JP5464462B2 - Nitric oxide measuring method and measuring apparatus - Google Patents

Description

  The present invention relates to a measurement method suitable for immediately detecting nitric oxide (NO), particularly a trace amount of NO in a sample gas, and a measurement apparatus therefor.

  Nitric oxide (NO) is one of the NOx that cause air pollution, and NO concentration in the atmosphere is measured as an environmental standard. On the other hand, NO is increased in the amount of exhaled air when inflammation occurs in the airway epithelium, and the amount correlates well with the degree of inflammation, so in recent years, it will be noted as a marker for airway diseases including asthma It has become. In 1997 the European Respiratory Society (Eur. Respir. J., 10, 1683-1693 (1997)) and in 1999 at the American Thoracic Society (Am. J. Respir. Crit. Care. Med., 160 , 2104-2117 (1999)), guidelines for monitoring exhaled NO are adopted.

  When measuring NO in gas samples such as air and exhaled air, ppb level measurement sensitivity is required. Conventionally, chemiluminescence using ozone is used as a dry NO measurement method, and as a wet NO measurement method, the target gas is taken into the gas absorption liquid and reacted with the Salzmann reagent. (Absorptiometry) is in practical use.

The chemiluminescence method is a method for measuring NO in a gas sample by using luminescence when the excited NO ₂ produced by reacting gaseous NO and ozone returns to the ground state (Palmer, RM , et al., Nature, 327, 524-526 (1987)). Although this method is very sensitive and can measure NO in the air in real time, it requires an ozone generator (ozonator) to generate ozone for use in the reaction. There is a problem that it takes time to warm up to obtain ozone conditions. In order to increase the sensitivity for measuring NO at the ppb level and to avoid the influence of quenching by carbon dioxide coexisting in the sample gas, it is necessary to measure under reduced pressure conditions using a vacuum pump. As described above, the NO measurement in a gas sample by the chemiluminescence method has a drawback that it is difficult to reduce the weight and volume of the measuring device, and the cost for the device and the measurement becomes high. For this reason, for example, it is not a big problem when measuring NO in the environment by installing a fixed point, but moving the NO in the environment in multiple places (points) where you can not enter by car It is difficult to measure, carry a measuring device to the bedside of a patient with respiratory illness such as bronchial asthma in the medical field, or carry the measuring device by itself to measure NO in exhaled breath The chemiluminescence method has a problem that the usable environment is limited due to the apparatus.

Salzmann method, the NO ₂ gas in a sample using such bubbler incorporated into the gas absorbing liquid aqueous, water (H ₂ O) is reacted with NO ₂ nitrite ions (NO ₂ ^-) cause Then, the NO ₂ concentration in the gas sample is measured by measuring the absorbance of the azo dye formed by reacting the NO ₂ ^- with sulfanilic acid to form a diazotized sulfanilate and then azo-coupling with N-1-naphthylethylenediamine. Is a method of measuring. However, since NO itself does not react with the Salzmann reagent consisting of sulfanilic acid, N-1-naphthylethylenediamine, and acetic acid, NO cannot be directly measured by the Salzmann method. In order to measure NO by the Salzmann method, it is necessary to first oxidize NO to NO ₂ with an oxidizing agent such as potassium permanganate. When NO gas and NO ₂ gas coexist in the measurement sample, NO Prior to the oxidation of gas to NO ₂ , it was necessary to perform measurement by separating and removing NO ₂ gas derived from the sample. In addition, the Salzmann method is a method that measures the color development of the dye, so the sensitivity is low and there is no problem when the measurement target (sample) is air, but it is measured when the sample is small and NO is low in concentration. There was a disadvantage that it was not possible.

There is also known a method (DAN method) that uses a fluorescent reagent 2,3-diaminonaphthalene (DAN) instead of the Salzmann reagent to measure NO about 100 times more sensitively than when using a Salzman reagent. . However, in the DAN method, NO in the sample is first converted to NO ₂ and then absorbed by an alkaline aqueous solution. The alkaline aqueous solution is made acidic at pH 2 or lower, reacted with DAN, and adjusted again to alkaline at pH 10 or higher. There is a problem that it is necessary to measure the fluorescent substance produced later, and the measurement operation becomes extremely complicated. As described above, although the DAN method is more sensitive than the Salzmann method, the conversion from NO to NO ₂ is essential for measurement as in the Salzman method, and the conversion efficiency (oxidation rate) from NO to NO ₂ varies. However, it has not been solved that causes the measurement reliability to be impaired.

