Download Advances in High-Throughput Screening

Combinatorial Chemistry & High Throughput Screening, 2003, 6, 777-787
777
Advances in High-Throughput Screening: Biomolecular Interaction
Monitoring in Real-Time with Colloidal Metal Nanoparticles
P. Englebienne1*,2, A. Van Hoonacker2, M. Verhas1 and N. G. Khlebtsov3
1*
Biocybernetics Unit, Laboratory for Experimental Medicine, and Department of Nuclear Medicine, Free
University of Brussels, Brugmann Hospital, Place Van Gehuchten 4, B-1020 Brussels, Belgium
2
Englebienne & Associates, Zingem, Belgium
3
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, Saratov,
Russia.
Abstract: The post-genomic era is revolutionizing the drug discovery process. The new challenges in the
identification of therapeutic targets require efficient technological tools in order to be properly addressed.
Label-free detection systems use proteins or ligands coupled to materials of which the physical properties are
measurably modified upon specific interactions. Among the label-free systems currently available, the use of
metal nanocolloids offers enhanced throughput and flexibility for real-time biomolecular recognition
monitoring at a reasonable cost.
Keywords: Colloidal gold; colloidal silver; localized surface plasmon resonance; biomolecular interaction; protein
conformation; affinity; real-time association; label-free detection.
INTRODUCTION
Recent advances in genomics and molecular biology have
introduced new frontiers in the drug discovery process by
producing an increasing number of possible drug molecular
targets. This trend, coupled with major increases in
compound libraries produced by combinatorial techniques,
has created important needs for improved high-throughput
screening (HTS) technologies [1-3]. These needs have been
addressed along three independent but complementary lines
of technological development involving respectively in
silico (computational) methods [4, 5], miniaturization and
automation of classical platforms [6-9] and the optimization
of non-radioactive signal detection systems [10-12]. Within
the scope of the latter approach, the resonance energy transfer
of fluorescent and bioluminescent labels has been
successfully applied to homogeneous biomolecular
interaction studies, both in solution [13] and living cells
[14]. However, despite the fact that key problems associated
with the detection of fluorescence signals such as quenching
and autofluorescence have now been overcome [15], such
labels suffer from two important intrinsic drawbacks:
“photobleaching”, i.e. the irreversible conversion from
excited fluorophore to nonfluorescent products by
photochemical processes [16] and “blinking”, i.e. the rapid
intermittence of emission [17]. As a consequence, other
optical detection systems have been investigated as possible
alternatives applicable in HTS, including label-free optical
detection which is currently a field of tremendous research
[18-20]. Label-free detection systems use so-called ‘smart
materials’ to monitor the interaction between two molecules.
The interaction occuring at the surface of the ‘smart material’
*Address correspondence to this author at the Biocybernetics Unit,
Laboratory for Experimental Medicine, and Department of Nuclear
Medicine, Free University of Brussels, Brugmann Hospital, Place Van
Gehuchten 4, B-1020 Brussels, Belgium; E-mail: [email protected]
1386-2073/03 $41.00+.00
induces a directly measurable change in one of its physical
properties, such as conductivity or optical characteristics.
Among the label-free optical detection methods, surface
plasmon resonance (SPR) in biosensing instruments has
held high promise that sofar has unfortunately not been held
[21]. As an alternative to gold films as the substrate for
generating the SPR effect, we have indicated quite early the
benefits that could be gained by substituting colloidal gold
nanoparticles instead [22, 23]. This timely review
summarizes the recent advances made in the application of
noble metal colloidal nanoparticles to real-time monitoring
of biomolecular interactions for HTS.
THE SPR EFFECT AT THE METAL SURFACE OF
FILMS AND PARTICLES.
The SPR phenomenon results from the creation of
collective oscillations in the conduction electrons at a metal
surface by an incident light beam (optical electric field). In
biosensors, the metal surface is made of a thin layer (usually
gold) and the SPR phenomenon occurs when an incident
beam of light strikes the surface at a given angle through a
prism. This results in the induction of photon-plasmon
electromagnetic waves at the metal-dielectric interface that
propagate along the interface. The associated optical electric
field (evanescent wave) decays exponentially away from the
surface which results in a reduced intensity of the reflected
light [24]. The reflectance of the light beam depends on the
dielectric constant at the metal surface and hence on the
refractive index. Consequently, when a sensing molecule is
attached to the metal surface, any interaction with a cognate
ligand modifies the refractive index at the surface and the
change is directly reported by a decrease in reflected light
intensity. When monochromatic light strikes the surface
with a broad distribution of incident angles, the reflected
light reaches the detector at different points. The detector
monitors continuously the position of reduced light
© 2003 Bentham Science Publishers Ltd.
