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gelatin nanocomposites with enhanced fluorescence and Raman scattering
Colloids and Surfaces A: Physicochem. Eng. Aspects 514 (2017) 117–125
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Synthesis and application of bifunctional gold/gelatin nanocomposites with enhanced fluorescence and Raman scattering Qian Cao 1 , Xiaoyu Wang 1 , Qianling Cui ∗ , Yu Yang, Lidong Li ∗ School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, PR China
g r a p h i c a l
a b s t r a c t
h i g h l i g h t s • A bifunctional gold/gelatin nanocomposite was fabricated with core-shell structure. • Enhanced fluorescence and Raman scattering signals were observed in this particle. • It shows good performance for fluorescence and SERS imaging of HeLa cells.
a r t i c l e
i n f o
Article history: Received 12 July 2016 Received in revised form 18 November 2016 Accepted 27 November 2016 Available online 28 November 2016 Keywords: Fluorescence Cell imaging Gold nanoparticles Metal-enhanced fluorescence Surface-enhanced Raman scattering
a b s t r a c t In recent decades, the preparation of multi-functional nanoplatforms that integrate two or more abilities into a single nanocomposite has attracted increasing interest and potential applications in many fields. Here, we report a novel hybrid core-shell structured nanocomposite integrating the capabilities of metal-enhanced fluorescence (MEF) and surface-enhanced Raman scattering (SERS). The nanocomposite consists of porous gold nanoparticle as a magnifying material for both MEF and SERS, 4-aminothiophenol (ATP) molecule as Raman probe, gold nanocluster as the fluorescent emitter, and gelatin as the protecting outer shell. In this nanocomposite, the fluorescence intensity of the gold nanoclusters was enhanced by about four times, while the Raman signal of the ATP molecules was increased by 4 orders of magnitude. Additionally, the nanocomposite shows low cytotoxicity and good performance for fluorescence and SERS imaging of HeLa cells. © 2016 Elsevier B.V. All rights reserved.
∗ Corresponding authors. E-mail addresses: [email protected] (Q. Cui), [email protected] (L. Li). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.colsurfa.2016.11.057 0927-7757/© 2016 Elsevier B.V. All rights reserved.
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1. Introduction The development of multifunctional core-shell nanoparticles that integrate individual functions into a single system has become one of the most important developing trends for many nanotechnology fields [1,2]. Combining the superior advantages of each function, possible synergistic effects, and increased suitability for complex biological environments, multifunctional nanoparticles have been extensively studied and applied in biological fields including drug delivery, biosensing, bioimaging, and theranostic [3–6]. Among them, gold nanoparticles (AuNPs) are excellent scaffolds for the fabrication of hybrid nanocomposites and have attracted particularly intense interest because of their distinct physical and optical properties [7–13]. These include localized surface plasmon resonance, surface-enhanced Raman scattering (SERS), metal-enhanced fluorescence (MEF), catalytic ability, photothermal effects, aggregation-induced colorimetric effects, and nonlinear optical properties [14–20]. In recent years, SERS technology has attracted much research interest and has thus developed very rapidly. Compared to other analysis methods, SERS shows superior advantages, such as offering fingerprint vibrational information on analytes, non-destructive analysis, and less interference in aqueous environments. More importantly, the acquired enormous enhancement factor of SERS, which is up to 1014 , makes it possible to make detections at the level of single molecule or nanoparticle [21]. Consequently, SERS has already become a powerful spectroscopic technique for chemical and biological analysis with high sensitivity and selectivity [22–26]. While its application to cellular imaging has also attracted some interest [27,28], a limited imaging resolution and long measurement time are still barriers to its practical use for cellular imaging. Apart from the Raman scattering signal, the fluorescence emission of a fluorophore can also be enhanced if it is placed in close proximity to plasmonic nanostructures, caused by interaction with the resulting enhanced electromagnetic field. This phenomenon is known as MEF [29,30]. Because fluorescence techniques are widely used for labeling, sensing, and imaging in many fields, the design of novel MEF-based fluorescent nanocomposites with enhanced emission properties shows specific meanings for practical application. To date, several different types of fluorescent species have been reported to be suitable for constructing MEF-based nanocomposites, including organic fluorophores [31,32], conjugated polymers [33,34], semiconductor quantum dots [35,36], and lanthanide-based upconversion nanoparticles [37,38], the fluorescence of which were enhanced by nearby plasmonic nanostructures. In recent years, sub-nanometer-sized metal nanoclusters have emerged as novel promising fluorescent emitters, and have attracted enormous attention owing to their good photostability and size-dependent photoluminescence from the near infrared to ultraviolet regions [39–42]. However, their use as a component of MEF-based fluorescent nanocomposites has rarely been reported. To further improve the sensitivity of spectroscopic investigations, it is highly desirable to integrate MEF and SERS effects in a single plasmonic nanoparticle [43,44]. To maximally realize MEF and SERS, it is better to choose AuNPs with anisotropic shapes, which would have abundant hot spots around their edges or corners where the localized electromagnetic field would be significantly concentrated. It has been demonstrated that both MEF and SERS show a strong distance dependency, but the optimal distance for each is quite different. SERS effects decrease exponentially with increasing distance away from the plasmonic surfaces, and thus the Raman reporters must be very close to the metallic surface [45]. In contrast, fluorescence quenching often occurs if the fluorophore is placed close to the metallic surface, and only the fluorescence signals of fluorophores at certain distances can be enhanced [46,47]. Thus, to design such a dual-modal plasmonic platform, it is crucial
to find a suitable assembly method to place the Raman/fluorescent probes at reasonable distances from the metal nanoparticles. Additionally, an ideal fluorescent nanocomposite always requires good reproducibility and stability. Therefore, a shell layer is always needed to be coated on the Raman reporter and fluorescent emitterlabeled metallic nanoparticles to protect the probes and improve their dispersability and stability. In this work, we fabricated a novel plasmonic platform providing dual-modal enhanced spectroscopic properties via SERS and MEF. As shown in Scheme 1, the proposed bifunctional nanocomposite was composed of four components. The inner core was a gold nanoparticle, which served as the magnifying substrate for both MEF and SERS. Gold nanoclusters and 4-aminothiophenol (ATP) were selected as the fluorescent species and Raman reporter molecule, respectively. Finally, gelatin biomacromolecules were coated on the outside of the shell, to ensure the stability and biocompatibility of the nanocomposite. As expected, both the fluorescence and Raman signals of the nanocomposite were found to be enhanced, by factors of 4 and 3.1 × 104 , respectively. Finally, the good fluorescence and SERS imaging performance of the nanocomposite was demonstrated by co-culture with HeLa cells.
2. Experimental 2.1. Materials Gelatin from cold water fish skin, chloroauric acid tetrahydrate (HAuCl4 ·4H2 O), 4-aminothiophenol (ATP), glutathione (GSH), l-ascorbic acid (AA), (1-hexadecyl)-trimethylammonium chloride (CTAC), glutaraldehyde, sodium borohydride (NaBH4 ), and phosphate buffered saline (PBS) tablet were purchased from SigmaAldrich. All chemical reagents were used as received without further purification. Ultrapure Millipore water (18.6 M) was used throughout the experiments.