  A technique of taking a measurement target gas into a gas absorption liquid and measuring the measurement target gas as a chemical, such as the Salzmann method, is simple and has been used for a long time. However, the amount of gas absorption liquid for absorbing the measurement target gas by bubbling the measurement sample is required to be at least about 10 to 50 mL, and the volume of the gas absorption liquid causes the measurement target to be diluted. Become. Therefore, when measuring components at the ppb level, it is necessary to sample the sample gas over a long period of time from several tens of minutes to several hours to increase the concentration of the measurement target in the gas absorption liquid, and it is impossible to measure changes over time. There is also a problem.

As a method for solving such a problem, Patent Documents 1 to 3 and Non-Patent Document 1 disclose a gas collector using a microchannel provided on a block (substrate), to a gas absorption liquid of a measurement target gas. It is described that the absorption time (concentration of the sample gas) can be shortened. However, in any of these documents, measurement gas (carbon dioxide, hydrogen sulfide, sulfur dioxide) having high solubility in water is taken into an aqueous gas absorption liquid and measured, and the solubility in water No example has been reported so far in which NO gas with a low concentration is taken directly into the gas absorbent without conversion to NO ₂ .
JP 2000-155116 A JP 2004-361244 A JP 2007-139733 A Lab Chip, 5, 1374-1379 (2005)

The problem to be solved by the present invention is a method for quickly measuring NO concentration by taking NO gas having low solubility in water directly into the gas absorption liquid in the state of NO without conversion to NO ₂ and this. It is to provide an apparatus for carrying out the measuring method.

As a result of earnestly researching the present inventors to solve the above problems, when NO is brought into contact with the NO fluorescent probe aqueous solution through the gas permeable membrane, NO is taken into the aqueous solution and dissolved oxygen is dissolved. It was found that the concentration of NO can be measured by reacting with a NO fluorescent probe in the presence of NO and emitting fluorescence. Furthermore, in the above measurement, if NO ₂ is added from outside to measure the concentration of the sample gas so that the concentration in the sample gas becomes a constant concentration, the concentration of NO is measured much more than when NO ₂ is not added. It was found that the sensitivity was improved.

The present invention is based on the above findings and is as follows.
(1) A sample gas containing nitric oxide (NO) and a gas absorbing liquid containing a NO fluorescent probe are brought into contact with each other through a gas permeable membrane, and nitrogen monoxide in the sample gas is taken into the gas absorbing liquid. A method for measuring nitric oxide, comprising measuring the amount of fluorescence in a gas absorbing solution.

(2) sample gas containing nitric oxide, method for measuring nitric oxide according to claim 1 is intended to include nitrogen dioxide (NO ₂₎ in the range of 20 to 3000 volume ppb.
(3) The method for measuring nitric oxide according to the above (1) or (2), wherein the sample gas containing nitrogen monoxide and the gas absorbing liquid are contacted with a microchannel scrubber having a honeycomb structure.
(4) The nitric oxide according to any one of (1) to (3), wherein the NO fluorescent probe is a fluorescein derivative or rhodamine derivative that emits fluorescence by forming a triazole ring structure in the molecule in the presence of NO. Nitrogen measurement method.
(5) The NO fluorescent probe is 4,5-diaminofluorescein (DAF-2), 3-amino-4- (methylamino) -2 ′, 7′-difluorofluorescein (DAF-FM), 3-amino- The method for measuring nitric oxide according to any one of (1) to (4) above, which is selected from the group consisting of 4- (methylamino) -tetramethylrhodamine (DAR-4M).
(6) The method for measuring nitric oxide according to any one of (1) to (5), wherein the pH of the gas absorbing solution is 5 or more.
(7) The method for measuring nitric oxide according to any one of (1) to (6), wherein the gas absorbing liquid contains dimethylformamide at a concentration of 10 to 40%.
(8) The method for measuring nitric oxide according to any one of (1) to (7), wherein the sample gas containing nitric oxide is the atmosphere.
(9) The method for measuring nitric oxide according to any one of (1) to (7), wherein the sample gas containing nitric oxide is exhaled air.

(10)
An apparatus for measuring nitric oxide comprising the following [1] to [3].
[1] a gas flow path through which a sample gas containing nitric oxide flows,
[2] A gas collector having a gas absorption liquid channel through which a gas absorption liquid including a NO fluorescent probe arranged so as to come into contact with the gas channel via a gas permeable membrane on the boundary surface;
[3] Fluorescence detector connected to the gas absorption liquid outlet side channel of the gas collector

(11) The measurement apparatus for nitrogen monoxide according to (10), wherein the gas collector is a microchannel scrubber having a honeycomb structure.

