JPS63106549A - Oxygen reaction container - Google Patents

Abstract

PURPOSE:To continuously measure the concn. of oxygen in a small amount of a liquid specimen with high accuracy, by providing a magnetic stirrer rotated by a magnetic field rotary means and a constant temp. heating means to the electrode unit constituting the bottom surface of a container. CONSTITUTION:The magnetic rotary device 24 provided to the underside of the electrode unit 22 fixed to the lower part of a glass container 27 is constituted so that the magnetic stirrer 30 in the container 27 is rotated by the rotary magnet 25 rotating by the motor 26 in said rotary device 26. Since a housing supporting a measuring electrode for measuring the concn. of oxygen and a reference electrode is heated to predetermined temp. and a protruded part 1a is provided to a container support 1, the reaction solution 110 stirred by the stirrer 30 in the container 27 is efficiently held to predetermined temp. By the apparatus thus simplified and miniaturized, the concn. of oxygen can be continuously measured with high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an oxygen reaction vessel for continuously measuring changes in oxygen concentration in a small amount of solution sample.

[Prior art] Conventionally, in order to understand the characteristics of reactions in which 02 is consumed or generated in a solution sample, an oxygen @ electrode is inserted into a solution sample 4, and changes in the oxygen concentration therein are continuously monitored. measurements are being carried out.

The oxygen electrode method is a type of polarography, in which 02 is electrolyzed by applying a constant voltage between the negative and positive electrodes.
This method measures the concentration of 2.

Generally, as a cathode (measuring electrode), a noble metal such as platinum, gold, Ir, etc. is sealed in glass or plastic, and the tip thereof is exposed. A calomel electrode (Hg/HgC1) or a silver/silver chloride electrode is used as the anode (control electrode). Now, when a voltage of approximately 0°6V is applied between these two electrodes, the following electrode reaction proceeds.

Cathode: 02+484+4e -2H20 (acidic solution)
02+2HzO+4e -40H- (alkaline solution)
Anode: 4Ag+4CI -4AgC1+4e (
Wide pH range) As a result of the above electrode reaction, 02 reaches the cathode.
A current proportional to the number of molecules flows between the two electrodes.

Since the number of O2 molecules reaching the cathode is proportional to the O2 concentration in the solution, the O2 concentration can be determined by measuring this current.

Oxygen electrodes can be classified into yin-yang separated type and yin-yang combined type based on how the electrodes are arranged. In the former method, both electrodes are placed directly in the sample solution, and the electrode reaction occurs within the sample.

In the latter case, both electrodes and the electrolyte for electrode reaction are hydrophobic and 02
It is wrapped in a transparent plastic thin film (electrode 1 [a) and combined as one body. In the latter case,
The sample to be measured comes into contact with the thin film, and 02 passes through the thin film and reaches the measurement electrode, where it reacts.

Instead of measuring 02 by applying an appropriate voltage between the negative and positive electrodes as described above, select a reference electrode with an appropriate potential (e.g., Pb/Pb (DH) 2 system) and measure it without applying a voltage. There is also a method (galvanic cell type) that allows measurement of 02, but in the present invention it is used in the same way as an oxygen electrode.

Now, either the oxygen electrode method or the galvanic cell method
Since the electrode unit for oxygen measurement is usually long and thin, it must be inserted from the side or diagonally upward into the reaction vessel, and it is extremely convenient to attach it to the bottom of the vessel. If this is done, it will not be possible to rotate the magnetic rotor inside the container by placing a magnetic rotator at the bottom.

[Problems to be Solved by the Invention] Generally, it is desirable for an oxygen reaction vessel to satisfy the following conditions.

First, the shape must be such that 02 in the atmosphere does not penetrate into the measurement liquid, in order to prevent errors in measurement values due to diffusion of 02 in the atmosphere. Furthermore, the shape must be such that it is easy to inject and remove the sample, and that air bubbles that enter during the operation do not stay inside but can easily escape upwards.

