated annular body as a whole, and two measurement windows 16 and 17 are provided in the same direction in the longitudinal direction. In the vicinity of the measurement window 16, a rubber tube 8 made of a gas permeable film covers the sensor body 1, and only the measurement window 16 is covered with the gas permeable film. A pH electrode (A) 6 is arranged inside the measurement window 16, and a pH electrode (B) 7 is arranged inside the measurement window 17, and the sample 11 to be measured is placed in both measurement windows 16. , 17 in contact with the measurement. The baseline solution is introduced from the inlet 4 and discharged from the outlet 5. The comparison electrode 9 is preferably arranged upstream of the pH electrode (A) 6 and the pH electrode (B) 7, but the positional relationship between the pH electrode (A) 6 and the pH electrode (B) 7. Any of these may be arranged on the upstream side. At this time, if the path length from the pH electrode (A) 6 to the pH electrode (B) 7 is sufficiently larger than the distance from the gas permeable membrane to the pH electrode (A) 6 as described above, the distance between both electrodes Even without the gas permeable membrane, the gaseous acid or alkali and the liquid acid or alkali can be separated and quantified. In the embodiment shown in FIG. 4, it is easy to reduce the capacity in the measurement chamber, which is also preferable from this point. In particular, when a part of the body is contacted and secretions from the body are measured, measurement in a short time is easy and this is a preferred embodiment.

  Further, when both the pH electrode (A) 6 and the pH electrode (B) 7 are made of semiconductor sensors such as ISFET and LAPS, it is also preferable that these are formed on a single semiconductor substrate. By doing so, it is possible to manufacture a very small pH measuring apparatus with high productivity. At this time, the comparison electrode 9, the reference pH electrode 12, or the pseudo comparison electrode 13 is also preferably formed on a single semiconductor substrate. It is possible to form these various electrodes on a single semiconductor substrate and provide wiring by adopting a normal semiconductor manufacturing process. The channels are formed so that these electrodes are disposed within the baseline solution channels. As a method for forming the flow path, for example, a method in which glass obtained by etching the flow path pattern is stacked and adhered to the semiconductor substrate is employed. As a method of disposing a gas permeable membrane between the pH electrode (A) 6 and the measurement target, a method of covering the pH electrode (A) 6 with a gas permeable membrane, or a method of arranging the gas permeable membrane in the vicinity of the pH electrode (A) 6 Various methods described above, such as a method of covering the measurement window with a gas permeable membrane, can be employed.

  The baseline solution used in the pH measuring apparatus of the present invention is released from the sample after the supply of the baseline solution is stopped while the periphery of the electrode is kept at the pH of the baseline solution during the delivery. The pH is required to change as much as possible depending on the acid or alkali. For this purpose, it is inconvenient if the pH buffering capacity of the baseline solution is too strong or too weak, and a buffering capacity of 10 to 500 μM / pH is preferable. When the pH buffering capacity is too strong, there is a possibility that the measurement sensitivity becomes dull, preferably 500 μM / pH or less, more preferably 250 μM / pH or less. In particular, when sensitivity is important, it is more preferably 100 μM / pH or less. Most commercially available buffers exceed 1000 μM / pH, and it is preferable to use a baseline solution having a weak buffer capacity. This is because in the pH measurement device of the present invention, since a small amount of acid or alkali is often quantified, sensitivity is regarded as important. On the other hand, if the pH buffering capacity is too weak, the baseline of the measured potential may fluctuate, preferably 10 μM / pH or more, more preferably 13 μM / pH or more.

  The baseline solution may contain conjugated ions for the gaseous acid or alkali to be measured. For example, hydrogen carbonate ions may be contained when the measurement object contains carbon dioxide gas, and ammonium ions may be contained when ammonia is contained. By doing so, the sensitivity to gaseous acids and alkalis can be improved (the principle of the Severinghaus gas sensor) in some cases. However, in this case, the lower limit concentration that can be measured often increases, which may not be preferable when the amount of acid or alkali generated from the measurement target is small. Further, when the sample requires nutrients or oxygen such as a cell, a cut organ, or the surface of a living body, it is preferable to contain a nutrient such as glucose and oxygen in the baseline solution.

