Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications - Advanced Fluorescence Reporters in Chemistry and Biology - page 442

5
Lysozyme
Thrombin
Trypsin
BSA

1.0

0.8

0.6

0.4

0.2

0.0

400 450 500 550 600 650

Wavelength (nm)

Fig. 10 Normalized PL spectra of 5 alone and 5 with FAM-labeled lysozyme aptamer preincu-
bated with lysozyme, thrombin, trypsin and BSA, respectively: [FAM-labeled lysozyme-aptamer]
¼ 4.5 10 8 M, [lysozyme] ¼ 2.4 mg/mL in 5 mM Tris buffer; [5] ¼ 4.6 10 7 Min5mM
Tris buffer; Excitation at 370 nm

FRET to occur, and thus no Fl emission is observed. The specificity of this
assay was also examined for mixed samples. The mixed lysozyme samples were
prepared in fetal bovine serum (FBS), human saliva and human urine. It was
found that FAM emission was still visible upon addition of each mixed sample,
implying that this assay has a great potential for the detection of real biological
samples. This study illuminates that introduction of specific aptamer/protein
interaction as the recognition event, and utilization of FRET as the signal
transduction channel, is an effective way to develop CPE-based protein sensors
with good specificity.
We also utilized CCP and aptamer-functionalized silica NPs to develop a
sandwich assay for optical detection of thrombin in biological media with high
sensitivity and selectivity [ 101 ]. CCP 10 and Fl were chosen as the donor and the
acceptor, respectively. The assay scheme is shown in Scheme 12 . The primary
thrombin-binding aptamer (TBA) is covalently attached to triazine-functionalized
silica NPs, which is followed by BSA blocking on the free sites of NP surface.
During the sensing process, TBA forms an intramolecular G-quartet (known as the
guanine tetrad that consists of four guanine bases in a square planar array arranged
in a cyclic hydrogen-bonding pattern, where each guanine is both the donor and
acceptor of two hydrogen bonds) and recognizes the active site of thrombin,
affording thrombin-bound TBA-NPs. The Fl-labeled secondary TBA is then added
to associate with the remaining thrombin binding sites, resulting in the dye-containing
NPs. Finally, addition of CCP 10 into the NP suspension induces FRET, leading to
amplified Fl emission. In contrast, in the presence of nonspecific proteins, the
primary TBA-immobilized NPs cannot capture the Fl-labeled secondary TBA