Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 78

Fig. 29 Protocol for anion sensing using mesoporous MCM-41-like solids (UVM-7) containing
nanoscopic binding pockets and suitable dyes
and often involve several steps which are time-consuming and error-prone so that
meticulous control of the conditions is necessary.
An alternative method for increasing sensitivity to those mentioned above is to
increase the number of fluorophores per binding site or biomolecule, i.e., the F / B
ratio. In this way, every binding event can be signaled by several hundreds or
thousands of fluorophores, realizing true fluorescence signal amplification (Fig. 30 ).
However, the use of conventional fluorescent systems might involve self-quenching
problems. For example, if more than 10-15 fluorophores are attached to one
antibody, their distance is close to or within the F¨rster radius, resulting in signifi-
cant loss of fluorescence emission intensity due to energy transfer. On the other
hand, biomolecules labeled with many fluorophores may also exhibit reduced
specificity and binding affinity [ 176 ]. To overcome these problems, the preparation