2.2. Synthesis of porous gold nanoparticles (AuNPs) Porous AuNPs were prepared by a seed-mediated growth method according to a procedure reported in the literature [48]. First, gold nanoseeds were made by the addition of a freshly prepared, ice-cold aqueous NaBH4 solution (10 mM, 0.30 mL) to an aqueous mixture of HAuCl4 (10 mM, 0.25 mL) and CTAC (0.1 M, 10 mL) under magnetic stirring. The seed solution was vigorously stirred for 1 min and kept at room temperature for 2 h, after which it was diluted 1000-fold with 0.1 M CTAC and used in the following growth process. The growth solution was prepared by mixing aqueous solutions of HAuCl4 (10 mM, 0.5 mL), CTAC (0.1 M, 10 mL), and AA (0.1 M, 0.1 mL). After gently mixing the growth solution for 30 s, 0.02 mL of the diluted Au seed solution was added. The reaction solution was immediately gently mixed for another 30 s and then left undisturbed at room temperature for at least 4 h. The as-obtained AuNPs were washed with water three times by centrifugation/redispersion, and finally redispersed in 5 mL of water.
2.3. Synthesis of gold nanoclusters (AuNCs) AuNCs were synthesized according to a previously reported method [49]. In brief, 1 mL of 20 mM HAuCl4 was added to 8.7 mL of water followed by addition of GSH solution (9.2 mg in 0.3 mL of water). The mixture was gently stirred for 5 min at room temperature, and then stirred at 70 ◦ C for 24 h. The AuNCs were obtained as a yellow solution.
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2.4. Preparation of nanocomposites One milliliter of as-prepared AuNPs was mixed with 10 L of ATP solution (0.01 M in ethanol), and sonicated for 5 min. Then, the free ATP molecules were removed through centrifugation, and the particles were dispersed in 1 mL of as-obtained AuNCs solution. The mixture was sonicated for another 5 min, and added to a preheated aqueous gelatin solution (50 mg in 9 mL of H2 O) at 50 ◦ C under vigorous stirring. The mixture was stirred at 50 ◦ C for 30 min, after which its pH was adjusted to ∼3.0 using 1 M HCl. Then, under constant stirring at 40 ◦ C, 30 mL of acetone was added dropwise over 15 min. Next, 50 L of 25% glutaraldehyde was admixed with the stirring mixture, which was stirred at 40 ◦ C for a further 1 h, followed by overnight incubation at room temperature. Finally, the NPs were collected by centrifugation, washed with water twice, and finally dispersed in 1 mL of H2 O. 2.5. Raman measurements Raman measurements were performed with a Renishaw Invia Raman microscope equipped with a laser excitation wavelength of 633 nm (He-Ne laser), and the laser power was controlled at 0.5 mW. All Raman spectra were acquired using a 50× objective. All SERS spectra reported here were the results of a single 10 s accumulation. The diameter of the laser spot area was about 1 m, and the penetration depth was about 25 m. The equipped charge-coupled device (CCD) camera was coupled to a spectrograph that in combination provided a 2 cm−1 spectral resolution. The silicon wafer was used as the substrate and its characteristic Raman peak at 520 cm−1 was employed to calibrate the Raman spectra. 2.6. Confocal Raman imaging Raman images were obtained using the Raman point-mapping method on a Renishaw Invia Raman microscope equipped under 633 nm excitation (He-Ne laser). Microscopy image and Raman image were acquired using a 50× objective. The laser power was set at 1 mW, and 3 s accumulation time was used for a single Raman spectra. A computer-controlled x-y and x-y translation stage was used in a 1 m setup. A Raman mapping experiment was carried out by measuring the Raman spectrum centered at 1300 cm−1 (∼600 nm bandwidth) at each pixel over the 2D area of a HeLa cell. Then, a Raman image was obtained using the highest peak intensity at 1580 cm−1 to create a color map to depict intensity variation. 2.7. Raman enhancement factor (EF) calculation The Raman band at 1580 cm−1 was chosen to estimate the SERS enhancement factor (EF), according to the definition EF = (ISERS /Nads )/(Ibulk /Nbulk ), where ISERS and Ibulk are the Raman intensity at 1580 cm−1 , and Nads and Nbulk are the numbers of adsorbed and solid ATP molecules in the laser illumination volume, respectively. For ATP solids, the diameter of the laser spot area was about 1 m, and the penetration depth was about 25 m. Nbulk was calculated as about 9.41 × 1010 molecules. We assumed that the ATP molecules formed one dense monolayer on the surface of the gold nanoparticles, and it is reported that each ATP molecule occupies 0.2 nm2 [50,51]. Therefore, the number of ATP molecules adsorbed in the laser illumination volume was calculated to be 3.925 × 106 . 2.8. Cytotoxicity assay by MTT method The cytotoxicity of the NPs was studied using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay. HeLa cervical carcinoma cells were grown in
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Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). The cells were seeded into 96-well plates at a density of 1 × 104 cells per well. After 24 h of incubation at 37 ◦ C in a humidified 5% CO2 atmosphere, the cells were treated with different amounts of nanocomposite dispersions (10 L, 20 L, 40 L, 60 L, 80 L, 100 L), and cultured for another 24 h. After pouring out the medium, 100 L of freshly prepared MTT (1 mg mL−1 in 10 mM PBS) was added to each well and incubated for 4 h. After removing the MTT medium solution, the cells were lysed by adding 100 L of DMSO. The plate was gently shaken for 5 min, and then the absorbance of purple formazan at 570 nm was monitored using a Spectra MAX 340PC plate reader. 2.9. Cellular imaging experiments 40 L of nanocomposite dispersion was added to 1 mL of DMEM medium containing HeLa cells in a 35 × 35 mm plate. The plate was then incubated for 10 h at 37 ◦ C in a humidified environment containing 5% CO2 . Then, the culture media was removed and the cells were washed with PBS buffer (10 mM, pH = 7.4) several times. Fluorescence images and phase contrast bright-field images of the cells were recorded on a confocal fluorescence microscope (Olympus FV1000-IX81). To observe the fluorescence signals of the nanocomposites, the excitation wavelength was set at 488 nm and the emission was collected in the range of 550–650 nm. 2.10. Characterization The size distributions and zeta-potentials of the colloidal suspensions were measured by dynamic light scattering with a Malvern Zetasizer Nano ZS90. The morphology of the nanoparticles was characterized by transmission electron microscope (TEM; Hitachi H-7650). Ultraviolet-visible absorption spectra were measured on a Hitachi U3900 spectrophotometer. The fluorescence spectra of the samples were obtained using a Hitachi F-7000 spectrometer. Fluorescence images were recorded using an Olympus FV1000-IX81 confocal laser scanning microscope through a 100× objective lens, with 330–380 nm excitation produced by a 100 W mercury lamp light source. 3. Results and discussion The fabrication procedure of the bifunctional nanocomposites is schematically illustrated in Scheme 1. The core-shell structure of the nanocomposites consisted of four components: an AuNP, ATP molecules, AuNCs, and gelatin, which acted as the magnifying substrate, Raman probe, fluorescent species, and the protective outer shell, respectively. Both the Raman signals of the ATP molecules and the emission properties of the AuNCs were expected to be enhanced by the inner porous AuNP. Porous AuNPs were chosen as the inner core and enhancing material owing to their superior properties. First, their nanoscale channels or pores and large surface roughness would provide sufficient space for adsorbing a large amount of organic molecules or small particles. Second, the highly concentrated electromagnetic field at the abundant hot spots formed in the pores and channels were expected to generate both higher MEF and SERS activity compared with those obtainable with solid spherical shapes. The porous AuNPs used in this work were synthesized using a seed-mediated growth method reported in a previous work [48]. In the synthesis process, (1-hexadecyl)-trimethylammonium chloride (CTAC) was used as the surfactant to facilitate and stabilize the growth of the nanoparticles, and was mostly removed by subsequent centrifugation. Zeta potential measurements of the obtained porous AuNP dispersion revealed that the surfaces of the AuNPs were highly positively charged, at about +40 mV, owing to their full coverage
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Scheme 1. Schematic illustration of the preparation of the hybrid nanocomposites.