  The means for supplying the baseline solution is not particularly limited, and various pumps can be used. In order to stop the baseline solution, a method of closing the valve of the flow path or stopping the operation of the pump is employed. After supplying the baseline solution for a predetermined time and replacing the periphery of the electrode with the baseline solution, the feeding is stopped and measurement of the pH change is started. Considering the stability of the detected potential, the time for replacement with the baseline solution is preferably 1 second or more, and more preferably 10 seconds or more. On the other hand, in order to shorten the time required for all measurements, the time for replacement is preferably 10 minutes or less, and more preferably 3 minutes or less. In addition, the time for measuring the change in pH after stopping the liquid feeding varies depending on the measurement object and the measuring device, but the acid that has passed through the gas permeable membrane in the baseline solution around the pH electrode (A). Or it is necessary to set it as time until an alkali can reach | attain. In order to improve the measurement accuracy, it is preferable to measure until the final equilibrium concentration reaches a concentration that can be estimated. The measurement time is preferably 1 second or longer, and more preferably 10 seconds or longer. On the other hand, if the measurement time is too long, particularly when the measurement is performed with a part of the body in contact, the burden on the measurement subject increases, which is not preferable. Therefore, it is preferably 20 minutes or less, and more preferably 5 minutes or less. The pH measuring device of the present invention can measure in a relatively short time. The supply and stop timing of the baseline solution, the processing of acquired data from the electrodes, and the like can be automatically performed using a computer.

The acid or alkali to be measured by the pH measuring device of the present invention is not particularly limited as long as it is an acid or alkali that can be dissolved in water. Depending on the permeability of the gas permeable membrane, it can be divided into those detected only by the pH electrode (B) and those detected by both the pH electrode (A) and the pH electrode (B). The acid and alkali detected by both the pH electrode (A) and the pH electrode (B) have a low molecular weight and insufficient dissociation into ions. Specifically, the compound has a molecular weight of 70 or less and an acid dissociation constant (Ka) or base dissociation constant (Kb) at 25 ° C. of 10 ⁻⁶ or less. Typically, carbon dioxide, ammonia, hydrogen sulfide and the like. In addition, methylamine, methyl mercaptan, formic acid, acetic acid, and the like are targeted depending on the configuration of the apparatus and measurement conditions. On the other hand, the acid and alkali detected only by the pH electrode (B) have a high molecular weight or are sufficiently dissociated into ions. Specifically, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium bicarbonate, lactic acid, pyruvic acid, fatty acid having 3 or more carbon atoms, and the like. Among these, in order to examine the state of energy metabolism of cells, it is particularly preferable to quantify lactic acid and carbon dioxide as metabolites.

  Examples of the sample to be measured by the apparatus of the present invention include one that elutes or secretes both a gaseous acid or alkali and a liquid acid or alkali. The acid or alkali secreted from the living tissue of a living organism may be directly quantified, or the acid or alkali secreted from the living tissue collected from the organism may be quantified. Specific examples of the form of the sample include cells, microorganisms, living tissues, and cut organs. When the sample is a cell or a microorganism, they may be suspended in the measurement chamber, or may be adhered to the flat plate surface and adhered to the measurement window to form a sample. When the measurement object is a living tissue, for example, human epidermis, it is appropriate to bring the epidermis to the sample and attach it to the measurement window.

  Although the purpose of the measurement is not particularly limited, it is suitable for quantifying the amount of acid or alkali secreted from a biological sample, etc., and is in the bio-industrial field such as medicine, physiology, cell biology, and also fermentation, brewing, culture, etc. Is preferably used. Quantifying acid or alkali secreted from human skin is a preferred embodiment of the present invention. In this case, it is possible to easily check the energy metabolism of epidermal cells without damaging the body. It is suitably used not only as a device for diagnosing skin diseases such as metabolic abnormalities but also as a device for judging skin conditions for cosmetic purposes. In particular, in the case of cosmetic purposes, since it is extremely important to be able to measure easily in a short time, the apparatus of the present invention is preferably used.

Example 1
The test was conducted using the pH measuring apparatus described in FIG. Each of the pH electrode (A) 6 and the pH electrode (B) 7 is an ISFET having a width of 0.45 mm, a thickness of 0.2 mm, and an exposed portion length of 1.5 mm. The reference electrode 9 is a silver wire having a diameter of 0.5 mm and a length of 3 mm, coated with silver chloride (silver / silver chloride wire). The tube 8 is a silicone tube having an inner diameter of 0.5 mm, an outer diameter of 0.54 mm, and a length of 15 mm. The path length from the pH electrode (A) 6 to the pH electrode (B) 7 is 15 mm, and the distance from the inner wall of the silicone tube to the pH electrode (A) 6 is about 0.2 mm.

