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Small molecule colloidal aggregation is the cause of promiscuous inhibition of enzymes, a common problem in high throughput biochemical screening assays for enzyme inhibitors.  We have devised a simple technique for detecting these aggregators. 

The method exploits the meniscus curvature changes in high density multi-well plates associated with colloidal changes in solution. The shape of the meniscus has a significant effect on fluorescence intensity when detected using a top read fluorescence plate reader, because of the effect of total internal reflection on fluorescence emission through a curved liquid surface. It turns out that fluorescence intensity is decreased as downward meniscus curvature (wetting) increases, reaching a minimum at the critical micelle concentration of the colloid. A small molecule aggregator causes wetting by virtue of its colloidal properties.

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It is possible to distinguish between a non-aggregator and an aggregator in small molecule libraries from a comparison of the fluorescence intensity of a fluorophore in a solution of the compound in the presence and absence of a detergent. The detergent causes maximum curvature and minimum fluorescence. In the absence of detergent, a non-aggregator will cause no meniscus curvature, and exhibit maximum fluorescence, while an aggregator will cause a reduction in fluorescence. The comparison of the two measurements eliminates any variations due to absorption of light by colored compounds.

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In more recent work, we demonstrated a method to determine CMC using absorbance measurements only, without requiring addition of fluorescent probe to the wells