KR100329121B1 - Detecting Method of toxicity in Gas Phase Using Immobilized Bioluminescent Bacteria - Google Patents
Detecting Method of toxicity in Gas Phase Using Immobilized Bioluminescent Bacteria Download PDFInfo
- Publication number
- KR100329121B1 KR100329121B1 KR1019990023325A KR19990023325A KR100329121B1 KR 100329121 B1 KR100329121 B1 KR 100329121B1 KR 1019990023325 A KR1019990023325 A KR 1019990023325A KR 19990023325 A KR19990023325 A KR 19990023325A KR 100329121 B1 KR100329121 B1 KR 100329121B1
- Authority
- KR
- South Korea
- Prior art keywords
- luminescent bacteria
- toxicity
- biosensor
- bacteria
- luminescent
- Prior art date
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 42
- 231100000419 toxicity Toxicity 0.000 title claims abstract description 17
- 230000001988 toxicity Effects 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 10
- 239000007789 gas Substances 0.000 claims abstract description 27
- 235000015097 nutrients Nutrition 0.000 claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 5
- 239000013307 optical fiber Substances 0.000 claims abstract description 5
- 238000004020 luminiscence type Methods 0.000 claims abstract description 4
- 238000012360 testing method Methods 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 9
- 231100000331 toxic Toxicity 0.000 claims description 9
- 230000002588 toxic effect Effects 0.000 claims description 9
- 239000004033 plastic Substances 0.000 claims description 8
- 239000003440 toxic substance Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 231100000167 toxic agent Toxicity 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 230000007423 decrease Effects 0.000 claims description 3
- 241000607565 Photobacterium phosphoreum Species 0.000 claims description 2
- 238000005415 bioluminescence Methods 0.000 claims description 2
- 230000029918 bioluminescence Effects 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 abstract description 3
- 231100001261 hazardous Toxicity 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract 2
- 239000000383 hazardous chemical Substances 0.000 abstract 1
- 230000003287 optical effect Effects 0.000 abstract 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 abstract 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 abstract 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 27
- 239000004743 Polypropylene Substances 0.000 description 7
- 229920001155 polypropylene Polymers 0.000 description 7
- 244000005700 microbiome Species 0.000 description 5
- -1 polypropylene Polymers 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000013500 data storage Methods 0.000 description 3
- 231100000614 poison Toxicity 0.000 description 3
- 239000002341 toxic gas Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000195628 Chlorophyta Species 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012855 volatile organic compound Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Virology (AREA)
- General Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
본 발명은 발광 박테리아를 이용한 유해가스의 독성 탐지방법에 관한 것으로서 보다 상세하게는 미생물 배지를 원심분리시켜 걸러낸 발광 박테리아를 젤(gel) 상태의 영양배지와 혼합한 후 외면에 나사선을 낸 투명 폴리프로필렌 튜브에 부어 상온에서 고형화하여 발광 박테리아를 바이오센서화하고 이것을 유해가스의 독성 탐지에 이용하는 것이다.The present invention relates to a method for detecting toxicity of harmful gases using luminescent bacteria, and more particularly, a transparent poly having a screw thread on the outer surface after mixing luminescent bacteria filtered through a microbial medium with a nutrient medium in a gel state. It is poured into a propylene tube and solidified at room temperature to biosensor luminescent bacteria and use it to detect the toxicity of harmful gases.
본 발명의 유해가스 독성 탐지방법은 발광 박테리아가 고정화된 바이오센서에 광섬유를 연결하고 가스상의 화학물질의 독성에 의해서 변화하는 발광 박테리아의 발광량을 광신호로 루미노미터에 전달함으로써 발광량의 변화에 의하여 독성 여부를 탐지할 수 있다.Hazardous gas toxicity detection method of the present invention by connecting the optical fiber to the biosensor in which the luminescent bacteria are immobilized and by the change in the amount of luminescence by transmitting the luminescence of the luminescent bacteria changed by the toxicity of the gaseous chemicals as an optical signal Toxicity can be detected.
