High Throughput Single Molecule Spectral Imaging of Photoactivated

Mar 24, 2009 - Real-time fluorescence spectra from photoactivated individual silver clusters on silver island film were obtained via inserting a trans...
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J. Phys. Chem. C 2009, 113, 5991–5997

5991

High Throughput Single Molecule Spectral Imaging of Photoactivated Luminescent Silver Clusters on Silver Island Films Lehui Xiao, Yan He,* and Edward S. Yeung* Biomedical Engineering Center, College of Chemistry and Chemical Engineering, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan UniVersity, Changsha 410082, Peoples Republic of China ReceiVed: NoVember 23, 2008; ReVised Manuscript ReceiVed: January 31, 2009

Real-time fluorescence spectra from photoactivated individual silver clusters on silver island film were obtained via inserting a transmission grating in the emission light path of an inverted microscope. The high throughput data collection ability allowed us to monitor photochemical dynamics of multiple silver clusters in two dimensions simultaneously and capture intermediates and rare events that can not be observed using traditional spectrometers. Neutral silver clusters were found to be the species that fluoresce on the film, and three major types of sliver clusters with emission maximums at 516, 530, and 550 nm were identified under the excitation of a 488 nm argon ion laser. In addition, randomly occurring events including reversible spectral shifts and abnormal fluorescence bursts from a few individual silver clusters were captured. Introduction Because of their unique optical and physical properties and potential applications in physics, chemistry, and biotechnology, metal nanostructures such as silver nanomaterials have attracted enormous interests recently.1-5 For example, because of their size-dependent plasmon resonance absorption and scattering, silver nanoparticles can display various bright colors under white light illumination. This property has been explored for biosensing.6,7 The strong increase of localized electromagnetic field produced near the metal surface leads to the widely studied surface-enhanced Raman Scattering (SERS) effect. The Raman signal of a reporter molecule could be significantly enhanced when it was in the vicinity of the nanoparticle surface.8,9 Similarly, metal-enhanced fluorescence (MEF) from dye molecules near the surface of silver islands was also observed because of the reduced radiationless energy transfer rate and fluorescence liftime.10-20 Besides strong SERS or MEF effects from reporter molecules on or near the surface of silver nanostructures, photoactivated fluorescence directly from individual silver nanoclusters has been reported recently.21,22 With dendrimers as the capping molecules, highly luminescent silver nanoclusters baring controllable emission maxima were synthesized using nitrate solution at ambient temperature. These fluorescent nanodots were ascribed to nanometer-sized clusters of several silver atoms. On adding silver ions into a colloidal silver solution stabilized by polyanions, intense fluorescence emission in the visible region was obtained, which is likely due to chemical reorganization of silver atoms at the surface of the silver nanoparticles.23 Colorful fluorescence was also observed from photoactivatied silver island films24 and silver oxide nanoparticles. 25,26 It was believed that neutral or charged silver clusters produced at the silver oxide surface during the photoactivation process were the main fluorescent species.25 These fluorescent silver nanoclusters could serve as potential biolabels. To gain more insights into their structural and spectroscopic * Corresponding author. Phone: +86 731 8821900. Fax: +86 731 8821904. E-mail: [email protected] (Y.H.) and yeung@ameslab. gov (E.S.Y.).

properties, it is important to develop new methods to monitor the real-time photochemical dynamics of individual silver clusters. During the past two decades, it has become possible to study the fundamental physical and chemical dynamic processes in solution at ambient temperature at the single molecule level.27-30 Previous works have used single molecule spectroscopy (SMS) to study the blinking of quantum dots31 or fluorescent protein molecules,32,33 conformational changes in single protein or RNA molecules,34,35 and so forth. Mechanisms of these fundamental physical processes have been successfully revealed. Moreover, the individual fluorescence spectrum and fluorescence lifetime from single dye molecules have been measured by coupling a fluorescence microscope with a spectrograph/CCD combination. The spectral diffusion of single rhodamine molecule from different meta-stable states or single quantum dots has been observed.36-38 However, only one spectral profile could be collected each time. The time-consuming data collection ability greatly limits its vast application in various areas. To capture rare and randomly occurring processes, it is necessary to record individual spectra in a large area at the same time. Previously, we have demonstrated that high throughput single molecule fluorescence spectra could be obtained simultaneously by inserting a transmission grating in the light collection path and have applied this technique to discriminate dye-labeled single DNA molecules in free solution.39,40 Here, using this imaging technique, the photoactivation process from silver islands was further investigated at the single molecule level. During the photoactivation process, multiple fluorescence spectra from spatially separated individual silver clusters were obtained at room temperature in a large area. Three types of silver clusters on excitation by a 488 nm argon ion laser were clearly identified. Rare events including randomly occurring reversible spectral shift on the silver island surface and strong fluorescence bursts from a few single silver clusters with intensity enhancement as high as 20 times were observed.

