JP7202592B2 - SKIN GAS MEASURING DEVICE AND SKIN GAS MEASURING METHOD - Google Patents

Description

TECHNICAL FIELD The present invention relates to a skin gas measuring device and a skin gas measuring method for measuring components of skin gas generated from the surface of a living body by a laser-induced fluorescence method.

Skin gas generated from the surface of the living body contains gas components generated in various metabolic processes, and in recent years, attempts have been made to analyze gas components as markers that reflect in vivo metabolic information.

Conventionally, skin gas is collected by sucking skin gas from the surface of the skin with a gas collecting means, and then introduced into a gas analyzer for measurement (for example, Patent Document 1). Apparatuses have been proposed that lead skin gases to a measuring apparatus (eg, Patent Document 2).

In particular, reactive oxygen species such as hydroxyl radicals (OH radicals) have attracted attention as indicators of oxidative stress. The gas components to be measured in the skin gas, such as reactive oxygen species, have extremely low concentrations, and the above-described method cannot measure them with high accuracy. It is known that the concentration of OH radicals in the ambient air in the flatlands is actually at the ppt level and accompanies diurnal fluctuations (Non-Patent Documents 1 and 2). The concentration of OH radicals released from human skin is also considered to be at approximately the same level (Non-Patent Document 3). Therefore, it is difficult to accurately measure OH radicals released from the skin under normal atmospheric conditions. Furthermore, sensitive analysis of skin gases other than reactive oxygen species is also susceptible to fluctuating environmental trace substances.

As a high-precision analysis technique, a method of measuring a target component of skin gas using a laser-induced fluorescence method has also been proposed (Patent Document 3). In the laser-induced fluorescence method, an atomic or molecular chemical species to be measured is excited by irradiating it with a laser beam corresponding to its excitation level, and the resulting fluorescence is measured to identify the chemical species. It is a quantitative method and a highly sensitive and selective analytical technique.

JP 2005-214855 A Japanese Patent Application Laid-Open No. 2002-195919 JP 2013-205061 A

C. C. Wang and L. I. Davis, "Measurement of hydroxyl concentrations in air using a tunable uv laser beam," Phys. Rev. Lett., vol. 32, no. 7, pp. 349-352, Feb. 1974. D. E. Heard and M. J. Pilling, “Measurement of OH and HO2 in the troposphere,” Chem. Rev., vol. 103, no. 12, pp. 5163-5198, Nov. 2003 Che DC, Shimouchi A, Mizukami T, Nose K, Seiyama A, Kasai T. Emanation of hydroxyl radicals from human skin. IEEE Sensors J 13(4):1223-1227, 2013.

However, with the above-described technique, measurement values fluctuate greatly depending on the measurement atmosphere and measurement temperature, making it difficult to stably and accurately measure skin gas components (reactive oxygen species).

Therefore, an object of the present invention is to provide a skin gas measuring device and a skin gas measuring method that can measure skin gas components, particularly reactive oxygen species, with high accuracy.

In order to achieve the above object, the invention according to claim 1 provides a skin gas measuring device for measuring reactive oxygen species in skin gas generated from the surface of a living body by a laser-induced fluorescence method, the device comprising: A measurement chamber that is brought into contact with the skin to release skin gas, a gas supply means that introduces atmospheric gas into the measurement chamber, and a gas that controls the temperature of the gas in the measurement chamber to a preset temperature higher than the body temperature of the subject. temperature control means; laser irradiation means for irradiating the skin gas discharged into the measurement chamber and mixed with the atmospheric gas with a laser beam that excites the skin gas component to be measured; and fluorescence generated by irradiating the skin gas with the laser beam. and a fluorescence measuring means for measuring the number of molecules of the skin gas component, wherein the measurement chamber has an opening , and is shut off from the outside by closing the opening. The internal atmosphere can be replaced with ambient gas, and the skin gas can be introduced into the measurement chamber by closing the opening with the skin of the person to be measured . Use means. Here, the term "reactive oxygen species" as used in the present invention is a general term for oxygen derivatives, and particularly refers to those produced in vivo, such as hydroxyl radicals (OH radicals), superoxides, and hydrogen peroxide.

According to the second aspect of the invention, in the skin gas measuring device according to the first aspect, a technical means is used in which the atmospheric gas is pure air.

According to the third aspect of the invention, in the skin gas measuring device according to the first or second aspect, a technical means is used in which the active oxygen species are hydroxyl radicals.

