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Metal Nanoparticle Plasmon-Enhanced Light-Harvesting in a Photosystem I Thin Film Iltai Kim,† Shana L. Bender,‡,§ Jasmina Hranisavljevic,† Lisa M. Utschig,‡ Libai Huang,†,|| Gary P. Wiederrecht,† and David M. Tiede*,‡ †
Center for Nanoscale Materials and ‡Chemical Sciences and Engineering Division, Argonne National Laboratory, Argonne, Illinois, United States
bS Supporting Information ABSTRACT: Silver metal nanoparticle (NP) enhanced fluorescence is investigated in thin films of cyanobacterial Photosystem I trimer complexes (PSI) by correlating confocal laser scanning microscopy, dark-field imaging, and fluorescence lifetime measurements. PSI represents an interesting light-harvesting complex with a 20 nm diameter that is not uniformly contained within the surface-localized plasmon field of the NPs. With weak farfield illumination, 5- to 20-fold fluorescence enhancement is observed for PSI complexes adjacent to NPs, arising from efficient nanoparticle light collection and subsequent localized, surface plasmon excitation of PSI. Enhanced PSI fluorescence is detected most prominently near “rafts” of aggregated NPs that more completely fill the confocal field of view. These results demonstrate opportunities to probe energy transfer within photosynthetic complexes using plasmonic excitation and to design nanostructures for optimizing artificial light-harvesting systems. KEYWORDS: Photosystem I, metal nanoparticle, plasmon-enhanced, fluorescence, light-harvesting, scanning confocal microscopy
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here is growing interest in the utilization of plasmonic structures as optical antenna and light concentrators in advanced thin film optoelectronic, solar devices24 and nanobiosensing5,6 The ability of plasmonic structures to achieve extreme light concentration at the nanometer scale creates opportunities to design efficient light-absorbing devices based on thin film lightconverters that by themselves have low optical absorption. Among the materials of interest for integration within optical devices are the molecular ensembles that function in photosynthesis. Photosystem I (PSI) is one the most robust and efficient lightharvesting complexes in nature for solar energy utilization. Because of its large voltage generation and nanoscale dimension, PSI has received attention as a prototype for a next-generation material with applications in photoelectronics7,8 including broadband enhancement of light absorption,9 photochemical cells,10 and hydrogen production.11,12 Light-harvesting enhancement is a key element for the design of an optimized thin film solar device. Efficient coupling of molecular and plasmonic optical resonances in hybrid structures have been characterized, including demonstrations of plasmon resonance energy transfer (PRET) and metal-enhanced fluorescence.9,1824 The magnitude of fluorescence enhancement is a function of the distance between the fluorophore and metal surface. For small molecular emitters that fit uniformly within the surface associated plasmon field, the distance dependence for metal-enhanced fluorescence have been resolved using single molecule and nanoparticle probe techniques.1,21,23,25,26 Fluorescence enhancement is found to be maximal with a separation of about 15 nm between discrete metal nanoparticles (NPs) and molecular emitters.1,21,23 At shorter r 2011 American Chemical Society
distances, strong dipole and multipole coupling between the molecular oscillator and the plasmonic resonance provides an efficient nonradiative energy transfer pathway from the molecular excited state to the metal that exceeds the molecular emission rate, and fluorescence quenching is observed. At longer distances, the metal quenching rate is attenuated and the molecular fluorescence enhancement tracks the attenuation length of the localized plasmon field.21,23 For small metal NPs the plasmon attenuation length is typically on the order of 515 nm (13% the plasmon resonance wavelength), while it can extend an order of magnitude further for planar and extended metal structures.26 Conforming to these observations, surface plasmon excitation of small, water-soluble peridininchlorophyllprotein (PCP)27 and phycobilisome light-harvesting complexes28 deposited onto silver island films show sample dependent 5- to 18-fold increases in metal-enhanced fluorescence, and accelerated, multiphasic fluorescence lifetime decays consistent with heterogeneous contacts that produce a distribution of metal-to-pigment distances.29 In contrast, PSI trimer complexes provide examples of large, efficiently coupled light energy transfer networks (∼20 nm diameter) that do not uniformly fit within a surface-localized plasmon field of single NPs. Recent experiments demonstrate a variety of metal-enhanced optical effects depending upon the method for preparation of NPPSI conjugates. Significant optical absorption enhancement was observed for colloidal Received: March 25, 2011 Revised: June 22, 2011 Published: June 29, 2011 3091