10.1021/jp8102904 CCC: $40.75  2009 American Chemical Society Published on Web 03/24/2009

5992 J. Phys. Chem. C, Vol. 113, No. 15, 2009 Experimental Methods Silver Island Film Preparation. The silver island film, a substrate commonly applied to metal enhanced fluorescence study, was prepared according to the method described elsewhere.20 In brief, cover glasses (22 × 22 mm2, Corning, NY) were dipped in piranha solution (mixture of H2SO4 (95-98%) and H2O2 (30%) with volume ratio 7:3) overnight to remove organic residues, then sonicated in 75% alcohol solution three times and followed by extensive rinsing with deionized water (MilliPore). Freshly prepared cover glasses were oven-dried immediately before silver island film preparation. The silver island film was chemically reduced onto the cover glass surface with D-glucose. First, 1.5 mL 5% NaOH solution was added dropwise into 45 mL 0.049 M silver nitrate solution with vigorously stirring. After sliver ions were completely precipitated, 1 mL 30% NH3•H2O was slowly added into the solution to dissolve the precipitate. The solution was cooled to 5 °C in an ice bath, and 11 mL of a freshly prepared D-glucose solution was added. Several pairs of cover glasses processed by the above method were dipped in the solution, so only one face of each cover glass would be covered with silver islands. The solution was kept at 5 °C for two more minutes and then warmed up to 30 °C over 5 min. Those freshly prepared silver island films were washed with deionized water three times and sonicated for 0.5 min to remove excess nonadhesive silver particles and other residues. The prepared silver island films were then stored in deionized water. All of the above procedures were performed in the dark. Flow Channel Preparation. In order to monitor the variation of fluorescence intensity of silver clusters with changing environment, a Y-shaped flow cell was made in house. Two microsyringes filled with water, 0.01 M NaBH4, or 1% mercaptoproponal (Sigma-Aldrich) were mounted on the two branches of the PDMS flow cell. By slowly injecting the solution into the channel, the fluorescence intensity fluctuation caused by changing of environment was monitored by the CCD camera. Imaging Setup. A Nikon TE2000-U inverted microscope operated under fluorescence imaging mode was used for all of the fluorescence measurements. Images were collected by a CoolSnap HQ2 CCD (Photometrics). The MetaVue (Universal Imaging Corp.) software was used to control the CCD camera. A 488 nm argon ion laser coupled with a single mode optical fiber with variable output power was used to photoactivate the fluorescence from silver islands. A 488 nm MaxLine Laserline Filter (Semrock Inc., Rochester, NY) was positioned before the fiber coupler to eliminate plasma light. Laser induced fluorescence from silver clusters were collected by a 60× TIRF objective (Plan Apo, NA 1.45, Nikon, Japan) and passed through a FITC filter set (Chroma Technology Corp., Rockingham). In order to obtain the full range of fluorescence spectra from different silver clusters, the emission bandpass filter was replaced by an ultrasteep 488 nm long pass filter (LP02-488RU25, Semrock Inc., Rochester, NY), which has a light transmission efficiency of less than 1 ppm at 488.0 nm and over 98% at 493.0 nm or beyond. A transmission grating beam splitter with 70 lines/mm (Edmund Scientific, Barrington, NJ) was mounted in front of the CCD camera. This transmission grating allows one portion of the incoming fluorescence light to pass undeviated to form the zero-order image and disperses another portion of the light to form the wavelength-resolved first-order image. When the distance between the grating and the CCD camera is set within an appropriate range, both the zero-order and firstorder images can be captured by the CCD camera. Image sequences were collected with 100 ms exposure time for each

