Surface-enhanced Raman imaging of intracellular bioreduction of chromate in Shewanella oneidensis.
Surface-enhanced Raman imaging of intracellular bioreduction of chromate in Shewanella oneidensis.
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DOI：
10.1371/journal.pone.0016634
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发表时间：
2011-02-25
期刊：
影响因子：
3.7
通讯作者：
Irudayaraj J
中科院分区：
文献类型：
--
作者：
Ravindranath SP;Henne KL;Thompson DK;Irudayaraj J
这项研究旨在利用新型纳米粒子传感器和组成表面增强拉曼光谱(SERS)和荧光寿命成像(FLIM)的光谱工具来研究单个生物修复微生物的细胞内化学活动。最大的挑战是通过化学和终身成像来了解修复细菌坚尼希瓦氏菌MR-1还原和定位铬酸盐的机理。MR-1因其在减少多种化学和金属电子受体方面的潜力而引起了研究界的广泛兴趣。虽然存在几种破译微生物还原机制的生物分子方法，但在将研究从基于群体的研究推进到单细胞水平的传感器平台方面存在着相当大的差距。这项研究是首次尝试结合表面增强拉曼散射成像来解决这一差距。首先，我们用透射电子显微镜和荧光寿命成像技术证明了铬酸盐修饰的纳米颗粒可以被细胞摄取，以证实金纳米探针的内在化。其次，我们展示了拉曼化学成像平台在单细胞内监测铬酸盐还原和定位的实用性。铬(VI)和铬(III)的拉曼特征的显著差异使得它们能够在单个细胞内从拉曼图像中进行空间识别。对毒性和细胞干扰实验进行的全面评估揭示了这些探针的惰性，并且它们是无毒的。我们的结果强烈表明存在内部还原机制，还原发生在细胞内的特定位置，而不是像以前报道的那样发生在整个细胞的分散还原位置。虽然本研究中使用的铬酸盐修饰的金纳米传感器为跟踪细胞内和细胞表面的特定铬酸盐相互作用提供了一种改进的手段，但我们预计我们的单细胞成像工具将扩展到监测其他有毒金属物种的相互作用。
This proposed research aims to use novel nanoparticle sensors and spectroscopic tools constituting surface-enhanced Raman spectroscopy (SERS) and Fluorescence Lifetime imaging (FLIM) to study intracellular chemical activities within single bioremediating microorganism. The grand challenge is to develop a mechanistic understanding of chromate reduction and localization by the remediating bacterium Shewanella oneidensis MR-1 by chemical and lifetime imaging. MR-1 has attracted wide interest from the research community because of its potential in reducing multiple chemical and metallic electron acceptors. While several biomolecular approaches to decode microbial reduction mechanisms exist, there is a considerable gap in the availability of sensor platforms to advance research from population-based studies to the single cell level. This study is one of the first attempts to incorporate SERS imaging to address this gap. First, we demonstrate that chromate-decorated nanoparticles can be taken up by cells using TEM and Fluorescence Lifetime imaging to confirm the internalization of gold nanoprobes. Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells. Distinctive differences in Raman signatures of Cr(VI) and Cr(III) enabled their spatial identification within single cells from the Raman images. A comprehensive evaluation of toxicity and cellular interference experiments conducted revealed the inert nature of these probes and that they are non-toxic. Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported. While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species.
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