JPH08220033A - Method for analyzing paramagnetic species - Google Patents

Abstract

PURPOSE: To selectively analyze a paramagnetic species existing in a very small area of a living body by detecting the electrical spin resonance of the paramagnetic species existing in an electric double layer from the variation of the impedance in the electric double layer. CONSTITUTION: A resonator 2 introduces electromagnetic waves of a fixed frequency generated from a signal source 1 and only efficiently radiates the magnetic field component of the electromagnetic waves upon a sample section 3. The section 3 is provided with a micro electrode 7 and reference electrode 8 inserted into a sample and a DC bias current flows to the electrodes from a power source 4. The sample section 3 is provided in a static magnetic field formed by a magnet 5 so that a sample can be put in a uniform static magnetic field. when the intensity of the magnetic field is slowly raised while the resonator 2 radiates the electromagnetic waves upon the section 3, a paramagnetic species existing in an electric double layer causes electric spin resonance when the intensity reaches a certain value and the impedance in the electric double layer varies. The paramagnetic species can be analyzed by detecting the impedance variation in the electric double layer as a signal after amplifying 6 the variation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing paramagnetic species having unpaired electrons and an analyzer used in the method. More specifically, the present invention relates to an analysis method capable of selectively analyzing paramagnetic species in a minute region and an apparatus used for the method.

[0002]

2. Description of the Related Art Paramagnetic species such as free radicals and transition metals existing in the living body are known to induce or aggravate various diseases such as cancer, inflammatory diseases, ulcers, cerebrovascular diseases, and myocardial infarction. ing. Therefore, the detection and analysis of paramagnetic species in the cellular level in the living body, that is, in the submicron microscopic region, is carried out to study the physiological action of paramagnetic species, and to diagnose, treat, and It is expected to be applicable to prevention.

An electron spin resonance method (ESR method) has been conventionally known as an excellent method for detecting unpaired electrons possessed by paramagnetic species in a substance in a highly sensitive and non-destructive state. According to the electron spin resonance method, it is possible to read various information of a substance having a paramagnetic species from the change and fluctuation range of the Larmor precession frequency of unpaired electrons in a magnetic field. However, the conventional electron spin resonance method merely observes the result (electron spin resonance absorption) of the electron spin resonance of the paramagnetic species present in the sample due to the microwave irradiated to the whole sample, It did not selectively obtain information on a specific minute area.

As a method for observing the electron spin resonance absorption at a specific site in a sample, there are used a Zougmatography using a magnetic field gradient and a microcoil with a miniaturized probe (Reference: High-sensitivity). broad ban
d microwave spectroscopy with small non resonant c
oils, Rev. Sci. Instrnm., 57 (6), 1986). However, it is practically impossible to achieve a spatial resolution in the submicron unit by these methods, and there is a problem that the measurement sensitivity decreases as the observation site (volume) is reduced. Another problem with these methods is that expensive microwave detectors are needed to observe microwave changes. In particular, a detector having high sensitivity and a wide dynamic range in a high frequency band close to a millimeter wave has not been known so far, which is a big problem in constructing the device.

On the other hand, when a bias voltage / current is applied to the microelectrode, it is known that an electric double layer is generated on the surface of the microelectrode and the interface thereof has a high resistance, so that the electric field concentrates on the interface. ing. As a result, the current flowing through the microelectrode reflects the characteristics of the interface, and therefore various measurement methods utilizing this phenomenon have been proposed in the fields of electrochemistry and the like. However, no example has been reported yet in which this phenomenon was applied to the electron spin resonance method to selectively obtain information on paramagnetic species having unpaired electrons.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for analyzing a paramagnetic species existing in a submicron minute region in a sample. More specifically, the present invention efficiently detects electron spin resonance absorption in a submicron microscopic region of a sample to detect, quantify, and identify paramagnetic species present in the microscopic region. It is intended to provide a method for performing analysis such as. From another point of view, it is also one of the objects of the present invention to provide a method for measuring electron spin resonance absorption of paramagnetic species in a minute region with high measurement sensitivity without requiring an expensive microwave detector. Is. Yet another object of the invention is to provide an apparatus for use in the above method.

