Kenneth E. Gonsalves, Craig R. Halberstadt, Cato T. Laurencin, Lakshmi S. Nair. Biomedical Nanostructures
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distance between closely spaced two-color nanoparticles [33]. In 2002,
Thompson et al. reported a framework for accurate localization of single
fluorescent molecules and tested it using 30-nm fluorescent beads. The authors
concluded that minimization of noise in the system and maximization of the
photons collected from each fluorophore was the key for high accuracy in
localization. Later, using photobleaching of one Cy3 dye molecule in a pair,
Selvin and coworkers were able to localize the dye molecules with a precision of
1.5 nm [41]. In the same year, using similar approach, Scherer and co workers
localized Cy3 molecules with precision of 2.5 nm [36]. More recently, Spudich
and co-workers have demonstrated use of Cy3 and Cy5 molecules for
measuring intermolecular distances [38] and Ram et al. have proposed new
resolution measures for locating single molecules with high accuracy [40]. In
several of these reports, imag e of a fluorophore is fit to a 2D Gaussian profile
and the center is estimated by finding the local maxima of the fit. The error in
position of a single molecule is then given by the equation shown below [37].
h DxðÞ
2
i¼
s
2
þ a
2
=12
N
þ
4
ffiffiffi
p
p
s
3
b
2
aN
2
ð8:2Þ
where Dx is the error in center localization, s is the standard deviation of a 2D
Gaussian kernel used to fit the intensity profile, a is the pixel size in the image, b
is the background noise, and N is the numbe r of photons co llected per
nanoparticle. While advances in optics and imaging instrumentation have
enabled data collection with minimal noise (b), high accuracy in localization is
largely determined by the number of photons co llected from the object being
imaged, meaning that brighter and more photostable fluorophores can be
localized with greater accuracy.
8.4 APPLICATIONS
8.4.1 Detection of Single Respiratory Syncytial Virus Particles
Respiratory syncytial virus (RSV) causes serious infection of the lower respiratory
tract in young children and the immune compromised adults. However, no RSV
vaccine is available at present, and diagnosis is often made based on symptoms or
using serological studies. Serological approaches take a long time for disease
diagnosis and may not be useful in acute illness. Thus, there is a pressing need to
develop sensitive and rapid methods for detection of viral particles.
RSV has surface proteins that are used to fuse and attach with the host cell .
Using antibodies against these proteins, RSV and the surface protein
expression on its surface could be detect ed [42]. In this work, nanoparticles
were functionalized with antibodies that specifically recognize either attach -
ment protein or the fusi on protein on the viral particle, as shown in Fig. 8.6.
Then, two probes carrying nanoparticles with different emission profiles were
allowed to form a sandwich with the viral particle. When RSV is present in the
216 BIOMEDICAL NANOSTRUCTURES
solution, the two nanoparticles pass through the confocal probe volume at the
same time and the photons emitted from them are coincident (Fig. 8.6b).
The SMD system used in the above studies not only removes the par-focality
issue by using nanoparticles but also provides a single test for detecting
presence of protein, DNA, or virus in solution in real time. Real-time data
FIGURE 8.6 Counting viral particles in solution. The 40-nm nanoparticles conjugated
to RSV anti-F or anti-G protein monoclonal antibodies were used to produce red or
green photon emission, respectively. RSVDG, used as a control, produced low green
photon counts as expected and did not show coincidence signals (a), while coincident
peaks were observed for RSV/A2 (b).
BIOCONJUGATED NANOPARTICLES FOR ULTRASENSITIVE DETECTION 217
processing with even counting and coincidence detection can enable multi-
plexed SMD and has been shown to provide wider dynamic range in
comparison with cross-correlation [43]. However, flow-based molecule count-
ing is slow because of the low sample throughput. For target concentrations
below the femtomolar level, signi ficant time may be required to achieve
confidence in the detection. As shown in the next section, imaging-based single-
molecule detection assays may provide a solution.
8.4.2 Single-Molecule Detection by Two-Color Imaging
Figure 8.7 shows the results obtained when complementary DNA sequences
are coupled with dual-color nanoparticles. Several yellow spots indicating
nanoparticle colocalization are seen only when the targets are complementary
(Fig. 8.7a and b). The DNA hybrid was 24 base pair long, which is smaller
than the persistence length of double-strand DNA [44]. Thus, it i s like a
‘‘rigid rod’’ and one would expect the center of the two beads to appear at
54 nm distance. By using the above localization scheme, we find that the two
beads are separated by 54-nm, which is consistent with the theoretical
estimates.