As mentioned previously, the NO gas in the sample is measured in a wet far, after removal of the NO ₂ coexisting, converts NO to NO _2, it has been measuring the NO ₂ indirectly. In contrast, according to the method of the present invention, NO can be directly measured with a measurement sensitivity of several tens of ppb by a combination of a gas collector and a NO fluorescent probe. Surprisingly, in the conventional measurement of NO gas, coexisting NO ₂ that had to be removed in advance is added to the sample gas from the outside so as to obtain a constant concentration instead of removing it. When the concentration of NO was measured, the detection sensitivity was remarkably improved compared to the case where NO ₂ was not added, and NO could be directly measured with higher sensitivity. Furthermore, the measurement device of the present invention can be reduced to 1/10 or less in weight and volume as compared with the conventional chemiluminescence device, and the measurement sensitivity can respond to NO of human breath. Therefore, it can be used as a portable and portable NO gas measuring device, and can be used for environmental measurement and evaluation of respiratory diseases.

In the present invention, the sample gas containing nitric oxide (NO) to be measured is not particularly limited by any gas, but the atmosphere or breath having a high necessity for measurement, or It is preferable to target automobile exhaust gas and the like. The sample gas containing nitric oxide removes other coexisting gases using commonly used methods, such as using a soda lime column to remove carbon dioxide gas from the sample gas, even if it is used for measurement. It may be used for the measurement after it has been performed, or may be used for the measurement after the other coexisting gas is converted into another chemical substance by an appropriate chemical substance or the like. The present invention also includes an invention relating to “a method of measuring nitric oxide in which the sample gas containing nitric oxide contains nitrogen dioxide (NO ₂ ) in the range of 20 to 3000 ppb”. Even when measuring the sample gas, the sample gas contains NO ₂ at a predetermined concentration at the time when the gas absorbing liquid of the present invention and the sample gas come into contact with each other through the gas permeable membrane. Further, it is not excluded that a step of removing NO ₂ in advance (ANALYTICAL CHIMICA ACTA, 603, 60-66 (2007), etc.) is included in a column packed with molecular sieve impregnated with triethanolamine.
Note that in this specification, the term “nitrogen monoxide” or “NO” is used to indicate the name of a chemical substance, or nitric oxide gas or NO gas.

  In the present invention, a fluorescein derivative or a rhodamine derivative that emits fluorescence by reacting with NO to form a triazole ring structure in the molecule in the presence of dissolved oxygen can be used as the NO fluorescent probe. For example, a compound represented by the following general formula 1; 4,5-diaminofluorescein (DAF-2) (JP-A-10-226688, etc.), 3-amino-4- (methylamino) -2 ′, 7′-difluoro Fluorescein (DAF-FM) (US6,441,197, etc.), 3-amino-4- (methylamino) -tetramethylrhodamine (DAR-4M) (WO99 / 1447, US6,756,231, etc.), as well as the carboxyl group of fluorescein NO fluorescent probe (WO2004 / 005917) in which two amino groups are substituted at adjacent positions on the phenyl ring using a fluorescein derivative (TokyoGreen: registered trademark) substituted with an alkyl group or the like as a nucleus, carboxyl of rhodamine A NO fluorescent probe (WO2005 / 085811) in which two amino groups are substituted at adjacent positions on the phenyl ring using a rhodamine derivative in which the group is substituted with an alkyl group as a nucleus is preferably used. It is, but is not limited thereto. The types of substituents that can be substituted for the fluorescein derivative and rhodamine derivative are described in the above-mentioned documents, and those skilled in the art can appropriately select them when used in the present invention.

  Among these, 4,5-diaminofluorescein (DAF-2) is excellent in fluorescence intensity and can be suitably used in the present invention. Taking DAF-2 as an example, the mechanism by which the NO fluorescent probe of the present invention reacts with NO in the presence of dissolved oxygen in an aqueous solution to generate fluorescence is shown in the following chemical formula 2.

The above mechanism will be described in more detail. When NO is taken into the aqueous solution, it reacts with dissolved oxygen to produce NO ₂ (Formula A). When this produced NO ₂ reacts with NO, N ₂ O ₃ is produced (formula B). This N ₂ O ₃ has a strong nitrosation ability and reacts instantaneously with two amino groups substituted at adjacent positions on the phenyl ring of DAF-2 to form a triazole ring structure in the molecule. Give the fluorescent substance DAF-2T. Thus, two molecules of NO are usually required to convert DAF-2 to the fluorescent substance DAF-2T using dissolved oxygen.
NO + 1/2 O ₂ → NO ₂ (Formula A)
NO + NO ₂ → N ₂ O ₃ (Formula B)
DAF-FM, DAR-4M, etc. also have two amino groups substituted at adjacent positions on the phenyl ring, like DAF-2, and reacts with NO in the presence of dissolved oxygen in aqueous solution to triazole. It has the property of forming a ring and emitting fluorescence.