본 발명은 발광 박테리아를 영양배지에 고정화한 바이오센서를 쉽게 휴대할 수 있고 가스상의 유해 화학물질의 독성을 직접적으로 신속하게 실시간으로 탐지하여 유해물질을 취급하는 현장의 작업환경을 감시하고 유해가스가 유출시 작업자가 환기 및 대피 등 신속한 안전조치를 취할 수 있게 하는 것을 목적으로 한다.The present invention can easily carry a biosensor immobilized luminescent bacteria in a nutrient medium and directly and quickly detect the toxicity of harmful chemicals in the gas in real time to monitor the working environment of the field handling hazardous materials and It is intended to enable workers to take prompt safety measures such as ventilation and evacuation in case of spill.
Description
본 발명은 발광 박테리아를 이용한 유해가스의 독성 탐지방법에 관한 것으로서 보다 상세하게는 미생물 배지를 원심분리시켜 걸러낸 발광 박테리아를 영양배지와 혼합한 후 외면에 나사선을 낸 투명 폴리프로필렌 튜브에 부어 상온에서 고형화하여 발광 박테리아를 바이오센서화하고 이것을 유해가스의 독성 탐지에 이용하는 것이다.The present invention relates to a method for detecting toxicity of harmful gases using luminescent bacteria, and more particularly, by mixing luminescent bacteria filtered through microbial medium with a nutrient medium and pouring them into a transparent polypropylene tube with a screw thread on the outer surface at room temperature. By solidifying, bioluminescent bacteria are biosensored and used for the detection of toxic gases.
종래의 유해 가스의 독성 탐지방법은 대기시료를 포집하여 가스크로마토그래피를 이용하여 분석하는 방법과 광이온 검출기(Photo ionization detector)를 이용하여 대기중의 농도를 탐지, 분석하는 방법이 있으나 이들은 포집 및 분석하는 과정이 복잡하고 장시간이 소요되어 실시간으로 오염정도를 측정할 수 없고 가격이 비싼 문제점이 있다.Conventional methods for detecting toxicity of harmful gases include collecting atmospheric samples and analyzing them using gas chromatography and detecting and analyzing atmospheric concentrations using a photo ionization detector. As the analysis process is complicated and takes a long time, it is impossible to measure the degree of contamination in real time, and there is a problem that the price is high.
이러한 문제점을 해결하기 위하여 유해가스의 독성에 민감한 반응을 나타내는 미생물을 생물학적 감지기, 즉 바이오센서(Biosensor)화하여 유해가스의 독성 탐지에 이용하고 있는데 유해가스의 독성을 탐지할 수 있는 바이오센서로는 마누스 (Manus J. Dennisson) 등이 발표한 효소의 반응에 의한 전위의 변화를 이용한 바이오센서(Manus J. Dennison, Jennifer M. Hall and Anthony P.F. Turner,1995, Gas-Phase Microbiosensor for Monitoring Phenol Vapor at ppb Levels, Anal. Chem. 67. 3922-3927), 이 정임(Jeong Im Lee) 등이 발표한 세포의 대사작용에 의한 산소 소모량을 이용한 바이오센서(Jeong Im Lee & Isao Karube,1996,Development of a biosensor for gaseous cyanide in solution, Biosensors & Bioelectronics, Vol. 11. No. 11. 1147-1154), 마틴(Martine Naessens) 등이 발표한 녹조류의 광합성 작용을 이용한 바이오센서(Martine Naessens & Canh Tran-Minh, 1998, Whole-cell biosensor for direct determination of solvent vapours, Biosensors & Bioelectronics, Vol. 13. No. 3-4. 341-346) 등이 연구되어 왔지만 아직까지 상용화된 기술은 없다.In order to solve this problem, microorganisms that are sensitive to the toxicity of harmful gases are used as biological sensors, that is, biosensors, to detect the toxicity of harmful gases. Manus J. Dennisson et al. Biosensors using changes in the potentials of enzymes (Manus J. Dennison, Jennifer M. Hall and Anthony PF Turner, 1995, Gas-Phase Microbiosensor for Monitoring Phenol Vapor at ppb) Levels, Anal.Chem. 67. 3922-3927), and Jung Im Lee, et al. (Jeong Im Lee & Isao Karube, 1996, Development of a biosensor) for gaseous cyanide in solution, Biosensors & Bioelectronics, Vol. 11. No. 11. 1147-1154), Martin Naessens et al., Biosensors using photosynthesis of green algae (Martine Naessens & Canh Tran-Minh, 19) 98, Whole-cell biosensor for direct determination of solvent vapours, Biosensors & Bioelectronics, Vol. 13. No. 3-4. 341-346).
