pubs.acs.org/Langmuir © 2009 American Chemical Society
Pluronic Block Copolymer-Mediated Interactions of Organic Compounds with Noble Metal Nanoparticles for SERS Analysis Timur I. Abdullin,*,† Oxana V. Bondar,†,‡ Yu. G. Shtyrlin,§ Mehmet Kahraman,‡ and Mustafa Culha*,‡ †
Faculty of Biology and Soil Sciences, Kazan State University, Kazan, Tatarstan, Russia, ‡Department of Genetics and Bioengineering, Yeditepe University, Istanbul, Turkey, and §A.M. Butlerov Chemistry Institute of Kazan State University, Kazan, Tatarstan, Russia Received September 25, 2009. Revised Manuscript Received November 11, 2009 The composite silver and gold nanoparticles (AgNPs and AuNPs) coated with nonionic amphiphilic block copolymers (Pluronics L121, F68, or F127) are prepared by their adsorption under critical micelle concentrations. It is found that Pluronics bind to the surface of metal NPs as a very thin film by the hydrophobic association through poly(propylene oxide) block of the copolymers. The modification increases the colloidal stability of NPs with increasing hydrophilic-lipophilic balance of Pluronics in the order of L121, F127, and F68. In order to investigate the potentials of polymer coated noble metal NPs as surface-enhanced Raman spectroscopy (SERS) probes, fluorescent dyes and doxorubicin are used as model compounds. It is found that Pluronic component promotes the adsorption of these compounds on the composite NPs resulting in a considerable increase of Raman signal. This effect is attributed to increased concentration of the analyte molecules on the composite surface due to the hydrophobic and charge-charge interactions between the analytes and the Pluronic coat, and the stabilization of NPs by poly(ethylene oxide) blocks. The copolymer coated AgNPs show higher SERS activity than the counterparts prepared with AuNPs. Among the prepared composites, the AgNPs modified with Pluronic F127 containing extended poly(propylene oxide) and poly(ethylene oxide) blocks exhibit maximal Raman activity using rhodamine 6G (Rh6G) with a EF of 9.04 106. The results show that the developed Pluronic-based SERS probes can be used for sensitive and selective analysis of organic analytes.
Introduction The development of inorganic nanoparticle probes is one of the main approaches in the analysis of biological matters in vitro and in vivo.1 Noble metal nanoparticles such as AuNPs and AgNPs are of considerable analytical interest due to their unique plasmonic properties, which strongly depend on particle size, shape, aggregation status and environment.2,3 These nanoparticles not only can be used for the direct detection of molecular interactions in solutions4 but also are compliant to controllable synthesis and chemical modifications to utilize in several applications such as cellular and biomedical.2,5-8 The most applications of metal nanoparticles generally require their surface modifications in order to improve the stability and analytical characteristics of the probes.9 The modification is also intended to control cellular effects of the nanoparticles as well as to introduce reactive groups onto the surface of the probes for covalent attachment of specific ligands or antibodies.10 To date, *Corresponding authors. E-mail:
[email protected] (T.A.); mculha1@ gmail.com (M.C.).
(1) De, M.; Ghosh, P. S.; Rotello, V. M. Adv. Mater. 2008, 20, 4225–4241. (2) Liao, H.; Nehl, C. L.; Hafner, J. H. Nanomedicine 2006, 1, 201–208. (3) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem., B. 2006, 110, 7238–7248. (4) Zhao, J.; Zhang, X.; Yonzon, C. R.; Haes, A. J.; Van Duyne, R. P. Nanomedicine 2006, 1, 219–228. (5) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293–346. (6) Hering, K.; Cialla, D.; Ackermann, K.; Dorfer, T.; Moller, R.; Schneidewind, H.; Mattheis, R.; Fritzsche, W.; Rosch, P.; Popp, J. Anal. Bioanal. Chem. 2008, 390, 113–124. (7) Kneipp, K.; Kneipp, H.; Kneipp, J. Acc. Chem. Res. 2006, 39, 443–450. (8) Kneipp, J.; Kneipp, H.; Rajadurai, A.; Redmond, R. W.; Kneipp, K. J. Raman Spectrosc. 2009, 40, 1–5. (9) Yang, M.; Chen, T.; Lau, W. S.; Wang, Y.; Tang, Q.; Yang, Y.; Chen, H. Small 2009, 5, 198–202. (10) Xu, T.; Zhang, N.; Nichols, H. L.; Shi, D.; Wen, X. Mater. Sci. Eng., C. 2007, 27, 579–594.