[0007]

As a result of intensive studies to solve the above-mentioned problems, the present inventor left a microelectrode in a sample containing a paramagnetic species, and an electric double layer formed on the surface of the electrode. When we observed a small current flowing in the magnetic field in a static magnetic field while applying a high-frequency electromagnetic wave, we found that the change in current due to electron spin resonance of paramagnetic species existing in the electric double layer can be detected. Further, the present inventor detects the change in impedance of the electric double layer of such a microelectrode,
It was found that the electron spin resonance of paramagnetic species can be selectively detected in an extremely small area in the electric double layer. The present invention has been completed based on these findings.

That is, the present invention is characterized in that the electron spin resonance of paramagnetic species existing in the electric double layer formed on the surface of the microelectrode inserted in the sample is detected by impedance change in the electric double layer. The present invention provides a method for analyzing paramagnetic species. According to a preferred embodiment of the present invention, the above method, wherein the electric double layer is formed by applying a bias voltage / current;
Also provided is the above method of detecting by a change in current.

According to another aspect of the present invention, there is provided a method for analyzing paramagnetic species, which comprises the steps of: (a) applying a bias voltage / current to microelectrodes inserted in a sample. A step of forming an electric double layer on the surface of the microelectrode; (b) a step of inducing electron spin resonance of a paramagnetic species present in the electric double layer; and (c) an electron spin resonance caused by impedance change in the electric double layer. Methods are provided that include the step of detecting.

According to yet another aspect of the present invention, there is provided an apparatus for use in each of the above methods. That is,
According to the present invention, a magnet 5 for forming a static magnetic field, a sample part 3 arranged in the static magnetic field, a resonator 2 for irradiating the sample part 3 with an electromagnetic wave in the static magnetic field, and a sample part 3 are inserted into the sample. Microelectrode 7 and reference electrode 8 for applying a bias voltage / current between the microelectrode 7 and the reference electrode 8 to form an electric double layer on the surface of the microelectrode 7.
An analyzer for paramagnetic species is provided, which comprises a power source 4 connected to the microelectrode 7 and an amplifier 6 connected to the microelectrode 7 for detecting a change in impedance of the electric double layer. According to a preferred embodiment of the present invention, there is provided the above device further including an electromagnetic wave signal source 1; and the above device in which the power supply 4 and the amplifier 6 are formed as an integrated electronic circuit.

Without being bound to any particular theory, in the case of microelectrodes, most of the electric field is concentrated in the electric double layer, so that when the method of the present invention detects a change in impedance in the electric double layer. , The results directly show that the characteristics of the electric double layer are changed by the electron spin resonance of the paramagnetic species in the electric double layer. In the present specification, the electric double layer means that, in a contact interface between two phases having different compositions, an extra positive charge is continuously distributed on one side of the interface and an extra negative charge is continuously distributed on the other side, but as a whole. Refers to a boundary layer that satisfies the condition of electrical neutrality.
For example, an electric double layer formed near the surface of an electrode when the electrode is charged by being inserted into an electrolyte solution is one example. According to the method of the present invention, the electron spin resonance of the paramagnetic species in the extremely small area in the electric double layer can be selectively extracted, and only the paramagnetic species existing in the minute area in the sample can be analyzed. It is possible. Needless to say, the paramagnetic species analysis of the present invention includes all analytical concepts such as identification, detection, and quantification of paramagnetic species.

The sample used in the analysis method of the present invention is not particularly limited, and in addition to the solution containing the paramagnetic species to be analyzed, tissues, body fluids, cells or suspensions thereof separated and collected from the living body. Alternatively, the living body itself can be used. As the solvent for forming the solution or suspension, for example, water, diethyl ether, dimethoxyethane, tetrahydrofuran, ethers such as tetrahydropyran, acetonitrile, dimethylformamide, dimethylsulfoxide, liquid ammonia, sulfuric acid and the like can be used. it can. Hereinafter, the method and apparatus of the present invention will be described more specifically with reference to the attached FIG. 1, but the scope of the present invention is not limited to these examples.

An electromagnetic wave having a constant frequency generated by the signal source 1 is introduced into the resonator 2, and the resonator 2 has a material and shape that can efficiently irradiate only the magnetic field component of the electromagnetic wave onto the sample portion 3. ing. As the resonator 2, for example, a cavity, a loop gap, a Fabry-Perot, a transmission line, or any other element that can be used by those skilled in the art can be used, and the person skilled in the art can appropriately select the element depending on the analysis target and purpose.

