Patent Application
20200348227
The present invention relates to a carbon isotope analysis device and a carbon isotope analysis method. In particular, the present invention relates to a light generator useful for analysis of radioactive carbon isotope 14C and the like, which generates narrow-line-width and high-intensity light, and a purifier and a method for a radioactive carbon isotope-containing gas as an analytical gas object, for use in a radioactive carbon isotope analysis device and a radioactive carbon isotope analysis method, by use of the light generator.
Carbon isotope analysis has been applied to a variety of fields, including assessment of environmental dynamics based on the carbon cycle, and historical and empirical research through radiocarbon dating. The natural abundances of carbon isotopes, which may vary with regional or environmental factors, are as follows: 98.89% for 12C (stable isotope), 1.11% for 13C (stable isotope), and 1×10−13% for 14C (radioisotope). These isotopes, which have different masses, exhibit the same chemical behavior. Thus, artificial enrichment of an isotope of low abundance and accurate analysis of the isotope can be applied to observation of a variety of reactions.
In the clinical field, in vivo administration and analysis of a compound labeled with, for example, radioactive carbon isotope 14C are very useful for assessment of drug disposition. For example, such a labeled compound is used for practical analysis in Phase I or Phase IIa of the drug development process. Administration of a compound labeled with radioactive carbon isotope 14C (hereinafter may be referred to simply as “14C”) to a human body at a very small dose (hereinafter may be referred to as “microdose”) (i.e., less than the pharmacologically active dose of the compound) and analysis of the labeled compound are expected to significantly reduce the lead time for a drug discovery process because the analysis provides findings on drug efficacy and toxicity caused by drug dispositon.
Examples of the traditional 14C analysis include liquid scintillation counting (hereinafter may be referred to as “LSC”) and accelerator mass spectrometry (hereinafter may be referred to as “AMS”).
LSC involves the use of a relatively small table-top analyzer and thus enables convenient and rapid analysis. Unfortunately, LSC cannot be used in clinical trials because of its low JAC detection sensitivity (10 dpm/mL). In contrast, AMS can be used in clinical trials because of its high 14C detection sensitivity (0.001 dpm/mL), which is less than one thousandth of that of LSC. Unfortunately, the use of AMS is restricted because AMS requires a large and expensive analyzer. For example, since only around fifteens of AMS analyzers are provided in Japan, analysis of one sample requires about one week due to a long waiting time for samples to be analyzed. Thus, a demand has arisen for development or a convenient and rapid method of analyzing 14C.
Some techniques have been proposed for solving the above problems (see for example, Non-Patent Document 1 and Patent Document 1).
I. Galli, et al. reported the analysis of 14C of a natural isotope abundance level by cavity ring-down spectroscopy (hereinafter may be referred to as “CRDS”) in Non-Patent Document 1, and this analysis has received attention.
Unfortunately, the 14C analysis by CRDS involves the use of a 4.5-μm laser source having a very intricate structure. Thus, a demand has arisen for a simple and convenient apparatus or method for analyzing 14C.
The present inventors have made studies in order to solve the above problems, and as a result, have proposed a simple and convenient carbon isotope analysis device and analysis method by use of an optical comb as a light source (see Patent Document 2).
However, there has arisen an additional object for an increase in partial pressure of carbon dioxide isotope in gas sent into an optical resonator for the purpose of further increases in sensitivity performance and analytical accuracy.
An object of the present invention is to provide a carbon isotope analysis device high in partial pressure of carbon dioxide isotope in gas sent into and mixed in an optical resonator, and high in sensitivity performance and analytical accuracy, and an analysis method by use of the carbon isotope analysis device.
The present invention relates to the following aspect:
The present invention provides a carbon isotope analysis device high in partial pressure of carbon dioxide isotope in gas sent into an optical resonator, and higher in sensitivity performance and analytical accuracy, and an analysis method by use of the carbon isotope analysis device.
The present invention will now be described by way of embodiments, which should not be construed to limit the present invention. In the drawings, the same or similar reference signs are assigned to components having the same or similar functions without redundant description. It should be noted that the drawings are schematic and thus the actual dimensions of each component should be determined in view of the following description. It should be understood that the relative dimensions and ratios between the drawings may be different from each other.
The carbon dioxide isotope generator 40 includes a combustion unit that generates gas containing carbon dioxide isotope from carbon isotope, a carbon dioxide isotope purifying unit, and a measurement unit of the amount of carbon, the measurement unit measuring the total amount of carbon from the amount of carbon dioxide.
The spectrometer 10 includes an optical resonator 11 having a pair of mirrors 12a and 12b, and a photodetector 15 that determines the intensity of light transmitted from the optical resonator 11.
Not only operation of the pump P, but also control of opening and closing of the valves 66a to 66d enables introduction of carbon dioxide isotope generated in the carbon dioxide isotope generator into the optical resonator 11 to be controlled.
In this embodiment, a radioisotope 14C, carbon isotope will be exemplified as an analytical sample. The light having an absorption wavelength range of the carbon dioxide isotope 14CO2 generated from the radioisotope 14C is light of a 4.5-μm wavelength range. The combined selectivity of the absorption line of the target substance, the light generator, and the optical resonator mode can achieve high sensitivity (detail is described later).
Throughout the specification, the term “carbon isotope” includes stable isotopes 12C and 13C and radioactive isotopes 14C, unless otherwise specified. In the case that the elemental signature “C” is designated, the signature indicates a carbon isotope mixture in natural abundance.
