JP2764540B2 - Dialysis membrane probe, connecting tube and continuous measuring device for glutamate in vivo - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dialysis membrane probe and a connecting tube capable of continuously measuring a substance collected over time from an object to be measured, and a continuous measuring apparatus for glutamic acid in a living body. In particular, a dialysis membrane probe for continuous measurement that enables stable and highly sensitive analysis without noise,
The present invention relates to a connecting pipe and a continuous measuring device.

[0002]

2. Description of the Related Art Conventionally, methods for measuring substances in a living body using various reactions have been used. Such measurement methods include, for example, a microdialysis method, a biosensor method,
There is a voltammetry method and the like. Of these measurement methods, the biosensor method must be converted into an electrical measurement system, so the measurement substances are limited.The voltammetry method measures the oxidation-reduction potential of the substance directly, The method cannot be applied to substances having no electric potential, and therefore, the microdialysis method is superior in general versatility.

[0003] In these measurement methods, a method is usually employed in which an object to be measured is collected for a certain period of time, and the collected substance is analyzed. The probe and connecting pipe used for the measurement are also used in such a method. Designed for the method. However, if the measurement of a substance can be performed in real time, it is easy to imagine that it is highly possible to discover a fact that could not be found by the above method. Therefore, it has been proposed to perform such continuous measurement (Analytica Chimica Acta, 2).
05 (1988) 53-59, Brain Research.
rch, 475 (1988) 58-63), and noise (pulsation) could not be stably analyzed with high sensitivity. This is because the probe and the connection pipe described above are not designed in consideration of continuous measurement, and there is a portion (air retention portion) where air serving as a noise source can accumulate.

FIG. 4 is a sectional view showing a conventional dialysis probe. The dialysis probe 4 includes a tubular main body 41,
A dialysis membrane tube 42 provided at one end of the main body 41 and having a closed end, and a thin tube 4 passing through the dialysis membrane 42 and the main body 41.
3a, a thin tube 43b reaching the middle of the main body, and respective thin tubes 43a, 43 to increase the outer diameter of the thin tube.
3b, the main body 41, the stainless steel tubes 44a, 44b, and the thin tubes 43a, 4b.
And a holding member 45 for holding 3b.

The capillary 43a of the dialysis probe 4
FIG. 5 shows the connection between the (43b) and the stainless steel pipe 44a (44b). The connection part is a stainless steel tube 44a (44
b) and the boundary between the thin tube 43a (43b) inserted into the stainless steel tube 44a (44b) and the adhesive member 46 on the surface.
a gap (air retention portion) D exists between the thin tube 43a (43b) and the stainless tube 44a (44b).

[0006] Such an air stagnation portion D is provided with a thin tube 43a.
It is present not only at the connection between the (43b) and the stainless steel tube 44a (44b) but also at the rear end of the adhesive member 46b fixing the dialysis membrane tube 42 in the main body 41. In the dialysis probe having the air stagnation portion as described above, air accumulates in the air stagnation portion at the time of measurement, and the measurement system responds to even a small change in water pressure to increase the change in flow velocity, so that accurate analysis cannot be performed. In addition, the air itself that has accumulated in the air stagnation section may flow out and cause noise.

Although there is a means of increasing the flow rate in order to suppress noise, new problems such as a cooling effect of the living body and a decrease in the sample recovery rate at the dialysis membrane caused by the high flow rate occur, and High sensitivity analysis cannot be performed while maintaining the specificity, specificity, and stability.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a high-recovery rate of a substance to be measured, a low rate of noise (pulsation), a low-cost, general-purpose, high-sensitivity, stable analysis. It is to provide a simple dialysis membrane probe. Another object of the present invention is to provide a connecting pipe which has a low noise generation rate and can perform highly sensitive and stable analysis. Still another object of the present invention is to provide a continuous measurement apparatus for in vivo glutamic acid which has a low generation rate of noise (pulse flow) and can perform highly sensitive and stable analysis.

[0009]

Means for Solving the Problems In view of the above problems, as a result of earnest studies, the present inventors have prepared a dialysis membrane probe and a connecting tube having no gap between a thin tube and a thick tube, and used them for measurement. For example, they found that the rate of noise generation was low, and high-sensitivity and stable analysis could be performed. Thus, the present invention was completed.

