KR102248839B1 - Dna aptamer specifically binding to lithium and using the same - Google Patents
Dna aptamer specifically binding to lithium and using the same Download PDFInfo
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- KR102248839B1 KR102248839B1 KR1020190162395A KR20190162395A KR102248839B1 KR 102248839 B1 KR102248839 B1 KR 102248839B1 KR 1020190162395 A KR1020190162395 A KR 1020190162395A KR 20190162395 A KR20190162395 A KR 20190162395A KR 102248839 B1 KR102248839 B1 KR 102248839B1
- Authority
- KR
- South Korea
- Prior art keywords
- lithium
- dna
- dna aptamer
- aptamer
- present
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Abstract
Description
본 발명은 리튬에 특이적으로 결합하는 DNA 앱타머 및 이의 용도에 관한 것이다.The present invention relates to a DNA aptamer that specifically binds to lithium and uses thereof.
리튬은 주기율표 1족 2주기에 속하는 알칼리 금속 원소로 은백색의 연질금속이지만 나트륨보다 단단하며 고체인 홑원소 물질 중에서 가장 가볍다. Lithium is an alkali metal element belonging to
리튬은 높은 반응성을 보여 자연계에서 원소 형태로 존재하는 경우가 거의 없으며, 잠재적인 리튬의 공급원으로는 점토(clay)와 해수(seawater)가 있다. 점토로부터 리튬을 회수하기 위해서는 석회석-석고 배소/선택 염소화 공정, 석회석-석고 배소-수용액 침출 공정과 같이 복잡한 공정을 거쳐야하고, 해수로부터 리튬을 회수하기 위해서는 이온교환수지, 용매추출, 공침, 막, 흡착기술 등이 이용된다. 그러나, 이들 공정은 상업화하기에 생산 비용이 너무 높다는 단점이 있다.Lithium is highly reactive and rarely exists in the form of an element in nature, and potential sources of lithium include clay and seawater. In order to recover lithium from clay, complicated processes such as limestone-gypsum roasting/selective chlorination process, limestone-gypsum roasting-aqueous solution leaching process are required.In order to recover lithium from seawater, ion exchange resin, solvent extraction, coprecipitation, membrane, Adsorption technology or the like is used. However, these processes have the disadvantage that the production cost is too high for commercialization.
금속 중에서 가장 가벼운 리튬은 노트북 컴퓨터와 같은 휴대용 장치나 전기 자동차에서 가장 중요한 전지에 필수적으로 사용되는 재료이다. 리튬의 에너지 밀도는 종래의 전지 재료들에 비해 50%보다 큰 160 Wh/㎏ 이상을 가지고 있어, 리튬이온전지의 지속적인 개발이 요구되고 있다.Lithium, the lightest among metals, is an essential material used in portable devices such as notebook computers and batteries, which are the most important in electric vehicles. Since lithium has an energy density of 160 Wh/kg or more, which is 50% higher than that of conventional battery materials, continuous development of lithium ion batteries is required.
최근 2차 전지를 중심으로 리튬의 수요가 연간 두자릿수의 성장을 보여왔으며, 특히 전기차 및 신재생 에너지 저장 장치에 사용되는 중대형 리튬이온 배터리 수요는 연간 20~30% 안팎으로 가파르게 성장하면서 리튬의 가격 또한 2015년 말부터 급격히 상승하였다. 그러나, 관련 산업의 발달로 리튬의 수요는 이후에도 지속적으로 증가할 것으로 예상되며, 가격 또한 지속적으로 상승할 것으로 예측된다.Recently, the demand for lithium, centered on rechargeable batteries, has shown a double-digit annual growth. In particular, the demand for mid- to large-sized lithium-ion batteries used in electric vehicles and renewable energy storage devices has grown sharply around 20-30% per year, and the price of lithium. It also increased sharply from the end of 2015. However, with the development of related industries, the demand for lithium is expected to continue to increase in the future, and the price is also predicted to continue to increase.
또한, 리튬이차전지의 사용이 증가함에 따라, 이의 재활용을 위해 리튬을 회수하는 기술도 지속적으로 연구되고 있다. 이와 관련하여, 국내에서 탄산리튬을 수세 및 농축한 뒤, 순도 99% 이상의 탄산리튬 분말로 회수하는 공정을 개발하였으나, 아직 상용화까지 이르지 못하였다. 또한, 대한민국 특허등록 제10-2020238호는 리튬 이차 전지의 폐 양극으로부터 획득된 폐 양극 활물질 혼합물을 유동층 반응기 내에서 반응시켜 예비 전구체 혼합물을 형성한 뒤, 이로부터 선택적으로 리튬 전구체를 회수하는 방법을 개시하고 있다.In addition, as the use of lithium secondary batteries increases, a technology for recovering lithium for its recycling is continuously being studied. In this regard, a process for recovering lithium carbonate as a lithium carbonate powder having a purity of 99% or higher after washing and concentrating lithium carbonate in Korea has been developed, but has not yet reached commercialization. In addition, Korean Patent Registration No. 10-2020238 discloses a method of reacting a waste positive electrode active material mixture obtained from a waste positive electrode of a lithium secondary battery in a fluidized bed reactor to form a pre-precursor mixture, and then selectively recovering a lithium precursor therefrom. It is starting.
이와 같이, 리튬의 재활용을 위해서는 공정의 원가를 낮춰 시장경쟁력을 높여야 하는데, 여러가지 공정 중 농축공정의 생산원가를 낮추기 위한 공정개발이 필요하다. 그러나, 최근 급증한 리튬의 수요로 인해 해수나 육상광물로부터 리튬을 생산하는 단가가 증가하고 있어, 더욱 경제적으로 리튬을 회수하기 위한 방법의 개발이 필요하다.As described above, in order to recycle lithium, it is necessary to increase market competitiveness by lowering the cost of the process. Among various processes, process development is required to lower the production cost of the concentration process. However, due to the recent rapid increase in demand for lithium, the cost of producing lithium from seawater or terrestrial minerals is increasing, and thus a method for more economically recovering lithium is required.
본 발명의 목적은 리튬에 특이적으로 결합하는 DNA 앱타머를 제공하는 것이다.An object of the present invention is to provide a DNA aptamer that specifically binds to lithium.
본 발명의 다른 목적은 상기 DNA 앱타머를 이용한 리튬의 검출 또는 제거용 조성물, 키트, 마이크로어레이 및 센서를 제공하는 것이다.Another object of the present invention is to provide a composition, kit, microarray, and sensor for detecting or removing lithium using the DNA aptamer.
본 발명의 또 다른 목적은 상기 DNA 앱타머를 이용하여 리튬을 검출 또는 제거하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for detecting or removing lithium using the DNA aptamer.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택되는 어느 하나의 염기서열로 구성되는 리튬에 특이적으로 결합하는 DNA 앱타머를 제공한다.In order to achieve the above object, the present invention provides a DNA aptamer that specifically binds to lithium consisting of any one nucleotide sequence selected from the group consisting of the nucleotide sequences described in SEQ ID NOs: 1 to 16.
또한, 본 발명은 상기 DNA 앱타머를 유효성분으로 함유하는 리튬 검출, 제거 또는 회수용 조성물을 제공한다.In addition, the present invention provides a composition for detecting, removing or recovering lithium containing the DNA aptamer as an active ingredient.
또한, 본 발명은 상기 DNA 앱타머를 유효성분으로 함유하는 리튬 검출, 제거 또는 회수용 키트를 제공한다.In addition, the present invention provides a kit for detecting, removing or recovering lithium containing the DNA aptamer as an active ingredient.
또한, 본 발명은 상기 DNA 앱타머가 고정된 기판을 갖는 리튬 검출용 마이크로어레이를 제공한다.In addition, the present invention provides a lithium detection microarray having a substrate to which the DNA aptamer is fixed.
또한, 본 발명은 상기 DNA 앱타머가 고정된 기판을 갖는 리튬 검출용 센서를 제공한다.In addition, the present invention provides a sensor for detecting lithium having a substrate on which the DNA aptamer is fixed.
또한, 본 발명은 상기 조성물을 시료와 반응시키는 단계를 포함하는 리튬의 검출방법을 제공한다.In addition, the present invention provides a method for detecting lithium comprising reacting the composition with a sample.
또한, 본 발명은 상기 조성물을 시료와 반응시켜 형성된 DNA 앱타머와 리튬의 복합체를 제거하는 단계를 포함하는 리튬의 제거방법을 제공한다.In addition, the present invention provides a method for removing lithium comprising the step of removing the complex of lithium aptamer and DNA formed by reacting the composition with a sample.
나아가, 본 발명은 상기 조성물을 시료와 반응시켜 형성된 DNA 앱타머와 리튬의 복합체를 회수하는 단계를 포함하는 리튬의 회수방법을 제공한다.Furthermore, the present invention provides a method for recovering lithium including recovering a complex of DNA aptamer and lithium formed by reacting the composition with a sample.
본 발명에 따른 DNA 앱타머는 리튬에 강하게 특이적으로 결합함으로써, 리튬을 검출, 제거 또는 회수하는데 유용하게 사용될 수 있다.The DNA aptamer according to the present invention can be usefully used to detect, remove, or recover lithium by strongly and specifically binding to lithium.
