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Intracellular pH Sensing Using p-Aminothiophenol Functionalized Gold Nanorods with Low Cytotoxicity Shenfei Zong, Zhuyuan Wang, Jing Yang, and Yiping Cui* Advanced Photonics Center, School of Electronic Science and Engineering, Southeast University, Nanjing 210096, Jiangsu, China ABSTRACT: We report an intracellular pH sensor based on surface enhanced Raman scattering (SERS) using the hydrochloric acid (HCl) treated gold nanorods (GNRs) as the SERS substrates and p-aminothiophenol (pATP) as the Raman reporter. Using the HCl treated GNRs previously reported by us, the biocompatibility and the SERS performance of GNRs have been greatly improved. Meanwhile, the adsorbed reporters are allowed to be directly exposed to the surrounding environments, which is very important for biosensors. It is found that the SERS spectrum of pATP is strongly dependent on the pH value. The intensities of SERS bands at 1142 cm1, 1390 cm1, and 1432 cm1 increased obviously with the pH value varying from 3.0 to 8.0. This pH-dependent SERS performance of pATP-functionalized HCl treated GNRs was well retained after the incorporation of the GNRs into living HeLa cells. Our experimental results indicate that such pATP-functionalized HCl treated GNRs can be used as an effective intracellular pH sensor. Thus, we show a good example that the bioapplications of the normal CTAB-stabilized GNRs can be expanded after the simple HCl treatment.
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unctionalized nanoprobes are expected to play key roles for the targeting, sensing, and imaging in the living bioenvironment. 1,2 Involved materials are magnetic nanocrystals, 3 silica nanoparticles,4 novel metal nanoparticles,5 and so on. Among these various materials, the novel metal species have received special attention due to their particular surface plasmon resonance (SPR) characteristic which is capable of generating great enhancement of optical signals, such as fluorescence and Raman scattering.6,7 Since its discovery, surface enhanced Raman scattering (SERS) has been used as a molecular and cellular analytical technology with extremely high sensitivity.8,9 The unique fingerprints of SERS spectra of different molecules make it possible to track the targeted probe in harsh living conditions.10 As one of its various applications, biological pH sensing based on SERS has recently been studied.1119 Usually, the fabrication and utilization of SERS pH sensor is based on the fact that the SERS signals of some Raman active molecules vary as the pH value of the surrounding circumstance changes. Two typical such molecules are 4-mercaptobenzoic acid (4MBA) and 2-aminothiophenol (2ATP). The carboxyl groups of 4MBA exhibit the COO form in alkaline solutions resulting in a higher sensitivity in the alkaline region;12 while the amino groups of 2ATP exhibit the NH3þ form in acidic solutions leading to a higher sensitivity in the acidic region.11 Hill et al. has reported that p-aminothiophenol (pATP), an isomer of 2ATP, also possessed a pH-dependent SERS performance.20 They proposed that the pH dependence of pATP originated from the transformation between two states of pATP: the aromatic state presented in acidic solutions and the quinonoidic one in neutral and alkaline solutions. r 2011 American Chemical Society
Up to date, the reported SERS pH sensors are based on two types of SERS substrates, namely, silver nanoparticles (Ag NPs) and gold nanospheres (GNSs). Despite their strong SERS enhancement, the clinical application of Ag NPs may be hindered by their intrinsic toxicity.2123 While for essentially nontoxic GNSs,24 their relatively weak enhancing ability requires a more sophisticated detection system especially for the in vivo applications. A compromise between the SERS enhancement and the biocompatibility is to use the rod shaped gold nanoparticles (GNRs), which can generate a great electric field enhancement at their tips, as the SERS substrates.25,26 However, the obvious cytotoxicity caused by the surfactant molecules cetyltrimethylammonium bromide (CTAB),27,28 used as a soft template in the seed-mediated growth of GNRs,29,30 becomes the most annoying obstacle lying in the way to the bioapplications of GNRs. This brings up massive attention concerned with how to get rid of the negative effect of CTAB.3133 Here, we demonstrate the realization of an intracellular pH sensor using pATP functionalized hydrochloric acid (HCl) treated GNRs based on SERS method. As we have reported previously, after being treated with HCl, most CTAB molecules around the GNRs could be removed, resulting in a greatly reduced cytotoxicity and an improved SERS activity.33 Although the cytotoxicity of CTAB-stabilized GNRs can also be reduced by encapsulating the GNRs inside a shell of other materials (such as silica, polymers, and so on), the coating shell makes it impossible for the GNRs or the adsorbed reporters to be directly exposed to Received: February 23, 2011 Accepted: April 22, 2011 Published: April 22, 2011 4178
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the surrounding environments. Thus, using HCl treated GNRs as the SERS substrate has the advantages of not only the reduced cytotoxicity but also the direct contact between the reporters and the surroundings, which are important requirements for the sensors used in biosystems. Our experimental results show that the SERS spectrum of pATP adsorbed on HCl treated GNRs changes drastically as the surrounding pH value increases from 3.0 to 8.0. After being incorporated into living human cervical cancer cells (HeLa cells), the pH sensitivity of pATP functionalized HCl treated GNRs is well retained.
