KR102690859B1 - Method for Detecting Hypoxanthine Based on Colorimetric Reaction by Peroxidase Activity - Google Patents
Method for Detecting Hypoxanthine Based on Colorimetric Reaction by Peroxidase Activity Download PDFInfo
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- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 title claims abstract description 164
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 title claims abstract description 82
- 102000003992 Peroxidases Human genes 0.000 title claims abstract description 55
- 108040007629 peroxidase activity proteins Proteins 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000000694 effects Effects 0.000 title claims description 13
- 238000006243 chemical reaction Methods 0.000 title abstract description 19
- 108010093894 Xanthine oxidase Proteins 0.000 claims abstract description 26
- 102100033220 Xanthine oxidase Human genes 0.000 claims abstract description 26
- 238000004040 coloring Methods 0.000 claims abstract description 19
- 230000034994 death Effects 0.000 claims description 24
- 238000002835 absorbance Methods 0.000 claims description 20
- 239000000758 substrate Substances 0.000 claims description 17
- PKYCWFICOKSIHZ-UHFFFAOYSA-N 1-(3,7-dihydroxyphenoxazin-10-yl)ethanone Chemical compound OC1=CC=C2N(C(=O)C)C3=CC=C(O)C=C3OC2=C1 PKYCWFICOKSIHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000003593 chromogenic compound Substances 0.000 claims description 11
- 210000004127 vitreous body Anatomy 0.000 claims description 10
- ZTOJFFHGPLIVKC-YAFCTCPESA-N (2e)-3-ethyl-2-[(z)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical group S\1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C/1=N/N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-YAFCTCPESA-N 0.000 claims description 8
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 claims description 6
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 claims description 6
- ZTOJFFHGPLIVKC-UHFFFAOYSA-N 3-ethyl-2-[(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound S1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C1=NN=C1SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-UHFFFAOYSA-N 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 229910021389 graphene Inorganic materials 0.000 claims description 6
- 239000002122 magnetic nanoparticle Substances 0.000 claims description 6
- 239000004480 active ingredient Substances 0.000 claims description 5
- IYXMNTLBLQNMLM-UHFFFAOYSA-N benzene-1,4-diamine;hydron;dichloride Chemical compound Cl.Cl.NC1=CC=C(N)C=C1 IYXMNTLBLQNMLM-UHFFFAOYSA-N 0.000 claims 2
- 238000004458 analytical method Methods 0.000 abstract description 10
- 238000005516 engineering process Methods 0.000 abstract description 8
- 239000000126 substance Substances 0.000 abstract description 2
- 238000011842 forensic investigation Methods 0.000 abstract 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 29
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 18
- 238000001514 detection method Methods 0.000 description 18
- 229940075420 xanthine Drugs 0.000 description 9
- 239000010839 body fluid Substances 0.000 description 6
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 5
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 5
- 210000001124 body fluid Anatomy 0.000 description 5
- 229940116269 uric acid Drugs 0.000 description 5
- RIIWUGSYXOBDMC-UHFFFAOYSA-N benzene-1,2-diamine;hydron;dichloride Chemical compound Cl.Cl.NC1=CC=CC=C1N RIIWUGSYXOBDMC-UHFFFAOYSA-N 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical group C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 3
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 3
- 229960001456 adenosine triphosphate Drugs 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 208000035992 Postmortem Changes Diseases 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000006395 oxidase reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
본 발명은 하이포잔틴을 검출하는 방법에 관한 것으로, 더욱 자세하게는 잔틴 산화효소(xanthine oxidase), 과산화효소(peroxidase) 또는 그 유사체 및 발색물질을 함유하는 조성물을 이용하여, 하이포잔틴을 검출하는 방법에 관한 것으로, 본 발명에 따른 잔틴 산화효소와 과산화효소 및 그 유사체를 이용하여 시료 내 하이포잔틴을 검출 또는 정량하는 방법은 하이포잔틴을 단시간 내에 육안으로 간단하게 확인할 수 있고, 발색반응 신호 기반의 간편한 분석방법으로써 법의학 수사를 비롯하여 다양한 분야의 현장 진단용 기술로 활용 가능하다.The present invention relates to a method for detecting hypoxanthine, and more specifically, to a method for detecting hypoxanthine using a composition containing xanthine oxidase, peroxidase or an analog thereof, and a coloring substance. Related to this, the method of detecting or quantifying hypoxanthine in a sample using xanthine oxidase, peroxidase, and their analogs according to the present invention allows hypoxanthine to be easily confirmed with the naked eye within a short period of time and is a simple analysis based on a color reaction signal. As a method, it can be used as an on-site diagnostic technology in various fields, including forensic investigation.