778
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8
intensity and calculates the angle of reflection, which
increases the sensitivity of detection [25].
In a similar way, when noble metal nearly monodisperse
spherical nanoparticles in solution are excited by
electromagnetic radiation, they exhibit collective oscillations
in their conduction electrons which result in wavelengthselective absorption and scattering of the incident radiation.
The extinction of such particles, provided that their size is
smaller than the wavelength of the incident light, is the sum
of the absorption and scattering components, both of which
are directly related to the particle polarizability. The
polarizability (α, at a given frequency ν=c/λ) of such metal
spheres is in direct relation with the dielectric constants (ε)
of both the metal (ε m ) and its surrounding medium (ε 0 ),
according to the relationship:
α(v) = f m
ε
m-
ε
m+
εo
kε o
,
(1)
where ƒm is the metal volume fraction and κ is a constant
characteristic of the form of the particles (κ=2 for spheres).
Englebienne et al.
The dielectric constants of noble metals is composed of
an imaginary and a real part. The imaginary component is
small and does not vary much with the wavelength in the
visible region. Counter to that, the real component is
negative and decreases with increasing wavelengths.
Therefore, the above relationship tells us that for a sphere
particle, at the wavelength where ε m = -2ε 0 , the particle
polarizability and hence the extinction become very large.
For colloidal gold or silver nanoparticles, this results in an
extinction spectrum exhibiting a single peak respectively
around 520 or 405 nm, which is termed the localized
(L)SPR [26]. The equation tells us also that if the dielectric
constant of the external medium is increased by a factor Δε0,
the εm term will have to be more negative by the same factor
for the LSPR condition to be met. Therefore, any tiny
increase of the refractive index of the medium surrounding
the particles (the refractive index equals the square root of
the dielectric constant) will induce a red shift of the LSPR
peak wavelength. This is exemplified in Fig. (1) for silver
and gold nanoparticles of the same diameter in an aqueous
solution of which the refractive index is progressively
Fig. (1). Linear relationship between the LSPR red shift of silver (triangles) or gold (circles) nanoparticles (50 nm diameter) in
solution and the increase in refractive index of the surrounding medium progressively changed by adding increasing proportions of
glycerol in water (open symbols and solid lines). These experimental data are in good agreement with data obtained by theoretical
simulation (solid symbols, dotted lines) according to the Maxwell-Garnet model [27, 28].
Advances in High-Throughput Screening
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8 779
modified by the addition of glycerol. As further shown in
Fig. (1), there is a good agreement between the experimental
and simulated data. In this example, the data have been
simulated according to the Maxwell-Garnett theory
summarized in equation (1) above, which is valid in the
limit where particle dimensions are small relative to the
wavelength [27]. This LSPR peak red-shift results in a
decrease in the extinction at the original LSPR wavelength
and in an increase in extinction at longer wavelengths. A
detailed comparison of the physical optical principles
underlying the SPR and LSPR phenomena is provided in a
recent review [28]. We use this LSPR property of colloidal
noble metals nanoparticles to analyze biomolecular
interactions. When such a particle is coated with a ligand or
a binding molecule, a first shift occurs in the LSPR peak as
the result of the refractive index change at the particle surface
and a further shift occurs when the coated particle interacts
with the cognate molecule. Therefore, any interaction is
transduced by a change in the extinction spectrum of the
colloid.
Fig. (2). Principles of kinetic measurements by SPR during biomolecular interactions using either a biosensor (A) or metal
nanocolloids (B), respectively. In the biosensor, a change in refractive index (ε 0) at the surface of the metal layer due to the progress
of the interaction during time is transduced by a change in the angle of light reflection (θ) with reduced intensity. With the
nanocolloids, the change in ε0 at the particle surface is transduced by a shift in maximal absorption wavelength.
780
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8
Fig. (2) illustrates and compares the principles of kinetic
SPR measurements of biomolecular recognition events
occuring on either a metal layer and recorded in a biosensor
(part A), or on colloidal metal nanoparticles and recorded in
a spectrophotometer (part B), respectively.
