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Fluorescence enhancement and lifetime modification of single nanodiamonds near a nanocrystalline silver surface. Tsong-Shin Lim,a Chi-Cheng Fu,b ...


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Fluorescence enhancement and lifetime modification of single nanodiamonds near a nanocrystalline silver surface Tsong-Shin Lim,a Chi-Cheng Fu,b Kang-Chuang Lee,b Hsu-Yang Lee,b Kowa Chen,b Wen-Feng Cheng,b Woei Wu Pai,c Huan-Cheng Chang*b and Wunshain Fannbd Received 6th October 2008, Accepted 27th November 2008 First published as an Advance Article on the web 20th January 2009 DOI: 10.1039/b817471g Fluorescent nanodiamond (FND) contains nitrogen-vacancy defect centers as fluorophores. The intensity of its fluorescence can be significantly enhanced after deposition of the particle (35 or 140 nm in size) on a nanocrystalline Ag film without a buffer layer. The excellent photostability (i.e. neither photobleaching nor photoblinking) of the material is preserved even on the Ag film. Concurrent decrease of excited state lifetimes and increase of fluorescence intensities indicate that the enhancement results from surface plasmon resonance. Such a fluorescence enhancement effect is diminished when the individual FND particle is wrapped around by DNA molecules, as a result of an increase in the distance between the color-center emitters inside the FND and the nearby Ag nanoparticles. A fluorescence intensity enhancement up to 10-fold is observed for 35 nm FNDs, confirmed by fluorescence lifetime imaging microscopy.

Introduction Fluorescent nanodiamond (FND) is emerging as a new type of nanomaterial that holds great promise for biological applications.1,2 Containing a high concentration of nitrogen-vacancy (N–V) defect centers as fluorophores, FND exhibits several remarkable features, including emission of bright photoluminescence in the extended red region, no photobleaching and photoblinking, and easiness of surface functionalization for specific or nonspecific binding with nucleic acids and proteins, etc.3,4 In particular, the capability of emitting light at B700 nm, where cell autofluorescence signal is low,5 makes FND wellsuited for cellular imaging application. These excellent photophysical properties, together with the good biocompatibility of the material,6 have enabled three-dimensional tracking of a single FND particle with a size of 35 nm in a live mammalian cell over a time period of more than 3 min using a wide-field fluorescence microscope.7 Although the observation of a single 35-nm FND can be readily made in solution and cells, improving the detection sensitivity is desirable to widen its biological applications.8 A technology based on the use of metallic nanostructures that interact with fluorophores to increase their emission intensity has long been recognized since its discovery in 1980.9 Experiments with dye-doped polymer films10 and dye-labeled oligonucleotides11 on nanocrystalline Ag surfaces indicated that the maximum fluorescence enhancement occurred at a separation of B3 and B9 nm, respectively, between metal and fluorophore.

It means that covering the metallic film with a buffer layer of a few nanometers in thickness is essential for achieving the largest fluorescence enhancement effect. Typically, a 2- to 10-fold enhancement in the fluorescence intensity is observed for fluorophores with low quantum yields,12 whereas no significant enhancement has been reported for high-quantum-yield fluorophores.13 A number of biotechnological applications taking advantage of this so-called metal-enhanced fluorescence (MEF) have been developed, such as, to improve the sensitivity of DNA hybridization assays.14 Aside from these applications, which were all conducted for an ensemble of molecules, the technique has also been applied to the detection of single quantum dots (such as CdSe and CdTe) on silver island films (SIFs), resulting in a 5-fold increase in fluorescence intensity.15,16 Herein, we show that the fluorescence intensity of (N–V) centers, which have near unity photoluminescence quantum efficiency in diamond,17 can also be significantly enhanced when FNDs are in proximity to Ag nanoparticles on a SIF. Moreover, for particles with sizes of 35 and 140 nm, no buffer layers are required since the (N–V) centers are embedded in the diamond lattice and are separated by several tens nm from the Ag surface. Such a surface enhancement effect is particularly evident for 35-nm FNDs, where a near 10-fold increase in fluorescence intensity was observed. Additionally, the enhancement effect is diminished nearly completely when FNDs are coated with layers of T4 DNA molecules, which act as a spacer that increases the separation between FNDs and SIF. To the best of our knowledge, this is the first time that such MEF is observed for single particles with sizes ranging from 35 to 140 nm.