도 1은 본 발명의 일 실시예에서 랜덤 DNA 라이브러리를 증폭한 PCR 산물(lane 1), 단일가닥 DNA를 증폭한 PCR 산물(lane 2) 및 회수된 ssDNA(lane 3)를 아가로스 겔 전기영동을 통해 확인한 결과 도면이다.
도 2는 본 발명의 일 실시예에서 수행된 SELEX의 과정을 나타낸 모식도이다.
도 3은 본 발명의 일 실시예에서 SELEX 수행 횟수에 따라 수득된 RNA 앱타머와 리튬의 결합력을 나노드랍 방법으로 확인한 결과 그래프이다.
도 4는 본 발명의 일 실시예에서 SELEX 수행 횟수에 따라 수득된 RNA 앱타머와 리튬의 결합력을 실시간 PCR 방법으로 확인한 결과 그래프이다.
도 5는 본 발명의 일 실시예에서 리튬과 높은 결합력을 보이는 것으로 확인된 3개 DNA 앱타머의 2차 구조를 확인한 결과 도면이다.
도 6은 본 발명의 일 실시예에서 리튬과 높은 결합력을 보이는 것으로 확인된 3개 DNA 앱타머의 리튬과의 결합을 주사전자현미경으로 관찰한 결과 사진이다(A: DNA 앱타머가 결합되지 않은 스트렙트아비딘 비드, B: DNA 앱타머가 결합된 스트렙트아비딘 비드, C: DNA 앱타머가 결합되지 않은 스트렙트아비딘 비드+리튬, D: Li-4 DNA 앱타머가 결합된 스트렙트아비딘 비드+리튬, E: Li-8 DNA 앱타머가 결합된 스트렙트아비딘 비드+리튬, F: Li-14 DNA 앱타머가 결합된 스트렙트아비딘 비드+리튬).1 shows a PCR product amplifying a random DNA library (lane 1), a PCR product amplifying a single-stranded DNA (lane 2), and a recovered ssDNA (lane 3) in an embodiment of the present invention by agarose gel electrophoresis. This is the result of checking through
2 is a schematic diagram showing the process of SELEX performed in an embodiment of the present invention.
3 is a graph showing the results of confirming the binding strength of RNA aptamer and lithium obtained according to the number of times SELEX is performed in an embodiment of the present invention by a nanodrop method.
4 is a graph showing the results of confirming the binding strength of RNA aptamer and lithium obtained according to the number of times SELEX is performed in an embodiment of the present invention by a real-time PCR method.
5 is a diagram showing the results of confirming the secondary structures of three DNA aptamers confirmed to exhibit high binding power with lithium in an embodiment of the present invention.
Figure 6 is a photograph of the result of observing the binding of the three DNA aptamers with lithium with a scanning electron microscope (A: Strept without DNA aptamer binding) in an embodiment of the present invention. Avidin beads, B: streptavidin beads with DNA aptamer bound, C: streptavidin beads without DNA aptamer bound + lithium, D: Li-4 streptavidin beads with DNA aptamer bound + lithium, E: Li -8 Streptavidin beads bound to DNA aptamer + lithium, F: Streptavidin beads bound to Li-14 DNA aptamer + lithium).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택되는 어느 하나의 염기서열로 구성되는 리튬에 특이적으로 결합하는 DNA 앱타머를 제공한다.The present invention provides a DNA aptamer that specifically binds to lithium consisting of any one nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 1 to 16.
본 명세서에서 사용된 용어, "리튬(lithium, Li)"은 원자번호 3번인 은색의 고체 금속 원소로서, 밀도가 가장 낮고 물과 반응성이 높아 자연에서 원소상태로 존재하기 어려우며 주로 인반석(LiAlPO4F)와 같이 여러 광물과 함께있는 상태로 존재한다. 상기 리튬의 녹는점은 180.5℃이고, 끓는점은 1,330℃이며, 25℃에서 밀도는 0.534 g/㎤이고, 리튬 이온 전지뿐만 아니라, 유리 및 도자기 산업이나 질병 치료제로서도 사용되고 있다.As used herein, the term "lithium (Li)" is a silver solid metal element with an atomic number of 3, and has the lowest density and high reactivity with water, making it difficult to exist in an elemental state in nature and is mainly made of phosphorus (LiAlPO4F). It exists in a state of being with several minerals. The melting point of lithium is 180.5° C., boiling point is 1,330° C., and density at 25° C. is 0.534 g/
본 명세서에서 사용된 용어, "앱타머(aptamer)"는 특정 물질에 고친화성 및 특이성으로 결합할 수 있는 DNA 핵산분자를 의미한다. 상기 앱타머는 통상의 기술분야에 잘 알려진 방법으로 제조될 수 있고, 상기 방법은 필요에 따라 적절히 변형될 수 있다.As used herein, the term "aptamer" refers to a DNA nucleic acid molecule capable of binding to a specific substance with high affinity and specificity. The aptamer may be prepared by a method well known in the art, and the method may be appropriately modified as necessary.
본 발명에 따른 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택되는 어느 하나의 염기서열로 구성될 수 있다. 상기 DNA 앱타머는 리튬에 특이적으로 결합하는 특성을 유지하는 한, 상기 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택되는 염기서열과 80% 이상, 90% 이상, 95% 이상, 97% 이상 또는 99% 이상 상동성을 가질 수 있다.The DNA aptamer according to the present invention may be composed of any one nucleotide sequence selected from the group consisting of the nucleotide sequences described in SEQ ID NOs: 1 to 16. The DNA aptamer is 80% or more, 90% or more, 95% or more, 97% with a base sequence selected from the group consisting of the base sequences described in SEQ ID NOs: 1 to 16, as long as it maintains the property of specifically binding to lithium. Or more or 99% or more homology.
상기 DNA 앱타머는 표적 물질에 대한 결합성 및 안정성을 높이기 위해, 각 뉴클레오티드의 당 잔기, 구체적으로 리보오스 또는 디옥시리보스가 수식될 수 있다. 상기 수식은 당 잔기의 2'위치, 3'위치 및 4'위치로 구성된 군으로부터 선택되는 어느 하나 이상의 산소 원자를 다른 원자로 치환한 것일 수 있다. 수식의 예로는 플루오로화, O-알킬화, O-알릴화, S-알킬화, S-알릴화, 아미노화 등이 포함될 수 있다. 상기와 같은 당 잔기의 변형은 통상적인 방법으로 수행될 수 있다.The DNA aptamer may be modified with a sugar residue of each nucleotide, specifically ribose or deoxyribose, in order to increase binding and stability to a target substance. The above formula may be obtained by substituting another atom for at least one oxygen atom selected from the group consisting of the 2'position, 3'position, and 4'position of the sugar moiety. Examples of the formula may include fluorination, O-alkylation, O-allylation, S-alkylation, S-allylation, amination, and the like. Modification of the sugar moiety as described above can be carried out in a conventional manner.
또한, 상기 DNA 앱타머는 표적 물질에 대한 결합성을 높이기 위해, 핵산염기가 변형될 수 있다. 상기 핵산염기의 변형은 5위치의 피리미딘 변형, 6위치의 푸린 변형, 8위치의 푸린 변형, 환외(環外) 아민에서의 변형, 4-티오우리딘으로의 치환, 5-브로모로의 치환 또는 5-요오드-우리실로의 치환 등을 포함할 수 있다.In addition, the DNA aptamer may have a nucleotide base modified in order to increase the binding property to the target substance. The modification of the nucleotide base is a pyrimidine modification at
또한, 상기 DNA 앱타머는 뉴클레아제 및 가수분해효소 등에 대해 내성을 갖도록 인산기가 변형될 수 있다. 예를 들면, 상기 인산기는 티오에이트, 디티오에이트, 아미데이트, 포름아세탈, 3'-아민 등으로 치환될 수 있다. 나아가, 상기 DNA 앱타머는 3' 말단 또는 5' 말단의 변형을 포함할 수 있고, 상기 변형은 캡핑이나, 바이오티닐, 폴리에틸글리콜, 아미노산, 펩티드, 핵산, 뉴클레오시드, 미리스토일(myristoyl), 리소콜릭-올레일(lithocolic-oleyl), 도코사닐(docosanyl), 라우로일(lauroyl), 스테아로일(stearoyl), 팔미토일(palmitoyl), 올레오일(oleoyl), 리놀레오일(linoleoyl), 지질, 스테로이드, 콜레스테롤, 카페인, 비타민, 색소, 형광물질, 항암제, 독소, 효소, 방사성 물질, 비오틴 등이 부가된 것일 수 있다.In addition, the DNA aptamer may have a phosphate group modified to have resistance to nucleases and hydrolytic enzymes. For example, the phosphoric acid group may be substituted with thioate, dithioate, amidate, formacetal, 3'-amine, or the like. Furthermore, the DNA aptamer may include a modification of the 3'end or 5'end, and the modification is capping, but biotinyl, polyethyl glycol, amino acid, peptide, nucleic acid, nucleoside, myristoyl , Lisocolic-oleyl, docosanyl, lauroyl, stearoyl, palmitoyl, oleoyl, linoleoyl , Lipids, steroids, cholesterol, caffeine, vitamins, pigments, fluorescent substances, anticancer agents, toxins, enzymes, radioactive substances, biotin, etc. may be added.