As the signal source 1, any source may be used as long as it can generate an electromagnetic wave having a stable frequency and amplitude and can finely adjust the frequency. For example, a synthesized signal generator is generally used, but other types of oscillators such as a Gunn oscillator and a gyrotron can also be appropriately used depending on the frequency and the like. Electron spin resonance of paramagnetic species is induced by irradiating an electromagnetic wave in a static magnetic field, and the frequency of the electromagnetic wave can be any frequency band from the HF band to the far infrared. The method of the present invention is characterized by detecting electron spin resonance of paramagnetic species by detecting impedance change in the electric double layer, and it is not necessary to directly detect electromagnetic waves causing electron spin resonance. Therefore, millimeter waves, far infrared rays, or the like, for which it is difficult to obtain a high-performance detector, may be used.

The sample section 3 has a structure in which a sample tube of an appropriate material and shape filled with the sample and a sample stand for mounting the sample can be arranged. Further, the sample section 3 is inserted into the sample. A microelectrode 7 for performing the operation, a counter electrode if necessary, and a reference electrode 8 are arranged. These electrodes are connected to a power supply 4, which allows a bias voltage / current, preferably a DC bias current, to flow between the electrodes.

As the microelectrode 7 used in the present invention, any member and shape may be used as long as an electric double layer having a thickness of about several angstroms can be formed on the surface by a bias voltage / current. For example, the microelectrode 7
As the counter electrode member, stainless steel, gold, carbon or the like having no magnetism is desirable. The shape of the microelectrodes is preferably, for example, disk type, ring type, array type, or the like. As a member of the reference electrode 8, for example, a silver / silver chloride electrode or a calomel electrode is used. The counter electrode may be omitted when the microelectrode is sufficiently smaller than the reference electrode.

The sample portion 3 is provided in the static magnetic field formed by the magnet 5 so that the sample is placed in a uniform static magnetic field. The resonator 2 is arranged so that the sample portion 3 can be irradiated with electromagnetic waves within the static magnetic field. Any magnet may be used as the magnet 5 as long as it can form a uniform magnetic field having a required strength. For example, an electromagnet,
A superconducting magnet, a permanent magnet, or the like can be used, and can be appropriately selected depending on the purpose of analysis, required magnetic field strength, homogeneity, and the like. In addition to the magnet 5 for generating the static magnetic field, a magnetic field sweep coil or a modulation coil may be used for fine adjustment of the magnetic field such as magnetic field sweep or modulation. It should be noted that the respective means of the magnet 5 and the resonator 2 and the method of arranging the respective means are known to those skilled in the art by the conventional ESR device.

In such a device, when the intensity of the magnetic field is slowly increased while irradiating the sample with the electromagnetic wave from the resonator 2, the electron spin resonance of the paramagnetic species existing in the electric double layer at a certain magnetic field intensity is generated. Occurs, which results in a change in impedance within the electric double layer. The paramagnetic species can be analyzed by amplifying this impedance change by the amplifier 6 connected to the microelectrode and preferably detecting it as a signal. Changes in impedance are usually observed by changes in voltage and / or current.

As the power source 4, a controllable bias power source capable of applying an arbitrary bias voltage / current to the microelectrode 7 is used in order to bring the electric double layer formed on the surface of the microelectrode 7 into an optimum state. Is preferred. In the method of the present invention, it is necessary to apply a constant DC voltage between the electrode and the sample in order to form a stable electric double layer between the sample and the electrode. Since the state of the electric duo changes depending on the value of the applied voltage, the applied voltage should be set to a value at which the best signal is observed by the method of the present invention. A weak current flows through the electric double layer when a voltage is applied in this way, but this current value is generally a function of the applied voltage, and conversely, when the current is applied at a constant value. If the voltage value is controlled, the voltage also becomes constant. In this specification, these applied voltages and currents are referred to as bias voltage / current.

Various methods are known for controlling or observing the voltage between the electrode and the sample. For example, based on the method of controlling with the above-mentioned current value or the voltage generated at the reference electrode different from the sample electrode. There are ways to control. These methods are known means in the field of electrochemistry and can be appropriately selected by those skilled in the art. The amplifier 6 is preferably an amplifier having a high internal resistance in order to detect a minute change in impedance. The power source 4 and the amplifier 6 are usually formed as a single electronic circuit that is inseparable, for example, an amplifier with a bias applying function, and is connected to the microelectrode 7 and the reference electrode 8.

