Microarrays for biotechnology: The imaging challenge

摘要

Our goal is to develop intelligent molecular diagnostic systems (IMDS) that can analyze liquid samples that contain a variety of biochemical compounds. In order to analyze the liquid samples we use dedicated microarrays. At this stage, these are basically miniaturized micro titer plates. Typical dimensions of a well are 200 x 200 x 20 μm~3. These dimensions may be varied and the shape of the wells can be modified with a result that the volume of a wells can be from 0.5 to 1.6 nl. For our experiments, we have used wells with the shape of a truncated pyramid. These wells are fabricated in silicon by a wet etching process. For testing purposes the wells are filled with a fluorescing dye (e.g. rhodamine) of various concentrations. To avoid evaporation, glycerol-water 1:1 v/v with a viscosity of 8.3 times the viscosity of water is used as solvent. To analyze the molecular detection capabilities of such a system, we have used an epi-illumination fluorescence microscope equipped with various objectives and a scientific CCD camera to collect the fluorescent light emitted from the solutions in the wells. We have, in particular, studied the effects of lens magnification and numerical aperture, NA. on the detection capabilities. From these experiments we have found that for this configuration the detection limit is on the order of nanomolar concentrations of fluorescing particles. This translates to 100,000 molecules per well. Further, we have used the digital analysis of images of the evaporation process to understand how fluorescing particles move during the evaporation process. Using digital images analysis of the interference fringes produced in this dynamic process, we have obtained an axial resolution of 70 water molecules.