또한, 본 발명은 상기 DNA 앱타머를 유효성분으로 함유하는 리튬 검출 또는 제거용 조성물 또는 키트를 제공한다.In addition, the present invention provides a composition or kit for detecting or removing lithium containing the DNA aptamer as an active ingredient.
본 발명에 따른 리튬 검출 또는 제거용 조성물 또는 키트에 포함되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다.The DNA aptamer included in the composition or kit for detecting or removing lithium according to the present invention may have the characteristics as described above. For example, the DNA aptamer may be selected from the group consisting of the nucleotide sequences described in SEQ ID NOs: 1 to 16.
본 발명에 따른 리튬의 검출 또는 제거용 조성물에 포함되는 DNA 앱타머는 발색효소, 형광물질, 방사성 동위원소 또는 콜로이드 등의 검출체로 표지된 접합체일 수 있다. 발색효소는 퍼록시다제, 알칼라인 포스파타제 또는 산성 포스파타제일 수 있고, 형광물질은 티오우레아(FTH), 7-아세톡시쿠마린-3-일, 플루오레신-5-일, 플루오레신-6-일, 2',7'-디클로로플루오레신-5-일, 2',7'-디클로로플루오레신-6-일, 디하이드로 테트라메틸로사민-4-일, 테트라메틸로다민-5-일, 테트라메틸로다민-6-일, 4,4-디플루오로-5,7-디메틸-4-보라-3a,4a-디아자-s-인다센-3-에틸 또는 4,4-디플루오로-5,7-디페닐-4-보라-3a,4a-디아자-s-인다센-3-에틸, Cy3, Cy5, 폴리 L-라이신-플루오레세인 이소티오시아네이트(FITC), 로다민-B-이소티오시아네이트(RITC), PE(Phycoerythrin) 또는 로다민일 수 있다.The DNA aptamer contained in the composition for detecting or removing lithium according to the present invention may be a conjugate labeled with a detection agent such as a color developing enzyme, a fluorescent substance, a radioactive isotope, or a colloid. The coloring enzyme may be peroxidase, alkaline phosphatase or acidic phosphatase, and the fluorescent substance is thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl , 2',7'-dichlorofluorescein-5-yl, 2',7'-dichlorofluorescein-6-yl, dihydro tetramethylrosamin-4-yl, tetramethylrhodamine-5-yl , Tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl or 4,4-difluoro Rho-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl, Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), Rhoda Min-B-isothiocyanate (RITC), PE (Phycoerythrin), or rhodamine.
또한, 상기 DNA 앱타머는 상기 DNA 앱타머에 특이적으로 결합할 수 있는 리간드를 포함할 수 있다. 상기 리간드는 발색효소, 형광물질, 방사성 동위원소 또는 콜로이드 등의 검출체로 표지된 접합체, 및 스트렙타비딘 또는 아비딘이 처리된 리간드일 수 있다. 본 발명의 검출 또는 제거용 조성물은 상기 서술한 바와 같은 시약 외에도 이들의 구조를 안정하게 유지시키는 증류수 또는 완충액을 더 포함할 수 있다.In addition, the DNA aptamer may include a ligand capable of specifically binding to the DNA aptamer. The ligand may be a conjugate labeled with a detection agent such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope or a colloid, and a ligand treated with streptavidin or avidin. In addition to the reagents as described above, the composition for detection or removal of the present invention may further contain distilled water or a buffer solution to stably maintain their structure.
본 발명에 따른 검출 또는 제거용 조성물을 포함하는 키트는 이에 포함되는 DNA 앱타머의 세척이나 복합체의 분리 등과 같은 이후 단계를 용이하게 하기 위해 고형 기질에 결합될 수 있다. 이때, 고형 기질로는 합성수지, 니트로셀룰로오스, 유리기판, 금속기판, 유리섬유, 미세구체 또는 미세비드가 사용될 수 있다. 또한, 합성수지로는 폴리에스터, 폴리염화비닐, 폴리스티렌, 폴리프로필렌, PVDF 또는 나일론 등이 사용될 수 있다.The kit comprising the composition for detection or removal according to the present invention may be bound to a solid substrate to facilitate subsequent steps such as washing of the DNA aptamer contained therein or separation of the complex. In this case, as the solid substrate, synthetic resin, nitrocellulose, glass substrate, metal substrate, glass fiber, microspheres or fine beads may be used. In addition, polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF or nylon may be used as the synthetic resin.
또한, 상기 키트는 당업자에게 알려진 종래의 제조방법에 의해 제조될 수 있으며, 버퍼, 안정화제, 불활성 단백질 등을 더 포함할 수 있다. 상기 키트는 시약의 양을 탐색하기 위해 검출체로서 부착된 형광물질의 형광을 검출함으로써 수행되는 형광법 또는 검출체로서 부착된 방사선 동위원소의 방사선을 검출함으로써 수행되는 방사선법을 통한 초고속 스크리닝(high throughput screening, HTS) 시스템, 검출체의 표지 없이 표면의 플라즈몬 공명 변화를 실시간으로 측정하는 SPR(surface plasmon resonance) 방법 또는 SPR 시스템을 영상화하여 확인하는 SPRI(surface plasmon resonance imaging) 방법을 이용할 수 있다.In addition, the kit may be prepared by a conventional manufacturing method known to those skilled in the art, and may further include a buffer, a stabilizer, an inactive protein, and the like. The kit includes high-speed screening through a fluorescence method performed by detecting the fluorescence of a fluorescent substance attached as a detection agent in order to detect the amount of reagent, or a radiographic method performed by detecting the radiation of a radioactive isotope attached as a detection agent. A screening, HTS) system, a surface plasmon resonance (SPR) method that measures changes in plasmon resonance of a surface in real time without labeling a detection object, or a surface plasmon resonance imaging (SPRI) method that confirms by imaging an SPR system can be used.
또한, 본 발명은 상기 DNA 앱타머가 고정된 기판을 갖는 리튬 검출용 마이크로어레이 또는 센서를 제공한다.In addition, the present invention provides a lithium detection microarray or sensor having a substrate on which the DNA aptamer is fixed.
상기 리튬 검출용 마이크로어레이 또는 센서에 포함되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다.The DNA aptamer included in the lithium detection microarray or sensor may have the above-described characteristics. For example, the DNA aptamer may be selected from the group consisting of the nucleotide sequences described in SEQ ID NOs: 1 to 16.
본 명세서에 사용된 용어, "마이크로어레이(microarray)"는 기판 상에 올리고뉴클레오티드 그룹이 높은 밀도로, 각각 일정한 영역에 고정화된 것으로, 유전자 칩(gene chip)과 동일한 의미로 사용된다. 상기 마이크로어레이는 유전자의 발현을 정성적 및 정량적으로 빠르게 분석할 수 있다는 점에서 다양한 분야에서 사용될 수 있다. 상기 마이크로어레이는 통상적인 방법으로 제조될 수 있고, 필요에 따라 적절히 변형되어 제조될 수 있다.As used herein, the term "microarray" refers to an oligonucleotide group immobilized on a substrate at a high density and in a certain region, and is used in the same meaning as a gene chip. The microarray can be used in various fields in that it can rapidly analyze gene expression qualitatively and quantitatively. The microarray may be manufactured by a conventional method, and may be manufactured by appropriately deforming as necessary.
상기 기판은 결합 특성을 보유하고, 결합의 배경 수준이 낮게 유지되는 조건하에 DNA 앱타머가 부착될 수 있는 임의의 기판일 수 있다. 일례로, 상기 기판은 미세역가(microtiter) 플레이트, 나일론 또는 니트로셀룰로오스 막, 비드, 슬라이드, 필터, 겔, 튜빙, 고분자 또는 칩을 포함할 수 있다. 본 발명에 따른 DNA 앱타머는 기판에 적용 또는 고정하기 전에 고정화를 촉진시키거나 결합효율을 개선시키기 위해 변형될 수 있다. 상기 변형은 단독중합체 테일링(homopolymer tailing), 지방족기, NH2기, SH기 및 카르복실기와 같은 상이한 반응성 작용기와의 커플링, 또는 바이오틴, 합텐 또는 단백질과의 커플링을 포함할 수 있다.The substrate may be any substrate to which a DNA aptamer can be attached under conditions that retain binding properties and maintain a low background level of binding. For example, the substrate may include a microtiter plate, nylon or nitrocellulose membrane, beads, slides, filters, gels, tubing, polymers, or chips. The DNA aptamer according to the present invention may be modified to promote immobilization or improve binding efficiency prior to application or immobilization on a substrate. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH 2 groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.
