Diffusion of microspheres in sealed and open microarrays.

In several experiments, we study the diffusion of microspheres with different radii in microarrays filled with a variety of aqueous solutions of ethylene glycol. We study diffusion in open and closed (sealed) microarrays. In sealed nanoliter wells, the tracers show pure diffusion, whereas in open reactors, a radial outward-directed evaporation-induced liquid flow is superimposed onto the diffusion. In general, one of the following quantities can be calculated if the others are known: the temperature, the viscosity of the medium, the radius of the microbeads, or the diffusion constant. The estimated diffusion constants in closed microarrays are in good agreement with theoretical predictions based on the Brownian motion. We monitor the motion of the microbeads under a microscope and extract their paths in time from the digital recordings. Ambiguous paths due to the crossing of two trajectories can be detected. We show that low microsphere concentrations or high viscosities do not hamper a robust estimation of the diffusion parameters.

Monitoring enzymatic reactions in nanolitre wells.

We have developed a laboratory-on-a-chip microarray system based on nanolitre-capacity wells etched in silicon. We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme-catalysed reactions. Such reactions can be found in the glycolytic pathway of yeast. We describe the design, construction and testing of our laboratory-on-a-chip. We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence is 5 micro m with a microscope-based system and 100 micro m for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 micro m for ATP/luciferase bioluminescence.