JP5877472B2 - Atmospheric nitrogen oxide measurement method and atmospheric nitrogen oxide measurement kit - Google Patents

Description

  The present invention relates to an atmospheric nitrogen oxide measurement method and an atmospheric nitrogen oxide measurement kit, and more particularly to an atmospheric nitrogen oxide measurement method and an atmospheric nitrogen oxide measurement kit using nitration modification of proteins in the atmosphere.

  Nowadays, the impact of air pollution in China has spread to Japan, and there is a general interest in the situation of air pollution.

  Among air pollution, measurement of air pollution by nitrogen oxides can be done by using a dedicated nitrogen oxide measuring machine, gas chromatograph, etc., but since specialized knowledge and training are required, it is more convenient. As a measuring method, a method using a colorimeter such as a Salzmann reagent and a colorimeter has been proposed.

  In particular, in the nitrogen oxide measuring method using a colorimeter, it has been proposed that a more simple and highly accurate measurement can be performed by improving the nitrogen oxide collector (see, for example, Patent Document 1). .)

JP 2004-069345 A

  The present inventor is studying the effects on the health of the atmospheric environment, and among the proteins bound to particulate matter suspended in the atmosphere, the amino acid tyrosine reacts with nitrogen oxides in the atmosphere. It was found that nitration was performed. Nitrogen oxide coexists in the atmosphere together with particulate matter, and the amount of 3-nitrotyrosine nitrated by tyrosine is considered to reflect the concentration of average nitrogen oxide in the atmosphere.

  In particular, when the amount of nitrogen oxides in the atmosphere is measured using a nitrogen oxide measuring device or a gas chromatograph, the amount of nitrogen oxides can be detected with high accuracy in a short time. Nitrogen oxidation based on the amount of 3-nitrotyrosine, while the concentration of sulfite tends to fluctuate due to the influence of wind direction and strength, and the measurement result may be problematic. By measuring the concentration of the object, it is easy to accurately reflect the concentration of nitrogen oxides, which is useful as a method for evaluating the atmospheric environment.

  Thus, this inventor can measure the density | concentration of the average nitrogen oxide in air | atmosphere from the quantity of 3-nitrotyrosine, and has comprised this invention.

  In the method for measuring atmospheric nitrogen oxides of the present invention, a sampling step for adhering airborne particulate matter to a predetermined filter, a transfer step for transferring proteins bound to the airborne particulate matter to a predetermined sheet, The amount of nitrogen oxides in the atmosphere can be determined by calculating the amount of 3-nitrotyrosine based on the binding step of binding the chemiluminescent substance to 3-nitrotyrosine in the transcribed protein and the amount of light emitted by the chemiluminescent substance. And a measuring step for measuring.

  Further, the atmospheric nitrogen oxide measurement method of the present invention is characterized in that, in the measurement step, the calculated amount of 3-nitrotyrosine is corrected according to the amount of suspended particulate matter collected in the collection step. It is what has.

  In the atmospheric nitrogen oxide measurement kit of the present invention, a filter for adhering airborne particulate matter, a sheet for transferring a protein bound to the airborne particulate matter attached to the filter, and a sheet transferred to the sheet are transferred. A binding agent that binds a chemiluminescent substance to 3-nitrotyrosine in a protein, and a calibration curve filter in which a plurality of 3-nitrotyrosine solutions having different concentrations are contained in a filter of the same material as the filter. It is.

  According to the present invention, by measuring the amount of nitrogen oxides in the atmosphere via 3-nitrotyrosine, it is possible to measure the amount of nitrogen oxides in the air during a certain period of time, relatively easily. You can determine the state of the atmospheric environment. In particular, it is less susceptible to sudden effects such as wind, and the pollution status of the air environment can be correctly evaluated.

2 is a developed photograph of an X-ray film. It is a graph of a calibration curve created using a calibration curve piece.

  The atmospheric nitrogen oxide measuring method and the atmospheric nitrogen oxide measuring kit of the present invention measure the amount of nitrogen oxides in the atmosphere using proteins attached to the suspended particulate matter in the atmosphere.

  That is, the suspended particulate matter in the atmosphere is attached to a predetermined filter in the sampling step which is the first step, and then the protein bound to the suspended particulate matter is transferred to the predetermined sheet in the second transfer step. Then, a chemiluminescent substance is bound to 3-nitrotyrosine in the protein transferred to the sheet in the third step of the binding step, and then based on the amount of light emitted by the chemiluminescent body in the fourth step of the measurement step. The amount of nitrogen oxides in the atmosphere is measured by calculating the amount of 3-nitrotyrosine.

  A plurality of 3-nitrotyrosine solutions having different concentrations for a calibration curve for calculating the amount of 3-nitrotyrosine together with a filter used in the collecting step, a sheet used in the transfer step, and a binder used in the binding step The atmospheric nitrogen oxide measurement kit is composed of the filter containing the.

  By using this atmospheric nitrogen oxide measurement kit, it is possible to measure the amount of nitrogen oxides in the atmosphere even in junior high school students or the upper grades of elementary school. It can make it easy to hold.