That is, the present invention comprises a dialysis membrane tube, one of which is closed, two thin tubes passing through the dialysis membrane tube, and a large tube connected to a liquid feed tube. A dialysis membrane probe comprising a connecting means for connecting the thin tube and the thick tube, wherein the connecting means connects the thin tube and the thick tube so that no gap is formed.

[0011] Further, the present invention provides a method for converting the flow of liquid flowing through n liquid feed pipes, which is an integer of at least two or more, into the flow of liquid flowing through one liquid feed pipe or one liquid feed pipe. A connection pipe for converting the flow of the liquid flowing through the pipe into the flow of the liquid flowing through n liquid feed pipes, which is an integer of at least 2 or more.
N thin tubes and n connected to each of the n thin tubes
In a connecting pipe having three thick pipes and a thick pipe connected to the bundle of n thin pipes, the n thin pipes and the (n +
1) The thick pipe is a connecting pipe for dialysis, which is connected without any gap. Further, the present invention provides a device for discharging a Ringer solution and an enzyme solution, a first injector and a second injector installed in the device for discharging the Ringer solution and the enzyme solution at a constant speed, The first dialysis membrane probe to be input, the first injector and the dialysis membrane probe are connected to a first liquid supply pipe through which a Ringer solution flows, and the second injector is connected to the enzyme solution, A second liquid supply pipe flowing therethrough, a third liquid supply pipe through which a Ringer's solution containing glutamic acid obtained from the living body through the dialysis membrane probe flows, and the second liquid supply pipe and the third liquid supply pipe. A connecting pipe that combines the liquid sending pipes into one pipe,
In a device for continuously measuring glutamic acid in a living body, comprising: a fourth liquid feed pipe combined by the connecting pipe, and a detector connected to the end of the fourth liquid feed pipe,
The dialysis membrane probe includes a dialysis membrane tube one of which is closed, two thin tubes passing through the dialysis membrane tube, and a thick tube connected to a liquid sending tube. Is a dialysis membrane probe connected so as not to form a gap, wherein the connecting tube is composed of two thin tubes, two thick tubes connected to each of the two thin tubes, and the two thin tubes. A thick pipe connected to a bundle of thin pipes, wherein the two thin pipes and the three thick pipes are connected pipes with no gap therebetween. It is a continuous measuring device for glutamate in the body.

Hereinafter, the present invention will be described in detail with reference to the drawings. [1] Dialysis Membrane Probe FIG. 1 is a schematic diagram showing an example of a dialysis membrane probe that can be used in the present invention. Dialysis membrane probe 1 of the present invention
Is a dialysis membrane tube 11 one of which is closed by a sealing material 14b.
And two thin tubes 12 passing through the dialysis membrane tube 11 and a large tube 13 fitted to each of the thin tubes 12 so as to be connected to the liquid feed tube. The sealing material 14a is filled between each of the thin tubes 12 and the thick tubes 13, and there is no gap. As described above, by eliminating the gap between the thin tube 12 and the thick tube 13, a portion where air can accumulate (air retention portion) is completely eliminated.

Further, in this embodiment, the opening of the dialysis membrane tube 11 is sealed by the sealing material 14b so that the thick tube 13 on the dialysis membrane tube 11 side can be used.
Of the dialysis membrane tube 11 and the thin tube 12 between the dialysis membrane tube 11 and the large tube 13 are sealed with a sealing material 14c. The tubule 12b has reached the tip, and the other thin tube 12b has reached halfway through the dialysis membrane tube 11.

In the present invention, "sealing" refers to bonding and fixing each member with a resin or the like so that there is no gap. Therefore, the sealing material 14c is also filled from the upper end of the dialysis membrane tube 11 to the substantially lower end of the thin tube 12b (A in the figure). The thickness and length of the dialysis membrane tube 11 in the dialysis membrane probe 1 used in the present invention are as follows:
It varies widely depending on the object, but usually the thickness is 0.1 to
It is preferably about 0.5 mm, and the length is 2 to 10 m
m is preferable.

