A Smart Triple-Signal Fluorescent Probe for Real-Time Differential Imaging of HClO, H2O2, and Their Mixture in Diabetic Models

Abstract

Diabetes is a complex metabolic disorder characterized by persistent hyperglycemia, which causes damage to multiple target organs and triggers a range of complications. Oxidative stress, driven by reactive oxygen species (ROS) such as hypochlorite (HClO) and hydrogen peroxide (H₂O₂), plays a crucial role in the onset and progression of diabetes and its associated complications. Therefore, the simultaneous and differential detection of HClO, H₂O₂, and their mixture is essential for accurately assessing oxidative stress status and understanding their synergistic roles in disease progression. In this study, we present a triple-signal fluorescent probe, probe 1, designed to simultaneously and selectively detect HClO, H₂O₂, and their combination with high specificity and sensitivity. The probe emits three distinct fluorescence signals, enabling precise real-time visualization of oxidative stress dynamics in complex biological systems. Probe 1 has been successfully applied to track both exogenous and endogenous levels of HClO and H₂O₂ in living cells and zebrafish models. Furthermore, its efficacy has been demonstrated in diabetic mouse models, where it facilitates the spatial and temporal monitoring of oxidative stress across different organs. These findings underscore the potential of probe 1 as a powerful tool for advancing the understanding of oxidative stress mechanisms and developing targeted therapeutic strategies for diabetes and related diseases.

Keywords: Diabetes; Fluorescent Probe; Hydrogen Peroxide; Hypochlorite; Oxidative Stress.

1. Rational Design Strategy for Probe 1, a Unique Type of a Single Fluorescent Probe That Can Report HClO, H₂O₂, and HClO/H₂O₂ with Three Different Sets of Fluorescence Signals: Green, Red, and Yellow

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(a) Fluorescence intensity spectra of probe (10 μM) upon the addition of HClO (10 equiv). λ_ex = 350 nm (b) Fluorescence intensity spectra of probe 1 (10 μM) upon the addition of H₂O₂. λ_ex = 430 nm (c) (d) Fluorescence intensity spectra of probe (10 μM) upon the addition of HClO and H₂O₂ in sequence (10 equiv). λ_ex = 360 nm. Conditions: DMSO-PBS (10 mM, pH = 7.4, v/v, 5/5) slit (nm): 2.5/2.5.

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(a) Fluorescence intensity spectra of probe (10 μM) in the presence of 0 μM to 100 μM of HClO. (b) The corresponding linear changes of the fluorescence intensity of probe at 504 nm and as a function of HClO concentration. λ_ex = 350 nm (c) Fluorescence intensity spectra of probe (10 μM) in the presence of 0 μM to 100 μM of H₂O₂. (d) The linear changes of the fluorescence intensity of probe at 640 nm and as a function of H₂O₂ concentration. λ_ex = 430 nm. Conditions: DMSO-PBS (10 mM, pH = 7.4, v/v, 5/5) slit (nm): 2.5/2.5.

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(a) Confocal fluorescence images of metabolisms of HClO and H₂O₂ in living RAW 264.7 cells. (A1–C1) Cells were only treated with probe 1 (10.0 μM). (A2–C2) Cells were incubated with probe 1 and then treated with NAC. (A3–C3) Cells were incubated with probe 1 and then treated with HClO. (A4-C4) Cells were incubated with probe 1 and then treated with H₂O₂. (b) The fluorescence intensity of the green, yellow and red channels in Figure . (A1–C4). Scale bar: 25 μm.

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(a) Confocal fluorescence images of metabolisms of HClO and H₂O₂ in zebrafish. (A1–A4) Zebrafish were incubated with probe 1 (10 μM) for 30 min, and finally imaged. (B1–B4) Zebrafish were pretreated with NAC (10 μM, 30 min), then incubated with probe 1 (10 μM) for 30 min, and finally imaged. (C1–C4) Zebrafish were pretreated with LPS and PMA (10 μL, 1 mg/mL, 30 min), then incubated with probe 1 (10 μM) for 30 min, and finally image. (D1–D4) Zebrafish were pretreated with NAC (10 μM, 30 min), then incubated with HClO (10 μM, 30 min), and finally incubated with probe (10 μM) for 30 min. (E1–E4) Zebrafish were pretreated with NAC (10 μM, 30 min), then incubated with H₂O₂ (10 μM, 30 min), and finally incubated with probe (10 μM) for 30 min. (b) The corresponding fluorescence intensity changes from the green, yellow and red channels in Figure a. (A1–E4). Scale bar: 0.5 mm.

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(a) Fluorescence imaging of different anatomical organs (heart, liver, spleen, lung, kidney) and tissue after intravenous injection of probe 1 in the diabetic mouse. (b) The fluorescence intensity statistics were output by five representative regions of three different channels. (Green channel: λ_ex = 420 nm, collected 520 nm. Yellow channel: λ_ex = 440 nm, collected 570 nm. Red channel: λ_ex = 440 nm, collected 620 nm.).