1,3-Dichloro-5,5-dimethylhydantoin as Triggering Reagent for Chemiluminescent Biochip Based on the Luminol-H2O2 Reaction

Abstract

A free chlorine donor, 1,3-dichloro-5,5-dimethylhydantoin (DDH), was used for the first time as triggering reagent for the chemiluminescent detection of enzymatically produced hydrogen peroxide in the presence of luminol. A biochip has been developed based on glucose oxidase (GOx) activity as model enzyme. Photopolymer-based sensing layer entrapping luminol immobilized on anion exchanger beads (DEAE-Sepharose) and GOx immobilized on chelating beads (IDA-Sepharose) were spotted as an array of 800 µm spots at the surface of a PVC substrate. Each spot could be incubated separately in a 5 µL solution of DDH and glucose or H2O2, and the different light intensities measured simultaneously on the entire array with a CCD camera. The CL reaction conditions were optimized in terms of DDH concentration: 200 µM and pH: 8.5. Using the selected conditions and with a 3 min measurement duration, hydrogen peroxide could be detected in the range 5 µM–1 mM with a limit of detection of 5 µM. Applied to the glucose determination, the biochip exhibited good performances with a limit of detection of 7.5 µM (Signal/Noise ratio of 3) and a detection ranging over three decades at least. Used for the measurement of glucose concentration in normal and pathological human serum, no interferences problem were encountered for serum dilution of 1:100. In those conditions, the measured concentrations compared well with the expected reference values.