As the material of the dialysis membrane forming the dialysis membrane tube 11, any material may be used as long as it is generally used, for example, a cellulosic membrane, various filtration membranes (polysulfone, polycarbonate, etc.), A sintered filter or the like can be used. Further, the thickness of the dialysis membrane and the size of the holes may be appropriately set depending on the type of the measurement target,
Usually, the thickness is about 5 to 20 μm, and the size (diameter) of the hole is about 0.1 to 20 μm.

A sealing material 14b for closing the tip of the dialysis membrane tube 11
May be made of any material. For example, an adhesive such as an epoxy resin or a synthetic resin such as Teflon can be used. The lower end of the thin tube 12a according to the present invention is shown in FIG.
However, the position of the lower end is not particularly limited as long as the distance from the other thin tube 12b can be maintained to some extent and the perfusion solution can sufficiently elute the measurement target.

Similarly, the position of the lower end of the other thin tube 12b is not particularly limited as long as the object to be measured can be sufficiently eluted into the perfusate while maintaining a certain distance from the thin tube 12a. In order to avoid stagnation, it is preferable to match the lower end of the sealing material 14c. Thin tube 12
a and 12b are dialysis membrane tubes 11 to facilitate piping.
Bend in opposite directions above
It is preferably in the shape of a letter.

The thickness (inner diameter) of the thin tube 12 is generally set to a value of 0.1.
It is about 0.5 to 0.2 mm. As the material of the thin tube 12, any material may be used as long as it is generally used. For example, Teflon, vinyl chloride, polyimide resin-coated quartz tube (PFS), fused silica, or the like may be used. Can be used. The upper end surface of the thick tube 13 is flush with the upper end surface of the thin tube 12 in FIG. 1, but if there is no gap between the thin tube 12 and the thick tube 13 and an air stagnant portion is not formed, the thick tube 13 Only 13 may protrude upward. The thickness (outer diameter) of the large pipe 13 is preferably about 0.2 to 0.8 mm so as to easily connect a liquid feed pipe that is usually used, and the large pipe 13 protrudes outside the sealing material 14a. The length of the thick pipe 13 is preferably about 3 to 10 mm.

The material of the thick tube 13 is not particularly limited as long as it can be jointed with the liquid sending tube, but it is preferable to use a synthetic resin such as stainless steel or Teflon in consideration of durability, adhesion and the like. Length A for sealing material 14c to seal dialysis membrane tube 11
Is not particularly limited as long as the dialysis membrane tube 11 and the thin tube 12 can be sufficiently fixed, and is preferably about 0.5 to 5 mm. The material of the sealing material 14c is used to bond and adhere each member so that there is no gap.
Any material can be used as long as it can be fixed. For example, epoxy resin, dental cement, various adhesives, and the like can be used, and they can be used in combination.

In the dialysis membrane probe 1 of this embodiment, the thick tube 1
Although the sealing material 14a is used to fill the gap between the thin tube 3 and the thin tube 12, the present invention is not limited to this. For example, if a thick tube having the same inner diameter as the outer diameter of the thin tube is used. There is no need to use a sealing material. In the dialysis membrane probe 1 of the present embodiment, the opening of the dialysis membrane tube 11, the end of the thick tube 13 on the dialysis membrane tube 11 side, and the thin tube 12 between the dialysis membrane tube 11 and the large tube 13 are sealed. Although the sealing is performed later by the adhesive material 14c, the present invention is not limited to this. For example, members other than the dialysis membrane tube are integrally formed at the same time, and this and the dialysis membrane tube are adhered. Is also good.

In order to use the dialysis membrane probe 1 of the present invention, for example, for a living body, first, a liquid feed pipe is connected to each of the thick pipes 13 and a dialysis membrane portion (from the tip of the dialysis membrane pipe to the lower end of the sealing material) B) in the figure is stimulated into the living body. Next, the perfusate flows into the thin tube 12a from one of the liquid feed tubes, and the inside of the dialysis membrane tube 11 is filled with the perfusate. The measurement target elutes into the perfusate through the dialysis membrane, and flows out from the other thin tube 12b together with the perfusate.