Stable isotopic oxygen includes 16O, 17O and 18O and the elemental signature “O” indicates an isotopic oxygen mixture in natural abundance.
The term “carbon dioxide isotope” includes 12CO2, 13CO2 and 14CO2, unless otherwise specified. The signature “CO2” includes carbon dioxide molecules composed of carbon isotope and isotopic oxygen each in natural abundance.
Throughout the specification, the term “biological sample” includes blood, plasma, serum, urine, feces, bile, saliva, and other body fluid and secretion; intake gas, oral gas, skin gas, and other biological gas; various organs, such as lung, heart, liver, kidney, brain, and skin, and crushed products thereof. Examples of the origin of the biological sample include all living objects, such as animals, plants, and microorganisms; preferably, mammals, preferably human beings. Examples of mammals include, but, should not be limited to, human beings, monkey, mouse, rat, guinea pig, rabbit, sheep, goat, horse, cattle, pig, dog, and cat.
The carbon dioxide isotope generator 40 may be of any type that can convert carbon isotope to carbon dioxide isotope. The carbon dioxide isotope generator 40 should preferably have a function to oxidize a sample and to convert carbon contained in the sample to carbon dioxide.
The carbon dioxide isotope generator 40 may be a carbon dioxide generator (G) 41, for example, a total organic carbon (TOC) gas generator, a sample gas generator for gas chromatography, a sample gas generator for combustion ion chromatography, or an elemental analyzer (EA).
Gas containing carbon dioxide isotope 14CO2 (hereinafter merely “14CO2”) can be generated through combustion of a pretreated biological sample; however, gaseous contaminants, such as CO and N2O are generated together with 14CO2 in this process. CO and N2O each exhibit a 4.5-μm wavelength range absorption spectrum as illustrated in
A typical process of removing CO and N2O involves collection and separation of 14CO2 as described below. The process may be combined with a process of removing or reducing CO and N2O with an oxidation catalyst or platinum catalyst.
As illustrated in
Preferably, the combustion tube 410 is configured from refractory glass (such as quartz glass) so as to be able to accommodate a sample therein and is provided with a sample port formed on a part thereof. Besides the sample port, a carrier gas port through which carrier gas is introduced to the combustion tube may also be formed on the combustion tube. Herein, not only such an aspect where the sample port and the Hike are provided on a part of the combustion tube, but also a configuration where a sample introducing unit is formed as a separate component from the combustion tube at an end of the combustion tube and the sample port and the carrier gas port are formed on the sample introducing unit, may be adopted.
Examples of the heater include electric furnaces, specifically tubular electric furnaces that can place and heat a combustion tube therein. A typical example of the tubular electric furnace is ARF-30M (available from Asahi Rika Seisakusho).
The combustion tube 410 should preferably be provided with a combustion oxidation unit 410 and/or a reduction unit 412 packed with at least one catalyst, downstream of the carrier gas channel. The combustion oxidation unit and/or the reduction unit may be provided at one end of the combustion tube 41 or provided in the form of a separate component. Examples of the catalyst to be contained in the combustion oxidation unit include copper oxide and a mixture of silver and cobalt oxide. The combustion oxidation unit can be expected to oxidize H2 and CO generated by combustion of a sample into H2O and CO2. Examples of the catalyst to be contained in the reduction unit include reduced copper and a platinum catalyst. The reduction unit can be expected to reduce nitrogen oxide (NOx) containing N2O into N2.
The carbon dioxide isotope purifying unit 43 may be a thermal desorption column. (CO2 collecting column) 432 of 14CO2 in a gas generated by combustion of a biological sample, for use in gas chromatography (GC). Thus, any influence of CO and/or N2O at the stage of detection of 14CO2 can be reduced or removed. A CO2 gas containing 14CO2 is temporarily collected in a GC column and thus concentration of 14CO2 is expected. Thus, it can be expected that the partial pressure of 14CO2 increases.
The carbon dioxide isotope purifying unit 43 should preferably include a 14CO2 adsorbent 431, for example, soda lime or calcium hydroxide. Thus, 14CO2 can be isolated in the form of carbonate to thereby allow the problem of gaseous contaminants to be solved. 14CO2 can be retained as carbonate and thus a sample can be temporarily reserved. Herein, phosphoric acid can be used in the discharge.
Such gaseous contaminants can be removed by any of or both (i) Collection and separation of 14CO2 by thermal desorption column and (ii) Separation of 14CO2 through trapping and discharge of 14CO2 with and from 14CO2 adsorbent.
(iii) Concentration (Separation) of 14CO2
14CO2 generated by combustion of the biological sample is diffused in piping. Therefore, 14CO2 may also be allowed to adsorb to an adsorbent and be concentrated, resulting in as enhancement is detection sensitivity (intensity). Such concentration can also be expected to separate 14CO2 from CO and N2O.
With reference to
A laser beam incident on and confined in the optical resonator 11 repeatedly reflects between the mirrors over several thousand to ten thousand times while the optical resonator 11 emits light at an intensity corresponding to the reflectance of the mirrors. Thus, the effective optical path length of the laser beam reaches several tens of kilometers, and a trace amount of analyte gas contained in the optical resonator can yield large absorption intensity.
The optical resonator may also be CRDS with fiber Bragg grating (FBG) and a gain-switched semiconductor laser or CRDS with an evanescent optical device.
