Biomedical Engineering Reference
In-Depth Information
the signal specificity. A more recent report by Genicon Sciences assessed the
dynamic range and limit of detection in gene expression studies using known
amounts of specific cDNA transcripts that were spiked into complex cDNA
samples [107]. The reported lower limit of detection (LLOD) was 8
.
2
10
6
×
copies (
170 fM, 80 uL) with a 3.3 log dynamic range. By comparison, a
Cy3 label had an LLOD of 2
.
8
∼
10
7
copies (
580 fM, 80 uL) with a 3.2 log
dynamic range. It should be noted that although a single color was reported
in this study, two color nanoparticle labelling is now available using silver and
gold particles [27].
In an approach similar to Genicon, Schultz and coworkers have utilized
large silver nanoparticles referred to as plasmon resonant particles (PRPs)
and resonant scatter based detection for microarray labelling [89]. The 40-
100 nm diameter silver particles are prepared by solution-based reduction of
silver onto small gold particle seeds (
∼
5 nm diameter). The PRPs scatter
light based on the position of the surface plasmon band as observed for gold
particles [90]. In this study, 55
×
∼
±
17 nm diameter particles which exhibit max-
imum scatter at
430 nm were utilized. The particles were derivatized with
mouse anti-biotin antibodies for detection of biotin labelled targets, Fig. 8.6a.
For detection, the slide is illuminated in dark field using a halogen lamp, and
a high resolution image of each microarray spot is captured using a CCD cam-
era through a 10
∼
dark-field/bright-field objective lens on an optical
microscope [89]. Individual plasmon resonant particles provide a scattering
signal that is distinguishable from other sources of scatter, thereby enabling
particle counting to be used for measuring the amount of total signal from each
microarray spot. This unique detection methodology was applied on a small
model array containing positive and negative control capture sequences. A bi-
otin labelled 30-mer target was hybridized to the array overnight followed by
overnight incubation with the antibiotin labelled silver particles. A detection
limit of 1
×
or 100
×
10
6
target copies (830 fM, 2 uL) was achieved, which was
×
∼
10-fold
10
7
copies)
using this illuminaton/detection technique. The improvement in sensitivity is
attributed to the elimination of background pixels that decrease the average
scattering signal on microarray spots that are not completely coated with par-
ticles. This labelling technology is under development at Seashell Corporation
for microarray applications [89].
The above cited literature clearly demonstrates that RLS labels hold
promise as high sensitivity labelling systems for gene expression. However,
the 3-4 fold increase in assay sensitivity observed with spiked transcripts
was significantly less than the expected 2-3 orders of magnitude improve-
ment predicted based on the theoretical RLS detection limit of 0.005 RLS
particles/
better than obtained by measuring average scatter intensity (1
×
m
2
. This significant disparity is likely attributed to the large size
of the gold particles required, which presents steric and kinetic limits to the
number of particles bound to each cDNA probe. In addition, the passive ad-
sorption of the antibiotin antibodies to the nanoparticle surface may be prone
to desorption [77]. Particle counting may be used to increase sensitivity in
µ