In the integrated amplifier with a bias applying function including the power supply 4 and the amplifier 6 as described above, a constant voltage bias system in which the electrode voltage is kept constant and the current change is amplified, or the current flowing through the electrode is controlled to be constant. One of the constant current bias methods for amplifying the voltage change can be used. The former is
Since the electrochemical characteristics often depend on the voltage value, it has the characteristic of being excellent in control characteristics, and the latter is characterized in that it is easy to build a reasonable amplification circuit that makes use of the high resistance of the sample. . Since the two have a complementary relationship, they may be appropriately selected according to the purpose of analysis and the type of device.

In order to reduce 1 / f noise,
As in the case of a normal ESR device, it is possible to use a means for sweeping a magnetic field while superimposing a low-frequency alternating magnetic field by a magnetic field modulation coil and detecting only a modulation frequency component of a current change with a lock-in detector. The frequency of the alternating magnetic field applied to the magnetic field modulation coil is about 0.1 Hz to 10 MHz, preferably 40 to
The frequency band of 80Hz can be used. Furthermore, in addition to constant frequency / amplitude waveforms as electromagnetic waves for irradiating the sample, time-varying information is extracted using amplitude-modulated, frequency-modulated, phase-modulated or pulse-modulated waveforms. Or 1 / f noise may be reduced.

[0023]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1 (1) Construction of a paramagnetic species analyzer An amplifier with a bias applying function was manufactured as follows. As a reference voltage source for the bias voltage, a low ripple power supply using two series constant voltage regulators in a cascade was introduced to the reference voltage generator to generate the reference voltage. Since the reference voltage element itself also generates noise, after reducing the noise with the voltage smoothing circuit, the power supply 4 capable of generating any stabilized voltage by the buffer circuit using the low-noise operational amplifier and the multi-turn volume control. Configured.

The amplifier 6 is a constant voltage bias type circuit, which amplifies a change of a minute current flowing through the minute electrode 7 to which a constant voltage is applied and converts it into a voltage signal. Since the amplifier 6 needs to have both a high input resistance and a low noise characteristic, the amplifier circuit is composed of an element having a super low bias current low noise characteristic of a monolithic operational amplifier. Further, output terminals via buffers are provided so that the bias voltage and current applied to the electrodes can be monitored.

Commercially available X-band ESR device (TE, manufactured by JEOL Ltd.)
200) was modified so that the external output of the magnetic field modulation component and the external input of the recorder were possible. The reference output of the lock-in detector was connected to the external output of the magnetic field modulation component, and the signal output of the lock-in detector was connected to the external input of the recorder. The signal input section of the lock-in detector was connected to the signal output section of the amplifier 6, and the microelectrode 7 and the reference electrode 8 were connected to the signal input section of the amplifier 6. Φ0.25 as microelectrode 7
A mm-coated stainless steel wire having a polished tip was used, and the reference electrode 8 was a φ0.7 mm silver / silver chloride electrode having a length of 2 cm. The counter electrode was not used because the reference electrode was large enough for the microelectrodes.

(2) Analysis of paramagnetic species Paramagnetic species were analyzed using the above apparatus. As a paramagnetic species, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (3-carbamoyl-2,2,5,5-tetrameth
ylpyrrolidin-1-yloxy: CarbamoylPROXYL) was used. Inner diameter of 75 mM carbamoyl PROXYL dissolved in physiological saline
40 μl was injected into a 1 mm capillary tube. The microelectrode 7 and the reference electrode 8 described above were inserted into the capillary tube, and the tip of the electrode was placed so as to be immersed in the carbamoyl PROXYL solution. This capillary tube functions as an electrolytic cell. This electrolytic bath was inserted into a quartz ESR sample tube having an inner diameter of 5 mm and left in the sample section 3 inside the resonator 2 of the above apparatus. An electromagnet was used as the magnet 5 of the above apparatus, and the magnetic flux density near the sample portion was about 335 mT.

