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