JP2005127974A - Apparatus and method for measuring hydrogen ion concentration - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately and quickly measure hydrogen ion concentrations of a plurality of specimens. <P>SOLUTION: A nozzle 2, a measuring chamber 3, a flow path 4, a reference chamber 5 and a flow path 8 are sequentially linked, and totally form one liquid flow path. A baseline solution accommodated in a vessel 11 is supplied from the flow path 8 to the liquid flow path by a pump 9. The liquid in the liquid flow path is sucked by the pump 9. A measuring hydrogen ion sensor 1 is disposed in the measuring chamber 3. A pseudocomparison electrode 7 and a reference hydrogen ion sensor 6 are disposed in the reference chamber 5. The measuring hydrogen ion sensor and the reference hydrogen ion sensor are pH sensitive ISFETs. A tantalum pentoxide membrane is formed as a pH sensitive membrane on a gate of the pH sensitive ISFET by a sputtering method. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a hydrogen ion concentration measuring apparatus and measuring method used in the medical or chemical measurement field.

The ion concentration in the solution is usually measured with an ion electrode. An ion electrode has an electrode potential that changes according to a specific ion concentration in a solution, and electrodes for various ions such as H ⁺ , Na ⁺ , K ⁺ , and Ca ²⁺ are known. In order to measure the ion concentration with these ion electrodes, it is necessary to use a comparison electrode whose potential does not vary depending on the ion concentration in the solution, and to measure the potential of the ion electrode with respect to the comparison electrode.

  As a comparative electrode often used, for example, a silver / silver chloride electrode is known. This reference electrode utilizes the fact that the potential of silver / silver chloride shows a constant value if the chlorine ion concentration in the solution is constant. In order to keep the potential of these comparison electrodes constant, It is necessary to immerse silver chloride in a solution of chlorine ions having a constant concentration, for example, saturated KCl solution.

  Therefore, this reference electrode usually takes the form of a liquid junction type reference electrode. In this case, a container having a liquid junction is filled with a solution (internal solution) of a certain concentration of chlorine ions, and a silver wire whose surface is chlorinated is immersed therein.

  With such a liquid junction type reference electrode, chlorine ions in the internal solution leak into the measurement solution from the liquid junction with use, and the concentration of chlorine ions in the internal solution changes.

  Also, the internal solution itself is reduced. Therefore, it is necessary to always replenish a new internal solution, and maintenance is troublesome in this sense, and this is the first problem in the conventional ion concentration measurement technique.

A second problem in the measurement of ion concentration with an ion electrode is that calibration is necessary. The zero point and sensitivity of the ion electrode vary over time. Therefore, prior to the measurement of ion concentration, it is necessary to perform zero point calibration and sensitivity calibration using a standard solution of ions.
When the fluctuation of the sensitivity of the ion electrode is small and the change of the zero point is large, only the zero point calibration is performed. Zero point calibration is performed using one standard solution with a known ion concentration. When both the zero point and the sensitivity change, calibration of both the zero point and the sensitivity is performed using two types of standard solutions having different ion concentrations.

  The third problem in the measurement of ion concentration with an ion electrode is that when measuring the ion concentration of a large number of samples, the measurement of one sample is completed, and the electrode is washed with pure water before moving to the next sample measurement. Furthermore, it is necessary to wipe off the pure water adhering to the electrode. If this operation is neglected, the subsequent sample is contaminated by the previous sample, and accurate measurement is hindered.

As described above, there are the following three problems in the measurement of ion concentration using a conventional ion sensor.
1) A liquid junction reference electrode, which is troublesome to maintain, was used as the reference electrode.
2) Prior to the measurement of ion concentration, calibration of the zero point and sensitivity was necessary.
3) When measuring a plurality of specimens, it was necessary to clean the ion sensor or wipe off the cleaning liquid.

  Therefore, an ion sensor for measurement is arranged on one end side of one flow path and a reference ion sensor is arranged on the other end side, and a substrate solution is supplied to the flow path from the other end side so as to overflow from the one end side. The flow path is washed, and then the sample solution is sucked into the flow path from the one end side to move the liquid in the flow path toward the other end side, and the ion sensor for measurement is in the sample solution However, the reference ion sensor is still in the substrate solution, and the ion concentration of the sample solution is measured from the outputs of the measurement and reference ion sensors at that time (see, for example, Patent Document 1).

However, the ion sensor used in such a conventional apparatus is selected as having no relationship between the strength and weakness of the pH buffering ability of the sample solution and further having no relationship between the response and the pH measurement accuracy. It was. For this reason, conventionally, it takes time to measure a sample solution having a weak pH buffering capacity, and the measurement result is inaccurate.
Further, in the conventional apparatus, when a large number of sample solutions are measured, since the operator carries and injects each sample to the sample intake port, it takes a long time for the measurement.
JP-A-10-332634 (pages 4-5, especially paragraph 0027, FIG. 1)

  As described above, conventionally, it takes a long time to measure a large number of samples, and the measurement result is inaccurate for a sample solution having a weak pH buffering ability. The present invention has been made in order to solve such a conventional problem, and an object of the present invention is to measure a large number of specimens in a short time and to accurately detect hydrogen regardless of the strength of pH buffering ability. It is to be able to measure the ion concentration.