As the sample 11, a human umbilical artery endothelial cell adhered and cultured on the inner wall of a glass tube having an inner diameter of 3 mm and a length of 50 mm was used. As a baseline solution, 2.7 g of D-glucose, 8.18 g of sodium chloride, 0.38 g of potassium chloride, 0.186 g of magnesium chloride hexahydrate in 1 L of solution, N- (2-hydroxyethylpiperazine) -N ′ -After dissolving 0.024 g of (2-ethanesulfonic acid), a solution having a pH adjusted to 7.4 by adding a small amount of sodium hydrogen carbonate was used. The buffer capacity β of this baseline solution was 200 μM / pH. That is, when hydrochloric acid was added to this baseline solution to 200 μM, the pH became 6.4. After the glass tube was sufficiently replaced with the baseline solution, the feeding of the baseline solution was stopped. FIG. 5 shows changes in ΔpH _T and ΔpH _G for 10 minutes after stopping the liquid feeding. At 10 minutes after stopping the feeding, ΔpH _T was −0.376, and ΔpH _G was −0.225.

From this, it acidified A _L by a gaseous acid acidification with (carbon dioxide) A _G and the liquid of the acid (mainly lactic acid),
A _G = ΔpH _G = −0.225
A _L = ΔpH _T −ΔpH _G = −0.376 − (− 0.225) = − 0.151
It becomes. From now on, the secreted concentrations C _G and C _L of gaseous acid and liquid acid are
C _G = β | A _G | = 45 μM
C _L = β | A _L | = 30 μM
Met. That is, in this cell, the secretory concentration of gaseous acid (carbon dioxide) in 10 minutes was 45 μM in terms of hydrochloric acid, and that of liquid acid was 30 μM. The secretory concentration calculated here is a hydrochloric acid equivalent value to the last. The carbon dioxide gas having a hydrochloric acid conversion value of 45 μM means “the concentration of carbon dioxide gas having the same effect as that of 45 μM hydrochloric acid with respect to fluctuations in pH”. Since carbon dioxide gas is much weaker than hydrochloric acid, the concentration of carbon dioxide gas having the same effect as 45 μM hydrochloric acid is considerably higher than 45 μM.

Example 2
The test was performed using the pH measuring apparatus described in FIG. The internal volume of the measurement chamber 3 is about 50 μL. Each of the pH electrode (A) 6 and the pH electrode (B) 7 is an ISFET having a width of 0.45 mm, a thickness of 0.2 mm, and an exposed portion length of 1.5 mm. The comparison electrode 9 has a surface of a silver wire having a diameter of 0.5 mm and a length of 3 mm coated with silver chloride. The tube 8 is a silicone tube having an inner diameter of 0.5 mm, an outer diameter of 0.54 mm, and a length of 15 mm. The path length from the pH electrode (A) 6 to the pH electrode (B) 7 is 15 mm, and the distance from the inner wall of the silicone tube to the pH electrode (A) 6 is about 0.2 mm.

The sensor window of FIG. 1 was brought into close contact with the outer arm of the healthy person, and the same baseline solution as in Example 1 was circulated for 1 minute at a flow rate of 3 mL / min, and then the liquid supply was stopped. The time-dependent changes in ΔpH _T and ΔpH _G for 10 minutes after stopping the liquid feeding were shown. ΔpH _T and ΔpH _G at 10 minutes after stopping the liquid feeding were −1.519 and −0.720, respectively. In the same manner from Example 1 This, when obtaining the C _G and C _L, were as follows.
C _G = 144 μM
C _L = 160 μM
That is, the secretory concentration of gaseous acid (carbon dioxide) from the epidermis over 10 minutes was 144 μM in terms of hydrochloric acid, and that of liquid acid (mainly lactic acid) was 160 μM.

Example 3
The test was conducted using a small-tube type pH measuring device described in FIG. The measurement chamber 3 is a polytetrafluoroethylene pipe having an inner diameter of 1.5 mm and a length of 50 mm, and its internal volume is about 50 μL. Each of the pH electrode (A) 6 and the pH electrode (B) 7 is an ISFET having a width of 0.45 mm, a thickness of 0.2 mm, and an exposed portion length of 1.5 mm. The comparison electrode 9 has a surface of a silver wire having a diameter of 0.5 mm and a length of 3 mm coated with silver chloride. The tube 8 is a silicone tube having an inner diameter of 0.5 mm, an outer diameter of 0.54 mm, and a length of 15 mm. The distance from the pH electrode (A) 6 to the pH electrode (B) 7 is 15 mm, and the distance from the inner wall of the silicone tube to the pH electrode (A) 6 is about 0.1 mm.

The fine tube type sensor shown in FIG. 4 is brought into close contact with the palm of a healthy person, and physiological saline (buffering capacity β is 20 μM / pH) is circulated for 1 minute at a flow rate of 0.5 mL / min. Stopped. FIG. 7 shows changes over time in ΔpH _T and ΔpH _G for 5 minutes after stopping the liquid feeding. ΔpH _T and ΔpH _G at 5 minutes after stopping the feeding were −1.451 and −0.714, respectively. In the same manner from Example 1 This, when obtaining the C _G and C _L, were as follows.
C _G = 14 μM
C _L = 15 μM
That is, the secretory concentration of gaseous acid (carbon dioxide) from the epidermis in 10 minutes was 14 μM in terms of hydrochloric acid, and that of liquid acid (mainly lactic acid) was 15 μM.

