Nanobiosensors - Nanomaterials for Medical Applications - page 144

4.6.2 Quantification of DNA Capture Probes on the Surface
of NMs
In DNA biosensors the capture probe that is immobilized on the NM surface is
an ONT that has complementary bases to the target DNA (tDNA) or the analyte.
Conjugation of the capture probe to the functional groups on the surface of the
NMs is the first step in the preparation of this type of nanobiosensors. The fol-
lowing sample illustrates the quantification of immobilized capture probes on
the surface of NPs.
4.6.2.1 Chemicals
(1) 12 mM mercaptoethanol.
(2) cy3.5-labeled oligonucleotide-AuNP (AuNP-thiolONT-cy3.5).
(3) 0.3 M PBS, pH 7.
4.6.2.2 Protocol 91
(1) Add mercaptoethanol to a final concentration of 12 mM to AuNP-
thiolONT-cy3.5 in 0.3 M PBS.
(2) Incubate for 18 h at RT with intermittent shaking.
(3) Separate the displaced ONTs from the AuNPs by centrifugation.
(4) Dilute the supernatant by twofold with 0.3 M PBS, pH 7.
(5) Measure the fluorescence maximum at 563 nm and convert this to molar
concentration using the calibration curve of the cy3.5-alkanethiol-modified
ONT.
(6) Prepare the standard curves with known concentrations of cy3.5-labeled
ONTs using identical buffer pH, salt, and mercaptoethanol concentrations.
(7) Keep the pH and ionic strength of the sample and calibration standard solu-
tions the same for all measurements due to the sensitivity of the optical
properties of cy3.5 to these conditions.
(8) Calculate the average number of ONTs per particle by dividing the mea-
sured ONT molar concentration with the original AuNP concentration.
(9) Calculate the normalized surface coverage values by dividing with the esti-
mated particle surface area (assuming spherical particles) in the NP solution.
The assumption of roundness is based on a measured average roundness factor
of 0.93. Roundness factor is computed as: (4 π (surface area))/(2 × perimeter).
4.6.2.3 AuNPs Dissolution with KCN to Establish Surface
Coverage with ONT
This process is used to evaluate the surface coverage of AuNPs with thiolated
ONTs. The process involves dissolution of the AuNPs to release the ONT.
Absorption at specific wavelengths is used to establish the concentration using
standard calibration curves.