At this time, since the dialysis membrane probe of the present invention having the above-described structure has no portion where air can accumulate (air retention portion), if the dialysis membrane probe is used, the dialysis membrane probe will The resulting noise generation rate is extremely low, and high sensitivity and stable continuous measurement can be performed. Further, the dialysis membrane probe of the present invention has a simple structure and can be easily manufactured at low cost. Furthermore, since the perfusate and the measurement object pass through the inactivated space and do not come into contact with metal or the like, there is no danger of their own denaturation.

By using the dialysis membrane probe of the present invention, it is possible to continuously measure chemical substances in a living body as described above. For example, measurement of glutamate in the brain, various other amino acids, neurotransmitters, neurotransmitter modifiers,
It is possible to measure substances leaked out of cells such as sugar metabolism-related substances. In addition, the present invention is not limited to experimental biology and clinical testing techniques for living organisms, but can be applied to various fields such as research on reaction processes of inorganic or organic chemistry and monitoring of environmental conditions. [2] Connecting pipe The connecting pipe of the present invention converts the flow of liquid flowing through a plurality of liquid feed pipes into the flow of liquid flowing through one liquid feed pipe into the flow of liquid flowing through the plurality of liquid feed pipes. For the sake of simplicity, the following description will be made taking a case of two flows as an example.

FIG. 2 is a schematic view showing an example of the connecting pipe of the present invention. The connecting pipe 2 of the present invention includes two thin tubes 21a, 2a.
1b, a thick pipe 22a externally fitted to the thin tube 21a and a thick pipe 22b externally fitted to the thin tube 21b, and a two thin tubes 21 in the side where the two thin tubes 21a and 21b are branched.
a, 21b has a thick tube 22c externally fitted to a bundle of two thin tubes 21a, 21b,
A sealing material 2 is provided between the thin tube 21a and the thick tube 22a, between the thin tube 21b and the thick tube 22b, and between the thin tubes 21a and 21b and the thick tube 22c.
3a is filled and there is no gap.

As described above, by eliminating the gap between each narrow tube and the thick tube, there is no portion where air can accumulate (air stagnant portion), and therefore, the rate of occurrence of noise caused by the air stagnant portion Is very low, and highly sensitive and stable continuous measurement can be performed. In the connecting pipe of this embodiment,
Thin tubes 21a, 21b connecting the ends of the thick tubes 22a, 22b, 22c to each other, and thick tubes 22a, 22b, 2
2c is sealed with a sealing material 23b,
Each of the thin and thick tubes is fixed.

The thickness (inner diameter) of the thin tubes 21a and 21b is generally preferably about 0.05 to 0.2 mm.
As the material of the thin tubes 21a and 21b, any material may be used as long as it is generally used. For example, Teflon, vinyl chloride, fused silica, P.I. F. S. Etc. can be used.

Although the end faces of the thick pipes 22a, 22b and 22c are on the same plane as the end faces of the thin pipes 21a and 21b in FIG. 2, there is no gap between the thin pipe and the thick pipe, and an air stagnation portion is formed. If not, only the thick tube may protrude. The thickness (outer diameter) of the thick pipe is preferably about 0.2 to 0.8 mm so that the commonly used liquid feed pipe can be easily connected, and the thick pipe that is exposed outside the sealing material is used. Length is 3-10m
m is preferable.

The material of the thick pipes 22a, 22b and 22c is not particularly limited as long as it can be connected to the liquid feed pipe. However, it is preferable to use a synthetic resin such as stainless steel or Teflon in consideration of durability and adhesion. The materials of the sealing materials 23a and 23b are as follows.
Any material can be used as long as each member can be bonded and fixed. For example, epoxy resin, dental cement, various adhesives, and the like can be used, and they can be used in combination.

In the connecting pipe 2 of this embodiment, the sealing material 23a is used to fill the gap between the thick pipe and the thin pipe. However, the present invention is not limited to this, and for example, the outside of the thin pipe may be used. If a large tube having the same inner diameter as the diameter is used, there is no need to use a sealing material. Further, in the connecting pipe of the present embodiment, the thick pipe and the thin pipe between the thick pipe are sealed later by a sealing material, but the present invention is not limited thereto, for example, The thick tube and the thin tube may be integrally formed at the same time.

