KR101620905B1 - Detecting method for gamma ray irradiation - Google Patents

Abstract

The present invention relates to a detecting method for radiation exposure and, more specifically, provides a detecting method for radiation exposure using infrared rays or Raman spectroscopy within 24 or 48 hours. According to the present invention, provided is a detecting method for gamma-ray exposure using spectroscopy, which comprises the following steps of: (a) measuring a Raman spectrum; and (b) detecting gamma-ray exposure.

Description

{Detecting method for gamma ray irradiation}

The present invention relates to a method of determining a radiation exposure, and more particularly, to a method of determining a radiation exposure or the like within 24 or 48 hours by using infrared or Raman spectroscopy.

Acute radiation syndrome caused by radiation exposure due to nuclear accident, war or terror is the first to destroy the hematopoietic system, the gastrointestinal system and the neurovascular system. Especially, the hematopoietic system is most sensitive to the radiation damage . It is difficult to determine the amount of exposure and the treatment method or prognosis for the light bulb or latency period after exposure.

Radiation workers are at risk of exposure to high-level radiation in industrial and medical fields, as well as in emergencies such as war and terrorism. There is no variety of treatments or treatment methods to prevent damage to the living body caused by such exposure or to be exposed to acute radiation. Therefore, it is urgent to develop digestive system, hematopoietic stem cell system, degree of damage of nervous system, and therapies that are caused by acute exposure to high-level radiation. An increase in the exposure dose will lead to a longer period of recovery after damage to the organism, resulting in various side effects. Therefore, it is urgent to develop a method for early diagnosis of exposure.

The degree of exposure to radiation is generally measured by measuring the number of leukocytes in the blood and predicting the extent of the exposure by the decreased number of leukocytes. It is common to express the clinical manifestations of a wide range of doses as well as the biological risk of exposure. When exposed to high - level radiation, it is known that it undergoes four stages of prolongation, latency, collapse, and death. However, there has not been developed a living body damage measurement system based on the degree of exposure and the time taken to diagnose after exposure. Actively treating the prophylactic and latent periods before the main symptom after the acute radiation exposure can reduce the degree of the radiation exposure syndrome, so it is necessary to standardize the exact exposure dose and thus the living body damage.

Acute radiation syndrome (ARS) refers to biologically hazardous effects that occur within 24 hours of exposure to large quantities of ionizing radiation. Exposure to ionizing radiation damages cells through damage to many biomolecules, including DNA, within the cell. Damage to DNA and other key molecules affects normal cell division capabilities, leading to serious health problems. These symptoms can begin within one to two hours and last for months. Therefore, acute radiation exposure syndrome can be defined as a clinical symptom including a prolonged period, which can lead to hematopoietic, gastrointestinal, skin or central nervous system syndrome. If the onset and type of symptom depends on the radiation dose, it can be categorized into three to four severity levels. In general, natural healing is possible when the patient is exposed to radiation at a relatively low level of less than 1.5 Gy. Exposure to radiation above 2 Gy causes acute radiation exposure syndrome. Exposure to less than 5 Gy of radiation can have a strong impact on very sensitive hematopoietic systems, and this effect can reduce white blood cell counts in a dose dependent manner. In addition, large quantities of radiation in excess of 8 Gy increase lethal dose and also increase the lethal dose of patients receiving intensive care. A relatively large amount of radiation affects the gastrointestinal system and the nervous system, leading to rapid death. Acute radiation exposure syndrome is diverse and nonspecific, so it can not be easily diagnosed. Because there are no symptoms of neurovascular disease, signs or symptoms to diagnose acute radiation exposure syndrome rarely occur until 24 or 48 hours after exposure. Typically, leukocyte counts, vomiting and mood swings within the first 24 or 48 hours are used to diagnose and determine the prognosis of acute radiation exposure syndrome. The absolute number of white blood cells is the fastest and easiest laboratory test to predict radiation exposure. Although other clinical or biological methods have been developed, there are few methods to assess the severity of acute radiation exposure syndrome.

Therefore, it is very important to develop faster and easier diagnostic methods after exposure in order to successfully treat acute radiation exposure syndrome and prolong the life span.

