Multiparametric microenvironment sensing via distinct molecular equilibria in a single cyanine dye
- 1Department of Chemistry, New York University, New York, New York, 10003, United States
Abstract
Reading both physical and chemical properties of a microenvironment from a single fluorophore remains a challenge. Here we demonstrate that two coexisting molecular equilibria within one near-infrared cyanine, CyC4, encode two mechanistically distinct ratiometric reporting channels. A meso-amino group and a pendant carboxylate form a tunable intramolecular hydrogen bond that toggles the dye between closed (700 nm) and open (780 nm) emissive conformers. Time-dependent density functional theory (TD-DFT) calculations show that the hydrogen bond raises the LUMO and blue-shifts the emission, establishing the 700/780 emission ratio as a local reporter of hydrogen bonding and polarity. Independently, the chromophore self-associates under crowding- and cosolvent-rich conditions into an aggregate with a blue-shifted, H-type absorption signature near 530-540 nm and a distinct emission near 610 nm upon 540 nm excitation. The intensity of this aggregate band relative to the monomer emission (Ra) serves as a ratiometric reporter of crowding and self-association. Because the two channels arise from distinct molecular equilibria (intramolecular hydrogen bonding vs. intermolecular self-association) they are largely decoupled: a glycerol titration series confirms that the self-association channel (Ra) can be moved while the hydrogen-bonding channel stays essentially fixed. Applied to protein-PEG biomolecular condensates, the two ratios move oppositely with increasing salt, showing that the interior's chemical (polarity, hydrogen bonding) and physical (packing, self-association) environments co-vary across the salt series; a single CyC4 measurement thereby maps this coupled microenvironment, providing a general strategy for multiparametric, ratiometric sensing of crowded microenvironments.
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BibTeX
@misc{bais2026multiparametric,
title = {{Multiparametric microenvironment sensing via distinct molecular equilibria in a single cyanine dye}},
author = {S. Bais and S. Westrey and C. Samaniego Lopez and M. V. Rivas and C. C. Spagnuolo and S. Saurabh},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.29.747692},
url = {https://www.biorxiv.org/content/10.64898/2026.08.29.747692v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Multiparametric microenvironment sensing via distinct molecular equilibria in a single cyanine dye
AU - S. Bais
AU - S. Westrey
AU - C. Samaniego Lopez
AU - M. V. Rivas
AU - C. C. Spagnuolo
AU - S. Saurabh
PY - 2026
DA - 2026/08/29
PB - bioRxiv
DO - 10.64898/2026.08.29.747692
UR - https://www.biorxiv.org/content/10.64898/2026.08.29.747692v1
AB - Reading both physical and chemical properties of a microenvironment from a single fluorophore remains a challenge. Here we demonstrate that two coexisting molecular equilibria within one near-infrared cyanine, CyC4, encode two mechanistically distinct ratiometric reporting channels. A meso-amino group and a pendant carboxylate form a tunable intramolecular hydrogen bond that toggles the dye between closed (700 nm) and open (780 nm) emissive conformers. Time-dependent density functional theory (TD-DFT) calculations show that the hydrogen bond raises the LUMO and blue-shifts the emission, establishing the 700/780 emission ratio as a local reporter of hydrogen bonding and polarity. Independently, the chromophore self-associates under crowding- and cosolvent-rich conditions into an aggregate with a blue-shifted, H-type absorption signature near 530-540 nm and a distinct emission near 610 nm upon 540 nm excitation. The intensity of this aggregate band relative to the monomer emission (Ra) serves as a ratiometric reporter of crowding and self-association. Because the two channels arise from distinct molecular equilibria (intramolecular hydrogen bonding vs. intermolecular self-association) they are largely decoupled: a glycerol titration series confirms that the self-association channel (Ra) can be moved while the hydrogen-bonding channel stays essentially fixed. Applied to protein-PEG biomolecular condensates, the two ratios move oppositely with increasing salt, showing that the interior's chemical (polarity, hydrogen bonding) and physical (packing, self-association) environments co-vary across the salt series; a single CyC4 measurement thereby maps this coupled microenvironment, providing a general strategy for multiparametric, ratiometric sensing of crowded microenvironments.
ER -