FIGURE 8.7 Dual-color imaging and colocalization of nanoparticle probes at
nanometer precision. (a and b) Nanoparticles form diffraction-limited pairs resulting
in color change only in presence of complementary sequences. (c) Image of a real RG
bead pair separated into red and green components that are convolved with a Gaussian
kernel to determine center. (d) High SNR >60 allows highly accurate measurements.
(e) Close-up of colocalization of the RG bead pair.
218
BIOMEDICAL NANOSTRUCTURES
When this assay is performed for over nine million bead pairs to detect a
segment of breast cancer antiestrogen resistance gene at picomolar concentra-
tions, the image processing can be performed in under a minute on a desktop
computer. The results are shown in Fig. 8.8. In the presence of the target
molecule, a clear peak is seen in the RG distance histogram which is absent
when the target is absent. This approach is not limited to nucleic acid molecules
but can be extended to protein and pathogen detection as well. Thus, single-
molecule imaging allows quantitative and high throughput detection of
biological targets. Since infectious agents lie in the same size range as
nanoparticles, the use of color-coded nanoparticles should allow rapid and
sensitive detection of infections disease biomarkers and intact viruses.
8.5 CONCLUSION AND OUTLOOK
Quantum dots and other fluorescent nanoparticles have emerged as a new class
of versatile biological probes for in vitro diagnostics [42] to molecular/cellular
imaging [25]. By combining magnetic and optical nanoparticles, multimodality
probes have also been prepared (Agrawal and Nie, unpublished data). Using
magnetic enrichment and separation along with fluorescent detection and
identification, highly multiplexed, ultrasensitive assays can be realized for
clinical use. Advances in nanoparticle synthesis and instrumentation are likely
to continue to open up new frontiers in ultrasensitive biological detection.
Miniaturization of instruments and availability of safe nanoparticle probes will
allow production of low cost, point-of-care, multifunctional diagnostic devices.
Practical applications of these multifunctional nanodevices will not come
without careful research, but the multidisciplinary nature of nanotechnology
may expedite these goals by combining the great minds from many different
fields. The results seen so far with QDs point tow ard the success of QDs in
biological systems and also predict the success of other nanotechnologies for
biomedical applications.
FIGURE 8.8 Rapid detection of DNA molecules. Nanoparticle labeled probes
colocalize in target-dependent manner. RG distance histogram (a) has a peak that
is missing in the control experiment (b, no target present).
BIOCONJUGATED NANOPARTICLES FOR ULTRASENSITIVE DETECTION 219
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PART II
Bio-Nano Interfaces
CHAPTER 9
ECM Interactions with Cells from the
Macro- to Nanoscale
STEVE MWENIFUMBO and MOLLY M. STEVENS
9.1 INTRODUCTION
Cells respond and interact with local chemical and topographical stimuli within
their environment that range from the macro- to nanoscale. The extracellular
matrix (ECM) plays a vital role in determining cell behavior through a melange
of carefully choreographed extrinsic signals that orchestrate cellular functions
in tissue development and homeostasis [1]. The significance of this role has
become apparent in recent years, as the discovery and characterization of new
ECM constituents, identification of novel receptors and signaling mechanisms,
and the characteriza tion of various effector molecules that regulate matrix
assembly and turnover have brought new insights into ECM function.
The constituent molecules and architecture of ECM provide a dynamic
environment that not only physically supports cells, but also guides cell
behavior [2–7]. The numerous variants of the constituent molecules, their
ratios, and relative arrangements give rise to considerable diversity in ECM
form, each adapted for tissue specific biological, structural, mechanical, and
functional requirements. This diversity generates a wealth of exposed and
encoded information that is interpreted by a large number of cellular receptors
such as transmembrane glycoproteins, proteoglycans, and glycolipids. A two-
way flow of information ensues, which mediates gene and consequently
protein expression that ultimately regulates cell processes such as cell
migration or ECM remodelling [4]. The regulation of these processes requires
both spatial and temporal coordination if cells are to form dynamic functional
tissues [6].
BiomedicalNanostructures, EditedbyKennethE.Gonsalves,CraigR.Halberstadt,CatoT.Laurencin,
and Lakshmi S. Nair
Copyright # 2008 John Wiley & Sons, Inc.
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