Second, the sample must be constantly stirred at a constant speed. This is necessary to quickly and uniformly mix the reagents when they are added at the start of the reaction or during the reaction, and to keep the sensitivity of the electrode constant. Electrode sensitivity differs significantly between when the liquid is stationary and when it is stirred and moving, and also varies somewhat depending on the strength of the stirring.

Thirdly, the structure must be such that the temperature of the sample can be maintained at a predetermined temperature. This is because the sensitivity of the oxygen electrode changes depending on the temperature, and the reaction rate also changes depending on the temperature.

Fourth, it is often preferable to have a small volume, which can save money when measuring samples (especially biological samples) or reaction materials are difficult to prepare or are made from aged sake. This is because it can be measured.

In addition to the above, it goes without saying that the oxygen electrode installed in the oxygen reaction vessel must have stable sensitivity and a sufficiently fast response speed.

In the case of separated oxygen@, poles, a container that satisfies all of the first to fourth conditions above was devised and commercialized by Hagiwara (Funji Hagiwara: A container that satisfies all of the first to fourth conditions above) (Funji Hagiwara: Oxygen concentration measuring device, filed in 1979-05
1817, Utsuko Sho 42-010978) (B, hagi
hara: Biochim, Biophis, A
cta, 46.134 (1961)) (B, Hag
ihara, F, l5hibashi, ni, 5asak
i & Y, Kamigawara: Anal, Bi
ochea+, 86, 417/431 (1978)),
In these cases, the microscopic measurement and the reference (or its salt 1) can be separated and placed at any position in the reaction vessel, so the first and second conditions above are satisfied.
Moreover, it was not difficult to manufacture a reaction vessel with a small capacity.

On the other hand, the measurement accuracy, particularly the stability of electrode sensitivity, is generally higher with composite electrodes than with separate electrodes.For this reason, various oxygen reaction vessels using composite electrodes have been devised, including 2.
3 are commercially available.

A typical example of an oxygen reaction vessel using these composite electrodes is shown in FIG. 7. In this example, a magnetic stirrer 6 is placed in the vessel in order to satisfy the second condition.

This magnetic stirrer 6 is rotated by a rotating magnet 8 provided at the bottom of the container 4, and stirs the sample 12. Composite electrode unit) 10 is yong! ! The electrode surface 10a is in contact with the sample 12. The outside of the container 4 is filled with circulating constant temperature water 14 to keep the temperature of the sample 12 constant.

Such conventional devices have the following drawbacks.

In the composite electrode unit, a measurement electrode and a reference electrode are integrally combined so as to be connected by WIN of the electrolyte. For this reason, including the structural support increases the diameter of the electrode unit, which inevitably increases the capacity of the container.

Furthermore, since the flat electrode surface is fitted into the cylindrical or spherical side wall of the container, air bubbles adhere to and around the electrode surface and are difficult to escape upwards. In particular, in the case of composite oxygen electrodes, the center of the electrode surface is made of a hydrophobic plastic film that covers the measurement electrode and is difficult to wet with water, making it easy for air bubbles to adhere to it.

Generally, the concentration of 02 dissolved in the test liquid in the oxygen reaction vessel is significantly lower than the concentration of 02 in the air.When an aqueous solution is equilibrated with air at 25°C, the dissolved 02 concentration is 246
Since it is μnole/l, if air (02 = 20
．． 7%) stays in the container, several tens of times the volume of 02 will be mixed into the bubbles. Therefore, even small bubbles can cause significant errors in measuring O2 consumption or generation reactions. For this reason, air bubbles within the container must be completely removed. Similarly, contact between the reaction solution and air can also cause large errors due to the components of the reaction solution (buffer, reaction gas, etc.).
Some of the biologically active substances, reagents, etc.) need to be injected into the container during the measurement, so it is necessary to provide an injection port in the container, but if 02 in the air enters from there, the measured value will change. This will cause an error.