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AuNPs and AgNPs have been modified with lipophilic compounds,11 oligo- and polysaccharides,12 PEG derivatives,13 and synthetic polyelectrolytes14 by chemosorption11,13 or noncovalent association.12,14 Another important class of surface modifiers are uncharged amphiphilic polymers. Particularly, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers, also known as Pluronics, are biocompatible polymers with hydrophilic-lipophilic balance (HLB) determined by the ratio of ethylene oxide and propylene oxide units.15 Both in unimer and micellar forms, Pluronics were shown to promote the intracellular transport of small drugs and nucleic acids in vitro and in vivo.16 Owing to these properties, Pluronics are of considerable interest in the development of customizable nanoparticle probes for biomedical applications. It was demonstrated that Pluronic copolymers can be used in the combination with metal nanoparticles.17,18 Specifically, the simple method of synthesis of AuNPs in the presence of Pluronics has been proposed.17 It is based on the reduction of gold ions in water by poly(ethylene oxide) component of Pluronics to produce stable and homogeneous colloids covered with the polymeric shell. Another approach is based on the association of presynthesized AuNPs with Pluronics resulted in the formation of (11) Chung, Y.-C.; Chen, I. H.; Chen, C.-J. Biomaterials. 2008, 29, 1807–1816. (12) Tan, W. B.; Zhang, Y. J. Biomed. Mater. Res., Part A. 2005, 75, 56–62. (13) Niidome, T.; Yamagata, M.; Okamoto, Y.; Akiyama, Y.; Takahashi, H.; Kawano, T.; Katayama, Y.; Niidome, Y. J. Controlled Release 2006, 114, 343–347. (14) Takahashi, H.; Niidome, T.; Kawano, T.; Yamada, S.; Niidome, Y. J. Nanopart. Res. 2008, 10, 221–228. (15) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189–212. (16) Batrakova, E. V.; Kabanov, A. V. J. Controlled Release 2008, 130, 98–106. (17) Sakai, T.; Alexandridis, P. Langmuir 2004, 20, 8426–8430. (18) Rahme, K.; Oberdisse, J.; Schweins, R.; Gaillard, C.; Marty, J. D.; Mingotaud, C.; Gauffre, F. ChemPhysChem 2008, 9, 2230–2236.
Published on Web 12/07/2009
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individual composite nanoparticles and their aggregates.18 Although these composite nanoparticles were found to exhibit some interesting structural properties, e.g., the dependence of their hydrodynamic diameter on the temperature, their possible applications remain unclear. Because of their amphiphilic nature, Pluronic copolymers are expected to reversibly interact with both hydrophilic and hydrophobic compounds in water solutions. Therefore, Pluronics can be potentially exploited to control the selectivity of metal nanoparticle probes toward analyzed species. To study this effect, we prepared composite nanoparticles composed of AuNPs and AgNPs covered with a thin Pluronic copolymer shell. The structure of the composite nanoparticles and their interactions with aromatic solutes were explored with the aid of UV-visible spectroscopy, dynamic light scattering, AFM, and SERS. Our results form a background for the development of new amphiphilic polymer-based nanoprobes for SERS analysis of biologically relevant organic compounds.
