A pyrene-based ratiometric fluorescent probe with a large Stokes shift for selective detection of hydrogen peroxide in living cells

Abstract

Hydrogen peroxide (H₂O₂) plays a significant role in regulating a variety of biological processes. Dysregulation of H₂O₂ can lead to various diseases. Although numerous fluorescent imaging probes for H₂O₂ have been reported, the development of H₂O₂ ratiometric fluorescent probe with large Stokes shift remains rather limited. Such probes have shown distinct advantages, such as minimized interference from environment and improved signal-to noise ratio. In this work, we reported a new pyrene-based compound Py-VPB as H₂O₂ fluorescent probe in vitro. The probe demonstrated ratiometric detection behavior, large Stokes shift and large emission shift. In addition, the probe showed high sensitivity and selectivity towards H₂O₂ in vitro. Based on these excellent properties, we successfully applied Py-VPB to the visualization of exogenous and endogenous H₂O₂ in living cells. Cell imaging study also showed that our probe was localized in the mitochondria. We envision that the probe can provide a useful tool for unmasking the biological roles of mitochondrial H₂O₂ in living systems.

Keywords: Fluorescent probe; Hydrogen peroxide; Large Stokes shift; Pyrene; Ratiometric detection.

Graphical abstract

Scheme 1

Schematic diagram of the detection mechanism of the probe Py-VPB.

Scheme 2

Synthetic route of probe Py-VPB for H₂O₂ detection.

Fig. 1

Quantitative measurements of Py-VPB (10 μM) fluorescence changes after addition of different concentrations of H₂O₂. (A) Fluorescence emission spectra of Py-VPB after addition of different concentrations of H₂O₂ (0–100 μM). (B) Plot of fluorescence intensity ratio changes (I₄₈₀/I₆₀₀) after incubation with increasing amounts of H₂O₂. Inset: Linear regression plot of fluorescence intensity ratio change (I₄₈₀/I₆₀₀) as a function of the concentration of H₂O₂ (0–45 μM). Ex = 380 nm.

Fig. 2

Time-dependent response of Py-VPB to H₂O₂. (A) Fluorescence emission spectra of Py-VPB after incubation with 100 μM H₂O₂ for different time intervals (0–45 min). (B) Plot of I₄₈₀/I₆₀₀ ratio changes after incubation with 100 μM H₂O₂ for different time intervals (0–45 min). Ex = 380 nm.

Fig. 3

Relative fluorescence responses of Py-VPB (10 μM) to various analytes (100 μM): (1) Blank, (2) Mg²⁺, (3) Ca²⁺, (4) Cu²⁺, (5) Fe²⁺, (6) Cr³⁺, (7) Co²⁺, (8) Zn²⁺, (9) F⁻, (10) Cl⁻, (11) I⁻, (12) PO₄³⁻, (13) NO₃⁻, (14) NO₂⁻, (15) SO₃²⁻, (16) GSH, (17) Hcy, (18) Cys, (19) ^.NO, (20) TBO^., (21) t-BuOOH, (22) ClO⁻, (23) HO·, (24) O₂^.-, (25) ONOO⁻, (26) H₂O₂.

Fig. 4

DLS data of Py-VPB (A) and Py-VP (B) in water/DMSO (4:1, V/V).

Fig. 5

(A-C) Confocal imaging of exogenous H₂O₂ in HeLa cells (A: 0 μM; B: 100 μM; C: 200 μM). (D) The ratio of channel 2/channel 1 fluorescence intensity when incubated with different concentrations of H₂O₂. The relative fluorescence intensity was analyzed by the ImageJ software. Every data point represents the mean of five fields of cells. Ex = 405 nm, channel 1: 580–630 nm (Py-VPB), channel 2: 450–500 nm (Py-VP). Scale bar = 20.0 μm.

Fig. 6

(A-B) Confocal imaging of endogenous H₂O₂ in RAW264.7 cells (A: blank; B: pretreated with PMA). (C) The ratio of channel 2/channel 1 fluorescence intensity. The relative fluorescence intensity was analyzed by the ImageJ software. Every data point represents the mean of five fields of cells. Ex = 405 nm, channel 1: 580–630 nm (Py-VPB), channel 2: 450–500 nm (Py-VP). Scale bar = 20.0 μm.