KR20120030264A - Ecotoxicity assay method using inhibition of dehydrogenase - Google Patents
Ecotoxicity assay method using inhibition of dehydrogenase Download PDFInfo
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- 101710088194 Dehydrogenase Proteins 0.000 title claims abstract description 26
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000000691 measurement method Methods 0.000 claims description 10
- 239000008363 phosphate buffer Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 239000003708 ampul Substances 0.000 claims description 4
- 239000002198 insoluble material Substances 0.000 claims description 4
- 238000011481 absorbance measurement Methods 0.000 claims description 2
- JORABGDXCIBAFL-UHFFFAOYSA-M iodonitrotetrazolium chloride Chemical compound [Cl-].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C=CC=CC=2)=N1 JORABGDXCIBAFL-UHFFFAOYSA-M 0.000 abstract description 21
- 239000000758 substrate Substances 0.000 abstract description 8
- 238000011156 evaluation Methods 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 3
- 231100000463 ecotoxicology Toxicity 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 21
- 239000003440 toxic substance Substances 0.000 description 18
- 231100000614 poison Toxicity 0.000 description 12
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 9
- 239000000126 substance Substances 0.000 description 8
- 231100000419 toxicity Toxicity 0.000 description 7
- 230000001988 toxicity Effects 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000010842 industrial wastewater Substances 0.000 description 4
- 231100000167 toxic agent Toxicity 0.000 description 4
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000006392 deoxygenation reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000002079 cooperative effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
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Abstract
Description
본 발명은 생태독성 측정방법에 관한 것으로, 특히 탈수소효소를 이용하여 생태독성을 측정할 수 있도록 된 탈수소효소를 이용한 생태독성 측정방법에 관한 것이다.
The present invention relates to a method for measuring ecotoxicity, and more particularly, to a method for measuring ecotoxicity using a dehydrogenase which is capable of measuring ecotoxicity using a dehydrogenase.
산업화로 인하여 생태계의 건전성에 영향을 미치는 독성물질의 종류와 양이 지속적으로 증가하고 있다. 현재 국내에서 상업적으로 유통되고 있는 화학물질의 수는 4만종 이상이며, 매년 400여종이 새로이 국내 시장으로 유입되는 등 화학물질의 사용이 꾸준히 증가하고 있다(환경부, 2009).Industrialization continues to increase the types and quantities of toxic substances that affect the health of ecosystems. At present, the number of commercial chemicals in Korea is over 40,000, and the use of chemicals is increasing steadily, with 400 new species entering the domestic market every year (Ministry of Environment, 2009).
화학물질들은 다양한 경로를 통하여 환경으로 유입되며, 산업폐수를 통하여 수생태계로 유입된 독성물질은 생물체 내에 축적되어 생태계를 파괴하고 수질 악화를 초래한다. 따라서 산업폐수 처리시설의 방류수에 포함된 독성물질의 양을 상시적으로 측정하여 처리효율을 평가하고 유해성을 저감시키는 적절한 조치를 취해야 한다.Chemicals enter the environment through various routes, and toxic substances entering the aquatic ecosystem through industrial wastewater accumulate in living organisms, destroying ecosystems and causing water deterioration. Therefore, it is necessary to constantly measure the amount of toxic substances contained in the discharged water of industrial wastewater treatment facilities to evaluate treatment efficiency and take appropriate measures to reduce the harmfulness.
환경시료에서 특정 독성 물질의 농도를 측정하기 위하여 널리 사용되고 있는 방법은 분석기기를 사용하는 것이다. 중금속의 분석은 주로 유도결합 플라스마 질량분석기(ICP-MS)를 이용하며, 기체크로마토그래피 또는 액체크로마토그래피는 휘발성 및 용존성 독성물질의 분석에 널리 사용되고 있다.A widely used method for measuring the concentration of certain toxic substances in environmental samples is to use an analyzer. Heavy metals are mainly analyzed by inductively coupled plasma mass spectrometry (ICP-MS), and gas chromatography or liquid chromatography is widely used for the analysis of volatile and dissolved toxic substances.