Experimental Section Reagents and Materials. Silver nitrate (AgNO3) and chloroauric acid (HAuCl4 3 3H2O) were purchased from Fluka. Poly(ethylene oxide)-poly(propylene oxide) block copolymers, rhodamine 6G, rhodamine B, aminofluorescein, and doxorubicin hydrochloride were obtained from Sigma-Aldrich. All solutions were prepared using deionized water and salts of analytical quality. Synthesis of Silver and Gold Nanoparticles. Both Ag and AuNPs were synthesized in water by the standard reduction of AgNO3 or HAuCl4 salts by sodium citrate as a reductant under heating.19 Briefly, sodium citrate was added to boiling solutions of 1.0 10-3 M AgNO3 or 0.25 10-3 M HAuCl4 under vigorous stirring to obtain final concentrations of the reductant of 0.9 10-3 and 1.5 10-3 M, respectively. Resultant solutions were boiled for 1 h in case of AgNPs and for 15 min in case of AuNPs to produce spherically shaped nanoparticles stabilized with citrate ion. We used mass concentrations of synthesized Au and AgNPs which, for simplification, were assumed equal to mass concentrations of corresponding metals in the suspensions. These concentrations were 180 μg mL-1 for AgNPs and 50 μg mL-1 for AuNPs. Adsorption of Pluronic Block Copolymers and Analytes. Synthesized AgNPs and AuNPs were modified with Pluronic copolymers (L121, F68, or F127) by the method commonly used to prepare colloidal gold-proteins conjugates.20 Table 1 summarizes the physicochemical properties of the block copolymers used in the study. Briefly, the suspension of AgNPs (AuNPs) was placed in a 96-well plate and mixed with an aliquot of Pluronic solution to obtain final concentration of the copolymers of 0.1-500 μg mL-1. The mixture was incubated for 1 h at room temperature allowing the copolymer to be adsorbed on the surface of the nanoparticles. The procedure resulted in the modification of AgNPs (AuNPs) and protected them against salt aggregation detected visually by the decolouration of nanoparticle suspension. Thus, we ascertained minimal concentrations of Pluronics required for the formation of stable modified nanoparticles which were resistant to the aggregation in 0.04 M sodium chloride solution (Table 2). Pluronic-modified nanoparticles, which contained minimal amount of unbound polymers, were further analyzed without a separation. UV-visible absorption spectra of the modified nanoparticles (150 μg mL-1) were registered on a spectrophotometer Multiskan Spectrum (Thermo Scientific). (19) Kahraman, M.; Yazici, M. M.; Sahin, F.; Culha, M. J. Biomed. Opt. 2007, 12, 054015. (20) Oliver, C. Methods Mol. Biol. 1999, 115, 331–334.
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Table 1. Physicochemical Characteristics of Block Copolymers of Poly(ethylene oxide) and Poly(propylene oxide) (Pluronics)15 copolymer L121 F68 F127
molecular mass
average number of EO/PO units
HLB
cmc (μg mL-1)
4400 8400 12 600
10.0/68.3 152.7/29 200.5/65.2
1 29 22
4.4 4032 35.3
AgNPs premodified with Pluronics were mixed with rhodamine dyes or other analytes (final concentration 1-1000 nM) in water or a buffer solution. The resultant mixture was incubated for 1 h at room temperature to produce Pluronic-nanoparticledye complexes which were analyzed using SERS.
Structural Characterization of Modified Nanoparticles. Average size and structure of AgNPs and their complexes with Pluronic copolymers were determined by an atomic force microscope (Park Systems XE-100). An aliquot of the suspension of bare or modified AgNPs with the nanoparticle concentration of 6, 32, and 162 μg mL-1 was evenly spread on the surface of freshly cleaved mica and air-dried. Both bare and modified AgNPs were found to be better distributed on the substrate at the concentration of 32 μg mL-1 which was further used for the sample preparation. Dry samples were analyzed in air using AFM noncontact mode. Size distribution and surface charge of AgNPs and AuNPs and their complexes with Pluronics in water suspension were measured by a Zetasizer NanoZS instrument (Malvern Instruments). The measurements were performed at 25 °C using six subruns for each scanning. Surface-Enhanced Raman Spectroscopy. Bulk-Raman and SERS spectra were obtained by an automated InVia microRaman system (Renishaw) precalibrated against monocrystalline silicon wafer peak (520 cm-1). An aliquot of bare or Pluronic-modified AgNPs with adsorbed fluorescent dye was cast on a calcium fluoride substrate and air-dried to obtain uniformly distributed nanoparticle aggregates on the substrate. The spectra were acquired in the range 400-1700 Raman shift (cm-1) using an objective 20 and a diode laser 830 nm (power 30 mW; exposure time 10 s). For the quantification of rhodamine 6G adsorbed on the surface of modified nanoparticles, the intensity of characteristic SERS peaks from 10 aggregates of similar size were measured in each sample. The reproducibility of the SERS spectra of Rh6G obtained from Ag-F127 complex was provided as Figure 1 in the Supporting Information. The percent coefficient of variation (CV%) for Ag-F127 complex was calculated as around 25 for the SERS spectra obtained from the ten different aggregates located under the 20 microscope objective.