By using the connecting pipe described above,
Continuous measurement of glutamic acid in a living body can be stably performed with high sensitivity. In addition, not only glutamic acid but also various other amino acids, neurotransmitters, neurotransmitter modifiers, glucose metabolism-related substances and other substances leaked out of cells can be measured. Experimental biology,
The present invention can be applied to various fields such as a study of a reaction process of inorganic or organic chemistry and a monitoring of an environmental condition without being limited to a clinical examination technique.

[3] Apparatus for continuous measurement of glutamate in vivo The apparatus for continuous measurement of glutamate in vivo according to the present invention comprises the above-described dialysis membrane probe of the present invention and a connecting tube. An example of the continuous measurement device for in vivo glutamic acid of the present invention will be described below. FIG. 3 is a schematic diagram showing an apparatus for continuously measuring the amount of glutamic acid in the brain of a rat by microdialysis in the brain.

The present apparatus is connected to a syringe pump 31 for discharging the Ringer solution and the enzyme solution, injectors 32a and 32b installed in the syringe pump 31 for discharging the Ringer solution and the enzyme solution at a constant speed. And a liquid sending pipe 33a through which the Ringer solution flows, and a liquid sending pipe 33b which is connected to the injector 32b and through which the enzyme solution flows.
A loop injector 34 provided in the middle of the liquid sending pipe 33a, a dialysis membrane probe 1 stimulated into a rat,
A dual sebel 35 for reciprocating the Ringer solution between the dialysis membrane probe 1 and a connecting pipe 2 for combining the liquid feeding pipe 33a from the dual sybel 35 and the liquid feeding pipe 33b from the injector 32b into one pipe; It has a liquid sending pipe 33c combined by the pipe 2, and a fluorescence detector 36 with a flow cell connected to the tip of the liquid sending pipe 33c.

A method for continuously measuring the amount of glutamate in the brain of a rat using the present apparatus will be described. First, the dialysis membrane probe 1 is inserted into the rat's inner frontal cortex and fixed by referring to the brain map. About 1 day after postoperative recovery, Ringer's solution is perfused from the injector 32a to the dialysis membrane probe 1 under the free movement of the rat. The Ringer's solution usually used conforms to the Pharmacopoeia Law of Japan, but may be a buffer of another biological system, and the flow rate is preferably about 0.5 to 2.5 μl / min.

The Ringer's solution is supplied to the loop injector 34.
And reaches the dialysis membrane probe 1 via the dual sebel 35. By using the loop injector 34, a pressure change which is a noise source can be reduced. Glutamic acid eluted through the dialysis membrane flows out of the dialysis membrane probe 1 together with the Ringer's solution, and travels to the connecting pipe 2 via the dual sieve 35. On the other hand, at the same time, the enzyme solution is poured into the connecting pipe 2 from the injector 32b,
It is combined with Ringer's solution containing glutamic acid and allowed to react for a certain period of time. The enzyme used here is glutamate dehydrogenase, and it is also possible to use glutamate oxidase. The flow rate of the enzyme solution may be the same as the flow rate of the Ringer's solution, and is preferably about 0.5 to 2.5 μl / min.

The reaction between the glutamic acid and the enzyme is carried out in
The reaction time is preferably about 1 to 5 minutes. By this glutamate dehydrogenation reaction, reduced β-nicotinamide adenine dinucleotide (NAD
Since H) is produced, the amount of glutamate in the brain can be measured and analyzed by continuously measuring the amount of NADH using the fluorescence detector 36 with a flow cell. The excitation wavelength in the fluorescence detector 36 is preferably about 340 nm, and the measurement wavelength is preferably about 450 to 460 nm. Further, the volume of the flow cell is preferably 5 μl or less.

In the continuous measuring device of the present invention, the use of the above-mentioned dialysis membrane probe and the connecting tube prevents generation of noise (pulse flow) and keeps the flow rate of the liquid feed (ringer solution, enzyme solution) constant. As a result, it is possible to suppress a change in the recovery rate of the measurement target due to a change in the flow rate of the perfusate, and to suppress the reaction solution (perfusate / enzyme solution containing the measurement target)
The change in the mixing ratio of each liquid due to the change in the flow rate can be suppressed, and highly reliable analysis can be performed in continuous measurement. Further, since there is almost no generation of noise (pulsating flow), there is no need to increase the flow velocity, and therefore, analysis with high sensitivity and excellent specificity can be performed.

