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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. 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