Fig. 1. (a) TEM image of synthesized porous AuNPs. (b) Magnified TEM image of individual porous AuNP. (c) TEM image of obtained nanocomposites and (d) gold nanoclusters in the gelatin layer of the nanocomposites.
of CTAC molecules. The transmission electron microscopy (TEM) image shown in Fig. 1a and the representative magnified TEM image of an individual particle shown in Fig. 1b demonstrate the highly porous structure of the AuNPs. It can be seen clearly that the AuNPs had an average size of around 60 nm, with nanoscale pores and channels randomly distributed over their surfaces. ATP is widely used as a Raman probe owing to its simple chemical structure and distinct Raman bands [44,52–54]. More importantly, it can tightly bind to gold surfaces through the strong Au-S affinity. As a benefit of their small size, ATP molecules were able to easily access the pores and channels of the AuNPs, and their Raman signals were thus expected to be enhanced enormously. Furthermore, having the ATP molecules in the pores of the AuNPs also kept them from being removed during the following modification and centrifugation procedures, and enhanced the reproducibility and stability of the Raman signals. Glutathione (GSH) capped AuNCs were fabricated as the fluorescent emitters according to a previously reported procedure [55].
Their mean diameter was 2–3 nm, as shown by the TEM image in Fig. 1d. The negative charge of the GSH capping agent meant that the AuNCs could easily attach to the positively charged surface of the porous AuNPs via electrostatic attraction. Furthermore, their extremely small size also made them easily access to the nanoscale channels or pores of the AuNPs. Of course, not all of the AuNCs could tightly bind with the larger nanoparticles, and most of them were dispersed more freely. After the addition of the gelatin solution, the free GSH-capped AuNCs bound with gelatin macromolecules, driven by their similar functional groups and electrostatic attraction. Finally, glutaraldehyde was used as a cross-linker between the amino groups of both the gelatin and the GSH molecules. Accordingly, the free AuNCs were fixed in the formed gelatin shell, as demonstrated by the TEM image of the obtained nanocomposites shown in Fig. 1c. Taking advantage of its excellent biocompatibility and abundant functional groups, gelatin, a type of thermally and hydrolytically denatured collagen, was used as the protecting shell. It has been
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Fig. 2. (a) Variation in the zeta potential of the obtained nanoparticles during the preparation process. (b) Size distribution of the porous AuNPs and nanocomposites in aqueous dispersion.
demonstrated that gelatin can easily be coated onto nanoparticles via a thermally induced, sol-gel phase transition [33,56]. Here, gelatin was employed as the outer shell to maintain the stability of the nanocomposites and prevent both desorption of the Raman reporters and AuNCs as well as adsorption of external species. It is worth noting that the gelatin shell was found to be composed of two layers. The inner shell was much darker than the outer shell, because many AuNCs were located in this region, as shown in Fig. 1d. From this TEM image, it can be seen that the 2–3 nm AuNCs were dispersed uniformly in this gelatin layer. To monitor the assembly process, the variation in surface charge of each stage of the nanocomposite fabrication was determined by zeta potential measurements, as shown in Fig. 2a. After the addition of ATP molecules, the zeta potential of the AuNPs increased from +40 mV to +50 mV, indicating the replacement of CTAC with ATP molecules because of the strong affinity of thiol group towards the gold surface. Once the small AuNCs were added, the zeta potential converted from a positive to a negative value, owing to the excess carboxylic acids groups of the GSH capping agent. This complete charge inversion implies that the AuNCs indeed obtained access to the AuNP-ATP surfaces as a result of electrostatic attraction and adsorption owing to their small size. After coating with the gelatin layer, the abundant amino groups from the arginine and lysine of the gelatin converted the surface charge to positive again. The following crosslinking reaction using glutaraldehyde as the cross-linker consumed most of the amino groups, leaving carboxylic acid groups (from aspartic acid and glutamic acid) and
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Fig. 3. (a) Ultraviolet-visible absorption spectra of AuNCs (black curve), porous AuNPs (red curve), and nanocomposites (blue curve) dispersed in water. (b) Fluorescence spectra of aqueous dispersions of AuNCs (black curve) and nanocomposites (red curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
subsequently a negative surface. These obvious changes in surface charge strongly demonstrated the successful assembly of each layer formed during the preparation process. The mean size of the bare AuNPs and nanocomposites determined by dynamic light scattering (Fig. 2b) was 60 nm and about 200 nm, respectively, in accordance with the TEM results. The observed narrow size distribution without any appearance of large aggregates also reflected the uniform dispersability of the nanocomposites in aqueous solution. These nanocomposites were dispersed in water and stored at 4 ◦ C, keeping its blue transparent appearance during one-month storage. They can also keep good stability in PBS buffer (10 mM, pH = 7.4) at least for two weeks, which is favorable for biological application. The optical properties of the as-obtained nanocomposites were determined by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy, as displayed in Fig. 3a and b, respectively. The bare AuNP dispersion showed a strong surface plasmon resonance band that covered a broad range from 520 nm to 800 nm with a center at around 633 nm. No obvious plasmon resonance peak was observed in the absorption spectrum of the AuNC dispersion except for a small shoulder peak at around 420 nm, owing to its small particle size. Compared with that of the bare AuNPs, the absorption band of the nanocomposites was lower and broader, with a 10-nm blue shift of the main peak ascribed to the change in the surrounding environment. The fluorescence emission spectra
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of the AuNCs and nanocomposites were also recorded in aqueous dispersion, as shown in Fig. 3b. Compared with that of the discrete AuNCs, the emission intensity of the AuNCs in the nanocomposites was obviously enhanced by a factor of about 4, caused by the magnifying effect of the inner porous AuNPs. It should be noted that only the AuNCs located at a suitable distance from the inner metallic core could be affected by the enhanced electromagnetic field of the porous AuNPs. The measured fluorescence intensity represents the combined signal from all AuNCs present, i.e., those that were quenched, those that were enhanced, and those that were unaffected by interaction with the plasmonic field. Therefore, we believe that the real MEF enhancement factor of the AuNCs at the optimal distance was much higher than the obtained value. The nanocomposites were observed using a fluorescence microscope under ultraviolet light excitation produced by a mercury lamp, and the captured fluorescent image is displayed in Fig. 4. It can be seen that the nanocomposites dispersed uniformly in water, and produced a bright yellow-orange emission. This demonstrates that the present hybrid nanocomposite would be an ideal imaging agent for cellular applications. To confirm the SERS ability of the nanocomposite, Raman spectra were recorded after each stage of the assembly process using a confocal Raman microscope. The 633 nm He-Ne laser was used as the excitation light source, because of its well overlap with the plasmon resonance peak of the nanocomposites. The measurement parameters used to collect Raman signals from each sample were kept constant throughout Fig. 5. Here, all the displayed Raman spectra only show the relevant range of 1000–1700 cm−1 . The spectrum of solid ATP (Fig. 5a) shows several characteristic Raman peaks assignable to the C H bending (1090 cm−1 , 1172 cm−1 ) and C C stretching (1596 cm−1 ) of the phenyl ring. On the bare AuNPs, the
Fig. 4. Fluorescence microscopy image of the hybrid nanocomposites in solution obtained under ultraviolet excitation produced by a mercury lamp.