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Nano Letters goldPSI hybrids synthesized by metal reduction in the presence of the PSI protein.9 The efficient optical coupling was modeled to be achieved through the creation of NPPSINP junctions in the colloidal aggregates that placed the PSI in “hot spots” with intense plasmonic fields but also resulted in the complete quenching of normal PSI fluorescence.9 PSI shows multiple sites for metal ion binding3033 that could potentially nucleate NP formation within the PSI protein at sites close to the light-harvesting and redox cofactors. The in situ NP synthesis approach provides a means to achieve exceptional optical coupling but also potentially creates paths for efficient optical quenching. Recent single-molecule spectroscopy of PSI in the presence of colloidal gold and on silver island films have measured a distribution of metal-enhanced PSI fluorescence factors that is also correlated with NP induced shifts in the emission spectra.34 Here, we present evidence for metal nanoparticle enhanced fluorescence emission in a PSI thin film that is deposited on surface-supported, preformed silver NPs using confocal laser scanning microscopy, dark-field imaging, single-particle darkfield spectroscopy, PSI fluorescence lifetime, and fluorescence measurements. Factors of 5 to 20-fold enhanced fluorescence is observed for PSI adjacent to NPs and is detected most prominently near “rafts” of aggregated nanoparticles that more completely fill the confocal field of view. Significantly, the metal NP-enhanced PSI fluorescence emission shows no alteration compared to PSI in areas of the films devoid of NPs. Simulations of enhancement factors35 suggest that the observed fluorescence yields are consistent with a 55 nm effective radius of the NP and separation distance of 3 nm from the emitting chlorophyll cofactors in PSI. These results demonstrate opportunities to probe energy transfer within photosynthetic complexes using plasmonic excitation and to design plasmon nanostructures for optimizing artificial light-harvesting systems. Experimental Section. PSI and Ag NP Preparation. Cells of Synechococcus leopoliensis (UTEX625) were grown in Ac medium36 at ∼40 °C. Trimeric PSI was purified as described previously.37,38 Purified trimeric PSI samples were dialyzed overnight in 5 mM HEPES-NaOH pH 7.5, 0.04% dodecyl-β-D-maltoside (LM) and were concentrated using an Amicon (Bedford, MA) Ultra 100 000 MWCO centrifugal filter device to a final concentration of 0.33 mg chl/ml. A colloid aqueous suspension of 20 nm diameter Ag nanoparticles was prepared by sodium borohydride reduction of AgNO3 in the presence of sodium polyphosphate as described in Supporting Information. PSI and Ag NP Thin Film Preparation. Glass coverslip substrates were cleaned using a piranha solution etched39 and the clean glass coverslips were immersed in a 1019 M Ag colloid solution for several hours and allowed to shake on a shaker overnight. (Warning: piranha solution reacts violently with organics and must be handled with extreme care.) The Ag NPcoated cover glass was rinsed with Milli-Q (Millipore) purified water and dried under nitrogen gas. Subsequently, a PSI film was deposited on top of the NP-coated slide by drop-casting a 20 μL drop of a PSI solution that had been diluted to 2.4 mg of Chl/ml using Milli-Q water. The PSI film/NP coated glass slide was held in the dark until the water was fully evaporated. Absorption and fluorescence spectra of PSI in a thin film are shown in Supporting Information Figure S1 that shows that the absorption peak of chlorophyll is located at 678 nm and the fluorescence peak is at 685 nm, as measured from a UVvisible spectrometer (PerkinElmer Lambda 950), respectively.