In the case of the absence of a light-absorbing substance in the optical resonator, the dotted curve in
The transmitted light leaked from the optical resonator is detected with the photodetector, and the concentration of 14CO2 is calculated with the arithmetic device. The concentration of 14CO2 is then calculated from the concentration of 14CO2.
The distance between the mirrors 12a and 12b in the optical resonator 11, the curvature radius of the mirrors 12a and 12b, and the longitudinal length and width of the body should preferably be varied depending on the absorption wavelength of the carbon dioxide isotope (i.e., analyte). The length of the resonator is adjusted from 1 mm to 10 m, for example.
In the case of carbon dioxide isotope 14CO2, an increase in length of the resonator contributes to enhancement of the effective optical path length, but leads to an increase in volume of the gas cell, resulting in an increase in amount of a sample required for the analysis. Thus, the length of the resonator is preferably 10 cm to 60 cm. Preferably, the curvature radius of the mirrors 12a and 12b is equal to or slightly larger than the length of the resonator.
The distance between the mirrors can be adjusted by, for example, several micrometers to several tens of micrometers through the drive of the piezoelectric element 13. The distance between the mirrors can be finely adjusted by the piezoelectric element 13 for preparation of an optimal resonance state.
The mirrors 12a and 12b (i.e., a pair of concave mirrors) may be replaced with combination of a concave mirror and a planar mirror or combination of two planar mirrors that can provide a sufficient optical path.
The mirrors 12a and 12b may be composed of sapphire glass, Ca, F2, or ZnSe.
The cell 16 to be filled with the analyte gas preferably has a small volume because even a small amount of the analyte effectively provides optical resonance. The volume of the cell 16 may be 8 ml to 1,000 ml. The cell volume can be appropriately determined depending on the amount of a 14C source to be analyzed. For example, the cell volume is preferably 80 mL to 120 mL for a 14C source that is available in a large volume (e.g., urine), and is preferably 8 mL to 12 ml, for a 14C source that is available only in a small volume (e.g., blood or tear fluid).
Evaluation of Stability Condition of Optical Resonator
The 14CO2 absorption and the detection limit of CRDS were calculated based on spectroscopic data. Spectroscopic data on 12CO2 and 13CO2 were retrieved from the high-resolution transmission molecular absorption database (HITRAN), and spectroscopic data on 14CO2 were extracted from the reference “S. Dobos, et al., 3. Naturforsch, 44a, 633-639 (1989)”.
A Modification (Δβ) in ring-down rate (exponential decay rate) caused by 14CO2 absorption (Δβ=β−β0 where β is a decay rate in the presence of a sample, and β0 is a decay rate in the absence of a sample) is represented by the following expression:
Δβ=σ14(λ,T,P)N(T,P,X14)c
where σ14, represents the photoabsorption cross section of 14CO2, N represents the number density of molecules, c represents the speed of light, and σ14 and N are the function of λ (the wavelength of laser beam), T (temperature), P (pressure), and X14=ratio 14C/TotalC.
If a modification (Δβ) in ring-down rate (corresponding to noise derived from the optical resonator) can be reduced to a level on the order of 101 s−1, the analysis could be performed at a ratio 14C/TotalC on the order of 10−11. Thus, cooling at about −40° C. is revealed to be most preferable during the analysis.
In the case of a ratio 14C/TotalC of 10−11 as a lower detection limit, the drawing suggests that requirements involve an increase (for example, 20%) in partial pressure of CO2 gas due to concentration of the CO2 gas and the temperature condition described above.
The cooler and the cooling temperature will be described in more detail in the section of a second aspect of the carbon isotope analysis device, described below.
The light generator 20A of
The light source 23 is preferably an ultrashort pulse generator. In the case of use of an ultrashort pulse generator as the light source 23, a high photon density per pulse enables a nonlinear optical effect to be easily exerted, simply generating light of a 4.5-μm wavelength range corresponding to an absorption wavelength of radioactive carbon dioxide isotope 14CO2. A flux of comb-like light beams uniform in width of each wavelength. (optical frequency comb, hereinafter may be referred to as “optical comb”.) is obtained, and thus the variation in oscillation wavelength can be negligibly small. In the case of a continuous oscillation generator as the light source, the variation in oscillation wavelength causes a need for measurement of the variation in oscillation wavelength with an optical comb or the like.
The light source 23 can be, for example, a solid-state laser, a semiconductor laser or a fiber laser that generates short pulse by mode-locking. In particular, a fiber laser is preferably used because a fiber laser is a practical light source that is compact and also excellent in stability to environment.
Such a fiber laser can be an erbium (Er)-based (1.55-μm wavelength range) or ytterbium. (Yb)-based (1.04-μm wavelength range) fiber laser. An Er-based fiber laser is preferably used from the viewpoint of economics, and an Yb-based fiber laser is preferably used from the viewpoint of an enhancement in intensity of light.
A plurality of optical fibers 21 and 22 can be a first optical fiber 21 that transmits light from the light source and a second optical fiber 22 for wavelength conversion, the second optical fiber splitting from the first optical fiber 21 and coupling with the first optical fiber 21 downstream. The first optical fiber 21 can be any one connected from the tight source to the optical resonator. A plurality of optical components and a plurality of optical fibers can be disposed on each path of the optical fibers.
It is preferred that the first optical fiber 21 can transmit high intensity of ultrashort light pulses without deterioration of the optical properties of the pulses. Specific examples can include a dispersion-compensating fiber (DC) and a double-clad fiber. The first optical fiber 21 should preferably be composed of fused silica.