’ EXPERIMENTAL SECTION Materials. Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 3 3H2O), sodium borohydride (NaBH4), and p-aminothiophenol (pATP) were purchased from Alfa Aesar. Cetyltrimethylammonium bromide (CTAB) was purchased from Sinopharm Chemical Reagent Co. Ltd. Silver nitrite (AgNO3) and citric acid were purchased from Shanghai Shenbo Chemical Co. Ltd. Ascorbic acid was purchased from Shanghai Shisihewei Chemical Co., Ltd. Hydrochloric acid (HCl) was purchased from Shanghai Zhongshi Chemical Co., Ltd. Disodium hydrogen phosphate (Na2HPO4 3 12H2O) was purchased from Nanjing Chemical Reagent Co., Ltd. All the reagents were used as received. Deionized water (Millipore Milli-Q grade) with resistivity of 18.2 MΩ was used in all the experiments. Synthesis of pATP Functionalized HCl Treated GNRs (Denoted as pATPGNRs). GNRs were synthesized by a previously published seed-mediated growth method.34 HCl treated GNRs were prepared as follows. HCl solution (10 M) was added to GNR solution until the pH reached 1.4. This solution was stirred at 60 °C for 3 h and then centrifuged twice at 10 000 rpm for 30 min. After each round of centrifugation, the precipitate was redispersed in deionized water. The final SERS pH sensor (pATPGNRs) was prepared by adding 5 μL of 10 mM pATP ethanol solution to 10 mL of aqueous solution of HCl treated GNRs; the mixture was aged for at least an hour before the SERS measurements. Culture of HeLa Cells and MTT Assay. HeLa cells were purchased from Nanjing KeyGen Biotech Co., Ltd. and cultured in medium (DMEM) under standard cell cultural condition (5% CO2, 37 °C). Media were supplemented with 10% heat-inactivated newborn calf serum (Hangzhou Every Green Organism Engineering Materials Co., Ltd.) and 1% penicillinstreptomycin (Nanjing KeyGen Biotech Co., Ltd.). The biocompatibility of HCl treated GNRs was examined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. HeLa cells (104/ml) were seeded onto 96-well plates (100 μL/hole) and incubated for 24 h at 37 °C under a 5% CO2 atmosphere. Then, 10 μL and 20 μL of the GNRs and HCl treated GNR solutions were added, respectively, and incubated for 36 h. After that, 50 μL of MTT solution (MTT buffer to dilution buffer 1:4) was added into each well and the plate was incubated for another 4 h. The reaction was terminated by adding 150 μL of DMSO after removing the supernatant medium. When the purple formazan crystals were dissolved by DMSO, the absorbances of the wells at 490 nm were measured with a microplate reader (Bio-Rad model 680). Cells incubated in the absence of nanoparticles were used as a control. SERS Measurements. Citric acidphosphate buffer solutions with well-defined pH ranging from 3.0 to 8.0 were prepared by mixing aqueous solutions of citric acid (0.1 M) and disodium
Figure 1. (a) TEM image of CTAB-stabilized GNRs, (b) TEM image of HCl treated GNRs.
