Article pubs.acs.org/Langmuir
Silver Nanocube-Enhanced Far-Red/Near-Infrared Fluorescence of Conjugated Polyelectrolyte for Cellular Imaging Jing Liang,† Kai Li,† Gagik G. Gurzadyan,‡ Xianmao Lu,† and Bin Liu*,† †
Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, 117576, Singapore Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, 637371, Singapore
‡
S Supporting Information *
ABSTRACT: We present the study of silver nanocube (Ag NC)enhanced fluorescence of a cationic conjugated polyelectrolyte (CPE) for far-red/near-infrared fluorescence cell imaging. Layer-bylayer self-assembly of polyelectrolytes on 78 nm Ag NCs is used to control CPE−metal distance and its effect on CPE fluorescence. The highest fluorescence enhancement factor (FEF) is obtained for Ag NCs with two bilayers, corresponding to a CPE−metal spacer thickness of ∼6 nm. At the optimal excitation wavelength, the FEF is 13.8 with respect to the control silica nanoparticles (NPs). The fluorescent NPs are further used for cellular imaging studies. The CPE-loaded Ag NCs with two bilayers exhibit excellent image contrast, superior to the control of CPE−silica NP at a similar uptake efficiency. The viability test indicates low cytotoxicity of the CPE-loaded Ag NCs, rendering them as promising cell imaging agents.
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CPE backbone18 or side chain11,19 modifications or molecular architecture alterations.20 This approach, however, requires sophisticated molecular design and tedious experimental procedures. Development of highly fluorescent CPEs with a versatile strategy remains in demand. Gold and silver nanoparticles (NPs) display unique surface plasmon resonance (SPR), which is the collective oscillation of surface electrons under light radiation, creating strong electric fields that can increase the excitation of a nearby fluorophore.21 The local fields can interact with the excited fluorophore in proximity, leading to enhanced excitation of the fluorophore. In addition, metal NPs can interact with the excited fluorophore and create plasmons, which radiate rapidly to the far-field (at least several wavelengths from the fluorophore), giving rise to increased emission and decreased lifetimes. This phenomenon is called metal-enhanced fluorescence (MEF), and its effect and mechanism have been widely studied for different metal structures as their surface plasmon properties are highly shape and size dependent.22,23 MEF gives rise to a number of useful effects, such as increased fluorescence intensity, increased photostability, and decreased lifetimes of the fluorophores, paving the way for its application in biosensors.24 These studies are largely focused on organic fluorophores,25 while very few
INTRODUCTION Bioimaging in the far-red/near-infrared (FR/NIR) region (650−1000 nm) has attracted substantial research interest due to its noninvasive nature, reduced autofluorescence, and deep specimen penetration.1 A wide range of materials have been employed for FR/NIR bioimaging, such as organic fluorophores,2 quantum dots,3,4 fluorescent proteins,5 and single walled carbon nanotubes.6 However, they have certain disadvantages, such as low fluorescence quantum yield, poor photostability, or cytotoxicity.7 Recently, conjugated polyelectrolytes (CPEs) have emerged as a useful alternative for bioimaging applications.8 CPEs are fluorescent macromolecules with π-conjugated backbones and charged side chains.9 They possess superior optoelectronic properties as compared to conventional small organic fluorophores, such as high optical absorption cross-section, good photostability, and large Stokes shift.10 The development of CPE-based fluorescent nanoparticles further enables their application in bioimaging as cell contrast agents,11−14 making them preferential over other fluorescent materials. The far-red and near-infrared fluorescent CPEs have been rarely reported, which often suffer from low quantum yield (QY), greatly limiting their applications in FR/NIR cell imaging.15,16 The low QY is mainly due to aggregation-induced fluorescence quenching or charge transfer between the media and polymer backbone with donor−acceptor units.17 A common strategy to enhance CPE fluorescence is through © 2012 American Chemical Society
Received: November 22, 2011 Revised: July 9, 2012 Published: July 12, 2012 11302