상기 고정은 화학적 결합방법 또는 UV와 같은 공유결합적 방법에 의해 수행될 수 있다. 예를 들어, 본 발명에 따른 DNA 앱타머는 에폭시 화합물 또는 알데히드기를 포함하도록 변형된 유리표면에 결합될 수 있고, 폴리라이신 코팅 표면에서 UV에 의해 결합될 수 있다. 또한, 상기 DNA 앱타머는 에틸렌글리콜 올리고머나 디아민과 같은 링커를 통해 기판에 고정될 수도 있다.The fixation may be performed by a chemical bonding method or a covalent bonding method such as UV. For example, the DNA aptamer according to the present invention may be bound to a glass surface modified to contain an epoxy compound or an aldehyde group, and may be bound by UV on a polylysine-coated surface. In addition, the DNA aptamer may be fixed to the substrate through a linker such as ethylene glycol oligomer or diamine.
또한, 본 발명은 상기 조성물을 시료와 반응시키는 단계를 포함하는 리튬의 검출방법을 제공한다.In addition, the present invention provides a method for detecting lithium comprising reacting the composition with a sample.
본 발명에 따른 리튬의 검출방법에 사용되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다.The DNA aptamer used in the method for detecting lithium according to the present invention may have the above-described characteristics. For example, the DNA aptamer may be selected from the group consisting of the nucleotide sequences described in SEQ ID NOs: 1 to 16.
상기 시료는 리튬을 검출하고자 하는 시료라면 모든 시료를 포함할 수 있다. 또한, 상기 시료는 음이온 교환 크로마토그래피, 친화도 크로마토그래피, 크기별 배제 크로마토그래피, 액체 크로마토그래피, 연속추출, 원심분리 또는 젤 전기영동 등의 방법을 이용하여 전처리될 수 있다.The sample may include all samples as long as it is a sample for detecting lithium. In addition, the sample may be pretreated using methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous extraction, centrifugation, or gel electrophoresis.
나아가, 본 발명은 상기 조성물을 시료와 반응시켜 형성된 DNA 앱타머와 리튬의 복합체를 제거 또는 회수하는 단계를 포함하는 리튬의 제거 또는 회수방법을 제공한다.Further, the present invention provides a method for removing or recovering lithium comprising the step of removing or recovering a complex of DNA aptamer and lithium formed by reacting the composition with a sample.
본 발명에 따른 리튬의 제거 또는 회수방법에 사용되는 DNA 앱타머는 상술한 바와 같은 특징을 가질 수 있다. 일례로, 상기 DNA 앱타머는 서열번호 1 내지 16으로 기재된 염기서열로 구성된 군으로부터 선택될 수 있다.The DNA aptamer used in the lithium removal or recovery method according to the present invention may have the above-described characteristics. For example, the DNA aptamer may be selected from the group consisting of the nucleotide sequences described in SEQ ID NOs: 1 to 16.
상기 시료는 리튬을 제거 도는 회수하고자 하는 시료라면 모든 시료를 포함할 수 있다. 또한, 상기 시료는 음이온 교환 크로마토그래피, 친화도 크로마토그래피, 크기별 배제 크로마토그래피, 액체 크로마토그래피, 연속추출, 원심분리 또는 젤 전기영동 등의 방법을 이용하여 전처리될 수 있다.The sample may include any sample as long as it is a sample to be recovered or removed from lithium. In addition, the sample may be pretreated using methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous extraction, centrifugation, or gel electrophoresis.
이하, 본 발명을 하기 실시예에 의해 상세히 설명한다, 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 이들에 의해 본 발명이 제한되는 것은 아니다. 본 발명의 청구범위에 기재된 기술적 사상과 실질적으로 동일한 구성을 갖고 동일한 작용 효과를 이루는 것은 어떠한 것이라도 본 발명의 기술적 범위에 포함된다.Hereinafter, the present invention will be described in detail by the following examples, provided that the following examples are for illustrative purposes only, and the present invention is not limited thereto. Anything that has substantially the same configuration as the technical idea described in the claims of the present invention and achieves the same operation and effects is included in the technical scope of the present invention.
실시예 1. 랜덤 DNA 라이브러리의 증폭Example 1. Amplification of random DNA library
리튬(lithium)에 특이적으로 결합하는 DNA 앱타머를 선별하기 위해, 먼저 하기에 기재된 바와 같이 DNA 라이브러리를 제조하였다.In order to select a DNA aptamer that specifically binds to lithium, a DNA library was first prepared as described below.
구체적으로, dA:dG:dC:dT가 1.5:1.15:1.25:1의 비율로 포함된 40개의 랜덤 염기서열(N40)을 포함하는 76 bp 크기의 주형 DNA와 이에 대한 정방향 프라이머(서열번호 17: 5'-ATACCAGCTTATTCAATT-3') 및 5'-말단이 비오티닐화된(biotinylated) 역방향 프라이머(서열번호 18: 5'-AGATTGCACTTACTATCT-3')를 바이오니아 사(한국)에 주문제작하였다. 1 ㎕의 주형 DNA, 5 ㎕의 10x PCR 완충용액, 4 ㎕의 2.5 mM dNTP 혼합물, 2 ㎕의 25 μM 정방향 프라이머, 2 ㎕의 25 μM 비오티닐화된 역방향 프라이머, 0.25 ㎕의 ExTaq 중합효소(Takara, 일본)(1 unit/㎕) 및 35.75 ㎕의 증류수를 혼합하여 PCR 반응물을 준비하였다. 준비된 반응물을 하기 표 1에 기재된 바와 같은 조건으로 PCR을 수행하였다. 수득된 PCR 산물을 2% 아가로스 겔 전기영동을 통해 확인하고, 그 결과를 도 1에 나타내었다. Specifically, dA:dG:dC:dT is a template DNA of 76 bp size containing 40 random nucleotide sequences (N 40) contained in a ratio of 1.5:1.15:1.25:1 and a forward primer therefor (SEQ ID NO: 17 : 5'-ATACCAGCTTATTCAATT-3') and 5'-end biotinylated reverse primers (SEQ ID NO: 18: 5'-AGATTGCACTTACTATCT-3') were custom-made by Bioneer (Korea). 1 μl of template DNA, 5 μl of 10x PCR buffer, 4 μl of 2.5 mM dNTP mixture, 2 μl of 25 μM forward primer, 2 μl of 25 μM biotinylated reverse primer, 0.25 μl of ExTaq polymerase (Takara , Japan) (1 unit/µl) and 35.75 µl of distilled water were mixed to prepare a PCR reaction product. The prepared reaction product was subjected to PCR under the conditions as described in Table 1 below. The obtained PCR product was confirmed through 2% agarose gel electrophoresis, and the results are shown in FIG. 1.
도 1에 나타난 바와 같이, 76 bp 크기의 PCR 산물을 확인하였고, 이를 PCR 정제키트(Qiagen, 미국)를 사용하여 정제하였다.As shown in FIG. 1, a PCR product having a size of 76 bp was identified, and it was purified using a PCR purification kit (Qiagen, USA).
실시예 2. 단일가닥 DNA의 증폭Example 2. Amplification of single-stranded DNA
상기 수득된 76 bp 크기의 PCR 산물로부터 DNA 앱타머를 제조하기 위해, 단일가닥 DNA(single-strand DNA, ssDNA)를 비대칭(asymmetric) PCR 방법으로 증폭시켰다.To prepare a DNA aptamer from the obtained 76 bp PCR product, single-strand DNA (ssDNA) was amplified by an asymmetric PCR method.
먼저, 1 ㎕의 실시예 1에서 수득된 PCR 산물, 10 ㎕의 10× PCR 완충용액, 8 ㎕의 dNTP 혼합물, 10 ㎕의 25 μM 정방향 프라이머(서열번호 17), 2 ㎕의 25 μM 비오티닐화된 역방향 프라이머(서열번호 18), 0.5 ㎕의 ExTaq 중합효소(Takara, 일본)(1 unit/㎕) 및 68.5 ㎕의 증류수를 혼합하여 PCR 반응물을 준비하였다. 준비된 반응물을 하기 표 2에 기재된 바와 같은 조건으로 PCR을 수행하였다. 수득된 PCR 산물을 2% 아가로스 겔 전기영동을 통해 확인하고, 그 결과를 도 1에 나타내었다.First, 1 μl of the PCR product obtained in Example 1, 10 μl of 10× PCR buffer, 8 μl of dNTP mixture, 10 μl of 25 μM forward primer (SEQ ID NO: 17), 2 μl of 25 μM biotinylation A PCR reaction product was prepared by mixing the reverse primer (SEQ ID NO: 18), 0.5 µl of ExTaq polymerase (Takara, Japan) (1 unit/µl) and 68.5 µl of distilled water. The prepared reaction was subjected to PCR under the conditions as described in Table 2 below. The obtained PCR product was confirmed through 2% agarose gel electrophoresis, and the results are shown in FIG. 1.
확인된 PCR 산물에 이의 1/100 부피의 tRNA(Sigma aldrich, 미국) 및 3 부피의 100% 에탄올을 첨가하고 -70℃에서 1시간 이상 반응시켰다. 반응 후, 이를 4℃, 13,000 rpm의 조건하에서 15분 동안 원심분리하고 펠렛을 수득하였다. 수득된 펠렛을 65℃에서 건조시킨 뒤, 50 ㎕의 증류수에 현탁하여 얻어진 ssDNA를 2% 아가로스 겔을 이용하여 확인하였다.1/100 of the volume of tRNA (Sigma aldrich, USA) and 3 volumes of 100% ethanol were added to the identified PCR product, followed by reaction at -70°C for 1 hour or more. After the reaction, it was centrifuged for 15 minutes under conditions of 4° C. and 13,000 rpm to obtain a pellet. After drying the obtained pellet at 65° C., ssDNA obtained by suspending in 50 μl of distilled water was confirmed using a 2% agarose gel.