하지만, 분석기기는 미지의 독성물질을 검출할 수 없으며, 두 종류 이상 독성물질의 협동작용 또는 길항 작용에 의한 유해성의 증가나 감소를 측정할 수 없는 단점이 있다. 특히, 산업폐수에는 다양한 종류의 생태독성 물질이 포함되어 있으며, 포함된 성분을 예측하기 힘들기 때문에 분석기기를 이용한 독성물질의 정량은 매우 어렵다. 이러한 문제를 해결하기 위하여 효소의 저해도를 평가함으로써 대상 시료에 생태독성 물질의 존재 여부 및 농도를 평가하는 방법이 개발되어 활용되고 있다.However, the analyzer can not detect the unknown toxic substances, there is a disadvantage that can not measure the increase or decrease of the harmfulness by the cooperative action or antagonism of two or more types of toxic substances. In particular, industrial wastewater contains various types of ecotoxic substances, and it is very difficult to quantify the toxic substances using an analyzer because the contained components are difficult to predict. In order to solve this problem, a method for evaluating the presence and concentration of ecotoxic substances in a target sample by evaluating the degree of inhibition of the enzyme has been developed and utilized.
효소를 이용하는 방법은 독성물질에 의한 효소 활성이 저해되는 정도를 측정하여 생태독성 정도를 간접적으로 평가하는 것으로, 수시료, 토양 시료 및 폐수에서 생태 독성을 평가하기 위하여 널리 사용되고 있다. 사용되는 효소의 종류는 탈수소효소 등이 있다.The method using enzyme is to indirectly evaluate the degree of ecotoxicity by measuring the degree of inhibition of enzymatic activity by toxic substances, and is widely used to evaluate ecotoxicity in samples, soil samples and wastewater. Examples of the enzyme used include dehydrogenase.
탈수소효소를 이용한 기존의 연구는 시료에 존재하는 미생물을 사용하기 때문에 효소활성도 변화를 측정하기 위하여 매우 오랜 시간이 걸린다. 또한, 시료에 존재하는 미생물의 종류에 따라 결과가 달라지기 때문에 실험의 재현성이 떨어지고, 표준화시키기 어려운 단점이 있어 산업폐수 처리 방류수의 생태독성 측정법으로 적합하지 않았다.
Existing studies using dehydrogenase use a microorganism present in the sample, so it takes a very long time to measure changes in enzyme activity. In addition, because the results vary depending on the type of microorganisms present in the sample, the reproducibility of the experiment is inferior and it is difficult to standardize, and thus it is not suitable for measuring the ecotoxicity of industrial wastewater treated effluent.
본 발명의 목적은 상술한 문제점을 해소하기 위해 안출된 것으로, 탈수소효소를 이용한 생태 독성 평가법의 단점을 보완하고 이를 폐수시설 방류수의 독성 평가에 사용하기 위하여 처리시간, 완충용액의 조성 등 독성 평가에 필요한 조건을 최적화시킬 수 있는 탈수소효소를 이용한 생태독성 측정방법을 제공하는데 있다.
An object of the present invention was devised to solve the above-mentioned problems, to compensate for the shortcomings of the ecotoxicity assessment method using dehydrogenase and to evaluate the toxicity, such as treatment time, the composition of the buffer solution, for use in the toxicity evaluation of the effluent from wastewater facilities. The present invention provides a method for measuring ecotoxicity using dehydrogenase that can optimize necessary conditions.