According to a fourth aspect of the invention, there is provided a skin gas measuring method for measuring reactive oxygen species in skin gas generated from the skin surface by a laser-induced fluorescence method, the method comprising an opening and closing the opening. The opening is isolated from the outside, the atmosphere inside can be replaced with ambient gas, and the skin gas can be introduced inside by closing the opening with the skin of the person to be measured. introducing atmospheric gas into a measurement chamber in which the measuring object is placed; controlling the temperature of the gas in the measurement chamber to a preset temperature higher than the body temperature of the person to be measured; and bringing the skin of the person to be measured into contact with the opening. irradiating the skin gas discharged into the measurement chamber and mixed with the ambient gas with a laser beam that excites the skin gas component to be measured; and irradiating the skin gas with the laser beam. and counting the number of molecules of the skin gas component by detecting the fluorescence produced by.

According to the skin gas measuring device and the skin gas measuring method of the present invention, the factor that the measurement atmosphere and the measurement temperature greatly affect the measurement accuracy is found, and the atmospheric gas is replaced and the temperature of the atmospheric gas is controlled. By adopting a configuration that strictly controls the measurement environment and eliminates its influence, skin gas can be measured noninvasively with high accuracy. In particular, it can be suitably used for measuring reactive oxygen species represented by hydroxyl radicals, which are important as oxidative stress markers but difficult to measure.

FIG. 2 is a diagram schematically illustrating the configuration of a skin gas measuring device; It is explanatory drawing which shows the structure of a measurement chamber and a peripheral device. FIG. 2(A) is a side view, and FIG. 2(B) is a side view of FIG. FIG. 4 is an explanatory diagram showing how skin gas generated from the palm of the hand is measured; FIG. 4 is an explanatory diagram showing the temperature dependence of the photon count number of hydroxyl radicals. FIG. 4 is an explanatory diagram showing reproducibility test results of background measurements and skin gas measurements;

(Skin gas measuring device)
The skin gas measuring device of the present invention will be described with reference to the drawings. Here, an apparatus configuration capable of measuring hydroxyl radicals will be described as an example.

The skin gas measuring device of the present invention measures the measurement target component of skin gas using a laser induced fluorescence method (hereinafter referred to as LIF method).

In the LIF method, an atomic or molecular chemical species to be measured is excited by irradiating it with a laser beam corresponding to its excitation level, and the amount of the chemical species is determined by measuring the resulting fluorescence. It is a method of measuring.

As shown in FIG. 1, the skin gas measuring apparatus 1 includes a laser irradiation means 10, a measurement chamber 20, a gas supply means 30, a gas temperature control means 40, and a fluorescence measurement means 50.

The laser irradiation means 10 is for generating and irradiating the skin gas introduced into the measurement chamber 20 with a laser beam that excites the components of the skin gas to be measured. A configuration used in a normal LIF method including an element 12 (beta barium borate crystal: BBO in this embodiment) and a bandpass filter 13 can be adopted.

Here, the wavelength of the laser light can be selected according to the type of the object to be measured, and a laser oscillator such as a pulse laser or a continuous wave laser can be used as the laser oscillator 11 . A variable wavelength laser light source can also be employed.

In this embodiment, the laser oscillator 11 is a semiconductor laser oscillator, and oscillates continuous light with a wavelength of 561 nm. The laser light oscillated by the laser oscillator 11 is doubled by the wave doubler 12 and has a wavelength of 280.5 nm. It is introduced into the measuring chamber 20 .

If the intensity of the laser beam is too high, stray light due to scattered light and multiphoton processes generate unwanted molecules to generate noise, and if the intensity is too low, sufficient signal intensity cannot be obtained. Therefore, a light amount adjusting filter or the like is used to adjust the laser light to a preferable intensity.

The measurement chamber 20 is a chamber configured to introduce skin gas into the interior, irradiate laser light, and measure fluorescence.

As shown in FIG. 2, the measurement chamber 20 includes a main body 21 having a cylindrical space with an inner diameter of about 40 mm and a depth of about 60 mm, into which skin gas is introduced. Hereinafter, the positional relationship will be described with reference to the directions shown in FIG. 2, but it is not limited to this.

An opening 22 for collecting skin gas is provided above the main body 21 . When the skin gas is measured, the opening 22 can be closed by bringing the skin surface of the palm of the hand into close contact with the main body 21 . A lid 23 is provided to close the opening 22 when skin gas is not collected.

A laser beam introduction window 24 for introducing a laser beam into the main body 21 is provided on the side of the measurement chamber 20, and a side surface adjacent to the side surface provided with the laser beam introduction window 24 is provided with a fluorescent light. A fluorescence detection window 25 for detecting is provided. The positional relationship between the laser light introduction window 24 and the fluorescence detection window 25 is arbitrary as long as stable fluorescence measurement is possible.

