Ratiometric Near-Infrared Probes Utilizing BODIPY Dyes for Viscosity and Protein Aggregation Sensing in Live Cells - This proposal aims to advance the detection of viscosity changes and protein aggregation in live cells through the development of sophisticated, self-calibrating near-infrared fluorescent probes. These innovative probes will quantitatively assess microviscosity variations and monitor protein misfolding and aggregation within essential organelles, including mitochondria, the endoplasmic reticulum, and lysosomes. To ensure optimal performance, our probes will be engineered for enhanced water solubility, stability, and cell permeability, thereby maximizing biocompatibility and selectivity for detecting both viscosity changes and protein aggregation. Utilizing near- infrared imaging (650–860 nm) will facilitate deep tissue penetration, minimize cellular damage, and reduce biological autofluorescence, resulting in clearer and more precise imaging. The implementation of ratiometric imaging, which capitalizes on pseudo-Stokes shifts and dual emissions, will further mitigate errors related to excitation light and scattered fluorescence, yielding reliable quantitative data. A pivotal innovation of this system is its self-calibrating ratiometric responses, designed to overcome the limitations inherent in intensity-based probes, such as fluctuations in excitation light, sample heterogeneity, and uneven probe distribution. This feature will enable accurate diagnosis and monitoring of diseases associated with altered cellular microenvironments and protein homeostasis. Our probes will specifically target biomarkers pertinent to neurodegenerative diseases and cancer. In neurodegenerative contexts, they will be responsive to oxidative stress, pH changes, and protein aggregation associated with conditions such as Alzheimer’s and Parkinson’s diseases. For cancer applications, the probes will detect hypoxia, acidic environments, oxidative stress, and increased viscosity, allowing for real- time imaging of critical disease markers. Additionally, this project will explore microviscosity dynamics, protein folding, and aggregation in live cells under various conditions. We will investigate how glycerol concentrations and protein aggregation influence the probes’ optical properties, emphasizing their sensitivity and specificity. Molecular dynamic studies on proteins amyloid-β (Alzheimer’s) and α-synuclein (Parkinson) and computational studies on the ratiometric fluorescent probes will provide a theoretical understanding of the experimental results and suggest the nature of the conformational changes. The study will also assess the effects of chemical interventions, including cancer therapeutics and oxidative stressors, while evaluating the roles of protein chaperonins and co-chaperones in ensuring proper protein folding and preventing aggregation. Ultimately, this research aims to uncover valuable insights into the diagnostic potential of abnormal cellular viscosity dynamics and protein aggregation in various disease processes. By enhancing early diagnosis and therapeutic monitoring of conditions such as cancer and neurodegenerative diseases, our pioneering approach will make significant contributions to the fields of cellular imaging and disease diagnostics.