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Scheme 1. Chemical Structures of PFVBT, PDDA, and PSS, and Schematic Illustration of Fluorescent NC Formation via Layerby-Layer Self-Assembly of Polyelectrolytes Followed by CPE Adsorption on Ag NCs
Technologies). Dulbecco’s modified essential medium (DMEM) is a commercial product of National University Medical Institutes (NUMI, Singapore), and phosphate-buffer saline (PBS, 10×, pH 7.4) is a commercial product of 1st BASE Singapore. Milli-Q water (18.2 MΩ) was used to prepare all stock solutions. MCF-7 breast cancer cells were provided by American Type Culture Collection. Characterization. The extinction spectra of Ag NCs and absorption spectra of PFVBT were measured using a UV−vis spectrometer (Shimadzu, UV-1700, Japan). PL measurements were carried out on a Perkin-Elmer LS-55 equipped with a xenon lamp excitation source and a Hamamatsu (Japan) 928 PMT, using 90° angle detection for solution samples. Transmission electron microscopy (TEM) images of Ag NCs were acquired on a JEOL JEM-2010F operating at 200 kV. The zeta potentials of the Ag NCs with different numbers of bilayers were measured using a zeta potential analyzer (ZetaPlus, Brookhaven Instruments Corp.) at room temperature. Inductively coupled plasma mass spectrometry (ICP-MS) measurements were conducted with an Agilent 7500 ICP-MS instrument. Samples were prepared by dissolving the nanocrystals with concentrated nitric acid followed by dilution in DI water. Fluorescence lifetime measurements were performed on a FluoTime 200 TCSPC fluorescence platform from Picoquant GmbH (Berlin, Germany). A titanium-sapphire 100 fs laser (Chameleon, Coherent) with secondand third-harmonic generation was used as the excitation source, and its excitation wavelength was 500 nm. In time correlated single photon counting (TCSPC) apparatus, the detector is a microchannel plate (MCP) PMT system HAM-R3809U-50 (Hamamatsu) with a spectral range from 160 to 850 nm and an instrument response of 30 ps. Fluorescence lifetimes were extracted from the decay curves using commercially available fluorescence lifetime analysis software (FluoFit Pro. PicoQuant GmbH). Fluorescence decay curves were fitted using a two or three exponential mode. Synthesis of Silver Nanocubes. Ag NCs were synthesized according to the published protocol with slight modification.35 Ethylene glycol (EG, 10 mL) was heated in a loosely capped 100 mL two-neck round-bottom flask under stirring at 140 °C for 1 h. HCl (2.1 mL, 3.02 mM) was then quickly added into the EG solution. After 10 min, AgNO3 (6 mL, 94 mM) in EG and PVP (6 mL, 147 mM) in EG solutions were slowly injected into the heated EG simultaneously using two 10 mL syringes in 6 min. The EG solution was continuously heated at 140 °C. After the solution turned greenish red, its UV−vis spectra were monitored regularly while heating. When the Ag NCs grew to the size with desired spectrum (with extinction maximum at ∼510 nm), the flask was immersed in an ice bath to quench the reaction immediately. The colloids were then washed with acetone
examples have been demonstrated for CPEs. In addition, most of the MEF examples reported use metals on solid substrates.26,27 In this contribution, we report silver nanocube (Ag NC)enhanced fluorescence of a cationic CPE in colloidal solution for FR/NIR cell imaging. The molecular structure of the CPE poly[9,9-bis(6′-(N,N,N-trimethylammonium)hexyl)fluorenyldivinylene-alt-4,7-(2,1,3-benzothiadiazole) dibromide] (PFVBT) is shown in Scheme 1. Ag NC is chosen as the fluorescence amplifying material for two reasons. First, silver NPs, especially with large size, are reported to exhibit high scattering cross-section as compared to gold NPs, which contributes significantly to effective fluorescence enhancement.28,29 Second, the plasmon band of Ag NCs is red-shifted as compared to spherical Ag NPs, leading to enhanced local field and reduced radiative damping that benefit fluorescence enhancement.30,31 A spacer layer is created between Ag NCs and PFVBT using multilayered polyelectrolytes with varying thicknesses, followed by adsorption of PFVBT on Ag NCs. The influence of spacer thickness on fluorescence enhancement factors (FEFs) is investigated with respect to the control sample with silica NP core. The fluorescent NPs are then applied for FR/NIR imaging of MCF-7 breast cancer cells, and their cytotoxicity is evaluated using MTT viability assay.