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Scanning Microscopy. The morphologies of Ag NPs are characterized by atomic force microscope (AFM) and scanning electron microscope (SEM). For AFM scanning, a Veeco Multimode AFM is used to scan Ag NPs deposited on a silicon substrate in tapping mode with an aluminum reflective coated tip (42 N/m, 320 kHz). For SEM scanning, a Jeol 7500 SEM is used to characterize Ag NPs on silicon substrates with a 10 kV illumination. Confocal Microscopy. A schematic of the experimental setup is shown in Supporting Information Figure S2. The microscopy system is modified to be able to illuminate the same sample region with a white light source from the top (Olympus halogen 100W) or bottom (Oriel Arc 75 W) in order to measure extinction and scattering, respectively. For the fluorescence enhancement experiments, 1 μW of the 482 nm output at 75 MHz from a frequency doubled TiSapphire laser system (Coherent Mira 900, in picosecond pulse mode) was used. The laser beam is expanded eight times through a magnifier (Special Optics) and focused on a sample through a microscope objective (5 or 100) mounted in an inverted microscope (Olympus IX 71). The illumination light preferentially excites the PSI molecules adjacent to the NPs, while the emitted photons are reflected back through the objective, dichroic cube and longpass-filter and detected by either a spectrograph (Andor SR303i), a charge-coupled device (PixelLINK CCD) camera, or an avalanche photodiode (APD), depending on the measurement purpose. The signal measured for the APD (MPD series) using a 690 nm band-pass filter was transferred to a time-correlatedsingle-photon-counting (TCSPC, Picoharp 300) unit for lifetime measurement or to a data acquisition unit (RHK Technology) for a confocal scanning enhanced fluorescence measurement. Hence, metal-enhanced fluorescence is characterized through an elaborate and integrated correlation between confocal laser scanning microscopy, dark-field imaging, dark-field scattering spectroscopy, emission lifetime measurements, and fluorescence measurements. A 5 objective is used for ensemble averaging and a 100 objective is used for single particle or spot detection. In single particle spectroscopy, the spectrometer measurement window is adjusted to be 1.5 μm 1.5 μm in width and height using the spectrometer slit width and CCD detector binning selection, respectively. The concentration of Ag NPs is controlled to have one single or individual spot in the spectrometer measurement window. The identification of locations of large Ag NPs within a the PSI film/NP coated glass slide was accomplished using dark-field imaging microscopy in an inverted microscope geometry, using with a high numerical aperture objective (NA 0.9, 100) that collected scattering contrast using white light in reflection mode.40 After finding single individual scatters through the dark-field imaging mode, the measurement mode is changed to the laser excitation and spectrometer detection mode with 635 nm longpass filter mounted after the objective within the same microscope. Fluorescence emission from the sample through a 635 nm long-pass filter or 690 nm band-pass filter is measured by an APD which is connected to a confocal data acquisition unit or a TCSPC unit or a spectrometer depending on measurement purpose. The integration time for each pixel in confocal scanning is 1.5 ms with 256*256 pixels. The sample is scanned with piezoelectric actuator (Mad City) with a 0.2 nm spatial resolution. A 65 μm diameter-sized fiber optic is located in the image plane after the objective to block the defocused light, which effectively 3092
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Figure 1. AFM (parts A and B) and SEM (parts C and D) images of surface supported Ag NPs showing representative regions containing examples both isolated small NPs with examples marked by circles labeled I in parts A and C, and larger, aggregated NPs, examples marked by circles labeled II.
functions as a 0.65 μm pinhole with a 100 objective. Noise signal from the instrument shows almost zero level and the signal from background region without metal nanoparticles shows almost uniform fluorescence which indicates a uniformly coated PSI thin film. Dark-field imaging is recorded by a CCD camera. For single-particle dark-field scattering, an arc lamp is used as an illuminating light source. Dark-field scattering signal is measured by a spectrometer in a single exposure for 120 s and later corrected with the reference signal from a broadband target (Labsphere).40 The scattering signal for the background region without metal NPs that has no enhanced fluorescence shows negligible scattering signal. The lifetime is measured by the TCSPC unit for 180 s using a 690 ( 10 nm band-pass filter. Enhanced metal fluorescence is measured in single track mode with 1 s exposure and 30 time accumulation through a spectrometer with the same microscope optics as in confocal scanning, dark-field scattering spectra, and dark-field imaging. Results and Discussion. Surface Absorbed Ag NP Characterization. AFM and SEM images of colloidal Ag NPs absorbed to glass sides, Figure 1, show that the surface absorbed Ag NPs consist of primarily