Raman signals of the ATP molecules were enhanced enormously, by a factor of 4.4 × 105 (Fig. 5b). It is worth noting that the strong new bands that appeared at 1386 and 1429 cm−1 were attributed to the formation of 4,4 -dimercaptoazobenzene (DMAB), which is the product of the photocatalyzed dimerization reaction of ATP molecules [57,58]. This side reaction always takes place on bare SERS-active surfaces when the ATP molecules are in direct contact with the plasmonic substrate. On the other hand, the Raman band caused by C C stretching also shifted from 1596 to 1580 cm−1 , which might be caused by the large variation in environment from
Fig. 5. (a) Raman spectrum of solid ATP. (b) SERS spectrum of ATP on bare porous AuNPs. (c) SERS spectrum of ATP on porous AuNPs in the presence of AuNCs. (d) SERS spectrum of ATP in the final hybrid nanocomposites.
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solid state to plasmonic surface in colloids. After the addition of the small AuNCs, the Raman enhancement factor was reduced to 2.5 × 105 (Fig. 5c). In the final hybrid nanocomposites, the enhancement factor was further decreased to 3.1 × 104 , but the Raman signal of the characteristic peaks was still strong enough to be identifiable (Fig. 5d). Interestingly, the strong characteristic Raman band of the DMAB side products was nearly invisible, suggesting that the unwanted photocatalyzed reaction was almost completely avoided in the hybrid nanocomposites. It should be noted that in the final nanocomposite, the emission intensity of the AuNCs was enhanced, while the Raman enhancement factor of ATP was reduced. This phenomenon is reasonable because the optimal distance for MEF and SERS is different. Both of the MEF and SERS effects show a close dependency on distance between the probe and metallic surface [46,47,59,60,61]. However, the Raman enhancement factor achieves the highest value when the probes get close to the metallic surface, and reduce quickly with distance as the electromagnetic field enhancement decays nearly exponentially. On the other hand, fluorescence would be quenched if the probe is attached on the metallic surface, and only be enhanced in a suitable distance (5–30 nm). In this case, the average emission intensity of AuNCs was enhanced in the final composites, because some of them located at a suitable region towards gold surface. Nevertheless, compared to that on bare porous AuNPs, the SERS enhancement factor reduced in the final nanocomposites, due to the loss and migration of some ATP molecules far away from the inner core in the assembly process. Besides, the reduc-
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Fig. 6. Cell viability results after incubation of HeLa cells with various volumes of nanocomposite dispersion.
tion in laser power caused by the presence of gelatin shell was also responsible for the decrease in SERS efficiency. Considering its gelatin shell, the present hybrid nanocomposites were expected to have good biocompatibility. Before investigation of its practical application in living cells, the cytotoxicity of the nanocomposites was evaluated using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Fig. 7. (a) Fluorescence image of HeLa cells without treatment of nanocomposites. (b) Fluorescence image and (c) bright-field image of HeLa cells co-cultured with nanocomposites for 10 h at 37 ◦ C. (d) Overlapped image of (b) and (c). The scale bars represent 20 m.
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bromide (MTT) cell-viability assay. The degree of activation of the cells can be reflected by the absorbance of MTT at 570 nm. The cell viability is defined by the ratio of the absorbance of cells incubated with nanocomposite dispersion to that of untreated control cells. Here, the cell viability was calculated using HeLa cells incubated for 24 h with various amounts of nanocomposite dispersion (10–100 L). As shown in Fig. 6, more than 77% of the cells remained alive in the tested concentration range of the nanocomposites, indicating their low cytotoxicity. The above results prove that this new nanocomposite is safe to be used in living cells. Taking advantage of its good emission properties and low cytotoxicity, the cell imaging performance of the nanocomposites was examined after co-culture with HeLa cells at 37 ◦ C for 10 h. After removal of the culture media and washing with PBS buffer (10 mM, pH = 7.4), the cultured cells were observed by confocal fluorescence microscope. Upon excitation by a 488 nm laser, the cells incubated with the nanocomposites exhibited a strong orange fluorescence (Fig. 7b). In contrast, the untreated control cells did not show any fluorescence signal (Fig. 7a). The overlapped image (Fig. 7d) of the fluorescence (Fig. 7b) and bright field (Fig. 7c) images shows that the fluorescence mostly surrounded the nucleolus and was located in the cytoplasm of the HeLa cells. Thus, it can be concluded that the nanocomposites were easily captured by the cells and entered the cytoplasm, which can be attributed to their biocompatible gelatin surface. These results demonstrate that the present hybrid nanocomposite is an ideal agent for imaging living cells. These HeLa cells co-cultured with nanocomposites were also placed under a confocal Raman microscope to observe their Raman signals. Fig. 8a shows a representative Raman spectrum collected from the HeLa cell, where the intense SERS characteristic signal was detected. The Raman peaks of the ATP molecules are clear and sharp with a high signal-to-noise, indicating a less interference from the cellular environment. In addition, the absence of characteristic Raman band of DMAB implies the ATP molecules were quite stable in the cell, and the unwanted side reaction was avoided. Insert in Fig. 8a is a microscopy image of a single HeLa cell, and the Raman mapping experiments were carried out on the area in red rectangle. Fig. 8b displays the false color SERS mapping image, which is calculated from the Raman spectrum at each pixel based on the strong characteristic peak of ATP at 1580 cm−1 . The cell shape and surface outline were clearly shown in the image, compared to the low background. The distribution of Raman signals across the cell revealed the well capture of nanocomposites by the cell, in consistent with the fluorescence imaging. In addition, the intense SERS signal observed in the cell indicated that this nanocomposite can serve as an ideal SERS probe for cellular imaging. 4. Conclusions In summary, we have fabricated a gold/gelatin core-shell nanocomposite integrating both MEF and SERS capabilities into a single system, in which AuNCs and ATP were employed as fluorescent and Raman probes, respectively. TEM images of the nanocomposites confirmed the successful preparation of the intended core-shell structure, with a middle gelatin shell containing a large amount of embedded AuNCs. The fluorescence intensity of the AuNCs in the nanocomposites were enhanced by about four times compared with that of the same amount of discrete AuNCs in solution, which is attributed to the MEF effect from the inner porous gold nanoparticles. Moreover, the composite also displayed an enormous enhancement of the Raman signal of ATP, which makes this composite a good candidate as a label-free Raman probe. Finally, taking advantage of its low toxicity towards living cells, the nanocomposite exhibited good fluorescence and SERS imaging performance after co-culture with HeLa cells.
Fig. 8. (a) A representative SERS spectrum of the HeLa cell incubated with the nanocomposites for 10 h at 37 ◦ C. Insert is the bright field microscopy image of a single HeLa cell. (b) SERS mapping image in false color of the single HeLa cell, created from the Raman spectrum at each pixel across the red rectangle area in Fig. 8a insert, using the Raman peak intensity at 1580 cm−1 . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Acknowledgments This work was supported by the National Natural Science Foundation of China (51503015), and the Fundamental Research Funds for the Central Universities (FRF-TP-15-003A1). The authors thank Dr. Libin Liu for giving support for the cellular experiments.