Infrared (IR) spectroscopy is a useful analytical method for studying protein structure changes. Amide I region, which is a characteristic band of proteins, contains a lot of information about protein structure. Therefore, it is possible to identify protein structure change through correlation between spectrum and protein structure.

In addition, surface-enhanced Raman scattering (SERS) spectroscopy increases the intensity of Raman scattering (10 ⁶ -10 ¹⁴ times) when molecules are adsorbed on metal nanostructures such as gold and silver As the spectroscopy used, it is expected to be highly sensitive technology that can measure only one molecule in combination with nanotechnology, which is currently developing at a very high speed. Recently, more active research is underway. The SERS phenomenon can provide information on the molecular structure provided by the conventional Raman spectroscopy. Therefore, the SERS phenomenon exhibits high selectivity and fixed bending property compared to conventional high sensitivity measurement techniques such as laser fluorescence analysis, is very sensitive to chemical analysis, Has been used in the field.

The present invention aims to provide a diagnostic method capable of promptly estimating the radiation dose after exposure in addition to acute radiation exposure syndrome caused by high-level radiation exposure, as well as long-term low-dose radiation exposure.

In order to develop a biosensor capable of quantitatively measuring the extent of injury of a hematopoietic system against acute radiation exposure, the present invention provides a method of spectroscopically detecting and quantifying a structural change of a hematopoietic system derived from a hematopoietic system To be provided.

In order to solve the above problems, the present inventors have detected spectroscopically the structural changes of damaged proteins according to the radiation exposure in cell and animal models.

In the present invention, a mouse serum within 24 hours of exposure to gamma rays was examined and a vibration spectrometer was used to detect radiation exposure early. Infrared spectroscopy and Raman spectroscopy techniques reveal structural changes in damaged proteins when cells or animals are exposed to radiation. Infrared (IR) spectroscopy is a very useful tool for studying protein structure changes. The IR spectrum analysis of the protein secondary structure is mostly based on the amide I region. The present inventors have focused only on the amide I band analysis of the IR spectrum, and the correlation between the spectrum and the protein structure for this bond is best known. Raman spectra are rich in information about side-chain oscillations and the vibrations of the polypeptide's main skeleton, so they can explain protein structure changes well and facilitate discussion. Thus, Raman spectra can provide alternative and supplementary information on IR spectra.

Figure 1 shows the IR spectra of mouse serum before and after 10 Gy irradiation and the second derivative spectra of IR spectra. The IR spectra before and after radiation exposure are very similar. Therefore, it is impossible to clarify the change in the spectrum induced by irradiation. Also, at about 1646 cm ^-1 , the broad amide I band is a very overlapping band, making it difficult to identify spectral changes due to changes in protein secondary structure. Secondary differential spectra of IR spectra were analyzed to distinguish fine spectral changes after radiation exposure. As shown in Fig. 1, in the secondary differential spectrum of the IR spectrum, there are two distinct bands in the amide I region. In the secondary spectrum of the IR spectrum, two bands are separated at 1653 and 1631 cm ^-1 , respectively, corresponding to the α-helical and β-sheet portions of the protein. In the IR spectrum, the relative intensity ratio I 1650 / I 1630 of the two bands before and after radiation exposure was 1.95 and 2.98, respectively. After the mice were exposed to the gamma rays, the intensity of the a-helical band increased more than the intensity of the β-sheet band. This finding suggests that the protein secondary structure of mouse serum is altered by radiation exposure. Thus, an increase in the relative intensity ratio I 1650 / I 1630 can provide a radiation exposure cue and can be used for early detection of radiation exposure.