상기 목적을 달성하기 위해 본 발명의 일 실시예에 따른 탈수소효소를 이용한 생태독성 측정방법은, (a) 대조시료가 함유된 반응액과 실험하고자 하는 시료가 함유된 반응액을 원심분리관에 각각 넣은 후 항온수조에서 진탕하면서 반응시키는 단계; (b) 상기 각 원심분리관을 항온수조에서 꺼낸 후 각 원심분리관에 INT를 첨가하는 단계; (c) INT가 첨가된 상기 각 원심분리관을 항온수조에서 진탕하면서 반응시키는 단계; (d) 상기 각 원심분리관을 항온수조에서 꺼낸 후 원심분리후 상등액을 제거하는 단계; (e) 상등액이 제거된 불용성 물질을 메탄올에 녹이는 단계; 및 (f) 원심분리 후 상등액만 취해 분광도계로 각 시료의 흡광도를 측정 비교하는 단계를 포함한다.Ecotoxicity measurement method using a dehydrogenase according to an embodiment of the present invention to achieve the above object, (a) the reaction solution containing the control sample and the reaction solution containing the sample to be tested in each centrifuge tube Reacting while shaking in a constant temperature bath; (b) removing each of the centrifuge tubes from the constant temperature bath and adding INT to each centrifuge tube; (c) reacting each centrifuge tube to which INT is added while shaking in a constant temperature water bath; (d) removing the supernatant after centrifugation by removing each centrifuge tube from the constant temperature water bath; (e) dissolving the insoluble material from which the supernatant is removed in methanol; And (f) taking only the supernatant after centrifugation and comparing the absorbance of each sample with a spectrophotometer.
상기 목적을 달성하기 위해 본 발명의 다른 실시예에 따른 탈수소효소를 이용한 생태독성 측정방법은, (a) 대조시료가 함유된 반응액과 실험하고자 하는 시료가 함유된 반응액을 원심분리관에 각각 넣은 후 항온수조에서 진탕하면서 반응시키는 단계; (b) 상기 각 원심분리관을 항온수조에서 꺼낸 후 각 원심분리관에 INT를 첨가하는 단계; (c) INT가 첨가된 상기 각 원심분리관을 항온수조에서 진탕하면서 반응시키는 단계; (d) 상기 각 원심분리관을 항온수조에서 꺼낸 후 각 원심분리관에 THF를 첨가하는 단계; 및 (e) THF가 첨가된 상기 각 원심분리관을 원심 분리시키후 분광도계로 각 시료의 흡광도를 측정 비교하는 단계를 포함한다.Ecotoxicity measurement method using a dehydrogenase according to another embodiment of the present invention to achieve the above object, (a) the reaction solution containing the control sample and the reaction solution containing the sample to be tested in each centrifuge tube Reacting while shaking in a constant temperature bath; (b) removing each of the centrifuge tubes from the constant temperature bath and adding INT to each centrifuge tube; (c) reacting each centrifuge tube to which INT is added while shaking in a constant temperature water bath; (d) removing each centrifuge tube from the constant temperature water bath and adding THF to each centrifuge tube; And (e) centrifuging each centrifuge tube to which THF is added and then measuring and comparing the absorbance of each sample with a spectrophotometer.
상기 실시예들에서 상기 반응액은 균이 들어 있는 앰플의 윗부분을 자른 다음, 앰플에 포스페이트 버퍼를 첨가하여 균을 완전히 현탁시켜 현탁액을 만들고, 원심분리관에 포스페이트 버퍼를 넣은 후 원심분리관에 상기 현탁액을 첨가하여 만들어진다.In the above embodiments, the reaction solution is to cut the upper part of the ampoule containing the bacteria, and then add phosphate buffer to the ampule to completely suspend the bacteria to make a suspension, put the phosphate buffer in the centrifuge tube, and then the centrifuge tube. It is made by adding a suspension.
상기 실시예들에서 분광광도계를 통한 흡광도 측정은 465nm에서 이루어진다.In the above examples, the absorbance measurement through the spectrophotometer is made at 465 nm.
상기 다른 실시예에서 상기 (a) 단계에서의 각 반응액은 1ml이고, 상기 (b) 단계에서의 INT량은 0.2ml이며, 상기 (d) 단계에서 첨가되는 THF의 양은 0.8ml이다.
In another embodiment, each reaction solution in step (a) is 1 ml, the INT amount in step (b) is 0.2 ml, and the amount of THF added in step (d) is 0.8 ml.
이와 같은 본 발명에 따른 탈수소효소를 이용한 생태독성 측정방법에 의하면 다음과 같은 효과들을 갖는다.According to the ecotoxicity measurement method using the dehydrogenase according to the present invention has the following effects.
