JPH06317548A - Sample tube unit for esr measurement and capillary for esr measurement used in the unit - Google Patents

Abstract

PURPOSE:To perform an ESR measurement by a simple operation by using a capillary unit for ESR measurement because the material of the unit used in a sample tube unit for ESR measurement is low-cost and disposable. CONSTITUTION:A sample tube unit for ESR measurement is composed of a blind pipe 8 having a shape suitable for being inserted into, and fixing, a cavity resonator 13 for an ESR measuring apparatus, of a capillary 9 which can be inserted into the blind pipe 8 and which is composed of a material selected from an acrylic resin, polycarbonate, Teflon, glass, quartz and the like and of a capillary end-part blockade material 10 which is composed of wax, polyvinyl chloride, a silicone resin, Teflon wax, their mixture, a mixture of them with fluid paraffin and the like. A capillary unit for ESR measurement is composed of the capillary 9 and the capillary end-part blockade material 10 which are used in the unit.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample tube unit for ESR measurement and an ESR measuring thin tube unit used in the unit.

[0002]

2. Description of the Related Art Free radicals such as active oxygen generated in the living body cause cancers and diseases, and have therefore been extensively studied in the medical field in recent years. A method of observing and analyzing free radicals using an electron spin resonance (EPR / ESR) method for an aqueous solution sample is widely used for the study of such free radicals.

The above-mentioned aqueous solution sample has a problem that the dielectric loss of microwaves is large during ESR measurement because it has a high dielectric constant of water as a solvent. Therefore, for the aqueous solution sample, a sample cell having a special shape has been used in order to obtain the necessary sensitivity while keeping the dielectric loss low. Specifically, a "flat cell" in which the sample holding portion is flat and a "cylindrical aqueous solution cell" in which the sample is held in a part of a thick quartz tube are used.

Of these, the flat cell includes a sample cell main body (1) having a flat sample holder (4) as shown in FIG. 1, a holder (2), an aspirator (not shown) and It consists of a cap (3). In use, the holder (2) is fixed to the upper part of the sample cell body by screwing, and the sample is sucked into the sample holding part by an aspirator,
The cap is fitted to the lower part and the cell is set in the measuring device, but at this time, the holder and the aspirator need to be fitted evenly on the left and right, and the handling is troublesome. is there. In addition, after the measurement, it is necessary to disassemble the cell to wash and dry it. Therefore, it is difficult to finish this within a limited time when measuring systematic samples or multiple samples.
Especially when measuring free radicals such as active oxygen having a relatively short lifetime, a great deal of skill is required for the experimental operation.
In addition, a flat cell requires a relatively large amount of sample, about 100 μl. Further, quartz is used as the flat cell material, resulting in a high cell unit price, and it is financially difficult to use many of them.

On the other hand, the "cylindrical aqueous solution cell" is, as shown in FIG. 2, a sample tube (5) and a cap (6) for closing both ends.
And a sample cell holder (7) fitted in the sample tube to determine the setting position on the ESR resonator.
The “cylindrical aqueous solution cell” requires a comparatively small amount of sample, but since quartz is used, the cell unit price is high, and since it is a thin tube, it is difficult to clean the inside.

Further, when the flat cell is inserted into a cavity resonator for ESR measurement (hereinafter referred to as "resonator"), the cell must be properly set in the resonator so that the signal intensity of the sample to be measured is maximized. I won't. Furthermore, when these cells are used for measurement of protein samples or the like, they gradually become soiled and must be washed with concentrated nitric acid, sodium hydroxide or the like. If it is dried without sufficient washing, proteins and the like adhere to it, making it difficult to remove.

[0007]

As a result of studying an ESR measurement sample tube unit that solves the above problems, the present inventors have found that the sample tubes are an internal sample tube (sample holding tube) and an external sample tube (resonator). The internal sample tube is removable, and the lower end of the internal sample tube is closed with a blocking material during measurement, resulting in extremely easy sample replacement. It has been found that the method is extremely excellent for measuring radicals having a relatively short life such as active oxygen such as OH. Also, at this time, there is no problem in ESR measurement as the internal sample tube, a disposable material is used, and a viscous resin or the like is used as the occluding material, which enables quick and highly accurate measurement. Was confirmed and the present invention was completed.

