A fluorescent MTP‐based Detection Platform for Hydrogen Peroxide, Glucose, and Lactate

Hydrogen peroxide (H2O2) is an important small metabolite often quantified with commercially available multistep fluorescence-based assays. A new microtiter plate (MTP)-based platform that allows a rapid, one-step assay with a ratiometric readout function is developed. Specifically, 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) in a polyurethane-based hydrogel sensor membrane is embedded. For a ratiometric set-up, the membranes are loaded with polystyrene nanoparticles containing a Cy5-based reference which allowed for the compensation for variations in membrane thickness. These knife-coated µm-thin films are mounted onto bottomless MTPs with double-sided adhesive tape. Optimized membranes provide measurement times of 3 min upon sample addition and a limit of detection (LOD) in phosphate-buffered saline that is 10x lower than that of the ADHP-using Amplex Red commercial kit of 100 nmol L−1 H2O2. These ADHP hydrogels can be stored at room temperature for at least 22 months. Horseradish peroxidase (HRP) is nanospotted alone or together with either lactate oxidase or glucose oxidase for the detection of H2O2, lactate, and glucose, respectively. With 50 v% glycerol as cryoprotectant in the spotting solution, the HRP ADHP platform is stable for at least 13 weeks at –20 °C. Enhanced simplicity and comparable performance to multistep assays suggest that the platform can simplify MTP-based assays in the future.