It is preferred that the second optical fiber 22 can efficiently generate ultrashort light pulses at a desired longer wavelength and transmit high intensity of ultrashort light pulses without deterioration of the optical properties of the pulses. Specific examples can include a polarization-maintaining fiber, a single-mode fiber, a photonic crystal fiber, and a photonic bandgap fiber. The optical fiber preferably has a length or several meters to several hundred meters depending on the amount of wavelength shift. The second optical fiber 22 should preferably be composed of fused silica.
The nonlinear optical crystal 24 is appropriately selected depending on the incident light and the emitted light. In the present Example, for example, a PPMgSLT (periodically poled MgO-doped Stoichiometric Lithium. Tantalate (LiTaO3)) crystal, a PPLN (periodically poled Lithium Niobate) crystal, or a GaSe (Gallium selenide) crystal can be used from the viewpoint that light of a about 4.5-μm wavelength range is generated from each incident light. Since a single fiber laser light source is used, perturbation of optical frequency can be cancelled out in difference frequency generation as described below.
The length in the irradiation direction (longitudinal direction) of the nonlinear optical crystal 24 is preferably longer than 11 mm, more preferably 32 mm to 44 mm, because a high-power optical comb is obtained.
Difference frequency generation (hereinafter may be referred to as “DFG”) can be used to generate difference-frequency light. In detail, the light beams of different wavelengths (frequencies) from the first and second optical fibers 21 and 22 transmit through the non-linear optical crystal, to generate difference-frequency light based on the difference in frequency. In the present. Example, two light beams having wavelengths λ1 and λ2 are generated with the single light source 23 and extracted into the nonlinear optical crystal, to generate light in the absorption wavelength of carbon dioxide isotope based on the difference in frequency. The conversion efficiency of the DFG using the nonlinear optical crystal depends on the photon density of light source having a plurality of wavelengths (λ1, λ2, . . . λx). Thus, difference-frequency light can be generated from a single pulse laser light source through DFG.
The resultant 4.5-μm wavelength range light is an optical comb composed of a spectrum of frequencies (modes) with regular intervals (fr) each corresponding to one pulse (frequency f=fceo+N·fr, N: mode number). CRDS using the optical comb requires extraction of light having the absorption wavelength of the analyte into an optical resonator including the analyte. Herein, f is cancelled out and thus fceo is 0 in the optical comb generated, according to a process of difference frequency generation.
The light source may generate laser beams having different wavelengths from two laser devices (Nd:YAG laser and external-cavity diode laser (ECDL)) and generate irradiation light having the absorption wavelength of the carbon dioxide isotope based on the difference in frequency between these laser beams.
The light generator is preferably configured from a single fiber laser light source, an optical fiber having a length of several meters, and a nonlinear optical crystal. The reason is because the light generator having such a configuration has a compact size and is easy to carry and operate. Since a plurality of light beams are generated from a single light source, these beams exhibit the same width and timing of perturbation, and thus the perturbation of optical frequency can be readily cancelled through difference frequency generation without a perturbation controller.
In some embodiments, a laser beam may be transmitted through air between the optical resonator and the coupling node of the first optical fiber with the second optical fiber. Alternatively, the optical path between the optical resonator and the coupling node may optionally be provided with an optical transmission device including an optical system for convergence and/or divergence of a laser beam through a lens.
The arithmetic device 30 may be of any type that can determine the concentration of a light-absorbing substance in the optical resonator based on the decay time and ring-down rate and calculate the concentration of the carbon isotope from the concentration of the light-absorbing substance.
The arithmetic device 30 includes an arithmetic controller 31, such as an arithmetic unit used in a common computer system (e.g., CPU) an input unit 32, such as a keyboard or a pointing device (e.g., a mouse) a display unit. 33, such as an image display (e.g., a liquid crystal display or a monitor); an output unit 34, such as a printer; and a memory unit 35, such as a ROM, a RAM, or a magnetic disk.
Although the carbon isotope analysis device according to the first aspect has been described above, the configuration of the carbon isotope analysis device should not be limited to the embodiment described above, and various modifications may be made. Other aspects of the carbon isotope analysis device will now be described by focusing on modified points from the first aspect.
<Cooler and Dehumidifier>
As illustrated in
The cooler for cooling the optical resonator 11 may be, for example, a liquid nitrogen vessel or a dry ice vessel besides the Peltier element 19. The Peltier element 19 is preferably used in view of a reduction in size of a spectrometer 10, whereas a liquid nitrogen vessel or a dry ice vessel is preferably used in view of a reduction in production cost of the device.
The vacuum device 18 may be of any type that can accommodate the optical resonator 11, apply irradiation light from the light generator 20 to the optical resonator 11, and transmit light transmitted, to the photodetector.
A dehumidifier may be provided. Dehumidification may be here carried out with a cooling means, such as a Peltier element, or by a membrane separation method using a polymer membrane, such as a fluorinated ion-exchange membrane, for removing moisture.
In the case that the carbon isotope analysis device 1 is used in a microdose test, the prospective detection sensitivity to the radioactive carbon isotope 14C is approximately 0.1 dpm/ml. Such a detection sensitivity “0.1 dpm/ml” requires not only use of “narrow-spectrum laser” as a light source, but also the stability of wavelength or frequency of the light source. In other words, the requirements include no deviation from the wavelength of the absorption line and a narrow line width. In this regard, the carbon isotope analysis device 1, which involves CRDS with a stable light source using “optical frequency comb light”, can solve such a problem. The carbon isotope analysis device 1 has an advantage in that the device can determine a low concentration of radioactive carbon isotope in the analyte.