본 발명은 발광 박테리아를 영양배지에 고정화한 바이오센서의 크기를 소형화하여 쉽게 휴대할 수 있게 하고 이를 가스 상의 유해 독성 화학물질을 직접적으로 신속하게 실시간으로 탐지하여 유해물질을 취급하는 현장의 작업환경을 감시하고 유해가스가 유출시 작업자가 환기 및 대피 등 신속한 안전조치를 취할 수 있게하는 것을 목적으로 한다.The present invention makes it easy to carry by miniaturizing the size of the biosensor immobilized luminescent bacteria in the nutrient medium and detect the harmful toxic chemicals in the gas directly and in real time to deal with the hazardous environment on-site working environment It is intended to monitor and enable workers to take prompt safety measures such as ventilation and evacuation in case of leakage of harmful gases.
도 1은 발광 박테리아를 영양배지에 고정화한 바이오센서의 단면도.1 is a cross-sectional view of a biosensor immobilized luminescent bacteria in a nutrient medium.
도 2는 발광 박테리아를 고정화한 바이오센서를 이용하여 유해가스의 독성을 탐지하는 시스템 구성도.Figure 2 is a system configuration for detecting the toxicity of harmful gases using a biosensor immobilized luminescent bacteria.
도 3은 4℃에서 보관한 바이오센서를 30℃로 유지되는 콘트롤 반응기에 넣었을 때 발광량 변화를 나타낸 그래프.Figure 3 is a graph showing the change in emission amount when the biosensor stored at 4 ℃ put into the control reactor maintained at 30 ℃.
도 4는 독성물질로 가스상의 벤젠을 테스트 반응기에 유입시켰을 때 독성물질을 유입하지 않은 콘트롤 반응기와 벤젠이 유입된 테스트 반응기의 발광량을 벤젠의 양에 따라 비교한 상대적 발광량을 나타낸 그래프이다.Figure 4 is a graph showing the relative amount of light emission compared to the amount of benzene in the amount of benzene when the gaseous benzene is introduced into the test reactor as a toxic substance in the control reactor and the benzene is introduced.
< 도면의 주요부분에 대한 부호의 설명 ><Description of Symbols for Major Parts of Drawings>
1: 발광 박테리아 2: 투명 폴리프로필렌 튜브1: luminescent bacteria 2: transparent polypropylene tube
3: 고형화된 배지 4: 나사선3: solidified medium 4: thread
5: 원통형 검정 플라스틱 6: 광섬유5: cylindrical black plastic 6: optical fiber
7: 바이오센서 8: 검정 플라스틱 캡7: Biosensor 8: Black plastic cap
9: 이중 항온 자켓 10: 가스 유입구9: dual constant temperature jacket 10: gas inlet
11: 가스 배출구 12: 항온수 유입구11: gas outlet 12: constant temperature water inlet
13: 항온수 배출구 14: 루미노미터13: constant temperature water outlet 14: luminometer
15: 데이터 저장 시스템 16: 콘트롤 반응기15: data storage system 16: control reactor
17: 테스트 반응기17: test reactor
본 발명의 발광 박테리아를 이용한 유해가스의 독성물질을 탐지하는 방법에 있어서 먼저 발광 박테리아를 바이오센서화 해야 하는데 다음과 같은 방법에 의해서 발광 박테리아를 바이오센서화 할 수 있다. 먼저 발광 박테리아를 배양시킨 후 원심분리기로 미생물만 걸러낸 후에 37℃∼40℃로 유지되며 1ℓ 증류수당 LB Broth (Difco, USA)와 Micro agar(DUCHEFA, Netherlands)가 5:3의 비로 혼합된 젤(gel) 상태의 영양배지에 발광 박테리아가 균일하게 분포되도록 혼합한 후 광섬유 연결을 위해 외면에 나사선(4)을 낸 투명한 폴리프로필렌(PP) 튜브(2)에 부어서 상온에서 식혀서 고형화시킨다. 이렇게 고정화된 발광 박테리아 바이오센서를 발광 박테리아의 성장을 억제시키고 수분유지, 산소공급 및 장기보관을 위해서 실리콘 튜브에 넣어 밀폐시킨 후 4℃의 온도로 유지되는 냉장고에 보관한다. 한편 발광 박테리아를 투명한 폴리프로필렌 튜브에 고정화시킴으로써 미생물이 내는 빛을 광섬유로 효과적으로 전송시킬 수 있으며, 취급이 간편하도록 크기를 소형화하여 바이오센서를 다량으로 저가에 제작할 수 있으므로 휴대용 바이오센서 장치의 소모부품으로 사용할 수 있다. 