Department of Physics, Tunghai University, Taichung, 407, Taiwan Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, 106, Taiwan. E-mail: [email protected]; Fax: (+886)2 23620200 c Center for Condensed Matter Sciences, National Taiwan University, Taipei, 106, Taiwan d Department of Physics, National Taiwan University, Taipei, 106, Taiwan b

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Materials and methods Production and surface modification of FNDs Synthetic type Ib diamond powders with a mean size of 140 nm (Micron+ MDA, Element Six, USA) and 35 nm This journal is


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(MSY, Microdiamant, Switzerland) were purified in concentrated H2SO4–HNO3 solution (3 : 1, v/v) at 90 1C for 30 min. After washing extensively in deionized water, about 5 mg of the diamond powders was deposited on a silicon wafer and allowed to dry in air to form a thin film (area B0.5 cm2 and thickness B50 mm). The dried diamond film was exposed to a 3-MeV proton beam from a NEC tandem accelerator (9SDH-2, National Electrostatics Corporation) at a dose of B1  1016 ions cm2. Formation of (N–V) defect centers was facilitated by annealing the proton-beam-treated nanodiamonds in vacuum at 800 1C for 2 h. The freshly prepared FNDs were finally treated in concentrated H2SO4–HNO3 mixtures to remove graphitic surface structures and simultaneously functionalize the diamond surfaces with carboxyl and other oxygen-containing groups.3 Poly-L-lysines (PLs) with a molecular weight of B30 000 were used to decorate FND surfaces with amino groups. This was done by mixing 8 mg of N-(3-dimethylaminopropyl)-Nethyl-carbodiimide hydrochloride (EDC, Sigma) with 6 mg of N-hydroxysuccinimide (Sigma) in a 5 mL solution containing 6 mg of acid-washed FNDs, followed by adding 3 mg of PLs into the suspension. After incubation of the mixture at room temperature for 24 h, the PL-coated FNDs were thoroughly washed in deionized water. Approximately 1 mg of the amineterminated FND particles suspended in 100 mL of deionized water were drop-cast on a glass plate with or without SIF coating for fluorescence microscopy measurements. Noncovalent conjugation of DNA to FND was made by mixing 3 mg of the PL-coated FND particles suspended in 200 mL of 0.5 TBE buffer (Invitrogen) with a solution containing 165.6-kb T4 phage DNA (Wako) at a molar ratio of FND : DNA = 1 : 8. The conjugation was established by pure electrostatic attraction. The mixture, after incubation at room temperature for 10 min, was diluted with 0.5 TBE buffer to a concentration suitable for single particle detection, followed by drop-cast of the particles on SIFs for fluorescence measurements. Preparation of SIFs SIFs were prepared by using the Tollens ‘‘mirror’’ reaction, following the procedures described in ref. 10. In brief, glass plates (25  25  1 mm) were first cleaned in H2SO4–H2O2 solution (3 : 1, v/v) and rinsed extensively in deionized water. About 2–3 mL of concentrated NH4OH was added to 150 mL of 0.1 M AgNO3 and after thorough stirring to dissolve any precipitate that might have formed, 75 mL of 0.8 M KOH was added to the mixed solution. To form SIFs, an equal amount of this solution and 0.5 M dextrose were mixed together for 10 s at room temperature. Droplets of the mixed solution were deposited on the acid-cleaned glass plate positioned on a hot plate for 1 min at 35 1C prior to removal of excess reagents. Wide-field fluorescence microscopy imaging Fluorescence images of FND particles and SIFs were acquired using a wide-field fluorescence microscope (IX70, Olympus) equipped with a frequency-doubled Nd : YAG laser (DPSS 532, Coherent) operating at 532 nm and an electron multiplying charge-coupled device (CCD, DV887DCS-BV, Andor) This journal is