A microwave of 200 mW of about 9 GHz is applied to the electrolytic cell through the resonator 2, a magnetic field of about 335 mT is further applied, and the microelectrode 7 is adjusted to 5 mV with respect to the reference electrode 8. A voltage was applied. When the current component locked in by 80 Hz magnetic field modulation was detected, the signal shown in the lower part of Fig. 2 was obtained (measurement temperature: room temperature). The magnetic field region in which this signal appears generally coincided with the magnetic field region in the ESR signal. The amount of unpaired electrons present in the electric double layer that was the target of measurement was calculated to be about 3.75x10 ^-16 mol. This amount can be measured using a normal ESR device using the carbamoyl PROXY
When L was measured at room temperature, it was much smaller than the required amount of unpaired electrons (about 1.4x10 ^-13 mol).

Further, the concentration of carbamoyl PROXYL was 50 mM.
When the amount of unpaired electrons was reduced to 2.5x10 ^-16 mol (corresponding to 2.5x10 ^-16 mol), the signal intensity decreased in proportion to the concentration (middle part of Fig. 2). Furthermore, the concentration of carbamoyl PROXYL was 10 mM (unpaired electron amount 0.5x10 ^-16 m
No signal was detected when the signal was reduced to (equivalent to ol) (upper part of Fig. 2). Therefore, it was concluded that the signal detected at a concentration of 50 mM or more was attributed to carbamoyl PROXYL.

[0030]

INDUSTRIAL APPLICABILITY By the method of the present invention, paramagnetic species existing in a test tube or in a minute region in a living body can be selectively and highly sensitively analyzed. Since the method of the present invention detects electron spin resonance of paramagnetic species by changing the impedance of the electric double layer, it can be easily carried out by making appropriate improvements to the conventional ESR device, and a special detection device is required. There is a feature that does not. Therefore, the present invention is extremely useful not only for physicochemical studies of paramagnetic species, but also for studies on physiological actions of paramagnetic species and for establishing methods for diagnosing, treating, and preventing diseases involving paramagnetic species. . Further, the device of the present invention is useful for simply carrying out the above method.

[Brief description of drawings]

1 of an analyzer for carrying out the method according to the invention, FIG.
It is a figure showing an example.

FIG. 2 is a diagram showing the results of analyzing carbamoyl PROXYL as a paramagnetic species in physiological saline by the method of the present invention. Unpaired electron amount is about 3.75x10 ^-16 mol (lower), about 2.5x
It is 10 ^-16 mol (middle) and about 0.5x10 ^-16 mol (upper).

[Explanation of symbols]

 1 Signal Source 2 Resonator 3 Sample Part 4 Power Supply 5 Magnet 6 Amplifier 7 Micro Electrode 8 Reference Electrode

Claims (9)

[Claims] 1. Paramagnetic properties characterized by detecting electron spin resonance of a paramagnetic species present in an electric double layer formed on the surface of a microelectrode inserted in a sample by impedance change in the electric double layer. Species analysis method. 2. The method according to claim 1, wherein the electric double layer is formed by applying a bias voltage / current. 3. The method according to claim 1, wherein a change in impedance is detected by a change in voltage and / or current. 4. A method for analyzing paramagnetic species, comprising the steps of: (a) forming an electric double layer on the surface of a microelectrode by applying a bias voltage / current to the microelectrode inserted in a sample. A method comprising: (b) inducing electron spin resonance of a paramagnetic species present in the electric double layer; and (c) detecting electron spin resonance by impedance change in the electric double layer. 5. A magnet 5 for forming a static magnetic field, a sample part 3 arranged in the static magnetic field, a resonator 2 for irradiating the sample part 3 with an electromagnetic wave in the static magnetic field, and a sample part inserted in the sample. Microelectrode 7 and a reference electrode 8 for applying a bias voltage / current between the microelectrode 7 and the reference electrode 8 to form an electric double layer on the surface of the microelectrode 7. An apparatus for analyzing paramagnetic species, comprising a power source 4 connected to the microelectrode 7, and an amplifier 6 connected to the microelectrode 7 for detecting a change in impedance of the electric double layer. 6. The apparatus according to claim 5, further comprising an electromagnetic wave signal source 1. 7. The device according to claim 5, further comprising a counter electrode. 8. The device according to claim 5, wherein the power supply 4 and the amplifier 6 are formed as an integrated electronic circuit. 9. A method as claimed in any one of claims 1 to 4 for use in a method as claimed in any one of claims 1 to 4.
The device according to item.