hydrogen phosphate (0.2 M) with different volume ratios. pATPGNRs solutions at different pH were prepared by mixing the pATPGNRs solutions with the corresponding buffer solutions (volume ratio pATPGNRs solutions/buffer = 1:2). After casting 3 μL of each pATPGNRs solution onto the glass slides, SERS spectra under different pH values were obtained. For in vitro SERS measurements, pATPGNRs solutions were added to the cell culture dishes (volume ratio pATPGNRs/culture medium = 1:3). After incubating for 3 h, the cell culture dishes were washed with phosphate buffered saline (PBS) for three times; then, the buffer solutions with different pH values were added into each culture dish, respectively, before each SERS measurement. The SERS measurements were repeated for 10 times for each specific pH environment to obtain the average results. Instruments. Extinction spectra were measured by a Shimadzu UV-3600 PC spectrophotometer with quartz cuvettes of 1 cm path length. A Sartorius PP-15 pH meter was employed for pH measurements. Transmission electron microscope (TEM) images were obtained with a FEI Tecnai G2T20 electron microscope operating at 200 kV. SERS measurements were performed with confocal microscope (FV 1000, Olympus, Japan) equipped with a spectrograph (sharmrock, Andor, UK) with Newton 303i CCD. HeNe laser with 632.8 nm radiation was used for excitation, and the laser power at the sample position was 2.3 mW; the integrating time of each SERS measurement was 60 s.
’ RESULTS AND DISCUSSION Characterization of HCl Treated GNRs. GNRs used in our study were synthesized through the seed-mediated growth method, which can produce GNRs with a very high yield.35 Figure 1a shows the TEM image of GNRs without HCl treatment. As shown in the picture, the average aspect ratio of the synthesized CTAB-stabilized GNRs was about 3.0 with a good rod shape. Figure 1b is the TEM image of the GNRs after being treated with HCl. Comparing the two pictures, a slight aggregation of the GNRs can be observed, which helped to improve the SERS activity of HCl treated GNRs due to more “hot spots”. Besides, the morphology of GNRs after being treated with HCl seemed to be slightly different from that without HCl treatment. According to the results in previously published literature, the morphology of GNRs is dependent on the temperature or pH value of the solution.3638 In our experiments, during the HCl treatment procedure, the GNR solution was kept at 60 °C for 3 h with the pH value being adjusted to 1.4. It is possible that the GNRs experienced slight morphology changes during the HCl treatment procedure. More detailed discussion about HCl 4179
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Analytical Chemistry treated GNRs has been provided in our previously published paper.33 The successful fabrication of GNRs can also be confirmed by the extinction spectra. As shown in Figure 2 (black curve), two
Figure 2. Extinction spectra of CTAB-stabilized GNRs, HCl treated GNRs, and pATPGNRs.
Figure 3. SERS spectra of pATPGNRs under pH = 3.0 and 8.0.
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SPR bands are observed, the longitudinal SPR band (LSPR) peak at 701 nm and the transverse SPR (TSPR) band at 514 nm. The appearance of a new SPR peak at longer wavelength compared with gold nanospheres is the most distinguishable difference between GNRs and other shaped nanoparticles. The LSPR is very sensitive to the dielectric constant of the surrounding media and can be tuned from visible to near-infrared region of the optical spectrum by changing the aspect ratio of GNRs.39 For HCl treated GNRs, the LSPR blue-shifted to 698 nm (the red curve in Figure 2). This is mainly due to the removal of the excess CTAB molecules, which results in a lower dielectric constant around GNRs.40 Besides, the HCl treated GNRs seemed to aggregate in a side-by-side fashion, which also contributed to the blue shift of LSPR.41 After the adsorption of pATP, the LSPR red-shifted to 702 nm while the TSPR remained almost unchanged. No broadening or tailing of the LSPR band was observed through both the HCl treatment and the pATP functionalization procedures, indicating that GNRs were not overaggregated. Besides, the HCl treated GNRs solution remained stable for more than 6 months without any sediment. SERS Experiments of pATPGNRs. When pATP molecules adsorb onto the surfaces of GNRs, they preferentially adopt a vertical orientation.20,42 There are two possible binding sites, the mercapto group and the amino group.43 Since the interaction between AuS is much stronger than that between AuN, the pATP molecules bind to the GNRs surface through the mercapto groups, leaving the amino groups exposed to the surrounding media. After the chemical adsorption of pATP molecules, they were brought into the close vicinity of GNRs, allowing large enhancement of the Raman scattering. For HCl treated GNRs, the adsorption of pATP became more efficient, since most of the CTAB molecules were removed and more surfaces of the GNRs were directly exposed to the surrounding environment. The limited aggregation of GNRs induced by the HCl treatment procedure can also contribute to the SERS enhancement because more hot spots were formed.33 Figure 3 shows the SERS spectra of the presented pH sensor (pATPGNRs) under pH 3.0 (bottom curve) and pH 8.0 (top curve), respectively. Obviously, there is a significant spectral difference between the two spectra. SERS peaks of the pATP molecules have been well assigned previously.42,46 In the SERS spectrum under pH 3.0, the Raman bands at 1007 cm1, 1076 cm1, 1180 cm1, and 1577 cm1 are attributed to the
Figure 4. (a) pH-dependent SERS spectra of pATPGNRs. All spectra are normalized by the intensity of 1076 cm1 and placed in parallel for clarity. (b) Normalized intensity of 1432 cm1 at different pH values. For each specific pH value, the SERS measurements were performed 10 times, and the average results were adopted. The error bars represent the standard derivation. 4180
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Figure 6. Bright field image of HeLa cells incubated with pATPGNRs.