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set at an exposure time of 0.1 s. In imaging 140-nm FNDs, the laser-excited fluorescence was collected with a 40, NA 0.75 objective (UPLFL 40, Olympus) and guided through a 565 nm long-pass filter (E565lp, Chroma Tech) before reaching the detector. As for the Ag nanoclusters and 35-nm FND particles, the fluorescence was collected by using a 100, NA 1.35 oil objective (UPLFL 100, Olympus) and selected by a 680–730 nm band-pass filter (HQ655/150 m, Chroma Tech) prior to detection. Measurements of fluorescence lifetimes and spectra Fluorescence lifetime measurements were performed with a modified confocal optical microscope (E600, Nikon) equipped with a 60, NA 0.7 objective (ELWD 60, Nikon) and a frequency-doubled picosecond Nd : YAG laser (IC-532-30, High Q Laser) operating at 532 nm and a repetition rate of 50 MHz.2,18 The picosecond-laser-excited fluorescence, after passing through a 565 nm long-pass filter (E565lp, Chroma Tech), was collected and detected by using either an avalanche photodiode (SPCM-AQR-15, Perkin-Elmer) or a GaAsP photon-counting photomultiplier tube (H7422P-40, Hamamatsu). The use of the latter allowed for fluorescence detection with a time resolution of B300 ps, limited by the instrument response function. Confocal fluorescence images were acquired for particles dispersed on either a glass plate or a SIF after a raster scan of the specimen using a piezo-driven nanopositioning and scanning system (E-710.4CL & P-517.3CL, Physik Instrument). By moving the particles of interest consecutively to the focal point of the microscope objective, both the fluorescence time traces and spectra of the individual particles were collected. The former were recorded with a time-correlated single photon counting module (SPC-600, Becker & Hickl) and the latter were obtained with a monochromator (SP300i, Acton Research) equipped with a liquid-nitrogen-cooled CCD camera (LN/CCD-1100-PB, Princeton Instruments). The intrinsic lifetime of the fluorescence was analyzed by using a commercial program (FAST, Fluorescence Analysis Software Technology), which deconvoluted the signal with the instrument response function and fit each time trace with a multi-exponential function as given by eqn (1). Fluorescence lifetime gating and imaging Fluorescence lifetime imaging microscopy (FLIM) was used to obtain lifetime-resolved images of 35-nm FNDs with a timecorrelated single photon counting system (PicoHarp 300, PicoQuant). Both the fluorescence decays of FNDs and Ag nanoclusters were measured and analyzed. To obtain the fluorescence images of 35-nm FNDs without the background signals of SIFs, proper time-gating windows were chosen. Time-gated counts were calculated by summing over all data points constituting the fluorescence decay curve within this selected window.

Results and discussion Fig. 1a shows a typical atomic force microscopy image of an as-grown SIF, composed of Ag nanoclusters with an average size of B80 nm. Optical spectroscopic measurements indicated Phys. Chem. Chem. Phys., 2009, 11, 1508–1514 | 1509

Fig. 1 (a) Typical atomic force microscopy image of SIF. (b) Comparison of the absorption spectrum of SIF (green) and the emission spectrum of 140-nm FNDs (red). (c) Wide-field epifluorescence image of SIF, obtained by laser excitation at 532 nm and emission collection at 680–730 nm. (d) Typical fluorescence decay curve of an Ag nanocluster.

that this film absorbs strongly in the wavelength range of 350–600 nm with a maximum at B450 nm (Fig. 1b). The band, originating from surface plasmon resonance (SPR), overlaps well with the absorption band of FNDs at B560 nm,19 which ensures resonant interaction. The SPR band, on the other hand, is fairly separated from the emission band (peaking at 680 nm) of FNDs. A close examination of the Ag nanoclusters revealed that their fluorescence spectra are highly heterogeneous. Each nanocluster shows a distinctly different spectrum (Fig. 2). In particular, some nanoclusters (such as particle 1 in Fig. 2) fluoresce in the spectral region overlapping with that of FNDs.20 They appear as bright red spots as undesirable backgrounds in the fluorescence image (Fig. 1c). A preliminary experiment showed that the fluorescence brightness of these Ag nanoclusters is B6 times higher than that of 35-nm FNDs deposited on a glass plate, but is roughly one order of magnitude lower than that of 140 nm FNDs. For the Ag nanoclusters prepared in this work, they all have a fluorescence decay lifetime of B300 ps, essentially limited by the instrument response time (Fig. 1d). The result is in accord with the findings of Dickson and coworkers,21 who reported picosecond fluorescence lifetimes for Ag nanoclusters. We started the MEF measurement with 140-nm FNDs surface-coated with PLs. Fig. 3a shows a fluorescence image along with its intensity histogram for single 140-nm FNDs dispersed on a bare glass plate. The fluorescence intensity distribution centers at the count number of B8000 with a distribution width of 3900. A 2-fold increase in both mean value and width of the distribution was observed when the same particles were deposited on a SIF and excited under the 1510 | Phys. Chem. Chem. Phys., 2009, 11, 1508–1514