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EXPERIMENTAL SECTION
Materials. Poly[9,9-bis(6′-(N,N,N-trimethylammonium)hexyl)fluorenyldivinylene-alt-4,7-(2,1,3-benzothiadiazole) dibromide] (PFVBT) was synthesized according to a previous report.32 Poly(diallyldimethylammonium chloride) (PDDA, MW 200 000−350 000, 20 wt % in water, Aldrich), poly(styrene sulfuric acid) sodium salt (PSS, MW 70 000, Alfa-Aesar), methanol (99.8%, Merck), and hydrochloric acid (HCl, 37%, AnalaR NORMAPUR) were used as received. Silica nanoparticles with a diameter of 100 nm and particle concentration of 1.67 nM were synthesized using a modified Stöber method according to the previous reports.33,34 Polyvinylpyrrolidone (PVP, MW 55 000), silver nitrate (AgNO3, >99.8%), ethylene glycol (EG), sodium chloride (NaCl, ≥99.5%), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin-streptomycin solution, and 4′,6-diamidino-2phenylindole (DAPI) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from Invitrogen (Life 11303
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uptake efficiency was defined as the ratio of the fluorescence in the sample wells to that of the positive control. Cytotoxicity of PFVBT-Loaded NPs. MCF-7 cells were seeded into a 96-well transparent plate (Costar, IL) with a density of 5 × 104 cells mL−1. After 24 h incubation, the medium was replaced with PFVBT-loaded Ag NCs and SNPs as well as PFVBT in 100 μL of FBS free DMEM medium at PFVBT concentrations of 125, 250, and 500 nM, respectively. After 24 h, the wells were washed twice with 1 × PBS buffer. Afterward, 100 μL of freshly prepared MTT (0.5 mg mL−1) solution in culture medium was added to each well. After 3 h incubation, the MTT medium solution was carefully removed, followed by addition of 100 μL of DMSO into each well to dissolve all precipitates formed. The absorbance of purple MTT at 570 nm was measured by the microplate reader (Genios Tecan). Cell viability was expressed by the ratio of absorbance of the cells incubated with NP suspension to that of the cells incubated with culture medium only.