NANOPARTICULATE NOBLE METALS FOR USE IN
MONITORING BIOMOLECULAR INTERACTIONS
Nanoparticles of gold and silver are synthesized by
reduction of an aqueous solution of the metal salt. Several
methods have been advocated of which the most popular are
the Frens’ reduction with sodium citrate and the borohydride
reduction process [22, 23]. These procedures allow to obtain
homeodisperse solutions of particles with diameters ranging
Englebienne et al.
from several nanometers up to micrometers. The molar ratio
of reductant over the metal salt and the pH of the solution
govern the size of the particles generated [23, 29]. The
synthetic process occurs by metal nucleation followed by
further crystal growth and particle maturation. The colloid
remains stable due to the repelling identical negative charges
present on the particle surfaces but is susceptible to
coagulation upon addition of small concentrations of salts.
Such nanoparticles present the particularity to remain
negatively charged over a wide range of pH and
consequently, a protein layer is easily coated on the particle
surface by charge adsorption. Once coated by a protein layer,
the particles are fully stabilized against salt coagulation,
which is verified by spectrophotometry after salt addition.
The process can be optimized by evaluating pH isotherms of
particle stabilization and reactivity in presence of a given
Fig. (3). Stability of colloidal metal nanoparticles coated with proteins by charge adsorption toward leakage during time. This is
exemplified by the small progressive reduction in performance of dose-response curves of an immunoassay for human ferritin using
colloidal gold coated with anti-ferritin antibody [31]. The curves shown have been obtained with the same batch of reagent at the time
of preparation (open circles), and after respectively 17 (open squares) and 47 (closed circles) months of storage at 2-8°C. The loss of
reactivity is less than 50% after close to 4 years of storage. The data shown are the average ± sd of duplicate measurements.
Advances in High-Throughput Screening
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8 781
protein concentration [22, 23]. Monitoring the reactivity
during the coating process is important because the
orientation of the binding molecule on the particle surface is
dictated by the relative isoelectric points of its molecular
parts and hence by the coating pH [22, 23]. Alternatively,
the particles can be coated and stabilized by an alkylthiol
layer which allows for further covalent attachment of the
molecule of interest [30]. The particles are coated noncovalently either with an excess of binding protein or ligand
which is eventually removed by centrifuging and washing
the particles [22], or with a limited protein or ligand
concentration sufficient to form the external layer [22, 23],
respectively. The latter procedure presents the advantage that
no purification is required. Such coated particles are
particularly stable over time and offer sustained performance
[23]. This is exemplified in Fig. (3) which shows the doseresponse relationships obtained with colloidal gold coated
with an anti-ferritin antibody and interacting with ferritin,
according to a published procedure [31]. The curves shown
have been obtained with the same batch of colloid either at
the time of preparation, or 17 and 47 months later,
respectively. The loss of reactivity is less than 50% after
close to four years of storage at 2-8 °C.
When nanoparticles coated with a binding protein or a
ligand interact with the cognate molecule, the change in
refractive index induced at the particle surface by the binding
event is reported by a change in the visible extinction
spectrum of the solution [22, 23, 28, 32]. This phenomenon
Fig. (4). Difference extinction spectra observed using silver (A) or gold (B) nanoparticles sensitized with the same anti-C-reactive
protein (CRP) polyclonal antibody (IgG fraction), as previously described [39]. The reaction mixture (1 ml) contains 1.3x101 2
nanoparticles, each coated in average by 5 IgG molecules. These are respectively reacted for 5 min. with 0.5, 1, 2.5 and 5 x 101 1
molecules of CRP. The reference cell contains no ligand. The change in refractive index at the particle surface induced by ligand
binding produces a decrease of extinction at the LSPR and an increase of extinction at longer wavelengths (arrows).
782
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8
is further exemplified by the difference spectra shown in Fig.
(4) which have been respectively obtained with silver and
gold nanoparticles coated with the same antibody and reacted
with increasing amounts of the ligand during a fixed period.
The optical reactivity of the sensitized nanoparticles is not
only dose-dependent as shown on Figs. (3) and (4), but is
also time-dependent at a given ligand dose and it allows
therefore to monitor the real-time kinetics of interaction
between the cognate biomolecular species respectively
present on the particle surface and in the surrounding
medium [23, 28, 32]. Optical LSPR permits also to monitor
protein conformational changes at the surface of the particles
[28], a particularly interesting tool as far as drug discovery is
concerned. In this application, the LSPR peak modifications
are also induced by changes in the size of the protein layer
surrounding the particles, which is dependent on the protein
conformational orientation at the surface. Such application is
similarly possible in SPR biosensors and has been validated
versus both circular dichroism and Fourier transform infrared
spectroscopies [33].