Xiao et al. frame and further analyzed by Image J software (NIH, http:// rsb.info.nih.gov/ij/). Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS). The fine nanostructure of the freshly prepared silver island film was characterized on a JSM-6700 (JEOL, Japan). The elements on the silver island were determined by energy dispersive X-ray spectroscopy (Oxford Instruments, Oxfordshire, U.K.). Results and Discussion Characterization of Fluorescent Silver Clusters. By inserting a transmission grating beam splitter into the emission light path of a fluorescence microscope, the fluorescence signal is separated into a nondispersed zero-order image and a wavelengthdispersed first-order image.39 The separation distance between the zero-order and the first-order image depends on the emission wavelength of the dye and the distance between the transmission grating and the front surface of the CCD chip. With the grating positioned 87.4 mm away from the CCD chip, one pixel on the CCD sensor represents 1.05 nm which indicates that one could readily discriminate molecules based on their differences in emission spectra. In order to demonstrate the accuracy of this setup, the fluorescence spectra from YOYO-1 labeled (one dye per five bases) single lambda DNA molecule was recorded (data not shown). The measured distance between the zero-order and the maximum of the first-order image is 482.5 ( 1.1 pixels. It agrees well with the calculated value of 482.0 pixels. The small standard deviation indicates that the spectrum measured based on this setup is highly reliable and accurate. Thus one could simultaneously distinguish different individual luminescent spots in a large area in the subsecond time scale. Figure 1 shows the fluorescence color image without the grating, fluorescence spectral image with the grating, and the SEM image of the silver island film. It can be seen that the silver island film was composed of 50∼100 nm irregular silver nanoparticles. When it was exposed to the 488 nm argon ion laser, multicolor fluorescent spots gradually grew on the surface. On elongation of the exposure time, the number of fluorescent spots increased in a slower rate compared with those on a silver oxide nanoparticle film.23 The majority of the dots on the surface showed typical blinking tracks. Figure 1b shows a fluorescence spectral image of these dots. The zero- and their corresponding first-order fluorescence images are labeled with single and double dots, respectively. It can be seen that the zero-order image from an individual dot is a focused spot and the firstorder image is a long streak. If the number density of fluorescent spots on the silver island film is not very high, there will be no overlap between zero-order and first-order images of different silver clusters and they can be readily separated. It is well-known that the optical properties of nanomaterials are greatly different from their bulk counterparts. The quantum efficiencies of nanomaterials exhibit strong size-dependent property. In the case of a large metal nanoparticle, with size greater than tens of nanometers, the motion of the electrons in the nanoparticle is not quantized. Energy absorbed by the electrons was used to enhance their motion. Thus it is very difficult for those relatively large nanoparticles to form electron and hole pairs (charge carriers). In other words, the fluorescence quantum efficiency from bulk metal would be extremely low. Consequently, those bright luminescent spots cannot be due to the relatively large silver nanoparticles. When the size of metal nanoparticles decreases to the range of the Fermi radius or even smaller, the energy levels between the top of the valence band and the bottom of the conduction band become more discrete

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Figure 1. Color image of photoactivated fluorescence from silver clusters on silver island excited by a 488 nm argon ion laser, scale bar 10 µm (a), zero- and first-order fluorescence image of silver clusters after inserting a transmission grating, scale bar 10 µm (b), SEM image of a silver island film (c), and the corresponding elemental analysis (EDS; d).

and more widely spaced; as a result, the motion of charge carriers (the excitons) is confined.3,5 Under this favorable size range, the excitons could be produced by irradiating the small metal dots with visible light (380-750 nm), and subsequently photons are emitted from these metal dots. Geddes et al.24 have also observed fluorescence from the silver island film, and they ascribed it to luminescent silver clusters. However, the formation mechanism of the silver clusters and what kinds of clusters existed on the surface were not discussed. Although colorful fluorescent spots on the surface of silver nanoparticles represent various kinds of silver clusters, it was demonstrated that luminescent silver clusters could not be directly produced on the surface of pure silver nanoparticles.25 As reported in earlier works,25,41 during the photoreduction process, luminescent silver clusters could be gradually formed from silver oxide, silver sulfide, or even silver halide but not from a pure silver film. To determine the components of the silver island film, energy dispersive X-ray spectrum (EDS) was used to characterize the elements on the silver island. Figure 1d shows the EDS spectrum of silver island film. There was no sulfide or halogen on the surface. Potassium and sodium peaks in the EDS spectrum originated from the glass substrate. Therefore, silver clusters were not produced through the decomposition of silver sulfide or silver halide. Since a thin layer of silver oxide would quickly form on the silver island surface when it was exposed to air, silver clusters on silver islands could be produced through the photoactivation process of silver oxide;26 that is, Ag2O f 2Ag + 1/2O2. When excited by a 488 nm argon ion laser, colorful silver clusters with core size ranging from 2 to 8 atoms could gradually form to emit