Results and Discussion Preparation and Optical Properties of Ag and AuNPPluronic Complexes. Synthesized AgNPs and AuNPs were modified with Pluronics (see Experimental Section) similarly to the procedure which is exploited to attach proteins to gold nanoparticles by means of hydrophobic association.20,21 Specifically, metal NPs were coincubated with Pluronics at different concentrations to produce modified nanoparticles which were more stable in salt solutions than unmodified ones. The fact that Pluronics improve colloidal stability of AgNPs upon salt aggregation assumes that the amphiphilic copolymers are absorbed on the surface of AgNPs to form polymernanoparticle complexes. The adsorption is presumably resulted from hydrophobic interactions between colloidal silver and poly(propylene oxide), PPO, block of the copolymers. Pluronics were (21) Abdullin, T. I.; Bondar, O. V.; Nikitina, I. I.; Bulatov, E. R.; Morozov, M. V.; Hilmutdinov, A. Kh.; Salakhov, M. Kh.; Culha, M. Bioelectrochemistry. 2009, 77, 37-42.
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Article Table 2. Effect of Pluronic Copolymers on Optical Properties of Silver and Gold Nanoparticles
nanoparticle
copolymer
MSC (μg mL-1)
absorbance increase (%)
AgNPsa
λmax shift (nm)
absorbance decreasec (%)
L121 1.5 F68 1.5 F127 1.5 L121 0.4 11.5 7 AuNPsb F68 3.0 2 F127 1.5 a AgNPs concentration 162 μg mL-1. b AuNPs concentration 45 μg mL-1. c In the presence of 0.04 M sodium chloride.
Figure 1. UV/vis absorption spectra of AgNP-Pluronic copolymer complexes in 0.04 M sodium chloride solution. Key: (1) unmodified AgNPs; (2) AgNP-L121; (3) AgNP-F127; (4) AgNP-F68.
reported to exhibit similar stabilizing effect when associated with AuNPs.18 Aggregation of plasmonic nanoparticles under different factors is known to accompany with a change of their visible spectra up to the disappearance of their characteristic color4. The addition of 0.04 M sodium chloride induced irreversible aggregation of prepared AgNPs due to screening of negative charge of citratecapped nanoparticles by sodium ions. This allowed us to estimate the efficiency of the modification of AgNPs with Pluronics which prevented nanoparticle aggregation in NaCl solution. By detecting decolouration of the suspension of AgNPs, we ascertained minimal stabilizing concentrations (MSC) of Pluronics required for stable modification of the nanoparticles. Although Pluronics used for the modification varied in molecular weight and HLB (Table 1), they exhibited the same MSC of 1.5 μg mL-1 (Table 2) indicating generally unspecific character of their interaction with colloidal silver. This MSC was further used to prepare AgNP-Pluronic complexes containing minimal amount of unbound copolymer. For comparison, Table 2 also represents Pluronic MSCs required for the modification of citrate-capped AuNPs (about 13 nm in diameter).21 For these nanoparticles, MSCs of the copolymers in NaCl solution were somewhat different from those observed for Pluronic-modified AgNPs. Particularly, hydrophobic Pluronic L121 (HLB 1) exhibited stabilizing effect at a concentration as low as 0.4 μg mL-1 whereas more hydrophilic Pluronics F127 (HLB 22) and F68 (HLB 29) stabilized AuNPs at higher MSCs of 1.5 and 3 μg mL-1, respectively (Table 2). The differences in MSCs of Pluronics observed for modified AgNPs and AuNPs show that Pluronic-nanoparticle interactions are affected by both the copolymer characteristics and the properties of metal colloids. In order to examine these interactions, we analyzed optical absorption spectra of silver and gold nanoparticles in the presence of Pluronic copolymers at their MSCs. We found that the Langmuir 2010, 26(7), 5153–5159
700 80 150 43 22 23
association of Pluronics with AgNPs did not result in a change of their typical plasmon spectra characterized by the absorption maximum at 410 nm. In case of AuNPs, only Pluronic L121 induced noticeable increase of the absorption maximum at 518 nm by almost 11.5% as well as a shift of its wavelength (λmax) by 7 nm toward the red region (Table 2). Similar changes in AuNP spectra were detected upon their conjugation with proteins21 that attributed to the alteration of dielectric properties of nanoparticle environment.4 Pluronic F127, which is of similar PPO block length with Pluronic L121 but of much higher HLB, did not result in a change of AuNPs spectra after the modification and, as indicated above, exhibited higher MSC value compared to L121 (Table 2). Altogether, these results show that hydrophobic Pluronic L121 stronger interacts with the surface of AuNPs than two other copolymers. This reveals the importance of hydrophobic interactions upon the association of amphiphilic copolymers with metal nanoparticles. In case of AgNPs, Pluronic L121 along with other Pluronics