Description
본 발명은 하이포잔틴을 검출하는 방법에 관한 것으로, 더욱 자세하게는 잔틴 산화효소(xanthine oxidase), 과산화효소(peroxidase) 또는 그 유사체 및 발색물질을 함유하는 조성물을 이용하여, 하이포잔틴을 검출하는 방법에 관한 것이다. The present invention relates to a method for detecting hypoxanthine, and more specifically, to a method for detecting hypoxanthine using a composition containing xanthine oxidase, peroxidase or an analog thereof, and a coloring substance. It's about.
시신의 사후 경과시간을 정확하게 판단하는 것은 사망사건의 수사에 있어 아주 중요한 일이다. 사후 경과시간의 추정을 위해 체온강하, 시반, 시체 강직, 부패 진행 등의 시체 현상 분석이 보편적으로 사용되고 있다. 그러나 시체 현상들은 외부 환경을 비롯하여 매우 다양한 인자들에 의해 민감하게 영향을 받고, 시신마다 개인차가 존재하기 때문에 정확한 사후 경과시간 추정을 어렵게 만든다.Accurately determining the time elapsed after death of a body is very important in the investigation of a death case. To estimate the time elapsed after death, analysis of corpse phenomena such as body temperature drop, body stiffness, corpse stiffness, and progression of decomposition are commonly used. However, corpse phenomena are sensitively affected by a wide variety of factors, including the external environment, and individual differences exist for each corpse, making it difficult to accurately estimate the elapsed time after death.
위와 같은 문제점을 해결하기 위하여, 뇌척수액(cerebrospinal fluid, CSF)이나 유리체액(vitreous humor, VH)과 같은 체액의 사후 성분 변화를 분석하여 사후 경과시간을 추정하는 방법이 제시되었다. 특히, 유리체는 해부학적으로 격리되어 있어(isolated) 외부 환경에 따른 오염이나 자가융해(autolysis) 같은 사후 변화가 다른 체액에서보다 천천히 일어나는 것으로 알려져 있어, 생화학 분석에 적합한 시료로서 연구되고 있다.In order to solve the above problems, a method of estimating the elapsed time after death by analyzing changes in postmortem components of body fluids such as cerebrospinal fluid (CSF) or vitreous humor (VH) was proposed. In particular, since the vitreous body is anatomically isolated, postmortem changes such as contamination from the external environment or autolysis are known to occur more slowly than in other body fluids, and it is being studied as a suitable sample for biochemical analysis.
하이포잔틴(hypoxanthine)은 아데노신 삼인산(adenosine 5'-triphosphate, ATP)이 분해되어 생성되는 퓨린 유도체로, 유리체액 내의 하이포잔틴 농도는 사망 직후부터 경과시간에 따라 일정하게 증가하므로, 하이포잔틴을 사후 경과시간 추정을 위한 바이오마커로서 활용할 수 있다 (Rognum et al., Forensic Science International, 51, 139-146, 1991).Hypoxanthine is a purine derivative produced by the decomposition of adenosine 5'-triphosphate (ATP). Since the concentration of hypoxanthine in the vitreous humor increases steadily with time elapsed immediately after death, hypoxanthine is used after death. It can be used as a biomarker for time estimation (Rognum et al ., Forensic Science International , 51, 139-146, 1991).
하이포잔틴의 검출 및 정량을 위해 가장 널리 사용되는 방법으로는 고성능 액체 크로마토그래피(high-performance liquid chromatography, HPLC), 액체 크로마토그래피-질량 분석법(liquid chromatography-mass spectrometry, LC-MS) 등이 있으나, 상기 방법은 고가의, 대형의 장비가 요구되며, 전문 연구소나 병원과 같은 대형 기관에서 사용되는 등 사건 현장에서 직접 사용하기에는 적합하지 않다는 한계를 가지고 있다.The most widely used methods for detection and quantitation of hypoxanthine include high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). The method requires expensive, large-scale equipment and has limitations in that it is not suitable for direct use at the scene of an incident, such as in large institutions such as specialized laboratories or hospitals.
이에, 본 발명자들은 상기 종래 기술들의 한계점을 해결하며 간편하게 하이포잔틴의 유무를 검출할 수 있는 방법을 개발하기 위하여 예의 노력한 결과, 하이포잔틴이 잔틴 산화효소의 작용에 의해 잔틴을 거쳐 요산으로 전환되며 발생하는 과산화수소가 과산화효소의 작용에 의해 발색기질을 산화시켜 흡광도가 변화하는 것을 이용하여 하이포잔틴을 간편하게 측정할 수 있다는 것을 확인하고, 본 발명을 완성하게 되었다. Accordingly, the present inventors have made diligent efforts to develop a method that can easily detect the presence or absence of hypoxanthine while solving the limitations of the above-described prior technologies. As a result, hypoxanthine is converted to uric acid via xanthine by the action of xanthine oxidase, and is generated. After confirming that hypoxanthine can be easily measured by using hydrogen peroxide to change absorbance by oxidizing the coloring substrate through the action of peroxidase, the present invention was completed.