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Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Synthesis and application of bifunctional gold/gelatin nanocomposites with enhanced fluorescence and Raman scattering Qian Cao 1 , Xiaoyu Wang 1 , Qianling Cui ∗ , Yu Yang, Lidong Li ∗ School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, PR China
g r a p h i c a l
a b s t r a c t
h i g h l i g h t s • A bifunctional gold/gelatin nanocomposite was fabricated with core-shell structure. • Enhanced fluorescence and Raman scattering signals were observed in this particle. • It shows good performance for fluorescence and SERS imaging of HeLa cells.
a r t i c l e
i n f o
Article history: Received 12 July 2016 Received in revised form 18 November 2016 Accepted 27 November 2016 Available online 28 November 2016 Keywords: Fluorescence Cell imaging Gold nanoparticles Metal-enhanced fluorescence Surface-enhanced Raman scattering
a b s t r a c t In recent decades, the preparation of multi-functional nanoplatforms that integrate two or more abilities into a single nanocomposite has attracted increasing interest and potential applications in many fields. Here, we report a novel hybrid core-shell structured nanocomposite integrating the capabilities of metal-enhanced fluorescence (MEF) and surface-enhanced Raman scattering (SERS). The nanocomposite consists of porous gold nanoparticle as a magnifying material for both MEF and SERS, 4-aminothiophenol (ATP) molecule as Raman probe, gold nanocluster as the fluorescent emitter, and gelatin as the protecting outer shell. In this nanocomposite, the fluorescence intensity of the gold nanoclusters was enhanced by about four times, while the Raman signal of the ATP molecules was increased by 4 orders of magnitude. Additionally, the nanocomposite shows low cytotoxicity and good performance for fluorescence and SERS imaging of HeLa cells. © 2016 Elsevier B.V. All rights reserved.
∗ Corresponding authors. E-mail addresses: [email protected] (Q. Cui), [email protected] (L. Li). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.colsurfa.2016.11.057 0927-7757/© 2016 Elsevier B.V. All rights reserved.
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1. Introduction The development of multifunctional core-shell nanoparticles that integrate individual functions into a single system has become one of the most important developing trends for many nanotechnology fields [1,2]. Combining the superior advantages of each function, possible synergistic effects, and increased suitability for complex biological environments, multifunctional nanoparticles have been extensively studied and applied in biological fields including drug delivery, biosensing, bioimaging, and theranostic [3–6]. Among them, gold nanoparticles (AuNPs) are excellent scaffolds for the fabrication of hybrid nanocomposites and have attracted particularly intense interest because of their distinct physical and optical properties [7–13]. These include localized surface plasmon resonance, surface-enhanced Raman scattering (SERS), metal-enhanced fluorescence (MEF), catalytic ability, photothermal effects, aggregation-induced colorimetric effects, and nonlinear optical properties [14–20]. In recent years, SERS technology has attracted much research interest and has thus developed very rapidly. Compared to other analysis methods, SERS shows superior advantages, such as offering fingerprint vibrational information on analytes, non-destructive analysis, and less interference in aqueous environments. More importantly, the acquired enormous enhancement factor of SERS, which is up to 1014 , makes it possible to make detections at the level of single molecule or nanoparticle [21]. Consequently, SERS has already become a powerful spectroscopic technique for chemical and biological analysis with high sensitivity and selectivity [22–26]. While its application to cellular imaging has also attracted some interest [27,28], a limited imaging resolution and long measurement time are still barriers to its practical use for cellular imaging. Apart from the Raman scattering signal, the fluorescence emission of a fluorophore can also be enhanced if it is placed in close proximity to plasmonic nanostructures, caused by interaction with the resulting enhanced electromagnetic field. This phenomenon is known as MEF [29,30]. Because fluorescence techniques are widely used for labeling, sensing, and imaging in many fields, the design of novel MEF-based fluorescent nanocomposites with enhanced emission properties shows specific meanings for practical application. To date, several different types of fluorescent species have been reported to be suitable for constructing MEF-based nanocomposites, including organic fluorophores [31,32], conjugated polymers [33,34], semiconductor quantum dots [35,36], and lanthanide-based upconversion nanoparticles [37,38], the fluorescence of which were enhanced by nearby plasmonic nanostructures. In recent years, sub-nanometer-sized metal nanoclusters have emerged as novel promising fluorescent emitters, and have attracted enormous attention owing to their good photostability and size-dependent photoluminescence from the near infrared to ultraviolet regions [39–42]. However, their use as a component of MEF-based fluorescent nanocomposites has rarely been reported. To further improve the sensitivity of spectroscopic investigations, it is highly desirable to integrate MEF and SERS effects in a single plasmonic nanoparticle [43,44]. To maximally realize MEF and SERS, it is better to choose AuNPs with anisotropic shapes, which would have abundant hot spots around their edges or corners where the localized electromagnetic field would be significantly concentrated. It has been demonstrated that both MEF and SERS show a strong distance dependency, but the optimal distance for each is quite different. SERS effects decrease exponentially with increasing distance away from the plasmonic surfaces, and thus the Raman reporters must be very close to the metallic surface [45]. In contrast, fluorescence quenching often occurs if the fluorophore is placed close to the metallic surface, and only the fluorescence signals of fluorophores at certain distances can be enhanced [46,47]. Thus, to design such a dual-modal plasmonic platform, it is crucial
to find a suitable assembly method to place the Raman/fluorescent probes at reasonable distances from the metal nanoparticles. Additionally, an ideal fluorescent nanocomposite always requires good reproducibility and stability. Therefore, a shell layer is always needed to be coated on the Raman reporter and fluorescent emitterlabeled metallic nanoparticles to protect the probes and improve their dispersability and stability. In this work, we fabricated a novel plasmonic platform providing dual-modal enhanced spectroscopic properties via SERS and MEF. As shown in Scheme 1, the proposed bifunctional nanocomposite was composed of four components. The inner core was a gold nanoparticle, which served as the magnifying substrate for both MEF and SERS. Gold nanoclusters and 4-aminothiophenol (ATP) were selected as the fluorescent species and Raman reporter molecule, respectively. Finally, gelatin biomacromolecules were coated on the outside of the shell, to ensure the stability and biocompatibility of the nanocomposite. As expected, both the fluorescence and Raman signals of the nanocomposite were found to be enhanced, by factors of 4 and 3.1 × 104 , respectively. Finally, the good fluorescence and SERS imaging performance of the nanocomposite was demonstrated by co-culture with HeLa cells.
2. Experimental 2.1. Materials Gelatin from cold water fish skin, chloroauric acid tetrahydrate (HAuCl4 ·4H2 O), 4-aminothiophenol (ATP), glutathione (GSH), l-ascorbic acid (AA), (1-hexadecyl)-trimethylammonium chloride (CTAC), glutaraldehyde, sodium borohydride (NaBH4 ), and phosphate buffered saline (PBS) tablet were purchased from SigmaAldrich. All chemical reagents were used as received without further purification. Ultrapure Millipore water (18.6 M) was used throughout the experiments.