[0008]

That is, the present invention relates to an ES including a blind tube having a shape adapted to be fixed in a resonator of an ESR measuring device, a thin tube insertable into the blind tube, and a narrow tube end blocker.
A sample tube unit for R measurement is provided. Further, the present invention is
Provided is an ESR measuring thin tube unit for use in the above unit, which comprises a thin tube and a tube end blocker.

The blind tube having a shape adapted to be fixed in the resonator of the ESR measuring device has an outer diameter and a shape which can be inserted into the resonator of the ESR measuring device to be used, and whether the lower end is closed. Any member can be used as long as it can be closed.
For example, a quartz sample tube for ESR measurement having a diameter of about 5 mm, which is a commercial product conventionally used for measuring an organic solution sample or a powder sample, is used. This is easy to set in the resonator and can be used as it is.

The thin tube used in the present invention has an effective length of about 3 cm as a sample liquid portion, and has a total length including a block portion by a occluding material, which will be described later, and a space portion interposed between the occluding material and the sample liquid. Any material having a length of 4 cm or more may be used. If the length is less than 4 cm, it is difficult to insert the sample and perform the clogging operation with the clogging material, and in terms of ESR measurement,
Sufficient sensitivity cannot be obtained. 5cm-10cm, especially 6c
It is preferably m to 8 cm.

The inner diameter (diameter) of the thin tube is preferably about 0.5 mm or more. If the inner diameter of the thin tube is less than 0.5 mm, sufficient sensitivity cannot be obtained in measuring ESR. If the inner diameter of the thin tube becomes large, the dielectric loss becomes large, and sufficient sensitivity cannot be obtained in measuring ESR. In the case of commonly used solvents, the upper limit of the inner diameter is about 2.0 mm. Outside diameter of thin tube
Is preferably as thin as it is possible to secure sufficient strength for performing the closing operation with the closing material.

The thin tube may be a simple cylindrical thin tube, but may be partially enlarged as long as it has a thin tube portion satisfying the above conditions over a length of 4 cm or more. For example, a thin tube whose lower end side is a thin tube portion and whose upper end side has a larger diameter than this is suitable for measuring a powder sample because it is relatively easy to insert a sample from the upper end of the tube. Further, when inserted into the blind canal, the ampulla abut against the inner wall of the blind canal to restrict movement in the blind canal, which is suitable for appropriately positioning the thin canal in the blind. The material of the thin tube is
There is no particular limitation as long as it does not output the ESR signal. Preferred materials include acrylic resins such as polymethacrylic acid ester (PMMA), polycarbonate,
Examples include Teflon, glass, and quartz. Acrylic resin is particularly preferable from the viewpoint that a tube having a relatively precise shape can be manufactured at low cost, and a disposable sample tube can be used, so that the labor for experiments such as washing and drying of the sample is extremely small.

A cap made of the same material as or a material similar to the capillaries may be used as the capillaries for the capillaries, but wax and wax-like resin can be preferably used. Specific examples thereof include wax, polyvinyl chloride, silicone resin, Teflon wax, and mixtures thereof, and mixtures of these with liquid paraffin. The ESR measurement using the measuring tube unit of the present invention can be performed as follows (see FIG. 3). First, one end of the thin tube (9) is immersed in a sample solution prepared according to a conventional method.
Due to the capillary phenomenon, the sample is sucked up into the capillary, but when the required amount (4.5 cm or more in length) of the sample is sucked up, pull the capillary out of the sample solution while closing the upper end of the capillary with your finger. . Next, when the capillary is made horizontal and the blockage on the upper end side is released, the sample layer as a whole moves to the upper end side, and a small amount of space (12) is formed on the lower end side. In a state where there is a space, the upper end of the thin tube is closed with a finger again, and the lower end of the thin tube in which the space is formed is pressed against the occluding material. When the thin tube is pulled up from the occluding material, a tubule containing a measurement sample whose lower end is closed by the occluding material (10) across a space (air layer) is formed. The air layer (1
2) prevents contact between the occluding material and the sample. Wipe the lower end of the thin tube in contact with the occluding material with Kimwipe (trade name, laboratory wipes, manufactured by Jujo Kimberley) or gauze, and insert it into a suitable blind tube (8) such as a general sample tube for EPR measurement. This is introduced into the EPR resonator (13) and used for measurement. FIG. 3 shows the drawing when set in the resonator. At this time, the closed portion at the lower end of the thin tube is to be outside the resonator. The determination of free radicals and the like may be performed according to a conventional method.