  The invention of claim 1 is formed by connecting the nozzle portion, the first storage chamber, the first flow path, the second storage chamber, and the second flow path in this order, and one liquid flow path as a whole. A liquid channel part formed with the liquid channel part connected to the second channel and supplying a liquid from the second channel into the liquid channel part or from the nozzle part into the liquid channel part A pump capable of both sucking a liquid, a measurement hydrogen ion sensor disposed in the first storage chamber for detecting hydrogen ions in the liquid, and a pseudo comparison electrode disposed in the second storage chamber And a reference hydrogen ion sensor disposed in the second storage chamber for detecting hydrogen ions in a liquid, wherein the measurement hydrogen ion sensor and the reference hydrogen ion sensor are: A pH sensitive ISFET, As pH sensitive membrane on the gate of the H-sensitive ISFET, 5 tantalum oxide film is characterized in that it is formed by sputtering.

  Invention of Claim 2 is a hydrogen ion concentration measuring method using the measuring apparatus of Claim 1, Comprising: By the said pump, the liquid with known hydrogen ion concentration is guide | induced into the said liquid flow-path part, At least the said The first storage chamber and the second storage chamber contain a liquid with a known hydrogen ion concentration, and the outputs of the measurement hydrogen ion sensor, the reference hydrogen ion sensor, and the pseudo comparison electrode at that time A calibration value is obtained based on the first step, and then the sample solution is guided into the liquid flow path by the pump, the sample solution is accommodated in the first accommodation chamber, and the second The storage chamber is in a state in which the liquid having a known hydrogen ion concentration is stored, and based on the output of the measurement hydrogen ion sensor, the reference hydrogen ion sensor and the pseudo comparison electrode and the calibration value at that time And having a measuring a hydrogen ion concentration of the second liquid.

  The invention of claim 3 is the hydrogen ion concentration measurement method according to claim 2, wherein the liquid having a known hydrogen ion concentration has a β value in the range of 0.04 to 0.13 mmole / L / pH. It is characterized by.

  According to a fourth aspect of the present invention, the nozzle portion, the first storage chamber, the first flow path, the second storage chamber, and the second flow path are connected in this order, and one liquid flow path is formed as a whole. A liquid flow path section formed with the liquid flow path section connected to the second flow path and supplying a liquid from the second flow path into the liquid flow path section, or from the nozzle section into the liquid flow path section. A pump capable of sucking a liquid into the first chamber, a measurement hydrogen ion sensor disposed in the first storage chamber for detecting hydrogen ions in the liquid, and a pseudo comparison disposed in the second storage chamber In the hydrogen ion concentration measuring apparatus, comprising: an electrode; and a reference hydrogen ion sensor that is disposed in the second storage chamber and detects hydrogen ions in the liquid, the liquid channel portion, and the measurement hydrogen ion sensor , The reference ion sensor and the pseudo-reference electrode A probe moving means that is integrated with a probe, includes a sample solution storage member having a plurality of sample solution storage units for storing a sample solution, and moves the probe with the nozzle portion facing downward; And / or a sample solution containing member moving means for moving the sample solution containing member.

  According to a fifth aspect of the present invention, in the apparatus according to the fourth aspect, the measurement hydrogen ion sensor and the reference hydrogen ion sensor are pH-sensitive ISFETs, and a pH-sensitive ISFET is provided on a gate of the pH-sensitive ISFET. As the film, a tantalum pentoxide film is formed by a sputtering method.

  According to the invention of claim 1, the hydrogen ion sensor for measurement and the hydrogen ion sensor for reference have a high-speed response to a solution having a strong pH buffer capacity and a solution having a weak pH buffer capacity, and Since the accuracy is high, the hydrogen ion concentration can be measured at high speed and with high accuracy regardless of the strength of the pH buffering ability of the solution to be measured.

  According to the second aspect of the invention, since the measurement work is simple, the automation of the apparatus is facilitated, which is effective for labor saving. This makes it easier to measure a large number of specimens at high speed.

  According to the invention of claim 3, the hydrogen ion concentration can be measured with higher accuracy in a shorter time.

  According to the fourth aspect of the present invention, the entire liquid flow path portion provided with the two hydrogen ion sensors and the pseudo comparison electrode is used as a probe, and this moves and / or the sample solution storage member moves. Since the sample solution can be sucked directly from the nozzle of the probe, the flow path of the sample solution can be shortened. For this reason, the volume of the sample required for the measurement can be reduced, and a large number of samples can be measured at high speed.