도 1은 이러한 발광 박테리아 바이오센서의 단면도를 나타내고 있다.In the method of detecting toxic substances of harmful gases using the luminescent bacteria of the present invention, bioluminescent bacteria should be biosensored, but bioluminescent bacteria can be biosensored by the following method. First, after culturing luminescent bacteria, only microorganisms were filtered through a centrifuge and maintained at 37 ° C. to 40 ° C., and the gel was mixed with LB Broth (Difco, USA) and Micro agar (DUCHEFA, Netherlands) at a ratio of 5: 3 per 1 liter of distilled water. After the luminescent bacteria are uniformly distributed in the gel nutrient medium, the mixture is poured into a transparent polypropylene (PP) tube (2) having a screw thread (4) on the outer surface thereof, and cooled and solidified at room temperature. The immobilized luminescent bacteria biosensor is kept in a refrigerator maintained at a temperature of 4 ° C. after being sealed in a silicone tube for inhibiting the growth of luminescent bacteria and maintaining moisture, supplying oxygen and long-term storage. On the other hand, by immobilizing luminescent bacteria in a transparent polypropylene tube, it is possible to effectively transmit light from microorganisms to optical fibers, and to reduce the size of the microorganisms for easy handling, so that biosensors can be manufactured at low cost in large quantities. Can be used. 1 shows a cross-sectional view of such a luminescent bacterial biosensor.
도 2는 발광 박테리아 바이오센서(7)를 원통형 검정 플라스틱(5)에 결합시키고 이것을 부착한 콘트롤 반응기(16), 테스트 반응기(17)와, 상기 원통형 검정 플라스틱에 결합된 발광 박테리아 바이오센서는 광섬유(6)를 거쳐 루미노미터(14)에연결된 독성 탐지장치의 개략도이다. 반응기는 발광 박테리아가 생존할 수 있는 적정한 온도를 유지하기 위해서 항온수가 순환되는 유입구(12)와 배출구(13)가 있는 이중 항온 자켓(9), 가스유입구(10) 및 가스배출구(11)로 구성되어 있다. 또한 발광 박테리아의 발광량 손실을 막기 위해 바이오센서를 나사선이 있는 원통형 검정 플라스틱(5)에 결합하고 외부의 빛이 차단되며 가스가 스며들 수 있는 검정 플라스틱 캡(8)을 바이오센서 끝 부분에 결합한다.2 shows a control reactor 16, a test reactor 17, to which a luminescent bacterial biosensor 7 is coupled to a cylindrical black plastic 5 and attached thereto, and a luminescent bacterial biosensor coupled to the cylindrical black plastic is an optical fiber ( 6 is a schematic diagram of a toxic detector connected to the luminometer 14 via a. The reactor consists of a double constant temperature jacket (9), a gas inlet (10) and a gas outlet (11) having an inlet (12) and an outlet (13) through which constant temperature water is circulated to maintain a suitable temperature for the luminescent bacteria to survive. It is. In addition, the biosensor is attached to a cylindrical black plastic with a thread (5) to prevent the loss of light emission of the luminescent bacteria, and a black plastic cap (8) to the end of the biosensor which blocks external light and seeps in gas is combined. .