A band-pass filter 13 is attached to the laser light introduction window 24, and a band-pass filter 51, which will be described later, is attached to the fluorescence detection window 25 so as to cover the respective windows.

At the side of the measurement chamber 20, an atmospheric gas introduction port 26 for introducing the atmospheric gas into the main body 21 and a gas outlet 27 for discharging the atmospheric gas are provided. The atmospheric gas introduction port 26 is connected to a gas supply means 30 for supplying atmospheric gas from a gas supply source such as a high-pressure gas cylinder via a mass flow controller, and the main body 21 is configured to be able to control the gas flow rate of the atmospheric gas. ing.

In the present embodiment, the atmospheric gas inlet 26 is provided above the center in the height direction of the main body 21 , and the gas outlet 27 is arranged below the atmospheric gas inlet 26 so as to face the atmospheric gas inlet 26 . is provided. This facilitates mixing of the atmospheric gas and the skin gas, and forms a measurement gas uniformly mixed with the skin gas.

A gas temperature control means 40 for adjusting the temperature of the gas in the measurement chamber 20 is provided below the main body 21 . The gas temperature control means 40 has a heater 41 and a temperature sensor 42, and controls the temperature of the gas in the measurement chamber 20 to a predetermined temperature by a controller (not shown). Here, it is preferable that the gas temperature control means 40 has a configuration capable of controlling the temperature of the atmosphere gas to about ±0.1° C. of the set temperature. Although the temperature sensor 42 is embedded in the main body 21 in this embodiment, a configuration for directly measuring the temperature of the gas in the measurement chamber 20 can also be adopted.

The fluorescence measurement means 50 includes a bandpass filter 51 for selecting a fluorescence wavelength suitable for the measurement component, a photomultiplier tube 52 for converting light energy into electrical energy, and a signal from the photomultiplier tube 52 directly or through an amplifier. A photon counter 53 for continuous counting and an analysis means 54 (FIG. 1) such as a personal computer for performing analysis based on the counted number are provided. In this embodiment, a photon counting head integrally including a photomultiplier tube 52 and a photon counter 53 is used. A band-pass filter 51 that selectively transmits fluorescence with a wavelength of 308 nm was used. The photon counter 53 preferably has a sufficiently fast response speed, for example, a resolution of about 100 to 500 MHz.

(Skin gas measurement method)
The skin gas measuring method of the present invention will be described by taking a hydroxyl radical measuring method as an example.

After the skin gas measuring device 1 is activated, the gas supply means 30 is operated in a state in which the opening 22 of the measuring chamber 20 is closed with the lid 23 to prevent outside air from entering the main body 21 . A predetermined flow rate of atmospheric gas is introduced into the measurement chamber 20, and the gas temperature control means 40 controls the atmospheric gas to a set temperature. Then, the atmosphere inside the main body 21 is replaced with the atmosphere gas, and the operation waits until the set temperature is reached. Here, it is preferable to set the flow rate of the atmosphere gas to a flow rate at which the skin gas stays in the main body 21, is uniformly mixed in the atmosphere gas, and does not form condensation.

Although the measurement itself by the LIF method can be performed in any atmosphere, for example, there are about 1 ppt of hydroxyl radicals in the air, and the daily fluctuation is large, so the background at the time of measurement fluctuates greatly, which is a big error factor. Become. By introducing the ambient gas, contaminants in the main body 21 are discharged, and at the same time, the gas in the main body 21 is agitated so as to be in a uniform state. This reduces the background level during measurement and improves the measurement sensitivity.

Inert gases such as high-purity nitrogen and argon can be used as atmospheric gases only for the purpose of reducing background levels and improving sensitivity. It is preferable to use pure air in order to grasp the in vivo situation more accurately, because the gas release state may be different from usual.

Here, pure air is air from which trace components present in the atmosphere have been removed. For example, it corresponds to Compressed Air Grade 1 manufactured by Taiyo Nippon Sanso Corporation.

As shown in the examples described later, in skin gas measurement, the temperature of the measurement atmosphere affects the amount of skin gas released, and the count number of the component to be measured varies greatly depending on the temperature of the gas in the measurement chamber 20. It is necessary to strictly control the temperature of the gas within the measurement chamber 20 . By tightly controlling the temperature of the gas within the measurement chamber 20, the reproducibility and accuracy of the measurements can be improved. Also, in order to facilitate the temperature control of the gas in the measurement chamber 20 and promote the release of skin gases such as perspiration, it is preferable to set the temperature to a temperature several degrees Celsius higher than the body temperature. Furthermore, in order to reduce the stress on the skin of the person being measured and to more accurately grasp the in vivo conditions, it is preferable to set the temperature to 50° C. or lower, for example, 40° C. is preferable. Also, if the supply of the atmospheric gas is stopped, the atmospheric gas will fill up in the main body 21, making it impossible to accurately control the temperature and humidity.