첫째, 탈수소효소의 기질용액으로 INT를 사용함으로써, 다른 기질에 비해 기질전환율이 높으며, 탈산소화와 같은 전처리가 필요 없기 때문에 독성 평가에 소요되는 처리시간을 줄일 수 있다.First, by using INT as a substrate solution of dehydrogenase, the substrate conversion rate is higher than that of other substrates, and pretreatment such as deoxygenation does not require the treatment time required for toxicity evaluation.
둘째, 대조시료의 흡광도를 측정하고 이를 실험하고자 하는 시료의 흡광도와 비교함으로써, 실험하고자 하는 시료에 함유된 독성물질의 양을 상대적으로 간편하게 측정할 수 있다.Second, by measuring the absorbance of the control sample and comparing it with the absorbance of the sample to be tested, the amount of the toxic substance contained in the sample to be tested can be relatively simply measured.
셋째, INT가 함유된 반응액에 포함되어 있는 불용성 물질을 무색 투명한 액체인 THF에 녹이면 1~2분 안에 전부 녹기 때문에 후속공정없이 바로 원심분리후 흡광도를 측정할 수 있다. 따라서, 독성평가에 소요되는 처리시간을 줄일 수 있다.
Third, when the insoluble substance contained in the INT-containing reaction solution is dissolved in THF, a colorless and transparent liquid, it is completely dissolved within 1 to 2 minutes, so the absorbance can be measured immediately after centrifugation without a subsequent process. Therefore, the treatment time for the toxicity evaluation can be reduced.
도 1은 본 발명의 일 실시예에 따른 탈수소효소를 이용한 생태독성 측정방법을 나타낸 플로우차트.
도 2는 본 발명의 다른 실시예에 따른 탈수소효소를 이용한 생태독성 측정방법을 나타낸 플로우차트.1 is a flowchart showing a method for measuring ecotoxicity using a dehydrogenase according to an embodiment of the present invention.
Figure 2 is a flowchart showing a method for measuring ecotoxicity using a dehydrogenase according to another embodiment of the present invention.
이하, 본 발명에 따른 바람직한 실시예를 첨부한 도면에 따라 상세하게 설명한다.
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
도 1은 본 발명에 따른 탈수소효소를 이용한 생태독성 측정방법을 나타낸 플로우차트이다.1 is a flowchart showing a method for measuring ecotoxicity using dehydrogenase according to the present invention.
본 발명에 따른 탈수소효소를 이용한 생태독성 측정방법은, (a) 반응액을 준비하는 단계(S10), (b) 반응액을 1차 진탕하는 단계(S20), (c) 반응액에 INT를 첨가하는 단계(S30), (d) INT가 첨가된 반응액을 2차 진탕하는 단계(S40), (e) 원심분리후 상등액을 제거하는 단계(S50), (f) 회수된 불용성 물질을 메탈올에 녹이는 단계(S60), 및 (g) 원심분리후 흡광도를 측정하는 단계(S70)를 포함한다.Ecotoxicity measurement method using a dehydrogenase according to the present invention, (a) preparing a reaction solution (S10), (b) first shaking the reaction solution (S20), (c) the INT in the reaction solution Step (S30), (d) the second step of shaking the reaction solution added INT (S40), (e) removing the supernatant after centrifugation (S50), (f) the recovered insoluble material metal Dissolving in a bowl (S60), and (g) measuring the absorbance after centrifugation (S70).
