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<p>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&#xa0;min for FITC), retaining 94% of its initial fluorescence after 60&#xa0;min of constant 23 mW/cm<sup>2</sup> irradiation, compared to only 52% for FITC. Direct singlet oxygen (<sup>1</sup>O<sub>2</sub>​) 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 (NaN<sub>3</sub>​) 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 (T<sup>1</sup>​). A quantitative Rehm–Weller analysis (ΔG<sub>PET</sub> ≈ − 0.76&#xa0;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.</p>

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Covalent ferrocene conjugation as an intramolecular strategy for photostability in fluorescein

  • 期刊论文

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  • 发表日期：

    2026年1月7日

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  • Gilbert K. Kosgei,
  • P. U. Ashvin Iresh Fernando,
  • Harley R. Mcalexander,
  • Afrachanna D. Butler

摘要

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.