2.2. Synthesis of porous gold nanoparticles (AuNPs) Porous AuNPs were prepared by a seed-mediated growth method according to a procedure reported in the literature [48]. First, gold nanoseeds were made by the addition of a freshly prepared, ice-cold aqueous NaBH4 solution (10 mM, 0.30 mL) to an aqueous mixture of HAuCl4 (10 mM, 0.25 mL) and CTAC (0.1 M, 10 mL) under magnetic stirring. The seed solution was vigorously stirred for 1 min and kept at room temperature for 2 h, after which it was diluted 1000-fold with 0.1 M CTAC and used in the following growth process. The growth solution was prepared by mixing aqueous solutions of HAuCl4 (10 mM, 0.5 mL), CTAC (0.1 M, 10 mL), and AA (0.1 M, 0.1 mL). After gently mixing the growth solution for 30 s, 0.02 mL of the diluted Au seed solution was added. The reaction solution was immediately gently mixed for another 30 s and then left undisturbed at room temperature for at least 4 h. The as-obtained AuNPs were washed with water three times by centrifugation/redispersion, and finally redispersed in 5 mL of water.
2.3. Synthesis of gold nanoclusters (AuNCs) AuNCs were synthesized according to a previously reported method [49]. In brief, 1 mL of 20 mM HAuCl4 was added to 8.7 mL of water followed by addition of GSH solution (9.2 mg in 0.3 mL of water). The mixture was gently stirred for 5 min at room temperature, and then stirred at 70 ◦ C for 24 h. The AuNCs were obtained as a yellow solution.
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2.4. Preparation of nanocomposites One milliliter of as-prepared AuNPs was mixed with 10 L of ATP solution (0.01 M in ethanol), and sonicated for 5 min. Then, the free ATP molecules were removed through centrifugation, and the particles were dispersed in 1 mL of as-obtained AuNCs solution. The mixture was sonicated for another 5 min, and added to a preheated aqueous gelatin solution (50 mg in 9 mL of H2 O) at 50 ◦ C under vigorous stirring. The mixture was stirred at 50 ◦ C for 30 min, after which its pH was adjusted to ∼3.0 using 1 M HCl. Then, under constant stirring at 40 ◦ C, 30 mL of acetone was added dropwise over 15 min. Next, 50 L of 25% glutaraldehyde was admixed with the stirring mixture, which was stirred at 40 ◦ C for a further 1 h, followed by overnight incubation at room temperature. Finally, the NPs were collected by centrifugation, washed with water twice, and finally dispersed in 1 mL of H2 O. 2.5. Raman measurements Raman measurements were performed with a Renishaw Invia Raman microscope equipped with a laser excitation wavelength of 633 nm (He-Ne laser), and the laser power was controlled at 0.5 mW. All Raman spectra were acquired using a 50× objective. All SERS spectra reported here were the results of a single 10 s accumulation. The diameter of the laser spot area was about 1 m, and the penetration depth was about 25 m. The equipped charge-coupled device (CCD) camera was coupled to a spectrograph that in combination provided a 2 cm−1 spectral resolution. The silicon wafer was used as the substrate and its characteristic Raman peak at 520 cm−1 was employed to calibrate the Raman spectra. 2.6. Confocal Raman imaging Raman images were obtained using the Raman point-mapping method on a Renishaw Invia Raman microscope equipped under 633 nm excitation (He-Ne laser). Microscopy image and Raman image were acquired using a 50× objective. The laser power was set at 1 mW, and 3 s accumulation time was used for a single Raman spectra. A computer-controlled x-y and x-y translation stage was used in a 1 m setup. A Raman mapping experiment was carried out by measuring the Raman spectrum centered at 1300 cm−1 (∼600 nm bandwidth) at each pixel over the 2D area of a HeLa cell. Then, a Raman image was obtained using the highest peak intensity at 1580 cm−1 to create a color map to depict intensity variation. 2.7. Raman enhancement factor (EF) calculation The Raman band at 1580 cm−1 was chosen to estimate the SERS enhancement factor (EF), according to the definition EF = (ISERS /Nads )/(Ibulk /Nbulk ), where ISERS and Ibulk are the Raman intensity at 1580 cm−1 , and Nads and Nbulk are the numbers of adsorbed and solid ATP molecules in the laser illumination volume, respectively. For ATP solids, the diameter of the laser spot area was about 1 m, and the penetration depth was about 25 m. Nbulk was calculated as about 9.41 × 1010 molecules. We assumed that the ATP molecules formed one dense monolayer on the surface of the gold nanoparticles, and it is reported that each ATP molecule occupies 0.2 nm2 [50,51]. Therefore, the number of ATP molecules adsorbed in the laser illumination volume was calculated to be 3.925 × 106 . 2.8. Cytotoxicity assay by MTT method The cytotoxicity of the NPs was studied using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay. HeLa cervical carcinoma cells were grown in
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Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). The cells were seeded into 96-well plates at a density of 1 × 104 cells per well. After 24 h of incubation at 37 ◦ C in a humidified 5% CO2 atmosphere, the cells were treated with different amounts of nanocomposite dispersions (10 L, 20 L, 40 L, 60 L, 80 L, 100 L), and cultured for another 24 h. After pouring out the medium, 100 L of freshly prepared MTT (1 mg mL−1 in 10 mM PBS) was added to each well and incubated for 4 h. After removing the MTT medium solution, the cells were lysed by adding 100 L of DMSO. The plate was gently shaken for 5 min, and then the absorbance of purple formazan at 570 nm was monitored using a Spectra MAX 340PC plate reader. 2.9. Cellular imaging experiments 40 L of nanocomposite dispersion was added to 1 mL of DMEM medium containing HeLa cells in a 35 × 35 mm plate. The plate was then incubated for 10 h at 37 ◦ C in a humidified environment containing 5% CO2 . Then, the culture media was removed and the cells were washed with PBS buffer (10 mM, pH = 7.4) several times. Fluorescence images and phase contrast bright-field images of the cells were recorded on a confocal fluorescence microscope (Olympus FV1000-IX81). To observe the fluorescence signals of the nanocomposites, the excitation wavelength was set at 488 nm and the emission was collected in the range of 550–650 nm. 2.10. Characterization The size distributions and zeta-potentials of the colloidal suspensions were measured by dynamic light scattering with a Malvern Zetasizer Nano ZS90. The morphology of the nanoparticles was characterized by transmission electron microscope (TEM; Hitachi H-7650). Ultraviolet-visible absorption spectra were measured on a Hitachi U3900 spectrophotometer. The fluorescence spectra of the samples were obtained using a Hitachi F-7000 spectrometer. Fluorescence images were recorded using an Olympus FV1000-IX81 confocal laser scanning microscope through a 100× objective lens, with 330–380 nm excitation produced by a 100 W mercury lamp light source. 3. Results and discussion The fabrication procedure of the bifunctional nanocomposites is schematically illustrated in Scheme 1. The core-shell structure of the nanocomposites consisted of four components: an AuNP, ATP molecules, AuNCs, and gelatin, which acted as the magnifying substrate, Raman probe, fluorescent species, and the protective outer shell, respectively. Both the Raman signals of the ATP molecules and the emission properties of the AuNCs were expected to be enhanced by the inner porous AuNP. Porous AuNPs were chosen as the inner core and enhancing material owing to their superior properties. First, their nanoscale channels or pores and large surface roughness would provide sufficient space for adsorbing a large amount of organic molecules or small particles. Second, the highly concentrated electromagnetic field at the abundant hot spots formed in the pores and channels were expected to generate both higher MEF and SERS activity compared with those obtainable with solid spherical shapes. The porous AuNPs used in this work were synthesized using a seed-mediated growth method reported in a previous work [48]. In the synthesis process, (1-hexadecyl)-trimethylammonium chloride (CTAC) was used as the surfactant to facilitate and stabilize the growth of the nanoparticles, and was mostly removed by subsequent centrifugation. Zeta potential measurements of the obtained porous AuNP dispersion revealed that the surfaces of the AuNPs were highly positively charged, at about +40 mV, owing to their full coverage
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Scheme 1. Schematic illustration of the preparation of the hybrid nanocomposites.