twice and water four times, and redispersed in 20 mL of milli-Q water. The final colloidal dispersion appeared greenish ocher. The Ag NCs have an average size of 78 nm as confirmed by TEM images, and the Ag atomic concentration ([Ag]) was measured to be 14.81 mM by ICP-MS. Layer-by-Layer Self-Assembly of Polyelectrolytes on Ag NCs. 600 μL of Ag NC dispersion ([Ag NC] = 3.7 mM) was added dropwise into 750 μL of positively charged PDDA ([repeat unit] = 10 mM in 50 mM NaCl aqueous solution) in a 2 mL tube under sonication. The dispersion was further sonicated for 5 min and then shaken by thermal shaker for 10 min at 600 rpm at room temperature. The dispersion was subsequently washed with milli-Q water three times and redispersed in 600 μL of milli-Q water. Between the washing steps, AgNC−polymer NPs were sonicated to be redispersed in water. The resulting dispersion was then added dropwise into 750 μL of negatively charged PSS ([repeat unit] = 10 mM in 50 mM NaCl aqueous solution) under sonication. After sonicating for 5 min and shaking for 10 min, the dispersion was washed three times with water. Multilayer assemblies of Ag NCs were prepared by consecutive absorption of oppositely charged polymers of PDDA and PSS. The samples of Ag NCs assembled with one, two, and three bilayers were designated as Ag-1bl, Ag-2bl, and Ag-3bl, respectively. Preparation of AgNC−CPE and SNP−CPE NPs. PFVBT (40 μL, 0.04 mM) was added to 475 μL of Ag-1bl, Ag-2bl, and Ag-3bl samples ([Ag NC] = 0.02 nM in 50 mM NaCl aqueous solution), under shaking at 600 rpm. After incubation for 20 min, the colloidal dispersions were centrifuged at 7500 rpm. The supernatant solutions that contain free CPEs were carefully removed and diluted for UV/vis measurement. The precipitated AgNC−CPE for each sample was redispersed in Milli-Q water for PL measurement. The absorbance of the supernatant CPE was compared to that before adsorption to calculate the adsorption efficiency. The NPs were adjusted to adsorb 30% of the CPE. On the basis of this ratio, 40 μL of PFVBT (0.04 mM) was added to the SNP dispersion (0.08 nM in 50 mM NaCl aqueous solution) with shaking for 20 min. The amount of SNPs was optimized to adsorb the same amount of CPEs with the same surface area as that of Ag NCs. PL measurement of each sample was carried out to compare the fluorescence intensity for enhancement factor calculation. Cell Cultures. MCF-7 breast cancer cells were cultured in DMEM medium containing 1% penicillin−streptomycin and 10% FBS at 37 °C in a humidified environment containing 5% CO2. Before experiment, the cells were precultured until confluence was reached. Cell Imaging. MCF-7 cells were cultured in the chambers (LABTEK, Chambered Coverglass System) at 37 °C. After 80% confluence, the medium was removed and the adherent cells were washed twice with 1 × PBS buffer. The PFVBT-loaded Ag NCs and SNPs in 400 μL of FBS free DMEM medium at PFVBT concentration of 125 nM were then added to the wells. After incubation for 2 h, the wells were washed three times with 1 × PBS buffer to carefully remove the free NPs. The cells were then fixed with 75% ethanol for 10 min and further washed twice with 1 × PBS buffer. The nuclei were stained with DAPI for 10 min. The cells were washed twice with 1 × PBS buffer and imaged by CLSM (Zeiss LSM 410, Jena, Germany) with imaging software (Fluoview FV1000). Cellular Uptake. MCF-7 cells were seeded into a 96-well black plate (Costar, IL) for quantitative study of cellular uptake. After the cells reached 80% confluence, the medium was replaced with the suspensions of PFVBT-loaded Ag NCs and SNPs in 100 μL of FBS free DMEM medium at 125 nM PFVBT. The cells were then cultured at 37 °C for 2.5 h. For each sample, six sample wells were washed three times with 50 μL of 1 × PBS buffer to remove free NPs or PFVBT, while another six wells were used as positive control without washing. 100 μL of DMEM medium was then added to each sample well. Next, a solution of 0.5% Triton X-100 in 0.2 N NaOH (50 μL) was added to both sample wells and control wells to lyse the cells. The fluorescence intensity of the samples in each well was then measured by the microplate reader (Genios Tecan) at excitation wavelength of 510 nm. The fluorescence intensity was collected at 670 nm. The cellular