  In the following, embodiments of the present invention will be described in more detail.

<Collecting step>
In the collection step, airborne particulate matter is adhered to a predetermined filter. Here, a quartz fiber filter was used as the filter. In order to effectively attach the suspended particulate matter to the filter, an air sampler (HV-1000R) manufactured by Shibata Science Co., Ltd. was used in this embodiment to attach the suspended particulate matter to the filter. In this embodiment, the air sampler was operated continuously for 168 hours to allow the suspended particulate matter to adhere to the filter.

  In this embodiment, the suspended particulate matter is adhered to the filter for a relatively long period of time, but an appropriate collection time may be used depending on the situation. Moreover, an appropriate dust collector may be used as the air sampler, or a self-made dust collector may be used.

  In the present embodiment, a test piece having a predetermined size is cut out by punching out a filter to which suspended particulate matter is attached with a perforator. It is desirable to create multiple test pieces.

  A calibration curve piece is created along with the test piece. That is, the calibration curve piece is made to have a predetermined size by containing a 3-nitrotyrosine solution having a predetermined concentration in a filter made of the same material as the filter used for collecting the suspended particulate matter and then punching with a perforator. It is formed by cutting out. In particular, a plurality of 3-nitrotyrosine solutions having different concentrations are prepared and respective calibration curve pieces are prepared. The calibration curve piece is not formed by punching a filter containing a 3-nitrotyrosine solution having a predetermined concentration with a perforator, but after punching a solid filter with a perforator into a predetermined shape. It may be formed by dipping in the 3-nitrotyrosine solution.

<Transfer step>
In the transfer step, the protein bound to the suspended particulate matter is transferred to a predetermined sheet. Specifically, the protein is transferred by electrophoresis by the same method as Western blotting.

  That is, first, a lower layer is formed using a filter paper infiltrated with an electrophoresis buffer. In the present embodiment, three filter papers are stacked, and an electrophoresis buffer is infiltrated to form a lower layer.

  Next, a nitrocellulose film frequently used in Western blotting is placed on the upper surface of the lower layer in an overlapping manner. This nitrocellulose membrane is a sheet to which proteins are transferred. A PVD (Polyvinylidene Difluoride) film may be used instead of the nitrocellulose film.

  Next, a test piece and a calibration curve piece are placed on the upper surface of the nitrocellulose membrane. At this time, the test piece is placed in contact with the nitrocellulose membrane on the surface to which the suspended particulate matter is attached.

  Next, an acrylamide gel film is placed on top of the nitrocellulose membrane on which the test piece and the calibration curve piece are placed.

  Next, a filter paper impregnated with electrophoresis buffer is placed on top of the acrylamide gel membrane and placed thereon to form an upper layer. In the present embodiment, three filter papers are overlapped, and an electrophoresis buffer is infiltrated to form an upper layer.

  In this way, the laminated body sandwiched between the lower layer and the upper layer is sandwiched between the electrode plates for electrophoresis, and by applying a predetermined voltage, the protein contained in the test piece and the calibration curve piece is converted into a nitrocellulose membrane. Transcript to. In this embodiment, the electrophoresis conditions are 100 V and 1 hour.

<Join step>
In the binding step, a chemiluminescent substance is bound to 3-nitrotyrosine in the protein transferred to the nitrocellulose membrane. Specifically, the chemiluminescent substance is not directly bonded to 3-nitrotyrosine, but the chemiluminescent substance is bonded to 3-nitrotyrosine via two types of antibodies as described later. .

  First, since the nitrocellulose membrane has a high protein adsorption power, a blocking layer is formed on the nitrocellulose membrane in order to prevent the antibody from adhering to unintended sites in future operations. That is, the blocking layer is formed on the surface of the nitrocellulose membrane by placing the nitrocellulose membrane in a buffer solution in which skim milk is dissolved. Here, the buffer solution is a 5 w / v% skim milk solution.

  The nitrocellulose membrane on which the blocking layer is formed is thoroughly washed to remove excess skim milk, and then immersed in a primary reaction solution containing an anti-nitrotyrosine antibody to remove proteins exposed from the blocking layer. Anti-nitrotyrosine antibody was bound to 3-nitrotyrosine. In this embodiment, the immersion time in the primary reaction solution was overnight. The liquid temperature of the primary reaction liquid was maintained at 4 ° C.

  The nitrocellulose membrane to which the anti-nitrotyrosine antibody is bound is thoroughly washed to remove the unreacted anti-nitrotyrosine antibody, and then immersed in a secondary reaction solution containing a labeled secondary antibody to which peroxidase is bound. Then, a labeled secondary antibody was bound to the anti-nitrotyrosine antibody. The immersion time in the secondary reaction solution was 1 hour in this embodiment. In addition, the liquid temperature of the secondary reaction liquid was maintained at room temperature.

  The nitrocellulose membrane to which the labeled secondary antibody is bound is thoroughly washed to remove the unreacted labeled secondary antibody, and the substrate is labeled secondary antibody using a chemiluminescent kit (Perkinelmer Western lightning kit). A chemiluminescent substance is bound to 3-nitrotyrosine by binding a chemiluminescent substance that reacts with peroxidase of the present invention and exhibits weak luminescence.