상기 (a) 단계에서는 독성을 평가하기 위한 반응액을 준비하게 된다(S10). 반응액을 준비하기 위해서는 먼저 균이 들어 있는 앰플의 윗부분을 자른 다음, 앰플에 Phosphate buffer(포스페이트 버퍼) 1ml를 첨가하여 균을 완전히 현탁시켜 현탁액을 만든다. 이어서 2ml 용량의 원심분리관을 시료 갯수에 2를 더한 수만큼 준비한다(만일 실험을 2배수로 하고자 한다면 원심분리관의 수도 2배로 준비한다). 원심분리관에 Phosphate buffer(0.1M, pH7.0~7.6)를 0.4ml씩 분주한다. 이어서 Phosphate buffer가 분주된 각 원심분리관에 현탁액을 0.1ml씩 더한다. 3개의 원심분리관중 어느 하나에는 증류수 0.5ml, 다른 한개의 원심분리관에는 Cu(1ml/L) 0.5ml, 나머지 원심분리관에는 실험하고자 하는 시료를 0.5ml 넣는다. 한편, Phosphate buffer는 완충액으로서 반응액의 산도를 일정하게 유지시키는 기능을 한다.In the step (a) to prepare a reaction solution for evaluating the toxicity (S10). To prepare the reaction solution, first cut the upper part of the ampoules containing the bacteria, and then add 1 ml of Phosphate buffer (phosphate buffer) to the ampoules to completely suspend the bacteria to form a suspension. A 2 ml centrifuge tube is then prepared by adding 2 to the number of samples (if you want to double the experiment, double the number of centrifuge tubes). Dispense 0.4ml of Phosphate buffer (0.1M, pH7.0 ~ 7.6) into the centrifuge tube. Subsequently, add 0.1 ml of suspension to each centrifuge tube dispensed with Phosphate buffer. 0.5 ml of distilled water is added to one of the three centrifuge tubes, 0.5 ml of Cu (1 ml / L) is added to the other centrifuge tube, and 0.5 ml of the sample to be tested is added to the remaining centrifuge tubes. Phosphate buffer, on the other hand, serves as a buffer to keep the acidity of the reaction constant.
상기 (b) 단계에서는 반응액이 들어 있는 각각의 원심분리관을 잘 섞은 후 37℃ 항온수조에서 100~150rpm으로 진탕하면서 10~60분간 반응시킨다(S20). 이 때, 진탕시간은 20분이 가장 바람직하다.In the step (b), each centrifuge tube containing the reaction solution is well mixed and then reacted for 10 to 60 minutes while shaking at 100 to 150 rpm in a constant temperature water bath at 37 ° C. (S20). At this time, the shaking time is most preferably 20 minutes.
상기 (C) 단계에서는 (b) 단계를 통해 진탕시킨 원심분리관을 항온수조에서 꺼낸 후 각각의 원심분리관에 탈수소효소의 기질용액인 0.1% INT(Iodonitrotetrazolium; 이오도니트로테트라졸리움)를 각각 0.2ml씩 첨가한 후 잘 섞어준다. 참고로, 탈수소효소의 기질로는 INT 외에도 TTC(Triphenyl tetrazolium chloride ; 트리페닐 테드라졸리움 클로라이드)가 있으나, TTC에 비해 INT의 기질 전환율이 높으며, INT를 사용할 경우 탈산소화와 같은 전처리가 필요 없기 때문에 이를 기질로 사용하는 것이 적합하다.In step (C), the centrifuge tubes shaken through step (b) are removed from the constant temperature bath, and 0.1% INT (Iodonitrotetrazolium; Iodonitrotetrazolium), which is a substrate solution of dehydrogenase, is respectively added to each centrifuge tube. Add ml and mix well. For reference, there are TTC (Triphenyl tetrazolium chloride) in addition to INT, but the substrate conversion rate of INT is higher than that of TTC, and since INT does not require pretreatment such as deoxygenation, It is suitable to use it as a substrate.
상기 (d) 단계에서는 (c) 단계를 거친 각각의 원심분리관을 37℃ 항온수조에서 100~150rpm으로 진탕하면서 1~240분 동안 반응시킨다. 이러한 반응을 통해 불용성 물질이 생성된다. 한편, 진탕시간은 30분이 가장 바람직하다.In the step (d), each centrifuge tube passed through the step (c) is reacted for 1 to 240 minutes while shaking at 100 to 150 rpm in a 37 ° C. constant temperature water bath. This reaction produces insoluble matter. On the other hand, the shaking time is most preferably 30 minutes.
