State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun 130022, People's Republic of China.
Anal Chem. 2014 May 6;86(9):4371-8. doi: 10.1021/ac500195u. Epub 2014 Apr 15.
A new label-free strategy for sensitive fluorometric biosensing based on perylene probe monomer-excimer transition has been developed. A negatively charged perylene probe (compound 1) was used. Compound 1 shows strong monomer fluorescence in an aqueous buffer solution. A cationic polymer could induce aggregation of compound 1 through noncovalent interactions. Compound 1 monomer emission was quenched, and meanwhile strong excimer emission was observed. Upon addition of a single-stranded DNA (an anionic polymer), strong electrostatic attractive interactions between the cationic polymer and the DNA weakened the binding of aggregates of compound 1 to the polycation. Compound 1 monomers were released, and excimer-monomer emission transition was detected. This observation formed the basis for DNA methyltransferase (MTase) activity detection. When the 3'-OH terminus of a duplex DNA was removed, the DNA strands could not be elongated by terminal deoxynucleotidyl transferase (TdT), and little restoration of compound 1 monomer emission was detected. However, in the presence of MTase and endonuclease, the DNA could be specifically methylated and then cleaved into single-stranded fragments. The DNA fragments contained newly generated 3'-OH termini, which could be elongated by TdT. An excimer-monomer transition signal could then be detected. Simple, sensitive, selective, and inexpensive sensing of DNA methylation was therefore established.
基于苝探针单体-二聚体转变的灵敏荧光无标记策略已被开发用于敏感的荧光生物传感。使用带负电荷的苝探针（化合物 1）。在水性缓冲溶液中，化合物 1 显示出强的单体荧光。阳离子聚合物可以通过非共价相互作用诱导化合物 1 的聚集。化合物 1 单体发射被猝灭，同时观察到强的二聚体发射。当加入单链 DNA（带负电荷的聚合物）时，阳离子聚合物和 DNA 之间的强静电吸引相互作用减弱了化合物 1 聚集物与聚阳离子的结合。化合物 1 单体被释放，并且观察到二聚体-单体发射转变。这一观察结果构成了 DNA 甲基转移酶（MTase）活性检测的基础。当双链 DNA 的 3'-OH 末端被去除时，末端脱氧核苷酸转移酶（TdT）不能延伸 DNA 链，并且检测到化合物 1 单体发射的恢复很少。然而，在 MTase 和内切酶存在的情况下，DNA 可以被特异性甲基化，然后被切割成单链片段。这些 DNA 片段包含新生成的 3'-OH 末端，可以被 TdT 延伸。然后可以检测到二聚体-单体转变信号。因此，建立了简单、灵敏、选择性和廉价的 DNA 甲基化传感方法。