Next, the laser irradiation means 10 irradiates the pure air introduced into the main body 21 with a laser beam having a wavelength of 280.5 nm, and the fluorescence measurement means 50 measures the count number of fluorescence having a wavelength of 308 nm. This count is taken as the background measurement.

Subsequently, the cover 23 is removed, and the skin surface of the palm or the like is brought into close contact with the opening 22 as shown in FIG. 3 to close the main body 21 .

Subsequently, the laser irradiation means 10 irradiates the measurement gas formed by mixing the skin gas and the ambient gas in the main body 21 with a laser beam having a wavelength of 280.5 nm, and the fluorescence measurement means 50 detects fluorescence having a wavelength of 308 nm. Measure the number of counts. Let this number of counts be the measured value of skin gas.

Measurement of the number of counts is performed in a stable state where fluctuations in the number of counts have settled down. At this time, the skin gas is sufficiently released and mixed with the ambient gas to be in a uniform state, so that the ambient gas temperature is stable and the humidity in the measurement chamber 20 is also stable.

The number of counts can be measured under conditions such as, for example, measuring for 10 minutes after closing the opening 22 with the skin surface in close contact, and using the data for the final 3 minutes.

Here, since the wavelengths of the excitation light and the fluorescence are different, measurement can be performed without being affected by irregular reflection of the excitation light. In addition, there is no need to use a shutter to switch the timing of irradiation of excitation light and measurement of fluorescence, and the configuration of the device can be simplified.

In addition, since continuous light is used, the energy is low and there is little effect even if the skin is irradiated. The laser light introduction window 24 is provided at a position where the laser beam is not irradiated even when the palm of the hand is pressed against the opening 22 and protrudes inside from the opening 22 .

Then, the difference between the measured value of the skin gas and the measured value of the background is calculated, and this calculated value is used as the measured value of the target skin gas component released from the skin gas.

After the measurement is completed, the opening 22 is again closed with the lid 23, and the inside of the measurement chamber 20 is replaced with pure air to prepare for the next measurement.

As skin gas components to be measured, there are components other than reactive oxygen species for which important findings can be obtained. For example, acetone is said to be effective in detecting diabetes, abnormalities in lipid metabolism, and the like. Acetone can be excited by laser light with a wavelength of 280.5 nm, emits fluorescence with a wavelength of 435 nm, and can be measured by the skin gas measuring device 1 and skin gas measuring method. The skin gas measuring device 1 and skin gas measuring method of the present invention can also be applied to the measurement of skin gas components such as carbon monoxide, nitric oxide, and ammonia, which are effective for understanding the in vivo conditions.

(Effect of Embodiment)
According to the skin gas measuring device 1 and the skin gas measuring method of the present invention, the factors of the measurement atmosphere and the measurement temperature that greatly affect the measurement accuracy are found, and the atmospheric gas is replaced, and the gas in the measurement chamber 20 is replaced. Since a configuration is adopted in which temperature control is performed to strictly control the measurement environment to eliminate its influence, skin gas can be measured noninvasively and with high accuracy. In particular, it can be suitably used to measure active oxygen represented by hydroxyl radicals, which are important as oxidative stress markers but difficult to measure.

Using the skin gas measuring device and skin gas measuring method of the present invention, hydroxyl radicals released from the palm were measured. Hydroxyl radicals are excited by light with a wavelength of 280.5 nm and emit fluorescence with a wavelength of 308 nm.

A continuous wave laser with a wavelength of 561 nm and an output of 75 mW was used as the laser light. The oscillated laser light was transmitted through a beta-barium borate crystal to generate a double wave with a wavelength of 280.5 nm. Further, the light was decomposed into component wavelengths using a Perrin-Brocker prism, and only the laser light with a wavelength of 280.5 nm was reflected by an ultra-wideband dielectric multi-film flat mirror and introduced into the measurement chamber. A 280 nm band-pass filter was installed in the laser light introduction window of the measurement chamber to selectively introduce only the laser light and to seal the measurement chamber. Pure air (compressed air Grade 1) was introduced into the measuring section at 100 mL/min using a mass flow controller. The inside of the measurement chamber was controlled at 40° C. by the gas temperature control means. In order to detect fluorescence emitted in the measurement chamber, a photomultiplier tube (sensitivity wavelength: 300 nm to 650 nm) was placed in close contact with the measurement chamber. The light-receiving surface of the photomultiplier tube was installed so that the light-receiving surface was parallel to the optical axis so as to be at the same height as the optical axis of the laser beam. A short wavelength cut filter of 300 nm was installed between the measuring section and the photomultiplier tube so as not to detect laser light.