상기 (e) 단계에서는 불용성 물질을 수확하기 위해 시료를 5분간 원심 분리한 후 상등액을 완전히 제거하게 된다.In step (e), the supernatant is completely removed after centrifuging the sample for 5 minutes to harvest the insoluble material.
상기 (f) 단계에서는 회수된 불용성 물질의 용해를 위해 2ml의 메탄올(100%)을 첨가한 후, 초음파세척기로 5분간 처리한다.In step (f), 2 ml of methanol (100%) is added to dissolve the recovered insoluble substance, and then treated with an ultrasonic cleaner for 5 minutes.
상기 (g) 단계에서는 각각의 원심분리관을 12,000rpm으로 10분간 원심분리한 후, 원심분리관을 1회용 큐벳에 꽂아서 불용성 물질의 양을 분광광도계를 이용하여 465nm에서 상등액의 흡광도를 측정하여 정량화하게 된다.In the step (g), each centrifuge tube was centrifuged at 12,000 rpm for 10 minutes, and the centrifuge tube was placed in a disposable cuvette to measure the absorbance of the supernatant at 465 nm using a spectrophotometer. Done.
상기와 같은 과정을 통해 증류수가 포함된 시료(대조시료1-독성물질이 포함되지 않은 시료)의 흡광도, Cu가 포함된 시료(대조시료2-독성물질의 시료)의 흡광도 및 실험하고자 하는 시료의 흡광도를 각각 구하게 된다. 이 때, 대조시료1의 흡광도(A)는 최대값을 나타내게 되며, 대조시료2의 흡광도(B)는 최소값을 나타내게 된다. 그리고 실험하고자 하는 시료(독성물질이 일부 함유된 시료)의 흡광도(C)는 A값과 B값 사이에 위치하게 된다.Absorbance of the sample containing the distilled water (control sample 1-no toxic substance), absorbance of the sample containing Cu (sample of the control sample 2-toxic substances) and the sample to be tested Each absorbance is obtained. At this time, the absorbance (A) of the control sample 1 shows the maximum value, and the absorbance (B) of the control sample 2 shows the minimum value. And the absorbance (C) of the sample to be tested (sample containing some toxic substances) is located between the A value and the B value.
따라서, A, B의 값과 C 값의 차이를 각각 구하여, 실험하고자 하는 시료가 대조시료1에 가까운지 대조시료2에 가까운지를 판단하게 된다. 즉, 대조시료1에 가깝게 되면 독성물질이 적게 함유되었음을 알 수 있으며, 대조시료2에 가깝게 되면 독성물질이 많이 함유되었음을 알 수 있게 된다.Therefore, by determining the difference between the values of A, B and C, it is determined whether the sample to be tested is close to control sample 1 or close to control sample 2. In other words, the closer to the control sample 1, the less toxic substances contained, and the closer to the control sample 2, the more toxic substances contained.
참고로, 상기와 같은 흡광도를 측정하지 않고 각 시료의 색깔을 통해 실험하고자 하는 시료의 독성 함유량을 상대적으로 비교할 수도 있다. 즉, 독성물질인 Cu가 함유된 대조시료2의 경우에는 무색을 띄게 되며, 독성물질이 포함되지 않은 증류수가 함유된 대조시료1의 경우 짙은 황색계통의 색을 띄게 된다. 따라서, 실험하고자 하는 시료가 무색에 가까우면 독성이 많이 함유되었음을 알 수 있으며, 짙은 황색계통에 가깝게 되면 독성이 적게 함유되었음을 알 수 있게 된다.
For reference, the toxic content of the sample to be tested may be relatively compared through the color of each sample without measuring the absorbance as described above. That is, the control sample 2 containing Cu as a toxic substance is colorless, and the control sample 1 containing distilled water containing no toxic substance has a dark yellow color. Therefore, when the sample to be tested is close to colorless, it can be seen that it contains a lot of toxicity, and when it is close to the dark yellow system, it can be seen that it contains less toxicity.