본 발명의 목적은 고가의 대형 장비를 필요로 하지 않는 과산화효소의 작용을 이용한 하이포잔틴의 검출 또는 정량방법을 제공하는데 있다.The purpose of the present invention is to provide a method for detecting or quantifying hypoxanthine using the action of peroxidase that does not require large, expensive equipment.
본 발명의 다른 목적은 하이포잔틴 검출용 조성물을 제공하는데 있다.Another object of the present invention is to provide a composition for detecting hypoxanthine.
본 발명의 또 다른 목적은 상기 하이포잔틴의 검출 또는 정량방법을 이용한 시신의 사후 경과시간을 측정하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method of measuring the time elapsed after death of a corpse using the above-described method of detecting or quantifying hypoxanthine.
본 발명의 또 다른 목적은 시신의 사후 경과시간 측정용 조성물을 제공하는데 있다. Another object of the present invention is to provide a composition for measuring the time elapsed after death of a corpse.
상기 목적을 달성하기 위하여, 본 발명은 다음 단계를 포함하는 하이포잔틴의 검출 또는 정량방법을 제공한다:In order to achieve the above object, the present invention provides a method for detecting or quantifying hypoxanthine comprising the following steps:
(a) 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 함유하는 하이포잔틴 검출용 조성물에 하이포잔틴 함유 시료를 첨가하여 반응시키는 단계; 및(a) adding and reacting a hypoxanthine-containing sample to a composition for detecting hypoxanthine containing xanthine oxidase, peroxidase or an analogue thereof, and a coloring substrate; and
(b) 상기 시료가 첨가된 조성물의 발색정도를 흡광도로 측정하여, 시료 내의 하이포잔틴을 검출 또는 정량하는 단계.(b) Measuring the degree of color development of the composition to which the sample is added using absorbance to detect or quantify hypoxanthine in the sample.
본 발명은 또한, 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 유효성분으로 포함하는 하이포잔틴의 검출용 조성물을 제공한다.The present invention also provides a composition for detecting hypoxanthine containing xanthine oxidase, peroxidase or an analog thereof, and a chromogenic substrate as active ingredients.
본 발명은 또한, 다음 단계를 포함하는 시신의 사후 경과시간을 측정하는 방법을 제공한다:The present invention also provides a method of measuring the time elapsed after death of a corpse comprising the following steps:
(a) 사체 유래의 체액을 포함하는 시료를 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 함유하는 하이포잔틴 검출용 조성물과 반응시키는 단계; 및(a) reacting a sample containing body fluid derived from a cadaver with a composition for detecting hypoxanthine containing xanthine oxidase, peroxidase or an analog thereof, and a chromogenic substrate; and
(b) 상기 시료가 첨가된 조성물의 색변화를 흡광도를 이용하여 측정하여, 시료 내의 하이포잔틴을 검출 또는 정량하여, 시신의 사후 경과시간을 측정하는 단계.(b) measuring the color change of the composition to which the sample is added using absorbance, detecting or quantifying hypoxanthine in the sample, and measuring the time elapsed after death of the body.
본 발명은 또한, 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 유효성분으로 포함하는 시신의 사후 경과시간 측정용 조성물을 제공한다. The present invention also provides a composition for measuring the time elapsed after death of a corpse, comprising xanthine oxidase, peroxidase or its analogue, and a coloring substrate as active ingredients.
본 발명에 따른 잔틴 산화효소와 과산화효소 및 그 유사체를 이용하여 시료 내 하이포잔틴을 검출 또는 정량하는 방법은 하이포잔틴을 단시간 내에 육안으로 간단하게 확인할 수 있고, 발색반응 신호 기반의 간편한 분석 방법으로써 법의학 수사를 비롯하여 다양한 분야의 현장 진단용 기술로 활용 가능하다.The method of detecting or quantifying hypoxanthine in a sample using xanthine oxidase, peroxidase, and their analogs according to the present invention allows hypoxanthine to be easily confirmed with the naked eye in a short period of time, and is a simple analysis method based on a color reaction signal, which can be used in forensic science. It can be used as an on-site diagnostic technology in various fields, including investigations.
도 1은 본 발명에 따른 하이포잔틴 검출과정을 도식화한 것이다. 시료 내 하이포잔틴이 존재하면 하이포잔틴이 잔틴 산화효소에 의해 산화될 때 과산화수소(H2O2)가 발생하고, 과산화수소와 과산화효소에 의해 발색기질이 산화되어 발색되면, 색변화를 흡광도 측정을 통해 분석한다.
도 2는 잔틴 산화효소, 과산화효소, 하이포잔틴의 유무 및 조합에 따른 흡광도 변화를 확인한 결과를 나타낸 것이다.
도 3은 본 발명에 따른 하이포잔틴 검출방법을 이용하여 하이포잔틴 검출 민감도를 확인한 결과를 나타낸 것이다.