Fig. 1. (a) TEM image of synthesized porous AuNPs. (b) Magnified TEM image of individual porous AuNP. (c) TEM image of obtained nanocomposites and (d) gold nanoclusters in the gelatin layer of the nanocomposites.
of CTAC molecules. The transmission electron microscopy (TEM) image shown in Fig. 1a and the representative magnified TEM image of an individual particle shown in Fig. 1b demonstrate the highly porous structure of the AuNPs. It can be seen clearly that the AuNPs had an average size of around 60 nm, with nanoscale pores and channels randomly distributed over their surfaces. ATP is widely used as a Raman probe owing to its simple chemical structure and distinct Raman bands [44,52–54]. More importantly, it can tightly bind to gold surfaces through the strong Au-S affinity. As a benefit of their small size, ATP molecules were able to easily access the pores and channels of the AuNPs, and their Raman signals were thus expected to be enhanced enormously. Furthermore, having the ATP molecules in the pores of the AuNPs also kept them from being removed during the following modification and centrifugation procedures, and enhanced the reproducibility and stability of the Raman signals. Glutathione (GSH) capped AuNCs were fabricated as the fluorescent emitters according to a previously reported procedure [55].
Their mean diameter was 2–3 nm, as shown by the TEM image in Fig. 1d. The negative charge of the GSH capping agent meant that the AuNCs could easily attach to the positively charged surface of the porous AuNPs via electrostatic attraction. Furthermore, their extremely small size also made them easily access to the nanoscale channels or pores of the AuNPs. Of course, not all of the AuNCs could tightly bind with the larger nanoparticles, and most of them were dispersed more freely. After the addition of the gelatin solution, the free GSH-capped AuNCs bound with gelatin macromolecules, driven by their similar functional groups and electrostatic attraction. Finally, glutaraldehyde was used as a cross-linker between the amino groups of both the gelatin and the GSH molecules. Accordingly, the free AuNCs were fixed in the formed gelatin shell, as demonstrated by the TEM image of the obtained nanocomposites shown in Fig. 1c. Taking advantage of its excellent biocompatibility and abundant functional groups, gelatin, a type of thermally and hydrolytically denatured collagen, was used as the protecting shell. It has been
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Fig. 2. (a) Variation in the zeta potential of the obtained nanoparticles during the preparation process. (b) Size distribution of the porous AuNPs and nanocomposites in aqueous dispersion.
demonstrated that gelatin can easily be coated onto nanoparticles via a thermally induced, sol-gel phase transition [33,56]. Here, gelatin was employed as the outer shell to maintain the stability of the nanocomposites and prevent both desorption of the Raman reporters and AuNCs as well as adsorption of external species. It is worth noting that the gelatin shell was found to be composed of two layers. The inner shell was much darker than the outer shell, because many AuNCs were located in this region, as shown in Fig. 1d. From this TEM image, it can be seen that the 2–3 nm AuNCs were dispersed uniformly in this gelatin layer. To monitor the assembly process, the variation in surface charge of each stage of the nanocomposite fabrication was determined by zeta potential measurements, as shown in Fig. 2a. After the addition of ATP molecules, the zeta potential of the AuNPs increased from +40 mV to +50 mV, indicating the replacement of CTAC with ATP molecules because of the strong affinity of thiol group towards the gold surface. Once the small AuNCs were added, the zeta potential converted from a positive to a negative value, owing to the excess carboxylic acids groups of the GSH capping agent. This complete charge inversion implies that the AuNCs indeed obtained access to the AuNP-ATP surfaces as a result of electrostatic attraction and adsorption owing to their small size. After coating with the gelatin layer, the abundant amino groups from the arginine and lysine of the gelatin converted the surface charge to positive again. The following crosslinking reaction using glutaraldehyde as the cross-linker consumed most of the amino groups, leaving carboxylic acid groups (from aspartic acid and glutamic acid) and
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Fig. 3. (a) Ultraviolet-visible absorption spectra of AuNCs (black curve), porous AuNPs (red curve), and nanocomposites (blue curve) dispersed in water. (b) Fluorescence spectra of aqueous dispersions of AuNCs (black curve) and nanocomposites (red curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
subsequently a negative surface. These obvious changes in surface charge strongly demonstrated the successful assembly of each layer formed during the preparation process. The mean size of the bare AuNPs and nanocomposites determined by dynamic light scattering (Fig. 2b) was 60 nm and about 200 nm, respectively, in accordance with the TEM results. The observed narrow size distribution without any appearance of large aggregates also reflected the uniform dispersability of the nanocomposites in aqueous solution. These nanocomposites were dispersed in water and stored at 4 ◦ C, keeping its blue transparent appearance during one-month storage. They can also keep good stability in PBS buffer (10 mM, pH = 7.4) at least for two weeks, which is favorable for biological application. The optical properties of the as-obtained nanocomposites were determined by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy, as displayed in Fig. 3a and b, respectively. The bare AuNP dispersion showed a strong surface plasmon resonance band that covered a broad range from 520 nm to 800 nm with a center at around 633 nm. No obvious plasmon resonance peak was observed in the absorption spectrum of the AuNC dispersion except for a small shoulder peak at around 420 nm, owing to its small particle size. Compared with that of the bare AuNPs, the absorption band of the nanocomposites was lower and broader, with a 10-nm blue shift of the main peak ascribed to the change in the surrounding environment. The fluorescence emission spectra
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of the AuNCs and nanocomposites were also recorded in aqueous dispersion, as shown in Fig. 3b. Compared with that of the discrete AuNCs, the emission intensity of the AuNCs in the nanocomposites was obviously enhanced by a factor of about 4, caused by the magnifying effect of the inner porous AuNPs. It should be noted that only the AuNCs located at a suitable distance from the inner metallic core could be affected by the enhanced electromagnetic field of the porous AuNPs. The measured fluorescence intensity represents the combined signal from all AuNCs present, i.e., those that were quenched, those that were enhanced, and those that were unaffected by interaction with the plasmonic field. Therefore, we believe that the real MEF enhancement factor of the AuNCs at the optimal distance was much higher than the obtained value. The nanocomposites were observed using a fluorescence microscope under ultraviolet light excitation produced by a mercury lamp, and the captured fluorescent image is displayed in Fig. 4. It can be seen that the nanocomposites dispersed uniformly in water, and produced a bright yellow-orange emission. This demonstrates that the present hybrid nanocomposite would be an ideal imaging agent for cellular applications. To confirm the SERS ability of the nanocomposite, Raman spectra were recorded after each stage of the assembly process using a confocal Raman microscope. The 633 nm He-Ne laser was used as the excitation light source, because of its well overlap with the plasmon resonance peak of the nanocomposites. The measurement parameters used to collect Raman signals from each sample were kept constant throughout Fig. 5. Here, all the displayed Raman spectra only show the relevant range of 1000–1700 cm−1 . The spectrum of solid ATP (Fig. 5a) shows several characteristic Raman peaks assignable to the C H bending (1090 cm−1 , 1172 cm−1 ) and C C stretching (1596 cm−1 ) of the phenyl ring. On the bare AuNPs, the
Fig. 4. Fluorescence microscopy image of the hybrid nanocomposites in solution obtained under ultraviolet excitation produced by a mercury lamp.