  According to the invention of claim 5, it is possible to perform a higher speed measurement.

FIG. 1 shows the basic configuration of the apparatus of the present invention. In FIG. 1, a nozzle 2 is provided in a measurement chamber 1 (first storage chamber). A measurement hydrogen ion sensor 3 is accommodated in the measurement chamber 1. The measurement chamber 1 communicates with the reference chamber 5 by a flow path 4 (first flow path), and a reference hydrogen ion sensor 6 and a pseudo comparison electrode 7 are accommodated in the reference chamber 5 (second storage room). ing. The reference chamber 5 is communicated with a pump 9 by a flow path 8 (second flow path). The pump 9 receives a baseline solution from a container 11 containing a baseline solution having a known hydrogen ion concentration via a flow path 10. It has come to be given.
A liquid flow path portion is formed from the nozzle 2, the measurement chamber 1, the flow path 4, the reference chamber 5 and the flow path 8.

The steps of measuring the ion concentration by the hydrogen ion concentration measuring apparatus shown in FIG. 1 are as follows.
1) Zero point calibration process By sending the baseline solution in the container 11 to the channel 8 by the pump 9, the entire channel of the liquid channel part is replaced with the baseline solution. Drain the line solution. When at least the reference chamber 5 and the measurement chamber 1 are filled with the baseline solution, the output Vm0 of the measurement hydrogen ion sensor 3 and the output Vr0 of the reference hydrogen ion sensor 6 at that time are measured, and the difference ΔV0 is obtained. . That is,
ΔV0 = Vm0−Vr0 (1)
Ask for. In this example, obtaining this ΔV0 (referred to as a calibration value) used for calibration later is a zero point calibration process.
The output of the measurement hydrogen ion sensor 3 and the output of the reference hydrogen ion sensor 6 are based on the output of the pseudo comparison electrode 7.

2) Sample Aspiration and Measurement Step Next, the pump 9 is stopped, the tip of the nozzle 2 is put into the sample solution, and the pump 9 is driven in reverse to aspirate the sample solution into the measurement chamber 1. The pump 9 is stopped so as not to flow. At this time, the output Vm1 of the measurement hydrogen ion sensor and the output Vr1 of the reference hydrogen ion sensor are measured, and the difference ΔV1 is obtained. That is,
ΔV1 = Vm1−Vr1 (2)
Ask for.
Since the outputs of the hydrogen ion sensors 3 and 6 are proportional to the logarithm of the hydrogen ion concentration (activity) as in the case of a normal ion electrode, if the ion concentration (activity) of the baseline solution is C0 mol / L, the sample solution The hydrogen ion concentration (activity) C1 mol / L is obtained from the following equation.
logC1 / logC0 = ΔΔV / S (3)
Here, ΔΔV is the difference between the differential outputs ΔV1 and ΔV0.
ΔΔV = ΔV1 -ΔV0 (4)
S is the sensitivity of the hydrogen ion sensor. When the hydrogen ion sensor is a normal ion electrode, S roughly matches the value obtained by the following Nernst equation.
S = RT / nF (5)
Here, R is a gas constant, T is an absolute temperature, n is a valence of an ion to be measured, and F is a Faraday constant. The theoretical value of S is about 60 mV / decade at 25 ° C. with monovalent ions.

  Assuming that S is known and does not change over time, C1 can be obtained from equation (3) by actually measuring ΔΔV. Further, by repeating the steps 1) and 2), it is possible to measure the pH of a large number of specimens.

In the above example, zero point calibration was performed based on the ion concentration of the baseline solution, but one or more ion concentration reference solutions were put as specimens, and the following calibration curve was obtained from ΔΔV for them. It is also possible to calculate the ion concentration.
logC1 = A + ΔΔV / S (6)
Here, A is a constant. When S is known, A is obtained by using one kind of solution as the ion concentration reference solution (one-point calibration). When both S and A are unknown, they are obtained using a two-concentration ion concentration reference solution (two-point calibration). Furthermore, it is also possible to obtain S and A by the least square method using an ion concentration reference solution having two or more concentrations.

  The baseline solution used in the present invention is a solution containing a known concentration of ions to be measured. As the concentration of hydrogen ions in the baseline solution, it is desirable to select a concentration close to the concentration range of hydrogen ions in the specimen. It is also important that the pH buffering capacity of the baseline solution is appropriate. If the pH buffering capacity of the baseline solution is too weak, it takes a long time for the measurement chamber pH to return to the baseline solution pH when the sample in the measurement chamber is pushed out with the baseline solution in the zero point calibration process. It becomes like this. On the other hand, if the pH buffering capacity of the baseline solution is too strong, when the sample solution is aspirated into the measurement chamber in the measurement process, the measured pH value is affected by the trace amount of the baseline solution remaining in the measurement chamber. An error occurs that is biased toward the pH of the solution. Therefore, as the baseline solution, it is necessary to use a solution having a pH buffering ability that allows zero-point calibration promptly and does not affect the pH of the specimen in the measurement process. In general, the β value (molar concentration of hydrochloric acid necessary to change the pH of the solution by 1) is used as a measure representing the pH buffering capacity. In the pH measurement system of the present invention, the β value of the baseline solution is Is preferably 0.04 to 0.13 mmole / L / pH as specifically shown in Example 3.