실시예 3. ssDNA의 회수Example 3. Recovery of ssDNA
상기에서 수득된 ssDNA 중에서 바이오틴이 결합된 이중가닥 DNA(dsDNA) 및 ssDNA를 제거하고 순수한 정방향의 ssDNA를 확보하기 위해, 하기와 같이 가열-냉각(heating-cooling) 기법을 수행하였다.In order to remove biotin-conjugated double-stranded DNA (dsDNA) and ssDNA from the ssDNA obtained above and to secure pure forward ssDNA, a heating-cooling technique was performed as follows.
구체적으로, 실시예 2에서 수득된 ssDNA에 50 ㎕의 증류수를 첨가한 후, 이를 85℃에서 5분 동안 반응시켜 dsDNA를 ssDNA로 변성시킨 후, 이를 4℃로 냉각하여 ssDNA를 수득하였다. 수득된 ssDNA는 즉시 4℃에 냉각시켰다. 100 ㎕의 냉각된 ssDNA에 50 ㎕의 스트렙타비딘 아가로즈 레진(streptavidin agarose resin, Pierce, 미국)을 첨가하여 상온에서 1시간 동안 반응시켰다. 이후, 상기 반응액을 4℃, 13,000 rpm의 조건하에서 15분 동안 원심분리하고, 상층액만을 취하였다. 수득된 상층액에 페놀:클로로포름:이소아밀알콜이 25:24:1의 부피비로 혼합된 PCI 용액을 PCR 산물과 동일 부피로 첨가하고, 강하게 교반하였다. 교반 후, 이를 4℃, 13,000 rpm의 조건하에서 15분 동안 원심분리하고 상층액을 수득하였다. 이후, 수득된 상층액에 실시예 2에 기재된 바와 동일한 조건 및 방법으로 에탄올 침전법을 수행하여 펠렛을 수득하였다. 이후, 수득된 펠렛을 65℃에서 건조시킨 뒤, 50 ㎕의 증류수에 현탁하여 ssDNA를 수득하여 2% 아가로스 겔 전기영동을 통해 확인하고, 그 결과를 도 1에 나타내었다.Specifically, 50 µl of distilled water was added to the ssDNA obtained in Example 2, and then reacted at 85° C. for 5 minutes to denature dsDNA into ssDNA, and then cooled to 4° C. to obtain ssDNA. The obtained ssDNA was immediately cooled to 4°C. 50 µl of streptavidin agarose resin (Pierce, USA) was added to 100 µl of cooled ssDNA and reacted at room temperature for 1 hour. Thereafter, the reaction solution was centrifuged for 15 minutes under conditions of 4° C. and 13,000 rpm, and only the supernatant was taken. To the obtained supernatant, a PCI solution in which phenol:chloroform:isoamyl alcohol was mixed in a volume ratio of 25:24:1 was added in the same volume as the PCR product, and stirred vigorously. After stirring, it was centrifuged for 15 minutes under conditions of 4° C. and 13,000 rpm to obtain a supernatant. Thereafter, the obtained supernatant was subjected to ethanol precipitation under the same conditions and methods as described in Example 2 to obtain a pellet. Thereafter, the obtained pellet was dried at 65° C., and then suspended in 50 μl of distilled water to obtain ssDNA, which was confirmed through 2% agarose gel electrophoresis, and the results are shown in FIG. 1.
실시예 4. 3차원 구조의 DNA 앱타머 제작Example 4. Preparation of a DNA aptamer with a three-dimensional structure
실시예 3에서 수득된 40 ㎕의 ssDNA에 60 ㎕의 증류수 및 100 ㎕의 2× DNA 앱타머 선별용액을 첨가하였다. 상기 혼합물을 85℃에서 5분 동안 끓여 변성시킨 후, 상온에서 1시간 이상 방치하여 서서히 식힘으로써, ssDNA로부터 3차원 구조의 DNA 앱타머를 제작하였다.To 40 µl of ssDNA obtained in Example 3, 60 µl of distilled water and 100 µl of 2x DNA aptamer selection solution were added. The mixture was denatured by boiling at 85° C. for 5 minutes, and then allowed to stand at room temperature for 1 hour or longer to cool slowly, thereby preparing a DNA aptamer having a three-dimensional structure from ssDNA.
실시예 5. 리튬에 특이적으로 결합하는 DNA 앱타머의 선별Example 5. Selection of DNA aptamer specifically binding to lithium
5-1. 리튬에 특이적으로 결합하는 DNA 앱타머의 선별5-1. Selection of DNA aptamers that specifically bind to lithium
리튬에 특이적으로 결합하는 DNA 앱타머를 SELEX 기법을 이용하여 선별하였다.DNA aptamer that specifically binds to lithium was selected using the SELEX technique.
먼저, 리튬이 고정된 탄소 멤브레인(에너지기술연구원, 한국)에 실시예 4에서 제작된 DNA 앱타머를 첨가하고, 25℃에서 1시간 동안 반응시켰다. 반응이 끝난 뒤, 상기 혼합물에 1 ㎖의 1× 앱타머 선별용액(10 mM Tris-HCl(pH 7.5), 1 mM EDTA, 150 mM NaCl)을 첨가하고 3회 세척하여 비특이적으로 결합된 DNA 앱타머를 제거하였다. 리튬에 결합한 DNA 앱타머는 400 ㎕의 1× 앱타머 용출 용액(10 mM Tris-HCl(pH 7.5), 1 mM EDTA)을 첨가하여 85℃에서 5분 동안 반응시켜 멤브레인으로부터 분리시켰다. 분리된 멤브레인을 제거하고, DNA 앱타머가 용출된 용액을 상기와 동일한 조건으로 원심분리하여 변성된 DNA 앱타머를 회수하였다. 회수는 2회 수행되었으며, 이후, 회수된 DNA 앱타머를 상기 서술한 바와 같이 PCI 추출 및 에탄올 침전법을 수행하여 최종적으로 50 ㎕의 증류수에 현탁된 DNA 앱타머를 회수하였다.First, the DNA aptamer prepared in Example 4 was added to a lithium-immobilized carbon membrane (Korea Institute of Energy Research, Korea), and reacted at 25° C. for 1 hour. After the reaction was completed, 1 ml of 1× aptamer selection solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl) was added to the mixture and washed 3 times to non-specifically bound DNA aptamer. Was removed. DNA aptamer bound to lithium was separated from the membrane by adding 400 μl of 1× aptamer elution solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) and reacting at 85° C. for 5 minutes. The separated membrane was removed, and the solution in which the DNA aptamer was eluted was centrifuged under the same conditions as above to recover the denatured DNA aptamer. Recovery was performed twice, and then, the recovered DNA aptamer was subjected to PCI extraction and ethanol precipitation as described above to finally recover the DNA aptamer suspended in 50 µl of distilled water.
5-2. 비특이적으로 결합하는 DNA 앱타머의 제거5-2. Removal of non-specifically bound DNA aptamer
리튬이 아닌 탄소 멤브레인에 결합하는 비특이적 DNA 앱타머를 제거하는 동시에 리튬에 결합하는 DNA 앱타머의 특이성을 향상시키기 위해 SELEX를 1회 수행할 때마다 네가티브(negative) SELEX를 수행하였다(도 2).In order to remove the non-specific DNA aptamer that binds to the carbon membrane other than lithium and at the same time improve the specificity of the DNA aptamer that binds to lithium, a negative SELEX was performed each time SELEX was performed (FIG. 2).
먼저, 실시예 5-1에서 수득된 DNA 앱타머를 리튬이 고정되지 않은 탄소 멤브레인과 반응시킨 것을 제외하고는 실시예 5-1과 동일한 방법 및 조건으로 SELEX를 수행하고, 탄소 멤브레인에 결합하지 않는 앱타머만을 수득하였다.First, SELEX was performed in the same manner and conditions as in Example 5-1, except that the DNA aptamer obtained in Example 5-1 was reacted with a carbon membrane to which lithium was not immobilized, and did not bind to the carbon membrane. Only the aptamer was obtained.