Fortunately, the spread of 02 molecules in stationary water is slow, so if an elongated tube-shaped opening is provided above the reaction vessel, the liquid within the tube remains unaffected by stirring. Therefore, it is possible to almost completely prevent 02 in the air from entering the reaction solution. Since this elongated opening must also serve as an opening for removing air bubbles within the container, the upper part of the container must have a conical shape and smoothly connect to the thin tube section.

An object of the present invention is to provide an oxygen reaction vessel with high measurement accuracy that satisfies the above conditions.

[Means for Solving the Problems] The oxygen reaction vessel according to the present invention includes a flat composite oxygen electrode unit provided on the bottom of the vessel with its reaction end face facing upward, and a magnetic field rotating under the electrode unit. rI the means! , a magnetic stirrer is placed on top of the electrode surface, which is rotated by the magnetic stirrer. Complex oxygen fI! The electrode surfaces of the poles are arranged to constitute the bottom surface of the container.

[Function] Since the composite oxygen electrode unit is made into a flat shape that is narrow in the vertical direction, the magnetic flux of the magnetic field rotation means provided at the bottom of the electrode can penetrate the electrode and rotate the magnetic stirring body.

Since the electrode unit is provided at the bottom, the height of the container and the diameter of the container can be made small regardless of the diameter of the electrode, and air bubbles will not adhere to the electrode surface or its surroundings and become difficult to escape to the top.

〔Example〕

An outline of the structure of an oxygen reaction vessel according to an embodiment of the present invention is shown in FIG. 1. A metal vessel support 1 is adhered to the lower side surface of a glass vessel 27. An electrode unit 22 is fixed to the lower part of the container support 1 with bolts through a waterproof backing film. It is being The motor 2 is inside the rotator 24.
A rotating magnet 25 is provided, which is rotated by a magnet 6, thereby causing a magnetic stirrer 30 within the container 27 to rotate.

In Figure 2, which shows an example of an exploded detailed view of the electrode unit 22, 58 is a measurement electrode, 59 is a reference electrode, 56 is an electrode film, 76 is an electrode film covering plate, 80 is a constant temperature heating metal part,
56 and 76 constitute an electrode surface. Electrode unit 2
2 is comprised of an f$, ffi body 51, a membrane holder 55, and an electrode covering key 54. The body 51 is equipped with a cathode, that is, a measuring electrode 58 (gold or platinum), and an lla electrode, that is, a reference electrode ff159(i). There is. The shape of the exposed end surface of the measurement electrode 5B may be a disk type, a straight type, a multi-point type, or
A ring type or the like can be used. The reference electrode 59 is made of a silver circular tube and becomes an Ag/AgC:I electrode during measurement and exhibits a constant potential.The measurement tffisa and the reference electrode 59 are insulated by an insulator 60 such as glass. An insulating plastic support 62 (polyacetal, 9 noble vinyl chloride, etc.) supports the poles 58 and 59 of 1 to the housing 80. The housing 80 is made of a thermally conductive metal (for example, brass), and is maintained at a predetermined temperature by a heater 70 and a →J'-mister 71, which are constant temperature heating means, as shown in FIG. The lower side of the casing 80 is made of epoxy resin 6.
3 is filled and covered with a plastic cover 64.

The membrane holder 55 is constructed by pasting an electrode membrane 56 on a plastic circle 75 (made of polyacetal, polycarbonate, polypropylene, etc.). The electrode film 56 is made of hydrophobic plastic with a thickness of 5 to 30 μm.
(films such as polypropylene, Teflon, Mylar, etc.)
is used.

? The It electrode V4 covering 54 is made of a non-reactive metal such as stainless steel, and has a membrane holder housing 76 and a screw hole 77. This tf! When assembling the i-unit 22, apply approximately 173 drops of electrolyte 57 (for example, KCI containing 8 oz ethylene glycol, 0.02M phosphate buffer) to the center of the electrode membrane 56 of the membrane holder 55, and then attach the electrode to the center of the electrode membrane 56 of the membrane holder 55. Insert the section 51 onto the upper surface of the membrane holder 55.
The electrode membrane covering 54 is fitted and fixed to the electrode main body 51 with bolts 65.

