SERS combined with PCR as a potent tool for detecting mutations: a case study of tomato plants.

Conventional methods of detecting economically essential mutations have several disadvantages. Even though fluorescence-based methods are still the best option, Surface-Enhanced Raman Spectroscopy (SERS) may soon emerge as an alternative to the current techniques for detecting these mutations, because of its ability to detect molecular vibrational signatures. We were able to identify and develop a PCR-based SERS assay that can relentlessly differentiate between different types of indels, and SNPs as demonstrated in the case of tomato genome related to tomato yellow leaf curl virus and root-knot nematodes, diseases that are economically significant to the global agriculture industry and where the selection of resistant crops is the best solution. This tri-primer assay utilizes mutation-specific forward primers and SERS probes tagged with FAM and Cy3 dyes, specific for each allele of a particular gene (Ty-3 and Mi-1). The unique Raman spectral features of these dyes enabled to perform of multiplexing, which made it possible to detect not only the indel type but also the zygosity in a single experiment. Moreover, this technique successfully differentiated between two different SNP-based alleles. Therefore, due to its efficient multiplexing capability and lack of the need for quenchers, it has the potential to become a powerful onsite and offsite screening tool in the not-too-distant future.

Insights on Aggregation of Hen Egg-White Lysozyme from Raman Spectroscopy and MD Simulations.

Protein misfolding and aggregation play a significant role in several neurodegenerative diseases. In the present work, the spontaneous aggregation of hen egg-white lysozyme (HEWL) in an alkaline pH 12.2 at an ambient temperature was studied to obtain molecular insights. The time-dependent changes in spectral peaks indicated the formation of ß sheets and their effects on the backbone and amino acids during the aggregation process. Introducing iodoacetamide revealed the crucial role of intermolecular disulphide bonds amidst monomers in the aggregation process. These findings were corroborated by Molecular Dynamics (MD) simulations and protein-docking studies. MD simulations helped establish and visualize the unfolding of the proteins when exposed to an alkaline pH. Protein docking revealed a preferential dimer formation between the HEWL monomers at pH 12.2 compared with the neutral pH. The combination of Raman spectroscopy and MD simulations is a powerful tool to study protein aggregation mechanisms.

Surface-Enhanced Raman Spectroscopy as a Tool for Distinguishing Extracellular Vesicles under Autophagic Conditions: A Marker for Disease Diagnostics.

Extracellular vesicles (EVs) laden with lipids, proteins, DNA, and micro-RNAs play important biological functions in intercellular communication and have pivotal roles in pathophysiological conditions. Characterization of the EVs has always been a multistep process involving large volumes, and they are heterogeneous in size and properties. A multitude of approaches is used to distinguish the EVs. Here, we report simple citrate reduced silver nanoparticles assisted surface-enhanced Raman spectroscopy (SERS) as a tool to distinguish EVs extracted from several cell lines isolated under autophagic conditions (nitrogen starvation). This study is the first report of its kind in characterizing EVs from cells under autophagic conditions using SERS. We used two cancerous cell lines, HeLa, its corresponding autophagy-deficient cell line (Atg5-/-), and a noncancerous cell line, HEK293, to isolate EVs. Our study helps in the facile detection and differentiation of EVs isolated between two closely related human cell lines that differ by their autophagic ability. The principal component analysis (PCA) of the SERS spectra of these EVs consistently showed the presence of distinct chemical compositions of the EVs. SERS of EVs can help in probing more into the molecular level information from EVs and could become a powerful tool once coupled with improved microscopy techniques for diagnosis and therapy.