Normally, when measuring NO gas by a wet method, it is necessary to measure NO ₂ in advance, but in the measurement method of the present invention, the concentration in the sample gas is a constant concentration relative to the sample gas. The detection sensitivity could be remarkably improved by adding NO ₂ from the outside. This means that when measuring NO in the presence of a certain amount of NO ₂ , DAF-2 is converted to fluorescent DAF-2T because the reaction of Formula B proceeds without the need for the reaction of Formula A This is probably because only one molecule of NO is required. Furthermore, the reaction rate of Formula A is very slow compared to the reaction rate of Formula B. As a result, when NO ₂ is not added, the production of N ₂ O ₃ that reacts with DAF-2 is slowed and NO and DAF-2 Before the reaction proceeds sufficiently, it is sent to the fluorescence detector and measured, but when NO ₂ is added, only the reaction of fast formula B needs to proceed, and NO and DAF-2 This is thought to be because the reaction proceeds rapidly and almost all NO reacts with DAF-2 and then sent to the fluorescence detector for measurement. The number of NO molecules in the above description is based on the above formulas A and B, and the number of molecules may be different depending on how the formula is written. The above mechanism is an example of a mechanism estimated by the inventor, and is not limited to this mechanism.

In the present invention, the NO fluorescent probe is dissolved in water and used as a gas absorbing solution. In this case, the concentration of the NO fluorescent probe can be appropriately set experimentally depending on the type of the NO fluorescent probe to be used and the NO concentration in the measurement sample. For example, in the case of DAF-2, the concentration is usually 0. It is preferably 1 to 50 μM, particularly 1 to 10 μM. Moreover, as this gas absorption liquid, since fluorescein and fluorescein derivatives such as DAF-2 emit fluorescence at pH 5 or higher, an aqueous solution of pH 5 or higher can be used. In order to improve the reaction of the NO fluorescent probe, the pH is preferably 7 or more, particularly preferably 10 or more. Gas absorption liquids include phosphate buffer, borate buffer, borax buffer, wide-area buffer for ultraviolet spectrum measurement (The Chemical Society of Japan, "Chemical Handbook Basics II", revised 3rd edition, 355, 1984), It is preferable to adjust to the suitable pH using an aqueous sodium hydroxide solution or the like. Furthermore, an organic solvent miscible with water may be added to the gas absorption liquid in order to improve the poor solubility of NO and increase the measurement sensitivity. For example, an organic solvent such as dimethylformamide can be added in an amount of 10 to 40%.
It should be noted that the dissolved oxygen in the gas absorption liquid is sufficient for the practice of the present invention as long as it is contained at a concentration of about 1 to 20 ppm that is normally dissolved in water in the atmosphere. An oxygen source such as may be added separately.
When measuring by adding NO ₂ , the NO ₂ concentration in the sample gas is 20 volume ppb or more, preferably from the outside so that the NO ₂ concentration is constant within the range of 20 to 300 volume ppb. Can be added. In particular, it is preferable to add so that it becomes 200 capacity ppb or more. However, when it is difficult to control the concentration range due to the design of the measurement device or the supply source of NO ₂ , up to 3000 capacities ppb is added and coexists, so long as it does not hinder NO measurement. It is also acceptable.

  In the present invention, the measurement object gas and the gas absorbing liquid are contacted via a gas permeable membrane. The gas permeable membrane can be made of a hydrophobic, porous polyolefin resin, fluororesin, or the like. Examples of polyolefin resins include polyethylene, polypropylene, poly-1-butene, and poly-4-methyl-1-pentene resins. Examples of fluororesins include polytetrafluoroethylene, tetrafluoroethylene / hexafluoropropylene copolymer, tetra Fluoroethylene / perfluoro (alkyl vinyl ether) copolymers, tetrafluoroethylene / ethylene copolymers, polychlorotrifluoroethylene, polyvinylidene fluoride, vinylidene fluoride / hexafluoropropylene copolymers, and the like are suitable.