도 4는 본 발명에 따른 하이포잔틴 검출방법을 이용하여 하이포잔틴 검출 선택도를 확인한 결과를 나타낸 것이다. Figure 1 schematically illustrates the hypoxanthine detection process according to the present invention. If hypoxanthine is present in the sample, hydrogen peroxide (H 2 O 2 ) is generated when hypoxanthine is oxidized by xanthine oxidase. When the color-generating substrate is oxidized by hydrogen peroxide and peroxidase and color is developed, the color change is measured through absorbance measurement. Analyze.
Figure 2 shows the results of confirming the change in absorbance according to the presence or absence and combination of xanthine oxidase, peroxidase, and hypoxanthine.
Figure 3 shows the results of confirming hypoxanthine detection sensitivity using the hypoxanthine detection method according to the present invention.
Figure 4 shows the results of confirming hypoxanthine detection selectivity using the hypoxanthine detection method according to the present invention.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.
하이포잔틴(hypoxanthine)의 농도는 사망 시신의 유리체액 내에서 사망 직후부터 경과시간에 따라 일정하게 증가하므로, 하이포잔틴을 사후 경과시간 추정을 위한 바이오마커로서 활용할 수 있으나, 현재까지 고성능 크로마토그래피(HPLC)나 액체크로마토그래피-질량분석법(LS-MS)등의 고가의 장비를 이용하여 전문연구소에서만 분석이 가능하였다. Since the concentration of hypoxanthine increases consistently with the elapsed time from immediately after death in the vitreous humor of a deceased body, hypoxanthine can be used as a biomarker for estimating the elapsed time after death. However, to date, high-performance chromatography (HPLC) ) or liquid chromatography-mass spectrometry (LS-MS), analysis was only possible at specialized laboratories using expensive equipment.
본 발명에서는 잔틴 산화효소 작용에 의해 시료 내에 존재하는 하이포잔틴의 산화, 이에 따른 과산화수소의 생성 및 과산화효소 작용에 의한 발색 기질의 산화에 따라 색변화 및 흡광도를 측정하여 시료 내 존재하는 하이포잔틴 존재 유무를 확인하고 정량하는 방법을 개발하였다.In the present invention, the presence or absence of hypoxanthine in the sample is determined by measuring the color change and absorbance according to the oxidation of hypoxanthine present in the sample by the action of xanthine oxidase, the resulting production of hydrogen peroxide, and the oxidation of the coloring substrate by the action of peroxidase. A method to identify and quantify was developed.
시료 내 하이포잔틴이 존재하면 하이포잔틴이 잔틴 산화효소에 의해 산화될 때 과산화수소가 발생하고, 과산화수소와 과산화효소에 의해 발색 기질이 산화되며, 이로 인해 발색반응을 유도하고 색변화를 흡광도 측정을 통해 측정하여 하이포잔틴을 정량할 수 있으며, 정량된 하이포잔틴의 농도를 통해 시신의 사망경과 시간을 측정할 수 있다.If hypoxanthine is present in the sample, hydrogen peroxide is generated when hypoxanthine is oxidized by xanthine oxidase. The coloring substrate is oxidized by hydrogen peroxide and peroxidase, which induces a color reaction and the color change is measured through absorbance measurement. Thus, hypoxanthine can be quantified, and the elapsed time of death of the body can be measured through the quantified concentration of hypoxanthine.
따라서, 본 발명은 일관점에서, 다음 단계를 포함하는 하이포잔틴의 검출 또는 정량방법에 관한 것이다:Accordingly, the present invention generally relates to a method for detecting or quantifying hypoxanthine comprising the following steps:
(a) 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 함유하는 하이포잔틴 검출용 조성물에 하이포잔틴 함유 시료를 첨가하여 반응시키는 단계; 및(a) adding and reacting a hypoxanthine-containing sample to a composition for detecting hypoxanthine containing xanthine oxidase, peroxidase or an analogue thereof, and a coloring substrate; and
(b) 상기 시료가 첨가된 조성물의 발색정도를 흡광도로 측정하여, 시료 내의 하이포잔틴을 검출 또는 정량하는 단계.(b) Measuring the degree of color development of the composition to which the sample is added using absorbance to detect or quantify hypoxanthine in the sample.
본 발명에서는 잔틴 산화효소, 과산화효소와 그 유사체 및 발색 기질을 포함하는 하이포잔틴 검출용 조성물에 검출 시료를 첨가하여, 시료 내 하이포잔틴을, 잔틴을 거쳐 요산으로 산화시켜 과산화수소를 발생시키고, 생성된 과산화수소가 과산화효소의 작용에 의해 발색기질을 산화시키며, 하이포잔틴 검출용 조성물의 색변화를 흡광도를 이용해 측정하여, 시료 내 하이포잔틴을 검출 또는 정량할 수 있다. In the present invention, a detection sample is added to a composition for detecting hypoxanthine containing xanthine oxidase, peroxidase, its analogs, and a coloring substrate, and the hypoxanthine in the sample is oxidized to uric acid via xanthine to generate hydrogen peroxide. Hydrogen peroxide oxidizes the coloring substrate through the action of peroxidase, and the color change of the composition for detecting hypoxanthine can be measured using absorbance to detect or quantify hypoxanthine in the sample.