Fig. 2

Emission spectra of three single Ag nanoclusters in SIF.

same conditions (Fig. 3b). Such an increased distribution width is attributed to the increase in heterogeneity of the MEF effect as well as the size distribution of the Ag nanoclusters in the island film. To further illustrate this MEF phenomenon, we increased the FND-SIF distance by overcoating the PL-conjugated FNDs with T4 DNA. As have been demonstrated elsewhere for single FND/DNA detection,2 the DNA coating does not result in any significant changes in the photoluminescence property of the material, since the red emission of FND originates from the (N–V) centers embedded in the diamond lattice and their photophysical characteristics are insensitive to surface functionalization. Furthermore, the individual 140-nm FND particle is wrapped around by the T4 DNA molecule, This journal is


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Fig. 3 Wide-field epifluorescence images (left) and corresponding intensity histograms (right) of (a) PL-coated FNDs on a glass plate, (b) PL-coated FNDs on SIF, and (c) PL-coated FNDs wrapped around by T4 DNA on SIF. Note the change of the contrast scale in (b).

which has an extended length of B60 mm and a radius of gyration of B2 mm. Fig. 3c shows a fluorescence intensity histogram of the FND/DNA complex. Indeed, the average fluorescence intensity dropped to its original value, accompanied with a narrower distribution width. Assuming that only one half of each DNA molecule is involved in the particle wrapping process and the other half is extended freely into solution, we estimate that the FND-SIF distance is increased by B1 mm, which essentially eliminates all the MEF effect. This distance-dependent measurement result supports our suggestion that the observed fluorescence enhancement is caused by SPR. The origin of the MEF effect can be further elucidated by fluorescence lifetime measurements. We compare in Fig. 4a the fluorescence lifetime histograms of 140-nm FNDs on a bare glass plate and on a SIF surface. Each fluorescence lifetime was obtained by analyzing the fluorescence decay curve in terms of a multi-exponential model after proper deconvolution of the instrument response function as,22 I¼


ai expðt=ti Þ;


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where ti is the lifetime of each component and ai is the corresponding amplitude with Sai = 1. The amplitudeweighted lifetime is finally determined as X hti ¼ ai ti ð2Þ i

By summing the results over 30 single particles (Fig. 4b), we found a shortening of the average fluorescence lifetime of FNDs on the SIF to hti = 14.1 ns, compared with hti = 23.7 ns of the same FNDs deposited on the glass plate. The near two-fold reduction in lifetime, together with the two-fold increase in fluorescence intensity, again suggests that the observed fluorescence enhancement derives from SPR. Conventionally, the MEF effect can be observed more readily for fluorophores with low quantum yields.23 The fluorescence enhancement process can be understood as a result of the quenching of a molecular excited state (exciton) by non-radiative transfer of energy from the exciton to surface plasmons supported by metallic structures in close proximity, followed by the re-radiation of this energy from the plasmons.24 Recently, there is a report on the fluorescence enhancement of high-quantum-yield fluorophores.25 However, Phys. Chem. Chem. Phys., 2009, 11, 1508–1514 | 1511

Fig. 4 (a) Histograms of fluorescence lifetimes and (b) averaged fluorescence decay curves of FNDs on a bare glass plate (blue) and on a SIF surface (red).

Fig. 5 Comparison of photostability of 140-nm FNDs (blue) and Ag nanoclusters (red) on SIF.

in that ensemble measurement, it is difficult to separate the SPR-associated enhancement from other effects such as the surface coverage effect. This work, combining single particle imaging with fluorescence lifetime measurements, unambiguously demonstrates that the intensity of the photoluminescence from fluorophores with near unity quantum yields can

be metal-enhanced. To account for our observations, we consider the spectral overlaps of the absorption and emission bands of the fluorophores with those of the underneath substrate. As shown in Fig. 1b, good spectral overlap was achieved between FNDs and SIFs, whereas the emission band of FND is well separated from the plasmon resonance. The mechanism of the fluorescence enhancement process observed herein is, therefore, most likely to involve excitation of SPR by incident light, followed by transfer of the energy nonradiatively to nearby FNDs, and finally radiation from the FND.26 It is an open question whether or not the excellent photostability of FND is preserved on the silver nanocrystalline film. To answer this question, we monitored the fluorescence intensities of the individual 140-nm FND particles over a time period of 30 s with 100-ms time resolution. Similar to that found previously for FNDs on a glass plate,2 excellent photostability was observed for the same particles on SIF. Under the excitation with a cw 532-nm laser at a power density of 2  103 W cm2, the fluorescence intensity of the individual FNDs stays essentially the same over 30 s and no sign of intermittency was found. In contrast, the underneath Ag nanoclusters exhibit distinct photoblinking behavior (Fig. 5). In accord with previous observations for the same FNDs on glass plates and in cells,2,7 no photobleaching was observed over an excitation time period of more than 3 min (data not