two types of morphologies; one is a single nanoparticle and the other is a cluster or “raft” of aggregated nanoparticles. This is illustrated by a wide-field (1.1 μm 1.1 μm) AFM tapping mode scanning image shown in Figure 1a. Examples of areas that have NPs distributed as approximately 20 nm diameter isolated single nanoparticles are marked by circles labeled “I”. In addition, other region labeled with “II” show a cluster of larger aggregates or rafts of nanoparticles with a composite diameter of at least 200 nm. Figure 1b shows a different AFM field of view having a large composite NP centered in the image. In addition, this field of view also shows a distribution of dispersed, single NPs. This combination of morphologies is also seen in SEM images, Figures 1c,d. For example, the SEM image in Figure 1c shows a combination of small particles consistent with the expected ∼20 nm Ag single NP (region “I”) and aggregated nanoparticles (region “II”) that correspond fairly well to the AFM images of Figure 1a. Figure 1d shows an SEM image
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of a single large composite NP, which corresponds well to the type of large NP seen in the AFM image, Figure 1b. The surface absorbed Ag NP films were characterized by lowmagnification (5) transmission extinction (absorption) and dark-field scattering measurements, Supporting Information Figure S3, that average spectra across a wide field of view with the diameter of 2 mm. A typical wide-field plasmon scattering spectrum for the Ag NP-coated slides was found to have a peak at 480 nm while the peak of the extinction spectra of Ag NPs was positioned at 454 nm. The scattering and extinction spectra were measured using the same optics, except for the location of the light source, as indicated in the instrumental layout in Supporting Information Figure S2. Background corrections were made using a clean glass coverslip for the extinction measurements, and a broadband target (Labsphere) for the scattering measurement. Spatially resolved imaging within this field show that the scattering arises from a distribution of point sources, each with a different spectral characteristic. For example a wide-field scattering images show a full range of blue to red scattering sources, presumably arising from either individual or clusters of particles, Supporting Information Figure S4. Strong scattering from a small, ∼20 nm NP with a blue plasmon peak would not be expected. However, this observation is consistent with the picture described above of a population of large, highly scattering NP aggregates or “rafts” that are composed of small NPs that the scattering spectra indicates retain the optical properties individual NPs, but scatter efficiently because of aggregation. PSI Coated, Surface-Absorbed Ag NP Film Characterization. A PSI film was deposited on top of the NP-coated slides by drop casting. Optical interferometry measurements show the PSI film to have an average thickness of about 30 nm (see more detail in Supporting Information), corresponding to 1 to 3 PSI molecular layers, depending upon the orientation and packing of the PSI complexes in the film. The PSI film/NP coated glass slides were characterized by a combination of confocal fluorescence scanning microscopy, dark-field imaging, and confocal microscopy fluorescence lifetime measurements. Confocal field-of-view measurements of uniform, thin film PSI fluorescence can be expected to be a combination of fluorescence produced relatively inefficiently by direct PSI excitation with the weak laser field, as well as enhanced fluorescence produced by PSI within the NP plasmon near-fields. In addition, confocal PSI fluorescence can be expected to differ significantly depending whether a single small NP or large NP aggregate is isolated within the field of view. Figure 2a,b shows schematic representations of the confocal laser measurements from areas of the PSI thin films that contain either a single small NP, or a NP aggregate with a dimension comparable to the confocal field of view, respectively. With an isolated, single small Ag NP, Figure 2a, the far-field illuminated laser beam area (diameter ∼λ/2300 nm) is an order of magnitude larger than that of the NP (∼20 nm). Hence, the enhancement of a uniform PSI film fluorescence by the NP will be reduced by the area fraction of the NP within the field of view. Conversely, for large aggregates, or rafts, of metal NPs that more closely fill the confocal field of view, illustrated in Figure 2b, the measured fluorescence can match that produced by NP enhancement. The following position-dependent measurements of PSI fluorescence from thin-films deposited on top of Ag NPs conform to this schematic view. Figure 3 compares scanning confocal PSI fluorescence (part A) and NP dark-field scattering (part B) images of a 20 μm 20 μm region of a dilute NP-supported slide covered with a PSI 3093
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Figure 2. Conceptual drawing of laser beam illumination of surface supported Ag NPs coated by a PSI thin film (∼30 nm thickness). Part A illustrates confocal laser illumination in the region with a single Ag NP, and part B, illumination of a region with aggregated Ag NPs.