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RESULTS AND DISCUSSION Characterization of PFVBT and Ag NCs. Figure 1A shows the UV−vis absorption and photoluminescence (PL) spectra of PFVBT in 50 mM NaCl solution. The polymer has two absorption maxima at 386 and 527 nm, which correspond to the π−π* transitions of vinyl fluorene segments and vinyl BT segments, respectively.32 The PL maximum of PFVBT is at 677 nm, with an emission tail extended to 860 nm, which corresponds to FR/NIR fluorescence. The Ag NCs were synthesized via the reduction of AgNO3 by ethylene glycol in the presence of PVP as the surfactant at elevated temperature. As the reaction proceeds, the Ag colloidal dispersion undergoes a series of color changes. Upon formation of small Ag NCs, extinction spectra were monitored in 10 min intervals as the NCs continue to grow. The growth was indicated by a continuous red-shift of the extinction maximum, and the reaction was quenched when the extinction spectrum of Ag NCs reached a maximum at 506 nm (Figure 1B). The extinction spectrum matches well with the absorption maximum of PFVBT to favor surface plasmon coupling and local field enhancement. The SPR peak at 506 nm is attributed to the excitation of dipolar charge distribution, while the other lower wavelength peaks can be attributed to higher multipolar charge distributions.36 The TEM image of the Ag NCs is shown in Figure 1C. The synthesized Ag NCs display high monodispersity with an average size of 78 nm in edge length. As measured by ICP-MS, the Ag colloidal solution has a Ag atomic concentration of 14.81 mM. On the basis of this concentration and particle size, the NP concentration is calculated to be 0.53 nM (see Supporting Information). Zeta potential of the Ag NCs was analyzed to be −25.8 mV, confirming negatively charged nature of the surface. Self-Assembly of Polyelectrolytes on Ag NCs. As the fluorescence properties of a fluorophore near metallic nanostructures are distance dependent, it is necessary to study the PL intensity of PFVBT at different CPE−metal distances.37 The layer-by-layer assembly is an effective and flexible technique to construct multilayer films through various methods,38,39 which may provide better thickness control as compared to the commonly used silica coating method.21,37 The conventional layer-by-layer technique relying on adsorption of alternately charged polyions is used to fine-tune the PFVBT−Ag NC distance due to its simplicity and suitability to incorporate the positively charged PFVBT.40 PDDA and PSS are used as the polycation and polyanion for the self-assembly, respectively (Scheme 1). The as-synthesized Ag NCs are protected by PVP, which carries a fractional negative charge on 11304
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PDDA and PSS polyelectrolytes to make the Ag-2bl and Ag-3bl NCs, respectively. The zeta potentials of Ag NCs with different polymer layers were monitored (Figure S1 in the Supporting Information). Charge reversal was observed for each new layer adsorption, indicating successful assembly of the oppositely charged polyelectrolytes. The optical properties of Ag NCs with varying bilayers were also examined. During the multilayer assembly, a single polymer chain may be adsorbed on two or more Ag NCs, leading to some degree of aggregation. This can be indicated as a red-shift of the major SPR peak and increased absorption at the longer wavelength. The aggregation is minimized by reducing Ag NC concentration, and increasing the homogeneity of colloids by performing the assembling process under sonication. Figure 2 shows the normalized extinction spectra of
Figure 2. Extinction spectra of Ag NCs with 0−3 bilayers.
various colloidal dispersions. As the number of bilayers increases, the major SPR peak remains nearly unchanged and only slightly broadens, indicating little degree of aggregation. From the TEM images in Figure 3, the average polymer layer thicknesses of Ag NCs with 0−3 bilayers are measured to be 1.5, 3, 5.2, and 7.5 nm, respectively. Ag NCs are initially stabilized by PVP, thus accounting for the 1.5 nm thickness before polymer assembly. There is a gradual thickness buildup with the increased numbers of bilayers. The thickness increment for the first bilayer appears smaller than the subsequent bilayers. This is due to the relatively small surface charge of Ag NCs that results in adsorption of less amount of PDDA to achieve saturation. The thickness increment for each bilayer from the TEM image is slightly smaller than that determined by elipsometry, which is 3 nm.44 The encapsulating polymer layers may look thinner under TEM than their real thickness due to polymer chain collapse during drying process and viewing in the high vacuum environment. PFVBT Assembly and Distance-Dependent PL Enhancement Factors. Because the optical properties of PFVBT in the solution are different from those on the surface, the fluorescence of PFVBT adsorbed on Ag NCs was compared to that on the nonmetal nanoparticle to evaluate the MEF of Ag NCs. Silica nanoparticles (SNPs) with a diameter of 100 nm were chosen as the reference particles. They are negatively charged with a zeta potential of −50.2 mV due to the presence
Figure 1. (A) Normalized absorption (blue) and PL (red) spectra of PFVBT in 50 mM NaCl solution upon excitation at 490 nm. (B) Normalized absorption spectrum of PFVBT and extinction spectrum of Ag NCs in 50 mM NaCl solution. (C) TEM image of Ag NCs. Inset shows the FESEM image of Ag NCs (scale bar: 100 nm).