도 2는 본 발명의 다른 실시예에 따른 탈수소효소를 이용한 생태독성 측정방법을 나타낸 플로우차트이다.2 is a flowchart showing a method for measuring ecotoxicity using dehydrogenase according to another embodiment of the present invention.
본 발명의 다른 실시예에 따른 탈수소효소를 이용한 생태독성 측정방법은, (a) 반응액을 준비하는 단계(S100), (b) 반응액을 1차 진탕하는 단계(S110), (c) 반응액에 INT를 첨가하는 단계(S120), (d) INT가 첨가된 반응액을 2차 진탕하는 단계(S130), (e) 원심분리관에 THF를 첨가하는 단계(S140), (f) 원심분리후 흡광도를 측정하는 단계(S150)를 포함한다.Ecotoxicity measurement method using a dehydrogenase according to another embodiment of the present invention, (a) preparing a reaction solution (S100), (b) first shaking the reaction solution (S110), (c) reaction Adding INT to the solution (S120), (d) secondly shaking the reaction solution to which INT is added (S130), (e) adding THF to the centrifuge tube (S140), (f) centrifugation And measuring absorbance after separation (S150).
상기 (a) 내지 (d) 단계는 본 발명의 일 실시예에서의 (a) 내지 (d) 단계와 동일하므로 그 설명을 생략하기로 한다.Steps (a) to (d) are the same as steps (a) to (d) in the embodiment of the present invention, and description thereof will be omitted.
상기 (e) 단계에서는 (d) 단계를 거친 각각의 원심분리관에 THF(Tetrahydro -furan;테트라히트로퓨란)을 각각 0.8ml씩 첨가하고 잘 섞어준다. 무색 투명한 액체인 THF는 INT가 함유된 반응액에 포함되어 있는 불용성 물질을 1~2분 안에 전부 녹여준다. In step (e), 0.8 ml of THF (Tetrahydro-furan; tetrahitfurfuran) is added to each centrifuge tube which has passed step (d) and mixed well. THF, a colorless and transparent liquid, dissolves all insoluble substances contained in the INT-containing reaction solution in 1-2 minutes.
상기 (f) 단계에서는 (e) 단계를 거친 각각의 원심분리관을 12,000rpm으로 10분간 원심분리한 후, 원심분리관을 1회용 큐벳에 꽂아서 분광도계로 465nm에서 각 시료의 흡광도를 측정한다.In the step (f), after centrifuging each of the centrifuge tubes passed through step (e) at 12,000 rpm for 10 minutes, the centrifuge tubes are placed in a disposable cuvette and the absorbance of each sample is measured at 465 nm with a spectrophotometer.
한편, 실험하고자 하는 시료에 함유되어 있는 독성물질의 양을 구하는 방법은 본 발명의 일 실시예서와 동일하므로 그 설명을 생략하기로 한다.
On the other hand, the method for obtaining the amount of toxic substances contained in the sample to be tested is the same as in the embodiment of the present invention will be omitted the description.
이상에서 설명한 바와 같이, 본 발명에 따른 바람직한 실시예들을 기초로 설명하였으나, 본 발명은 특정 실시예들에 한정되는 것은 아니며, 해당분야 통상의 지식을 가진 자가 특허청구범위 내에서 기재된 범주 내에서 변경할 수 있다.As described above, the present invention has been described based on the preferred embodiments, but the present invention is not limited to the specific embodiments, and those skilled in the art may change within the scope described in the claims. Can be.