The reaction container 27 is fixed to the electrode unit 22.
This is the device shown in Figure S1. In FIG. 1, reaction vessel 2
The upper part 28 of 7 is a thin tube with a diameter of about 1.5 mm. This is to prevent 02 from entering the reaction solution in the atmosphere.The 0 reaction container 27 is fixed to the container support 1 with a waterproof adhesive (waterproof epoxy, etc.). A plastic membrane 53 (for example, a single-sided adhesive Teflon film of approximately 100 μm) for water leakage prevention is attached to the lower surface of the container support l. It is for the purpose of Syringe 3
2 is a syringe equipped with a plastic thin tube for sucking out the reaction liquid, and the reaction vessel sealing stopper 33 is a thin tube-like part of the reaction vessel in order to completely prevent 02 from entering from the atmosphere during long-term reactions. According to this embodiment, the electrode unit is equipped with a heater 70 and a thermistor 71 and is kept at a constant temperature, so that the reaction solution 110 can be kept at a predetermined temperature.That is,
Heat from the heater 70 installed in the electrode unit 22 is transferred to the reaction solution 1 via the electrode membrane covering plate 76 and the container support 1.
Warm 10. In particular, as shown in FIG. 1, by providing the protrusion 1a on the container support l, the side wall of the reaction container 27 can be efficiently heated. Note that this protrusion 1
A is made of a non-reactive metal and is provided inside the side wall of the reaction vessel 27. According to this implementation, it is not necessary to submerge the entire vessel in constant temperature water as in the conventional case. Therefore, the device can be simplified and downsized.

In addition, as another embodiment, the thermistor 71 and the heater 70 may be installed inside the reaction vessel support instead of providing the temperature adjustment mechanism inside the electrode unit as shown in FIG. Compared to the case shown in the figure, there is greater tolerance regarding the shape and size of the heater and temperature measuring element used. However, in the case of FIG. 1, the reaction liquid was heated from both the bottom surface and the side wall of the reaction vessel, but in this case, only the side wall is heated. However, by increasing the heating area of the side wall, the temperature of the reaction solution can be sufficiently maintained.

FIG. 4 shows an example in which a constant temperature heating means is not provided in the electrode unit 22, but a constant temperature heating means is provided inside the reaction vessel support 1, where 70 is a heater and 71 is a thermistor. The first protruding portion 1a is provided so as to cover the side surface of the reaction chamber 527 from the outer periphery. In the case of this embodiment, it is effective to form the 44-pole membrane cover plate 76 from a thermally conductive material, since heating is also carried out from the electrode surface.

Note that according to this embodiment, the following effects can be obtained.

First, since it is not necessary to provide constant temperature heating means in the electrode unit 22 itself, the structure of the electrode is simplified. Therefore, the electrode unit 22 is less likely to fail, and the electrode unit 2
The manufacturing cost of 2 can be lowered.

Secondly, in the case where the electrode unit 22 is equipped with constant temperature heating means, the electrode unit 22 has to be large, and it is difficult to significantly reduce the diameter of the reaction vessel 27. On the other hand, according to the above embodiment, the electrode unit 22
can be made smaller, and a significantly thinner reaction vessel 27 can be manufactured. Furthermore, since the constant-temperature heating means is provided within the reaction vessel support l, which is not limited in size, there is no need to use minute and expensive elements.

Figure 6A shows an example of the results of measuring the mitochondrial oxygen consumption reaction at 25°C using the oxygen reaction vessel shown in Figure 1.The straight line A at the right end of this graph shows the relationship between air and air at 25°C. Equilibrated reaction solution (IQmHIJ)
6 songs, 11 IC. shows the change in oxygen concentration when succinic acid (respiratory substrate) equivalent to 0.1M is added at the point of mouth.The zero curve shows the oxygen concentration after adding ADP equivalent to 50μN at point C. This oxygen consumption rate, which shows the course of change in , slows down midway as shown by curve E. This is the ADP added in step C.