its dipolar imide group.41 This enables electrostatic interaction between the PVP layer and PDDA polycation. During the selfassembly, 600 μL of Ag NCs (3.7 mM) was added dropwise to 750 μL of PDDA (10 mM) in 50 mM NaCl solution under sonication. The presence of NaCl can screen the charge of polyelectrolyte and reduce electrostatic repulsions, leading to thicker multilayer buildups.42,43 The mixture was sonicated for 5 min and further shaken for 10 min to allow for effective adsorption. The excess free polymer in the supernatant was removed by centrifugation. The particles were redispersed in water by sonication after each removal of the supernatant to prevent aggregation. The above procedures were repeated for adsorption of PSS. Similarly, the second and third bilayers were assembled onto Ag-1bl NCs by consecutive adsorption of 11305
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Figure 3. TEM images of Ag NCs with 0 (A), 1 (B), 2 (C), and 3 bilayers (D).
Figure 4. PL spectra of the same amount of PFVBT loaded on Ag NCs with 0bl, 1bl, 2bl, and 3bl and SNPs, respectively, after subtraction of the respective NP background (excitation at 490 nm).
of surface silanol and siloxane groups, which are ready to adsorb the positively charged PFVBT. To study the distancedependent fluorescence enhancement effect of Ag NCs, Ag NCs with 0−3 bilayers as well as SNPs were assembled with PFVBT through electrostatic adsorption. All four types of Ag NC colloids were controlled to have a similar adsorption efficiency of ∼30% with the same amount of PFVBT. The amount of SNPs was adjusted to adsorb the same amount of polymer per unit surface area as that of Ag NCs. The purified Ag NCs with different bilayers and SNPs were redispersed in 1 mL of water for PL measurement. Fluorescence enhancement factor is defined as FEF =
PL peak height of AgNC-PFVBT PL peak height of SNP-PFVBT
By comparison of the PL intensity of PFVBT-loaded Ag NCs and SNPs (Figure 4), the FEFs are calculated to be 8.9, 11.0, 13.1, and 12.4 for Ag NCs with 0bl, 1bl, 2bl, and 3bl, respectively, with respect to the reference. To explore the origin of MEF, the fluorescence decay of PFVBT-loaded SNPs and Ag NCs with different numbers of bilayers as well as unbound PFVBT was investigated. As shown in Figure 5, the fluorescence of PFVBT on Ag NCs decays much faster than unbound PFVBT and that on SNPs, leading to shortened amplitude weighted average fluorescence lifetimes from 130 ps for unbound polymer and 210 ps for polymer on SNPs to 20, 50, 80, and 80 ps for polymer on Ag NCs with zero, one, two, and three bilayers, respectively. The shortened lifetime resulted from the interaction of the excited polymer and the Ag NCs through modification of decay rate, which gives rise to modified fluorescence quantum yields.45 In addition, the lifetimes increase slightly with the number of bilayers, indicating a reduced plasmon coupling effect at larger metal−fluorophore distance. The fluorescence decay rates and quantum yields of various samples were studied on the basis of measured lifetimes. The detailed calculation and simulation are provided in the Supporting Information. Table 1 summarizes the lifetimes and fluorescence quantum yields of PFVBT in unbound and bound forms. The low quantum yield of PFVBT in water (0.005) is attributed to charge transfer characteristics of the
Figure 5. Fluorescence decay of PFVBT loaded SNPs and Ag NCs and unbound polymer in water solution. Instrument response function (IRF) is also indicated.