실시예 6. 친화성이 높은 DNA 앱타머 풀의 선별Example 6. Selection of high affinity DNA aptamer pool
9회의 SELEX를 통해 각 라운드에서 수득된 DNA 앱타머의 리튬에 대한 친화성을 나노드랍 방법으로 확인하였다. 구체적으로, 실시예 5에서 각 회별로 수득된 DNA 앱타머 샘플의 농도를 나노드롭(Nanodrop 2000 Micro spectrophotometer, Thermo scientific)을 이용하여 제조사의 프로토콜에 따라 측정하였다.The affinity for lithium of the DNA aptamer obtained in each round through 9 SELEX was confirmed by the nanodrop method. Specifically, the concentration of the DNA aptamer sample obtained each time in Example 5 was measured using a nanodrop (
그 결과, 도 3에 나타난 바와 같이, 8회에 수득된 DNA 앱타머의 농도가 3505.9 ng/㎕로 가장 높았고, 9회에 수득된 DNA 앱타머의 농도가 2937.2 ng/㎕로 확인되었다. 따라서, 상기로부터 SELEX를 8회 수행한 뒤 수득된 DNA 앱타머 풀(pool)이 리튬과 가장 특이적으로 결합함을 알 수 있었다.As a result, as shown in FIG. 3, the concentration of the DNA aptamer obtained in the 8th cycle was the highest at 3505.9 ng/µl, and the concentration of the DNA aptamer obtained in the 9th time was confirmed to be 2937.2 ng/µl. Therefore, it was found that the DNA aptamer pool obtained after performing
실시예 7. DNA 앱타머의 서열 확인Example 7. Sequence confirmation of DNA aptamer
상기에서 리튬과 가장 친화성을 높은것으로 확인된 9회의 SELEX 수행을 통해 얻은 DNA 앱타머의 서열을 다음과 같은 방법으로 확인하였다.The sequence of the DNA aptamer obtained through 9 SELEX runs, which was found to have the highest affinity with lithium, was confirmed by the following method.
먼저, 상기 실시예 5에서 SELEX를 8회 수행하여 수득된 DNA 앱타머를 주형으로 사용하고, 정방향(서열번호 17) 및 역방향(서열번호 18) 프라이머를 이용하여 PCR을 수행하고, 이중가닥 형태의 DNA를 수득하였다. 수득된 dsDNA는 T-블런트 클로닝 키트(SolGent, 한국)를 이용하여 제조사의 프로토콜에 따라 T-벡터에 클로닝되었다.First, using the DNA aptamer obtained by performing
구체적으로, 1 ㎕의 T-벡터(10 ng/㎕), 4 ㎕의 PCR 산물(20 ng/㎕) 및 1 ㎕의 6x T-블런트 완충액을 25℃에서 5분 동안 반응시켰다. 6 ㎕의 상기 반응물을 100 ㎕의 DH5α 수용성 균주와 혼합하고, 42℃에서 30초 동안 열충격을 가해 형질전환시켰다. 이를 2분 동안 얼음에 방치하고, 900 ㎕의 SOC 배지(2% 트립톤, 0.5% 효모 추출물, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 및 20 mM 글루코스)를 첨가하여 37℃에서 1시간 동안 배양하였다. 200 ㎕의 배양액을 50 ㎍/㎖의 암피실린(Sigma Aldrich, 미국), 50 ㎍/㎖의 카나마이신(Sigma Aldrich, 미국), 50 ㎍/㎖의 X-갈(X-gal, Sigma Aldrich, 미국)및 5 ㎍/㎖의 IPTG(Thermo Scientific, 미국)가 포함된 LB 배양 플레이트에 스프레딩하였다. 이를 37℃에서 15시간 배양한 뒤, 생성된 콜로니 중 50개의 흰색 콜로니만 선별하여 통상적인 방법으로 DNA를 추출하고, 그 염기서열을 Solgent(한국) 사에 의뢰하여 확인한 결과를 표 3에 나타내었다.Specifically, 1 µl of T-vector (10 ng/µl), 4 µl of PCR product (20 ng/µl), and 1 µl of 6x T-blunt buffer were reacted at 25° C. for 5 minutes. 6 µl of the reaction was mixed with 100 µl of DH5α water-soluble strain, and transformed by applying heat shock at 42° C. for 30 seconds. This was left on ice for 2 minutes, and 900 μl of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 and 20 mM glucose) was added thereto. Incubated for 1 hour at 37 ℃. 200 µl of the culture medium was added 50 µg/ml of ampicillin (Sigma Aldrich, USA), 50 µg/ml kanamycin (Sigma Aldrich, USA), 50 µg/ml X-gal (X-gal, Sigma Aldrich, USA) It was spread on an LB culture plate containing 5 μg/ml of IPTG (Thermo Scientific, USA). After incubating this at 37°C for 15 hours, only 50 white colonies were selected out of the generated colonies to extract DNA by a conventional method, and the nucleotide sequence was requested to Solgent (Korea), and the results are shown in Table 3. .
표 3에 나타낸 바와 같이, 리튬에 결합하는 16개의 DNA 앱타머 서열을 확인하였다.As shown in Table 3, 16 DNA aptamer sequences that bind to lithium were identified.
실시예 8. 실시간 PCR 방법을 이용한 친화성 확인Example 8. Affinity check using real-time PCR method
실시예 7에서 확인된 16개의 DNA 앱타머를 이용하여 실시간 PCR 방법을 통해 리튬에 대한 친화성을 재확인하였다.Using the 16 DNA aptamers identified in Example 7, the affinity for lithium was reconfirmed through a real-time PCR method.
먼저, 표 3에 기재된 것과 동일한 염기서열을 갖는 16개의 DNA 앱타머를 각각 풀로 제작하였다. 이들 앱타머 풀을 동일한 농도로 준비하여 실시예 5-1에 기재된 조건 및 방법으로 SELEX를 1회 더 수행하고 회수된 DNA 앱타머의 농도를 실시간 PCR 방법으로 확인하였다. 구체적으로, 실시간 PCR은 iQ SYBR Green Supermix(Bio-rad, 미국)를 이용하여 통상적인 방법으로 수행되었다. 이때, PCR 조건은 하기 표 4에, 그 결과 수득된 C(t) 값을 도 4에 나타내었다.First, 16 DNA aptamers having the same nucleotide sequence as described in Table 3 were each prepared as a pool. These aptamer pools were prepared at the same concentration, SELEX was performed once more by the conditions and methods described in Example 5-1, and the concentration of the recovered DNA aptamer was confirmed by a real-time PCR method. Specifically, real-time PCR was performed by a conventional method using iQ SYBR Green Supermix (Bio-rad, USA). At this time, PCR conditions are shown in Table 4 below, and the C(t) values obtained as a result are shown in FIG. 4.
도 4에 나타난 바와 같이, 16개의 DNA 앱타머가 모두 리튬에 특이적으로 결합하였으나, 특히 Li-4, Li-8 및 Li-14의 DNA 앱타머가 다른 앱타머에 비해 더욱 낮은 C(t) 값을 나타냄으로써, 리튬에 더욱 특이적으로 결합함을 확인하였다.As shown in FIG. 4, all 16 DNA aptamers specifically bind to lithium, but in particular, the DNA aptamers of Li-4, Li-8 and Li-14 have lower C(t) values than other aptamers. By showing, it was confirmed that it binds more specifically to lithium.
실시예 9. DNA 앱타머의 구조 확인Example 9. Structure confirmation of DNA aptamer
실시예 8에서 리튬에 가장 높은 친화력으로 결합하는 것으로 확인된 3개 DNA 앱타머의 2차원 구조는 DNA mfold 프로그램(Rensselear polytechnic institute)을 이용하여 이미지화하고, 예측된 구조를 도 5에 나타내었다.The two-dimensional structures of the three DNA aptamers identified as binding with the highest affinity to lithium in Example 8 were imaged using a DNA mfold program (Rensselear polytechnic institute), and the predicted structure is shown in FIG. 5.
실험예 1. 리튬에 대한 DNA 앱타머의 친화력 확인Experimental Example 1. Confirmation of affinity of DNA aptamer for lithium
1-1. 리튬 코팅 센서 칩의 제작1-1. Fabrication of lithium coated sensor chip
표면 플라즈마 공명(surface plasmon resonance, SPR) 실험을 통해 선별된 DNA 앱타머의 결합력을 정량적으로 확인하기 위해 DNA 앱타머가 코팅된 센서 칩을 다음과 같은 방법으로 제작하였다.In order to quantitatively check the binding force of the DNA aptamer selected through surface plasmon resonance (SPR) experiments, a sensor chip coated with DNA aptamer was fabricated in the following manner.
먼저, 스트렙트아비딘으로 표면이 코팅된 센서 칩 SA(GE healthcare, 영국)에 1 M의 NaCl 및 50 mM의 NaOH 혼합액을 10 ㎕/min의 속도로 흘려주어 표면을 활성화시켰다. 한편, 10 ng/㎖의 농도로 상기 확인된 3개의 DNA 앱타머가 희석된 HBS-EP 완충액(GE Healthcare, 영국)을 활성화된 센서 칩 SA의 표면에 10 ㎕/min의 속도로 15분 동안 흘려줌으로써, DNA 앱타머가 표면에 코팅된 센서 칩 SA를 제작하였다. 이후, 상기 센서 칩 SA에 1 M의 NaCl 및 50% 이소프로판올 혼합액을 5 ㎕/min의 속도로 10분 동안 흘려주어 센서 칩 표면에 비특이적으로 결합한 DNA 앱타머를 제거하였다.First, the surface was activated by flowing a mixture of 1 M NaCl and 50 mM NaOH at a rate of 10 μl/min to a sensor chip SA (GE healthcare, UK) coated with streptavidin. On the other hand, by flowing the HBS-EP buffer (GE Healthcare, UK) in which the 3 DNA aptamers were diluted at a concentration of 10 ng/ml on the surface of the activated sensor chip SA at a rate of 10 µl/min for 15 minutes. , To fabricate a sensor chip SA coated on the surface of the DNA aptamer. Thereafter, a mixture of 1 M NaCl and 50% isopropanol was flowed on the sensor chip SA at a rate of 5 μl/min for 10 minutes to remove the DNA aptamer non-specifically bound to the sensor chip surface.
