Work

High-Sensitivity, Dual Ratiometric and Turn-on ABATaRs for Multiplexed Temporal Imaging of Ferroptosis Hallmarks in Living Cells

Year: 2026

Type: preprint

Abstract: We present a computationally-guided workflow for designing dual turn-on and ratiometric Raman sensors. The workflow has been used to rationally design activity-based alkyne tagged Raman, ABATaR , sensors that overcome the inherently low Raman sensitivity via strategic placement of electron-donating and electron-withdrawing groups. An ABATaR sensing scaffold designed via the workflow afforded ~ 9000 times higher Raman scattering cross-section than the dimethylsulfoxide C-H stretching-mode. The sensitivity of the scaffold was compared to that of 5-ethynyl-2'-deoxyuridine ( EdU ) to determine Raman intensity versus EdU , RIE, a parameter used to benchmark Raman probes. An RIE value of 170 was obtained which is one of the highest amongst previously reported monoyne and diyne-based Raman probes. The sensitive ABATaR -scaffold was developed into a dual turn-on and ratiometric Fe 2+ -sensor that was applied to distinctly quantify endogenous cellular labile Fe 2+ ion levels. In a key result, live-cell, temporal imaging with the Fe 2+ - ABATaR sensor revealed that intracellular labile Fe 2+ levels progressively increase with ferroptosis while the total Fe-level remains constant. To demonstrate the generalizability of the design workflow, we synthesized an orthogonal ABATaR -sensor for H 2 O 2 . The H 2 O 2 -sensor was applied together with the Fe 2+ -sensor along with inherent Raman peaks for cytochromes to enable simultaneous, multiplexed spontaneous Raman imaging of three distinct ferroptosis hallmarks—labile Fe 2+ , H 2 O 2 , and redox-sensitive cytochrome Raman signals—in living cells. Importantly, the ability to pre-design orthogonal ratiometric Raman sensors and simultaneously track three regulators of ferroptosis via discrete non-overlapping Raman peaks highlights the tremendous potential of Raman sensors toward attaining live cell multiplexed imaging and quantification.

Source: ChemRxiv

DOI: 10.26434/chemrxiv.15010169/v1

Authors: Sujit K Das, Heqi Xi, Saurabh Kumar, Katsumasa Fujita, Ankona Datta

Institutions: Tata Institute of Fundamental Research, The University of Osaka, Institute for Open and Transdisciplinary Research Initiatives

Language: English

Topic: Molecular Sensors and Ion Detection

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funders: Tata Institute of Fundamental Research, Department of Atomic Energy, Government of India

Awards: G2555966067, G3584033455

OpenAlex ID: W7221105602

Cites:

Cited by:

Related to: 10