Imaging of intracellular protein aggregates through plasmon-assisted clusteroluminescence.

The formation of clusters in non-aromatic molecules can give rise to unconventional luminescence or clusteroluminescence. Typically containing heteroatoms without extended conjugation or aromatic rings, these molecules have drawn much attention owing to the prospects of label-free biological imaging. However, their applications have been limited due to the lack of knowledge of the underlying mechanism. Herein, we have elucidated the mechanism of clusteroluminescence from proteins, which were explicitly aggregated using plasmonic silver nanoparticles. The nanoparticles promoted protein aggregation and induced nitrile formation on the surface, which, along with other lone-pair-containing heteroatoms, contributed to enhanced emission in the visible range. Remarkably, this makes imaging of proteins possible with visible excitations, as co-factor-lacking proteins generally undergo electronic transitions only in the ultraviolet range. Furthermore, the inherent protein-aggregating behaviour of plasmonic nanoparticles was harnessed for imaging of intracellular Huntingtin protein aggregates overexpressed in HeLa cells through clusteroluminescence. Significant plasmon-enhanced and red-shifted fluorescence emission was observed, which helped in the imaging and localization of the intracellular aggregates. Density functional theory calculations and transient absorbance spectroscopy were used to probe the molecular interactions at the protein-nanoparticle interface and the charge transfer states, further elucidating the role of nanoparticles and the emission mechanism. This technique thus opens alternate avenues for label-free fluorescence bioimaging.

Bridging Soft Interaction and Excluded Volume in Crowded Milieu through Subtle Protein Dynamics.

The impact of macromolecular crowding on biological macromolecules has been elucidated through the excluded volume phenomenon and soft interactions. However, it has often been difficult to provide a clear demarcation between the two regions. Here, using temperature-dependent dynamics (local and global) of the multidomain protein human serum albumin (HSA) in the presence of commonly used synthetic crowders (Dextran 40, PEG 8, Ficoll 70, and Dextran 70), we have shown the presence of a transition that serves as a bridge between the soft and hard regimes. The bridging region is independent of the crowder identity and displays no apparent correlation with the critical overlap concentration of the polymeric crowding agents. Moreover, the dynamics of domains I and II and the protein gating motion respond differently, thereby bringing to the fore the asymmetry underlying the crowder influence on HSA. In addition, solvent-coupled and decoupled protein motions indicate the heterogeneity of the dynamic landscape in the crowded milieu. We also propose an intriguing correlation between protein stability and dynamics, with increased global stability being accompanied by eased local domain motion.

Photoactivated plasmonic nanohybrid fibers with prolonged trapping of excited charge carriers for SERS analysis of biomolecules.

The quest to enhance Raman spectroscopic signals through the rational design of plasmonic substrates has enabled the detection and characterization of pharmaceutically important molecules with low scattering cross-sections, such as amino acids and proteins, and is helping in making forays into the diverse field of biomedical sciences. This work presents a simple strategy for synthesizing silver nanoparticles-incorporated alumina nanofibers (Ag-AlNFs) utilizing controlled microwave synthesis for enhancing the surface-enhanced Raman chemical enhancement factor through photo-induced charge accumulation at the plasmonic-dielectric interface. The plasmonic-dielectric fibers serve as excellent charge carrier trappers, as evident from the ultrafast transient absorption spectroscopy studies. Apart from chemical enhancement, the increase in electronic surface charge also enables the protein disulfide bonds to capture these electrons and form a transient disulfide electron adduct radical, which converts to free thiol radical on dissociation. This allows protein molecules to bind to the nanoparticle's surface with the favorable silver thiol bond leading to greater surface affinity and larger SERS enhancement. The proposed Ag-AlNFs represent a cost-effective material that can be potentially used to probe biological systems in a label-free manner by photoactivating the SERS substrate for obtaining higher enhancement factors.