C _G and C _L in any of Examples 1 to 3 are of the same order, secretion acid is clearly to be composed of both humoral and gaseous.

It is a schematic diagram of the composite sensor part of an example of the pH measuring apparatus of this invention which has a measurement chamber and a measurement window. It is a schematic diagram of the composite sensor part of an example of the pH measuring apparatus of this invention in which a reference electrode is comprised from the combination of a reference pH electrode and a pseudo-reference electrode. It is a schematic diagram of the composite sensor part of an example of the pH measuring apparatus of this invention which does not have a measurement chamber. It is a schematic diagram of the composite sensor part of an example of the pH measuring apparatus of the present invention, wherein the entire measurement chamber is formed of an elongated annular body. In Example 1, it is a graph showing changes in delta pH _T and delta pH _G liquid supply stop after 10 minutes. In Example 2, a graph showing changes in delta pH _T and delta pH _G liquid supply stop after 10 minutes. In Example 3, it is a graph showing changes in delta pH _T and delta pH _G liquid supply stop after 5 minutes.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sensor body 2 Front chamber 3 Measurement chamber 4 Baseline solution inflow port 5 Baseline solution outflow port 6 pH electrode (A)
7 pH electrode (B)
8 Tube 9 Reference electrode 10, 16, 17 Measurement window 11 Sample 12 Reference pH electrode 13 Pseudo comparison electrode 14 Upper end of tube 15 Lower end of tube

Claims (12)

A pH measuring device comprising a plurality of pH electrodes and means for supplying and stopping a baseline solution;
A pH electrode (A) and a pH electrode (B) are disposed in the flow path of the baseline solution,
The comparative electrode is arranged at a position where the liquid electrode can be connected to the pH electrode (A) and the pH electrode (B),
A gas permeable membrane is disposed between the pH electrode (A) and the measurement target,
A gas permeable membrane is not arranged between the pH electrode (B) and the measurement target,
While the supply of the baseline solution is stopped, the pH electrode (A) changes the pH of the baseline solution derived from the acid or alkali passing through the gas permeable membrane, and the pH electrode (B) A pH measurement apparatus comprising means for simultaneously measuring pH changes of the baseline solution derived from an acid or an alkali. The pH measuring device according to claim 1, wherein the pH electrode (A) is covered with a tube made of a gas permeable membrane, and the baseline solution flows through the tube. 3. A measurement chamber in which a pH electrode (A) and a pH electrode (B) are incorporated, and the measurement chamber has an inlet and an outlet for a baseline solution, and a measurement window in contact with a measurement object. PH measuring device. The measurement chamber has two measurement windows, one of the measurement windows is covered with a gas permeable membrane and a pH electrode (A) is arranged inside thereof, and the other measurement window is not covered with a gas permeable membrane, The pH measuring device according to claim 3, wherein a pH electrode (B) is disposed inside. The pH measuring device according to claim 1, wherein both the pH electrode (A) and the pH electrode (B) are formed on a single semiconductor substrate. The pH measuring device according to any one of claims 1 to 5, wherein a path length from the pH electrode (A) to the pH electrode (B) is at least five times a distance from the gas permeable membrane to the pH electrode (A). The pH measuring device according to claim 1, wherein a reference electrode is disposed upstream of the pH electrode (A) and the pH electrode (B) in the flow path of the baseline solution. 8. The pH measuring device according to claim 7, wherein the reference electrode is composed of a combination of a reference pH electrode and a pseudo-reference electrode. The pH measuring device according to any one of claims 1 to 8, wherein the pH buffering capacity of the baseline solution is 10 to 500 µM / pH. The pH electrode (A) and the pH electrode (B) are arranged in the flow path of the baseline solution, the comparison electrode is arranged at a position where the pH electrode (A) and the pH electrode (B) can be in liquid junction, and the pH electrode ( A) A base solution having the same composition is circulated around and replaced around the pH electrode (B), and then the base solution is stopped from flowing. The pH electrode (A) passes through the gas permeable membrane generated from the measurement target. A pH measurement method characterized by simultaneously measuring pH changes derived from an acid or an alkali, and pH changes derived from all acids or alkalis generated from a measurement object at the pH electrode (B). The secretion quantification method which quantifies the acid or alkali secreted from a biological tissue with the pH measurement method of Claim 10. The method for quantifying a secretion according to claim 11, wherein the amount of carbon dioxide and the amount of lactic acid are each quantified.

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