To identify other characteristics that could be used for detection of radiation exposure in mice within 24 hours of exposure to gamma rays, the present inventors analyzed the Raman spectrum of mouse serum before and after radiation exposure. The Raman spectrum is shown in FIG. The band intensities at 2930, 1562 and 1002 cm ^-1 correspond to the CH stretching mode of the protein after irradiation, the amino acid residues corresponding to the amide III region, that is, tryptophan and phenylalanine. The relative intensity ratios I 2930 / I 2889 and I 1562 / I 1547 of these bands in the Raman spectrum increase after radiation exposure. The CH stretching mode of protein after radiation exposure increases I (asymmetric CH stretching mode) / I (symmetric CH stretching mode). In addition, the 1339 cm ^-1 band in the Raman spectrum corresponds to the tryptophan of the protein, and the relative intensity ratio, I (the amino acid residue corresponding to the amide III region at high frequency) / I (Amide III at low frequency corresponding to the region of amino acid residues), and specifically, the I (1330 ~ 1340) / I (1370 ~ 1380), more specifically, increase after the I 1339 / I 1374 exposure. These results clearly show that the protein structure of the mouse serum changes after the exposure, indicating the damage caused by the exposure.

This result provides a new way to assess the extent of damage in mammals after radiation exposure. Up to now, biological approaches have been reported as methods for diagnosing acute radiation exposure syndrome, but there has been no method of diagnosis from body fluids using IR and Raman spectra.

The present invention

(A) measuring the body fluids of the test subject and the normal control by an infrared spectrum, respectively;

(B) generating a second derivative spectrum for the two infrared spectra; And

(C) The two differential spectra obtained above are compared to compare the intensities of the bands corresponding to the a-helix and the b-sheet to obtain relative intensity ratios I (a-helix) / I ) Is larger than the value of the normal control group, it is judged that the test subject is exposed to the gamma ray, and a method of judging the gamma ray exposure using the spectroscopic method.

The present invention also relates to a method for determining a gamma ray exposure using the spectroscopic method, wherein the step (a) is performed within 24 hours after the gamma ray exposure.

In addition, the present invention can be applied to the above-mentioned (a) and (b)

(a) measuring Raman spectra for the body fluids of the subject and the normal control, respectively; And

(b) comparing the two obtained spectra, when the relative intensity ratio I (1330 ~ 1340) / I ( 1370 ~ 1380) of the corresponding band of the amino acid residues corresponding to the amide III region is larger than the value of the normal control group, And judging that the subject is exposed to the gamma ray, and a method for determining the gamma ray exposure using the spectroscopic method.

Also, the step (a) is performed within 24 hours after the gamma ray exposure.

The present invention also relates to a method for determining a gamma ray exposure using the spectroscopic method, wherein the body fluid of step (a) is serum, blood, plasma, saliva, sweat or urine.

In addition,

(A) measuring Raman spectra for the body fluid of the test subject and the normal control, respectively; And

(B) Comparing the two obtained spectra, when the relative intensity ratio I (1330 ~ 1340) / I ( 1370 ~ 1380) of the corresponding band of the amino acid residues corresponding to the amide III region is larger than the value of the normal control group, And judging that the subject is exposed to the gamma ray, and a method for determining the gamma ray exposure using the spectroscopic method.

In addition, the present invention is characterized in that the step (a) is performed within 24 hours after the gamma ray exposure.

The body fluid may be serum, blood, plasma, saliva, sweat or urine.

In addition,

(A) measuring Raman spectra for the body fluid of the test subject and the normal control, respectively; And

(B) Comparing the two obtained spectra, when the relative intensity ratio I (asymmetric CH stretching mode) / I (symmetric CH stretching mode) of the band corresponding to the CH stretching mode is larger than the value of the normal control group, And judging that the subject has been exposed to the radiation. The present invention also relates to a method for determining a gamma ray exposure using a spectroscopic method.

Further, the present invention is characterized in that the body fluid is serum, blood, plasma, saliva, sweat or urine.

In addition,

(A) measuring a plurality of standard test groups irradiated with gamma rays of different intensity and a body fluid of a control group not irradiated with gamma rays, respectively, by an infrared spectrum;

(B) generating a second derivative spectrum for the infrared spectrum;

(C) a relative intensity ratio I (? -Heat) / I (? -Sheet) obtained by comparing the intensity of the band corresponding to the? -Heat with the intensity of the band corresponding to the? Generating a standard graph from the intensity ratio values of the gamma-ray untreated control group; And

(D) measuring the body fluids to be tested by an infrared spectrum, generating a secondary differential spectrum from the measured values, and comparing the obtained secondary differential spectrum with the standard graph of (c) And a method for determining a gamma ray exposure using a spectroscopic method.