[0037]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but these examples do not limit the scope of the present invention in any way.

Example 1 In this example, a dialysis membrane probe as shown in FIG. 1 was prepared. Dialysis membrane tube made of regenerated cellulose having a total length of 7 mm and an outer diameter of 0.23 mm (Asahi Kasei Co., Ltd., molecular weight cut: 5
One end of the (10,000) was closed with an epoxy-based adhesive (Chemedine, High Super 5). From the other end of the dialysis membrane tube, two thin tubes (made by ACOM Co., Ltd.) made of fused silica having an inner diameter of 0.1 mm, a total length of 20 mm, and 17 mm were inserted, and the portion A shown in FIG. Sealing was performed using a sealing material. The length of A was 4 mm.

The two thin tubes protruding from the dialysis membrane tube are passed through stainless steel tubes (manufactured by Terumo Corporation) each having a total length of 10 mm and an outer diameter of 0.55 mm, and the space between the two tubes is filled with the epoxy adhesive and protruded. Cut off the part. The two thin tubes and the dialysis membrane tube were sealed with a sealing material made of the above-mentioned epoxy adhesive so as to form a Y-shape, and then fixed with dental cement (manufactured by GC Dental Co., Unifast). . The length of the stainless steel tube coming out of the sealing material is
Each was about 5 mm.

Example 2 In this example, a connecting pipe as shown in FIG. 2 was manufactured. Two thin tubes (made by ACOM) made of fused silica having an inner diameter of 0.1 mm and a total length of 25 mm are passed through a stainless steel tube (manufactured by Terumo) having a total length of 10 mm and an outer diameter of 0.55 mm, and the narrow tube and the stainless tube are interposed. Was filled with an epoxy adhesive (High Super 5 manufactured by Cemedine Co., Ltd.), and the protruding portion was cut off.

Similarly, the two thin tubes protruding from the stainless steel tube are passed through a stainless steel tube having a total length of 10 mm and an outer diameter of 0.55 mm, and the space between the two is filled with the epoxy-based adhesive. Cut out. The thin tube and the stainless tube were sealed with a sealing material made of the above-mentioned epoxy adhesive so as to form a Y-shape, and then fixed with dental cement (manufactured by GC Dental Co., Unifast). The length of the stainless steel tube coming out of the sealing material is
Each was about 5 mm.

(Embodiment 3) This embodiment was performed with a configuration as shown in FIG. Before actually starting the experiment, a calibration curve of this measurement system was prepared as follows. The dialysis membrane tube portion of the dialysis membrane probe obtained in Example 1 was placed in a glutamic acid solution having a predetermined concentration,
A Ringer's solution (Ringer's solution, manufactured by Otsuka Pharmaceutical Co., Ltd.) was perfused at a flow rate of 2.5 μl / min from a syringe for high performance liquid chromatography (Model No. 9125, manufactured by Leodyne) to the dialysis membrane probe. In addition, as a syringe pump,
B. A. A CMA102 micro-injection pump manufactured by S Company, a Teflon tube (inner diameter: 0.1 mm) manufactured by Acom Co., Ltd. was used as a liquid sending tube, and 375 / D / 22QE manufactured by Instech Co., Ltd. was used as a dual sebel.

On the other hand, 2.5 μl of a glutamate dehydrogenase solution (127086, Boehringer Mannheim) was added.
The solution was fed at a flow rate of / min, combined with the above Ringer's solution (containing glutamic acid), reacted for 3 minutes, and passed through a 5 µl fluorescence detector equipped with a flow cell (manufactured by JASCO Corporation, 821FP). At this time, the excitation wavelength was set to 340 nm, and the measurement wavelength was set to 450 nm. The content of glutamic acid in the solution was adjusted to 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 3 μM
M, 3 μM, 3 μM, 3 μM and 3 μM over time.
The voltage was changed and the voltage detected by the fluorescence detector was printed out by a recorder connected to the fluorescence detector. FIG. 6 shows the results.