  The hydrogen ion concentration measuring apparatus of the present invention has several features. The first is that the measuring instrument of the present invention is a double differential ion sensor. As is apparent from the above description, the hydrogen ion concentration measuring apparatus of the present invention is a differential hydrogen ion sensor in that the measurement and reference hydrogen ion sensors are used. Furthermore, it is a differential type in terms of using a differential output difference ΔΔV before and after sample aspiration, and can be said to be a differential type hydrogen ion sensor in a double sense. By adopting the double differential output, it is possible to cancel the baseline drift of the hydrogen ion sensor after the one-point calibration or the two-point calibration and other various noises, and perform highly accurate measurement.

  The second feature is that a reference hydrogen ion sensor 6 and a pseudo comparison electrode 7 are used as a reference sensor instead of the conventional liquid junction type reference electrode. Thereby, it is not necessary to replenish the internal solution of the liquid junction type reference electrode.

  The third feature is that the zero point calibration of the hydrogen ion sensor can be automatically performed. That is, zero point calibration of the reference and measurement hydrogen ion sensors can be performed by feeding the baseline solution to the reference chamber and the measurement chamber.

  Next, each part of the hydrogen ion concentration measuring apparatus of the present invention will be described. First, the measurement chamber 1 and the nozzle 2 are spaces in which, when the sample solution is aspirated, the baseline solution that has existed until then is replaced by the sample solution. Therefore, the smaller the internal volume of this portion, the smaller the amount of specimen required for measurement.

  The dead volume inside the measurement chamber and the nozzle is preferably less than 2 μL. Furthermore, it is desirable that it be 1 μL or less. By doing so, the hydrogen ion concentration can be measured by aspirating a minute sample solution of about 1 μL.

  As the hydrogen ion sensors 3 and 6, an ion sensitive field effect transistor (ISFET) or the like is used.

  As described above, in order to measure the hydrogen ion concentration of a small amount of the sample solution, it is desirable that the hydrogen ion sensor is also minute.

  As the pseudo comparison electrode 7, a metal electrode such as gold, silver, platinum, or a conductive carbon electrode is used.

  The flow path 4 also has a role of separating the measurement chamber and the reference chamber. That is, it is necessary to secure a sufficient volume so that the sample in the measurement chamber does not diffuse naturally and reach the reference chamber while the sample solution is sucked into the measurement chamber and the output of the hydrogen ion sensor is read.

  As the pump 9, various pumps such as a roller tube pump, a plunger pump, and a syringe pump are used.

  It is desirable to use a pump having both functions in order to feed the baseline solution at a relatively fast flow rate during zero point calibration and to aspirate a small amount of sample solution during measurement. As a baseline solution of ions to be measured, it is desirable to use a solution having a concentration as close as possible to the concentration of the hydrogen ions in the specimen.

  An outline of an apparatus for measuring the hydrogen ion concentration of a large number of specimens according to the present invention is shown in FIG. In FIG. 2, the same elements as those shown in FIG.

  Here, the measurement chamber 1, nozzle 2, measurement hydrogen ion sensor 3, flow path 4, reference chamber 5, reference hydrogen ion sensor 6, pseudo comparison electrode 7 and flow path 8 are integrated to form a probe 20. Yes. A baseline solution is supplied to the flow path 8 via a pump (not shown).

  The gantry 12 shown in FIG. 2 includes a microplate 14, a waste liquid well 16, and a waste liquid path 17 that communicates above the waste liquid well 16. The microplate 14 is provided with a plurality of wells 15 for storing the sample solution. The number of wells 15 provided on the microplate 14 is, for example, 96, 384, and the like.

  The gantry 12 is supported horizontally, and the probe 20 is held above the microplate 14 with the nozzle 2 facing downward.

  FIG. 3 shows the relative positions of the probe 20 and the gantry 12 during zero point calibration. Here, the nozzle 2 is inserted into the waste liquid well 16. First, in this state, the baseline solution is supplied to the flow path 8. At this time, the baseline solution is delivered from the nozzle 2 into the waste liquid well 16 through the reference chamber 5, the flow path 4 and the measurement chamber 1. When the baseline solution sent to the waste liquid well 16 becomes full there, it is discharged from the waste liquid path 17. In this way, the measurement chamber 1 and the reference chamber 5 are filled with the baseline solution, and the zero point calibration described in the above 1) is performed and the baseline solution that overflows the waste liquid well 16 adheres to the outer wall of the nozzle 2. Wash the sample solution of the previous measurement.