본 발명의 하이포잔틴 검출방법은 도 1에 나타낸 반응에 의하여 수행되며, 본 발명의 상기 (a)단계는 하기와 같은 반응단계를 포함한다:The hypoxanthine detection method of the present invention is carried out by the reaction shown in Figure 1, and step (a) of the present invention includes the following reaction steps:
(i) 잔틴 산화효소에 의해 하이포잔틴이 잔틴으로, 연속하여 잔틴이 요산으로 산화되며 과산화수소가 생성되는 단계; 및 (ii) 과산화효소에 의해 과산화수소가 환원됨과 동시에 발색 기질이 산화하여 흡광도가 변화하는 단계.(i) a step in which hypoxanthine is oxidized to xanthine and subsequently xanthine to uric acid by xanthine oxidase, thereby generating hydrogen peroxide; and (ii) a step in which hydrogen peroxide is reduced by peroxidase and at the same time the coloring substrate is oxidized and the absorbance changes.
본 발명의 (b) 단계에서는 상기 (a) 반응을 통해 변화한 흡광도를 측정하고, 흡광도의 변화 정도를 통하여 시료 내 하이포잔틴을 검출 및 정량할 수 있다. In step (b) of the present invention, the absorbance changed through the reaction (a) is measured, and hypoxanthine in the sample can be detected and quantified through the degree of change in absorbance.
본 발명에서 사용되는 "잔틴 산화효소"는 하이포잔틴을 잔틴으로 전환시키며 과산화수소를 부산물로 생성시키며, 상기 생성된 잔틴을 다시 요산으로 산화시키며 과산화수소를 생성시키는 기능을 한다. “Xanthine oxidase” used in the present invention converts hypoxanthine into xanthine and produces hydrogen peroxide as a by-product, and oxidizes the produced xanthine back to uric acid to produce hydrogen peroxide.
본 발명에서 사용되는 "과산화효소"는 상기 생성된 과산화수소를 물로 전환시키면서 발색물질에 전자를 전달하여 발색시키는 기능을 한다. “Peroxidase” used in the present invention converts the generated hydrogen peroxide into water and transfers electrons to the coloring material to develop color.
본 발명에서 사용되는 과산화 효소는 호스래디쉬 과산화효소, 콩 과산화효소 , 땅콩 과산화효소, 애기장대 과산화효소 등을 사용할 수 있으나 이에 한정되는 것은 아니다. The peroxidase enzyme used in the present invention may include, but is not limited to, horseradish peroxidase, soybean peroxidase, peanut peroxidase, and Arabidopsis peroxidase.
본 발명에 있어서, 상기 과산화효소 또는 그 유사체는 과산화효소, 과산화효소 활성을 가지는 자성 나노입자 및 과산화효소 활성을 가지는 산화 그래핀으로 이루어진 군에서 선택되는 것을 사용할 수 있고, 상기 발색 기질은 ABTS(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB( 3,3',5,5'-tetramethylbenzidine), OPD(o-phenylenediamine dihydrochloride), DAB(3,3'-diaminobenzidine) 및 ADHP(10-acetyl-3,7-dihydroxyphenoxazine)로 이루어진 군에서 선택되는 것을 사용할 수 있으나 이에 한정되는 것은 아니다. In the present invention, the peroxidase or its analogue may be selected from the group consisting of peroxidase, magnetic nanoparticles with peroxidase activity, and graphene oxide with peroxidase activity, and the coloring substrate is ABTS (2 ,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB(3,3',5,5'-tetramethylbenzidine), OPD( o -phenylenediamine dihydrochloride), DAB(3,3'-diaminobenzidine) and ADHP (10-acetyl-3,7-dihydroxyphenoxazine) may be used, but are not limited thereto.