As is apparent from the graph of FIG.
Even if the measurement of glutamic acid is performed a plurality of times, almost the same results are obtained, and thus it can be said that the present measurement system has extremely high accuracy. FIG. 7 shows a calibration curve created from the graph of FIG.

Example 4 The dialysis membrane probe obtained in Example 1 was stimulated into the medial frontal cortex of a rat and fixedly connected to the skull. Postoperative recovery 1
One day later, 2.5 μl / Linger's solution was added to the rat under free movement.
The mixture was perfused at a flow rate of 1 min, and the glutamate dehydrogenase solution was combined with the Ringer's solution (containing glutamic acid from the brain) at a flow rate of 2.5 μl / min and reacted for 3 minutes.

60 minutes after the start of the measurement, the rats were given one of the stimulants.
1 seed of veratridine (VER) (manufactured by Sigma)
2.5 μl was injected, and 80 minutes after the start of measurement, sodium nitroprusside (SNP) (Wako), which is a reagent for changing nerve transmission efficiency, and 12.5 μl of VER were injected. 12.5μ
1 was injected. FIG. 8 shows the time course of the amount of glutamate secreted in the rat brain.

(Example 5) In Example 4, the second injection was SNP 12.5 μm.
Continuous measurement was performed in the same manner except that only 1 was used. FIG. 8 shows the results.

Example 6 In Example 4, the second injection was prepared by boiling SNPs.
Continuous measurement was performed in the same manner except that the VER was 12.5 μl. FIG. 8 shows the results. As is clear from the graph of FIG. 8, in the continuous measurement system using the dialysis membrane probe and the connecting pipe obtained in Examples 1 and 2, there is no generation of noise (pulsation flow), and high-sensitivity and stable analysis can be performed. Could be done.

Example 7 Continuous measurement was performed in the same manner as in Example 4, and a change with time in voltage was measured. FIG. 9 is a graph showing the relationship between the time and the detected voltage.

Comparative Example 1 Continuous measurement was performed in the same manner as in Example 7, except that the conventional dialysis probe shown in FIG. 4 was used. FIG. 10 is a graph showing the relationship between the time and the detected voltage. As is clear from the graphs of FIGS. 9 and 10, when the conventional dialysis probe is used, noise (pulsation flow) is generated and accurate measurement cannot be performed. On the other hand, the dialysis membrane probe obtained in Example 1 is used. Then, stable analysis with high sensitivity and little noise can be performed.

[0051]

According to the present invention, a dialysis membrane probe and a connecting pipe which can suppress generation of noise (pulsation flow) and enable high-sensitivity and stable continuous measurement can be easily obtained at low cost. The dialysis membrane probe and connecting tube of the present invention are used for experimental biology and clinical tests for measuring glutamate in the brain and blood, and measuring extracellular substances such as amino acids, neurotransmitters, and neurotransmitter modifiers. The present invention can be applied to various fields such as technology, research on reaction processes of inorganic or organic chemistry, and monitoring of environmental conditions. Moreover, according to the continuous measurement apparatus for in vivo glutamic acid of the present invention, generation of noise (pulsation) can be suppressed, and highly sensitive and stable analysis can be performed.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a dialysis membrane probe of the present invention.

FIG. 2 is a schematic view showing a connecting pipe of the present invention.

FIG. 3 is a schematic view showing a continuous measuring device for glutamic acid in a living body according to the present invention.

FIG. 4 is a sectional view showing a conventional dialysis probe.

FIG. 5 is a schematic view showing a connection portion between a thin tube and a stainless steel tube in a conventional dialysis probe.

FIG. 6 is a graph showing the relationship between the amount of glutamic acid and the detection voltage (temporal change) in Example 3.

FIG. 7 is a calibration curve showing the relationship between the amount of glutamic acid and the detection voltage in Example 3.

FIG. 8 is a graph showing the change over time in the amount of glutamic acid in Examples 4 to 6.

FIG. 9 is a graph showing a change over time of a detection voltage in Example 7.

FIG. 10 is a graph showing a change over time of a detection voltage in Comparative Example 1.

