Micro-injection of β-D-glucose standards and Amplex Red reagent on micro-arrays

Intelligent Molecular Diagnostic Systems (IMDS)- The objective of this multidisciplinary research program is to design and develop an analytical system that is able to measure and interpret concentrations of various analytes which are dispensed on a micro-array. The analytes are detected by means of fluorescence or (chemi)luminescence measurement. Furthermore, the collected data are combined and interpreted using modern reasoning techniques. Micro-injection- Dispensing picoliters (pl) of reagents (enzymes, antibodies, etc.) and liquid samples on a micro-array requires special techniques. At the moment we are working on a technique which will allow for accurately dispensing liquid volumes less than 100 pl on a micro-array. Detection of beta-D-glucose- beta-D-glucose standards are dispensed on a micro-array, after which a solution of Amplex Red reagent, horse radish peroxidase (HRP), and glucose oxidase in a mixture of ethylene glycol and water is added. Ethylene glycol is added to prevent evaporation. The beta-D-glucose reacts with glucose oxidase to D-gluconolactone and H2O2. The H2O2 reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) with a 1:1 stoichiometry to produce highly fluorescent resorufin. The formation of resorufin with time is followed with a Zeiss Axioskop microscope equipped with a KAF Photometrics CCD camera, in order to determine the sensitivity, concentrations, and volumes associated with the dispensed fluids.