did not affect the optical spectra of AgNPs and exhibited no difference in MSCs (Table 2). The fact can be explained, e.g., by weaker hydrophobic binding of Pluronic copolymers to AgNPs probably due to the presence of an oxide layer on the surface of colloidal silver.22 We also compared optical spectra of Pluronic-modified AgNPs and AuNPs in sodium chloride solution in order to evaluate stabilizing effect of the copolymers. The stabilization of AgNPs by Pluronics prevented the disappearance of optical absorption spectra of the nanoparticles. In particular, in the presence of NaCl, the complexes of Pluronics with AgNPs were still characterized by a well-defined absorbance peak at about 410 nm (Figure 1). This absorbance peak was almost nonshifted compared to the spectra of as-synthesized AgNPs (not shown). However, the peak was considerably decreased by height in NaCl solution to different extent depending on Pluronics used for the modification. In particular, the optical absorbance of AgNPs-Pluronic complexes decreased distinctly in the order of Pluronics F68, F127, and L121 (Figure 1). Whereas the copolymers themselves did not affect the optical properties of AgNPs (Table 2), this demonstrates that Pluronics differ in their protection of the nanoparticles against salt aggregation. The more hydrophilic Pluronic F68 exhibited the highest stabilizing effect on AgNPs whereas hydrophobic L121 showed the lowest one. Similar results were obtained for AuNPs which exhibited better colloidal stability in 0.04 M NaCl after the modification with Pluronics F68 or F127 compared to Pluronic L121 (Table 2). These results demonstrate that, although Pluronics of higher HLB seem to interact weaker with metal nanoparticles, these copolymers provide better stabilization of the colloids than Pluronics of lower HLB. Specifically, after the modification with Pluronic L121 (HLB 1) both AgNPs and AuNPs are more prone to aggregate in NaCl solution apparently due to L121 promotes (22) Quaroni, L.; Chumanov, G. J. Am. Chem. Soc. 1999, 121, 10642–10643.
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hydrophobic interactions between modified nanoparticles. In comparison with Pluronic L121, Pluronic F127 (HLB 22) and especially Pluronic F68 (HLB 29) with relatively long PEO blocks impart higher hydrophilicity to AgNPs reducing their nonspecific interactions. Structural Characterization of Ag and AuNP-Pluronic Complexes. The structure of metal NP-Pluronic complexes were analyzed by means of atomic force microscopy (AFM) as well as dynamic light scattering (DLS) and laser Doppler electrophoresis. Figure 2 shows representative AFM images of bare AgNPs and Pluronic-modified AgNPs spread on a mica substrate. Unmodified AgNPs appeared on the substrate in a form of aggregated complexes composed of three or more spherical particles (Figures 2A). Such aggregates are apparently formed as a result of sticking of AgNPs upon drying of the sample. Two particle fractions of AgNPs were generally revealed on AFM images; the smaller nanoparticles were 13.1 ( 4.7 nm and the larger ones were 32.7 ( 7.7 nm in size as measured by their height. Modification of AgNPs with Pluronics F127 or F68 resulted in a partial decrease of the aggregation extent of the nanoparticles on the substrate. The resulting AgNP-F127 and AgNP-F68 complexes represented well-defined aggregates consisting mainly of 2-3 particles (Figure 2, parts B and C). The mean particle size of smaller/larger fractions was 13.5 ( 6.6/31.6 ( 5.5 nm for AgNP-F127 and 14 ( 4.1/37.4 ( 13.1 nm for AgNP-F68 complexes. No relevant changes in the size of AgNPs were revealed after their modification with Pluronics F127 and F68. Unlike to these complexes, AgNPs modified with Pluronic L121 formed amorphous and relatively massive aggregates with mean particle size of 16.6 ( 4.5 and 37.4 ( 6.6 nm for smaller and larger fractions, respectively (Figure 2D). Thus, certain increase of the size of AgNP-L121 was observed in comparison with bare AgNPs and AgNPs modified with Pluronics F127 and F68. This can be explained by hydrophobic nature of Pluronic L121 which promotes the aggregation of AgNP-L121 complexes upon sample preparation. Based on DLS analysis, both AgNPs and Pluronic-modified AgNPs exhibited good colloidal properties. Specifically, the mean hydrodynamic diameter (Z-average) of unmodified AgNPs was of 54.3 nm with polydispersivity index (PdI) of 0.31 (see Supporting Information for details). Light scattering profile of the nanoparticles contained two distinct peaks with a maximum at about 84.9 and 11.5 nm indicating that AgNPs consisted of two particle fractions. Hydrodynamic diameter of the larger fraction was almost three-times higher than the size of large AgNPs measured by AFM. This probably arises from curtain