Biomedical Engineering Reference
In-Depth Information
4.6.2.3.1 Chemicals
(1)
0.2 M KCN and 2 mM K
3
Fe(CN)
6.
(2)
AuNPs.
(3)
3'-propanethiol, 5'-fluorescein 12-mer ONTs.
4.6.2.3.2 Protocol
(1)
Add aqueous 0.2 M KCN and 2 mM K
3
Fe(CN)
6
in water to 16.5 ± 1.2 nm
AuNPs previously modified with 3'-propanethiol, 5'-fluorescein 12-mer
ONTs in 0.3 M PBS to a final concentration of 0.08 M KCN and 0.8 mM
K
3
Fe(CN)
6
at 3.9 nM AuNPs.
(2)
Mix thoroughly for a few minutes until a pale yellow solution forms.
(3)
Establish the absorption of the excess ferricyanide at 420 nm and of the
fluorescein-labeled ONT at 490 nm.
(4)
Prepare a calibration curve by adding known amounts of 3'-propanethiol,
5'-fluorescein-12-mer ONT to AuNP solutions of known concentrations in
0.3 M PBS.
(5)
Calculate the normalized surface coverage as in the previous section.
4.6.2.4 Quantification of the Hybridized Target Surface Density
on AuNP Modified with the Capture ONT
The amount of ONT that is hybridized on the surface of the NMs containing
the capture ONT can be established following protocols that may be used for
each particular particle depending upon their respective properties. Below is
a protocol for the quantification of hybridized ONT on the surface of capture
ONT-modified NP, AuNP-thiolONT-dylight550.
4.6.2.4.1 Chemicals
(1)
AuNP-thiolONT-dylight550.
(2)
Target ONT-C6QD620 (this is the ONT that is complimentary to the ONT
on the AuNP; this ONT is labeled with a QD, Ocean NanoTech catalog #
QSH620, which absorbs at 610 nm and emits at 620 nm allowing for both
absorbance and fluorescence detection).
(3)
0.3 M PBS, pH 7.
(4)
1 M HCl.
(5)
1 M NaOH.
4.6.2.4.1 Procedure for the AuNP-Based Detection of ONT
(1)
Take AuNP-thiolONT-dylight550 and combine with equivalent amount
of the complementary target ONT-QSH620 (ONT-C6QD620) at 3 µM in
0.3 M PBS, pH 7.
