Department of Chemical and Biomolecular Engineering (BK 21+ program), KAIST, Daehak-ro 291, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, Republic of Korea.
Mikrochim Acta. 2019 May 6;186(6):330. doi: 10.1007/s00604-019-3453-2.
A simple and sensitive method is described for the determination of DNA. It relies on the use of (a) an invasive reaction that is catalyzed by flap endonuclease 1 (FEN 1), and (b) graphene oxide (GO)-based fluorescence signalling. The presence of target DNA mediates the formation of the invasive structure, and this induces FEN 1 to catalyze multiple cycles of cleavage reaction at the junction, thereby liberating numerous fluorophore-labeled flaps. The released flaps are intentionally designed too short to be adsorbed onto GO. Hence, intense green fluorescence whose maximum emission is observed at 520 nm after excitation at 480 nm is restored even in the presence of GO. The method can be applied to the determination of target DNA from Chlamydia trachomatis, one of the major pathogenic bacteria causing sexually transmitted diseases. The assay is sensitive and specific with the limit of detection of 6.7 pM, and was applied to reliable determination of Chlamydia trachomatis DNA in human serum. Graphical abstract Flap endonuclease 1 (FEN 1)-catalyzed invasive reaction and graphene oxide (GO)-based fluorescence signalling are integrated to develop a novel and sensitive target DNA detection method.
一种简单灵敏的 DNA 测定方法。它依赖于（a）由核酸内切酶 1（FEN 1）催化的侵入反应，和（b）基于氧化石墨烯（GO）的荧光信号。靶 DNA 的存在介导了侵入结构的形成，这诱导 FEN 1 在连接点处催化多个循环的切割反应，从而释放出许多荧光标记的瓣。释放的瓣被有意设计得太短，无法被 GO 吸附。因此，即使存在 GO，也能恢复最大发射峰在 520nm 处、激发波长在 480nm 处的强烈绿色荧光。该方法可用于检测引起性传播疾病的主要病原菌沙眼衣原体的靶 DNA。该测定法具有灵敏度和特异性，检测限为 6.7 pM，并已应用于人血清中沙眼衣原体 DNA 的可靠测定。
