Monitoring enzyme-catalyzed reactions in micromachined nanoliter wells using a conventional microscope based micoarray reader

Abstract

Yeast-Saccharomyces cerevisiae - it widely used as a model system for other higher eukaryotes, including man. One of the basic fermentation processes in yeast is the glycolytic pathway, which is the conversion of glucose to ethanol and carbon dioxide. This pathway consists of 12 enzyme-catalyzed reactions. With the approach of microarray technology we want to explore the metabolic regulation of this pathway in yeast. This paper will focus on the design of a conventional microscope based microarray reader, which is used to monitor these enzymatic reactions in microarrays. These microarrays are fabricated in silicon and have sizes of 300 by 300 micrometers 2. The depth varies from 20 to 50micrometers. Enzyme activity levels can be derived by monitoring the production or consumption rate of NAD(P)H, which is excited at 360 nm and emits around 450 nm. This fluorophore is involved in all 12 reactions of the pathway. The microarray reader is equipped with a back-illuminated CCD camera in order to obtain a high quantum efficiency for the lower wavelengths. The dynamic range of our microarray reader varies form 5 (mu)Molar to 1mMolar NAD(P)H. With this microarray reader enzyme activity levels down to 0.01 unit per milliliter can be monitored. The acquisition time per well is 0.1s. The total scan cycle time for a 5 X 5 microarray is less than half a minute. The number of cycles for a proper estimation of the enzyme activity is inversely proportional to the enzyme activity: long measurement times are needed to determine low enzyme activity levels.