In the measurement of a sample that requires variable temperature (for example, 0 ° C. to 30 ° C.), a heat medium (for example, glycerin etc.) is previously put in the blind tube, and a thin tube containing the sample is introduced into this. If necessary, a thin tube can be fixed to the blind tube by using bamboo rod or aluminum rod, etc., and it becomes possible to study the generation or change of radicals or the dynamics of radicals by temperature. ESR measurement of radicals generated by light irradiation can be performed in the same manner as in the ordinary method by using quartz as the material of the thin tube.

[0015]

As described above, the ESR measuring thin tube unit used in the ESR measuring sample tube unit of the present invention is
Since the material is inexpensive, it does not need to be washed and used repeatedly, it can be disposable, and can be used in large quantities in experiments. By using this unit, it is possible to perform measurement with simple operation, so it is particularly effective for continuous measurement of multiple samples such as systematic measurement and measurement of free radicals with a relatively short life. . In addition to this, the sample volume is about 1/3 of that of the conventional flat cell, which is also useful for measuring an expensive sample. The sample tube unit for ESR measurement of the present invention not only solves the problems of the conventional method, but also has various application fields in addition to the usage of the conventional method. When using a chemical system other than biology or medicine, for example, an organic solvent or a mixed solvent with an organic solvent, or for measuring a polymer system or a powder solid sample in which an unpaired electron is involved, the capillaries and the occluding material of the present invention are used. By selecting within the range, it becomes possible to detect radicals and paramagnetic species in the sample.

[0016]

EXAMPLES The present invention will be specifically described below with reference to examples, but these are examples for the purpose of description and the present invention is not limited thereto.

Example 1 PMMA having an inner diameter of 0.8 mm, an outer diameter of 1.3 mm and a length of 6.5 cm
A sample tube unit of the present invention in which a sample tube unit composed of a capillarity made of a mixture of polyvinyl chloride, silicone resin and liquid paraffin, and a commercially available quartz sample tube for measuring 5 mm diameter ESR and a conventional quartz tube are combined. As shown in FIG. 3, each of the flat cells (ESR sample cell of FIG. 1, LLC04A, manufactured by Labotech Co., Ltd.) manufactured by Japan Electronics Co., Ltd. was set in a measurement device (JES RE3X electron spin resonance device manufactured by JEOL Ltd.). ES of nitroxide radical
R measurement was performed. Sample solutions are 1, 1,
5,5-tetramethyl-4-hydroxypiperidine-1
Although a 5 mM aqueous solution of -N-oxide (TEMPOL) was used, 100 µl of the sample was used for the flat cell, whereas the unit of the present invention used only 30 µl of the sample. Other measurement conditions (resonance frequency: 9.1GH
z, microwave output: 10 mW, magnetic field modulation width: 0.1 m
T) are the same. The results are shown in FIGS. 4 and 5. Note that the gain is common to 1 × 1.6 in FIGS. 4 and 5. Although an inexpensive acrylic resin was used as the thin tube and the sample volume was set to about 1/3 of that of the conventional flat cell, the signal intensity was only reduced by about 10% as compared with the case of using the flat cell.

Example 2 Using the same sample tube unit of the present invention as in Example 1, 5 μ
An attempt was made to measure a low concentration TEMPOL aqueous solution of M at room temperature (measurement conditions are as follows. Resonance frequency: 9.105G
Hz, microwave output: 10 mW, magnetic field modulation width: 0.1
mT, gain: 5 × 10 ² ). Results are shown in FIG. When the sample tube unit of the present invention is used as shown in the figure,
It is possible to easily measure low-concentration radicals.

Example 3 Using a sample tube unit similar to that of Example 1, superoxide (hypoxanthine (HPX)) in a biological system was used.
/ Xanthine oxidase (XOD) system) spin adduct was measured (measurement conditions are as follows: resonance frequency: 9.101 GHz, microwave output: 10 m)
W, magnetic field modulation width: 0.1 mT, gain: 5 × 10 ² ).
HPX (2 mM) 50 μl, ligand: diethylenetriaminepentaacetic acid (DETPAC) (5.5 mM) 35 μl
1, spin trap agent: 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (9.2M) 10 μl,
Enzyme: Xanthine oxidase (XOD) ~ 0.6 Uni
E of reaction product (spin adduct) of superoxide and DMPO produced in a mixed system of 50 μl of t / ml
The SR spectrum is shown in FIG. 7.