Figure 5. Schematic illustration of the structure of pATPGNRs in acidic solution and the formation of DMAB in alkaline solution.
a1 modes of the pATP molecules while those at 1140 cm1, 1390 cm1, and 1433 cm1 are attributed to the b2 modes of pATP. In the SERS spectrum under pH 8.0, the Raman bands at 1007 cm1, 1076 cm1, 1190 cm1, and 1576 cm1 are assigned to the a1 modes while those at 1142 cm1, 1304 cm1, 1390 cm1, and 1432 cm1 are assigned to the b2 modes. Interestingly, the SERS intensities of b2 modes exhibit an obvious pH dependence, which is in consistence with the results previously reported.20 We then examined the pH-dependent SERS performance of pATPGNRs. As shown in Figure 4a, when the pH value of the surrounding solution gradually increased, a significant increase in the intensities of the b2 modes (1142 cm1, 1390 cm1, and 1432 cm1) was observed. An early study reported by Hill et al. has pointed out that pATP exhibits two structures under different pH values, the aromatic state in acidic solutions and the quinonoidic one in neutral and alkaline solutions.20 These two structures are responsible for the observed difference in SERS spectra under different pH values. They suggested that, as the pH value became lower, the quinonoidic surface imine form of pATP was protonated to the aromatic amine form, which was then further changed to the ammonium form. Zhou et al. fabricated a metalmoleculemetal nanosystem using pATP as the bridge molecules between silver and gold nanoparticles.43 They also mentioned the two resonance structures of pATP and concluded that b2 modes of pATP were strongly enhanced by charge transfer from silver to gold tunneling through pATP. It has been widely accepted that the b2 modes of pATP are enhanced by a charge transfer process and the a1 modes are enhanced by electromagnetic mechanism.42,44 However, this conclusion has recently been questioned. Huang et al. have proven that the b2 modes actually originate from the oxidated form of pATP, 4,40 dimercaptoazobenzene (DMAB) molecules.45 According to their results, the three Raman peaks at 1142 cm1, 1390 cm1, and 1432 cm1 are assigned to the ag modes of DMAB and are much stronger than the a1 modes of pATP. The ag modes of
DMAB can also generate Raman bands similar to the a1 modes of pATP, with comparable intensity and a slight shift in frequency. Similar results have been reported by other groups.4649 In a word, the b2 modes are generated by DMAB molecule, which is transformed from pATP by a possible nanoparticle catalyzed photochemical reaction during SERS measurements. In our SERS experiments, the excitation power is kept the same for all samples. If the formation of DMAB is only due to the photochemical reaction, the SERS spectra should be the same, which is definitely different from the experimental results. Thus, it is reasonable to conclude that the transformation of pATP to DMAB is also closely related with the pH value of the surrounding media. In the SERS spectra acquired from the solutions with a higher pH value, the “b2 modes” (or actually ag modes) showed higher intensities, which means that more pATP were transformed to DMAB. According to Huang et al, there are two possible structures of DMAB (the cis-DMAB and the transDMAB).48 The cis-DMAB is “π” shaped, and the trans-DMAB is near linearly shaped. The SERS spectral profile of the cis-DMAB differs from those in Figure 4a, while that of trans-DMAB is quite similar to those in Figure 4a, so we deduce that the DMAB molecules exhibit the trans form and may be formed from pATP molecules adsorbed on two different GNRs (shown in Figure 5). Considering all the factors mentioned above, the pH-dependent SERS performance of our sensor can be described as follows. When being exposed in a relatively low pH value solution, more pATP molecules adsorbed on HCl treated GNRs exhibit the aromatic state, and few pATP molecules are photochemically transformed to DMAB during SERS measurements; thus, the SERS signals of “b2 modes” are relatively weak. However, when the pH value increases, more pATP molecules begin to change to DMAB and the intensities of “b2 modes” gradually increase. The Raman band at 1076 cm1 is due to the γCS vibration, which can be generated by both pATP and DMAB with a comparable intensity. Thus, the intensity of this Raman band can be used as a control to compare the intensity changes of other bands.4446 The strong peaks at 1390 cm1 and 1432 cm1 are related to the NdN vibration of DMAB, so their intensity can best describe the formation of DMAB.45,48 The normalized intensity of Raman band at 1432 cm1 (I1432/I1076) against the 4181
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Figure 7. (a) SERS spectra of pATPGNRs inside HeLa cells placed in pH 3.0 and 6.0 buffer solutions. (b) Normalized intensity of 1432 cm1 acquired from pATPGNRs loaded HeLa cells placed in different pH value buffer solutions. For each specific pH value, SERS spectra were obtained from 10 different HeLa cells, and the average results were used. The error bars represent the standard derivation.