도면부호 15는 루미너미터에서 기록된 발광량을 저장 및 보관하는 데이터 저장시스템이다.Reference numeral 15 denotes a data storage system for storing and storing the amount of light emitted by the luminometer.
가스유입구를 통해 테스트 반응기에 유입된 유해가스는 검정 플라스틱 캡을 통해 바이오센서의 발광 박테리아와 반응하게 되며 발광 박테리아는 유해가스의 독성에 의해서 발광량이 점차 감소하고 이러한 발광량의 감소는 광섬유를 통해서 루미너미터에서 측정됨과 동시에 데이터 저장시스템에 저장된다. 유해가스가 도입되지 않는 콘트롤 반응기의 발광 박테리아 발광량과 유해가스가 도입된 테스트 반응기의 발광 박테리아의 발광량을 서로 비교함으로써 가스의 독성 정도를 알 수 있다.The harmful gas introduced into the test reactor through the gas inlet reacts with the luminescent bacteria of the biosensor through the black plastic cap, and the luminescent bacteria gradually decreases the emission amount due to the toxicity of the harmful gas. It is measured by the meter and stored in the data storage system. The degree of toxicity of the gas can be determined by comparing the amount of light emitted by the light emitting bacteria in the control reactor into which no harmful gas is introduced and the amount of light emitted by the light emitting bacteria in the test reactor into which the harmful gas is introduced.
한편 본 발명에 사용된 발광 박테리아는 생명공학연구소(KRIBB)에서 연구목적으로 1999년 4월 6일 분양 받은Photobacterium Phosphoreum(균주번호: KCTC 2852)으로 평시에는 다량의 빛을 내다가 독성 물질과 반응하면 발광량이 감소하는 성질을 가지고 있다.On the other hand, the luminescent bacteria used in the present invention are Photobacterium Phosphoreum (KCTC 2852), which was distributed on April 6, 1999 for research purposes by the KRIBB (Research Institute of Biotechnology). It has the property of decreasing the amount of emitted light.
이하 본 발명을 다음의 실시예에 의하여 설명하고자 한다. 그러나 이들이 본발명의 기술적 범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described by the following examples. However, these do not limit the technical scope of the present invention.
< 실시예 1 > 바이오센서의 발광량 비교<Example 1> Comparison of the emission amount of the biosensor
발광 박테리아를 배양시킨 후 원심분리기로 미생물만 걸러낸 후에 38℃∼39℃로 유지되며 1ℓ 증류수당 25g의 LB Broth(Difco, USA)와 15g의 Micro agar (DUCHEFA, Netherlands)가 혼합된 젤(gel) 상태의 영양배지에 발광 박테리아가 균일하게 분포되도록 혼합한 후 광섬유 연결을 위해 외면에 나사선을 낸 투명한 폴리프로필렌(PP) 튜브에 부어서 상온에서 식혀서 고형화시킨다. 이렇게 고정화된 발광 박테리아 바이오센서 3개를 30℃로 유지된 각각의 콘트롤 반응기에 넣은 후 발광 박테리아의 발광량의 변화량을 측정하였다. 발광량 측정 시작 후 50분 이내에 일정한 발광량을 유지하는 정상상태에 도달하였고 이러한 정상상태는 도 3에 나타낸 것처럼 100분을 전후하여 지속되었다.After culturing luminescent bacteria, only microorganisms were filtered through a centrifuge and maintained at 38 ° C to 39 ° C. A gel containing 25 g of LB Broth (Difco, USA) and 15 g of Micro agar (DUCHEFA, Netherlands) per 1 liter of distilled water was mixed. After luminescent bacteria are uniformly distributed in the nutrient medium in the) state, poured into a transparent polypropylene (PP) tube with a screw thread on the outer surface for fiber connection, and cooled and solidified at room temperature. Three immobilized luminescent bacteria biosensors were placed in each control reactor maintained at 30 ° C., and then the amount of change in luminescence of luminescent bacteria was measured. The steady state was maintained within 50 minutes after the start of the emission amount measurement, and this steady state was continued for about 100 minutes as shown in FIG. 3.