  For the contact between the gas to be measured and the gas absorbing liquid, it is preferable to use a microchannel scrubber having a honeycomb structure developed by the present inventor (see Patent Document 3 and Non-Patent Document 1) that can collect gas with high efficiency. .

  In the present invention, the fluorescence amount can be measured using a small fluorescent detector that is generally commercially available. Further, a commercially available LED lamp may be used as a light source, and a fluorescence detector may be combined with a small photomultiplier module.

Next, the measuring apparatus of the present invention will be described with reference to FIGS.
FIG. 1 is a schematic diagram showing the configuration of the measuring apparatus and its flow path.

This measuring apparatus includes (1) a gas flow path 1 through which a sample gas containing nitric oxide flows, and (2) NO disposed so as to be in contact with the gas flow path 1 through a gas permeable membrane 3 on the boundary surface. A gas collector 4 provided with a gas absorbing liquid flow path 2 through which a gas absorbing liquid containing a fluorescent probe flows, and (3) a fluorescence detector 5 provided in the gas absorbing liquid outlet side flow path of the gas collector 4 It has been.

In the measurement using this apparatus, the gas absorbing liquid containing the NO fluorescent probe is supplied from the storage tank 6 to the gas collector 4 by the pump 7 and passes through the gas absorbing liquid channel 2 in the gas collector 4. Then, NO is taken in through the gas permeable membrane 3. The gas absorbing liquid that has taken in NO is sent to the fluorescence detector 5 where the amount of fluorescence is measured and discharged. On the other hand, a sample gas containing nitric oxide is supplied from a sample gas supply unit (not shown) through the measurement sample inflow passage 11 from the gas inlet 8, passes through the gas passage 1, and passes through the gas permeable membrane 3. Then, after coming into contact with the gas absorbing liquid, the gas is discharged from the gas outlet 9. When adding nitrogen dioxide to the sample gas, the gas containing nitrogen dioxide is passed through the additive gas inflow passage 12 from a gas supply device (not shown in the drawing, which is different from the above-mentioned sample gas supply device). To the mass flow controller 10, and the flow rate is adjusted by the mass flow controller 10 so that the nitrogen dioxide concentration in the sample gas containing nitric oxide becomes constant. As a gas supply device for the gas containing nitrogen dioxide, in addition to a standard gas cylinder of NO ₂ , a permeation tube in which the amount of liquefied gas permeating and diffusing in the tube becomes constant when held at a constant temperature can be used.

  FIG. 2 is a schematic view showing a microchannel scrubber as an example of a gas collector. As shown in FIG. 2, the microchannel 14 (one aspect of the gas absorption liquid flow path 2) having a depth of 20 to 200 μm and a width of 100 to 1000 μm is formed on the substrate 13, and a gas permeation having a thickness of 30 μm or less is formed. The film 3 is fixed to the substrate 13 to form a gas absorption liquid channel. Note that the microchannel 14 is formed so that the area of the gas absorption liquid flow path contacting the gas flow path through the gas permeable membrane is 20% or more of the total area of the absorption liquid flow path section. The microchannel 14 may be of any other structure as long as the gas absorption liquid flows uniformly. However, the microchannel 14 is meandered or folded in order to increase the area of the gas absorption liquid flow path in contact with the gas absorption film. It is preferable to bend or provide an island for rectification to branch the gas absorption liquid flow path, and it is particularly preferable to be a honeycomb-structured microchannel. By fixing a block provided with a gas flow path on this microchannel scrubber (note that FIG. 1 is drawn upside down from FIG. 2), the microchannel scrubber can absorb NO in the sample gas. it can. In addition, the material of the board | substrate 13 can be suitably selected with reference to the description of patent document 3, etc. An example of a suitable material is polydimethylsiloxane.

  FIG. 3 shows a particularly preferable honeycomb structure as the structure of the microchannel scrubber. When such a microchannel scrubber having a honeycomb structure is used as a gas collector, the flow rate of the gas absorbing liquid is preferably 5 to 200 μL / min, and the gas flow rate is preferably 100 to 400 mL / min.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[Example 1] Compositional examination of gas absorption liquid (pH)
After a sample NO gas was passed through the gas absorbing solution by a bubbling method using a suction filter, the fluorescence intensity of the gas absorbing solution was measured to examine the composition (pH) of the gas absorbing solution of the present invention.