  FIG. 4 shows the arrangement of the nozzles 2 in the specimen suction and measurement process. At this time, with the supply of the baseline solution stopped, the nozzle 2 is inserted into a selected one of the wells 15 of the microplate 14, and then the baseline solution in the probe 20 is aspirated from the channel 8. Then, a predetermined amount of the sample solution is aspirated from the well 15. The suction amount is set so that a part or all of the baseline solution in the measurement chamber 1 is replaced with the sample solution but does not reach the reference chamber 3. Thereafter, the suction is stopped and the sensor output is read and calculated as described in 2) above to obtain the ion concentration C1.

  By repeating this 1) zero point calibration process and 2) suction and measurement process for each well 15 of the microplate 14, the hydrogen ion concentration of the sample solution in each well 15 is measured sequentially. For this purpose, a device for driving the probe 20 back and forth, left and right, up and down, or a device for driving the gantry 12 carrying the microplate 14 and the waste well 16 up and down, left and right, and up and down may be provided. Alternatively, a device may be provided in which the gantry 12 is driven back and forth and left and right, and the probe 20 is driven only up and down. In addition to the microplate 14, a sample tube may be used as a container for a large number of specimens. In that case, you may use a turntable as a drive stand.

  As described above, in the present invention, the probe including the parts including the respective sensors provided in the flow path is integrated into a probe. In particular, a large and heavy microplate, turntable, etc. (sample solution) If the housing member is not moved up and down at least, it is possible to simplify the drive mechanism for driving each part, speed up the multiple sample measurement process, and reduce the energy consumption.

  The hydrogen ion concentration measuring apparatus of the present invention is suitable as an apparatus for measuring the hydrogen ion concentration of a large number and a very small amount of specimen at high speed. In order to further enable high-speed measurement, it is desirable to use a hydrogen ion sensor having a high response speed. When the object to be measured is pH, not only is it difficult to measure a small amount of sample due to its large size when using a normal glass electrode, but in particular, the sample of a sample having a weak pH buffering ability has a long time of several minutes to 10 minutes. Cost. Therefore, it is difficult to apply a normal glass electrode to a high-speed pH measurement system. The present inventor has studied a pH-sensitive ISFET suitable for a high-speed pH measurement system. As a result, the pH-sensitive ISFET has been found to have a fast response and a slow response to a weak buffer capacity solution. It was. FIG. 5 shows the result.

  In the test of FIG. 5, in the measurement apparatus as shown in FIG. 2, a commercially available solution (pH 6.12) obtained by diluting a commercially available 6.86 pH buffer solution 1000 times with physiological saline is used as a baseline solution. This was done using a 1.68 pH buffer and a solution diluted 125-fold with saline. Before the time 0, the pump was rotated forward, the baseline solution was passed through the probe, and zero point calibration was performed.

  The pump was stopped at time 0 second, the pump was reversed at time 10 seconds, about 1 μL of the sample solution was sucked, and the pump was rotated forward at time 46 seconds. In FIG. 5, the solid line used is an ISFET formed by sputtering using a tantalum pentoxide target as a pH-sensitive film at the gate portion according to the method of Japanese Patent Application Laid-Open No. Sho 62-245958 (hereinafter referred to as Type A ISFET). Called). On the other hand, in the broken line, ISFET (hereinafter referred to as type B ISFET) in which tantalum pentoxide is formed on the gate by a chemical vapor deposition (CVD) method using tantalum alkoxide as a raw material was used. The following is clear from FIG.

  (1) When a type A ISFET is used, a specimen having a strong pH buffering capacity (pH 1.68 buffer, buffering capacity β = 50 mmole / L / pH) as well as a specimen having a weak pH buffering capacity (pH 1.68 buffer) A response of about 90% is obtained 2 seconds after sample aspiration, even for a solution obtained by diluting the solution 125 times (buffer capacity β = 0.25 mmole / L / pH). When the pump is rotated forward after the measurement is completed, the output returns to the baseline within 6 seconds after the start of forward rotation.

  (2) In contrast, when a type B ISFET is used, a sample with a strong pH buffering ability (pH 1.68 buffer solution) responds quickly, and a 100% response can be obtained within 4 seconds after the sample is aspirated. A sample having a weak ability (a solution obtained by diluting 125 times the pH 1.68 buffer solution) has a very slow response, and it takes 17 seconds or more to obtain a response of about 90%. Further, the response at the time of 5 seconds after the sample suction is 20 mV, and the response at 46 seconds after the suction is about 1/5 of 102 mV. Moreover, the return of the output after the forward rotation of the pump is slow, and an output of about 2 mV remains even 14 seconds after the forward rotation.

