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도파민 압타머의 혼성화에 따른 새로운 접근의 샌드위치 분석법
정희재,한수정,김진아,이인숙 서울여자대학교 자연과학연구소 2013 자연과학연구논문집 Vol.25 No.-
Aptamers, single-stranded oligonucleotides selected by SELEX, have been turned out as a novel molecular recognizer in immunoassay with inherent advantages. Dopamine (DA) is one of the most important catecholamine as neurotransmitter molecules in the central nervous system. The deficiency of DA causes Parkinson’s disease, dementia and attention-deficit hyper activity disorder. Here, based on enzyme-linked aptamer assay for DA in serum a sandwich method has been optimized and validated by using two aptamers. However, the results were quite opposite from normal sandwich assay results which we had expected. From these, we supposed that two aptamers could make duplex with their base pairs even if they all do not match. Since they were selected from same RNA pool by SELEX, they might have same binding site for DA. In this regard, if one aptamer binds DA, the other one would not be able to bind DA but could make duplex with free aptamer. To confirm this presumption, we applied different molecules as a target molecule instead of DA and used difference sequences of oligonucleotide. The several optimizations for this assay were performed. This method gives 2.78×10-11M of LOD in optimized condition; Absorbance=-0.1569×log[DA]—0.7545 in diluted serum (R2=0.9914). From these results we prove that the newly approached sandwich assay is favorable and available to detect DA.
RNA Aptamer를 사용하여 아스코르빈산과 요산이 존재하는 뇨 중에서 도파민의 선택적인 검출을 위한 효소-결합 압타머 분석법
박호영,김은혜,정희재,홍수연,정소희,이인숙 서울여자대학교 자연과학연구소 2011 자연과학연구논문집 Vol.23 No.-
A selective screening method based on a competitive enzyme-linked aptamer assay (ELAA) for dopamine (DA) in urine has been optimized and validated. Generally electroactive DA, uric acid (UA) and ascorbic acid (AA) are co-present in biological fluids such as blood and urine. Thus, it is important to develop a technique to selectively detect DA in the presence of AA and UA. In this paper, we report advantageous sensitivity and specificity of aptamer assays as compared to the existing antibody based-immunoassay. The RNA aptamer (67 mer) was immobilized via site-directed immobilization with biotin both at the 3’-end on aptamer and at neutravidin plate. Assay was performed with 0.01 μg mL-1 of aptamer and 1.205 × 10-7 M DA-HRP conjugate using the optimized method. A dose-response curve was constructed, and the limit of detection and a dynamic range for the DA were determined as 2.5 × 10-11 M and 4.0 × 10-7 M ~ 1.0 × 10-10 M, respectively. Competitive ELAA showed no matrix effect about AA and UA, which found in urine. Therefore, this paper describes usefulness of the aptamer assay in monitoring DA in urine. Key words:aptamer, dopamine, urine, immunoassay, horseradish peroxidase.
류지은,정소희,김은혜,정희재,박호영,이인숙 서울여자대학교 2010 자연과학연구논문집 Vol.22 No.-
The major distinguishing feature of the avidin/biotin system is the extraordinary affinity (K_(a) = 10^(15) M^(-1)) that characterizes the complex formed between the vitamin, biotin and the egg white protein, avidin. Interaction is so strong that even biotin (or avidin) coupled to proteins is available for binding by avidin (or biotin). Thus avidin/biotin system has become so popular in biological sciences. This work proposed the possibility of non-specific binding of free avidin to protein such as bovine serum albumin.