Raman signals of the ATP molecules were enhanced enormously, by a factor of 4.4 × 105 (Fig. 5b). It is worth noting that the strong new bands that appeared at 1386 and 1429 cm−1 were attributed to the formation of 4,4 -dimercaptoazobenzene (DMAB), which is the product of the photocatalyzed dimerization reaction of ATP molecules [57,58]. This side reaction always takes place on bare SERS-active surfaces when the ATP molecules are in direct contact with the plasmonic substrate. On the other hand, the Raman band caused by C C stretching also shifted from 1596 to 1580 cm−1 , which might be caused by the large variation in environment from
Fig. 5. (a) Raman spectrum of solid ATP. (b) SERS spectrum of ATP on bare porous AuNPs. (c) SERS spectrum of ATP on porous AuNPs in the presence of AuNCs. (d) SERS spectrum of ATP in the final hybrid nanocomposites.
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solid state to plasmonic surface in colloids. After the addition of the small AuNCs, the Raman enhancement factor was reduced to 2.5 × 105 (Fig. 5c). In the final hybrid nanocomposites, the enhancement factor was further decreased to 3.1 × 104 , but the Raman signal of the characteristic peaks was still strong enough to be identifiable (Fig. 5d). Interestingly, the strong characteristic Raman band of the DMAB side products was nearly invisible, suggesting that the unwanted photocatalyzed reaction was almost completely avoided in the hybrid nanocomposites. It should be noted that in the final nanocomposite, the emission intensity of the AuNCs was enhanced, while the Raman enhancement factor of ATP was reduced. This phenomenon is reasonable because the optimal distance for MEF and SERS is different. Both of the MEF and SERS effects show a close dependency on distance between the probe and metallic surface [46,47,59,60,61]. However, the Raman enhancement factor achieves the highest value when the probes get close to the metallic surface, and reduce quickly with distance as the electromagnetic field enhancement decays nearly exponentially. On the other hand, fluorescence would be quenched if the probe is attached on the metallic surface, and only be enhanced in a suitable distance (5–30 nm). In this case, the average emission intensity of AuNCs was enhanced in the final composites, because some of them located at a suitable region towards gold surface. Nevertheless, compared to that on bare porous AuNPs, the SERS enhancement factor reduced in the final nanocomposites, due to the loss and migration of some ATP molecules far away from the inner core in the assembly process. Besides, the reduc-
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Fig. 6. Cell viability results after incubation of HeLa cells with various volumes of nanocomposite dispersion.
tion in laser power caused by the presence of gelatin shell was also responsible for the decrease in SERS efficiency. Considering its gelatin shell, the present hybrid nanocomposites were expected to have good biocompatibility. Before investigation of its practical application in living cells, the cytotoxicity of the nanocomposites was evaluated using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Fig. 7. (a) Fluorescence image of HeLa cells without treatment of nanocomposites. (b) Fluorescence image and (c) bright-field image of HeLa cells co-cultured with nanocomposites for 10 h at 37 ◦ C. (d) Overlapped image of (b) and (c). The scale bars represent 20 m.
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bromide (MTT) cell-viability assay. The degree of activation of the cells can be reflected by the absorbance of MTT at 570 nm. The cell viability is defined by the ratio of the absorbance of cells incubated with nanocomposite dispersion to that of untreated control cells. Here, the cell viability was calculated using HeLa cells incubated for 24 h with various amounts of nanocomposite dispersion (10–100 L). As shown in Fig. 6, more than 77% of the cells remained alive in the tested concentration range of the nanocomposites, indicating their low cytotoxicity. The above results prove that this new nanocomposite is safe to be used in living cells. Taking advantage of its good emission properties and low cytotoxicity, the cell imaging performance of the nanocomposites was examined after co-culture with HeLa cells at 37 ◦ C for 10 h. After removal of the culture media and washing with PBS buffer (10 mM, pH = 7.4), the cultured cells were observed by confocal fluorescence microscope. Upon excitation by a 488 nm laser, the cells incubated with the nanocomposites exhibited a strong orange fluorescence (Fig. 7b). In contrast, the untreated control cells did not show any fluorescence signal (Fig. 7a). The overlapped image (Fig. 7d) of the fluorescence (Fig. 7b) and bright field (Fig. 7c) images shows that the fluorescence mostly surrounded the nucleolus and was located in the cytoplasm of the HeLa cells. Thus, it can be concluded that the nanocomposites were easily captured by the cells and entered the cytoplasm, which can be attributed to their biocompatible gelatin surface. These results demonstrate that the present hybrid nanocomposite is an ideal agent for imaging living cells. These HeLa cells co-cultured with nanocomposites were also placed under a confocal Raman microscope to observe their Raman signals. Fig. 8a shows a representative Raman spectrum collected from the HeLa cell, where the intense SERS characteristic signal was detected. The Raman peaks of the ATP molecules are clear and sharp with a high signal-to-noise, indicating a less interference from the cellular environment. In addition, the absence of characteristic Raman band of DMAB implies the ATP molecules were quite stable in the cell, and the unwanted side reaction was avoided. Insert in Fig. 8a is a microscopy image of a single HeLa cell, and the Raman mapping experiments were carried out on the area in red rectangle. Fig. 8b displays the false color SERS mapping image, which is calculated from the Raman spectrum at each pixel based on the strong characteristic peak of ATP at 1580 cm−1 . The cell shape and surface outline were clearly shown in the image, compared to the low background. The distribution of Raman signals across the cell revealed the well capture of nanocomposites by the cell, in consistent with the fluorescence imaging. In addition, the intense SERS signal observed in the cell indicated that this nanocomposite can serve as an ideal SERS probe for cellular imaging. 4. Conclusions In summary, we have fabricated a gold/gelatin core-shell nanocomposite integrating both MEF and SERS capabilities into a single system, in which AuNCs and ATP were employed as fluorescent and Raman probes, respectively. TEM images of the nanocomposites confirmed the successful preparation of the intended core-shell structure, with a middle gelatin shell containing a large amount of embedded AuNCs. The fluorescence intensity of the AuNCs in the nanocomposites were enhanced by about four times compared with that of the same amount of discrete AuNCs in solution, which is attributed to the MEF effect from the inner porous gold nanoparticles. Moreover, the composite also displayed an enormous enhancement of the Raman signal of ATP, which makes this composite a good candidate as a label-free Raman probe. Finally, taking advantage of its low toxicity towards living cells, the nanocomposite exhibited good fluorescence and SERS imaging performance after co-culture with HeLa cells.
Fig. 8. (a) A representative SERS spectrum of the HeLa cell incubated with the nanocomposites for 10 h at 37 ◦ C. Insert is the bright field microscopy image of a single HeLa cell. (b) SERS mapping image in false color of the single HeLa cell, created from the Raman spectrum at each pixel across the red rectangle area in Fig. 8a insert, using the Raman peak intensity at 1580 cm−1 . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Acknowledgments This work was supported by the National Natural Science Foundation of China (51503015), and the Fundamental Research Funds for the Central Universities (FRF-TP-15-003A1). The authors thank Dr. Libin Liu for giving support for the cellular experiments.
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