Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University , Chongqing 400715, People's Republic of China.
Anal Chem. 2017 Mar 7;89(5):2866-2872. doi: 10.1021/acs.analchem.6b04082. Epub 2017 Feb 13.
In this work, an "off" to "on" surface-enhanced Raman spectroscopy (SERS) platform was constructed for ultrasensitive detection of microRNA (miRNA) by using a magnetic SERS substrate (Co@C/PEI/Ag) and padlock probe-based exponential rolling circle amplification (P-ERCA) strategy. Herein, miRNA 155 could act as primers to initiate rolling circle amplification (RCA) for producing a long repeat sequence, and then the obtained DNA would be cleaved into two kinds of single-stranded DNAs in the presence of nickase. One of the DNAs can be a new primer to initiate new cycle reactions for obtaining large numbers of the other one (trigger DNA), consequently leading to an exponential amplification. On the other hand, the hairpin DNA (H1), with a Raman label (Cy5) at one end, would form a hairpin structure to make the Cy5 closer to the SERS substrates, which could produce a strong SERS signal ("on" status). Then placeholder DNA (P2) partly hybridized with H1 to open the hairpin structure making Cy5 far away from substrates with a decreased signal ("off" status). Next, the obtained trigger DNA can complement with P2 to make the Raman label reclosed to the SERS substrates with a strong SERS signal ("on" status). From this principle, the strategy could achieve the change from "off" to "on" status. This SERS strategy exhibited a wide linear range of 100 aM to 100 pM with a low detection limit of 70.2 aM, which indicated the proposed SERS platform has potential application value for ultrasensitive bioassay of miRNA.
在这项工作中，构建了一种“关闭”到“开启”表面增强拉曼光谱（SERS）平台，用于通过使用磁性 SERS 基底（Co@C/PEI/Ag）和基于发夹探针的指数滚环扩增（P-ERCA）策略超灵敏检测 microRNA（miRNA）。在这里，miRNA 155 可以作为引物启动滚环扩增（RCA）以产生长重复序列，然后在核酸内切酶存在下，所得 DNA 会被切割成两种单链 DNA。其中一种 DNA 可以作为新的引物，启动新的循环反应，获得大量另一种（触发 DNA），从而导致指数扩增。另一方面，发夹 DNA（H1）的一端带有拉曼标记（Cy5），会形成发夹结构，使 Cy5 更接近 SERS 基底，从而产生强 SERS 信号（“开启”状态）。然后，占位 DNA（P2）部分与 H1 杂交以打开发夹结构，使 Cy5 远离基底，信号减弱（“关闭”状态）。接下来，获得的触发 DNA 可以与 P2 互补，使拉曼标记重新闭合到 SERS 基底，产生强 SERS 信号（“开启”状态）。基于此原理，该策略可以实现从“关闭”到“开启”状态的转变。该 SERS 策略表现出 100 aM 至 100 pM 的宽线性范围，检测限低至 70.2 aM，表明所提出的 SERS 平台具有用于超灵敏生物测定 miRNA 的潜在应用价值。
