Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications - Advanced Fluorescence Reporters in Chemistry and Biology - page 446

Scheme 14 Schematic representations of the assay for nucleases and protease detection based on
the complex of ACP, DNA-TR and peptide-FI
of the data. The fluorescence pattern and the PCA score plots enabled to effectively
distinguish 18 proteins from each other. Although this array cannot be used for
protein quantification and is less suitable for mixed samples, it has good reliability
and versatility for the detection of purified proteins.
Recently, we reported a complex-based FRET assay capable of detecting prote-
ase and nuclease in one solution, which relied on a peptide-mediated combinatorial
FRET between an ACP and TR-labeled ssDNA [ 104 ]. The working mechanism of
the assay is shown in Scheme 14 . A solution of 5, peptide-1 (Arg-Arg-Arg-Arg-
Arg-Arg-Arg-Arg-Arg-Arg), peptide-2 (Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
Arg-Fl), and ssDNA 8 -TR is prepared, within which FRET among 20, Fl, and TR
occurs because of the formation of multicomponent complexes. Before enzyme
digestion, excitation of 5 at 380 nm leads to dominant TR emission (red) in the
solution fluorescence as shown in Fig. 12 . In the presence of trypsin, the peptides
are digested into fragments, giving rise to relatively weak electrostatic attraction
between peptide fragments and 5. Under these conditions, the complexes become