The earlier literature (Hiromoto Kazuo et al., “Designing of 14C continuous monitoring based on cavity ring down spectroscopy”, preprints of Annual Meeting, the Atomic Energy Society of Japan, Mar. 19, 2010, p. 432) discloses determination of the concentration of 14C in carbon dioxide by CRDS in relation to monitoring of the concentration of spent fuel in atomic power generation. Although the signal processing using the fast Fourier transformation (FFT) disclosed in the literature has a high processing rate, the fluctuation of the baseline increases, and thus a detection sensitivity of 0.1 dpm/mi cannot be readily achieved.
However, as described above, the present invention allows the partial pressure of carbon dioxide isotope 14CO2 is sample gas to be enhanced to thereby allow the prospective detection sensitivity to the radioactive carbon isotope 14C to be enhanced, thereby enabling a detection sensitivity of “0.1 dpm/ml” to be achieved.
Since the absorption intensity of 14CO2 has temperature dependence, the temperature in the optical resonator 11 is preferably adjusted to a minimum possible level. In detail, the temperature in the optical resonator 11 is preferably adjusted to 273K (0° C.) or less. The temperature may have any lower limit. In view of cooling effect and cost, the temperature in the optical resonator 11 is adjusted to preferably 173K to 253K (−100° C. to −20° C.), more preferably about 233K (−40° C.)
The spectrometer may further be provided with a vibration damper. The vibration damper can prevent a perturbation in distance between the mirrors due to the external vibration, resulting in an improvement in analytical accuracy. The vibration damper may be an impact absorber (polymer gel) or a seismic isolator. The seismic isolator may be of any type that can provide the spectrometer with vibration having a phase opposite to that of the external vibration.
As illustrated in
While such a delay line is exemplified as the wavelength filter, a dispersion medium may also be used without any limitation thereto.
In the aforementioned embodiment, the distance between the mirrors is adjusted with the piezoelectric element 13 for generation of ring-down signals in the spectrometer 10. For generation of ring-down signals, a light shield may be provided in the light generator 20 for ON/OFF control of light incident on the optical resonator 11. The light shield may be of any type that can promptly block light having the absorption wavelength of the carbon dioxide isotope. The excitation light should be blocked within a time much shorter than the decay time of light in the optical resonator.
A carbon isotope analysis device 10 is obtained by replacing the light generator 20A in
The light generator 20C in
The light generator includes a first amplifier that is disposed between the splitting node and the coupling node of the first optical fiber 21, a second amplifier that is disposed between the splitting node and the coupling node of the second optical fiber and that is different in band from the first amplifier, and a nonlinear optical crystal through which are allowed to propagate through to thereby generate light at an absorption wavelength of the carbon dioxide isotope, due to the difference in frequency.
The amplifier, for example, a first amplifier 25 disposed on the route of the first optical fiber 21 is preferably an Er-doped optical fiber amplifier, and a second amplifier 26 disposed on the route of the second optical fiber 22 is preferably a Tm-doped optical fiber amplifier.
The first optical fiber 21 should preferably further include a third amplifier, more preferably a third amplifier between the first amplifier 21 and the coupling node, because the intensity of light obtained is enhanced. The third amplifier should preferably be an Er-doped optical fiber amplifier.
The first optical fiber 21 should preferably further include a wavelength-shifting fiber, more preferably a wavelength-shifting fiber between the first amplifier and the coupling node, because the intensity of light obtained is enhanced.
The light source used is a high repetition rate ultrashort pulse fiber laser by use of a single-wall carbon nanotube (SWNT) and 980-nm LD as an excitation laser, where the wavelength of light emitted is 1.55 μm and the repeated frequency is 160 MHz. The light emitted from the light source is input as seed light, amplified by an Er-doped fiber amplifier (EDEA) and split to two beams by a polarization beam splitter (PBS).
Chirped pulse amplification is performed by an amplifier (DCF-Er-amp) using a dispersion-compensating fiber (DCF), EDFA, and an Er:Yb-doped double-clad fiber on one shorter wavelength route (first optical fiber). The delay line illustrated can also be subjected to fine correction of the wavelength.
The following is performed on other longer wavelength route (second optical fiber): the dispersion of light pulses amplified by use of a large-mode-area photonic crystal fiber (LMA-PCF) is compensated, ultrashort light pulses high in intensity are generated, the wavelength is then shifted to about 1.85 μm by a small core polarization-maintaining fiber (Small core PMF), and the light is amplified by a Tm-doped fiber amplifier (TDFA). Furthermore, wavelength conversion (expansion) is performed by a polarization maintaining highly nonlinear dispersion shifted fiber (PM-HE-DSP).
As described above, supercontincum (SC) light having an average output of 300 mW and expanding in a wavelength range from 1700 to 2400 nm (1.7 to 2.4 μm) can be generated.
Finally, difference frequency generation is performed by making each light output from the two routes, incident perpendicularly to the S1 surface of a nonlinear optical crystal (PPM SLT manufactured by Oxcie Corporation (Nonlinear Coefficient (deff)>7.5 pm/V, Typical PMT 44+/−5 degree C., AR. Coat. S1&S2 R<0.5% at 1064/532 nm, Crystal Size (T×W) 1 mm×2 mm, Crystal Length (L) 40 mm)) having a length in the longitudinal direction of 40 mm. As described above, a mid-infrared optical frequency comb of a wavelength range from 4400 to 4800 nm (4.5 μm) can be emitted from the S2 surface.