thin-film. The field of view in Figure 3A shows one bright spot (region “I”) with PSI fluorescence intensity that is a factor of more than 5 higher than the background PSI fluorescence. This position was also seen to correspond to a bright spot in the darkfield scattering, image shown in Figure 3B, attributable to the presence of a strongly scattering NP. The dark-field NP scattering spectrum measured for this spot, Supporting Information Figure S5, has two peaks. One is located close to 600 nm and the other is a small peak around 390 nm, suggesting the possibility that the plasmon resonance at 600 nm might arise from a coupling effect of aggregated Ag NPs and the small peak at 390 nm is assigned to the plasmon resonance of the individual Ag NP. Note that background region indicates pure PSI thin filmcoated area without observable metal NP scattering. The NP-induced PSI fluorescence enhancement, lifetime decay, and spectrum, compared to the PSI film background are shown in Figure 3c,d, respectively. Lifetime decays (Figure 3c) were measured using a 690 nm band-pass filter with a timecorrelated-single photon-counting (TCSPC) unit for the same spot and background regions shown in panels a and b. The amplitude of the PSI fluorescence was 7-fold in the presence of the NP compared to the adjacent areas of the PSI film. An estimate of the lifetime decays was obtained by fitting the TCSPC signals with a sum of exponentials convoluted with the instrument response function (fwhm ∼60 ps, Figure 3 and Supporting Informatin Figure S6). The fastest component of PSI lifetimes is estimated as 12 ps ( 2.4 and 16 ps ( 3.2 ps for the bright spot and background, respectively. Although the fast decays compared to the instrument response function clearly make the 25% NP-induced fluorescence lifetime reduction a tentative measurement, numerous measurements consistently yielded a similar result. After 1 ns, fluorescence signals converge to a long-lived component for both cases of PSI with and without metal NPs. The 16 ps time constant for the fast decay component that is estimated here for the PSI films in the absence of plasmon excitation is somewhat shorter than the 2040 ps values typically measured in other strains of cyanobacteria using ultrafast techniques.41,42 Investigation of these fast decay processes for
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the S. leopoliensis PSI preparations in the presence and absence of near-field plasmon excitation is being pursued in ongoing work using ultrafast techniques. There are recent observations regarding a slight change of lifetime with chromium43 and zinc44 metals but not with silver metal. According to refs 43 and 44, the reason for the slight change of lifetime with chromium and zinc metals conjugated with fluorophores is explained as being caused from an weak induced plasmon.24,43 However, silver is known to have good plasmon characteristics and fluorophores with low quantum yield show a significant change of lifetime in the presence of silver nanoparticles in previous reports.45,46 Thus, we suggest that the lack of noticeable shortening of lifetime of PSI in the presence of metal nanoparticles results from ultrafast energy transfer within the PSI complex that efficiently removes excitons away from the NP interface with a rate faster than the energy transfer from PSI to metal nanoparticles. The weakness of PSI fluorescence (quantum yield, Y, estimated to be ∼0.003, see Calculations below) prevented spectra from being measured by spatially resolved confocal microscopy. Instead, Figure 3d shows fluorescence spectrum for PSI thin films measured in the absence and presence Ag NPs. The results are averaged over 3040 measurements of different areas for each spectra. Figure 3d shows a 8.5-fold enhancement of fluorescence due to Ag nanoparticles (red line) at the fluorescence peak location of 684 nm with shoulder peak around 730 nm. The PSI on bare glass (blue line) exactly corresponds to the measured enhanced fluorescence spectra for PSI on Ag nanoparticles (red line) when multiplied by a factor of 8.5 (green line). No fluorescence signal was observed from pure Ag nanoparticles, which is not presented in the figure. In addition, a linear laser intensity dependence of fluorescence47 is confirmed in the experiment by varying laser