본 발명에서는 잔틴 산화효소에 의해서 하이포잔틴이 잔틴으로, 연속하여 잔틴이 요산으로 산화되어 과산화수소가 발생하고, 상기 반응과 동시에 과산화효소에 의해서 잔틴 산화효소 반응으로 생성된 과산화수소는 다시 환원되고, 발색 기질이 산화된다. 시료 내 상기 두 효소 중 하나 이상 또는 하이포잔틴이 존재하지 않는 경우, 발색 기질의 산화가 진행되지 않아 흡광도가 변하지 않는 반면, 상기 두 효소와 하이포잔틴이 모두 존재하는 경우에만, 발색 기질이 산화하여 흡광도가 증가한다. 이러한 현상을 통하여 본 발명에 따른 잔틴 산화효소와 과산화효소 조합을 이용하여 하이포잔틴의 존재 유무를 흡광도를 측정하여 판단할 수 있다는 것을 규명하였다 (도 2).In the present invention, hypoxanthine is oxidized to xanthine by xanthine oxidase, and xanthine is subsequently oxidized to uric acid to generate hydrogen peroxide, and at the same time as the above reaction, hydrogen peroxide generated by xanthine oxidase reaction is reduced again by peroxidase, and a coloring substrate is produced. This is oxidized. When at least one of the two enzymes or hypoxanthine is not present in the sample, oxidation of the chromogenic substrate does not proceed and the absorbance does not change. However, only when both enzymes and hypoxanthine are present, the chromogenic substrate is oxidized and the absorbance increases. increases. Through this phenomenon, it was established that the presence or absence of hypoxanthine can be determined by measuring absorbance using the combination of xanthine oxidase and peroxidase according to the present invention (Figure 2).
다른 관점에서, 본 발명은 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 유효성분으로 포함하는 하이포잔틴의 검출용 조성물에 관한 것이다.From another perspective, the present invention relates to a composition for detecting hypoxanthine containing xanthine oxidase, peroxidase or an analog thereof, and a chromogenic substrate as active ingredients.
본 발명에 있어서, 상기 과산화효소 또는 그 유사체는 과산화효소, 과산화효소 활성을 가지는 자성 나노입자 및 과산화효소 활성을 가지는 산화 그래핀으로 이루어진 군에서 선택되는 것을 사용할 수 있고, 상기 발색 기질은 ABTS(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB( 3,3',5,5'-tetramethylbenzidine), OPD(o-phenylenediamine dihydrochloride), DAB(3,3'-diaminobenzidine) 및 ADHP(10-acetyl-3,7-dihydroxyphenoxazine)로 이루어진 군에서 선택되는 것을 사용할 수 있으나 이에 한정되는 것은 아니다. In the present invention, the peroxidase or its analogue may be selected from the group consisting of peroxidase, magnetic nanoparticles with peroxidase activity, and graphene oxide with peroxidase activity, and the coloring substrate is ABTS (2 ,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB(3,3',5,5'-tetramethylbenzidine), OPD( o -phenylenediamine dihydrochloride), DAB(3,3'-diaminobenzidine) and ADHP (10-acetyl-3,7-dihydroxyphenoxazine) may be used, but are not limited thereto.
또 다른 관점에서, 본 발명은 또한, 다음 단계를 포함하는 시신의 사후 경과시간을 측정하는 방법에 관한 것이다:In another aspect, the present invention also relates to a method of measuring the time elapsed after death of a corpse comprising the following steps:
(a) 사체 유래의 체액을 포함하는 시료를 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 함유하는 하이포잔틴 검출용 조성물과 반응시키는 단계; 및(a) reacting a sample containing body fluid derived from a cadaver with a composition for detecting hypoxanthine containing xanthine oxidase, peroxidase or an analog thereof, and a chromogenic substrate; and
(b) 상기 시료가 첨가된 조성물의 색변화를 흡광도를 이용하여 측정하여, 시료 내의 하이포잔틴을 검출 또는 정량하여, 시신의 사후 경과시간을 측정하는 단계.(b) measuring the color change of the composition to which the sample is added using absorbance, detecting or quantifying hypoxanthine in the sample, and measuring the time elapsed after death of the body.
본 발명에서 "시신의 사후 경과시간"은 사망한 후 경과된 시간을 의미한다. In the present invention, “postmortem time of the body” means the time elapsed after death.
본 발명에 있어서, 상기 체액은 사체유래의 유리체액인 것을 특징으로 할 수 있다. 유리체액 내의 하이포잔틴 농도는 사망 직후부터 경과시간에 따라 일정하게 증가하여, 하이포잔틴의 농도를 측정하면 사후 경과시간을 추정할 수 있다. In the present invention, the body fluid may be characterized as vitreous body fluid derived from a cadaver. The concentration of hypoxanthine in the vitreous humor steadily increases with the elapsed time immediately after death, so the elapsed time after death can be estimated by measuring the concentration of hypoxanthine.
아울러, 본 발명의 다른 실시예에서는, 서로 다른 농도의 표적 하이포잔틴을 첨가하였을 때 흡광도 변화를 측정하였다. 본 발명의 하이포잔틴 검출 한계 (limit of detection, LOD)가 10.81 nM인 것을 확인하였으며, 이러한 결과는 본 발명의 하이포잔틴 검출 기술이 유리체액 내의 하이포잔틴을 검출 및 정량하기에 충분한 검출 민감도를 가지는 것을 의미한다 (도 3).In addition, in another example of the present invention, the change in absorbance was measured when different concentrations of target hypoxanthine were added. It was confirmed that the limit of detection (LOD) of hypoxanthine of the present invention was 10.81 nM, and these results show that the hypoxanthine detection technology of the present invention has sufficient detection sensitivity to detect and quantify hypoxanthine in vitreous humor. means (Figure 3).