A half-value width is narrower and an intensity is higher than those in a light spectrum diagram of a mid-infrared comb, created by a conventional method. A polarization maintaining highly nonlinear dispersion shifted fiber is added to a rear stage of TDI-A to thereby not only enhance the selectivity of light of an objective wavelength, but also efficiently provide desired light having a high intensity.
Since an optical comb may be obtained in the carbon isotope analysis within the scope where the wavelength region for analysis of 14C as an analyte is covered, the present inventors have focused on the following: obtaining higher-power light with a narrower oscillation spectrum of an optical comb light source. A narrower oscillation spectrum can allow for amplification with amplifiers different in band and use of a nonlinear optical crystal long in length. The present inventors have then made studies, and as a result, have conceived that high-power irradiation light having the absorption wavelength of carbon dioxide isotope is generated based on the difference in frequency, by (A) generating a plurality of light beams different in frequency, from a single light source, (B) amplifying intensities of the plurality of light beams obtained, by use of amplifiers different in band, respectively, and (C) allowing the plurality of light beams to propagate through a nonlinear optical crystal longer in length than a conventional nonlinear optical crystal, in generation of an optical comb by use of a difference frequency generation method. The present invention has been completed based on the above finding. There has not been reported any conventional difference frequency generation method that amplifies the intensity of light with a plurality of amplifiers different in band and provides high-power irradiation light obtained by use of a crystal long in length.
Absorption of light by a light-absorbing material, in the case of a high intensity of an absorption line and also a high intensity of irradiation light, is remarkably decreased in low level corresponding to the absorption of light and appears to be saturated with respect to the effective amount of light absorption (called saturation absorption). According to a SCAR theory (Saturated Absorption CRDS), in the case where light of a 4.5-μm wavelength range, high in intensity of an absorption line, is applied to a sample such as 14CO2 in an optical resonator, a large saturation effect is initially exhibited due to a high intensity of light accumulated in an optical resonator and a small saturation effect is subsequently exhibited due to a gradual reduction in intensity of light accumulated in an optical resonator according to progression of decay, with respect to a decay signal (ring-down signal) obtained. Thus, a decay signal where such a saturation effect is exhibited is not according to simple exponential decay. According to such a theory, fitting of a decay signal obtained in SCAR enables the decay rate of a sample and the decay rate of the back ground to be independently evaluated, and thus not only the decay rate of a sample can be determined without any influence of the variation in decay rate of the back ground, for example, due to the parasitic etalon effect, but also absorption of light by 14CO2 can be more selectively measured due to the saturation effect of 14CO2 larger than that of a gaseous contaminant. Accordingly, use of irradiation light higher in intensity is more expected to result in an enhancement in sensitivity of analysis. The light generator of the present invention can generate irradiation light high in intensity, and thus is expected to result in an enhancement in sensitivity of analysis in the case of use for carbon isotope analysis.
<Light Generator Including Light Source Other than Optical Comb, as Main Light Source>
It has been conventionally considered that, since a quantum cascade laser (QCL) has perturbation of oscillation wavelength and absorption wavelengths of 14C and 13C are adjacent, the QCL is difficult to use as a light source of a carbon isotope analysis device for use in 14C analysis. Thus, the present inventors have uniquely developed an optical comb light source that generates an optical comb from a single light source and thus have completed a compact and convenient carbon isotope analysis device (see Patent Document 2).
The present inventors have completed a light generator that generates narrow-line width and high-output (high-intensity) light, in order to achieve a further enhancement in analytical accuracy of a carbon isotope analysis device.
The present inventors have made studies about a further application of the light generator, and as a result, have conceived that perturbation of oscillation wavelength of light generated from QCL is corrected by a beat signal measurement device where narrow-line width light generated from the light generator is used as a frequency reference. The inventors have progressively made studies based on the finding, and as a result, have completed a compact, convenient, and highly-reliable light generator where a light source other than an optical comb is adopted as a main light source, and a carbon isotope analysis device by use of the light generator.
The light generator 50 includes:
The main light source of the carbon isotope analysis device 1C including the light generator 50 is not limited to an optical comb, can be a general-purpose light source such as QCL, and thus is increased in flexibilities of design and maintenance of the carbon isotope analysis device 1C.
The light generator 50 illustrated in
The present invention enables accurate measurement to be realized in a simple and convenient measurement system, although no phase-locking is daringly performed by an optical comb.
The cat eye 25 allows the dependence of back reflection affecting angle adjustment to be decreased, and thus enables light to be readily again incident on QCL. The optical isolator 29 enables light to be shielded.
The light source 23 may be a mid-infrared quantum cascade laser (Quantum Cascade Laser: QCL).
It is preferred that the optical fiber 21 can transmit high intensity of ultrashort light pulses without deterioration of the optical properties of the pulse. The optical fiber 21 should preferably be composed of fused silica.
In the fourth embodiment, it is preferable to generate a laser beam from the light source 23 and transmit such light obtained, to the optical fiber 21; to split the light from the light source 23 by use of a splitter 28; to focus the light split, on a condenser lens 25b and reflect the light focused, by use of a mirror 25a; and to send the light back to the light source 23 via the mirror 25a and the splitter 28 (feedback step).
The present inventors have proposed a carbon isotope analysis device that can allow for convenient and rapid analysis of 14C, and a carbon isotope analysis method by use of the carbon isotope analysis device (see Patent Document 2). Thus, studies about microdose with 14C can be conveniently and inexpensively performed.