heterogeneity of AgNPs synthesized by citrate reduction and also from reversible interparticle association in the suspension. We found that hydrodynamic diameter of AgNPs was almost unchanged after their modification with Pluronic copolymers (see Supporting Information). We also performed DLS measurement of the bare AuNPs and AuNP-Pluronics complexes to compare the size distribution after the modification with Pluronics (Data is not shown). This is in good agreement with AFM data revealed no relevant difference in the size of bare and modified metal NPs. This implies that under experimental conditions Pluronic copolymers seem to form very thin layers around nanoparticles. According to electrophoresis data, the mean zeta potential of unmodified AgNPs was -42.2 mV (pH g 7) as if synthesized AgNPs formed relatively stable negatively charged colloidal system. After the association with Pluronics, the zeta potential of AgNPs reduced (from -37 to -35 mV) indicating that Pluronics affect in some extent the surface properties of AgNPs (see Supporting Information). It is important to note that 5156 DOI: 10.1021/la9036309
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Pluronic copolymers have found to induce more noticeable change in the surface charge of AuNPs, so that their zeta potential (-44.8 mV) has reduced after the modification (from -22.2 to -16.4 mV). The data show that Pluronics interact more strongly with AuNPs than AgNPs presumably due to colloidal gold provides better surface for hydrophobic association with amphiphilic copolymers. This is in agreement with the results on optical properties of the nanoparticles as well as Pluronic MSCs (Table 2). In the case of AuNPs, the properties were shown to be dependent on characteristics of the copolymers as hydrophobic Pluronics were able to interact with AuNPs more specifically to alter their dielectric environment. Dissimilar to the AuNPs, the properties of the AgNPs were almost unaffected upon the modification with Pluronic copolymers. It is noteworthy to compare our results with those reported recently for hybrid complexes of AuNPs with Pluronic copolymers.18 In that study, the adsorption of Pluronics resulted in the formation of thick hydrated shells around the AuNPs. The concentrations of Pluronics used to prepare the hybrid nanoparticles were higher than critical micelle concentrations (CMC) of the copolymers.18 In order to prepare our composite AgNPs, we utilized Pluronics at MSCs, which were below their CMCs (see Tables 1 and 2). In this colloidal system, the AgNPs are expected to interact with the unimer form of amphiphilic Pluronics to produce surfacecovered nanoparticles rather than the nanoparticles imbedded in the polymer matrix. Altogether, our results allow us to propose the structural model of AgNP-Pluronic complexes that are composed of colloidal core surrounded by very thin polymeric shell. The shell is presumably formed as a result of the interaction of hydrophobic PPO block of Pluronic copolymers with the surface of AgNPs whereas hydrophilic PEO blocks are expected to be oriented in water phase (Figure 3). Interaction of Organic Compounds with Ag and AuNPPluronic Complexes. Above results demonstrated that the association of AuNPs and AgNPs with Pluronic copolymers at MSCs resulted in the formation of composite nanoparticles. These nanoparticles exhibited reproducible optical properties which were dependent on physicochemical characteristics of modified copolymers. Next, we applied surface-enhanced Raman spectroscopy (SERS) in order to ascertain how the copolymer component affects the adsorption of organic species on modified AuNPs and AgNPs. We studied xanthene dyes as model compounds commonly used in bioanalytical applications in the combination with nano- and microparticles.23 The possibility of using polymer coated metal NPs as SERS probes was investigated. Since the SERS activity of AuNPs was poor due to poor plasmonic property at the size it was used,8 the use of AgNPs coated with the Pluronics was explored. It was observed that there was no spectral contribution from Pluronics. This could be due to low Raman activity of the copolymers consisting of PEO and PPO blocks. After coincubation of AgNP-Pluronic complexes (150 μg/mL) with 50-1000 nM rhodamine 6G (Rh6G) well-defined Raman spectra of Rh6G appeared. Figure 4 represents typical SERS spectra of AgNPs and AgNP-L121 with adsorbed Rh6G. Specifically, for AgNP-L121 complexes registered Rh6G peaks gradually increased with the concentration increase from 10 nM (detection limit) to 500 nM. At the higher concentrations of Rh6G induced the aggregation of the complexes evidently as a result of the sticking of the aromatic dye to the surface of (23) Zhong, W. Anal. Bioanal. Chem. 2009, 394, 47–59.
