ApHID: pH-Sensitive Fluorescent Dye Resistant to Photobleaching and Oxidation

Background & Unmet Need

• Human intercellular activities are often facilitated by movement of membrane-bound vesicles such as late endosomes and lysosomes (LE/Ly)
• LE/Ly dysfunction has been linked to several diseases and disorders including Alzheimer’s disease and Tay-Sachs disease
• The ability to precisely measure LE/Ly activities under different cellular environments is important for the understanding of these diseases, and their potential diagnosis
• However, existing commercial fluorescence lack the photo intensity and chemical stability in highly acidic and reactive cellular environments within LE/Ly vesicles
• Unmet Need: A fluorescence probe that is pH-sensitive and able to withstand oxidation and photobleaching while maintaining structural integrity in vivo

Technology Overview

• The Technology: Acidic pH indicator Dye (ApHID) with high resistance to oxidation and photobleaching
• ApHID is composed of a BODIPY core and determines pH of the environment using an aniline moiety that has two methyl groups attached
• Optimized for use between pH 4.0 – 6.0, ApHID’s fluorescence emission increases sharply in amplitude with increasing acidity
• ApHID has pKa of 5.4 and excitation max at 506 nm
• PoC Data: ApHID fluorescence is 12-fold greater at pH 4.0 relative to pH 6.0
• ApHID fluorescence output showed no decrease after photobleaching in live cells and decreased by only 12% in fixed cells, compared to an 83% and 82% decrease with fluorescein and Oregon Green
• ApHID exhibits the greatest fluorescent dynamic range at the physiological pH range of LE/Lys compared to currently available commercial dyes

Technology Applications

• Fluorescent dye for LE/Ly research and experiments, including pH and live cell imaging
• Labeling of Aβ aggregates to image digestive exophagy
• Tracking efficacy of drugs for neurodegenerative diseases
• Tool for cancer research and drug development

Technology Advantages

• Greater brightness and sensitivity to acidity than existing dyes
• Highly resistant to photobleaching and highly concentrated reactive oxygen species
• Stable in living cells over 15 hours+ while emitting strong fluorescent signal
• Enables detection of pH differences within cells and real-time changes in pH