  FIG. 6 shows a specific example of the probe in the measuring apparatus of the present invention. The probe 40 is a differential pH sensor including a pH-sensitive ISFET 22 for measurement, a pH-sensitive ISFET 25 for reference, and a pseudo comparison electrode 28. In the probe 40, the nozzle 21, the measurement chamber 32, the flow channel 33, the reference chamber 34, and the flow channel 31 are formed of synthetic resin and integrated. The ISFETs 22 and 25 used here are of the type disclosed in Japanese Examined Patent Publication No. 57-43863 in which the entire circumference of the tip is insulated with silicon oxide / silicon nitride. There are bonding pads 24 and 27 on the other end side of the ISFETs 22 and 25, and a lead wire 30 is connected thereto. The ISFETs 22 and 25 have a width of 450 μm, a thickness of 180 μm, and a length of 550 μm. The bonding pads 24 and 27 and part of the lead wires 30 of the ISFETs 22 and 25 are sealed with an insulating resin 29. Tantalum pentoxide is formed as a pH sensitive film on each of the ISFETs 22 and 25 by a sputtering method using tantalum pentoxide as a target according to the method disclosed in Japanese Patent Laid-Open No. 62-245958 (type A). As the pseudo comparison electrode 28, a silver wire whose surface was converted to AgCl was used. In the zero point calibration step, the baseline solution is pumped from the flow path 31 and discharged from the nozzle 21 via the reference chamber 34, the flow path 33, and the measurement chamber 32. In the sample aspiration and measurement process, about 1 μl of the sample solution is aspirated from the nozzle. This aspiration amount is a value selected as an amount that the sample solution just fills the measurement chamber 32 but does not enter the reference chamber 34.

  Using the probe 40 shown in FIG. 6, a system for measuring the pH of the sample solution in a plurality of wells of the microplate 46 was manufactured as shown in FIG. In FIG. 7, the probe 40 can be driven in the vertical direction (Z direction) by the vertical movement drive unit 41. On the other hand, the microplate 46 and the waste well 47 are mounted on the table 42. The table 42 is driven so as to move on a horizontal plane (XY plane) by a table driving unit 42A. A baseline solution is sent from the container 45 to the probe 40 by a pump 43. As the pump 43, a tube pump driven by a microstepping motor is used, and thereby the baseline solution is fed and the sample solution is sucked. The entire system is controlled by a control device 44 including a CPU and a memory. The control device 44 controls the vertical movement drive unit 41, the table drive unit 42A, and the pump 43 based on an instruction given from the input unit 44A, and reads the outputs of the sensors and the pseudo comparison electrodes in the probe 40. The data processing is performed based on the above and the result is output to the display device 44B.

  The operation of this system will be described. The controller 44 is instructed in advance the order of wells to be processed in the microplate 46. And the control apparatus 44 sets the table 42 and the probe 40 to a reference position according to the instruction | indication of a measurement start. That is, the control device 44 moves the table 42 so that the waste liquid well 47 reaches directly below the nozzle 21 of the probe 40, moves the probe 40 downward, and the nozzle 21 is inserted into the waste liquid well 47. To do. Hereinafter, the zero point calibration process and the specimen aspiration and measurement process will be described with reference to the flowchart of FIG.

1) Zero point calibration process The control device 44 rotates the pump 43 in the normal direction and sends the baseline solution in the container 45 to the probe 40 for 10 seconds (step 101). At this time, the baseline solution overflows from the nozzle 21 and is stored in the waste liquid well 47. At the final stage of this process, the controller 44 reads the differential output ΔV0 of the two sensors (step 102).

2) Specimen Aspiration and Measurement Step Next, the controller 44 stops the pump 43, pulls up the probe 40 from the waste liquid well 47 and returns it upward (step 103), and then moves the table 42 to specify The first well is moved directly below the nozzle 21 of the probe 40, and the nozzle 21 is lowered into the well (step 104). The time required for this movement is 3 seconds. Next, the control device 44 reverses the pump 43 by a predetermined step to flow 1 μL of the sample solution back into the sensor (step 105), reads the differential output ΔV1 3 seconds after the start of the backflow (step 106), and ΔΔV = ΔV1−ΔV0 is obtained (step 107), and further
pHa = pHb + ΔΔV / S (7)
To obtain and display the pH (pHa) of the sample solution (step 108). Here, pHb is the pH of the baseline solution, and S is the pH sensitivity of the ISFET 22 for measurement. Next, the control device 44 pulls up the nozzle 21 from the well, drives the horizontal drive table 42, moves the waste liquid well 47 directly below the nozzle 21, lowers the probe 40, and inserts the nozzle 21 into the waste liquid well. (Step 109). This movement takes 3 seconds.

