Readily Accessible Fluorescent Probes for Sensitive Biological Imaging of Hydrogen Peroxide

Abstract

Hydrogen peroxide (H2O2) is a major component of oxygen metabolism in biological systems that, when present in high concentrations, can lead to oxidative stress in cells. Non-invasive molecular imaging of H2O2 using fluorogenic systems represents an effective way to detect and measure the accumulation of this metabolite. Herein, we detail the development of robust H2O2-sensitive fluorescent probes using a boronic ester trigger appended to the fluorophore via a benzyl ether linkage. A major advantage of the probes presented here is their synthetic accessibility, with only one step needed to generate the probes on the gram scale. The sensitivity of the probes was evaluated in simulated physiological conditions, showing micromolar sensitivity to H2O2. The probes were tested in biological model systems, demonstrating effective imaging of unstimulated, endogenous H2O2 levels in RAW 264.7 cells and murine brain tissue.

Factors Associated with Nitric Oxide-mediated β2 Integrin Inhibition of Neutrophils

“…Because NOX activity would result in elevations of H 2 O 2 , we also performed studies with 50 M FBBBE, an agent viewed as highly specific for H 2 O 2 versus DCF-DA, which reacts with a relatively wide variety of reactive species (40). As shown in Table 2, exposure to DENO resulted in a near 3-fold elevation in the rate of FBBBE fluorescence, and the response was inhibited by the NOX inhibitor, Nox2ds, the iNOS inhibitor 1400W, and the HSP90 inhibitor, geldanamycin.…”

Possibilities and Challenges for Quantitative Optical Sensing of Hydrogen Peroxide

“…Usually, a 'selectively' cleavable group is attached to an indicator dye molecule such as coumarin, anthracene, BODIPY, cyanine, fluorescein, resorufin or rhodamine [48][49][50][51] (Figure 3). The selected dye determines the spectral range of the probe.…”

“…The selected dye determines the spectral range of the probe. While a large variety of probes exist that emit throughout the entire electromagnetic spectrum from UV to near infra-red (NIR), it is rather surprising that many recently published probes still rely on inherently pH-sensitive indicators such as fluorescein (phenolic Chemosensors 2017, 5, 28 4 of 23 pK a 6.31-6.80 of the monoanion and dianione; two relevant species in aqueous solutions [52]) and resorufin (pKa 5.8; fluorescent at pH > 7) [53] Usually, a 'selectively' cleavable group is attached to an indicator dye molecule such as coumarin, anthracene, BODIPY, cyanine, fluorescein, resorufin or rhodamine [48][49][50][51] (Figure 3). The selected dye determines the spectral range of the probe.…”

“…Without simultaneous determination of cellular pH, the quantification of H2O2 concentrations with such probes is thus prone to large uncertainty. When it comes to choice of the oxidative cleavage groups (OCGs), there are also many Usually, a 'selectively' cleavable group is attached to an indicator dye molecule such as coumarin, anthracene, BODIPY, cyanine, fluorescein, resorufin or rhodamine [48][49][50][51] (Figure 3). The selected dye determines the spectral range of the probe.…”

Microfabricated needle for hydrogen peroxide detection

“…The FBBBE is a probe which is specically designed for measuring intracellular H 2 O 2 as it is capable of penetrating the cell wall. 26 The uorescence of the FBBBE molecule (with Ex/Em 480/512) increases with the amount of H 2 O 2 (from 0 to 150 mM). For calibration, 30% hydrogen peroxide (H 2 O 2 ) was used to make different concentrations of H 2 O 2 samples; 50 mM HEPES buffer (pH ¼ 7.2) was used to dilute the H 2 O 2 and the biosensor.…”