CAJ | 학술논문

利用目标蛋白、适体探针、挂锁探针和与适体探针匹配的互补序列之间的竞争反应,发展了一种基于滚环扩增的高灵敏荧光适配体传感器。在不含目标蛋白时,互补序列由于与适体探针杂交形成双链,因此不能与挂锁探针杂交以触发连接-滚环放大反应。相反,有目标蛋白存在时,由于目标蛋白与适体探针结合,使得互补序列被置换下来,从而可以与挂锁探针杂交。在DNA连接酶的作用下,挂锁探针被进一步环化并在Phi 29 DNA聚合酶的作用下发生滚环扩增反应。扩增产物含有许多能与分子信标检测探针环状部分杂交的重复序列,从而分子信标被打开产生荧光信号。对互补序列的长度及挂锁探针的浓度进行了考察。在优化的实验条件下,本传感系统可以实现对的凝血酶高灵敏检测,线性范围为0.067~32 nmol/L,检出限为0.03 nmol/L(约90 amol目标分子)。通过设计不同的适体探针和相关核酸序列,本传感系统可以作为一种通用型方法用于其它目标物的分析。
이용목표단백、괄체탐침、괘쇄탐침화여괄체탐침필배적호보서렬지간적경쟁반응,발전료일충기우곤배확증적고령민형광괄배체전감기。재불함목표단백시,호보서렬유우여괄체탐침잡교형성쌍련,인차불능여괘쇄탐침잡교이촉발련접-곤배방대반응。상반,유목표단백존재시,유우목표단백여괄체탐침결합,사득호보서렬피치환하래,종이가이여괘쇄탐침잡교。재DNA련접매적작용하,괘쇄탐침피진일보배화병재Phi 29 DNA취합매적작용하발생곤배확증반응。확증산물함유허다능여분자신표검측탐침배상부분잡교적중복서렬,종이분자신표피타개산생형광신호。대호보서렬적장도급괘쇄탐침적농도진행료고찰。재우화적실험조건하,본전감계통가이실현대적응혈매고령민검측,선성범위위0.067~32 nmol/L,검출한위0.03 nmol/L(약90 amol목표분자)。통과설계불동적괄체탐침화상관핵산서렬,본전감계통가이작위일충통용형방법용우기타목표물적분석。
Based on the competition reaction of target protein, aptamer probe, padlock probe and complementary sequence, a highly sensitive fluorescent aptasensor was developed in this study in combination with rolling circle amplification. In the absence of target protein, the ligation-rolling circle amplification reaction was repressed because the complementary sequence hybridized with aptamer probe to form double-stranded duplex. While in the presence of target protein, the target molecules bound specifically with aptamer probe, inducing displacement of the complementary sequence and hybridization with padlock probe. The padlock probe was circularized with the assistance of E. coli DNA ligase, and the rolling circle amplification process could be accomplished by Phi 29 DNA polymerase. The amplification product contained thousands of repeated sequences which could hybridize with the loop of molecular beacon ( the detection probes) , resulting in a significant fluorescence signal. The effects of length of complementary DNA ( CDNA ) sequence and concentration of padlock probe were investigated. Under the optimized experimental conditions, the model target protein thrombin could be highly sensitively detected by the proposed aptasensing system in a linear range of 0 . 067-32 . 4 nmol/L with a detection limit of 0 . 03 nmol/L ( approximately 90 amol target molecules). Moreover, the presented sensing method was universal for other target analysis by skillfully design of the sequence of aptamer probe and related oligonucleotides.