As already apparent in Fig. (1), the data presented in Fig.
(4) further show that the LSPR phenomenon is reported
differently for sensitized silver and gold nanoparticles upon
biomolecular recognition, the former displaying a larger
change in extinction at the LSPR (405 nm), although the
latter display the larger change in extinction at longer
wavelengths (600 nm). These differences result from
differences in particle polarizability due to the evolution of
the respective metal dielectric constants with wavelength. As
the data of Fig.(5) further show, the real part of the dielectric
constant of silver decreases monotonically with increasing
Englebienne et al.
wavelength in the visible spectrum. For gold, the decrease is
less monotonous and the slope is particularly high in the
LSPR region of the spectrum. Consequently, for an identical
change in refractive index, the LSPR peak shift is much
higher for silver than gold.
Besides the metallic composition of the nanoparticles,
their size play an important role in their capacity to report
biomolecular recognition by LSPR [34]. As shown in Fig.
(6), there is a quite fair agreement between theoretical
simulation and observed experimental data to indicate that
particles with a diameter of 50-60 nm provide with the best
analytical response. In our photometric applications, we use
particles of that size.
HIGH-THROUGHPUT SCREENING APPLICATIONS OF LSPR WITH NANOPARTICLES
The application of the SPR technology in biosensors
which arose in the early nineties, allowed for the labelingfree monitoring of molecular interactions in real-time [35].
The application of this technology led to the development of
commercial optical biosensing instruments which are
playing a significant role in basic research and
pharmaceutical sciences [36]. The relatively high cost and
limited throughput of such instruments has stimulated a new
field of research for the evaluation of cheaper optical
substrates and techniques capable to substitute for SPR
biosensors. Representative examples of these efforts are the
reflectometric interference spectroscopy [18] and the
colorimetric resonant reflection technique [19, 20]. Over the
Fig. (5). Evolution of the imaginary (ε”, open symbols) and real (ε’, closed symbols) parts of the dielectric constant of gold (circles)
and silver (triangles) as a function of the wavelength in the visible spectrum. The high negative slope of ε’Au at the LSPR wavelength
does not allow for such a high wavelength shift upon a given change of the refractive index at the particle surface, when compared to
that of silver of which the ε’ decreases monotonically (arrows). Data are from the Sopra S.A. database (www.sopra-sa.com/indices.htm).
Advances in High-Throughput Screening
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8 783
Fig. (6). Agreement between theoretical simulation and experimental observation for the ability of colloidal nanoparticles of various
sizes to report biomolecular recognition by LSPR. In the theoretical model, the sizes of the particles correspond to the original
diameter of gold to which is added a primary two-layered shell of recognizing molecules (thickness 2.5 nm each). The change in
absorbance (ΔA, triangles) at the LSPR peak is computed according to Mie’s theory [27] for the addition of two-layers (total
thickness 5 nm) corresponding respectively to a first shell formed by the antibody and a second shell of interacting molecules. The
experimental data have been obtained with gold particles coated with an anti-human chorionic gonadotropin (hCG) monoclonal
antibody (1.4 x 1012 molecules in the reaction mixture) and the size indicated correspond to the coated particles. The ΔA (circles) has
been measured at 600 nm after interaction with 4.5 x 1010 molecules of the ligand in a final volume of 200 µl, using an immunoassay
protocol identical to that described in ref. [31].
years, the sensitivity of SPR biosensors to biomolecular
interactions has been improved by several means, including
the use of a further layer of colloidal gold nanoparticles on
the metal film, which results in a tremendous signal
amplification [37, 38]. The phenomenon was attributed to
the significant absorption cross section of the nanoparticulate
layer, resulting in absorption of plasmon energy and
damping of the surface mode [38]. At that same period, we
showed that solutions of colloidal gold nanoparticles could
be used in a common spectrophotometer for the real-time
monitoring of association and dissociation kinetics between
interacting biomolecules [32]. The specificity of the
measurement was ascertained by verifying the absence of
significant signal upon interaction of particles sensitized
with receptors and non-cognate ligands, in comparison to the
signal observed with the cognate ligand [39], as well as by
correlation studies with other techniques [31, 32, 39]. We
showed also [32, 39] that the LSPR shift phenomenon that
we observed was different from that resulting from particle
agglutination which had previously been applied to solparticle immunoassays [40] and colorimetric detection of
DNA hybridization [41, 42]. The best proofs that the signal
observed does not result from particle agglutination have
been provided by monitoring the interaction of haptenic
ligands such as digoxin or theophylline with nanoparticles
coated with their respective monoclonal antibodies [39].