1-2. DNA 앱타머의 친화력 확인1-2. Confirmation of affinity of DNA aptamer
상기 선별된 DNA 앱타머의 리튬에 대한 친화력을 SPR 방법을 이용하여 정량적으로 확인하였다.The affinity of the selected DNA aptamer for lithium was quantitatively confirmed using the SPR method.
먼저, 리튬을 0, 50, 100, 200, 300, 400, 600, 800 또는 1,000 μM의 농도가 되도록 HBS-EP 완충액에 현탁하여 준비하였다. 실험은 BIAcore 3000(BIACORE)을 사용하여 제조사의 프로토콜에 따라 수행되었으며, 실험예 1-1에서 제작된 DNA 앱타머가 코팅된 센서 칩 SA에 대한 리튬의 결합력과 아무것도 코팅되지 않은 센서 칩 SA에 대한 리튬의 결합력 차이를 확인하였다. 이때, 속도 변수는 BIA 평가프로그램(BIACORE)을 이용하여 수득 및 정량하고 그 결과는 표 5에 나타내었다.First, lithium was prepared by suspending it in HBS-EP buffer to a concentration of 0, 50, 100, 200, 300, 400, 600, 800 or 1,000 μM. The experiment was performed according to the manufacturer's protocol using BIAcore 3000 (BIACORE), and the binding force of lithium to the DNA aptamer-coated sensor chip SA prepared in Experimental Example 1-1 and lithium to the sensor chip SA that is not coated with anything The difference in bonding strength was confirmed. At this time, the rate variable was obtained and quantified using the BIA evaluation program (BIACORE), and the results are shown in Table 5.
실험예 2. 리튬에 대한 DNA 앱타머의 결합 특이성 확인Experimental Example 2. Confirmation of binding specificity of DNA aptamer to lithium
실험예 1에서 리튬에 가장 높은 친화력으로 결합하는 Li-14 앱타머의 리튬에 대한 결합 특이성을 다음과 같이 확인하였다. 실험은 리튬을 HBS-EP 완충액에 현탁한 현탁액 대신 1 M의 LiCl, NdCl3, ArHNa2O4, CdCl2, CuCl2, MgCl2, FeCl3, NaCl 또는 NiCl2 수용액을 20 ㎕/min의 속도로 10분 동안 흘려준 것을 제외하고는, 상기 실험예 1과 동일한 조건 및 방법으로 수행하였다. 그 결과, Li-14 앱타머의 다양한 중금속에의 결합력을 확인하여 표 6에 나타내었다.In Experimental Example 1, the binding specificity for lithium of Li-14 aptamer, which binds with the highest affinity to lithium, was confirmed as follows. In the experiment, instead of a suspension in which lithium was suspended in HBS-EP buffer, 1 M of LiCl, NdCl 3 , ArHNa 2 O 4 , CdCl 2 , CuCl 2 , MgCl 2 , FeCl 3 , NaCl or NiCl 2 aqueous solution was added at a rate of 20 μl/min. Except for flowing for 10 minutes, it was carried out in the same conditions and method as in Experimental Example 1. As a result, the binding strength of Li-14 aptamer to various heavy metals was confirmed and shown in Table 6.
표 6에 나타난 바와 같이, Li-14 앱타머는 리튬에 특이적으로 결합하였으며, 다른 중금속에 대해서는 결합력이 높지 않았다.As shown in Table 6, Li-14 aptamer specifically binds to lithium, and does not have high binding strength to other heavy metals.
실험예 3. DNA 앱타머의 리튬 검출 확인Experimental Example 3. Confirmation of lithium detection of DNA aptamer
3-1. DNA 앱타머의 리튬 검출 확인3-1. Confirmation of lithium detection of DNA aptamer
실시예 8에서 높은 친화력으로 리튬에 결합하는 것으로 확인된 3개의 DNA 앱타머의 리튬 검출여부를 다음과 같이 확인하였다.In Example 8, it was confirmed whether or not lithium was detected by the three DNA aptamers, which were confirmed to bind to lithium with high affinity.
먼저, Li-4, Li-8 및 Li-14의 DNA 앱타머 각각이 고정된 스트렙트아비딘 기반의 비드가 충전된 컬럼을 제조하였다. 구체적으로, 상기 5개의 DNA 앱타머의 5' 말단을 바이오틴으로 변형(modification)시킨 뒤, 85℃에서 5분 동안 반응시키고, 실온에서 서서히 식혀 2차 구조를 형성시켰다. 2차 구조가 형성된 상기 비드를 스트렙트아비딘이 충전된 컬럼에 주입한 뒤, 여기에 10 ㎖의 증류수를 흘려주어 스트렙트아비딘과 결합되지 않은 DNA 앱타머를 제거하였다. 그 결과, 3개의 DNA 앱타머가 각각 고정된 스트렙트아비딘 컬럼을 제작하였다. 상기 제작된 컬럼에 리튬을 흘려주어 리튬을 검출할 수 있는지 여부를 확인하였다. 이때, 대조군으로서는 DNA 앱타머가 고정되지 않은 스트렙트아비딘 비드를 사용하였다. 본 발명에 따른 DNA 앱타머와 리튬과의 결합은 주사전자현미경(scaning eletron microscope, SEM)을 사용하여 관찰하였고, 그 결과를 도 6에 나타내었다.First, a column filled with streptavidin-based beads to which each of the DNA aptamers of Li-4, Li-8, and Li-14 is immobilized was prepared. Specifically, the 5'ends of the 5 DNA aptamers were modified with biotin, reacted at 85° C. for 5 minutes, and slowly cooled at room temperature to form a secondary structure. The beads having a secondary structure were injected into a column filled with streptavidin, and then 10 ml of distilled water was poured thereto to remove DNA aptamers not bound to streptavidin. As a result, a streptavidin column in which each of the three DNA aptamers was immobilized was prepared. It was confirmed whether or not lithium could be detected by pouring lithium into the prepared column. At this time, as a control, streptavidin beads to which DNA aptamer was not immobilized were used. The binding of the DNA aptamer and lithium according to the present invention was observed using a scanning electron microscope (SEM), and the results are shown in FIG. 6.
도 6에 나타난 바와 같이, 스트렙트아비딘 컬럼에 리튬을 처리한 대조군과는 달리, 본 발명에 따른 3개의 DNA 앱타머가 고정된 스트렙트아비딘 컬럼의 표면이 더욱 밝은 것으로 확인되었다. 이는 DNA 앱타머에 결합된 리튬의 영향인 것으로 판단되며, 따라서 본 발명에 따른 DNA 앱타머는 리튬의 검출에 사용될 수 있음을 알 수 있었다.As shown in Figure 6, unlike the control group treated with lithium on the streptavidin column, it was confirmed that the surface of the streptavidin column to which the three DNA aptamers according to the present invention were immobilized was brighter. This is judged to be the effect of lithium bound to the DNA aptamer, and thus it was found that the DNA aptamer according to the present invention can be used for detection of lithium.
3-2. DNA 앱타머와 리튬의 결합 효율 확인3-2. Confirmation of the binding efficiency of DNA aptamer and lithium
실험예 3-1에서 제작된 본 발명의 DNA 앱타머가 고정된 스트렙트아비딘 컬럼을 이용하여 DNA 앱타머와 리튬 사이의 결합효율을 확인하였다. 구체적으로, DNA 앱타머가 고정된 스트렙트아비딘 컬럼에 리튬을 흘려준 뒤, 컬럼에 결합한 리튬에 10 ppm의 Li+를 처리하여 용출액을 수득하였다. 이후, 컬럼을 통과하기 전 리튬의 농도(초기 리튬 농도) 및 상기 용출액에 포함된 리튬의 농도(리튬의 제거량)를 유도결합 플라즈마 분광기를 이용하여 측정하였다. 이들 측정값으로부터 하기 표 7과 같이 리튬의 결합 효율을 확인하였다. 이때, 대조군으로서는 DNA 앱타머가 고정되지 않은 스트렙트아비딘 비드를 사용하였다.The binding efficiency between the DNA aptamer and lithium was confirmed using a streptavidin column to which the DNA aptamer of the present invention was immobilized prepared in Experimental Example 3-1. Specifically, after flowing lithium into a streptavidin column on which DNA aptamer is immobilized, 10 ppm of Li + was treated with lithium bound to the column to obtain an eluate. Then, before passing through the column, the concentration of lithium (initial lithium concentration) and the concentration of lithium contained in the eluate (amount of lithium removal) were measured using an inductively coupled plasma spectrometer. From these measured values, the binding efficiency of lithium was confirmed as shown in Table 7 below. At this time, as a control, streptavidin beads to which DNA aptamer was not immobilized were used.
