Biomedical Engineering Reference
In-Depth Information
diagnostics applications. 158 These provide early diagnosis even before the onset of
clinical symptoms. A few nanobiosensors for DNA detection are discussed below.
The ONT-mediated AuNPs aggregation process has been extensively used
for the colorimetric screening of DNA binders and triplex DNA binders. The
applications of AuNP-based DNA colorimetric methods for disease diagnosis
and gene expression have been reported and reviewed. 21,72,73 Because AuNPs
have unique optical/electrical properties, various detection techniques have
been applied in the AuNP-based DNA assay. A one-step homogeneous DNA
detection method with high sensitivity was developed using AuNPs coupled
with dynamic light scattering (DLS) measurement. 159 Single-base pair-mis-
matched DNAs can be readily discriminated from perfectly matched tDNAs
using this assay. This DLS one-step homogeneous DNA detection method with
high sensitivity was developed using AuNPs. Citrate-AuNPs with a diameter of
30nm were initially functionalized with two sets of ssDNA probes before these
were used as optical probes for DNA detection. The hybridization between the
tDNA and the two AuNP probes led to the formation of NP dimers, trimers, and
oligomers. The NP aggregation increased the average diameter of the NP popu-
lation that was monitored by DLS. A quantitative correlation was established
between the average diameter of the resulting NPs and the tDNA concentration.
The detection limit of this facile DLS-based assay that requires no additional
separation and amplification steps was around 1 pM, which is four orders of
magnitude better than that of light absorption-based methods.
A novel approach using ClearRead™ that utilizes total human RNA as tar-
get nucleic acid without the need for labeling or amplification steps for target
preparation was reported. 160 In this study, the RNA is hybridized to an ONT
microarray and the bound molecules are detected in a second hybridization step
using ONT (oligo-dT20)-modified AuNP probes. The second probe hybridizes
through their oligo-dT20 sequences to the poly-A tail of the captured mRNA
molecules. The poly-A tail is a unique feature of eukaryotic mRNA molecules
(except the animal histone mRNAs), conferring an increased stability to the
mRNA in the cytoplasm. 161 Even the most abundant source of RNA in the cell
which are the ribosomal transcripts, also lack the poly-A tail. The oligo-dT20
NPs used in this study specifically detect only expressed coding sequences.
The light scatter ability of the AuNPs bound to the microarray is improved by
autometallography which involves a short step where Ag+ ions are reduced to
elementary silver that deposit around the AuNPs. The silver-coated AuNPs have
an increased extinction coefficient, leading to about 1000-fold increase in the
scatter signal. 106 The light scattering is captured with a photosensor of an imag-
ing system (Verigene ID) that has been previously shown to be 1000 times more
sensitive than fluorescent-based detection methodologies. 106 Using this method
for gene expression analysis, Huber et al. were able to detect expressed genes
as low as 0.5 µg of unamplified total human RNA in a 2-h hybridization assay.
A fluorescence QD-based ultrasensitive SMD method for quantification of
DNA coupled with TIRFM was developed. 50 The capture DNA was immobilized
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