Meanwhile, we have adapted the technique to HTS using a
clinical chemistry automated analyzer which allows to
handle over 3,000 samples a day in a walk-away mode [22,
23, 28, 32, 39]. The technique can be similarly adapted to
784
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8
higher throughput clinical analyzer or plate reader platforms.
As an example, Fig. (7) shows such association-dissociation
kinetics measured in a clinical chemistry analyzer with
colloidal gold nanoparticles sensitized with a monoclonal
antibody, during their interaction with increasing doses of
the cognate ligand, namely troponin I (a small protein) [39].
As mentioned above (see Fig. (4)), there is a difference in
LSPR reactivity between gold and silver. Because of the
large absorbance increase at longer wavelengths with gold,
we use to follow the kinetics at 600 nm with this metal,
where a significant positive signal can be observed during
association (see Fig. (7)). With colloidal silver however, the
higher signal sensitivity occurs at the original LSPR
wavelength (see Fig. (4)) and therefore, we follow the
kinetics at 400 nm, a wavelength where association kinetics
are translated by a progressive decrease in the optical signal,
as exemplified by the association curves shown in Fig. (8A).
Most clinical chemistry analyzers and plate readers are
equipped with filters permitting to record absorbances at
these wavelengths. In the example of Fig. (8A), the silver
particles have been coated with an anti-C-reactive protein
(CRP) polyclonal antibody and stabilized as previously
described for gold [32, 39]. The association kinetics
displayed have been observed with increasing CRP
Englebienne et al.
concentrations and it is worth noting that while for the
highest concentration used, the number of CRP molecules
reacted exceeds the number of IgG antibody molecules
available on the particles, the signal is still indicative of a
maximal saturation, and hence of absence of agglutination,
as further illustrated in Fig. (8 B ). Other groups have
meanwhile applied the LSPR technology with colloidal gold
or silver nanoparticles in a solid-phase format to interrogate
biomolecular interactions in real-time [43 - 46]. The solidphase format presents an advantage over the liquid format
because the former can easily be adapted to microarray
detection and imaging. Classical SPR biosensors have been
recently applied in such a format for the imaging detection
of either peptide-antibody [47] or nucleic acid-nucleic acid
[30] interactions. Such format could therefore be considered
in the very near future as particularly appealing for highthroughput screening in microarray photometric readers.
In our previous reports [32, 39], we have demonstrated
that the affinity constants calculated from LSPR data of
colloidal gold nanoparticles in solution were in good
agreement with those evaluated by other techniques
including SPR biosensors such as the BiaCore. As
mentioned elsewhere [21, 48], despite reports indicating a
possible parallelisation of measurements [49], the
Fig. (7). Association-dissociation kinetics observed by LSPR in a clinical chemistry analyzer as described previously [39]. Colloidal
gold nanoparticles coated with an anti-troponin I monoclonal antibody (cat. 10022, OEM concept, Toms River, N.J.; 1.9 x 101 2
molecules in the reaction mixture [final volume 200 µl], with an average of 3.8 molecules per particle) are reacted with the cognate
ligand at three different doses (0.25, 0.5 and 1x1012 molecules in the reaction mixture, respectively diamonds, triangles and circles)
and the association (solid symbols) is followed during 10 min. The dissociation (open symbols) is induced by injecting an 100 fold
excess of ligand (arrow). Data are the average ± sd of duplicates.
Advances in High-Throughput Screening
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8 785
Fig. (8). Association kinetics (part A) monitored in a clinical analyzer according to ref. [39], by the decrease in A400nm of a solution of
colloidal silver nanoparticles coated with a layer of anti-CRP polyclonal antibody (Midland Bioproducts, Boone, IA; 1.5 x 1013 IgG
molecules in reaction mixture [final volume 165 µl] with an average of 5 IgG molecules per particle), interacting with increasing
concentrations of the ligand (0.12, 0.5, 2.5 x 1011 and 5.1 x 1013 molecules in reaction mixture, respectively triangles, diamonds,
circles and squares). Data are the average ± sd of duplicates. Comparison (part B) between the simulated Heidelberger curve according
to Hill model equations [23] that would occur in case of agglutination (triangles and solid curve), and the actual dose-response data
(circles and dashed curve, average ± sd) indicative of full particle saturation, even beyond the equivalence point.