visible light at ambient temperature. This phenomenon is similar to photoinduced fluorescence on silver oxide nanoparticles.23,25,26 To further investigate this process, mercaptopropanol was passed over a photo saturated silver island film. Since mercaptopropanol is a strong oxygen free radical scavenger, reduction of free radicals in the photoactivation process would favor the photochemical reaction. In other words, the fluorescence intensity on the film would grow gradually. After flowing 1% mercaptopropanol through the silver island film, it can be seen that the overall fluorescence intensity decreased slightly initially, and then increase monotonically (Figure 2a). Since water is a Lewis acid, it has a quenching effect on the photoactivated silver clusters.42 The slight decrease of fluorescence intensity in the early stage could be attributed to the quenching effect from water. Although the photoactivated fluorescence on silver islands could be assigned to photoinduced silver clusters, whether neutral silver clusters, charged silver clusters, or both of them contribute to the fluorescence is unknown. It was reported by Dickson et al.25 that neutral or charged silver clusters, such as Ag3 or Ag3+, can be produced during the photoreduction process on the surface of silver oxide nanoparticles. If positively charged silver cluster was the fluorescent product on the surface of silver island here, the fluorescence intensity should be quenched by adding a strong reducing agent. On slowly injecting 0.01 M (a relatively very high concentration) NaBH4 into one of the flow channels to replace the pure water, there was no fluorescence intensity variation as seen in Figure 2b. This is different from a previous report that fluorescence from positively charged silver cluster on silver nanoparticles would be totally quenched by

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Figure 2. Variation of fluorescence intensity after passing over of 1 mM mercaptoethanol solution (a) and 0.01 M NaBH4 solution (b).

adding NaBH4.23 Another explanation is that the charged silver clusters were still luminescent after being reduced, so the observed fluorescence intensity does not change after adding NaBH4. Provided this assumption is true, the fluorescence spectra from those silver clusters should change with the reduction process because the energy gap within an ionized metal cluster and within a neutral metal cluster is different, which leads to different emission wavelengths. In the course of injecting NaBH4 solution, there was no apparent spectral shift. One reasonable interpretation of this phenomenon is that neutral silver clusters were the main luminescent species on the silver islands. Static and Dynamic Spectral Variations. Figure 3 shows the blinking track and fluorescence spectrum of an individual silver cluster. The stepwise blinking track gives us a clear confirmation that the fluorescence is from a single silver cluster.27 With Lorentz fitting, the emission maximum is determined to be 513.9 nm. The fluorescence spectral distribution of silver clusters was then obtained by randomly selecting 96 spectral profiles and analyzed by Lorentz fitting. Three major types of spectra, 516 ( 3nm, 51.1%, 531 ( 2nm, 12.5%, and 553 ( 6nm, 36.4%, existed on the silver islands when excited with a 488 nm argon ion laser. These varieties may be silver clusters with different nuclear composition, structural isomers, or ionized particles. Since there is no fluorescence intensity fluctuation and large scale spectral diffusion on the silver film after injecting NaBH4, as discussed above, these silver clusters cannot be differently charged species with the same nucleation number. If there are coexisting stable and meta-stable structural isomers, the fraction of these three spectra must be changed with time. In other words, large scale spectral shift toward a certain direction (irreversible) should be observed because of the structural changes from a meta-stable state to a stable one.43 However, no large scale irreversible spectral diffusion was

Figure 3. Fluorescence blinking track from a single silver cluster (a) and its corresponding fluorescence spectrum (doted curve) after Lorentz fitting (solid curve; b).