power. Confocal imaging of many PSI/NP thin films revealed NP associated spots with more than a 20-fold enhanced PSI fluorescence compared to adjacent PSI film areas, Supporting Information Figures S7 and S8. For a big cluster of Ag NPs characterized as in Figure 1b,d, fluorescence is increased more than 100 fold (Supporting Information Figure S9). Simulations suggest that this wide variation in fluorescence yield enhancement can arise from variations in the morphology of the NP aggregates and described below. The significant enhancement found here (5 to 20-fold) is in contrast the recent report that showed strong quenching of PSI florescence that occurred with in situ synthesized Au or Ag nanoparticles.9 Further, we note that NP enhanced emission is observed without measurable perturbation of the normal PSI photochemistry as indicated by the similarities in the lifetime and spectra for the enhanced and native PSI fluorescence. These observations contrast with recent measurements of significant alteration of PSI elicitation energy transfer pathways observed with PSI and 100 nm gold NP and PSIsilver island films measured at low temperature.34 These results indicate that the impact of plasmonic coupling to photosynthetic complexes varies significantly depending upon the details of the NP protein interactions. We also note here that with the highest fluorescence enhancement observed by confocal microscopy is correlated with large NPs aggregates that most completely fill the field of view. The leaves open the possibility that part of the enhancement might correlated with “hot spots” in near field intensity occurring at the junction of two or more NPs. Contributions of this sort are not resolved in the present measurements, and ongoing work is addressing this issue. Calculations. Excited state dynamics calculations were conducted to investigate the mechanism of metal-enhanced fluorescence. 3094
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Figure 3. Correlation of PSI fluorescence and metal nanoparticle dark-field scattering. (A) Confocal scanning microscope image of a 20 μm 20 μm region of dilute NP-supported slide overlaid with a 30 nm thick PSI film. (B) NP dark-field scattering image for the same field of view as part A. Region I marks a bright PSI fluorescence spot and region II marks the background used in panel C. (C) Time correlated single photon counting (TCSPC) fluorescence amplitude enhancement and lifetime measurements for the PSI thin film at spots corresponding to the regions marked I and II in panels A and B. (D) Enhanced fluorescence for a PSI thin film shown by comparing PSI fluorescence emission occurring with a single individual Ag NP (red trace) with the PSI thin film fluorescence emission on a bare glass substrate (blue trace). The green trace shows the PSI thin film fluorescence on bare glass multiplied by a factor of 8.5. In each case, fluorescence measured by excitation at 482 nm with 100 objective and laser power of 5 μW.
The emission intensity of PSI in the vicinity of metal NPs and the electric field enhancement factor (P) due to a metal were simulated electric field enhancement factor, P as a function of wavelength, λ, following Govorov’s model.27,35,48 Figure 4 shows the enhancement factor, P27,35,48 as a function of wavelength with a chosen effective radius, RNP. To illustrate, this simulation uses RNP = 55 nm to account for the NP aggregate effect, and it uses a NP surface to PSI chlorophyll separation distance, Δ of 3 nm, which is calculated using the estimated lifetimes based on Govorov’s model.27,35,48 The radius of 55 nm is assumed for the effective diameter of the individual Ag NPs that compose the extended aggregate or raft. This value agrees well with the reported data for different sized NPs.49 The emission intensity and enhancement factor are expressed as27,35,48 Iemiss ¼ YIabs ¼ ¼
0 Pðλemiss Þγ0rad Pðλexc ÞIabs γrad Iabs ¼ γtot γtot
0 Pðλemiss ÞPðλexc Þγ0tot Iemiss γtot
Pðλemiss ÞPðλexc Þ ¼
Iemiss γtot Iemiss τ0tot ¼ 0 0 0 Iemiss γtot Iemiss τtot
ð1Þ ð2Þ
where Y is an fluorescence quantum yield ratio of PSI, λemiss and λexc are emission and excitation wavelength, γ0tot, γtot =