Table 1. Summary of Lifetimes (τ) and Quantum Yields (QY) of PFVBT Samples sample
τ (ps)
QY
PFVBT SNP-PFVBT Ag-0bl-PFVBT Ag-1bl-PFVBT Ag-2bl-PFVBT Ag-3bl-PFVBT
130 210 20 50 80 80
0.005 0.04 0.16 0.47 0.59 0.61
polymer excited states, which leads to quenched fluorescence in polar media.32 The quantum yield of PFVBT assembled on SNPs should be equal to that of the polymer in solid film (0.04) as they are both in aggregated states. Previous studies have revealed that both radiative and nonradiative decay rates of a fluorophore near metal NP show distance depend11306
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Figure 6. CLSM images of MCF-7 breast cancer cells incubated with PFVBT-loaded Ag-2bl sample (A), PFVBT-loaded SNPs sample (B) at PFVBT concentration of 125 nM, and that without sample incubation (C). Excitation of PFVBT at 543 nm (1.5% laser power) and collection of fluorescence with BA560IF filter.
ence,46−48 which can be used to monitor the fluorescence quantum yield as a function of metal−fluorophore distance. Hence, a simplified model (shown in the Supporting Information) was proposed to calculate the quantum yields of PFVBT on Ag NCs with different bilayers. It shows that the quantum yield of the polymer is greatly enhanced by the Ag NCs, mainly attributed to the increasing radiative decay rate.29 This observation provides evidence for the enhanced fluorescence due to increased emission. As the metal−polymer distance decreases, nonradiative decay rate increases along with radiative decay rate, but at a much higher speed as most energy is dissipated into metal, leading to a quenched emission at short distances,46 which is also witnessed in Figure S2 in the Supporting Information. As expected, the quantum yield of polymers on metal with fewer bilayers is smaller than those with more bilayers. There is another factor that contributes to the mechanism of MEF, which is the enhanced absorption due to electric field enhancement. The effect of electric field can be verified by monitoring the MEF of polymer on Ag NCs with different excitation wavelengths. Metal NPs can interact strongly with the incident light to create localized electric field around them, which in turn enhances the absorption of a fluorophore in close proximity.45 FEFs were evaluated with a series of wavelengths (490, 500, 510, 520, 530, and 540 nm) for PFVBT-loaded Ag2bl colloids (Figure S3 in the Supporting Information). Results indicate that the maximum enhancement of 13.8 is observed at ∼500 nm, close to the λmax of Ag-2bl NPs. This can be understood in that the strongest electric field is generated when the metal colloid is in resonance with the incident light, leading to the largest absorption enhancement. As the electric field is inversely proportional to the metal−fluorophore distance,45 the absorption enhancement displays decreasing profile with the increase of metal−fluorophore distance; thus the combination of quantum yield modification and absorption enhancement gives rise to the highest fluorescence enhancement at an optimal distance. In our system, the highest FEF is achieved for Ag NCs with 2 bilayers that correspond to a spacer distance of 5.2 nm as measured from TEM images, in accordance with both theoretical simulations and previous reports.26,27,31 Application of PFVBT-Loaded Ag NCs in FR/NIR Fluorescence Imaging. To demonstrate the applicability of PFVBT-loaded Ag NCs in FR/NIR cellular imaging, we applied
