RESUMO
Conventional enzymatic assays for alcohol dehydrogenase, pyruvate kinase, and enolase performed in 96-well microtiter plates were compared with assays monitored in 25-well nanoarrays. All miniaturized reactions could be performed in maximum volumes of 6.3-8 nL and were read out with a conventional fluorescence microscope system equipped with a scientific grade CCD camera. Substrate and cofactor were already present inside the wells after having been presprayed, or they were applied in solution to the wells of the nanoarray shortly before the assays started. For all of the assays, commercially available enzymes and enzymes present in cell-free extracts were used. Assays carried out in premixed nanoarrays gave results comparable to those performed in presprayed nanoarrays. Enzyme activities determined in nanoarrays by using two different methods were in good agreement with assays performed in microtiter plates. Also, good correspondence was found between expected and observed enzyme levels. In short, enzymatic assays performed in premixed and in particular in presprayed nanoarrays are a promising low-volume and low-reagent- and sample-consuming alternative to current methodology and could find applications in many different areas of analytical chemistry.