This is because the respiration returned to the state before the addition of ADP because all of it became ATP and disappeared. These measurement results show that by using the oxygen reaction vessel of the present invention, it is possible to extremely accurately measure the complex oxygen consumption situation of biological samples, such as mitochondria, whose oxygen consumption rate changes depending on the conditions.

As described above, according to the present invention, it is not necessary to immerse the entire container in constant temperature water, so it is easy to attach an optical fiber or the like to the side surface of the glass container 27. Therefore, it is possible to provide an apparatus that performs optical measurement at the same time as oxygen concentration measurement.

FIG. 5 shows an example in which optical measurements (spectroscopic optical measurements, fluorescence optical measurements, etc.) are performed simultaneously with the measurement of oxygen concentration. It is stored in 94. At the bottom of the case 94 is an externally threaded introduction tube 85a.

85b is provided. A light transmitting light pipe 98a and a light receiving light pipe 98b are inserted into the introduction pipes 85a and 85b, a luminous O-ring 97 is fitted, and the threaded pipe 96
It is constrained by. Both the light transmitting optical conduit 98a and the light receiving optical conduit 98b are made by bundling optical fibers with an elastic binding tube 99 made of soft plastic.
The part to be inserted into is a metal tube 100 that is slightly thinner than the introduction tube.
a, 100b are fitted. Note that an M2O is provided in the upper part of the case 94, and a small hole 91 for passing a syringe or the like is provided in this i90. The actual measurement depends on the intensity of the light sent to the reaction vessel, the brightness of the room, the wavelength range of the light to be measured, etc.
In some cases, the entire container may be removed, in some cases, only the upper part 190 may be removed, and in some cases, the sample may be added through the small hole with the lid closed.

The results of measuring the relationship between the degree of binding between hemoglobin and oxygen and the oxygen concentration using this device are shown in Figures 6B and C. The hemoglobin reductase system (NADPH + ferredoxin - NADPI reductase + ferredoxin) is used to maintain the hemoglobin reductase system (NADPH + ferredoxin - NADPI reductase + ferredoxin) using a phosphoric acid titanium solution (pH 7.0), and the concentration of hemoglobin is 83μ.
K, temperature was 30°C. From the light transmitting optical conduit 98a, the monochromatic light was scanned over a wavelength range of 450 nm to 650 nm for 5 seconds, and was incident on the solution to be measured. In this case, the optical path length of the reaction solution 110 is 10 mm, and the transmitted light that passes through it is received by the light receiving light guide tube 98b, and the photomultiplier tube (
(not shown).

The experiment was first carried out using a reaction solution 110 that did not contain hemoglobin to memorize the intensity of transmitted light at each wavelength, and then a similar measurement was carried out using a reaction solution containing hemoglobin to determine the transmitted light at each wavelength. The ratio of the intensities was processed according to Beer's law and displayed on a data recorder (not shown) or the like as a graph showing the difference between wavelength and absorbance, that is, an absorption spectrum. Note that during this experiment, the sample solution was constantly stirred, and its 02 concentration was always recorded on a separate recorder, marking the time point at which the spectrum was measured. The recording curve for this 02 density is shown in FIG. 6C. In this experiment, we first drew a spectrum of a reaction solution that did not contain hemoglobin, which naturally became a straight line, and in Figure 6B, the spectrum was shown by straight line A.
Next, hemoglobin (j% initial concentration 83 μM) was added to the reaction solution, a small amount of the solution was removed with a microsyringe, the solution was brought into contact with the gas phase under the capillary of the container, and N2 gas was bubbled through the gas phase. By stirring and removing 02 from the solution, an oxygen-free hemoglobin solution is created. The spectrum of this state is B in this figure.
The spectrum recorded at the point (marked point C in FIG. 6C) where the 02 concentration of the solution rose slightly by aerating the 02 mixed gas was curve C, and the 02ia degree at this time was 11°3 μX.