There is increasingly demanded a distributed-feedback (DFB) quantum-cascade laser (hereinafter may be referred to as “QCL”) system as one aspect of a mid-infrared (MIR) laser for use in 14C analysis. The reason for this is because such a system is commercially available and is easily handled due to a broad mode-hop-free tuning range of several nanometers and monomode emission of a line width of typical several MHz.
Although sufficient in the above performance in many spectroscopic applications, such a QCL system has been demanded to have a line width of 100 kHz or less laser in coupling with a high-finesse optical resonator (reflectance R>99.9%) for use in CRDS. A solution for solving the problem of such a decrease in line width is, for example, high-speed electrical signal feedback (for example, PDH lock) with a frequency discriminator, and has the problems of a need for a high-speed signal processing system and of being expensive. Furthermore, there is a need for high bandwidth modulation in a laser light source.
Thus, it has been demanded to further improve stability of a light source in 14C analysis.
The present inventors have made studies, and as a result, have focused on a method using optical feedback known as delayed self-injection, as an alternative of high-speed electrical signal feedback with a frequency discriminator. It has found that such passive feedback can be applied to QCL to thereby allow the line width of a laser to be reduced by the minimum cost. That is, the fourth embodiment described above provides a carbon isotope analysis device improved in stability of a light source, and a carbon isotope analysis method by use of the carbon isotope analysis device.
The carbon dioxide trapping system (purifier) and the light source are also described through the description of the first to fourth aspects of the carbon isotope analysis device. Both the purifier and the light source each have a compact and space-less, simple configuration. An increase in freedom of the layout of the purifier and the light source can result in a significant decrease in volume of the entire carbon isotrope analysis device.
[Carbon Isotope Analysis Method]
The analysis of radioisotope 14C as an example of the analyte will now be described. Although the carbon isotope analysis method includes no pretreatment (step (A)) of a biological sample, carbon isotope analysis is preferably performed after a pretreatment of a biological sample is performed.
(A) Biological samples, such as blood, plasma, urine, feces, and bile, containing 14C are prepared as radioisotope 14C sources. The prepared biological sample is deproteinized and thus to remove the biological carbon source. The pretreatment of the biological sample is categorized into a step of removing carbon sources derived from biological objects and a step of removing or separating the gaseous contaminant in a broad sense this embodiment, the step of removing carbon sources derived from biological objects will now be mainly described.
A microdose test analyzes a biological sample, for example, blood, plasma, urine, feces, or bile containing an ultratrace amount of 14C labeled compound. Thus, the biological sample should preferably be pretreated to facilitate the analysis. Since the ratio 14C/TotalC of 14C to total carbon in the biological sample is one of the parameters determining the detection sensitivity in the measurement due to characteristics of the CRDS unit, it is preferred to remove the carbon source derived from the biological objects contained in the biological sample.
Examples of deproteinization include insolubilization of protein with acid or organic solvent; ultrafiltration and dialysis based on a difference in molecular size; and solid-phase extraction. As described below, deproteinization with organic solvent is preferred, which can extract the 14C labeled compound and in which the organic solvent can be readily removed after treatment.
The deproteinization with organic solvent involves addition of the organic solvent to a biological sample to insolubilize protein. The 14C labeled compound adsorbed on the protein is extracted to the organic solvent in this process. To enhance the recovery rate of the 14C labeled compound, the solution is transferred to another vessel and fresh organic solvent is added to the residue to further extract the labeled compound. The extraction operations may be repeated several times in the case that the biological sample is feces or an organ such as lung, which cannot be homogeneously dispersed in organic solvent, the biological sample should preferably be homogenized. The insolubilized protein may be removed by centrifugal filtration or filter filtration, if necessary.
The organic solvent is then removed by evaporation to yield a dry 14C labeled compound. The carbon source derived from the organic solvent can thereby be removed. Preferred examples of the organic solvent include methanol (MeOH), ethanol (EtOH), and acetonitrile (ACN). Particularly preferred is acetonitrile.
(B) Carbon isotope analysis device 1 illustrated in
(C) Moisture is preferably removed from the resultant 14CO2. For example, moisture can be removed from the 14CO2 gas in the carbon dioxide isotope generator 40 by allowing the 14CO2 gas to pass through a drying unit 44 and/or pass through a desiccant 46 (e.g., calcium carbonate). In addition, moisture can also be removed by cooling the 14CO2 gas for moisture condensation. For example, moisture condensation can be made by inserting cold water into a U-shaped supply tube 48 illustrated in
(D) Trap tube 61 is inserted into a Dewar flask 63 including liquid nitrogen. 65, and thus the trap tube 61 is cooled to 0° C. or less. The generated 14CO2 is then sent into the trap tube 61, together with a carrier gas lower in freezing point than the 14CO2. The carrier gas may be, for example, helium gas. Carbon dioxide isotope is condensed in the trap tube 61. After the 14CO2 is condensed, gas in the trap tube 61 is removed. For example, helium gas in the trap tube 61 can be removed by closing valves 66a and 66b illustrated in
(E) The optical resonator 11 is filled with the gasified 14CO2. The optical resonator 11 can be filled with the gasified 14CO2 by opening the valves 66a, 66h, 66c and 66d with the pump P being operated. The 14CO2 is preferably cooled to 273 K (0° C.) or less. The 14CO2 can be cooled by cooling the optical resonator 11 by a Peltier element 19 to enhance the absorption intensity of excitation light. The optical resonator 11 is preferably maintained under vacuum because a reduced effect of the external temperature on the optical resonator improves analytical accuracy.
