RESUMEN
A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of â¼1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.