commercial SPR biosensors do not match the current
throughput requirements of the pharmaceutical industry,
unless a tremendous increase in the number of instruments is
considered worth the expenditure. Therefore, the LSPR
system with colloidal noble metal nanoparticles offers more
flexibility in this respect. Another problem faced by the
SPR biosensors is their relatively low analytical sensitivity
in terms of detection limit, particularly when the interactions
between low molecular weight ligands and weak affinity
binders are considered [50]. An improvement of detection
limits is considered as a need for future development in the
field of SPR biosensing instruments [51]. SPR biosensors
can resolve smaller changes in refractive index [51] than do
LSPR with nanoparticles [43]. The electromagnetic field due
to the surface plasmon phenomenon extends approximately
to 200 nm from the metal film in biosensors [28] whereas
this extention is much smaller for nanoparticles [52].
However, in conventional SPR biosensors, the chip surface
available for interaction is about 25 mm2 although in a
typical experiment with nanoparticles, the surface available,
and hence the total surface coverage of biomaterial, is much
higher by the thousand order of magnitude. Consequently,
the use of LSPR with colloidal metal nanoparticles can offer
some improvement for weak affinity calculations [28, 39].
786
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8
The challenges currently facing HTS technology involve
not only a constant quest for higher throughput in terms of
biomolecular interaction analysis in vitro, but also the need
to evaluate the interactions in vivo, i.e. within the cell [11,
14, 53]. Colloidal gold probes have been in use for many
years as cellular component identifiers and markers detected
by transmission electron microscopy [54]. However, the cell
denaturing treatment required before the observation can be
made precludes to monitor any kinetic development of the
interaction during observation. Such probes have also been
studied by light microscopy but sofar, optical detection was
limited to aggregated nanoparticles and the performance of
detection did not compete with fluorescence [1, 54]. Recent
key advances in the field have however brought colloidal
nanoparticles back at the forefront of the optical microscope.
By elegant theoretical [55] and experimental [56] approaches,
Yguerabide and Yguerabide have demonstrated that due to
their light-scattering properties, resonant particles could be
treated and used as fluorescent analogs in cell and molecular
biology studies. In a recent review [57], Schultz claims that
under the same excitation conditions, a plasmon resonant
particle of 100 nm diameter has a brightness equivalent to
approximately 100,000 fluorescein molecules. The particles
are viewed using a microscope configured for various modes
of darkfield illumination [57]. The optical scattering
characteristics of the colloids can further be adapted
according to the shape of the particles. When compared to
fluorescent tracers, the plasmon resonant particles offer the
advantage that they do not undergo photobleaching [57].
This new application of the SPR properties of metal
nanoparticles in microscopy looks very promising in the
drug discovery process, particularly for new lead target
identification and for the intracellular real-time tracking of
molecules.
CONCLUSIONS AND PROSPECTS
The application of the optical properties of colloidal
noble metal nanoparticles to the drug discovery process has
now passed its infancy and has reached a full maturation
period. The numerous advantages (simple particle synthesis,
derivatization and handling, stability during storage, simple
optical label-free detection system, ease of automation and
low cost) over more sophisticated technologies renders the
technique appealing for various applications. The colloidal
noble metal LSPR technology offers a high level of
flexibility which allows for protein conformation and
biomolecular interaction studies in liquid or solid-phase
formats by visible spectroscopy, as well as cellular target
identification by light microscopy. Further evolution of the
technology will most probably occur along several lines of
development. A first line will see new biosensing platforms
developed, such as optical fibers [58], in order to enhance
the throughput capacity. A second line will take advantage
of the differential optical reactivity of particles with different
sizes and shapes in order to allow for multiplexed analysis
of different interacting systems. Along that line, a recent
publication [59] reports the synthesis of cylindrical nanorods
with alternating submicrometer striping patterns made of
different noble metals. Because of their pattern containing
alternating metals with different optical properties, such
nanorods can be distinguished from one another by
Englebienne et al.
microscopic examination exactly like barcode labels. A third
line will explore the capacity of composite or modified
particles to enhance the optical signal sensitivity to changes
in refractive index at the particle surface. As a recent
publication demonstrates [60], the LSPR peak shift of gold
nanoshells is 5-10 fold more sensitive to external refractive
index changes when compared to nanoparticles of identical
size. Finally, a fourth line of research will focus on the
improvement of classical labels performance by their
combination with colloidal noble metal nanoparticles. A
recent example of this trend of research is the development
of radiative decay engineering [61], which by combining
fluorophores with colloidal silver, allows to increase their
fluorescence intensity by several decades. The current and
foreseeable future developments of the LSPR technology
with noble metal nanoparticles deserve undoubtedly focused
attention for their possible applications in the drug discovery
process.