The signal from the photomultiplier tube was processed with a photon counter. The photon count was measured 600 times at 1 second intervals. When measuring the pure air and the measurement gas, the measurement was performed for 10 minutes, and the average photon count value per second for 3 minutes (7 to 10 minutes) before the end was used as the measurement value.

FIG. 3 shows the results of measuring the hydroxyl radicals released from the palm of the subject while changing the temperature of the measurement gas from 37° C. to 40° C. in 1° C. increments. Photon counts represent skin gas measurements minus background measurements. The number of photon counts increased as the temperature increased, resulting in a difference of about 1.7 times between 37°C and 40°C. This confirms that the temperature of the measurement atmosphere has a great effect on the measured values.

FIG. 4 shows the reproducibility test results of background measurements and skin gas measurements. Here, the photon count at each point indicates an integrated value for one minute. The control temperature of the atmosphere gas was 40° C., and the measurements were repeated in the order of 5 minutes of background measurement→10 minutes of skin gas measurement→5 minutes of background measurement→10 minutes of skin gas measurement→5 minutes of background measurement. Good reproducibility was shown for both background and skin gas measurements.

An embodiment of the skin gas measurement device of the present invention was compared with a conventional measurement method in which the measurement environment was not controlled. Table 1 shows the results. In the conventional measurement method, a pulse laser is used as a laser light source, and both the excitation wavelength and the measurement wavelength are set to 308 nm, so a shutter is provided to switch between excitation and fluorescence measurement. Fluorescence is emitted from hydroxyl radicals by short-time pulsed laser irradiation, but since the excitation wavelength and the measurement wavelength match, the fluorescence detected after switching the shutter is regarded as the LIF signal, and the order of the measured value is is different.

The relative standard deviation of LIF measured values in repeated measurements of blanks, the relative standard deviation of LIF measured values in repeated measurements of hydroxyl radicals, and the ratio (S / N ratio) of the LIF signal and the noise signal (noise width) are shown in Comparative Examples. Both were significantly improved, and it was confirmed that the measurement accuracy and sensitivity were improved.

REFERENCE SIGNS LIST 1 skin gas measuring device 10 laser irradiation means 11 laser oscillator 12 harmonic element 13 bandpass filter 20 measurement chamber 21 main body 22 opening 23 lid 24 laser light introduction window 25 fluorescence Detection window 26 Atmosphere gas introduction port 27 Gas discharge port 30 Gas supply means 40 Gas temperature control means 41 Heater 42 Temperature sensor 50 Fluorescence measurement means 51 Bandpass filter 52 Photomultiplier tube 53 Photon counter

Claims (4)

A skin gas measurement device for measuring reactive oxygen species in skin gas generated from the surface of a living body by a laser-induced fluorescence method, comprising: a measurement chamber in which the skin of a subject to be measured is brought into contact with the skin to release the skin gas; gas supply means for introducing an atmospheric gas; gas temperature control means for controlling the temperature of the gas in the measurement chamber to a preset temperature higher than the body temperature of the person to be measured; laser irradiation means for irradiating the skin gas with a laser beam that excites the skin gas component to be measured; means, wherein the measurement chamber has an opening, is isolated from the outside by closing the opening, and is capable of replacing an atmosphere inside the measurement chamber with atmospheric gas; A skin gas measuring apparatus, characterized in that the skin gas can be introduced into the measuring chamber by closing the measuring chamber with the skin of the person to be measured. 2. The skin gas measuring device according to claim 1, wherein the atmospheric gas is pure air. 3. The skin gas measuring device according to claim 1, wherein said active oxygen species is a hydroxyl radical. A skin gas measurement method for measuring reactive oxygen species in skin gas generated from the skin surface by a laser-induced fluorescence method, which has an opening, is blocked from the outside by closing the opening, and the inside The atmosphere can be replaced with the atmosphere gas, and the atmosphere gas is introduced into the measurement chamber configured so that the skin gas can be introduced into the interior by closing the opening with the skin of the person to be measured in close contact. controlling the temperature of the gas in the measurement chamber to a preset temperature higher than the body temperature of the person to be measured; and bringing the skin of the person to be measured into contact with the opening to release skin gas. a step of irradiating the skin gas discharged into the measurement chamber and mixed with the ambient gas with a laser beam that excites the skin gas component to be measured; and measuring the number of molecules of the component.

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