< 실시예 2 > 독성 유해 가스의 농도에 따른 발광량의 변화<Example 2> Change in the amount of emitted light according to the concentration of toxic harmful gas
상기 실시예 1에서 독성물질과 접촉하지 않은 각각의 바이오센서 발광량과 독성물질을 접촉한 바이오센서의 발광량을 측정하고 이들을 서로 비교하여 상대적 발광량을 구하여 독성물질의 농도에 따른 반응 정도를 알아보고자 하였다.In Example 1, the amount of light emitted from each biosensor not in contact with the toxic material and the amount of light emitted from the biosensor in contact with the toxic material were measured and compared with each other to obtain a relative amount of light emission.
가스상의 독성물질을 주입하지 않은 콘트롤 반응기와 독성물질을 주입한 테스트 반응기의 발광량을 비교하여 상대적 발광량(Relative Bioluminescence, RBL)을 아래의 식(1)을 이용하여 구하였고 이를 이용하여 독성물질의 농도에 따른 반응정도를 알 수 있었다. 독성물질 주입전 RBL은 1을 유지하다가 독성물질과 반응하면 RBL<1이 되며 특히 독성물질의 농도가 높을수록 RBL의 수치가 작아진다.Relative bioluminescence (RBL) was calculated by using Equation (1) below by comparing the emission levels of the control reactor without the gaseous toxic substance and the test reactor with the toxic substance. The degree of reaction was found. RBL remains 1 before toxic injection, and RBL <1 when reacting with toxic substances. In particular, the higher the concentration of toxic substances, the lower the RBL level.
......(1) ......(One)
도 4(a)∼4(c)는 독성 가스물질로 벤젠을 발광 박테리아에 무해하고 벤젠을 잘 녹이는 올레익산(Oleic acid)과 혼합하여 액상으로 테스트 반응기에 주입하고 테스트 반응기내에서 벤젠가스를 형성시킴으로써 테스트 반응기의 발광량 감소를 기록한 그래프이다. 액상의 벤젠 농도를 각각 0.1, 1, 10%로 조절하여 주입하였을 때 벤젠의 농도가 높을수록 발광 박테리아의 발광량이 보다 급격하게 감소하는 것을 알 수 있었다.4 (a) to 4 (c) show benzene as a toxic gas material, which is harmless to luminescent bacteria and mixed with oleic acid, which dissolves benzene well, into the test reactor in the liquid phase to form benzene gas in the test reactor. It is a graph recording the decrease in the light emission amount of the test reactor. When the benzene concentration was adjusted to 0.1, 1, and 10%, respectively, the higher the concentration of benzene, the more rapidly the emission of luminescent bacteria was found.
