Chemistry Reference
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Optimization of the Coupling of Target
Recognition and Signal Generation
Ana B. Descalzo, Shengchao Zhu, Tobias Fischer, and Knut Rurack
Abstract Fluorescent reporters that literally “light up” upon binding of a target
species are particularly interesting from an analytical point of view. This contribu-
tion introduces the main signaling concepts in fluorescent probe research, discusses
strategies toward their optimization in terms of signal output, and highlights the
wealth of alternative protocols that has been realized in the past two decades to
create signaling systems with luminescence amplification features.
Keywords Dyes
Fluorescence
FRET
Molecular Recognition
Nanoparticles
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2 Channels of Communication between Binding Site and Fluorophore . . . . . . . . . . . . . . . . . . . . . 44
2.1
-Conjugated Binding Site and Fluorophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.2 Spacer-Separated Binding Site and Fluorophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3 Strategies of Signal Optimization in Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.1 Combinatorial Synthesis of Functional Fluorophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.2 Design of Communication Channels for “Light-Up” Probes . . . . . . . . . . . . . . . . . . . . . . . . . 57
4 Strategies of Signal Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.1 Chemical Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.2 Displacement of Fluorophores from Binding Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4.3 Increasing the Number of Fluorophores per Binding Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.4 Involving Fluorophore Communication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.5 Resonance Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6 Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
p
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