Claims (5)
(b) 상기 각 원심분리관을 항온수조에서 꺼낸 후 각 원심분리관에 INT를 첨가하는 단계;
(c) INT가 첨가된 상기 각 원심분리관을 항온수조에서 진탕하면서 반응시키는 단계;
(d) 상기 각 원심분리관을 항온수조에서 꺼낸 후 원심분리후 상등액을 제거하는 단계;
(e) 상등액이 제거된 불용성 물질을 메탄올에 녹이는 단계; 및
(f) 원심분리 후 상등액만 취해 분광도계로 각 시료의 흡광도를 측정 비교하는 단계를 포함하는 탈수소효소를 이용한 생태독성 측정방법.(a) putting the reaction solution containing the control sample and the reaction solution containing the sample to be tested into a centrifuge tube and reacting with shaking in a constant temperature water bath;
(b) removing each of the centrifuge tubes from the constant temperature bath and adding INT to each centrifuge tube;
(c) reacting each centrifuge tube to which INT is added while shaking in a constant temperature water bath;
(d) removing the supernatant after centrifugation by removing each centrifuge tube from the constant temperature water bath;
(e) dissolving the insoluble material from which the supernatant is removed in methanol; And
(f) Ecotoxicity measurement method using a dehydrogenase comprising the step of taking only the supernatant after centrifugation and comparing the absorbance of each sample with a spectrophotometer.
(b) 상기 각 원심분리관을 항온수조에서 꺼낸 후 각 원심분리관에 INT를 첨가하는 단계;
(c) INT가 첨가된 상기 각 원심분리관을 항온수조에서 진탕하면서 반응시키는 단계;
(d) 상기 각 원심분리관을 항온수조에서 꺼낸 후 각 원심분리관에 THF를 첨가하는 단계; 및
(e) THF가 첨가된 상기 각 원심분리관을 원심 분리시키후 분광도계로 각 시료의 흡광도를 측정 비교하는 단계를 포함하는 탈수소효소를 이용한 생태독성 측정방법.(a) putting the reaction solution containing the control sample and the reaction solution containing the sample to be tested into a centrifuge tube and reacting with shaking in a constant temperature water bath;
(b) removing each of the centrifuge tubes from the constant temperature bath and adding INT to each centrifuge tube;
(c) reacting each centrifuge tube to which INT is added while shaking in a constant temperature water bath;
(d) removing each centrifuge tube from the constant temperature water bath and adding THF to each centrifuge tube; And
(e) Ecotoxicity measurement method using a dehydrogenase comprising the step of centrifuging each centrifuge tube to which THF is added and then measuring the absorbance of each sample with a spectrophotometer.
상기 반응액은, 균이 들어 있는 앰플의 윗부분을 자른 다음, 앰플에 포스페이트 버퍼를 첨가하여 균을 완전히 현탁시켜 현탁액을 만들고, 원심분리관에 포스페이트 버퍼를 넣은 후 원심분리관에 상기 현탁액을 첨가하여 만들어지는 것을 특징으로 하는 탈수소효소를 이용한 생태독성 측정방법.The method according to claim 1 or 2,
The reaction solution, after cutting the upper part of the ampoule containing the bacteria, and added the phosphate buffer to the ampoule to completely suspend the bacteria to make a suspension, put the phosphate buffer in the centrifuge tube and add the suspension to the centrifuge tube Ecotoxicity measurement method using a dehydrogenase characterized in that it is made.
분광광도계를 통한 흡광도 측정은 465nm에서 이루어지는 것을 특징으로 하는 탈수소효소를 이용한 생태독성 측정방법.The method according to claim 1 or 2,
Absorbance measurement using a spectrophotometer is an ecotoxicity measurement method using a dehydrogenase, characterized in that at 465nm.
상기 (a) 단계에서의 각 반응액은 1ml이고, 상기 (b) 단계에서의 INT량은 0.2ml이며, 상기 (d) 단계에서 첨가되는 THF의 양은 0.8ml인 것을 특징으로 하는 탈수소효소를 이용한 생태독성 측정방법.The method of claim 2,
Each reaction solution in step (a) is 1ml, the INT amount in step (b) is 0.2ml, and the amount of THF added in step (d) is 0.8ml, using a dehydrogenase. Ecotoxicity Measurement Method.
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| KR100550405B1 (en) * | 2003-06-14 | 2006-02-08 | 임상빈 | Method for extracting beta-cryptoxanthin from citrus press cakes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101415951B1 (en) * | 2012-06-22 | 2014-07-04 | 이손이엔엘 (주) | Estimation Method for ecotoxicity of copper and mercury using inhibition of Iodonitrotetrazolium-dehydrogenase |
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