본 발명에 있어서, 상기 과산화효소 또는 그 유사체는 과산화효소, 과산화효소 활성을 가지는 자성 나노입자 및 과산화효소 활성을 가지는 산화 그래핀으로 이루어진 군에서 선택되는 것을 사용할 수 있고, 상기 발색 기질은 ABTS(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB( 3,3',5,5'-tetramethylbenzidine), OPD(o-phenylenediamine dihydrochloride), DAB(3,3'-diaminobenzidine) 및 ADHP(10-acetyl-3,7-dihydroxyphenoxazine)로 이루어진 군에서 선택되는 것을 사용할 수 있으나 이에 한정되는 것은 아니다. In the present invention, the peroxidase or its analogue may be selected from the group consisting of peroxidase, magnetic nanoparticles with peroxidase activity, and graphene oxide with peroxidase activity, and the coloring substrate is ABTS (2 ,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB(3,3',5,5'-tetramethylbenzidine), OPD( o -phenylenediamine dihydrochloride), DAB(3,3'-diaminobenzidine) and ADHP (10-acetyl-3,7-dihydroxyphenoxazine) may be used, but are not limited thereto.
또 다른 관점에서, 본 발명은 또한, 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 유효성분으로 포함하는 시신의 사후 경과시간 측정용 조성물에 관한 것이다. From another perspective, the present invention also relates to a composition for measuring the time elapsed after death of a corpse, comprising xanthine oxidase, peroxidase or an analog thereof, and a coloring substrate as active ingredients.
본 발명에 있어서, 상기 과산화효소 또는 그 유사체는 과산화효소, 과산화효소 활성을 가지는 자성 나노입자 및 과산화효소 활성을 가지는 산화 그래핀으로 이루어진 군에서 선택되는 것을 사용할 수 있고, 상기 발색 기질은 ABTS(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB( 3,3',5,5'-tetramethylbenzidine), OPD(o-phenylenediamine dihydrochloride), DAB(3,3'-diaminobenzidine) 및 ADHP(10-acetyl-3,7-dihydroxyphenoxazine)로 이루어진 군에서 선택되는 것을 사용할 수 있으나 이에 한정되는 것은 아니다. In the present invention, the peroxidase or its analogue may be selected from the group consisting of peroxidase, magnetic nanoparticles with peroxidase activity, and graphene oxide with peroxidase activity, and the coloring substrate is ABTS (2 ,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB(3,3',5,5'-tetramethylbenzidine), OPD( o -phenylenediamine dihydrochloride), DAB(3,3'-diaminobenzidine) and ADHP (10-acetyl-3,7-dihydroxyphenoxazine) may be used, but are not limited thereto.
이하, 본 발명을 실시예에 의하여 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
[실시예][Example]
실시예 1. 하이포잔틴 검출 및 정량을 위한 반응 조건Example 1. Reaction conditions for hypoxanthine detection and quantification
본 하이포잔틴 검출 기술 반응 용액 제조 과정은 다음과 같으며, 이에 한정되지는 않는다. 하이포잔틴 검출 및 정량을 위한 반응용액 90μL를 제조하였다. 반응 용액은 포스페이트 버퍼(50mM, pH 7.4) 60μL, 호스래디쉬 과산화효소(20μg/mL, Sigma-Aldrich) 10μL, 잔틴 산화효소 (25U/mL, Sigma-Aldrich) 10μL, ABTS(10mM, Sigma-Aldrich) 10μL를 포함한다. 상기 제조된 반응용액에 하이포잔틴을 포함하는 분석시료 10μL를 넣어주고, 37℃에서 10분 동안 반응을 진행시킨다. 반응이 완료된 후, 파장에 따른 흡광도를 측정함으로써, 발색 기질 ABTS의 흡수파장 420nm에서 높은 흡광도를 나타내는 것을 확인하였다.The process for preparing the hypoxanthine detection technology reaction solution is as follows, but is not limited thereto. 90 μL of reaction solution for hypoxanthine detection and quantification was prepared. The reaction solution contained 60μL of phosphate buffer (50mM, pH 7.4), 10μL of horseradish peroxidase (20μg/mL, Sigma-Aldrich), 10μL of xanthine oxidase (25U/mL, Sigma-Aldrich), and ABTS (10mM, Sigma-Aldrich). ) contains 10 μL. Add 10 μL of the analysis sample containing hypoxanthine to the reaction solution prepared above, and proceed with the reaction at 37°C for 10 minutes. After the reaction was completed, by measuring the absorbance according to the wavelength, it was confirmed that the chromogenic substrate ABTS showed high absorbance at an absorption wavelength of 420 nm.
