Premium
A New Fluorescent PET Probe for Hydrogen Peroxide and its Use in Enzymatic Assays for L ‐Lactate and D ‐Glucose
Author(s) -
Groegel Dominik B. M.,
Link Martin,
Duerkop Axel,
Wolfbeis Otto S.
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100561
Subject(s) - chemistry , horseradish peroxidase , detection limit , fluorophore , hydrogen peroxide , fluorescence , glucose oxidase , redox , luminophore , peroxidase , moiety , nuclear chemistry , enzyme , chromatography , photochemistry , biochemistry , stereochemistry , inorganic chemistry , luminescence , physics , optoelectronics , quantum mechanics
We present a new probe for the determination of hydrogen peroxide (HP). It is based on the yellow fluorophore 4‐amino‐1,8‐napththalimide, coupled to p ‐anisidine (as a redox‐active group) to form a probe that is based on photoinduced electron transfer (PET). The preparation of the probe (which we refer to as “HP Green”) was accomplished in four steps with good yield. Its fluorescence is independent of pH in the physiological range and quenched by a PET process that occurs between the p ‐anisidine redox moiety and the naphthalimide luminophore. If the p ‐anisidine group is oxidized by HP, PET is suppressed and fluorescence intensity is strongly increased. Addition of horseradish peroxidase (HRP) enhances the oxidation of HP Green and further improves the detection limit of HP. The use of HRP and HP Green enables the determination of HP concentration in a range of 0.1 to 5 μ M , with a limit of detection (LOD) as low as 64 n M (16 pmol per well in microtiter plates). HP Green and HRP also enable sensitive enzymatic assays of oxidase substrates in a kinetic format, as shown for L ‐lactate and D ‐glucose. L ‐Lactate concentration can be rapidly determined between 0.5 and 10 μ M after 6 minutes of incubation at 30 °C, with an LOD of 164 n m (41 pmol per well). This LOD is more than sixfold lower than that of the best commercial assays for lactate. The detection range for D ‐glucose is 2 to 30 μ m, and the LOD is 644 n M (16 1 pmol per well). These are among the lowest concentrations detectable for oxidase‐based assays. The hexanoic acid moiety in HP Green may be further used to immobilize the probe in order to obtain sensor layers for continuous assays.