Bulgarian Academy of Sciences , Institute of Chemical Engineering , Sofia 1113 , Bulgaria.
Biosciences Division , Argonne National Laboratory , Argonne , Illinois 60439 , United States.
ACS Appl Mater Interfaces. 2019 Feb 27;11(8):7743-7754. doi: 10.1021/acsami.8b19977. Epub 2019 Feb 12.
Monitoring gene expression within whole plants is critical for many applications ranging from plant biology to agricultural biotechnology and biofuel development; however, no method currently exists for in vivo monitoring of genomic targets in plant systems without requiring sample extraction. Herein, we report a unique multimodal method based on plasmonic nanoprobes capable of in vivo imaging and biosensing of microRNA biotargets within whole plant leaves by integrating three different and complementary techniques: surface-enhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The method developed uses plasmonic nanostars, which not only provide large Raman signal enhancement but also allow for localization and quantification by XRF and plasmonics-enhanced TPL, owing to gold content and high two-photon luminescence cross sections. Our method uses inverse molecular sentinel nanoprobes for SERS bioimaging of microRNA within Arabidopsis thaliana leaves to provide a dynamic SERS map of detected microRNA targets while also quantifying nanoprobe concentrations using XRF and TPL. The nanoprobes were observed to occupy the intercellular spaces upon infiltration into the leaf tissues. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants, a tool that will revolutionize bioengineering research by allowing the study of these biotargets with previously unmet spatial and temporal resolution, 200 μm and 30 min, respectively.
监测整个植物中的基因表达对于从植物生物学到农业生物技术和生物燃料开发的许多应用都至关重要；然而，目前尚无无需提取样本即可在植物系统中对基因组靶标进行体内监测的方法。在此，我们报告了一种独特的基于等离子体纳米探针的多模态方法，该方法能够通过整合三种不同且互补的技术：表面增强拉曼散射（SERS）、X 射线荧光（XRF）和等离子体增强双光子荧光（TPL），对整个植物叶片中的 microRNA 生物靶标进行体内成像和生物传感。所开发的方法使用等离子体纳米星，由于金含量和高光子双光子荧光截面，它们不仅提供了大的拉曼信号增强，而且还允许通过 XRF 和等离子体增强 TPL 进行定位和定量。我们的方法使用反向分子哨兵纳米探针对拟南芥叶片中的 microRNA 进行 SERS 生物成像，以提供检测到的 microRNA 靶标的动态 SERS 图谱，同时还使用 XRF 和 TPL 定量纳米探针浓度。渗透到叶片组织中后，观察到纳米探针占据了细胞间隙。该报告为在整个植物中使用等离子体纳米探针进行核酸生物靶标的体内功能成像奠定了基础，这一工具将通过允许以前无法满足的空间和时间分辨率（分别为 200μm 和 30min）来研究这些生物靶标，从而彻底改变生物工程研究。