the AgNC-PFVBT to visualize MCF-7 breast cancer cells. The cell imaging of PFVBT-loaded Ag-2bl was investigated due to its high fluorescence, and PFVBT-loaded SNPs were used as the control. Both NPs were incubated in MCF-7 cells for 2 h at the same PFVBT concentration of 125 nM. The cells were then fixed, and their nuclei were stained with DAPI. The confocal laser scanning microscopy (CLSM) images of the samples are shown in Figure 6. The images were collected above 560 nm upon excitation at 543 nm. Both images (Figure 6A and B) show red fluorescence in the cytoplasm around nuclei, indicating that the PFVBT-loaded NPs are internalized by cells. The cells stained with PFVBT-loaded Ag NC-2bl sample (Figure 6A) appear much brighter than those stained with PFVBT-loaded SNPs (Figure 6B). Given the similar uptake efficiency (∼40%) for both, the brighter fluorescence of PFVBT-loaded Ag-2bl sample is mainly attributed to the metal-enhanced fluorescence. Cytotoxicity of PFVBT-Loaded NPs. The cytotoxicity of PFVBT-loaded Ag-2bl is evaluated by the MTT viability assay of MCF-7 cells after incubation with the NCs at various polymer concentrations for 24 h. Figure 7 shows the viability of cells incubated with PFVBT-loaded Ag NCs and free PFVBT at polymer concentrations of 125, 250, and 500 nM after 24 h,
Figure 7. Metabolic viability of MCF-7 cells after 24 h incubation with PFVBT-loaded Ag NCs and free PFVBT at polymer concentrations of 125, 250, and 500 nM, respectively. 11307
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respectively. The PFVBT-loaded Ag NCs have high viability at low polymer concentrations, and the viability slowly drops to a moderate value as the concentration increases. In comparison, pure PFVBT polymer shows little cytotoxicity even at the high concentration. The concentration-dependent toxicity is thus proposed to be mainly caused by incubation of NPs in high dosages, which may retard cell proliferation and damage cell membranes.49 To mitigate this effect, the fluorescent AgNCPFVBT in our system can be formulated with higher polymer loading to achieve the same brightness with lower dosages of NPs.
CONCLUSION We have successfully demonstrated the application of a cationic conjugated polyelectrolyte PFVBT for FR/NIR cell imaging utilizing silver nanocube enhanced fluorescence. A spacer layer is built up by layer-by-layer self-assembly of alternately charged polyelectrolytes to control the metal−polymer distance. The fluorescence of PFVBT is greatly enhanced in the vicinity of Ag NCs due to metal-enhanced fluorescence. This leads to an FEF of 13.8 at optimum metal−polymer distance of ∼6 nm at the excitation wavelength close to the Ag NC extinction maximum. The mechanism of MEF has been revealed to be attributed to both local field enhancement and plasmon coupling, which ensues enhanced absorption and emission. When PFVBTloaded Ag NCs are used for MCF-7 cell imaging, the cytoplasm stained with the fluorescent NPs displayed much higher brightness as compared to the control and showed low cytotoxicity. This strategy has provided an effective and versatile alternative to amplify CPE fluorescence for application in cell imaging. In future, the size and morphology of metal nanostructure can be tuned to have the proper plasmonic properties to interact with other CPEs in proximity. Further functionalization of the fluorescent NCs with biomolecule targeting groups will make them suitable for targeted imaging applications. ASSOCIATED CONTENT
S Supporting Information *
Details of the Ag NCs concentration calculation, zeta potential of Ag NCs with varying numbers of polymer layers, fluorescence quantum yield and decay rate calculation and simulation of PFVBT, and fluorescence enhancement factors of Ag NC-2bl at different excitation wavelengths. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
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[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the TDSI (R-279-000-305-592, R-279-000305-422, and R-279-000-305-232), Singapore Ministry of Defence (R279-000-301-232), National University of Singapore (R279-000-301-646), and Singapore National Research Foundation (R-279-000-323-281) for financial support. J.L. thanks the National University of Singapore for support via a research scholarship. 11308
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