# Covalent ferrocene conjugation as an intramolecular strategy for photostability in fluorescein

## TL;DR

This paper introduces a new method to make fluorescent dyes more resistant to fading by linking them to a redox-active compound called ferrocene.

## Contribution

The study presents a novel intramolecular photostabilization strategy using covalent ferrocene conjugation to enhance fluorescence durability.

## Key findings

- The Fc–FITC conjugate showed an 11-fold increase in photobleaching half-life compared to FITC.
- The conjugate retained 94% of its initial fluorescence after 60 minutes of irradiation, versus 52% for FITC.
- The PET mechanism was confirmed to suppress the formation of the destructive triplet state.

## Abstract

Photobleaching severely limits the utility and long-term reliability of fluorescence-based measurements. To address this long-standing limitation, we synthesized a conjugate that covalently links fluorescein isothiocyanate (FITC) to ferrocene (Fc), a redox-active metallocene. Our two-step synthesis involved reduction of ferrocene methylene azide followed by formation of a stable thiourea linkage. Photophysical characterization confirmed highly efficient intramolecular quenching, evidenced by an 81.5% reduction in quantum yield (Φ) and a shortened lifetime (τ = 3.2 ns vs. 4.1 ns for FITC). The Fc–FITC conjugate exhibited an 11-fold increase in photobleaching half-life (693 vs. 63 min for FITC), retaining 94% of its initial fluorescence after 60 min of constant 23 mW/cm2 irradiation, compared to only 52% for FITC. Direct singlet oxygen (1O2​) quantification using Singlet Oxygen Sensor Green (SOSG) confirmed that Fc conjugation reduces the photosensitization rate to only 28% of that of native FITC. Sodium azide (NaN3​) quenching assays further validated the suppression of reactive oxygen species (ROS), as the Fc–FITC system exhibited negligible quenching (4.6%) compared to the significant response of native FITC (32.5%). This stabilization arises from a Photoinduced Electron Transfer (PET) mechanism that suppresses formation of the destructive triplet state (T1​). A quantitative Rehm–Weller analysis (ΔGPET ≈ − 0.76 eV) and direct ROS validation establish a robust mechanistic basis for this photoprotective effect. Together, these findings establish a unique intramolecular photostabilization strategy where signal durability and quantitative precision are prioritized over peak brightness, offering a framework for designing robust hybrid redox–fluorophore probes suited for persistent sensing and long-term quantitative analysis.

The online version contains supplementary material available at 10.1038/s41598-025-34817-3.