Fig. 6 (a) Confocal fluorescence image of 35-nm FNDs on a glass plate and (b) comparison of confocal fluorescence image (left) and FLIM image (right) of 35-nm FNDs on SIF. Six bright spots are marked and compared between these two images in (b). Note that the scale bars in the confocal fluorescence image and the FLIM image are given in intensity (counts) and lifetime (ns), respectively.

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Fig. 7

FLIM images of 35-nm FNDs on SIF obtained at four different gating times.

shown). The exceptionally high photostability of FND is a key property for the application of this nanomaterial in single particle imaging and long-term tracking of a single particle interacting with biomolecules on surface as well as in cells. The success of enhancing the fluorescence intensity of 140-nm FNDs with the Ag nanostructures leads us to the suggestion that smaller FND particles (such as the 35 nm ones) should show more dramatic fluorescence enhancement. However, in accord with their volume ratio, the fluorescence intensity of 35-nm FNDs is B60-fold weaker (Fig. 6a).2,7 Detecting these particles thus becomes problematic owing to the large fluorescence background from SIF.20 Fortunately, time domain measurements provide a solution to overcome this problem. As shown in Fig. 1d, the fluorescence decay lifetimes of Ag nanoclusters are typically less than 300 ps, which is much shorter than that of FNDs on SIF. It is therefore possible to separate the signal of FND from that of the Ag nanoclusters using a lifetime gating technique, or FLIM.27 Fig. 6b (left) displays a fluorescence image of 35-nm FNDs dispersed on a SIF surface, obtained by using a confocal fluorescence microscope. Several bright spots appear in the image; however, the image alone does not allow us to tell which is FND and which is Ag nanocluster. Only with the use of the lifetime gating technique, they can be readily distinguished. Fig. 6b (right) shows the fluorescence lifetimeresolved image of the same specimen. Of the 6 particles circled in the image, spots 1, 2, and 3 have significantly shorter fluorescence lifetimes and they belong to the Ag nanoclusters, whereas spots 4, 5, and 6 have longer fluorescence lifetimes and they correspond to FND particles. Compared with the same particles deposited on a coverglass slide and This journal is


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excited under the same experimental conditions (Fig. 6a), the FNDs on SIF exhibit a B10-fold increase in fluorescence intensity. In Fig. 7, we also display the FLIM images of 35-nm FNDs on SIF obtained at four different gating times of 0.0, 1.6, 3.2, and 4.8 ns. The distinct changes of the images with the gating time allow us to distinguish clearly FNDs from Ag nanoclusters in the specimen.

Summary and conclusion We have observed significant fluorescence enhancements of FNDs (35 and 140 nm in size) on SIF surfaces. The enhancement observed for the 140-nm FNDs is about 3-fold smaller than that detected for single quantum dots of B5 nm in diameter.15,16 The difference is easily comprehensible since these FND particles are much larger and only B10% of the fluorophores (i.e. N–V centers) inside the diamond lattices are estimated to be located within the enhancement region (B10 nm) above the metal surface. Although the fluorescence background of the SIF makes it difficult to distinguish 35-nm FNDs from nearby Ag nanoclusters, the former can still be identified by using a time gating technique, thanks to the excellent photostability and the long fluorescence lifetime of the material. Further improvement of the detection sensitivity is possible if FNDs containing a higher concentration of (N–V) centers are available. Such a MEF effect, in combination with the easiness of surface modification, renders FND a potentially useful tool for long-term observation of DNA–protein and protein–protein interaction on surface at the single particle level. Phys. Chem. Chem. Phys., 2009, 11, 1508–1514 | 1513

Acknowledgements This work was supported by Academia Sinica and the National Science Council (Grant No. NSC 94-2120-M-002009- and NSC 96-2120-M-001-008-) of Taiwan. We thank one of the referees for pointing out a possible mechanism for the fluorescence enhancement observed in this experiment.

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