1/τtot are the total relaxation rates in the absence and presence of metal nanoparticles, and P the is electric field enhancement factor due to metal nanparticles adjacent to PSI, respectively. P can be estimated as 6 Px þ Py þ Pz RNP εNPP ðλÞ ε0 ð3Þ ¼1 þ 2 6 P ¼ 3 d εNP ðλÞ þ 2ε0 where R NP is the radius of the NP, d is the distance between the center of the Ag NP and PSI molecule (d = R NP + Δ + RPSI, where RPSI ∼ 10 nm), Δ is the separation distance between the surface of metal nanoparticle and PSI, and εNP and ε0 are the dielectric constants of nanoparticle and surrounding medium of PSI, respectively. The data for εNP is from Johnson and Christy50 and ε0 is assumed to be 2.2 higher than water considering biological components in PSI. 35 The calculated enhancement factors are P (λexc = 482 nm) ∼ 4.5 and P (λemiss = 684 nm) ∼ 2.1. With the variation of the effective radius of metal NPs from 10 to 100 nm, the enhancement factor at excitation wavelength, P(λexc) changes from 1.1 to 6.7 and P(λemiss =684 nm) changes from 1.0 to 2.7. Effective enhancement factors defined by P(λexc)P(λemiss) is 9 for R NP = 55 nm and changes from 1.1 to 20 with 10 nm < R NP < 100 nm. This calculation indicates that enhancement of fluorescence for PSI is primarily attributed to the increased probability of photon absorption, P(λexc) 3095
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Nano Letters because the excitation wavelength (482 nm) is located at the plasmon peak wavelength range (480 nm). This result agrees with previous observation.27 As a separation distance, Δ varies from 2 to 20 nm, P(λexc)P(λemiss) changes from 10 to 3 with RNP = 55 nm. Taking the fluorescence decay ratio, γtot/γ0tot = τ0tot/τtot ∼ 1.3 and the fluorescence enhancement ratio, Iemiss/I0emiss ∼ 7 (Figure 3c), from experiment, effective enhancement factor, P(λexc)P(λemiss) ∼ 9.3 is obtained, which corresponds well with the calculated result of 9.7 (4.6 2.1). A fluorescence quantum yield of PSI can be calculated by assuming that the radiative decay rate of PSI in the absence of a metal follows the general molecular radiative lifetime;35,51 γ0rad = 1/τ0tot = 8π(ε0)1/2w3exce3d2exc/3hc3 where dexc is the dipole moment of PSI. With a typical molecular radiative time τ0rad ≈ 5 ns, we get dexc ≈ 2.6 Å and a fluorescence quantum yield of PSI, Y = γ0rad/γ0tot = τ0tot/τ0rad ≈ 0.003, which agrees fairly well with the previous observation.41,42 Since the radiative decay rate is proportional to the initial fluorescence rate (Iemiss(t = 0)),52,53 γrad = Iemiss(t = 0)/I0emiss(t = 0)γ0rad. Here, fluorescence intensity, Iemiss at time = 0 can be obtained from TCSPC data. In Figure 3c, Iemiss(t = 0)/I0emiss(t = 0) ∼ 7, and the fluorescence quantum yield in the presence of a metal nanoparticle is Ymetal = γrad/γtot = Iemiss(t = 0)/I0emiss(t = 0)γ0rad/γtot ∼ 0.02. Thus, the ratio of Ymetal/Y ∼ 7, which is
Figure 4. Simulated electric field enhancement factor, P for Ag nanoparticles with effective radius (Reff) of 55 nm and a separation distance (Δ) of 3 nm.
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close to Iemiss(t = 0)/I0emiss(t = 0) ∼7. The quantum yield of PSI emission in the presence of metal nanoparticles increases by 7 fold. PRET. Plasmon resonance energy transfer (PRET) or plasmonic-molecular resonance coupling has been observed by alteration of the Rayleigh scattering spectrum for a variety of NPbiomolecule conugates.1317 We have observed similar phenomena from dark-field scattering measurements of surface absorbed NP coated with PSI thin films. PRET is readily observed in ensemble averaging mode. For example, Figure 5a shows darkfield scattering spectra surface supported Ag NPs measured with relatively wide field of view (5 objective) in the absence of an overlying PSI film (black trace), and in the presence of a PSI film (green trace). The scattering spectra of Ag NPs coated with PSI molecules shows dips at 438, 500, 590, and 629 nm which correspond to peaks in the PSI absorption spectra (red trace). The concentration of the PSI solution used to make the thin film was 2.4 mg/mL. Figure 5b show the corresponding darkfield image. Figure 5c shows that PRET is detected in the weakly scattering spectra for isolated or small clusters of NPs measured using with confocal microscopy. Absorption by PSI molecules is strong enough compared with weakly scattered NPs to make discernible dips in the scattering spectrum. The dotted black line is an estimated