Biomedical Engineering Reference
In-Depth Information
(3)
10 mM phosphate buffer, pH 7, from 0.3 M stock solution of PBS.
(4)
0.3 M NaCl.
4.5.2.2 Protocol
The following protocol was used by Demers and Mirkin
91
in the preparation of
AuNP-based DNA sensor.
(1)
Add the 10 nM aqueous AuNPs to a 3-µM final concentration of fluorescein-
alkanethiol ONTs.
(2)
After 24 h, buffer the solution with 10 mM phosphate at pH 7.
(3)
Add NaCl to a final concentration of 0.1 M.
(4)
Incubate the solution for 40 h.
(5)
Remove excess reagents by centrifugation for 30 min at 14,000 rpm.
(6)
Carefully remove the supernatant.
(7)
Wash the red oily precipitate with 0.3 M NaCl and 10 mM phosphate buf-
fer (pH 7) solution.
(8)
Repeat the isolation, washing, centrifugation, and redispersion of the oily
precipitate.
(9)
Redisperse in fresh 0.3 M PBS.
(10)
Establish the AuNP-SAM concentration by a combination of Transmission
electron microscopy (TEM), Inductively Coupled Plasma Atomic Emission
Spectroscopy (ICP-AES), and UV-vis. The extinction coefficient of the
AuNP-SAMs was earlier established at 4.2 × 10
8
M/cm for 15.7 ± 1.2 nm-
diameter particles.
4.6 QUANTIFICATION OF BIOMOLECULES LOADED ON NM
s
Surface modification of NMs is one of the preliminary steps in the preparation
of nanobiosensors. Complete coverage of the NM surface with the biomolecules
and the blocking agent is essential to eliminate non specific adsorption (NSA) of
analytes on the exposed functional groups. Thus, it is very important to know the
amount of bioreceptors on the surface of NMs before capture of the analyte of inter-
est. The number of immobilized bioreceptors on the NM's surface is theoretically
directly related to the amount of analyte that bins with the capture probe. Although,
this is so, a calibration curve still needs to be established for each nanobiosensor.
4.6.1 Quantification of Protein Capture Probes on the Surface
of NMs
In protein immunoassays, the capture probe that is immobilized on the NM
surface can be an antibody, a hapten, or an immunogen. Taking a protein immu-
noassay for the detection of an antigen as a model, conjugation of the capture
antibody to the functional groups on the surface of the NMs is the first step in