  The gas absorption solution used was a 10 mM UV spectrum wide-area buffer containing 2 μM DAF-2 (pH 7-12, edited by the Chemical Society of Japan, “Basic Guide to Chemistry II”, revised 3rd edition, 355, 1984). . A NO standard gas was used as the sample NO gas, and a gas whose flow rate was adjusted to 1 ppm by mass with a mass flow controller was bubbled for 10 minutes against 15 mL of each gas absorbent. For each gas absorbing solution at each pH, fluorescence intensity was measured at an excitation wavelength of 495 nm and a fluorescence wavelength of 515 nm using a fluorescence spectrophotometer (Shimadzu RF-5300PC) before bubbling and 5 minutes after bubbling was completed. The amount of increase in fluorescence intensity was determined by subtracting the fluorescence intensity before bubbling from the fluorescence intensity after minutes. The amount of increase in fluorescence intensity obtained was shown as a relative value where the amount of increase in fluorescence intensity in the gas absorption liquid at pH 7 was 1.

  The measurement results are shown in FIG. When the amount of increase in relative fluorescence intensity at each pH was compared, a remarkable increase was observed at pH 10 or higher after showing a substantially constant value between pH 7 and 9. From the above, it was found that when DAF-2 is used as the NO fluorescent probe, the pH of the gas absorbing solution is particularly preferably pH 10 or higher.

[Example 2] Measurement of NO by the method and apparatus of the present invention As the gas collector, the depth of the gas absorption liquid channel is 70 µm and the width is 200 µm, and it contacts the gas channel via the gas permeable membrane. A microchannel scrubber having a honeycomb structure in which the area of the gas absorption liquid flow path is 30% or more of the entire absorption liquid flow path portion was used. As the gas absorbing solution, 0.01 M NaOH aqueous solution (pH 11.7) containing 10 μM DAF-2 was used. The sample NO gas was prepared by adjusting the flow rate of NO standard gas using a mass flow controller and adjusting the NO concentration to 50 to 250 ppb by changing the dilution rate with air containing no NO and NO ₂ . Sample NO gas was supplied to the microchannel scrubber at 200 mL / min. The gas absorption liquid was adjusted to a flow rate of 10 μL / min with a pump and introduced into a microchannel scrubber. The fluorescence intensity of DAF-2T produced by reaction with DAF-2 was measured with a fluorescence detector. As the fluorescence detector, a 460 nm LED (20 mA) was used as the light source, and a small photomultiplier module was adopted as the photodetector. The light detector was equipped with a 520 nm cut filter to prevent the influence of excitation light. The sample NO gas was always monitored simultaneously with an apparatus based on the conventional method (chemiluminescence method).

  The measurement results are shown in FIG. The upper part of FIG. 5 shows the measurement results with the apparatus of the present invention, and the lower part of FIG. 5 shows the results of simultaneous monitoring with a chemiluminescence (CL) apparatus. The response in the upper diagram and the response in the lower diagram are in agreement, and it was confirmed that NO gas can be measured with the apparatus of the present invention. Further, when the NO concentration obtained by the conventional method in the lower stage is plotted as X, and the response intensity in the present invention in the upper stage is plotted as Y, a straight line having a correlation coefficient R = 0.9993 is obtained as shown in FIG. Thus, it was confirmed that the method of the present invention can obtain a response depending on the NO concentration.

Example 3 Measurement of NO in Breath Sample Using 0.01 M NaOH containing 10 μM DAF-2 as a gas absorbing solution, this was introduced into the microchannel scrubber at a flow rate of 10 μL / min. On the other hand, the breath of a healthy person was collected in a mylar bag, sucked from the bag at a timing of 900 seconds, 1500 seconds, and 2100 seconds and introduced into the microchannel scrubber at 200 mL / min for measurement.

  The measurement results are shown in FIG. A response consistent with the timing of exhalation was confirmed. From the above, it was confirmed that the method and apparatus of the present invention can detect NO during expiration.

[Example 4] Composition examination of gas absorption liquid (organic solvent)
The sample gas was passed through the gas absorption liquid by a bubbling method using a suction filter, and then the fluorescence intensity of the gas absorption liquid was measured to examine the composition (organic solvent) of the gas absorption liquid of the present invention.

  As the gas absorbing solution, a 10 mM ultraviolet spectrum measurement wide-area buffer (pH 11) containing 2 μM DAF-2 and 0 to 60% (v / v) dimethylformamide (DMF) was used. NO standard gas was used as test NO, and a gas whose flow rate was adjusted to 1 ppm by mass with a mass flow controller was bubbled for 10 minutes against 15 mL of each gas absorbent. For each gas absorbent, the amount of increase in fluorescence intensity was determined in the same manner as in Example 1. The amount of increase in the fluorescence intensity obtained was shown as a relative value with the amount of increase in the fluorescence intensity in the gas absorption liquid without addition of DMF (0%) being 1.

