Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications - Advanced Fluorescence Reporters in Chemistry and Biology - page 443

Primary TBA Block Thrombin

NP (100 nm)

h n

Secondary TBA CCP

h n ′

thrombin BSA 10

Primary TBA: 5'-amino-T 8 AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3'
or 5'-amino-T 8 GGTTGGTGTGGTTGG-3'

Secondary TBA: 5'-FAM-GGTTGGTGTGGTTGG- 3'

Scheme 12 Working principle of CCP-amplified NP-based thrombin detection

and no polymer-sensitized Fl emission is observed after addition of 10. There-
fore, thrombin detection can be easily realized by monitoring the amplified Fl
emission of NPs. On the basis of this assay, a thrombin detection limit of
1.06 nM is obtained. Moreover, the NP-based assay can be conducted in
blood serum, which benefits from its solid-state detection platform that allows
eliminating nonspecific adsorption through centrifugation-washing-redispersing
circles. On the other hand, the successful detection of lysozyme in a similar
manner also highlights the generality of this protein assay scheme.
Protein biochip for thrombin detection was also reported by Leclerc's group
using the complex of a cationic polythiophene (20) and dye-labeled ssDNA apta-
mer as the FRET-based probe as illustrated in Scheme 13 [ 102 ]. The complex probe
was prepared by stoichiometrically mixing CCP 20 with 3 0 -Cy3-labeled ssDNA
aptamer. Two ssDNA sequences were used, one was the specific thrombin aptamer,
ssDNA 9 -Cy3 (5 0 -NH 2 -C 6 -GGTTGGTGTGGTTGG-Cy3-3 0 ), and the other was the
random ssDNA, ssDNA 10 -Cy3 (5 0 -NH 2 -C 6 -GGTGGTGGTTGTGGT-Cy3-3 0 ). The
presence of amine group at the 5 0 -end of the ssDNA aptamer allows the complex of
20/ssDNA to covalently binding onto treated glass slides to form the solid-state
array. In the presence of thrombin, a significant fluorescence intensity increase is
observed for the spots with 20/ssDNA 9 -Cy3 complexes (binding sequence). In the
presence of two nonspecific proteins, BSA and immunoglobulin E (IgE), the
fluorescence intensities of the spots remain low, revealing an excellent specificity
of the detection with respect to the target. On the other hand, the spots with
20/ssDNA 10 -Cy3 complexes (nonbinding sequence) do not exhibit fluorescence
enhancement for human thrombin, confirming the specificity of the detection in
terms of the complex probe. Correlation of the fluorescence intensities as a function
of protein concentration using the spots of 20/ssDNA 9 -Cy3 reveals a limit of
detection of 10 7 molecules for human thrombin (i.e., 6.2 10 11 Min

1.5

0.4 L) together with a very good specificity. This work highlights that biochips can

m