Furthermore, the spectrum measured at the point where the O2 concentration increased (point D) is curved! lID, the 02 concentration at this time is 26.2μ
Next, change the ventilation gas which was N2 to 82-92gN2 to increase the concentration of 02 in the solution and reach point E (02 = 41.8 μM).
The spectrum obtained at curve 11E is curve 11E, and the spectrum obtained at the point where the 02 concentration further increased (point F, 02 = 64.1 μM) is curve F.0 Next, the ventilation gas was changed to 20.7202 (air). Then 0 points (02 = 173.3μM)
The spectrum obtained is the song 41i1G, and no more than 02
The shape of the spectrum did not change as the concentration increased, so
This curve was found to be a spectrum of 100% 02 summated hemoglobin. Through these experiments, we were able to accurately measure the relationship between the degree of oxygenation of hemoglobin (oxygen saturation) and oxygen concentration.

According to this embodiment, it is possible to accurately perform optical measurement at the same time as measuring the oxygen concentration of the sample solution.

[Effect of the invention] According to this invention, the following effects can be obtained.

Since the composite oxygen electrode is provided at the bottom of the container, the container can be shaped so that air bubbles can easily escape to the top. Furthermore, since the diameter and height of the container can be made small regardless of the diameter of the electrode, it is possible to significantly save the measurement sample. This is particularly effective when using medical or biological samples that can be obtained in very small quantities, or when using expensive reagents.

Furthermore, since a thin composite oxygen electrode is used, the magnetic stirring body inside the container can be rotated by the magnetic field rotation means provided under the electrode.

In addition, complex oxygen! Since the pole is installed at the bottom of the container,
It is easy to provide a light guide for optical measurements on the side of the container.

Furthermore, when adopting the arrangement and shape of the present invention, if a constant temperature heating means is provided on the electrode or the reaction vessel support, the sample solution in the reaction vessel can be easily maintained at a constant temperature. The complexity of using a tank can be omitted.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an oxygen reaction vessel according to an embodiment of the present invention, FIG. 2 is a diagram showing details of the electrode unit, FIG. 3 is a plan sectional view of the electrode unit, and FIG. FIG. 5 is a diagram showing another embodiment in which optical measurements are simultaneously performed. FIG. 6A is a diagram showing the oxygen consumption reaction of mitochondria using the oxygen reaction container of FIG. 1. Figure 6B is a diagram showing the absorption spectrum of the red blood cell dispersion solution measured using the oxygen reaction vessel in Figure 5, Figure 6C is a diagram showing changes in oxygen concentration, and Figure 7 is a diagram showing the results. FIG. 1 is a diagram showing a conventional oxygen reaction vessel. 22 is an electrode unit, 27 is a container, and 30 is a magnetic stirring body. Note that the same reference numerals in each figure indicate the same or corresponding parts. Agent: Patent attorney Ryuji Kurushima (i-Chiku＼2 stones) 1st r.
M Figure 4 Figure 5 02 Drifting cliff (/JM)

Claims (5)

[Claims] (1) A container for storing a reaction liquid to be measured with a capillary opening at the top, a flat electrode unit having a composite oxygen measurement electrode arranged so that the electrode surface forms the bottom surface of the container, and an electrode unit. A magnetic field rotation means placed under the magnetic field rotation means, and a magnetic rotor placed unfixed on the electrode unit and rotated by the magnetic field rotation means to stir the solution to be measured in the container. Oxygen reaction vessel. (2) The electrode unit is characterized in that at least a part thereof is made of a thermally conductive material that is in thermal contact with the reaction solution in the container, and is provided with constant temperature heating means therein. An oxygen reaction vessel according to claim 1. (3) The container is made of a material that is in thermal contact with the reaction solution, is provided with a container support that is in thermal contact around the container, and is provided with constant temperature heating means within the container support. The oxygen reaction vessel according to claim 1, characterized in that: (4) The oxygen reaction container according to any one of claims 1 to 3, wherein the container is made of glass or plastic. (5) The container is housed in a light-shielding container, a light transmitting means for optical measurement is provided on the side surface of the container, and a light receiving means for optical measurement is provided on the side surface facing the light transmitting means. An oxygen reaction vessel according to any one of claims 1 to 4.