MicroRNAs (miRNAs) have increasingly become an important biomarker target for applications ranging from clinical diagnostics to biofuel production monitoring. However, the current state of the art for the detection of such markers requires tedious processing and amplification techniques such as polymerase chain reaction (PCR). In an effort to create a relatively simple biosensing platform, we have developed a combined plasmonic biosensing method based on a Surface-Enhanced Raman Spectroscopy (SERS) platform called the inverse Molecular Sentinel (iMS) to directly detect in vivo miRNA such as miR858a. With Shifted Excitation Raman Difference Spectroscopy (SERDS), we can remotely detect these targets in the field in the presence of interfering background signal. The application of such technology can pave the way not just for biofuel monitoring but early and non-invasive disease detection and diagnostics.
Plant biotechnology and biofuel research is critical in addressing increasing global demands for energy. Further understanding of biomass producing associated metabolic pathways in plants can be used to exploit and increase the production of biomass for energy purposes. In vivo detection of biomarkers associated with plant growth for bioenergy has proved to be limited due to complex sample preparation required by traditional methods. In addition, genetic transformation and biomolecule monitoring inside plant cells is regulated by diameter and size exclusion limits of the plant cell wall (5 - 20 nm). Currently limited methods exist for enabling direct entry into plant cells. Moreover, these methods, such as biolistic particle delivery and electroporation use mechanical force that causes damages to the plant tissue. Nanoparticles could serve as promising platforms for probes to characterize intercellular and intracellular plant biomarkers and pathways. Bi-metallic nanostars are a plasmonics-active nanoplatform capable of high surface-enhanced Raman scattering (SERS) which can enter plant cells and have the future potential for nucleic acid sensing. Imaging technologies such as SERS mapping, confocal imaging, X-ray fluorescence imaging, multi-photon imaging, and transmission electron microscopy have been utilized to determine the compartmentalization and location of the SERS iMS biosensors inside Arabidopsis plants.
Our group has integrated surface-enhanced Raman scattering (SERS) silver coated gold nanostars on an optical fiber. Fiber-based sensors are an in-situ technology that can simultaneously bring the sensor and light to the sample without disturbing the environment. This technology is a multi-use method that does not require complex sample preparation. Fiber sensors or optrodes, enable the detection of analytes in samples that are difficult to access. Additionally, optrodes allow for specific detection while evading background signals from non-target regions. The fiber-optrode was used to detect miRNA and illegal food additives.
Further understanding of biomass producing associated metabolic pathways in plants can be used to increase the production of biomass. In vivo detection of these markers has proved to be limited due to complex sample preparation required by traditional methods. Recently the Vo-Dinh group has designed a platform to detect nucleic acid targets in biological systems called inverse molecular sentinels which utilize surface-enhanced Raman scattering. These multimodal probes were shown to detect and image key microRNA within whole plants in vivo. This work lays the foundation for detecting and imaging biological markers in plants with enhanced spatial and temporal resolution.
Molecular analysis has revolutionized many applications, including bio-safety, bio-engineering and biofuel research; however, there are limited practical tools for in situ detection during field work. New technology is needed to translate molecular advances from laboratory settings into the practical realm. The unique characteristics of plasmonic nanosensors have made them ideal candidates for field-ready sensing applications. Herein, we discuss the development of a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of miRNA targets, which are detected by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars to detect target nucleic acids using the surface-enhanced Raman scattering sensing mechanism of nanoprobes referred to as inverse molecular sentinels. The fiber sensors were tested in extracts from leaves of plants that were induced to have different miRNA expression levels. The results indicate that the fiber sensors developed have the potential to be a powerful tool for field analysis.
Gene expression monitoring within whole plants is critical for many applications ranging from plant biology to biofuel development. Herein, we report a unique multimodal method for in vivo imaging and biosensing of nucleic acid biotargets, specifically microRNA, within whole plant leaves by integrating three complementary techniques: surface-enhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The method described utilizes plasmonic nanostar-based inverse molecular sentinel (iMS) nanoprobes, which not only provide large Raman signal enhancement upon target binding, but also allow for localization and quantification by XRF and plasmonics-enhanced TPL. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants.
The ability to monitor gene expression within living plants is of importance in many applications ranging from plant biology research to biofuel development; however, no method currently exists without requiring sample extraction. Herein, we report a multimodal imaging method based on plasmonic nanoprobes for in vivo imaging and biosensing of microRNA biotargets within whole plant leaves. This method integrates three different but complementary techniques: surfaceenhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The multimodal method utilizes 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. For the sensing mechanism, inverse molecular sentinel (iMS) nanoprobes are used 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. 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 allow the study of these biotargets with previously unmet spatial and temporal resolution.
The knowledge over gene expression dynamics and location in plants is crucial for applications ranging from basic biological research to agricultural biotechnology (e.g., biofuel development). However, current methods cannot provide in vivo dynamic detection of genomic targets in plants. This limitation is due to the complex sample preparation needed by current methods for nucleic acids detection, which disrupt spatial and temporal resolution. We report the development of a unique multimodal method based on plasmonics-active nanoprobes, referred to inverse molecular sensitnels capable of in vivo imaging and biosensing of microRNA biotargets within whole plant using surface-enhanced Raman scattering (SERS) detection. This work lays the foundations for in vivo functional imaging of RNA biotargets in plants with previously unmet spatial and temporal resolution for many applications ranging from agricultural biotechnology to biofuel research.
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