observed on the film. One possible interpretation is that three kinds of neutral silver clusters with different nucleation numbers contribute to these three different spectra. Another explanation is that multiple types of stable structural isomers were produced during the photoactivation process. More studies in addition to fluorescence spectral imaging are needed to elucidate this phenomenon. With continuous excitation of the 488 nm laser, reversible spectral shift (Figure 4) at some individual silver clusters was observed. This type of event occurred rarely and randomly and constituted about 2% of the total population of fluorescent spots at an excitation power of 1.0 mW. It is short-lived with a duration of just about 500 ms. It is also short-ranged, that is, between 516 and 531 nm, as compared with a previous report on long-range (over 100 nm) and irreversible directional spectral diffusion (from blue to red) on silver oxide nanoparticles.25 To our knowledge there is no report on this type of reversible spectral shift on silver island so far. Many factors such as photoionization, compositional variation, or structural changes between isomers could lead to spectrum diffusion on silver clusters.25,43-45 As discussed earlier, the silver island film mainly contains neutral silver clusters with different fluorescence emission maxima. Thus the reversible spectral shift could not be induced by photoionization between neutral and charged silver clusters. Moreover, the blinking trajectory indicates that this process did not originate from multiple different silver clusters residing closely in space. That there is no correlation

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Figure 5. Number of reversible spectral shift events versus laser power.

Figure 4. Single silver cluster undergoing reversible spectral shift. Its blinking trajectory (a), the spectral profiles during the reversible shift (b), and the fluorescence emission maximum and intensity variation with time (c).

between the fluorescence intensity and variation of emission wavelength also confirms this argument (Figure 4c). One possible explanation is that under strong laser illumination, one or two silver atoms could temporarily dissociate from the silver cluster due to thermal motion and then fuse back with other atoms quickly, resulting in a transient reversible spectral variation. It has been reported previously that active silver clusters could undergo either photofragmentation or photoagglomeration under light illumination.44,46,47 Another reasonable interpretation is laser-induced configuration adjustment. It has been demonstrated that silver clusters with different configurations would have different fluorescence spectra.43 Both assumptions are related to laser illumination. Thus at high laser powers, more energy would be provided for either silver atom dissocia-

tion/association or configuration adjustment process. This should lead to an increase in the spectral shift events. Figure 5 indeed shows an increasing trend of spectral shift events with the increase of laser power. Cluster with Anomalous Intensities. In part because of the enhanced localized electromagnetic field, a Raman signal with an enhancement factor of 1014-1015 on irregular silver nanostructures was observed.8 Similar fluorescence enhancement effect has also been demonstrated; for example, five times enhancement of quantum dots near the silver film was founded by single molecule analysis.20 Here, a strong fluorescence enhancement effect from single silver cluster was observed on silver island film. During the photoreduction process, a few extraordinarily bright fluorescence bursts randomly appeared on the silver film. The integrated intensity is over 20 times higher than the average intensity from 10 randomly selected single silver clusters on the silver island (Figure 6). This phenomenon could be caused by two scenarios. One is that multiple fluorescent silver clusters positioned closely in space were produced simultaneously, and they appear at the same pixel of the CCD chip because of limited optical resolution of the imaging system. The other explanation is that only one luminescent silver cluster was produced, but its fluorescence intensity was greatly amplified by the strong localized electromagnetic field generated by nearby silver nanostructures. 16 From the first-order fluorescence image, only one emission maximum was observed on the spectral profile, and there were no spectral diffusions during the course of the burst. When a single luminescent object is irradiated by a laser, the photons are emitted from this object via a stochastic process. If there are several silver clusters photoactivated in a small region within the diffraction limit of the optical system, these objects should not be turned on or off simultaneously. Thus, multistep photoactivation or photobleaching or multiple emission maximum should be observed. However, that is not the case as shown in Figure 6. The blinking trajectory and the fluorescence spectral profile indicate that the burst originated from a single silver cluster. Moreover, the “on” time of the burst is significantly longer than other blinking silver clusters. This phenomenon is similar to the fluorescence enhancement effect of QDs or dyes on the silver island film. According to earlier reports by others, optimal fluorescence enhancement effect would be achieved when the separation distance between the dye molecule and the metal particle is approximately 10 nm away.48 Here, the silver clusters were directly grown on the surface of silver islands. Although there could be a thin silver oxide film between the silver clusters and the silver island, the space between them still could not reach 10 nm. Therefore, this enhancement effect is likely caused by strong electromagnetic field coupling between neighboring irregular silver island tips and the photoactivated

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Figure 6. Three dimensional image of a bright fluorescent burst (a) and a regular silver cluster (b), and the corresponding blinking trajectory of the same burst (c) and the cluster (d). The insert in c is the fluorescence spectrum at two different times (41.2 and 46.6 s).