ACKNOWLEDGEMENTS
NGK was partly supported by RFBR grant 01-03-33130a
and CRDF grant REC-006.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
Hertzberg, R.P.; Pope, A.J. Curr. Opin. Chem. Biol., 2000, 4, 445.
Auld, D.S.; Diller, D.; Ho, K-K. Drug Discov. Today, 2002, 7,
1206.
Landro, J.A.; Taylor, I.C.A.; Stirtan, W.G.; Ostermen, D.G.;
Kristie, J.; Hunnicutt, E.J.; Rae, P.M.M.; Sweetnam, P.M. J.
Pharmacol. Toxicol. Methods, 2000, 44, 273.
Fay, N.; Ullmann, D. Drug Discov. Today, 2002, 7, S181.
Rodrigues, A.D.; Lin, J.H. Curr. Opin. Chem. Biol., 2001, 5, 396.
Battersby, B.J.; Trau, M. Trends Biotechnol., 2002, 20, 167.
Huels, C.; Muellner, S.; Meyer, H.E.; Cahill, D.J. Drug Discov.
Today, 2002, 7, S119.
Stockman, B.J.; Dalvit, C. Prog. NMR Spectrosc., 2002, 41, 187.
Kyranos, J.M.; Cai, H.; Wei, D.; Goetzinger, W.K. Curr. Opin.
Biotechnol., 2001, 12, 105.
Boute, N.; Jockers, R.; Issad, T. Trends Pharmacol. Sci., 2002, 23,
351.
Schwille, P.; Kettling, U. Curr. Opin. Biotechnol., 2001, 12, 382.
Bazin, H.; Préaudat, M.; Trinquet, E.; Mathis, G. Spectrochim.
Acta Part A, 2001, 57, 2197.
Mere, L.; Bennett, T.; Coassin, P.; England, P.; Hamman, B.; Rink,
T.; Zimmerman, S.; Negulescu, P. Drug Discov. Today, 1999, 4,
363.
Taylor, D.L; Woo, E.S.; Giuliano, K.A. Curr. Opin. Biotechnol.,
2001, 12, 75.
Grepin, C.; Pernelle, C. Drug Discov. Today, 2000, 5, 212.
Boyer, D.; Tamarat, P.; Maali, A.; Lounis, B.; Orrit, M. Science,
2002, 297, 160.
Nirmal, M.; Dabbousi, B.O.; Bawendi, M.G.; Macklin, J.J.;
Trautman, J.K.; Harris, T.D.; Brus, L.E. Nature, 1996, 383, 802.
Kröger, K.; Bauer, J.; Fleckenstein, B.; Rademann, J.; Jung, G.;
Gauglitz, G. Biosens. Bioelectron., 2002, 17, 937.
Lin, B.; Qiu, J.; Gerstenmeier, J.; Li, P.; Pien, H.; Pepper, J.;
Cunningham, B. Biosens. Bioelectron., 2002, 17, 827.
Cunningham, B.; Li, P.; Lin, B.; Pepper, J. Sensors Actuators B,
2002, 81, 316.
Gauglitz, G. Curr. Opin. Chem. Biol., 2000, 4, 351.
Englebienne, P. J. Mater. Chem., 1999, 9, 1043.
Englebienne, P. Immune and Receptor Assays in Theory and
Practice, CRC Press: Boca Raton, 2000.
Frutos, A.G.; Corn, R.M. Anal. Chem., 1998, 70, 449A.
Wilson, W.D. Science, 2002, 295, 2103.
Malinsky, M.D.; Kelly, K.L.; Schatz, G.C.; Van Duyne, R.P. J.
Phys. Chem. B, 2001, 105, 2343.
Advances in High-Throughput Screening
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
Combinatorial Chemistry & High Throughput Screening, 2003, Vol. 6, No. 8 787
Foss, C.A. Jr.; Hornyak, G.B.; Stockert, J.A.; Martin, C.R. J. Phys.
Chem., 1994, 98, 2963.
Englebienne, P.; Van Hoonacker, A.; Verhas, M. Spectroscopy
Int. J., 2003, 17, 255.