Example 4 An ESR measurement of an organic solvent sample was conducted using the same sample tube unit as in Example 1 (measurement conditions are as follows: resonance frequency: 9.106 GHz, microwave output: 2 mW,
Magnetic field modulation width: 0.05 mT, gain: 3.2 × 1
0 ² ). Specifically, 48 μM of 1,1,5,5-tetramethylpiperidine-1-N-oxide (TEMPO)
An ethanol solution was used as a sample. The results are shown in Fig. 8.

Example 5 A thin tube portion (inner diameter 0.8 mm, outer diameter 1.8 m) as shown in FIG.
m, length 5 cm) and enlarged part (inner diameter 2 mm, outer diameter 3 mm,
ESR measurement of a solid sample by filling a powder sample from the upper part using an acrylic resin thin tube having a length of 3 cm (measurement conditions are as follows: resonance frequency: 9.105 GHz,
Microwave output: 2 mW, magnetic field modulation width: 0.1 mT, gain: 4 × 10 ² ) were performed. As a sample, about 25 μg of a mixed crystal (Cu complex content: about 1%) consisting of a Zn-cimetidine complex and a Cu-cimetidine complex was used. The results are shown in Fig. 10.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a sample tube unit having a conventional flat cell structure for performing ESR measurement.

FIG. 2 is an explanatory diagram of a conventional sample tube unit having a cylindrical aqueous solution cell structure for performing ESR measurement.

FIG. 3 is an explanatory diagram showing a method of using a sample tube when performing ESR measurement using the sample tube unit of the present invention.

FIG. 4 is an ESR spectrum of a nitroxide radical aqueous solution sample obtained by using the sample tube unit of the present invention.

FIG. 5 is an ESR spectrum of a nitroxide radical aqueous solution sample obtained using a conventional flat cell.

FIG. 6 is an ESR spectrum of a low concentration nitroxide radical aqueous solution sample obtained by using the sample tube unit of the present invention.

FIG. 7 is an ESR spectrum of a biological sample obtained by using the sample tube unit of the present invention.

FIG. 8 is an ESR spectrum of an organic solvent sample obtained by using the sample tube unit of the present invention.

FIG. 9 is a cross-sectional view of a sample tube unit according to one embodiment of the present invention.

FIG. 10 is an ESR spectrum of a mixed crystal sample (Cu-cimetidine complex) obtained by using the sample tube unit of the present invention.

[Explanation of symbols]

 1 Conventional technology ESR sample tube (flat cell) main body 2 Holder 3 Cap 4 Sample holding part 5 Conventional technology ESR sample tube (aqueous solution cell) main body 6 Cap 7 Sample tube holder 8 Blind tube 9 Sample insertion thin tube 10 Capillary occlusion material 11 sample 12 space 13 resonator

Claims (6)

[Claims] 1. A sample tube unit for ESR measurement comprising a blind tube having a shape adapted to be inserted and fixed in a cavity resonator of an ESR measuring device, a thin tube insertable into the blind tube, and a narrow tube end blocker. 2. The sample tube unit for ESR measurement according to claim 1, wherein the thin tube is made of a material selected from acrylic resin, polycarbonate, Teflon, glass or quartz. 3. The E according to claim 2, wherein the capillaries are made of polyacrylic acid ester or polymethacrylic acid ester.
Sample tube unit for SR measurement. 4. The occlusive material is made of a material selected from wax, polyvinyl chloride, silicone resin, Teflon wax, a mixture thereof, or a mixture of these and liquid paraffin. The sample tube unit for ESR measurement according to 1. 5. The thin tube has an inner diameter of 0.5 to 2.0 mm and 4.5 cm.
The sample tube unit for ESR measurement according to any one of claims 1 to 4, which has the above length. 6. An ESR comprising the capillary tube and a capillary tube end blocker according to claim 2, which is used in the sample tube unit for measuring ESR according to claim 1.
Capillary unit for measurement.