본 발명에 의한 독성 가스 탐지용 바이오센서는 기존의 장비에 비해 간편하고 쉽게 제작이 가능할 뿐만 아니라 현장에서 신속하고 민감하게 독성을 탐지할 수 있는 장점이 있다. 또한 본 발명의 독성 탐지방법을 이용하여 휘발성 유기화합물등 유독가스가 고농도로 유출되는 작업장의 대기환경을 감시하여 작업자의 불안감을 해소하고 작업자의 안전과 생산성을 높일 수 있게 해주며 유독가스의 유출시 경보를 발생시켜 작업자가 환기, 대피 등 안전조치를 취할 수 있다.Biosensor for toxic gas detection according to the present invention has the advantage that can be easily and easily manufactured compared to the existing equipment as well as detect the toxicity quickly and sensitively in the field. In addition, by using the toxicity detection method of the present invention to monitor the atmospheric environment of the workplace where toxic organic compounds, such as volatile organic compounds are released at high concentrations, it can solve the worker's anxiety, increase the safety and productivity of workers, By generating an alarm, the operator can take safety measures such as ventilation and evacuation.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019990023325A KR100329121B1 (en) | 1999-06-21 | 1999-06-21 | Detecting Method of toxicity in Gas Phase Using Immobilized Bioluminescent Bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019990023325A KR100329121B1 (en) | 1999-06-21 | 1999-06-21 | Detecting Method of toxicity in Gas Phase Using Immobilized Bioluminescent Bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20010003154A KR20010003154A (en) | 2001-01-15 |
| KR100329121B1 true KR100329121B1 (en) | 2002-03-21 |
Family
ID=19593966
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1019990023325A KR100329121B1 (en) | 1999-06-21 | 1999-06-21 | Detecting Method of toxicity in Gas Phase Using Immobilized Bioluminescent Bacteria |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100329121B1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102009014B1 (en) * | 2012-11-08 | 2019-08-08 | 고려대학교 산학협력단 | A Dip-stick Toxicity Biosensor Using Smart Functional Microbeads |
| KR101944418B1 (en) * | 2017-03-30 | 2019-01-31 | 주식회사 네오엔비즈 | Apparatus for toxicity identification evaluation comprising organic pollutants and volatile toxicants in unknown water samples |
-
1999
- 1999-06-21 KR KR1019990023325A patent/KR100329121B1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| KR20010003154A (en) | 2001-01-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2862556B2 (en) | Apparatus and device for detecting microorganisms | |
| Preininger et al. | Optical fiber sensor for biological oxygen demand | |
| Bjerketorp et al. | Advances in preservation methods: keeping biosensor microorganisms alive and active | |
| Kroneis et al. | A fluorescence-based sterilizable oxygen probe for use in bioreactors | |
| EP2245177B1 (en) | Systems and methods for identifying a culture as positive for microorganisms with high confidence | |
| Ge et al. | High-stability non-invasive autoclavable naked optical CO2 sensor | |
| FI97548C (en) | Device for the detection of micro-organisms | |
| Naessens et al. | Whole-cell biosensor for direct determination of solvent vapours | |
| Sipior et al. | Phase fluorometric optical carbon dioxide gas sensor for fermentation off‐gas monitoring | |
| CN205581046U (en) | Torrid zone aquaculture water body BOD surveys device on line based on little biosensor | |
| Damgaard et al. | Use of an oxygen-insensitive microscale biosensor for methane to measure methane concentration profiles in a rice paddy | |
| Voraberger et al. | Novel oxygen optrode withstanding autoclavation: technical solutions and performance | |
| KR100329121B1 (en) | Detecting Method of toxicity in Gas Phase Using Immobilized Bioluminescent Bacteria | |
| CN101477105A (en) | Rapid measuring method for high-salt trade waste BOD | |
| Eltzov et al. | Fiber-optic based cell sensors | |
| Lam et al. | Optical instrumentation for bioprocess monitoring | |
| KR100305218B1 (en) | Automatic measuring apparatus for toxic substances of water system using immobilized bioluminescent organism | |
| Chan et al. | Application of a luminescence-based pH optrode to monitoring of fermentation by Klebsiella pneumoniae | |
| KR100994691B1 (en) | Method of manufacturing thereof for carbon dioxide sensing membrane containing fluorescent basic dyes | |
| KR100355757B1 (en) | Device for monitoring toxicity in contaminated soils and method for monitoring toxicity in contaminated soils using same | |
| US20070212748A1 (en) | Detection of microbiological growth in a sealed container using a poising agent | |
| CN113574365A (en) | Device, system and method for continuous real-time blood culture measurement | |
| EP0486443A1 (en) | Method of detecting toxic compositions in water by monitoring the metabolism of selected living cells as microorganisms | |
| KR100316153B1 (en) | METHOD FOR MEASURING TOXICITY MATERIALS USING ELIMINATED LIGHT EMITTING MICROORGANISM AND BIOSENSOR KIT | |
| KR100392065B1 (en) | Water Toxicity Analysis Method Using Immobilized Bioluminescent Bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20111227 Year of fee payment: 11 |
|
| FPAY | Annual fee payment |
Payment date: 20121211 Year of fee payment: 12 |
|
| LAPS | Lapse due to unpaid annual fee |