Next, the control device 44 sequentially repeats steps 101 to 109 in the above steps for each of the second and subsequent wells, and measures the pH of the sample solution in each well. The measurement time per specimen solution is about 20 seconds. As a baseline solution, a solution obtained by diluting a commercially available pH 6.86 buffer solution 1000 times with physiological saline (pH = 6.12 at 25 ° C.) was used. As the specimen, pH buffering solution of commercially available pH9.18,7.41,4.01 and 1.68 were diluted further 9.18 and 1.68 buffer with pure water up to five times to 5 ^quadruple solution Was used. The measurement results are shown in Table 1 as pHobs. The time required for this measurement was 4 minutes. In Table 1, pHtheol is a pH value measured using a commercially available glass electrode pH meter and spending 10 minutes per sample solution.

  As apparent from Table 1, not only a commercially available pH buffer solution but also a solution diluted with pure water to weaken the buffer capacity, the pH is accurately measured by using the measuring device of the present invention. It is possible. The average absolute value of the measurement error for the 12 specimens in Table 1 was 0.09 pH.

[Comparative Example 1]
As the pH sensitive ISFET, a tantalum pentoxide film formed by chemical vapor deposition (CVD) method using tantalum alkoxide as a raw material on the gate (type B) was used, and the other solutions were prepared under the same conditions as in the examples. The pH was measured. The results are shown in the column of pHobs in Table 2. The reason for this pH-sensitive ISFET is unknown, but as shown in FIG. 5, the response to a solution with a weak buffer capacity is slow. As is apparent from Table 2, when this ISFET was used, the pH was measured with a relatively small error for the 9.18, 7.41, 4.01 and 1.68 buffers, but the 9.18 buffer. liquid large error arises a solution diluted 5 ⁴ times 5, and 1.68 buffer solution obtained by diluting 5 ⁴ times 5 ^3. The average absolute value of measurement error for 12 specimens in Table 2 was 0.31 pH. Thus, it has been found that when an ISFET having a slow response to a solution having a weak pH buffering ability is used, the pH measurement of a specimen having a weak buffering ability becomes inaccurate.

A commercially available pH 1.68 buffer under the same conditions as in Example 1 except that a physiological saline solution (pH 5.41, β = 0.13 mmole / L / pH) containing 2 mmole / L ammonium chloride was used as the baseline solution. the liquid and solution it was diluted 5-5 ^4-fold as a sample, it was measured three times each, examine its reproducibility, and the results are shown in Table 3. FIG. 9 shows a pH response curve in the repeated measurement of specimen numbers 13 and 14. As a result, it was found that the reproducibility of the pH measurement system of the present invention was good with a standard deviation of 0.020 pH or less.

  As a baseline solution, BS1 is 2 mmole / L ammonium chloride (NH4Cl) / saline solution, BS2 is 0.1 mmole / L ammonium chloride solution, BS3 is 6.86 buffer diluted 1000 times with RO water, BS4 is 6 .86 buffer was diluted 100 times with RO water, BS5 was diluted 6.86 buffer with RO water 10 times, BS6 was 15 mmole / L potassium chloride (KCl), and BS7 was physiological saline. Using the same sample as the sample of Example 1, using a sample 1, 6, 7, 8 (black), a calibration curve created by the least square method, and diluted samples 2, 3, 4, 5, 9, 10, Measurement points 11 and 12 (white marks) are shown in FIGS. A and S inserted in each figure are the intercept and slope of the calibration curve (see equation (6)), and r is the correlation coefficient of the least square method. The value of the intercept A theoretically corresponds to the pH of the baseline solution, and the slope S corresponds to the sensitivity of the pH sensor for measurement. As a result, as shown in FIG. 10, when BS1 is used as the baseline solution, the deviation of the diluted specimen from the calibration curve is relatively small. On the other hand, for example, when BS5 shown in FIG. 14 is used as the baseline solution, the deviation of the diluted specimen from the calibration curve is large. Table 4 shows the results of evaluating the baseline solution based on the amount of deviation from the calibration curve of such a diluted sample. Table 4 also shows the pH value (value measured with a glass electrode) and β value of each baseline solution. From this result, it can be seen that the buffer capacity of the baseline solution is preferably in the range of 0.04 to 0.13 mmole / L.