(F) A mid-infrared optical frequency comb of a wavelength range from 4.5 μm to 4.8 μm is generated as irradiation light at an absorption wavelength of the carbon dioxide isotope.
(G) The carbon dioxide isotope 14CO2 is in resonance with the light. To improve analytical accuracy, the external vibration of the optical resonator 11 is preferably reduced by a vibration absorber to prevent a perturbation in distance between the mirrors 12a and 12h. During resonance, the downstream end of the first optical fiber 21 should preferably abut on the mirror 12a to prevent the light from coming into contact with air. The intensity of light transmitted from the optical resonator 11 is then determined. As illustrated in
(H) The concentration of carbon isotope 14C is calculated from the intensity of the transmitted light.
An evaluation test o£ the basic performance of a carbon dioxide trapping system illustrated in
[Operation Procedure]
1. Sample (Rat Urine Sample)
Three rats were prepared, a cage was washed with a small amount of distilled water every 24 hours, and such distilled water used for the washing was combined to 100 g and defined as a urine sample (500 uL/collection) from each of the rats. Such a urine sample was collected from each of the rats at 8 time points in total. Such 24 samples obtained were subjected to the following experiment.
2. Carbon Dioxide Isotope Generation
Each sample was incorporated into a tin capsule or tin foil, and then oxidized and combusted in the following carbon dioxide isotope generation conditions, by use of an organic elemental analyzer (hereinafter may be referred to as “EA”, trade name: “Vario MICRO cube” manufactured by Elementar), thereby providing carbon dioxide isotope.
<Carbon Dioxide Isotope Generation Conditions>
3. Obtaining of Carbon Dioxide Isotope Partial Pressure
After sample gas was purified by use of a carbon isotope analysis device including a carbon dioxide trapping system illustrated in
The average values of the resultant measurement results of the 24 samples (3 individuals×8 time points) were as follows: the average amount of carbon: 2.2 mgC/500 uL and the average partial pressure: 80.4%.
[Operation Procedure]
1. Sample (Glucose Sample)
Each glucose sample having an amount of carbon of 0 to 96.2 (mgC), described below, was prepared as a sample.
2. Carbon Dioxide Isotope Generation and Obtaining of Partial Pressure
Carbon dioxide isotope was generated from each glucose sample in the same manner as in Examples except that no sample gas purification was performed by use of the carbon dioxide trapping system, and the partial pressure of the carbon dioxide isotope in the optical resonator was then measured.
The resultant partial pressure value of the carbon dioxide isotope relative to the amount of carbon, with respect to each sample, is shown in Table 1.
The results obtained in Examples and Comparative Examples are collectively illustrated in
It was confirmed from the foregoing that the partial pressure of carbon dioxide isotope in the optical resonator was increased by performing sample gas purification by use of the carbon dioxide trapping system.
Although the carbon isotope analysis method according to the first aspect has been described above, the configuration of the carbon isotope analysis method should not be limited to the embodiment described above, and various modifications may be made. Other aspects of the carbon isotope analysis method will now be described by focusing on modified points from the first aspect.
Although the embodiment of the present invention has been described above, the descriptions and drawings as part of this disclosure should not be construed to limit the present invention. This disclosure will enable those skilled in the art to find various alternative embodiments, examples, and operational techniques.
The carbon isotope analysis device according to the embodiment has been described by focusing on the case where the analyte as a carbon isotope is radioisotope 14C. The carbon isotope analysis device can analyze stable isotopes 12C and 13C besides radioisotope 14C. In such a case, excitation light of 2 μm or 1.6 μm is preferably used in, for example, absorption line analysis of 12CO2 or 13CO2 based on analysis of 12C or 13C.
In the case of absorption fine analysis of 12CO2 or 13CO2, the distance between the mirrors is preferably 10 to 60 cm, and the curvature radius of the mirrors is preferably equal to or longer than the distance therebetween.
Although the carbon isotopes 12C, 13C, and 14C exhibit the same chemical behaviors, the natural abundance of 14C (radioisotope) is lower than that of 12C or 13C (stable isotope). Artificial enrichment of the radioisotope 14C and accurate analysis of the isotope can be applied to observation of a variety of reaction mechanisms.
The carbon isotope analysis device according to the embodiment may further include a third optical fiber configured from a nonlinear fiber that splits from a first optical fiber and couples with the first optical fiber, downstream of a splitting node. Such first to third optical fibers can be combined to thereby generate two or more various light beams different in frequency.
A medical diagnostic device or environmental measuring device including the configuration described above in the embodiment can be produced as in the carbon isotope analysis device. The light generator described the embodiments can also be used as a measuring device.
An optical frequency comb corresponds to a light source where longitudinal modes of a laser spectrum are arranged at equal frequency intervals at a very high accuracy, and is expected to serve as a novel, highly functional light source in the fields of precision spectroscopy and high-accuracy distance measurement. Since many absorption spectrum bands of substances are present in the mid-infrared region, it is important to develop a mid-infrared optical frequency comb light source. The above light generator can be utilized in various applications.
As described above, the present invention certainly includes, for example, various embodiments not described herein. Thus, the technological range of the present invention is defined by only claimed elements o£ the present invention in accordance with the proper claims through the above descriptions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-007874 | Jan 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/001906 | 1/22/2019 | WO | 00 |