[Explanation of symbols]

1 ... dialysis membrane probe 11, 42 ... dialysis membrane tube 12, 12a, 12b, 21a, 21b, 43a, 43
b: Thin tubes 13, 22a, 22b, 22c ... Thick tubes 14a, 14b, 14c, 23a, 23b, 23c ...
-Sealing material 2-Connecting pipe 31-Syringe pump 32a, 32b-Injector 33a, 33b, 33c-Liquid feeding pipe 34-Loop injector 35-Dual sebel 36-・ Fluorescence detector 4 ・ ・ ・ Dialysis probe 41 ・ ・ ・ Main body 44a, 44b ・ ・ ・ Stainless steel tube 45 ・ ・ ・ Holding member 46a, 46b ・ ・ ・ Adhesive member

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI G01N 1/00 101 G01N 1/00 101F 1/10 1/10 V 21/78 21/78 C 33/68 33/68 (56 References JP-A-59-111736 (JP, A) JP-A-54-26783 (JP, A) JP-A-58-21847 (JP, U) WO 93/5701 (WO, A) BRAIN RESEARCH BULLETIN , VOL. 10, PP. 567-571, FIG. 1,1983, ANKHO INTERNATIONAL (58) Fields investigated (Int. Cl. ⁶ , DB name) A61B 5/00 A61B 5/14 310 A61B 10/00 103

Claims (6)

(57) [Claims] 1. A dialysis membrane tube, one of which is blocked, two thin tubes passing through the dialysis membrane tube, and a thick tube connected to a liquid sending tube, wherein the thin tube and the thick tube A dialysis membrane probe provided with connection means for connecting the thin tube and the thick tube so that no gap is formed. 2. The method according to claim 1, wherein an opening of the dialysis membrane tube, an end of the thick tube on the dialysis membrane tube side, and the thin tube between the dialysis membrane tube and the large tube are sealed. The dialysis membrane probe according to claim 1, wherein 3. The method according to claim 1, wherein the flow of the liquid flowing through the n number of liquid feeding pipes, which is an integer of at least two or more, is changed to the flow of the liquid flowing through one liquid feeding pipe, or the The flow
A connection pipe for converting a liquid flow flowing through n liquid feed pipes that is an integer of at least 2 or more, wherein the n thin pipes and the n thick pipes connected to the n thin pipes, respectively. In a connecting pipe having a thick pipe connected to the bundle of n thin pipes, the n thin pipes and the (n + 1) thick pipes are connected without any gap. Characterized by a connecting tube for dialysis. 4. The connecting pipe for a dialysis device according to claim 3, wherein the thin pipe connecting ends of the thick pipe and an end of the thick pipe are sealed. 5. A device for discharging a Ringer solution and an enzyme solution, a first injector and a second injector installed in the device for discharging the Ringer solution and the enzyme solution at a constant speed, A dialysis membrane probe to be connected, a first liquid supply pipe connected to the first injector and the dialysis membrane probe, through which a Ringer solution flows, and a second liquid injector connected to the enzyme solution. A second liquid supply pipe flowing through the dialysis membrane probe, a third liquid supply pipe through which a Ringer's solution containing glutamic acid obtained from the living body through the dialysis membrane probe flows, and the second liquid supply pipe and the third liquid supply pipe. A production pipe comprising a connecting pipe that combines the liquid feeding pipes into one pipe, a fourth liquid feeding pipe that is collected by the connecting pipe, and a detector connected to the end of the fourth liquid feeding pipe. An apparatus for continuously measuring glutamic acid in the body, comprising: The membrane probe includes a dialysis membrane tube one of which is closed, two thin tubes passing through the dialysis membrane tube, and a thick tube connected to a liquid sending tube, wherein the thin tube and the thick tube are connected to each other. A dialysis membrane probe connected so as not to form a gap, wherein the connecting tube is two thin tubes, two thick tubes connected to each of the two thin tubes, and the two thin tubes. In vivo glutamic acid, characterized in that the two thin tubes and the three thick tubes are connected tubes without any gaps. Continuous measuring device. 6. The method according to claim 1, wherein a loop injector is provided in the middle of the first liquid sending pipe, and a dual sebel is provided in the middle of the first and third liquid sending pipes. 6. The continuous measuring apparatus for glutamic acid in a living body according to claim 5.