Figure 8. Viability of HeLa cells incubated with HCl treated GNRs and nontreated GNRs. HeLa cells incubated without nanoparticles were used as a control.
pH value is plotted in Figure 4b. I1432/I1076 increases with the increase of the pH value, which reveals that the intensity of 1432 cm1 can be used as an indicator of the pH of the surrounding environment. Besides, our pH sensor is more sensitive in the low pH region. In Vitro SERS Experiments. To evaluate the performance of our pH sensor inside living cells, HeLa cells were incubated with pATPGNRs. Then, the culture media in six culture dishes containing pATPGNRs loaded HeLa cells were replaced by six buffer solutions with pH values stepped by 1.0 pH unit ranging from pH 3.0 to 8.0. The SERS spectra inside HeLa cells were recorded, respectively. Uptake of pATPGNRs was confirmed by the bright field images, as shown in Figure 6. pATPGNRs appeared as the black dots, which mainly distributed in the cytoplasm region. The incorporation of pATPGNRs did not bring a significant change to the cell morphology. During the SERS measurements, no obvious cell death was found. Figure 7a shows two typical SERS spectra acquired from HeLa cells placed in pH 3.0 and pH 6.0 buffer solutions. pATPGNRs exhibited a similar pH-dependent SERS performance as that in
pure buffer solutions. This indicates that the structure of the proposed pH sensor remained stable after being taken up by living cells. At pH 3.0, the SERS spectrum was dominant by the a1 modes of pATP (1077 cm1, 1182 cm1, 1580 cm1). For cells placed in the pH 6.0 buffer solution, the SERS spectrum was dominant by the ag modes (1140 cm1, 1390 cm1, 1432 cm1) of DMAB, very similar to that described in SERS Experiments of pATPGNRs. The normalized intensity of Raman band at 1432 cm1 (I1432/I1077) collected from pATPGNRs loaded HeLa cells against the pH value is plotted in Figure 7b. The pH sensitivity of pATPGNRs was well preserved after being incorporated into living cells. Cytotoxicity of HCl Treated GNRs Used in the pH Sensor. During the HCl treatment, most of the free CTAB molecules were removed, leaving a small proportion of CTAB binding tightly to the GNR surface, which helped to maintain the stability of HCl treated GNRs. According to Alkilany et al., the toxicity of CTAB-stabilized GNRs was due to the free CTAB molecules in the solution while CTAB tightly bound to GNR surfaces is far less toxic than the free CTAB.27 Thus, HCl treated GNRs were supposed to show a greatly reduced cytotoxicity. The results of MTT assay further confirmed this result. In the experiments, HeLa cells were incubated with HCl treated GNRs and GNRs without HCl treatment at the concentration of 10 μg/mL and 20 μg/mL for 36 h, respectively. As shown in Figure 8, the viability of HeLa cells incubated with HCl treated GNRs is about 3 times higher than nontreated GNRs at both concentrations. Thus, through the simple HCl treatment, biocompatibility of GNRs is conspicuously improved. At the same time, pATP molecules are allowed to be directly exposed to the surrounding circumstances, which is very important for biosensors.
