Development of a Modular Ratiometric Fluorescent Probe for the Detection of Extracellular Superoxide

Abstract

Extracellular detection of endogeneous analytes (e.g., superoxide) can provide important insights into mechanisms of homeostasis and diseases, such as tumorigenesis. A ratiometric probe with a fluorescent reference and an analyte-specific switch-on dye was developed. Detection of ROS in the extracellular milieu was ensured by connecting the two fluorophores with a modular peptide-nucleic-acid-based linker. The ROS-sensing ability was assessed and validated in cell-free assays and in cell culture.

Near‐Infrared Fluorescence Hydrogen Peroxide Assay for Versatile Metabolite Biosensing in Whole Blood

“…S7 preserved its fluorescence emission in the presence of a 100‐fold excess of the superoxide radical (Figure 2D), a degradation product of SOTS‐1. [ 24 ] However, S7 and the investigated control dyes were degraded by another ROS, hypochlorite, a bactericidal molecule secreted by immune cells [ 6 ] (Figure S11, Supporting Information).…”

“…Superoxide was generated in accordance with the literature. [ 24 ] In brief, SOTS‐1 (Cayman Chemicals, Ann Arbor, MI) was dissolved in dimethylformamide (Sigma‐Aldrich) and added to a near‐infrared fluorescent dye‐containing (final near‐infrared dye concentration 7.5 × 10 −6 m ; dihydroethidium (DHE) Thermofisher Scientific concentration 10 × 10 −6 m ) isotonic phosphate buffer 50 × 10 −3 m at pH 7.5 (final SOTS‐1 concentration 62.5 or 125 × 10 −6 m ) and protected from light. After 30 min at room temperature, the fluorescence intensity was determined by a plate reader (BioTek NEO2) at the fluorescence excitation and emission wavelength provided by the manufacturer.…”

Data on the removal of peroxides from functionalized polyethylene glycol (PEG) and effects on the stability and sensitivity of resulting PEGylated conjugates

“…Cy5 and Cy5-PEG were converted to their reduced forms (H-Cy5 and H-Cy5-PEG) using a previously described procedure [3] . Unconjugated Cy5 (1 mg, 1.93 μmol, 1 eq) was dissolved in 300 μL of MeOH and 500 μL of a 1 mg/mL solution of NaBH 4 in MeOH (13.2 μmol, 6.8 eq) was added.…”

“…The re-oxidation of H-Cy5 and H-Cy5-PEG was carried out by adapting a previously described procedure [3] . A 10 μM solution of H-Cy5 or H-Cy5-PEG was prepared in H 2 O. FeSO 4 and H 2 O 2 were added, yielding final concentrations of 10–75 μM and 100–750 µM respectively.…”

“…H-Cy5 can then be converted back to its fluorescent oxidized form when exposed to reactive oxygen species (ROS), allowing it to act as a highly sensitive, high wavelength fluorescent ROS sensor [2] . However, H-Cy5 is a small, poorly water-soluble molecule, and is rapidly taken up into cells in vivo , preventing its use for sensing extracellular ROS, which are implicated in inflammation, wound healing, and other processes [3] , [4] , [5] , [6] . A solution to this lies in the conjugation of Cy5 to a polyethylene glycol (PEG) polymer, increasing its solubility [7] .…”