(㎎/ℓ)Initial lithium concentration
(Mg/ℓ)
(㎎/ℓ)Lithium removal
(Mg/ℓ)
(%)Coupling efficiency
(%)
표 7에 나타난 바와 같이, 본 발명에 따른 3개의 DNA 앱타머는 높은 결합 효율로 리튬과 결합하였으며, 특히, Li-14 DNA 앱타머는 약 70%의 결합 효율을 보였다.As shown in Table 7, the three DNA aptamers according to the present invention bound lithium with high binding efficiency, and in particular, the Li-14 DNA aptamer showed a binding efficiency of about 70%.
<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> DNA APTAMER SPECIFICALLY BINDING TO LITHIUM AND USING THE SAME <130> DP-2019-0381-KR <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-1 aptamer <400> 1 ataccagctt attcaattcg ttaccagcta cggattacct gtgtcaattt ctgttaggag 60 atagtaagtg caatct 76 <210> 2 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-2 aptamer <400> 2 ataccagctt attcaattac cgcttttgtg gccataagtg atcgattagt taagtgtgag 60 atagtaagtg caatct 76 <210> 3 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-3 aptamer <400> 3 ataccagctt attcaattcg aggttggatt acgcgttgta ctttatagtt gtctatccag 60 atagtaagtg caatct 76 <210> 4 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-4 aptamer <400> 4 ataccagctt attcaattgg tagtatatcg caagctgtca tgttcggcat acgttagcag 60 atagtaagtg caatct 76 <210> 5 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-5 aptamer <400> 5 ataccagctt attcaattat agcacaatcc gcctttccgg tgagtaattg tgttgattag 60 atagtaagtg caatct 76 <210> 6 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-6 aptamer <400> 6 ataccagctt attcaattcg tcgtatctag gcttttgaat gctcattatg tcgcatccag 60 atagtaagtg caatct 76 <210> 7 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-7 aptamer <400> 7 ataccagctt attcaattgc cgcatcgaag ctcaaagatt catctcattg tccttttgag 60 atagtaagtg caatct 76 <210> 8 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-8 aptamer <400> 8 ataccagctt attcaattcc tgtatgtatg acgtcatacg tttgacgagt ccgctcttag 60 atagtaagtg caatct 76 <210> 9 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-9 aptamer <400> 9 ataccagctt attcaatttc actagccagt accatggagt gtacgttcac cgttacgcag 60 atagtaagtg caatct 76 <210> 10 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-10 aptamer <400> 10 ataccagctt attcaattct gatgacatgc gggagctgct cgtgatacgt ttattgttag 60 atagtaagtg caatct 76 <210> 11 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-11 aptamer <400> 11 ataccagctt attcaattac catctttata ggcggatccc gctagtctct tttcctgtag 60 atagtaagtg caatct 76 <210> 12 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-12 aptamer <400> 12 ataccagctt attcaattag tctagagatg gaacaatccc cgttactata acaacgctag 60 atagtaagtg caatct 76 <210> 13 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-13 aptamer <400> 13 ataccagctt attcaattct tggcagggac tggttagttt atcgtggtct caccttttag 60 atagtaagtg caatct 76 <210> 14 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-14 aptamer <400> 14 ataccagctt attcaattga taaggggaat atataattat tgtcctttgt gagatagaag 60 atagtaagtg caatct 76 <210> 15 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-15 aptamer <400> 15 ataccagctt attcaattcg aagtttgtcc tttctcttgt ccttttatgc atattgtcag 60 atagtaagtg caatct 76 <210> 16 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-16 aptamer <400> 16 ataccagctt attcaattaa cgttcttcca tttaccatat aactgtcggc gccttttaag 60 atagtaagtg caatct 76 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 ataccagctt attcaatt 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 agattgcact tactatct 18 <110> Chungbuk National University Industry-Academic Cooperation Foundation <120> DNA APTAMER SPECIFICALLY BINDING TO LITHIUM AND USING THE SAME <130> DP-2019-0381-KR <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-1 aptamer <400> 1 ataccagctt attcaattcg ttaccagcta cggattacct gtgtcaattt ctgttaggag 60 atagtaagtg caatct 76 <210> 2 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-2 aptamer <400> 2 ataccagctt attcaattac cgcttttgtg gccataagtg atcgattagt taagtgtgag 60 atagtaagtg caatct 76 <210> 3 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-3 aptamer <400> 3 ataccagctt attcaattcg aggttggatt acgcgttgta ctttatagtt gtctatccag 60 atagtaagtg caatct 76 <210> 4 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-4 aptamer <400> 4 ataccagctt attcaattgg tagtatatcg caagctgtca tgttcggcat acgttagcag 60 atagtaagtg caatct 76 <210> 5 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-5 aptamer <400> 5 ataccagctt attcaattat agcacaatcc gcctttccgg tgagtaattg tgttgattag 60 atagtaagtg caatct 76 <210> 6 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-6 aptamer <400> 6 ataccagctt attcaattcg tcgtatctag gcttttgaat gctcattatg tcgcatccag 60 atagtaagtg caatct 76 <210> 7 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-7 aptamer <400> 7 ataccagctt attcaattgc cgcatcgaag ctcaaagatt catctcattg tccttttgag 60 atagtaagtg caatct 76 <210> 8 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-8 aptamer <400> 8 ataccagctt attcaattcc tgtatgtatg acgtcatacg tttgacgagt ccgctcttag 60 atagtaagtg caatct 76 <210> 9 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-9 aptamer <400> 9 ataccagctt attcaatttc actagccagt accatggagt gtacgttcac cgttacgcag 60 atagtaagtg caatct 76 <210> 10 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-10 aptamer <400> 10 ataccagctt attcaattct gatgacatgc gggagctgct cgtgatacgt ttattgttag 60 atagtaagtg caatct 76 <210> 11 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-11 aptamer <400> 11 ataccagctt attcaattac catctttata ggcggatccc gctagtctct tttcctgtag 60 atagtaagtg caatct 76 <210> 12 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-12 aptamer <400> 12 ataccagctt attcaattag tctagagatg gaacaatccc cgttactata acaacgctag 60 atagtaagtg caatct 76 <210> 13 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-13 aptamer <400> 13 ataccagctt attcaattct tggcagggac tggttagttt atcgtggtct caccttttag 60 atagtaagtg caatct 76 <210> 14 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-14 aptamer <400> 14 ataccagctt attcaattga taaggggaat atataattat tgtcctttgt gagatagaag 60 atagtaagtg caatct 76 <210> 15 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-15 aptamer <400> 15 ataccagctt attcaattcg aagtttgtcc tttctcttgt ccttttatgc atattgtcag 60 atagtaagtg caatct 76 <210> 16 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Li-16 aptamer <400> 16 ataccagctt attcaattaa cgttcttcca tttaccatat aactgtcggc gccttttaag 60 atagtaagtg caatct 76 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 ataccagctt attcaatt 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 agattgcact tactatct 18
Claims (8)
DNA aptamer specifically binding to lithium consisting of the nucleotide sequence set forth in SEQ ID NO: 4, 8 or 14.
A composition for detecting, removing or recovering lithium containing the DNA aptamer of claim 1 as an active ingredient.
A kit for detecting, removing or recovering lithium containing the DNA aptamer of claim 1 as an active ingredient.
A microarray for detecting lithium having a substrate on which the DNA aptamer of claim 1 is fixed.
A sensor for detecting lithium having a substrate on which the DNA aptamer of claim 1 is fixed.
A method for detecting lithium comprising reacting the composition of claim 2 with a sample.
A method for removing lithium comprising the step of removing the complex of the DNA aptamer and lithium formed by reacting the composition of claim 2 with a sample.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120090477A (en) * | 2011-02-08 | 2012-08-17 | 경희대학교 산학협력단 | Dna aptamer specific for uranyl ion |
| KR20120126431A (en) * | 2011-05-11 | 2012-11-21 | 충북대학교 산학협력단 | DNA Aptamer Specifically Binding to Copper and Uses Thereof |
| KR101460450B1 (en) * | 2013-09-05 | 2014-11-12 | 충북대학교 산학협력단 | DNA aptamer specifically binding to cadmium and uses thereof |
| KR20150120003A (en) * | 2014-04-16 | 2015-10-27 | 서울대학교산학협력단 | High-Performance Graphene Aptasensor Based on Field Effect Transistor for Mercury Detection |
| KR102020238B1 (en) | 2018-04-09 | 2019-09-10 | 에스케이이노베이션 주식회사 | Method of recycling active metal of lithium secondary battery |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120090477A (en) * | 2011-02-08 | 2012-08-17 | 경희대학교 산학협력단 | Dna aptamer specific for uranyl ion |
| KR20120126431A (en) * | 2011-05-11 | 2012-11-21 | 충북대학교 산학협력단 | DNA Aptamer Specifically Binding to Copper and Uses Thereof |
| KR101460450B1 (en) * | 2013-09-05 | 2014-11-12 | 충북대학교 산학협력단 | DNA aptamer specifically binding to cadmium and uses thereof |
| KR20150120003A (en) * | 2014-04-16 | 2015-10-27 | 서울대학교산학협력단 | High-Performance Graphene Aptasensor Based on Field Effect Transistor for Mercury Detection |
| KR102020238B1 (en) | 2018-04-09 | 2019-09-10 | 에스케이이노베이션 주식회사 | Method of recycling active metal of lithium secondary battery |
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