Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 253

A Intramolecular ratiometric strategy—single probe biosensor-wavelength shift

Phosphorylation

P

Peptide substrate

B Intramolecular ratiometric strategy—dual probe biosensor

Phosphorylation

P

Peptide substrate

Inert probe Inert probe

Figure 6.12 Ratiometric strategies. (A) Intramolecular ratiometric strategy—single-probe
biosensor based on a coumarin/Cd 2 þ -cyclen probe that undergoes a wavelength shift
upon phosphorylation (from 360 to 410 nm) due to chelation of the phosphate group
by the Cd 2 þ center—anion sensor-based ratiometric probe. 132

(B) Intramolecular rati-

ometric strategy — based on incorporation of two different fluorescent probes on a single

biosensor scaffold one involved in the sensing process, the other serving as an internal

control, such as the Src merobody biosensor based on a fibronectin monobody that is
conjugated to a merocyanine dye and fused with a mCerulian which serves as an internal
control. 112

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3.5. Optimizing peptide biosensors through quenching and
caging strategies
3.5.1 Intramolecular quenching
One of the means of optimizing the signal-to-noise ratio in applications
involving the use of peptide or protein kinase biosensors consists in silencing
the basal fluorescence of the fluorescent probe, thanks to a quencher moiety
conjugated within the same biosensor. This strategy implies that the
quencher is in spatial proximity with the fluorescent probe and that
the quenching mechanism will be reversed upon phosphorylation of the
biosensor by the kinase. Different quencher strategies have been employed
( Fig. 6.13 ). The simplest strategy involves incorporation of a synthetic
quencher into the peptide backbone so that it quenches the fluorescent
probe until phosphorylation of the biosensor allows the disruption of the
interaction between the quencher and the fluorophore, much in the same
way as tryptophan or tyrosine residues quench proximal fluorophores 126,141
( Fig. 6.13A ). An alternative approach, which consists in intramolecular
homoquenching between two fluorophores

(through homo- or