In addition, the present invention can be applied to the above-mentioned steps (a) to (d)

(a) measuring Raman spectra for a plurality of standard test groups irradiated with different intensity gamma rays and a body fluid of a control group not irradiated with gamma rays, respectively;

(b) comparing the obtained spectra to generate a Raman spectrum standard graph from the relative intensity ratio I (1330 to 1340) / I ( 1370 to 1380) of the corresponding band of amino acid residues corresponding to the amide III region; And

(c) measuring the body fluids to be tested by Raman spectroscopy, and comparing the obtained spectrum with the Raman spectrum standard graph of (b) to determine the degree of exposure. will be.

In addition,

(a) measuring Raman spectra for a plurality of standard test groups irradiated with different intensity gamma rays and a body fluid of a control group not irradiated with gamma rays, respectively;

(b) comparing the obtained spectra to generate a Raman spectrum standard graph from the relative intensity ratio I (1330 to 1340) / I ( 1370 to 1380) of the corresponding band of amino acid residues corresponding to the amide III region; And

(c) measuring the body fluids to be tested by Raman spectroscopy, and comparing the obtained spectrum with the Raman spectrum standard graph of (b) to determine the extent of the exposure, and a method of determining a gamma ray exposure using the spectroscopic method .

In addition, the present invention

(a) measuring Raman spectra for a plurality of standard test groups irradiated with different intensity gamma rays and a body fluid of a control group not irradiated with gamma rays, respectively;

(b) comparing the obtained spectrum to generate a Raman spectrum standard graph from the relative intensity ratio I (asymmetric CH stretching mode) / I (symmetric CH stretching mode) value of the band corresponding to the CH stretching mode; And

(c) measuring the body fluids to be tested by Raman spectroscopy, and comparing the obtained spectrum with the Raman spectrum standard graph of (b) to determine the degree of exposure. will be.

That is, the method for determining the gamma ray exposure using the infrared spectroscopy and the method for determining the gamma ray exposure using the Raman spectroscopy may be used independently of each other, or two methods may be used to determine the gamma ray exposure.

According to the present invention, a system for diagnosis of radiation exposure can be constructed to quickly and accurately diagnose a risk of acute radiation exposure, which may be caused by routine examination of a radiation risk group such as medical personnel or radiation workers, It is possible to improve the preventive and therapeutic effect on radiation exposure.

In addition, according to the present invention, it is possible to measure the degree of radiation exposure of the normal tissues accompanied with the chemotherapy radiation therapy, and to improve the efficiency of the chemotherapy treatment through the initial response to the side effect reduction.

Figure 1 is the IR spectra of the mouse serum before and after radiation exposure of 10 Gy and the secondary differential spectra of the IR spectra.
Figure 2 is a Raman spectrum of mouse serum before and after radiation exposure of 10 Gy.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the scope of the embodiments.

6-8 week old male C57BL / 6 mice were purchased from Pyeong-Taek, Korea. All mice were treated according to guidelines approved by Kangwon National University Experimental Animal Care and Use Committee. Mice were irradiated (10 Gy; dose rate of 0.6 Gy / min) to the whole body using a cesium-137 gamma cell unit (Gamma cell 3000 Elan; Best Theratronics Ltd, Kanata, ON, Canada). After irradiation for 30 minutes to one hour, the mice were anesthetized and blood was drawn with a cardiac puncture. Total blood samples were placed in tubes and centrifuged at 12,000 rpm for 15 minutes to separate plasma. Fourier transform infrared spectroscopy (FTIR) spectra were measured on a Nicolet 6700 FT-IR spectrometer (Marietta, OH, USA) equipped with a mercury cadmium telluride detector cooled with liquid nitrogen. We collected 1024 scans from each spectrum with a spectral resolution of 4 cm ^-1 . A drop of protein solution or water was placed on the surface of a diamond crystal plate (face angle: 45 °; Pike Technologies, Inc., Fitchburg, Wis., USA) for attenuated total reflection (ATR) measurements. The sample temperature was maintained at 25 ° C during the scan. In IR spectra, baseline correction by linear function and normalization of constant total area were performed in the analysis part.