The figure which shows the basic composition of the hydrogen ion concentration measuring apparatus of this invention. The figure for demonstrating the hydrogen ion concentration measurement of the test substance in each well of a microplate. The figure for demonstrating the hydrogen ion concentration measurement of the test substance in each well of a microplate. The figure for demonstrating the hydrogen ion concentration measurement of the test substance in each well of a microplate. The figure which shows the response curve of pH sensitive ISFET (solid line) with a quick response with respect to the test substance with a weak buffer capacity (solid line), and slow pH sensitive ISFET (dashed line). The figure which shows the specific example of a probe. The block diagram of the system which measures automatically the sample of each well of a microplate. The flowchart for demonstrating operation | movement of the apparatus shown in FIG. The figure which shows the response curve in the repeated measurement of the pH measurement system of this invention. The figure which shows various baseline solutions and the calibration curve created using it. The figure which shows various baseline solutions and the calibration curve created using it. The figure which shows various baseline solutions and the calibration curve created using it. The figure which shows various baseline solutions and the calibration curve created using it. The figure which shows various baseline solutions and the calibration curve created using it. The figure which shows various baseline solutions and the calibration curve created using it. The figure which shows various baseline solutions and the calibration curve created using it.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Measurement chamber 2 Opening nozzle 3 Measurement hydrogen ion sensor 4 Channel 5 Reference chamber 6 Reference hydrogen ion sensor 7 Pseudo comparison electrode 8 Channel 9 Pump 10 Channel 11 Container 12 Base 14 Microplate 15 Well 16 Waste liquid well 17 Waste channel 20 Hydrogen ion sensor 21 Nozzle 22 pH sensitive ISFET for measurement
23, 26 pH sensitive parts 24, 27 Bonding pad 25 Reference pH sensitive ISFET
28 Pseudo-reference electrode 29 Insulating resin 30 Lead wire 31 Channel 32 Measurement chamber 33 Channel 34 Reference chamber 40 Probe 41 Vertical movement drive unit 42 Table 43 Pump 44 Controller 45 Container 46 Microplate 47 Waste fluid well

Claims (5)

A liquid flow in which a nozzle portion, a first storage chamber, a first flow path, a second storage chamber, and a second flow path are connected in this order, and one liquid flow path is formed as a whole. The road,
A pump connected to the second flow path and capable of either supplying a liquid from the second flow path into the liquid flow path section or sucking a liquid from the nozzle section into the liquid flow path section. When,
A hydrogen ion sensor for measurement that is arranged in the first storage chamber and detects hydrogen ions in the liquid;
A pseudo-reference electrode disposed in the second storage chamber;
A hydrogen ion sensor for reference that is arranged in the second storage chamber and detects hydrogen ions in the liquid;
In a hydrogen ion concentration measuring apparatus comprising:
The measurement hydrogen ion sensor and the reference hydrogen ion sensor are pH-sensitive ISFETs, and a tantalum pentoxide film is formed on the gate of the pH-sensitive ISFET as a pH-sensitive film by a sputtering method. A hydrogen ion concentration measuring device characterized by. It is a hydrogen ion concentration measuring method using the hydrogen ion concentration measuring apparatus according to claim 1, wherein the pump introduces a liquid having a known hydrogen ion concentration into the liquid flow path section, and at least the first accommodation. Calibration is performed based on outputs of the measurement hydrogen ion sensor, the reference hydrogen ion sensor, and the pseudo comparison electrode at that time, with the liquid having a known hydrogen ion concentration being contained in the chamber and the second accommodation chamber. A first step for determining a value;
Next, the sample solution is guided into the liquid flow path portion by the pump, the sample solution is stored in the first storage chamber, and the hydrogen ion concentration is known in the second storage chamber. And measuring the hydrogen ion concentration of the second liquid based on the output and the calibration value of the measurement hydrogen ion sensor, the reference hydrogen ion sensor, and the pseudo comparison electrode at that time. A method for measuring a hydrogen ion concentration, comprising two steps.   The method for measuring a hydrogen ion concentration according to claim 2, wherein the liquid having a known hydrogen ion concentration has a β value in the range of 0.04 to 0.13 mmole / L / pH. A liquid flow in which a nozzle portion, a first storage chamber, a first flow path, a second storage chamber, and a second flow path are connected in this order, and one liquid flow path is formed as a whole. The road,
A pump connected to the second flow path and capable of either supplying a liquid from the second flow path into the liquid flow path section or sucking a liquid from the nozzle section into the liquid flow path section. When,
A hydrogen ion sensor for measurement that is arranged in the first storage chamber and detects hydrogen ions in the liquid;
A pseudo-reference electrode disposed in the second storage chamber;
A hydrogen ion sensor for reference that is arranged in the second storage chamber and detects hydrogen ions in the liquid;
In a hydrogen ion concentration measuring apparatus comprising:
The liquid channel section, the measurement hydrogen ion sensor, the reference ion sensor, and the pseudo comparison electrode are integrated to form a probe,
A sample solution storage member having a plurality of sample solution storage units for storing the sample solution;
A hydrogen ion concentration measuring apparatus comprising probe moving means for moving the probe in a state where the nozzle portion is directed downward and / or a sample solution containing member moving means for moving the sample solution containing member.   The measurement hydrogen ion sensor and the reference hydrogen ion sensor are pH-sensitive ISFETs, and a tantalum pentoxide film is formed on the gate of the pH-sensitive ISFET as a pH-sensitive film by a sputtering method. The hydrogen ion concentration measuring apparatus according to claim 4.

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