A Recyclable Magnetic‑Silica Hybrid Probe for Fe³⁺ Detection and Live‑Cell Imaging

Abstract

We engineered a multifunctional BHN‑Fe₃O₄@SiO₂ nanostructure that selectively quenches fluorescence upon binding Fe³⁺. The probe is magnetically recoverable and fully reversible with EDTA, enabling repeated use. It offers a linear response from 0 to 20 µM with a detection limit of 1.25 × 10⁻⁸ M. Importantly, the nanostructure images Fe³⁺ in live HeLa cells, underscoring its biocompatibility and potential for bioimaging applications.

Background

Iron (Fe³⁺) is essential for myriad cellular processes—brain function, gene transcription, immune defense, and reproduction. Imbalances in Fe³⁺ levels are linked to metabolic disorders and neurodegenerative diseases. Conventional detection techniques include atomic absorption and chromatography, but fluorescence sensing stands out for its simplicity, sensitivity, and low detection limits.

Typical organic fluorophores, however, suffer from toxicity, limited recyclability, and poor reusability. Incorporating inorganic supports—especially magnetic Fe₃O₄@SiO₂ nanoparticles—addresses these shortcomings. Fe₃O₄ cores provide superparamagnetism for easy magnetic separation, while SiO₂ shells offer low toxicity, high biocompatibility, and ample surface area for functionalization.

Methods

Synthesis of Fe₃O₄@SiO₂ Nanoparticles

Fe₃O₄ cores (10–15 nm) were coated with a thin amorphous SiO₂ shell (average diameter 50–60 nm) via a modified Stöber process using TEOS in an ethanol/water mixture. The resulting core–shell particles served as the scaffold for the fluorescent probe.

Synthesis of BHN‑Fe₃O₄@SiO₂

The 1,8‑naphthalimide derivative BHN was first synthesized (see literature) and then reacted with 3‑isocyanatopropyl‑triethoxysilane (IPTES) to generate BHN‑IPTES. The silane-functionalized probe was grafted onto the SiO₂ surface via a hydrolysis–condensation reaction in anhydrous toluene at 110 °C under N₂. Excess BHN‑IPTES was removed by centrifugation and ethanol washes, yielding the final BHN‑Fe₃O₄@SiO₂ nanostructure.

Characterization

Transmission electron microscopy confirmed a well‑defined core–shell architecture. X‑ray diffraction showed the characteristic magnetite peaks, while a broad amorphous SiO₂ band appeared between 20° and 30°. FT‑IR spectra revealed Si–O–Si vibrations and the aromatic/alkyl C–H stretches of BHN, confirming successful grafting. Thermogravimetric analysis estimated a BHN loading of 3.34 × 10⁻⁵ M per gram of probe. Vibrating sample magnetometry demonstrated superparamagnetic behavior (saturation magnetization ≈ 4.0 emu g⁻¹) and negligible coercivity, enabling rapid magnetic recovery.

Fluorescence Response

In a 1:1 CH₃CN/H₂O buffer (pH 7.36) at 0.2 g L⁻¹, the probe exhibited intense emission at 518 nm upon 415 nm excitation. Addition of Fe³⁺ (5 × 10⁻⁵ M) caused immediate fluorescence quenching within 2 min, whereas other tested metal ions (Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, K⁺, Li⁺, Mg²⁺, Mn²⁺, Na⁺, Pb²⁺, Zn²⁺) produced negligible changes.

Fluorescence titration revealed a 1:1 binding stoichiometry with Fe³⁺ (Job plot). The association constant log β = 8.23. The linear range (0–20 µM) yielded a detection limit of 1.25 × 10⁻⁸ M (S/N = 3). UV‑vis absorption increased at 250 nm and 350 nm upon Fe³⁺ addition, indicating complex formation.

Reversibility and Reusability

Treating the Fe³⁺‑bound probe with 2.5 × 10⁻⁵ M EDTA restored fluorescence to baseline, confirming a reversible “on/off” system. Five successive Fe³⁺–EDTA cycles caused <5 % loss in response, demonstrating excellent reusability. Magnetic separation was achieved within 10 min by placing a magnet adjacent to the dispersion, after which the probe could be re‑dispersed by gentle agitation.

pH Dependence

Fluorescence quenching remained robust across pH 5.8–10.5, with optimal sensitivity near neutral pH. Under acidic conditions, protonation of the naphthalimide nitrogen reduces Fe³⁺ coordination, attenuating quenching.

Bioimaging in HeLa Cells

MTT assays confirmed low cytotoxicity of the probe at concentrations up to 0.2 g L⁻¹. Confocal microscopy of HeLa cells incubated with the probe (0.2 g L⁻¹) for 30 min displayed bright green fluorescence. Subsequent addition of 5 × 10⁻⁵ M Fe(ClO₄)₃ for 30 min resulted in a pronounced fluorescence decrease, validating the probe’s capability to monitor intracellular Fe³⁺ dynamics.

Conclusion

We have developed a safe, magnetic‑silica hybrid probe that selectively and sensitively detects Fe³⁺, offers reversible fluorescence modulation, and can be magnetically recovered for repeated use. Its low detection limit and compatibility with live‑cell imaging make it a promising tool for biomedical research and potential clinical diagnostics.