silver cluster. To further prove the enhancement mechanism on this irregular silver island film, we compared it with a low density silver colloid film (about 12 nm silver nanoparticles selfassembled on to an APTS functionalized cover glass surface). When it was exposed to the same laser beam, colorful fluorescence spots could also be observed on the surface. But there were no fluorescence bursts on the low density film compared with the irregular silver island film. We believe that the long distance between neighboring silver colloids greatly reduces the localized electromagnetic field coupling effect, and it does not favor the fluorescence enhancement process. This enhancement process is a randomly occurring process. A spectrometer cannot achieve such a spatial and temporal resolution to capture these randomly distributed fluorescence events on the surface. Thus, single molecule TCSPC cannot be used to measure the fluorescence lifetime of these silver clusters. Further study is needed to get a deeper understanding of this enhancement effect. Conclusions A transmission grating based single molecule imaging method enables us to directly monitor the photodynamic process of silver clusters on the silver island film in real time. The photoactivation process of luminescent silver clusters was studied in detail, and a neutral silver cluster was founded to be the main product of this process. Some rarely and randomly occurring events were recorded in a subsecond time scale, and a vast amount of information was extracted from those data. Reversible spectral shifts from single silver clusters and a strong fluorescence enhancement effect on the surface of silver islands were observed. This technique could be further used to monitor randomly occurring dynamical processes at the single molecule level in various environments.

Acknowledgment. This work was supported by NSFC (20605008), Program for New Century Excellent Talents in University and Hunan University 985 fund. E.S.Y. thanks the Ames Laboratory for partial support of this work. References and Notes (1) Rosi, N. L.; Mirkin, C. A. Chem. ReV. 2005, 105, 1547. (2) El-Sayed, M. A. Acc. Chem. Res. 2004, 37, 326. (3) Hodes, G. AdV. Mater. 2007, 19, 639. (4) Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chem. ReV. 2005, 105, 1025. (5) Kamat, P. V. J. Phys. Chem. B 2002, 106, 7729. (6) Mock, J. J.; Barbic, M.; Smith, D. R.; Schultz, D. A.; Schultz, S. J. Chem. Phys. 2002, 16, 6755. (7) Huang, T.; Nallathamby, P. D.; Gillet, D.; Xu, X.-H. N. Anal. Chem. 2007, 79, 7708. (8) Nie, S.; Emory, S. R. Science 1997, 275, 1102. (9) Emory, S. R.; Haskins, W. E.; Nie, S. J. Am. Chem. Soc. 1998, 120, 8009. (10) Gersten, J. J. Chem. Phys. 1980, 73, 3023. (11) Weitz, D. A.; Garoff, S.; Gersten, J. I.; Nitzan, A. J. Chem. Phys. 1983, 78, 5324. (12) Wokaun, A.; Lutz, H.-P.; King, A. P.; Wild, U. P.; Ernst, R. R. J. Chem. Phys. 1983, 79, 509. (13) Moskovits, M. ReV. Mod. Phys. 1985, 57, 783. (14) Sokolov, K.; Chumanov, G.; Cotton, T. M. Anal. Chem. 1998, 70, 3898. (15) Geddes, C. D.; Lakowicz, J. R. J. Fluoresc. 2002, 12, 121. (16) Lakowicz, J. R. Anal. Biochem. 2005, 337, 171. (17) Aslan, K.; Gryczynski, I.; Malicka, J.; Matveeva, E.; Lakowicz, J. R.; Geddes, C. D. Curr. Opin. Biotechnol. 2005, 16, 55. (18) Aslan, K.; Huang, J.; Wilso, G. M.; Geddes, C. D. J. Am. Chem. Soc. 2006, 128, 4206. (19) Pompa, P. P.; Martiradonna, L.; Torre, A. D.; Sala, F. D.; Manna, L.; Vittorio, M. D.; Calabi, F.; Cingolani, R.; Rinaldi, R. Nature Nanotech. 2006, 1, 126. (20) Ray, K.; Badugu, R.; Lakowicz, J. R. J. Am. Chem. Soc. 2006, 128, 8998. (21) Zheng, J.; Dickson, R. M. J. Am. Chem. Soc. 2002, 124, 13982.

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