SERS Nanoprobe for Intracellular Monitoring of Viral Mutations
SERS Nanoprobe for Intracellular Monitoring of Viral Mutations
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DOI：
10.1021/acs.jpcc.9b09253
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发表时间：
2020-02-06
影响因子：
3.7
通讯作者：
Fabris, Laura
中科院分区：
文献类型：
--
作者：
Dardir, Kholud;Wang, Hao;Fabris, Laura
利用表面增强拉曼光谱（Sers）技术，将3'末端带有拉曼活性荧光染料的巯基化DNA发夹功能化的金纳米星用于鉴定和定量甲型流感病毒（IAV）基因组中的RNA突变。DNA发夹结构被设计成在病毒RNA靶的不存在/存在下选择性地延伸/折叠，导致荧光团远离或靠近金纳米星表面，导致Sers信号的“关-开”切换。首先在缓冲液中进行了可切换Sers nanostar探针的验证，表明检测是序列特异性的，并且这些Sers探针提供的高灵敏度允许在单个颗粒水平上进行目标检测。我们还证明了信号恢复的程度可以与基因突变的数量密切相关。用掺有RNA寡核苷酸的HeLa细胞裂解物进行的进一步实验证明，这些纳米探针的功能性不受复杂基质的影响。作为概念证明，我们还通过特异性靶向用编码HA或另外两个IAV片段PB 1和PB 2的质粒转染的活HeLa细胞中的血凝素（HA）片段作为阴性对照，在体外测试了这些纳米颗粒探针。单个转染的HeLa细胞中的细胞内Sers响应证明了HA片段的探针的高序列选择性，表明这些探针用于多重检测和定量单个细胞中的病毒RNA的方法的适用性，该方法可以解释病毒群体的多样性。这也代表了第一次基于分子信标的Sers探针已被用于检测完整个体细胞中的病毒RNA靶。
Gold nanostars, functionalized with thiolated DNA hairpins bearing a Raman-active fluorescent dye at the 3' terminus, were engineered to identify and quantify RNA mutations in the influenza A virus (IAV) genome employing surface enhanced Raman spectroscopy (SERS). The DNA hairpin structure was designed to selectively extend/fold in the absence/presence of the viral RNA targets, resulting in the fluorophore being brought away from or close to the gold nanostar surface, leading to an "OFF-ON" switching of the SERS signal. Validation of the switchable SERS nanostar probes was first carried out in buffer, showing that the detection is sequence-specific and that the high sensitivity provided by these SERS probes allows target detection at the single particle level. We also demonstrate that the degree of signal recovery can be closely correlated with the number of genetic mutations. Further experiments carried out with HeLa cell lysate spiked with RNA oligonucleotides demonstrate that the functionality of these nanoprobes were not detrimentally affected by the complex matrix. As a proof of concept, we also tested these nanoparticle probes in vitro by specifically targeting the hemagglutinin (HA) segment in live HeLa cells transfected with plasmids coding for either HA or two other IAV segments, PB1 and PB2, as negative controls. The intracellular SERS response in individual transfected HeLa cells demonstrates high sequence-selectivity of the probes for the HA segment, suggesting the applicability of these probes for multiplexed detection and quantification of viral RNAs in individual cells with an approach that can account for the viral population diversity. This also represents the first time that molecular beacon-based SERS probes have been employed to detect viral RNA target in intact individual cells.