Development of a near-infrared fluorescent probe for in situ monitoring of hydrogen peroxide in plants

Abstract

In plants, hydrogen peroxide (H₂O₂), one of the significant reactive oxygen species, plays a dual function. Investigating its concentration is essential for understanding its production and scavenging mechanisms in plants. In this study, a near-infrared fluorescent probe (Cy-Bo) was developed, which is based on the hemicyanine compound. By introducing indole salts into the oxygenated anthraquinone structure, the conjugated system is expanded, enabling the probe to emit long-wavelength fluorescence in the near-infrared region, thereby minimizing interference from other biomolecules in plant tissues (λ_ex = 650 nm, λ_em = 720 nm). As for the specific recognition of H₂O₂, the pinacol phenylborate ester was selected to be the recognition group. It shows good linearity (R² = 0.998) in the concentration range of 0.5–100 μM, with a detection limit of 0.07 μM. Furthermore, this probe Cy-Bo has been used for in vivo fluorescence imaging in plants due to its good bio-penetration and in-situ imaging capabilities. The results reveal a significant increase in H₂O₂ concentration in Arabidopsis thaliana under progressively increasing drought, high-temperature, and salt stress. This tool provides a non-invasive, in situ imaging method for detecting H₂O₂ in plants, which has a fast response, easy operation, and high sensitivity. It enables visual monitoring of H₂O₂ fluctuations and aids in advancing physiological and pathological studies related to H₂O₂.