’ CONCLUSIONS We have successfully fabricated a pH sensor based on pATP functionalized HCl treated GNRs using SERS technique. The transformation of pATP molecule between two states under different pH conditions makes it suitable to be used as a pH indicator by measuring the SERS signals of pATPGNRs. When being incorporated into living cells, the pH sensor is able to deliver pH information of the surrounding circumstance. 4182
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Analytical Chemistry In summary, reducing the cytotoxicity of GNRs by a simple HCl treatment method may enlarge the bioapplications of GNRs. The proposed pH sensor should have great potential in the study of pathological changes or other biological processes at a single cellular level due to its excellent biocompatibility and good SERS enhancing capability.
’ AUTHOR INFORMATION Corresponding Author
*Tel.: þ86-25-83601769 ext. 828; Fax: þ86-25-83601769 ext. 838. E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by Natural Science Foundation of China (NSFC) (Nos. 60708024, 60877024) and the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) (Nos. 20070286058, 20090092110015). ’ REFERENCES (1) Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.; Zink, J. I. ACS Nano 2008, 2, 889–896. (2) Wang, Z. Y.; Zong, S. F.; Yang, J.; Li, J.; Cui, Y. P. Biosens. Bioelectron. 2011, 26, 2883–2889. (3) Chen, Y.; Chen, H. G.; Zhang, S. J.; Chen, F.; Zhang, L. X.; Zhang, J. M.; Zhu, M.; Wu, H. X.; Guo, L. M.; Feng, J. W.; Shi, J. L. Adv. Funct. Mater. 2011, 21, 270–278. (4) Rosenholm, J. M.; Meinander, A.; Peuhu, E.; Niemi, R.; Eriksson, J. E.; Sahlgren, C.; Linden, M. ACS Nano 2009, 3, 197–206. (5) Durr, N. J.; Larson, T.; Smith, D. K.; Korgel, B. A.; Sokolov, K.; Yakar, A. B. Nano Lett. 2007, 7, 941–945. (6) Geddes, C. D.; Cao, H. S.; Gryczynski, I.; Gryczynski, Z.; Fang, J. Y.; Lakowicz, J. R. J. Phys. Chem. A 2003, 107, 3443–3449. (7) Jana, N. R.; Pal, T. Adv. Mater. 2007, 19, 1761–1765. (8) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667–1670. (9) Nie, S. M.; Emory, S. R. Science 1997, 275, 1102–1106. (10) Wang, Y. L.; Seebald, J. L.; Szeto, D. P.; Irudayaraj, J. ACS Nano 2010, 4, 4039–4053. (11) Wang, Z. Y.; Bonoiu, A.; Samoc, M.; Cui, Y. P.; Prasad, P. N. Biosens. Bioelectron. 2008, 23, 886–891. (12) Talley, C. E.; Jusinski, L.; Hollars, C. W.; Lane, S. M.; Huser, T. Anal. Chem. 2004, 76, 7064–7068. (13) Pallaoro, A.; Braun, G. B.; Reich, N. O.; Moskovits, M. Small 2010, 6, 618–622. (14) Kneipp, J.; Kneipp, H.; Wittig, B.; Kneipp, K. Nanomed.: Nanotechnol., Biol., Med. 2010, 6, 214–226. (15) Ando, R. A.; Pieczonka, N. P. W.; Santos, P. S.; Aroca, R. F. Phys. Chem. Chem. Phys. 2009, 11, 7505–7508. (16) Nowak-Lovato, K. L.; Rector, K. D. Appl. Spectrosc. 2009, 63, 387–395. (17) Zhao, L. P.; Shingaya, Y.; Tomimoto, H.; Huang, Q.; Nakayama, T. J. Mater. Chem. 2008, 18, 4759–4761. (18) Kneipp, J.; Kneipp, H.; Witting, B.; Kneipp, K. Nano Lett. 2007, 7, 2819–2823. (19) Lim, J. K.; Joo, S. W. Appl. Spectrosc. 2006, 60, 847–852. (20) Hill, W.; Wehling, B. J. Phys. Chem. 1993, 97, 9451–9455. (21) Morones, J. R.; Elechiguerra, J. L.; Camacho, A.; Holt, K.; Kouri, J. B.; Ramirez, J. T.; Yacaman, M. J. Nanotechnology 2005, 16, 2346–2353. (22) Poon, V. K. M.; Burd, A. Burns 2004, 30, 140–147. (23) AshaRani, P. V.; Mun, G. L. K.; Hande, M. P.; Valiyaveettil, S. ACS Nano 2009, 3, 279–290. (24) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Small 2005, 1, 325–327.
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