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Figure 2. Topographic AFM-mages of AgNPs and their complexes with Pluronic copolymers on mica substrate. Key: (A) unmodified AgNPs; (B) AgNP-F127; (C) AgNP-F68; (D) AgNP-L121; (A0 -D0 ) magnified sections of the images. Langmuir 2010, 26(7), 5153–5159
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Figure 3. Schematic representation of the structure of AgNP with adsorbed Pluronic copolymers and small solutes in water. Key: (1) nanoparticle core; (2) Pluronic PPO blocks adsorbed on the nanoparticle by hydrophobic interaction; (3) water exposed Pluronic PEO blocks; (4) adsorbed solute.
Figure 4. SERS spectra of rhodamine 6G adsorbed on AgNPs (1) and their complexes with Pluronic L121 (2). Concentration of rhodamine 6G is 250 nM.
modified-AgNPs. This is accompanied by a certain decrease of Rh6G peaks height probably due to the retardation of Rh6G diffusion to aggregated nanoparticles. When bare AgNPs were used for the same SERS experiments, a concentration of 500 nM Rh6G was necessary for a SERS spectrum to appear. This indicates that Pluronic-modified AgNPs can easily detect the much lower concentrations of Rh6G due to the increased concentration of Rh6G molecules due to the strong interactions with the polymer surfaces (Figure 4). The peak at 1360 cm-1 was used to calculate the enhancement factor (EF) using the bulk Raman spectrum of Rh6G and SERS spectra obtained from AgNPs-Pluronic composites. The EFs were estimated using the formula (ISERS/IBulk) (NBulk/NSERS), where ISERS and IBulk are the scattering intensities for SERS and bulk Raman measurements, respectively, and NBulk and NSERS are the number of the analytes under the laser spot.24 Figure 5A shows the bulk-Raman spectrum obtained from the spot of 5 μL solution of 10 mM Rh6G located on CaF2. The intensity of the peak at 1360 cm-1 on bulk-Raman spectrum was 179. A proper amount of Rh6G was added into the colloidal suspension system of each AgNP- Pluronics to complete the Rh6G concentration to 250 nM. From this suspension, a 5 μL was placed onto CaF2 slide. With a laser power of 30 mW on the dried spot, the ten spectra from separate aggregates located under the 20x objective were acquired. Figure 5 B shows the average of the ten SERS spectra (24) Kahraman, M.; Tokman, N.; Culha, M. ChemPhysChem 2008, 9, 902–910.
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Figure 5. Bulk-Raman spectrum of 10 mM rhodamine 6G (A), SERS spectra of 250 nM rhodamine 6G adsorbed on AgNPPluronic F68 (1), AgNP-Pluronic L121 (2), and AgNP-Pluronic F127 (3) complexes (B), and enhancement factors of the Ag nanoparticles and its pluronic complexes (C).