Raman spectra were measured using a Jobin Yvon / HORIBA LabRam ARAMIS Raman spectrometer (Villeneuve-d'Ascq, France). A diode pumped solid state laser (532 nm) was used as the excitation energy source. Raman scattering was detected using 180 ° geometry detection and multi-channel air-cooled (-70 ° C) charge-coupled device (CCD) cameras (1024 × 256 pixels). Typical exposure times for each Raman measurement in this example were 20 seconds per measurement. All Raman spectra were also measured at 25 占 폚. Baseline correction by linear function and standardization using standard band at 674 cm ^-1 were performed for Raman spectra.

Claims (14)

(a) measuring Raman spectra for the body fluids of the subject and the normal control, respectively; And
(b) comparing the two obtained spectra, when the relative intensity ratio I (1330 ~ 1340) / I ( 1370 ~ 1380) of the corresponding band of the amino acid residues corresponding to the amide III region is larger than the value of the normal control group, And judging that the sample is exposed to the gamma ray.
The method according to claim 1,
In addition to steps (a) and (b)
(A) measuring the body fluids of the test subject and the normal control by an infrared spectrum, respectively;
(B) generating a second derivative spectrum for the two infrared spectra; And
(C) The two differential spectra obtained above are compared to compare the intensities of the bands corresponding to the a-helix and the b-sheet to obtain relative intensity ratios I (a-helix) / I ) Is greater than the value of the normal control group, determining that the test subject is exposed to the gamma ray, and determining the gamma ray exposure using the spectroscopic method.
The method according to claim 1,
Wherein the step (a) is performed within 24 hours after the exposure to the gamma ray.
The method of claim 2,
Wherein the step (a) is performed within 24 hours after the exposure to the gamma ray.
The method according to claim 1 or 2,
Wherein the body fluid is serum, blood, plasma, saliva, sweat or urine.
(A) measuring a plurality of standard test groups irradiated with gamma rays of different intensity and a body fluid of a control group not irradiated with gamma rays, respectively, by an infrared spectrum;
(B) generating a second derivative spectrum for the infrared spectrum;
(C) a relative intensity ratio I (? -Heat) / I (? -Sheet) obtained by comparing the intensity of the band corresponding to the? -Heat with the intensity of the band corresponding to the? Generating a standard graph from the intensity ratio values of the gamma-ray untreated control group; And
(D) measuring the body fluids to be tested with an infrared spectrum, generating a second differential spectrum from the measured values, comparing the obtained second differential spectrum with the standard graph of (c), and determining the degree of exposure;
In addition to the above (A) to (D)
(a) measuring Raman spectra for a plurality of standard test groups irradiated with different intensity gamma rays and a body fluid of a control group not irradiated with gamma rays, respectively;
(b) comparing the obtained spectra to generate a Raman spectrum standard graph from the relative intensity ratio I (1330 to 1340) / I ( 1370 to 1380) of the corresponding band of amino acid residues corresponding to the amide III region; And
(c) measuring the body fluids to be tested by Raman spectroscopy, and comparing the obtained spectrum with the Raman spectrum standard graph of (b) to determine the extent of the exposure; and determining the gamma ray exposure using the spectroscopic method.
(a) measuring Raman spectra for a plurality of standard test groups irradiated with different intensity gamma rays and a body fluid of a control group not irradiated with gamma rays, respectively;
(b) comparing the obtained spectra to generate a Raman spectrum standard graph from the relative intensity ratio I (1330 to 1340) / I ( 1370 to 1380) of the corresponding band of amino acid residues corresponding to the amide III region; And
(c) measuring the body fluids to be tested by Raman spectroscopy, and comparing the obtained spectrum with the Raman spectrum standard graph of (b) to determine the degree of exposure. delete delete delete delete delete delete delete

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