실시예 2. 하이포잔틴 검출의 민감도 및 선택도 검증Example 2. Verification of sensitivity and selectivity of hypoxanthine detection
실시예 1에서 언급된 반응조건을 이용하여 분석시료에 포함된 하이포잔틴의 농도를 달리하여, 반응 후 흡광도를 측정하여 민감도 검증 실험을 진행하였다. 다양한 농도(0, 1, 2, 5, 7, 10, 20, 50, 70, 100μM)의 하이포잔틴을 포함하는 분석시료(10 μL)를 제조한 후, 본 하이포잔틴 검출 기술 반응을 수행한 결과, 본 기술의 표적 하이포잔틴 검출 한계(limit of detection, LOD)는 10.81nM인 것을 확인하였다 (도 3).A sensitivity verification experiment was conducted by varying the concentration of hypoxanthine contained in the analysis sample using the reaction conditions mentioned in Example 1 and measuring the absorbance after the reaction. Results of performing this hypoxanthine detection technology reaction after preparing an analysis sample (10 μL) containing hypoxanthine at various concentrations (0, 1, 2, 5, 7, 10, 20, 50, 70, 100 μM) , it was confirmed that the target hypoxanthine detection limit (LOD) of this technology was 10.81nM (Figure 3).
또한, 하이포잔틴을 포함하는 분석시료 10μL를 대신하여, ATP, GTP, CTP, TTP 및 UTP 중 1종을 포함하는 분석시료(10μL)를 이용하여 본 하이포잔틴 검출 기술 반응을 수행한 결과, 오직 하이포잔틴을 포함하는 분석시료에서만 발색 기질의 흡수파장인 420nm에서 증가한 흡광도 변화를 확인하였다 (도 4).In addition, as a result of performing this hypoxanthine detection technology reaction using an analysis sample (10 μL) containing one of ATP, GTP, CTP, TTP, and UTP instead of 10 μL of an analysis sample containing hypoxanthine, only hypoxanthine was detected. An increased absorbance change was confirmed at 420 nm, which is the absorption wavelength of the chromogenic substrate, only in the analysis sample containing xanthine (Figure 4).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it will be clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention thereby. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (13)
(a) 사체 유래의 유리체액을 포함하는 시료를 잔틴 산화효소, 과산화효소 또는 그 유사체 및 발색기질을 함유하는 하이포잔틴 검출용 조성물과 반응시키는 단계; 및
(b) 상기 시료가 첨가된 조성물의 색변화를 흡광도를 이용하여 측정하여, 시료 내의 하이포잔틴을 검출 또는 정량하여, 시신의 사후 경과시간을 측정하는 단계;
여기서, 상기 과산화효소 또는 그 유사체는 과산화효소, 과산화효소 활성을 가지는 자성 나노입자 및 과산화효소 활성을 가지는 산화 그래핀으로 이루어진 군에서 선택되는 것을 특징으로 함.
How to measure the time elapsed after death of a body, including the following steps:
(a) reacting a sample containing vitreous humor derived from a cadaver with a composition for detecting hypoxanthine containing xanthine oxidase, peroxidase or an analog thereof, and a chromogenic substrate; and
(b) measuring the color change of the composition to which the sample is added using absorbance, detecting or quantifying hypoxanthine in the sample, and measuring the time elapsed after death of the body;
Here, the peroxidase or its analogue is characterized in that it is selected from the group consisting of peroxidase, magnetic nanoparticles with peroxidase activity, and graphene oxide with peroxidase activity.
The method of claim 7, wherein the coloring substrate is ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), TMB (3,3',5,5'-tetramethylbenzidine), OPD ( o - A method of measuring the postmortem time of a corpse, characterized in that it is selected from the group consisting of phenylenediamine dihydrochloride), DAB (3,3'-diaminobenzidine), and ADHP (10-acetyl-3,7-dihydroxyphenoxazine).
여기서, 상기 과산화효소 또는 그 유사체는 과산화효소, 과산화효소 활성을 가지는 자성 나노입자 및 과산화효소 활성을 가지는 산화 그래핀으로 이루어진 군에서 선택되는 것을 특징으로 함.
A composition for measuring the time elapsed after death of a corpse by measuring hypoxanthine in the vitreous humor of a corpse, comprising xanthine oxidase, peroxidase or an analogue thereof, and a chromogenic substrate as active ingredients;
Here, the peroxidase or its analogue is characterized in that it is selected from the group consisting of peroxidase, magnetic nanoparticles with peroxidase activity, and graphene oxide with peroxidase activity.
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| Shuisheng Hu et al., 'A sensing platform for hypoxanthine detection based onamino-functionalized metal organic framework nanosheet withperoxidase mimic and fluorescence properties', Sensors and Actuators B 267 (2018) 312-319 1부.* |
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