  The measurement results are shown in FIG. Comparing the amount of increase in relative fluorescence intensity in the gas absorption liquids of each DMF concentration, a significant increase in the amount of increase in relative fluorescence intensity was observed with the addition of DMF in the range of 10 to 40%. At this time, in the range of 10 to 40% DMF, the increase was 5 times or more with respect to the non-addition. From the above, it was found that when DAF-2 is used as a NO fluorescent probe, it is preferable that 10 to 40% DMF coexist in the gas absorbing solution.

[Example 5] Confirmation of NO ₂ coexistence effect (constant NO concentration)
Various concentrations of NO ₂ were added to measure NO. Adjust the standard gas of NO ₂ and standard gas of NO with the mass flow controller so that NO ₂ is 0 to 300 volume ppb and NO is 100 volume ppb respectively, and supply to the microchannel scrubber of the gas collector at 200 mL / min did. The gas absorption solution is 10 μM DAF-2 / 0.01 M NaOH, introduced into a microchannel scrubber at a flow rate of 20 μL / min, and the fluorescence intensity of DAF-2T produced by reacting with DAF-2 is detected by a fluorescence detector. Measured with

The results are shown in FIG. As the NO ₂ concentration increased, the response to 100 capacity ppb NO increased, and it almost reached a plateau above 200 capacity ppb. At this time, a response about 10 times that obtained when NO ₂ was not added was obtained, and it was found that NO can be measured with higher sensitivity when NO ₂ is added to the sample gas.

[Example 6] Confirmation of effect of NO ₂ coexistence (constant NO ₂ concentration)
NO was measured by adding 200 ppb of NO ₂ . NO ₂ standard gas and NO standard gas are respectively adjusted by the mass flow controller and the dilution ratio is changed to change NO ₂ to 200 capacity ppb for 125 min from the start of measurement (NO ₂ ppb for NO ₂ after 125 min). NO) in order 10 capacity ppb (2 times measurement), 20 capacity ppb (2 times measurement), 40 capacity ppb (2 times measurement), 60 capacity ppb (2 times measurement), 80 capacity ppb (2 times measurement) ), 100 capacity ppb (measured 4 times when NO ₂ is 200 capacity ppb and measured 4 times when NO ₂ is 0 capacity ppb after 125 min), and microchannel of gas collector at 200 mL / min The scrubber was fed. The gas absorption solution was introduced into the microchannel scrubber at a flow rate of 20 μL / min using 10 μM DAF-2 / 0.01 M NaOH. The test gas was continuously monitored by a conventional method (chemiluminescence method).

The measurement results are shown in FIG. (A) The upper part of the figure shows the results of measurement by the method and apparatus of the present invention, and the lower part shows the result of simultaneous analysis using a chemiluminescence (CL) apparatus. (B) is a comparison of the response to NO at a concentration of 100 ppb by overlapping the measurement results of 80-120 min and 125-165 min of (a). When NO ₂ is not added in (a), the response to NO at a concentration of 100 ppb is slight, but when NO ₂ is added, it is also to NO at a concentration of 10 ppb. Sufficient response is obtained. In (b), when NO ₂ is not added, the response to NO at a concentration of 100 ppb is slight at about 0.15V, but when NO ₂ is added, the response is 1.5V or more. It can be seen that a response of 10 times or more can be obtained by adding NO ₂ . Thus, it was found that NO can be measured with higher sensitivity by adding NO ₂ .

[Example 7] Measurement of NO in breath sample (in the presence of NO ₂ )
The NO sample of the breath sample was measured by adding 200 ppb of NO ₂ . 10 μM DAF-2 / 0.01 M NaOH was used as a gas absorbing solution, and this was introduced into the microchannel scrubber at a flow rate of 30 μL / min. Meanwhile adjustment, healthy subjects three exhalation (Breatha, B and C) were collected into Mylar bags respectively, the mass flow controllers standard gas NO ₂ as NO ₂ concentration aspirated breath from the bag is 200 volume ppb And added. The exhaled breath sample to which NO ₂ was added was introduced into the microchannel scrubber at 200 mL / min and measured.

The measurement results are shown in FIG. In the figure, the center line is the NO measurement result by the method and apparatus of the present invention, the upper line is the result of simultaneous analysis of NO _{2 with} a chemiluminescence (CL) apparatus, and the lower line is the chemical result. It is the result of having performed simultaneous analysis of NO with a luminescence method (CL) device. Exhaled breath (Breath A, B and C) of three healthy subjects measured with the method and apparatus of the present invention shows a response correlated with the value measured with a chemiluminescence (CL) apparatus and depends on the NO concentration in the expired air It was confirmed that the obtained response was obtained.

