基于核酸适配体和氧化石墨烯的单靶向及双靶向共价分子信标用于细胞内外ATP和钾离子检测研究

For the physically adsorbed sensor of aptamer and graphene oxide have non-specific desorption and false positive signal phenomena,and our previous study showed that the covalent binding of DNA probes on graphene oxide can reduce non-specific desorption,this project aims to use FAM modified ATP aptamer and Cy5 modified potassium aptamer to covalently bind on to the graphene oxide,respectively,forming the covalent molecular beacon.Through exploring the detection performance of the molecular beacons,such as the detection linear relationship,detection limits,non-specific desorption,detection specificity and stability,studing the cell uptake efficiency,the anti-degrading ability of the aptamers,intracellular fluorescence imaging,quantification of the intracellular fluorescence and in situ semiquantification of the fluorescence in living cells,and clarify the impact factors and the basic law in the detection process,thus to establish a new method to detect ATP and K+ with high specificity.Based on this,a dual-targeted molecular beacon will be developmented to simultaneous detection of ATP and K+.Through obtain the controllable preparation method,investigate the detection performance and the influencing factors, optimize the experimental conditions and reduce the mutual interference between the analytical signal,to achieve high specific and simultaneous detection of ATP and K+ in both intra- and extracellular,and to lay the foundation and provide new ideas for the design of multi-targeted molecular beacon.

本项目针对核酸适配体和氧化石墨烯的物理吸附传感器存在非特异性解吸附和假阳性信号现象，以及我们前期研究发现将DNA探针和氧化石墨烯共价结合可减少非特异性探针解吸附，提出采用FAM修饰的ATP核酸适配体和Cy5修饰的钾离子核酸适配体，分别将其共价键合至氧化石墨烯上形成共价分子信标，通过探索分子信标的检测性能，如检测线性关系、检测限、非特异性解吸附、检测特异性和稳定性，研究分子信标细胞摄取效率、适配体抗降解能力、细胞内荧光成像、细胞内荧光定量分析和细胞内荧光原位半定量分析，阐明检测过程中的影响因素和基本规律，从而建立ATP和K+特异性检测的新方法。在此基础上发展能同时检测ATP和K+的双靶向分子信标，通过获得其可控制备的方法，考察其检测性能及影响因素，优化实验条件，减少分析信号间的相互干扰，实现细胞内外ATP和K+的高特异性和同时检测，并为多靶向分子信标的设计奠定基础及提供新的思路。

近年来，氧化石墨烯（GO）物理吸附适配体探针已经成为有用的传感平台。信号由目标物诱导的探针解吸附产生。为了解决非特异性探针解吸，我们构建了用于ATP检测的共价连接的适配体探针。FAM和氨基双修饰的适配体通过EDC偶联至羧基化的GO上，制备得到的偶联效率为50±4％。我们进一步研究了共价信标的细胞外内ATP检测的线性，特异性和稳定性，并与物理吸附探针进行比较。两种传感器具有相似的灵敏度，但是共价信标对蛋白质的非特异性探针置换更具抗性。共价传感器具有0.125mM-2mM ATP的动态检测范围，并且对DNA酶具有抗降解性。用共价信标实现了细胞内ATP原位成像，而物理吸附传感器产生低得多的信号。通过采用5mM Ca2+刺激细胞产生ATP后，细胞内信号增强31.8％。这项工作显示了制备共价适配体传感器使用GO作为淬灭剂和用于细胞内代谢物检测的递送载体的优点。