  The binding agent constituting the atmospheric nitrogen oxide measurement kit is a buffer solution in which skim milk is dissolved, a primary reaction solution containing an anti-nitrotyrosine antibody, a secondary reaction solution containing a labeled secondary antibody bound to peroxidase, It consists of a chemiluminescent kit.

<Measurement step>
In the measurement step, the amount of nitrogen oxides in the atmosphere is measured by calculating the amount of 3-nitrotyrosine based on the amount of light emitted by the chemiluminescent material.

  That is, an X-ray film is superposed on a nitrocellulose membrane to which a chemiluminescent material is bonded, the X-ray film is exposed to light emitted from the chemiluminescent material, and then the X-ray film is developed. An exposure time of about several tens of seconds to several minutes is sufficient. FIG. 1 is a developed photograph of an X-ray film. The upper three rows are calibration curve pieces, and the lower three rows are three test pieces taken from one filter.

  A calibration curve shown in FIG. 2 is prepared using a calibration curve piece, and the amount of 3-nitrotyrosine in each filter is calculated based on this calibration curve. The amount of nitrogen oxide in the atmosphere can be estimated from the calculated amount of 3-nitrotyrosine.

  Of course, the measurement method of the present invention is affected not only by the amount of nitrogen oxides in the atmosphere but also by the concentration of suspended particulate matter in the atmosphere.

  Therefore, it is desirable to measure the amount of suspended particulate matter adhering to the test piece and to correct the amount of 3-nitrotyrosine calculated according to the amount of suspended particulate matter.

  Specifically, it can be corrected by measuring the weight of the filter used in the collection step before and after collection and estimating the amount of suspended particulate matter from the weight difference.

  Alternatively, as a method of measuring the amount of suspended particulate matter with higher accuracy, the amount of suspended particulate matter is estimated by measuring the amount of protein adsorbed on the suspended particulate matter adhering to the filter in the sampling step. You can also

  That is, when the test piece is cut out from the filter using a perforator in the sampling step, the test piece for measuring the amount of protein is also cut out, put into the microtube, and further, the thermoscientific is placed in the microtube. Incubate with Fick BCA protein analysis reagent working reagent. Incubation conditions are 37 ° C. for 30 minutes.

  After the incubation is completed, the supernatant is recovered by centrifuging to remove the solids dropped from the filter. Here, the centrifugation conditions were about 9000 × g at about 4 ° C. and about 10 minutes.

  The collected supernatant is dispensed onto a microplate and the absorbance at a wavelength of 562 nm is measured to calculate the protein concentration.

  Here, for the calibration by absorbance, a plurality of types of protein standard solutions having a predetermined concentration are prepared, these standard solutions are put in a microtube, and a working reagent of the BCA protein analysis reagent is further added to the microtube. The standard curve is prepared by incubating at 37 ° C. for 30 minutes, dispensing the solution on a microplate, and measuring the absorbance.

  By calculating the protein concentration using a calibration curve, the amount of protein adsorbed on the suspended particulate matter adhering to the test piece can be determined by calculation. Moreover, as a result of the inventors' research, the amount of this protein has been confirmed to be strongly correlated with the amount of suspended particulate matter adhering to the test piece with a correlation coefficient r = 0.836.

  Therefore, the amount of suspended particulate matter adhering to the test piece can be calculated fairly accurately using the amount of protein adhering to the test piece. By performing correction based on the amount of the suspended particulate matter, the amount of nitrogen oxides in the atmosphere can be measured with higher accuracy.

  Thus, according to the present invention, the amount of nitrogen oxides in the atmosphere during a certain period can be measured by measuring the amount of nitrogen oxides in the atmosphere via 3-nitrotyrosine. The state of the atmospheric environment can be determined relatively easily.

  Moreover, by setting it as the atmospheric nitrogen oxide measurement kit of the present invention, junior and senior high school students, and in some cases, elementary school high school children can be implemented as chemical experiments, and have high educational value.

Claims (3)

A sampling step for attaching suspended particulate matter in the atmosphere to a predetermined filter;
A transfer step of transferring the protein bound to the suspended particulate matter to a predetermined sheet;
A binding step of binding a chemiluminescent substance to 3-nitrotyrosine in the protein transferred to the sheet;
A measurement step of measuring an amount of nitrogen oxide in the atmosphere by calculating an amount of the 3-nitrotyrosine based on an amount of light emitted by the chemiluminescent material.   The atmospheric nitrogen oxide measurement method according to claim 1, wherein in the measurement step, the calculated amount of 3-nitrotyrosine is corrected according to the amount of the suspended particulate matter collected in the collection step. A filter that attaches suspended particulate matter in the atmosphere;
A sheet for transferring the protein bound to the suspended particulate matter attached to the filter;
A binder that binds the chemiluminescent substance to 3-nitrotyrosine in the protein transferred to the sheet;
An atmospheric nitrogen oxide measurement kit comprising a calibration curve filter in which a plurality of 3-nitrotyrosine solutions having different concentrations are contained in a filter of the same material as the filter.