  The measuring device of the present invention is 1/10 or less in weight and volume as compared with conventional chemiluminescent devices, and responds to NO of human breath. Therefore, it can be used as a portable and portable NO gas measuring device, and can be used for environmental measurement and evaluation of respiratory diseases.

It is the schematic which shows the structure of the NO measuring apparatus of one embodiment of this invention, and its flow path. It is the schematic which showed the microchannel scrubber as an example of a gas collector. It is the figure which showed the gas absorption liquid flow path of the microchannel scrubber of a honeycomb structure. It is the graph which showed the response with respect to NO when the pH of a gas absorption liquid was variously changed in the measurement by a bubbling method. It is the graph which showed the response characteristic with respect to NO in the measurement by a microchannel scrubber. It is the graph which showed the correlation of response intensity and NO concentration in the measurement by a microchannel scrubber. It is the graph which showed the response characteristic with respect to exhalation in the measurement by a microchannel scrubber. It is the graph which showed the response with respect to NO when the dimethylformamide density | concentration of a gas absorption liquid was 0, 10, 20, 30, 40, 60% in the measurement by a bubbling method. In measurement by the microchannel scrubber is a graph showing the response characteristics for NO of 100 volume ppb at various NO ₂ concentration. In measurement by the microchannel scrubber is a graph showing the response characteristics to NO in NO ₂ concentration of 200 volume ppb. In measurement by the microchannel scrubber, to breath sample is a graph showing the response characteristics for breath when NO ₂ was added so that the concentration of the breath sample is 200 volume ppb.

Explanation of symbols

1. 1. Gas flow path 2. Gas absorption liquid flow path 3. Gas permeable membrane 4. Gas collector Fluorescence detector 6. 6. Storage tank Pump 8. 8. Gas inlet Gas outlet 10. Mass flow controller 11. Measurement sample inflow path 12. Additive gas inflow path 13. substrate

Claims (10)

A sample gas containing nitrogen monoxide (NO) , nitrogen dioxide (NO ₂ ) in the range of 20 to 3000 ppb, and a gas absorbing solution containing a NO fluorescent probe are brought into contact with each other through a gas permeable membrane, and A method for measuring nitric oxide, comprising incorporating nitrogen monoxide into the gas absorbing solution and measuring the amount of fluorescence in the gas absorbing solution. The method for measuring nitric oxide according to claim 1, wherein the sample gas containing nitrogen monoxide and the gas absorbing liquid are contacted by a microchannel scrubber having a honeycomb structure. The method for measuring nitric oxide according to claim 1 or 2 , wherein the NO fluorescent probe is a fluorescein derivative or a rhodamine derivative that emits fluorescence by forming a triazole ring structure in the molecule in the presence of NO. The NO fluorescent probe is 4,5-diaminofluorescein (DAF-2), 3-amino-4- (methylamino) -2 ′, 7′-difluorofluorescein (DAF-FM), 3-amino-4- ( The method for measuring nitric oxide according to any one of claims 1 to 3 , wherein the method is selected from the group consisting of (methylamino) -tetramethylrhodamine (DAR-4M). The method for measuring nitric oxide according to any one of claims 1 to 4 , wherein the pH of the gas absorption liquid is 5 or more. The method for measuring nitric oxide according to any one of claims 1 to 5 , wherein the gas absorption liquid contains dimethylformamide at a concentration of 10 to 40%. The method for measuring nitric oxide according to any one of claims 1 to 6 , wherein the sample gas containing nitric oxide is air. Sample gas containing nitric oxide, method for measuring nitric oxide according to any one of claims 1 to 6 which is a breath An apparatus for measuring nitric oxide comprising the following (1) to (3).
(1) a gas flow path through which a sample gas containing nitric oxide is provided with a gas inlet at one end and a gas outlet at the other end ;
(2) A gas absorbing liquid including an NO fluorescent probe disposed so as to be in contact with the gas flow path through a gas permeable film on the boundary surface, provided with a gas absorbing liquid inlet at one end and a gas absorbing liquid outlet at the other end. A gas collector having a flowing gas absorption liquid channel,
(3) Fluorescence detector connected to the gas absorption liquid outlet side channel of the gas collector The apparatus for measuring nitric oxide according to claim 9 , wherein the gas collector is a microchannel scrubber having a honeycomb structure.