Chem 8157

Answers to comment questions are below. For whatever reason, I cannot post these as comments in the blog thread so I am doing it this way.

1. The proportionality constants were determined using a fluorescence microscope. The overall details are sparse in the supplementary information. My understanding is that they performed measurements varying the concentration of both ions and measuring the fluorescence intensity. The proportionality factors were then determined using nonlinear regression with three variables.

2. If one wished to determine the association and dissociation rates, a feasible method is immobilize the reporter onto the walls of a flow-cell and pass solutions of known concentrations through the cell. Much like the k and k-1 determination done with SPR, an ion-free solution could be passed through the cell followed by solution loaded with a known concentration of analyte. Once saturation is reached, analyte-free solution can be again passed through the cell. Using nonlinear fitting, you could determine the constants.

With regards to Nic's question, cleavage of the ester bonds is spontaneous in the intracellular matrix. Going back to undergraduate organic chemistry, many ester bonds are cleaved spontaneously in aqueous solution. This cleavage must take a reasonable amount of time, otherwise loading of the cell would not be possible. Once introduced into the sample, a 15 minute wait time was used to ensure relatively complete cleavage of the ester.