obtained from AgNP-L121, AgNP-F127, AgNP-F68 composites and the intensity of the peak at 1360 cm-1 was calculated from these spectra as 26778, 87165, 10132, respectively. It should be noted that the intensity of this peak was 1089 for the unmodified AgNPs (see Figure 4). The size of the laser spot on top of the sample was estimated as 2 μm using a 20 objective with a 0.4 NA. When a 5 μL of 10 mM Rh6G solution was spotted on the CaF2 slide, the diameter of the spot was measured as 2 mm. Assuming that the Rh6G molecules homogeneously distributed on the substrate surface, the number of Rh6G can be calculated as 3.02 1010 in the area of 2 μm. In order to estimate the number of Rh6G molecules in the aggregates of AgNP-Pluronic composites, from the concentration Rh6G in the suspension, the number of Rh6G molecules per particle was estimated as 4.70 102. The number of AgNPs in a 1 mL suspension was taken approximately to be 3.2 1011. Then, the number of the AgNPs in 2 μm was Langmuir 2010, 26(7), 5153–5159
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estimated as 3.46 103 assuming the size of AgNPs as around 35 nm. From these assumptions, one can calculate the number of Rh6G molecules under the laser light as 1.63 106. Using the formula provided above, the EFs were 9.04 106 for AgNPF127, 2.78 106 for AgNP-L121, 1.05 106 for AgNP-F68, and 1.12 105 for AgNPs (see Figure 5C). The results demonstrate that Raman enhancing effect of Pluronic modified nanoparticles for Rh6G depends on the copolymer properties. In particular, the signal intensity of adsorbed Rh6G correlates with HLB and molecular weight of the copolymers. Pluronics L121 and F127, with average PPO block length of 68 and 65, respectively, allowed higher signal amplification of Rh6G compared to Pluronic F68 (PPO block length is about 29). Apparently, the extended PPO component of Pluronics L121 or F127 forms hydrophobic environment that favors the adsorption of aromatic dye on the surface of modified nanoparticles. Much more hydrophilic Pluronic F68 seems not to provide such an environment and therefore does not promote the interaction of Rh6G with AgNPs. At the same time, Pluronic F127 was found to enhance Raman signal of Rh6G more than three times higher than Pluronic L121. This indicates that PEO component of Pluronics also participates in the interaction of dye molecules with modified AgNPs. The extended PEO component is expected to stabilize the probe from possible flocculation in the presence of Rh6G thereby facilitating its surface reactions with the dye. The results revealed the effect of Pluronic copolymers properties, such as HLB, on the association of Rh6G with modified AgNPs. We assumed that the association is also affected by the properties of Rh6G, first of all, its hydrophilicity and charge. To ensure that Rh6G molecules, containing an imino group, were in protonated or deprotonated forms, we adjusted pH of the probe solution to pH 2.0 and 8.5, respectively. When Rh6G was adsorbed on the probe at pH 8.5, Raman signal was several times higher than at pH 2.0 (3.8 and 5.9 times higher for AgNP-L121 and AgNP-F127 complexes, respectively). This indicates that the charging of Rh6G upon protonation at pH 2.0 inhibits the binding of the analyte to modified nanoparticles. On the contrary, deprotonation of Rh6G seems to facilitate Pluronicmediated adsorption of the uncharged dye on the modified nanoparticles. Furthermore, rhodamine B containing unesterified carboxylic group was found to produce poorly defined Raman spectra in
Langmuir 2010, 26(7), 5153–5159
Article
contact with AgNP-Pluronic complexes both at pH 2.0 and 8.5. Altogether, these results demonstrate that the charge of analytes impairs their adsorption on the complexes resulting in the significant decrease of Raman signal. This further supports our model that nonionic Pluronic copolymers mediate the binding of aromatic species to the surface of modified nanoparticles by hydrophobic PPO component (Figure 3). Our results reveal how Pluronics copolymers can improve the sensitivity and specificity of SERS probes toward hydrophobic substances of biological interest, e.g., reporter molecules and drugs. In addition, we found that developed probes exhibit much more pronounced Raman enhancement for aminofluorescein and anticancer drug doxorubicin compared to AgNPs without Pluronics (see Supporting Information). Subsequent study will be performed elsewhere in order to ascertain the possibility of using Pluronic-based nanoprobes for SERS analysis of such compounds in complex matrixes including living cells.
Summary and Conclusions Nonionic amphiphilic polymers, such as Pluronic block copolymes, are a relevant material for the development of composite nanoparticles for biomedical applications. We showed that Pluronic unimers can be controllably adsorbed on the surface of AgNPs to produce new class of composite SERS probes. The modification does not alter the size of AgNPs but promotes hydrophobic association of resultant probes with certain organic compounds to considerably enhance their Raman signal. Pluronic-mediated interactions can be modulated by both physicochemical properties of the copolymers and the charge of analyzed solutes. Thus, Pluronic component allows improving the colloidal stability and analytical characteristics of metal nanoparticles as SERS probes. The results can be extended to develop efficient probes for SERS analysis of drugs and biomolecules in complex matrixes. Acknowledgment. The financial support of Yeditepe University and The Scientific and Technological Research Council of Turkey (TUBITAK) during this study is gratefully acknowledged. Supporting Information Available: A table of dynamic light scattering data and ζ potentials and figures showing SERS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la9036309
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