Applications of Fluorescent Displacement-Based ... - Jacobs University

CHAPTER 1
Introduction
Scheme 1.4.2. The concept of supramolecular tandem assays, which allow timeresolved
monitoring of changes in analyte concentration during enzymatic
transformations.
Whereas in most assays a strong specificity of the receptor for the analyte
is required, this is not the case for supramolecular tandem assays. Here, it is only
a differential binding between two analytes, i.e., the substrate and the product of
the enzymatic reaction, towards a macrocyclic receptor in combination with a
fluorescent dye that is necessary. This way, the assay is performed using a
reporter pair, which is responsive to the concentration changes caused by the
enzymatic reaction (Scheme 1.4.2). Two sensing principles can be differentiated.
First, the formation of a strongly binding product can be monitored. In this case,
the dye resides in its complexed form before the enzymatic reaction is initiated.
The addition of the enzyme triggers a continuous displacement of the dye caused
by the higher affinity product (product-selective assay). Conventional indicator
displacement assays, especially antibody-based ones are conducted in this mode.
The second possibility relates to the situation in which the substrate binds more
strongly with the macrocyclic receptor as compared to the enzymatic product. In
this scenario, the reaction is followed via an uptake of the dye into the
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