Goia, D.V.; Matijevic, E. Colloids and Surfaces A, 1999, 146, 139.
Wegner, G.J.; Lee, H.J.; Corn, R.M. Anal. Chem., 2002, 74, 5161.
Englebienne, P.; Van Hoonacker, A.; Valsamis, J. Clin. Chem.,
2000, 46, 2000.
Englebienne, P. Analyst, 1998, 123, 1599.
May, L.M.; Russel, D.A. Analyst, 2002, 127, 1589.
Khlebtsov, N.G.; Bogatyrev, V.A.; Dykman, L.A.; Krasnov, Y.M.;
Melnikov, A.G. Izv. Vuz. Applied Nonlinear Dynamics, 2002, 10,
172.
Malmqvist, M. In Immunotechnology; Gosling J.P. and Reen, D.J.
Eds.; Portland Press: London, 1993, pp. 61-75.
Baird, C.L.; Myszka, D.G. J. Mol. Recognit., 2001, 14, 261.
Lyon, L.A.; Musick, M.D.; Natan, M.J. Anal. Chem., 1998, 70,
5177.
Lyon, L.A.; Peña, D.J.; Natan, M.J. J. Phys. Chem. B, 1999, 103,
5826.
Englebienne, P.; Van Hoonacker, A.; Verhas, M. Analyst, 2001,
126, 1645.
Gribnau, T.C.; Leuvering, J.H.; van Hell, H. J. Chromatog., 1986,
376, 175.
Mirkin, C.A.; Letsinger, R.L.; Mucic, R.C.; Storhoff, J.J. Nature,
1996, 382, 607.
Elghanian, R.; Storhoff, J.J.; Mucic, R.C.; Letsinger, R.L.; Mirkin,
C.A. Science, 1997, 277, 1078.
Nath, N.; Chilkoti, A. Anal. Chem., 2002, 74, 504.
Received: 24 April, 2003
Accepted: 25 September, 2003
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
Jensen, T.R.; Malinsky, M.D.; Haynes, C.L.; Van Duyne, R.P. J.
Phys. Chem. B, 2000, 104, 10549.
Haes, A.J.; Van Duyne, R.P. J. Am. Chem. Soc., 2002, 124, 10596.
Riboh, J.C.; Haes, A.J.; McFarland, A.D.; Yonzon, C.R.; Van
Duyne, R.P. J. Phys. Chem. B, 2003, 107, 1772.
Nelson, B.P.; Grimsrund, T.E.; Liles, M.R.; Goodman, R.M.; Corn,
R.M. Anal. Chem., 2001, 73, 1.
Rademann, J.; Jung, G. Science, 2000, 287, 1947.
Berger, C.E.H.; Beumer, T.A.M.; Kooyman, R.P.H.; Greve, J.
Anal. Chem., 1998, 70, 703.
Strandh, M.; Persson, B.; Roos, H.; Ohlson, S. J. Mol. Recognit.,
1998, 11, 188.
Homola, J.; Yee, S.S.; Gauglitz, G. Sensors and Actuators B, 1999,
54, 3.
Jensen, T.; Kelly, L.; Lazarides, A.; Schatz, G.C. J. Cluster Sci.,
1999, 10, 295.
Chanda, S.K.; Caldwell, J.S. Drug Discov. Today, 2003, 8, 168.
Beesley, J.E. Colloidal Gold: A New Perspective for Cytochemical
Marking. Oxford University Press and Royal Microscopical
Society, Oxford, 1989.
Yguerabide, J.; Yguerabide, E.E. Anal. Biochem., 1998, 262, 137.
Yguerabide, J.; Yguerabide, E.E. Anal. Biochem., 1998, 262, 157.
Schultz, D.A. Curr. Opin. Biotechnol., 2003, 14, 13.
Cheng, S-F; Chau, L-K. Anal. Chem., 2003, 75, 16.
Walton, I.D.; Norton, S.M.; Balasingham, A.; He, L.; Oviso, D.F.
Jr.; Gupta, D.; Raju, P.A.; Natan, M.j.; Freeman, R.G. Anal.
Chem., 2002, 74, 2240.
Sun, Y.; Xia, Y. Anal. Chem., 2002, 74, 5297.
Lakowicz, J.R.; Malicka, J.; Gryczynski, Z.; Roll, D.; Huang, J.;
Geddes, C.D.; Gryczynsky, I. PharmaGenomics, 2003, 3, 38.