scattering spectrum for a Ag NP in the absence of an overlying PSI film, which is close to the scattering spectra measured in ensemble averaging mode (Supporting Information Figure S3) indicating that there are a single or a few Ag NPs (region “I” in Figure 1a,c and Figure 2a). Scattered spectra measured for the weakly scatting NPs using a lower PSI concentration of to cast a film (0.3 mg/mL) also show comparable effects, Supporting Information Figure S5. Conclusions. We demonstrate that a PSI thin film with a low fluorescence quantum yield shows significant metal-enhanced fluorescence of more than 20-fold with aggregated Ag nanoparticles as verified through correlation between confocal laser scanning microscopy, dark-field imaging, single particle darkfield spectroscopy, emission lifetime, and enhanced metal fluorescence measurements. The observed fluorescence enhancement is mainly attributed to the increased excitation rates of PSI. The enhanced fluorescence of PSI demonstrates the opportunity to use plasmonic nanostructures as optical antenna to
Figure 5. PRET from Ag nanoparticles to PSI. (A) Ag NP scattering spectra measured with a wide field of view (5 objective). The black line trace shows the scattering spectrum for surface supported NPs in the absence of a PSI film layer. The green line trace shows the scattering spectrum for surface supported NPs coated with a PSI thin film layer. (B) Dark-field scattering image measured with the same optics and field of view (200 μm 200 μm) as in panel A. (C) PRET measured in the vicinity of a single NP using confocal magnification (100). 3096
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Nano Letters achieve a more efficient excitation of PSI in thin film device applications
’ ASSOCIATED CONTENT
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Supporting Information. Absorption and fluorescence spectra of PSI, schematic layout of integrated optical system, Extinction and scattering spectra for Ag NPs, wide-field scattering image for a Ag NP coated slide, Single particle dark-field scattering spectra corresponding to Figure 3, fluorescence normalized to the signal of Spot I, enhanced fluorescence of PSI correlated with confocal scanning microscope, dark-field imaging and scattering spectra measurement, Fluorescence enhancement correlated with lifetime, dark-field imaging, and dark-field scattering measurement, Fluorescence enhancement by more than 100 fold for a raft of Ag NPs. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Address: Chemical Sciences and Engineering Division/200, 9700 South Cass Ave., Argonne National Laboratory Argonne, Il 60439. Phone: 1-630-252-3539. Present Addresses §
)
Biofuels Research and Development, 331 PL, Bartlesville, OK 74004. Radiation Laboratory, Notre Dame University, Notre Dame, IN 46556.
’ ACKNOWLEDGMENT This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy under Contract DEAC02-06CH11357. Use of the Center for Nanoscale Materials was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. The authors would like to thank Dr. David Gosztola and Dr. Alexandre Bouhelier for their advice and discussion on the optical microscopy measurements. ’ REFERENCES (1) K€uhn, S.; Hakanson, U.; Rogobete, L.; Sandoghdar, V. Enhancement of Single-Molecule Fluorescence Using a Gold Nanoparticle as an Optical Nanoantenna. Phys. Rev. Lett. 2006, 97 (1), 017402. (2) Atwater, H. A.; Polman, A. Plasmonics for improved photovoltaic devices. Nat. Mater. 2010, 9 (3), 205–213. (3) Schuller, J. A.; Barnard, E. S.; Cai, W. S.; Jun, Y. C.; White, J. S.; Brongersma, M. L. Plasmonics for extreme light concentration and manipulation. Nat. Mater. 2010, 9 (3), 193–204. (4) Standridge, S. D.; Schatz, G. C.; Hupp, J. T. Distance Dependence of Plasmon-Enhanced Photocurrent in Dye-Sensitized Solar Cells. J. Am. Chem. Soc. 2009, 131 (24), 8407. (5) Lee, S. E.; Lee, L. P. Biomolecular plasmonics for quantitative biology and nanomedicine. Curr. Opin. Biotechnol. 2010, 21 (4), 489–497. (6) Kim, I.; Kihm, K. D. Unveiling Hidden Complex Cavities Formed during Nanocrystalline Self-Assembly. Langmuir 2009, 25 (4), 1881–1884. (7) Terasaki, N.; Yamamoto, N.; Hattori, M.; Tanigaki, N.; Hiraga, T.; Ito, K.; Konno, M.; Iwai, M.; Inoue, Y.; Uno, S.; Nakazato, K.
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